MICROFLUIDIC-BASED MULTIPLEX CELL ASSAY FOR DRUG COMPOUND

TESTING

FEDERALLY SPONSORED RESEARCH

This invention was made with government support under U01 HL114476 awarded by the National Institutes of Health. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

A major challenge with in vitro investigations of the pathophysiological processes (e.g., in sickle cell disease) has been the lack of a well-controlled microenvironment to mimic in vivo circulating conditions. For example, painful vasoocclusions, one of the hallmarks of sickle cell disease, are associated with rheological changes in blood flow resulting in blockage of microvasculature. The polymerization of hemoglobin S in deoxygenated environments and its resultant sickling and decrease in deformability leads to this increase in blood viscosity. Other factors, however, such as cell adhesion also contribute to blockage of blood flow, for example, by increasing the sickle RBC transit times in vessels, increasing the chances of sickling and reduction of deformability. A better understanding of how both deformability and adhesion contribute to vasoocclusions may offer therapeutic drug alternatives. Indeed, studies in deformability and adhesion are not limited to sickle cell disease and may prove useful for other diseases or disorders (e.g., cancer) where changes in adhesion play a pivotal role.

SUMMARY OF THE INVENTION

Cell adhesion is a form of communication and regulation between cells and their in vivo microenvironment. Often times dysfunction in cell specific adhesion is a sign of disease e.g. cancer, inflammatory diseases such as arthritis, sickle cell disease, etc. Detection and quantification of these adhesion specific changes would greatly aid in disease management. In addition, adhesion studies of cell populations can aid in the development of better tissue engineering alternatives and better drug treatment. In parallel, cell rigidity, stiffness, modulus or deformability is an indication of disease states, in many diseases such as cancer, malaria, sickle cell disease, leukemia, etc.

Provided herein are methods and conducted quantitative microfluidic measurements under controlled conditions. Controlled conditions may involve gas partial pressure modulation (diffusion rate control) using one or more gas mixtures, observing cell property (e.g. , adhesion, deformability) changes with different microenvironment conditions, such as precise shear stress modulation imposed by liquid flow profile, using different adhesive proteins (biophysical interaction between the cell/cell and/or cell/protein coated microchannel walls), under different temperatures, and osmolality control (constant or modulated osmolality).

The disclosure provides potential applications in patient specific disease diagnostics, screening drugs, studying drug efficacy, tissue engineering, etc.

Two pressure-driven flow systems were developed that can be paired with a two channel microfluidic -based platform to noninvasively study attachment and detachment of sickle cells, as one disease application, in a controlled microenvironment. One system utilizes changes in gravitational potential while the other uses pressurized liquid or air to precisely control flow velocity or pressure experienced by cells. In some embodiments, the

microfluidic platform provided herein also has gas control to enable the study of normoxic and hypoxic microenvironments, important for diseases such as sickle cell disease and cancer. Temperature and fluid and gas flow sensing capabilities also contribute to the controlled microenvironment. Finally, in some aspects, the cell/fluidic channel in the device has been designed to resemble the size scale of post-capillary venules, sites associated with vasoocclusions. A protein functionalization scheme was used for the cell/fluidic channel to ensure resilient spatially homogeneous binding of protein to the channel that can be easily imaged throughout the assay.

It is believed that, characterization of adhesive properties (e.g, of a cell binding to a protein coated substratum in a microfluidic channel) of sickle cells in the deoxygenated environment using microfluidics has not been reported. Early findings indicate differences in adhesion properties of sickle cells in a deoxygenated microenvironment as compared to the oxygenated microenvironment. Differences in mechanical properties of adherent sickle cells in the deoxygenated microenvironment as compared to an oxygenated microenvironment were observed. Furthermore, differences in adhesion for sickle cells depending on the extracellular matrix protein present in the environment and its concentration as well as differences for sickle cells from on or off drug treated patients were observed. In addition, density fractionation was used to separate cells based on morphological and mechanical properties and characterize adhesion associated with less or more rigid sickle cells. These studies have also indicated density specific adhesion differences between sickle cells.

The devices and systems provided herein offer the ability to study adhesion noninvasively in a well-controlled microenvironment that can be easily customized in terms of gas, temperature, protein, protein amount and flow rate experienced by cells to resemble the desired in vivo environment.

The customized, high throughput nature of microfluidic platforms make them an ideal platform to study sickle cell disease and hematologic pathophysiology. Several platforms have specifically focused on quantification of red blood cell deformability and adhesion (e.g., to a protein coated substratum) for diseases such as sickle cell disease. These platforms however have not accounted for hypoxia, which is central to recapitulating the sickle cell vasoocclusive microenvironment, and its role in deformability and adhesion.

Provided herein is a microfluidic platform that has improved on current microfluidic devices in its ability to control the gas mixture and its diffusion rate into the cellular microenvironment using a deformable PDMS membrane. This improvement enables the study of hypoxic microenvironments relevant for sickle cell disease and other diseases such as cancer. Other advantageous features of the platform include the ability to customize the flow profile to pulsatile or continuous flow to simulate the precise shear rate experienced by sickle cells at sites of vasoocclusion and ability to modulate temperature and geometry of a microfluidic channel. A protein functionalization scheme that can be quantified and imaged and is robust enough to stay intact during flow rate modulation has also been utilized.

Uses of the methods, devices and systems of the present disclosure include recapitulating complex in vivo microenvironments for tissue engineering and drug testing applications though systematic study of individual parameters such as gas, geometry, shear stress, protein and how they contribute to diseases such as cancer or blood-related diseases. Furthermore, image analysis can be time-intensive and may be automated to facilitate analysis. This can be done by using available high-end commercial software packages, or by developing customized image analysis packages.

Some aspects of the disclosure relate to assessing red blood cells. Red blood cell

(RBC) diseases, especially those that involve hemolysis, are often closely linked to RBCs' fragility or susceptibility to hemolysis. Commonly used fragility testing methods, including the osmotic fragility test and various forms of mechanical fragility tests, are not single-cell assays; they cannot be used to obtain the exact pressure or stress under which individual RBCs lyse, and they all require relatively large blood volume (-3-20 mL). Currently there is no high throughput method that can accurately test individual RBCs' fragility with a minimal amount of required blood volume (-100 μί). Accordingly, provided herein are microfluidic based assays for testing mechanical fragility at single cell level requiring a small blood volume. For validation purposes the assay can be designed to handle 20-100 cells per test; however due to the inherent advantages of microfluidic processability utilizing automated RBC loading and fluidic multiplexing, it can be easily extended with further engineering development for hemolytic screening of 2,000-10,000 cells in less than an hour. The setup can also be combined with a precise control of the varying gas microenvironment (including oxygen, nitric oxide etc.) for RBCs being tested. This setup, is expected to have high impact in providing a fundamentally new microfluidic -based assay for RBC mechanical fragility profiling at single-cell level requiring only a small blood volume (-100 μί), which can be used for being part of the assessment of the damage in stored blood (blood storage lesion), for the diagnosis of hemolytic diseases/disorders such as hereditary spherocytosis, and for providing new fundamental insights for various hemolytic diseases such as sickle cell anemia.

In some aspects, the invention is a high throughput method of measuring a

morphological and/or mechanical property of an individual cell under controlled gas conditions comprising: flowing a fluid comprising a plurality of cells through a channel comprising a wall, wherein at least a portion of the wall is coated with at least one protein, obtaining at least one measurement of a cell in the fluid; and regulating a level of gas in the fluid.

In some embodiments the property is a morphological property. In other embodiments the morphological property is cell shape. In another embodiment the cell shape is abnormal. In other embodiments the cell shape is round, disk shaped, biconcave, oblong, or sickle shaped.

In some embodiments the morphological property is cell texture. In another embodiment the cell texture is abnormal. In other embodiments the cell texture is smooth, coarse, or spiky.

In some embodiments the measurement is a fraction of cells with an abnormal shape or texture. In another embodiment the measurement is a delay time of an abnormal cell shape change. In another embodiment the measurement is a delay time of recovering from an abnormal shape change. In yet another embodiment the cell shape change is sickling or unsickling.

In some embodiments the cells are bound to the portion of the wall that is coated with at least one protein. In other embodiments the cells are not bound to the portion of the wall that is coated with at least one protein. In some embodiments the measurement is used to determine a proportion of the cells that have an abnormal shape or texture at a certain temperature, flow rate and/or gas concentration.

In some embodiments the property is a mechanical property. In other embodiments the mechanical property is adhesiveness. In yet another embodiment the mechanical property is adhesiveness to the portion of the wall that is coated with at least one protein.

In some embodiments the cell is bound to a fixed position on the portion of the wall that is coated with at least one protein. In other embodiments the cell slides, rolls, or tumbles along the portion of the wall that is coated with at least one protein.

In some embodiments the measurement is a number of and/or fraction of cells that bind to the portion of the wall that is coated with at least one protein. In other embodiments the measurement is a rate at which cells bind to the portion of the wall that is coated with at least one protein. In another embodiment the measurement is a speed that a cell slides, rolls, or tumbles along the portion of the wall that is coated with at least one protein. In yet another embodiment the measurement is a distance that a cell slides, rolls, or tumbles along the portion of the wall that is coated with at least one protein.

In some embodiments the measurement is a number of and/or a fraction of cells that detach from the portion of the wall that is coated with at least one protein. In another embodiment the measurement is used to determine a number of and/or fraction of cells that bind to the portion of the wall that is coated with at least one protein at a certain temperature, flow rate and/or gas concentration. In other embodiments the measurement is used to determine a rate at which cells bind to the portion of the wall that is coated with at least one protein at a certain temperature, flow rate and/or gas concentration.

In some embodiments the measurement is used to determine an average speed that a cell slides, rolls, or tumbles along the portion of the wall that is coated with at least one protein at a certain temperature, flow rate and/or gas concentration. In other embodiments the measurement is used to determine an average distance that a cell slides, rolls, or tumbles along the portion of the wall that is coated with at least one protein at a certain temperature, flow rate and/or gas concentration. In another embodiment the measurement is used to determine a number of and/or a fraction of cells that detach from the wall that is coated with at least one protein at a certain temperature, flow rate and/or gas concentration.

In some embodiments the method comprises contacting the cell with the wall that is coated with at least one protein while the fluid is flowing through the channel. In other embodiments the method further comprises stopping the flow of the fluid through the channel.

In some embodiments the method comprises contacting the cell with the wall that is coated with at least one protein while the fluid is not flowing through the channel. In other embodiments the method further comprises starting the flow of the fluid through the channel.

In some embodiments the mechanical property is deformability.

In some embodiments the cell is bound to a fixed position on the portion of the wall that is coated with at least one protein.

In some embodiments the measurement is an amount that the cell deforms. In other embodiments the measurement is the distance that a cell stretches. In another embodiment the measurement is a ratio of the length versus the width of the cell. In another embodiment the measurement is used to determine an amount a cell deforms at a certain temperature, flow rate and/or gas concentration. In yet another embodiment the measurement is used to determine an average amount that cells deform at a certain temperature, flow rate and/or gas concentration.

In some embodiments the cells are from a subject. In another embodiment the cells are from a blood sample. In other embodiments the cells comprise red blood cells, white blood cells, stem cells or epithelial cells. In another embodiment the cells are red blood cells. In yet another embodiment the cells comprise one or more tumor cells.

In some embodiments the gas is selected from the group consisting of oxygen, nitrogen, carbon dioxide, nitric oxide, carbon monoxide, nitrous oxide, nitrogen dioxide and/or methane. In other embodiments the gas is oxygen. In another embodiment the level of the gas in the fluid is regulated to be at a concentration of less than 5%. In another embodiment the level of the gas in the fluid is regulated to be at a concentration from 5% to 20%. In other embodiments the level of the gas in the fluid is regulated to be at a

concentration from 20% to 40%. In other embodiments the level of the gas in the fluid is regulated to be at a concentration from 40% to 60%. In other embodiments the level of the gas in the fluid is regulated to be greater than 60%. In another embodiment the level of the gas in the fluid is regulated to be at a concentration of about 20%. In another embodiment the level of the gas in the fluid is regulated to be at a concentration of about 5%. In other embodiments the level of the gas in the fluid is regulated to be at a concentration of about 2%. In other embodiments the level of the gas in the fluid is regulated to be at a concentration of about 20% oxygen, 5% carbon dioxide and about 75% nitrogen. In another embodiment the level of the gas in the fluid is regulated to be at a concentration of about 5% oxygen, 5% carbon dioxide and about 90% nitrogen. In yet another embodiment the level of the gas in the fluid is regulated to be at a concentration of about 2% oxygen, 5% carbon dioxide and about 93% nitrogen.

In some embodiments the property is measured at two or more different gas concentrations. In other embodiments the gas concentration is increased. In another embodiment the gas concentration is decreased. In yet another embodiment the property is measured as a function of time and as a function of gas concentration.

In some embodiments the cells are from a subject having or suspected of having a condition or disease selected from the group consisting of sickle cell disease (SCD), sickle cell trait (SCT), spherocytosis, ovalocytosis, alpha thalassemia, beta thalassemia, delta thalassemia, malaria, anemia, diabetes and leukemia. In some embodiments the cells are from a subject having or suspected of having sickle cell disease.

In some embodiments the fluid comprising the cells is flowed at a predetermined flow rate. In another embodiment the flow rate is in a range of about 0.01 μΐ/min to about 1000 μΐ/min. In another embodiment the flow rate is in a range of about 0.1 μΐ/min to about 50 μΐ/min. In other embodiments the flow rate is in a range of about 0.1 μΐ/min to about 10 μΐ/min. In yet another embodiment the flow rate is in a range of about 0.1 μΐ/min to about 1 μΐ/min.

In some embodiments the fluid comprising the cells is flowed at a predetermined pressure gradient. In another embodiment the pressure gradient is in a range of about 0.01 Pa/μιη to 10 Pa/μιη. In other embodiments the pressure gradient is in a range of about 0.1 Pa/μιη to 5 Pa/μιη. In yet another embodiment the pressure gradient is in a range of about 0.1 Pa/μιη to 2 Pa/μιη.

In some embodiments the flow rate or pressure gradient is increased relative to the predetermined flow rate or predetermined pressure gradient. In other embodiments the flow rate or pressure gradient is decreased relative to the predetermined flow rate or predetermined pressure gradient. In another embodiment the flow rate or pressure gradient is ceased. In other embodiments the flow rate or pressure gradient is continuous. In another embodiment the flow rate or pressure gradient is not continuous. In yet another embodiment the fluid is pulsed through the channel.

In some embodiments the property is measured after one or more reoxygenation (ReOxy) cycles. In other embodiments the property is measured after at least 5, 10, 20, 50, or 100 reoxygenation (ReOxy) cycles. In another embodiment the property is measured after one or more deoxygenation (DeOxy) cycles. In yet another embodiment the property is measured after at least 5, 10, 20, 50, or 100 deoxygenation (DeOxy) cycles.

In some embodiments the fluid comprising the cells is flowed at a predetermined temperature. In other embodiments the temperature is a physiological temperature.

In some embodiments the protein comprises a cell surface protein or extracellular matrix (ECM) protein. In other embodiments the cell surface protein is a cell adhesion molecule. In another embodiment the cell surface protein is an integrin, a cadherin, a selectin, a receptor tyrosine kinase, or a G-protein coupled receptor. In another embodiment the ECM protein is collagen, laminin, or fibronectin. In yet another embodiment the protein comprises an antibody.

In some embodiments the fluid comprising a plurality of cells is flowed through the device described herein. In some aspects, the invention is a microfluidic device comprising: a structure defining a microfluidic channel that comprises a first wall adjacent to the microfluidic channel, wherein at least a portion of the first wall is coated with at least one protein; and a second wall adjacent to the microfluidic channel, wherein at least a portion of the second wall comprises a gas permeable membrane or film.

In some embodiments the first wall is a top wall, a bottom wall, and/or a side wall. In other embodiments the second wall is a top wall, a bottom wall, and/or a side wall. In other embodiments the first wall and the second wall are different walls of the microfluidic channel. In another embodiment the first wall and the second wall are the same wall of the microfluidic channel. In yet another embodiment at least 10%, 20%, 30%, 40%, 50%, 60% 70%, 80%, or at least 90% of the second wall comprises a gas permeable membrane or film.

In some embodiments the second wall is a top wall. In other embodiments the second wall is a bottom wall or a side wall. In other embodiments the entire second wall comprises a gas permeable membrane or film. In another embodiment the second wall is a top wall. In yet another embodiment the second wall is a bottom wall or a side wall.

In some embodiments the first wall is a bottom wall. In other embodiments the first wall is a top wall, or a side wall. In another embodiment at least 10%, 20%, 30%, 40%, 50%, 60% 70%, 80%, or at least 90% of the first wall is coated with at least one protein. In other embodiments the first wall is a bottom wall. In another embodiment the first wall is a top wall or a side wall. In some embodiments the entire wall is coated with at least one protein.

In some embodiments the first wall is a bottom wall. In other embodiments the first wall is a top wall or a side wall.

In some embodiments the channel comprises a substantially planar transparent first wall and/or second wall. In other embodiments the first wall is a substantially planar transparent wall. In another embodiment the second wall is a substantially planar transparent wall. In another embodiment the substantially planar transparent wall is glass or plastic. In other embodiments the substantially planar transparent wall has a thickness in a range of 0.05 mm to 0.2 mm. In another embodiment the substantially planar transparent wall permits observation into the microfluidic channel by microscopy.

In some embodiments the protein comprises a cell surface protein or extracellular matrix (ECM) protein. In other embodiments the cell surface protein is a cell adhesion molecule. In another embodiment the cell surface protein is an integrin, a cadherin, a selectin, a receptor tyrosine kinase, or a G-protein coupled receptor. In other embodiments the ECM protein is collagen, laminin, or fibronectin. In yet another embodiment the protein comprises an antibody.

In some embodiments at least one measurement of cell that passes through the microfluidic device can be obtained. In other embodiments the microfluidic channel has a height from a top wall to a bottom wall in a range of 1 μιη to 50 μιη. In another embodiment the microfluidic channel has a height from a top wall to a bottom wall in a range of 1 μιη to 20 μηι. In other embodiments the microfluidic channel has a height from a top wall to a bottom wall in a range of 5μιη to 20 μιη. In another embodiment the microfluidic channel has a height from a top wall to a bottom wall in a range of 10 μιη to 20 μιη. In another embodiment the microfluidic channel has a height from a top wall to a bottom wall of 15 μιη.

In some embodiments the microfluidic channel has a width from a first side wall of the microfluidic channel to a second side wall of the microfluidic channel in a range of 0.1 mm to 3 mm. In other embodiments the microfluidic channel has a width from a first side wall of the microfluidic channel to a second side wall of the microfluidic channel in a range of 1 mm to 2 mm. In another embodiment the microfluidic channel has a width from a first side wall of the microfluidic channel to a second side wall of the microfluidic channel of 1.3 mm.

In some embodiments the microfluidic channel has a length in a range of 1 mm to 10 mm. In other embodiments the microfluidic channel has a length in a range of 2 mm to 5 mm. In another embodiment the microfluidic channel has a length of 3 mm. In some embodiments the microfluidic channel comprises at least one inlet at a first end of the microfluidic channel and/or at least one outlet at a second end of the microfluidic channel. In other embodiments the microfluidic channel comprises at least one inlet at a first end of the microfluidic channel. In another embodiment the microfluidic channel comprises at least one outlet at a second end of the microfluidic channel.

In some embodiments the microfluidic device further comprises a reservoir fluidically connected with the microfluidic channel. In other embodiments the microfluidic device further comprises a pump that is connected to the reservoir, and is configured to perfuse fluid from the reservoir to the microfluidic channel. In another embodiment the reservoir comprises an inlet at a first end of the reservoir and an outlet at a second end of the reservoir.

In some embodiments the microfluidic device further comprises a microscope configured to permit observation within the microfluidic channel through the first wall of the microfluidic device. In another embodiment at least one measurement of a cell that passes through one of the microfluidic channels can be obtained.

In some embodiments the microfluidic device further comprises a heat transfer element that directly or indirectly contacts the microfluidic device. In another embodiment the heat transfer element is configured to maintain a fluid in the microfluidic channel at a predetermined temperature. In other embodiments the predetermined temperature is a physiologically relevant temperature. In another embodiment the physiologically relevant temperature is in a range of 30 °C to 45 °C. In other embodiments the physiologically relevant temperature is 37 °C. In another embodiment the physiologically relevant temperature is 41 °C.

In some embodiments the microfluidic device further comprises a gas channel, wherein the gas channel comprises a wall that contacts the second wall of the microfluidic device, and wherein the gas channel contacts at least a portion of the gas permeable membrane or film of the microfluidic device. In other embodiments the gas channel contacts the entire portion of the gas permeable membrane or film. In another embodiment the gas channel comprises an inlet at first end of the gas channel. In other embodiments the gas channel comprises an outlet at a second end of the gas channel. In other embodiments the gas permeable membrane or film comprises polydimethylsiloxane (PDMS), hydroxypropyl cellulose (HPC), hydroxypropyl methylcellulose (HPMC), cellulose triacetate (CTA), or poly(methyl methacrylate) (PMMA). In yet another embodiment the gas permeable membrane or film comprises polydimethylsiloxane (PDMS). In some aspects, the invention is a method for identifying a therapeutic agent, the method comprising: perfusing a fluid comprising one or more cells through the device described herein; administering one or more compounds to the fluid, or wherein the fluid comprises the one or more compounds; determining a property of one or more of the cells; and comparing the property to an appropriate standard, wherein the results of the comparison are indicative of the therapeutic effectiveness of the compound.

In some embodiments two or more of the compounds are administered to the fluid sequentially. In other embodiments two or more of the compounds are administered simultaneously. In another embodiment the method further comprises identifying an effective therapeutic agent based on the comparison above.

In some embodiments the cells are from a subject. In other embodiments the method further comprises administering the effective therapeutic agent to the subject.

In some embodiments the compounds are from a library of compounds. In other embodiments the compounds are candidate therapeutic agents.

In some aspects, the invention is a method for analyzing a condition or disease in a subject, the method comprising: perfusing a fluid comprising one or more cells from the subject through the device described herein; determining a property of one or more of the cells; and comparing the property to an appropriate standard, wherein the results of the comparison are indicative of the status of the condition or disease in the subject.

In some embodiments the method of analyzing the condition or disease is a method for detecting the presence or absence of the condition or disease in the subject, and wherein the property is indicative of the presence of the condition or disease in the subject. In other embodiments the method of analyzing the condition or disease is a method for detecting the presence or absence of the condition or disease in the subject, and wherein the property is indicative of the absence of the condition or disease in the subject. In another embodiment the method of analyzing the condition or disease is a method for determining the severity of a condition or disease in the subject, and wherein the property is indicative of the severity of the condition or disease in the subject. In other embodiments the method of analyzing the condition or disease is a method for predicting vaso-occlusion crises in a subject, and wherein the property is indicative of a likelihood that the subject will undergo vaso-occlusion crisis.

In some embodiments the cells comprise blood cells. In some embodiments the condition or disease is selected from the group consisting of sickle cell disease (SCD), sickle cell trait (SCT), spherocytosis, ovalocytosis, alpha thalassemia, beta thalassemia, delta thalassemia, malaria, anemia, diabetes and leukemia. In another embodiment the condition or disease is sickle cell disease.

In some embodiments the property is a mechanical property. In another embodiment the property is deformability, or adhesiveness.

In some embodiments the property is deformability. In other embodiments the property is adhesiveness.

In some aspects, the invention is a method for monitoring the effectiveness of a therapeutic agent for treating a disease or condition in a subject comprising: (a) perfusing a fluid comprising one or more cells from the subject through the device described herein; (b) determining a property of one or more of the cells; (c) treating the subject with the therapeutic agent; and (d) repeating steps (a) and (b) at least once wherein a difference in the property of one or more cells is indicative of the effectiveness of the therapeutic agent.

In some aspects, the invention is a method for determining the effectiveness of a therapeutic comprising: (a) obtaining a biological sample from a subject comprising a cell; (b) perfusing a fluid comprising one or more cells from the subject through the device described herein; (c) determining a property of one or more of the cells; (d) contacting the biological sample comprising a cell with the therapeutic; (e) perfusing a fluid comprising the product of (d) through the device described hereon; (f) determining a property of one or more of the cells from (e); and (g) comparing the property of one or more cells from (c) with the property of one or more cells from (f), wherein the results of the comparison are indicative of the effectiveness of the therapeutic.

In some embodiments the therapeutic is for treating sickle cell disease.

In some embodiments the therapeutic is hydroxyurea (HU) or 5- hydroxymethylfurfural (Aes-103).

In some aspects, the invention is a real-time method for quantifying cell

morphological kinetics in response to varying levels of gas comprising: (a) perfusing a fluid comprising one or more blood cells through the device described herein, wherein the fluid has a first level of gas; (b) determining a property of one or more of the cells from (a); (c) perfusing a fluid comprising one or more cells through the device described herein; wherein the fluid has a second level of gas that is different from the first level; (d) determining a property of one or more of the cells from (c); and (e) quantifying the cell morphological kinetics of the cells from (b) and (d).

In some embodiments the cell morphological kinetics are cell sickling and/or unsickling kinetics.

In some aspects, the invention is a system comprising any of the microfluidic devices provided herein and a gas source, wherein the gas source is connected to the gas channel of the microfluidic device via one or more supply lines.

In some embodiments the gas source is configured to flow gas from the gas source into the gas channel of the microfluidic device. In another embodiment the gas source comprises oxygen, nitrogen, and/or carbon dioxide. In other embodiments the gas source is connected to the gas channel of the microfluidic device via a gas flow sensor and/or a gas flow regulator.

In some embodiments the system further comprises a microscope configured to observe one or more cells within the microfluidic device. In another embodiment the system further comprises a reservoir that is fluidically connected to the microfluidic channel of the microfluidic device via one or more supply lines. In other embodiments the reservoir comprises a fluid, and wherein the fluid comprises a plurality of cells. In another embodiment the reservoir is connected to the microfluidic channel via a flow sensor. In other

embodiments the reservoir is connected to the microfluidic channel via a pressure regulator and or a fluid flow sensor. In yet another embodiment the pressure regulator is configured to pulsate the fluid from the reservoir into the microfluidic device.

In some embodiments the system further comprises a temperature regulator that is configured to regulate the temperature of a fluid within the microfluidic device.

In some embodiments the system further comprises a computer in electronic communication with the gas flow sensor, the gas flow regulator, the pressure regulator, and/or the fluid flow sensor, wherein the computer comprises an input interface configured to receive information from the fluid flow sensor and/or the gas flow sensor, and an output interface configured to provide an output signal to the pressure regulator and/or the gas flow regulator. In other embodiments the computer is in electronic communication with the microscope, and wherein the input interface is configured to receive information from the microscope, and wherein the output interface is configured to provide an output signal to the microscope. In another embodiment the computer is in electronic communication with the temperature regulator, and wherein the input interface is configured to receive information from the temperature regulator, and wherein the output interface is configured to provide an output signal to the temperature regulator. In some aspects, the invention is a kit comprising a microfluidic device, wherein the microfluidic device comprises: a structure defining a microfluidic channel that comprises a first wall adjacent to the microfluidic channel; and a second wall adjacent to the microfluidic channel, wherein at least a portion of the second wall comprises a gas permeable membrane or film.

In some aspects, the invention is a kit comprising any of the systems described herein In some embodiments least a portion of the first wall is coated with at least one protein. In other embodiments the protein is in a container. In another embodiment the protein is lyophilized. In other embodiments the protein is in solution. In another embodiment the protein comprises a cell surface protein or extracellular matrix (ECM) protein. In other embodiments the cell surface protein is a cell adhesion molecule. In other embodiments the cell surface protein is an integrin, a cadherin, a selectin, a receptor tyrosine kinase, or a G- protein coupled receptor. In another embodiment the ECM protein is collagen, laminin, or fibronectin. In yet another embodiment the protein comprises an antibody.

In some embodiments the kit further comprises instructions for coating a wall of the microfluidic channel with the protein.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings are not intended to be drawn to scale. In the drawings, each identical or nearly identical component that is illustrated in various figures is

represented by a like numeral. For purposes of clarity, not every component may be labeled in every drawing. In the drawings:

FIG. 1- shows a non-limiting schematic representation of a system configuration for a microfluidic device.

FIGs. 2A-2C - show an example of protein functionalization and quantification. FITC-labeled fibronectin (FIG. 2A) and Rhodamine-labeled laminin (FIG. 2B) functionalized microfluidic channels at 100 μg/ml concentration imaged with a lOx objective lens. Protein quantification of a fibronectin functionalized channel using a Coomassie Blue assay (FIG. 2C). Mean +/- standard deviation.

FIGs. 3A-3B - are exemplary data showing that lighter density fraction sickle RBCs are more adherent to fibronectin. Density fractionation of sample showing four bands of increasing density (FIG. 3A). Adhesion percentage of individual density fractions under oxygenated and low flow conditions (FIG. 3B).

FIGs. 4A-4B - show exemplary detachment profiles for sickle cells adherent to fibronectin (FIG. 4A) and laminin (FIG. 4B) (50 μg/ml) after 15 minutes of static incubation. Times and shear rates corresponding to cells detached are provided. Percentage of cells detached are indicated by blue bars. Shear rate is indicated by a red dotted line.

FIG. 5 -shows examples of increased tethering of deformable sickle RBCs with increasing flow rates. Numbers listed to the right indicate time. Blue triangle represents increasing flow rate. Orange line represents length of tether as tethers cannot be observed at 63x objective magnification. For this specific attachment assay, sickle RBCs were incubated statically with 40 μg/ml of laminin functionalized microfluidic device for 15 min. After incubation, flow rate was steadily increased to detach adherent cells.

FIGs. 6A-6B - show exemplarydetachment profiles for sickle cells adherent to fibronectin under oxygenated (FIG. 6A) and deoxygenated (FIG. 6B) conditions after 15 minutes of static incubation. Red dotted line corresponds to pressure (mbar) cells experience for detachment.

FIG. 7 - shows exemplary hypoxia and dose-specific adhesion of sickle RBCs. Sickle RBCs were incubated for 15 min statically under oxygenated or deoxygenated conditions with fibronectin at the indicated concentrations.

FIGs. 8A-8B - show exemplary elongation ratio measurements in a deformable sickle cell at normoxia. Images of cell depicting (FIG. 8A) ratio of major to minor axes initially and (FIG. 8B) ratio of major to minor axes of right before detachment.

FIG. 9 - shows exemplary relaxation ratio measurements in deformable sickle cells. Time course images showing response of deformable sickle cell to perturbation (left; shaded in blue) and relaxation (right).

FIGs. 10A-10B - show an exemplary elongation response of same cell to 2000 mbar pressure perturbation under oxygenated (FIG. 10A) and deoxygenated (FIG. 10B) conditions.

FIG. 11 - is an exemplary schematic representation of the device design. FIG. 12 - is an exemplary schematic depicting a closed loop gravitational potential driven flow design setup.

FIG. 13 - is an exemplary schematic depicting open loop gravitational potential driven flow design setup.

FIG. 14 - is an exemplary schematic depicting pneumatic flow achieved through pressurized container or under vacuum reservoir.

FIG. 15 - is an exemplary schematic depicting pneumatic flow achieved through pressurized reservoir and single syringe as a source of back pressure.

FIG. 16 - is an exemplary schematic depicting pneumatic flow achieved through two pressurized reservoirs.

FIG. 17 - is an exemplary shape classification of oxygenated and deoxygenated sickle cells for fibronectin and laminin adhesion studies. Cell shapes are traditional classifications in sickle cell literature with elongated and irreversibly sickled cells having increasing aspect ratios as compared to discocyte populations. At least 100 cells were counted for each condition. Mean +/- standard deviation.

FIG. 18 - shows gas pressure-induced hemolysis (gas mixture of 20% 0 ₂ , 5% C0 ₂ , and 75%N ₂ ). Panels show exemplary images of red blood cells within the flow channel using an exemplary microfluidic device. Characteristic instances of red blood cell (RBC) lysis events are shown with dotted ovals. In the first image (t = 0) RBCs experience initial pressure under normoxic conditions, P _m i _t -n _o r _mox i _a ; from that point onward the increase in the control channel pressure is ~ 6 psi/min. Four images are shown, which represent red blood cells under pressure at zero seconds (t = 0 s), thirteen seconds (t = 13 s), twenty-two seconds (t = 22 s), and forty-four seconds (t = 44 s); left to right, respectively. At (t = 44 s) approximately one third of the RBCs within the field of view have lysed. The dotted oval with an asterisk indicates a lysing red blood cell. At t = 0, nine cells are highlighted with circles, which are labeled 1-9. In panels labelled t = 13 s, t = 22 s, and t = 44 s, circled cells, which are numbered, indicate cells that are undergoing lysis.

FIG. 19 - shows a magnified view of gas pressure-induced hemolysis of Fig. 18. Panels show exemplary images of red blood cells within the flow channel using an exemplary microfluidic device. In the first image (where t = 0 signifies the moment just prior to cell lysing) the red blood cell shown experiences initial pressure under normoxic conditions, Pini _t n _o r _mox i _a ; from that point onward the increase in the control channel pressure is ~ 6 psi/min. Four images are shown, which represent red blood cells under pressure a zero seconds (t = 0 s), 0.2 seconds (t = 0.2 s), 0.6 seconds (t = 0.6 s), and eight seconds (t = 8 s); left to right, respectively. At t = 0.2 s RBC membrane rupture in a single spot and consequent abrupt (~ milliseconds) release of hemolysis products is shown. At t = 0.6 s, hemolysis product diffusion within the microchannel is shown. There is an observable contrast change of the red blood cell image with respect to the background as the contents of the red blood cell are released. See from lysis initiation at t = 0 s up to t = 8 s.

FIG. 20 - shows a cross-sectional schematic representation of an exemplary micro- hemolytic device. The dual-layer design comprises a flow channel, where one or more cells (e.g. a red blood cell in suspension) flow through the flow channel. The design also comprises a control channel, where the pressure can be modulated (e.g., increased) to deform the membrane, thereby narrowing the flow channel. The narrowing of the flow channel may exert a force onto one or more cells flowing through the flow channel. The figure shows representative RBCs in the flow channel, which may be observed under a microscope objective lens. In the exemplary device shown, a PDMS membrane under pressure is deformed to narrow the flow channel.

FIG. 21 - shows gas pressure-induced hemolysis under hypoxic (DeOxy) gas conditions (gas mixture of 2% 0 ₂ , 5% C0 ₂ , 93% N ₂ ) under the identical experimental conditions as Figs. 18 & 19; and control channel pressure rate ~ 10 psi/min. Panels a-j show high speed imaging snapshots of RBC lysis over time. Abrupt sickled RBC membrane rupture and consequent release of visible clusters of polymerized HbS and hemolysis products under steady state deoxygenation. At t = 0 s the pressure at the sickle RBC is ~1 psi. At t = 24.8 s critical pressure for rupture recorded ~ 4.5 psi.

FIG. 22 - shows morphological heterogeneity of adherent sickle cells under shear flow and steady state hypoxia. The image shows adherent sickle cell heterogeneity after approximately 10 minutes of shear flow under hypoxic conditions. Walls of the flow channel are coated with fibronectin (FN-coated). Cells a-n are labelled.

FIG. 23 - shows the transition from single point (weak) adhesion to multiple point (firm) adhesion under shear flow and steady state hypoxia. Panels show successive images of the motion of "cell g," as indicated in FIG. 22, at the onset of attachment and after two minutes following attachment (t = 0 s). The cell attaches to the surface while forming a protruding membrane edge (dotted circle). At 1.5 seconds (t = 1.5 s) the cell has flipped around the attachment point. Following attachment, at 34 seconds (t = 34 s),the cell exhibits a jiggling motion under constant shear flow, indicative of having a single attachment point. At 2 minutes (t = 2 min), cell jiggling ceases and the cell appears firmly in place, indicating that the cell develops additional attachment sites while adherent. Reorganization of polymerized content within the cell via protrusion of pointy edges, presumably polymerized HbS fibers, are indicated with solid circles.

FIG. 24 - shows time-dependent adherent sickle shape change under shear flow and steady state hypoxia of cells "i", "f ' and "n" of Fig. 22. These characteristic snapshots showcase cells with minimal shape change and non-protruded polymer fibers outwards of the cell. In cell "i" between 0 s < t < 4min there is noticeable reorganization of polymerized content; the cell tends to form a tri-lobe after ~9 min. In cells "f ' and "n" the changes in morphology are minimal indicating the stable morphological steady state under

deoxygenation.

FIG. 25 - shows time-dependent adherent sickle shape change under shear flow and steady state hypoxia of an adhered reticulocyte. Panels show successive images of the cell shape change of "cell b," as indicated in FIG. 22. The image at t = 0 (panel a) shows the initial reticulocyte shape after attachment. From t = 9 s to t=54 seconds (panels b-f) gradual protrusion of polymer fibers (arrow heads) and an apparent increase of cell-surface contact area are observed. From t = 54 s to t = 7.9 min (panels f-j) polymer fibers continue to grow outward from the bulk of the cell. Estimated projected surface area increase between panel a and panel j is greater than approximately 15%.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION

Described herein are devices, methods, and systems for assessing cell properties, such as adhesion (e.g., to a protein coated on a substrate), morphology, and deformability under controlled gas environments. Accordingly, a microfluidics-based model was developed to quantify properties relating to the pathophysiology of disease (e.g., sickle cell disease (SCD), spherocytosis, ovalocytosis, alpha thalassemia, beta thalassemia, delta thalassemia, and anemia) under environments that mimic physiological conditions. For example, the devices provided herein may be used to mimic shear stress imposed by liquid flow, ambient gas partial pressures, gas content, osmolality, biophysical interaction between cells or between a cell and a substrate (e.g., a protein coated microchannel wall). This in vitro model enabled quantitative investigations of the kinetics of cell adhesion (e.g., attachment and detachment), cell morphology changes, such as cell sickling and unsickling, and cell deformability, for example of an adhered cell in response to a shear stress and/or in response to a change in a gas content. Exemplary uses of the devices provided herein included in the description, claims and Examples below. However, these uses are not meant to be limiting and additional uses would be apparent to the skilled artisan based on this disclosure. The Examples provided herein relate to SCD in order to demonstrate the effectiveness of the devices, systems and methods described herein. However, the invention is not limited to SCD. Other pathologies, for example cancer, may be examined using the devices, methods and systems provided herein. Briefly, the Examples demonstrate that the devices, systems and methods can be used to sequentially (i) identify and classify a cell shape and/or type within a cell population, (ii) assess cell attachment, (iii) assess adherent cell deformability, and (iv) assess cell detachment with "on demand" cell microenvironment modulation. For example under conditions of normoxia and hypoxia. These results point to the use of these devices, systems, and methods for a number of applications, including but not limited to, screening for and identifying therapeutics, assessing a patient response to a therapeutic, determining the effectiveness of a therapeutic, diagnosing a subject with a disease or condition, quantifying the kinetics of an adherent cell in response to changes in gas composition, as well as research applications. Microfluidic Devices

Devices are provided herein for evaluating, characterizing, and/or assessing properties of cells, such as cell adhesion, cell deformability and/or cell shape, under controlled gas conditions. In particular, devices are provided for measuring, evaluating and characterizing dynamic mechanical responses of biological cells, e.g. , red blood cells, to changes in the level of a gas (e.g., oxygen) and or in response to contacting a substrate comprising a protein (e.g., a laminin coated surface). The devices are typically designed and configured to permit measurements of cell shape, adhesion, and/or deformability in a high throughput manner. For example, by measuring the shape of a cell adhered to a wall of the microfluidic device in response to changes in temperature, osmolality, gas concentration or content, and/or fluid pressure.

The devices typically include a structure defining a microfluidic channel through which a fluid that comprises one or more cells may pass. The microfluidic channel of the devices typically include a wall, where at least a portion of the wall is coated with at least one protein. Any suitable protein may be coated onto at least a portion of the wall of the microfluidic channel. The protein used to coat at least a portion of the wall of the

microfluidic channel may depend on a number of factors, including but not limited to, the intended use of the device. It should be appreciated that methods for adhering (e.g., coating) a protein onto a substrate (e.g. , a wall of a microfluidic device) Any suitable protein may be used in accordance with this disclosure. In some embodiments, at least a portion of a wall of any of the microfluidic devices, provided herein, is coated with at least one protein. A wall of the microfluidic channel may be coated with any suitable protein or molecule comprising a protein. For example, the protein can be any protein that interacts (e.g., binds to) with one or more cells within the microfluidic channel of the microfluidic device. In some embodiments, the protein is a naturally-occurring protein (e.g., fibronectin or laminin). In some embodiments, the protein is a non-naturally occurring protein, for example, a variant of a naturally-occurring protein (e.g., fibronectin fused to a FITC molecule). In some embodiments, the non-naturally occurring protein comprises a naturally-occurring protein or a portion of a naturally-occurring protein with additional moieties (e.g., a protein tag, a sugar moiety, or a fluorescent molecule), which yields a protein that does not occur in nature. In some embodiments, the protein is fibronectin fused to FITC. In some embodiments, the protein is laminin fused to rhodamine. ECM Proteins:

Any number of ECM proteins, or variants thereof, may be used in accordance with this disclosure. In some embodiments, at least a portion of a wall of any of the microfluidic devices, provided herein, is coated with at least one ECM protein. In some embodiments, at least a portion of a wall is coated with an extracellular matrix (ECM) protein or variant thereof. The term "extracellular matrix protein, or ECM protein", as used herein, refers to a protein that typically occupies an extracellular space of a tissue (e.g., in an animal). In general, ECM proteins are a collection of extracellular molecules secreted by cells that provide structural or biochemical support to the surrounding cells. In some embodiments, the ECM protein is an ECM protein from an animal, for example, an interstitial matrix protein or a basement membrane protein. In some embodiments, the ECM protein is an interstitial matrix protein. Without wishing to be bound by any particular theory, interstitial matrix proteins are found in the interstitial matrix, which is present between various animal cells (e.g., the intercellular spaces). Gels of polysaccharides and fibrous proteins fill the interstitial space where they can act as a compression buffer against a stress placed on an extracellular matrix (ECM). In some embodiments, the ECM protein is a basement membrane protein. Without wishing to be bound by any particular theory, basement membrane proteins are proteins found in sheet-like depositions of ECM on which cell types (e.g., epithelial cells) rest. In some embodiments, at least a portion of a wall of the microfluidic device is coated with an ECM protein that comprises a carbohydrate polymer, (e.g., a glycosaminoglycan). In some embodiments, the ECM protein is a proteoglycan. In some embodiments, the ECM protein comprises a glycosaminoglycan. Examples of polysaccharides that may be bound to an ECM protein include, without limitation, heparan sulfate, chondroitin sulfate and keratin sulfate. However, it should be appreciated that additional polysaccharides may be bound to any of the ECM proteins provided herein and would be apparent to the skilled artisan. In some embodiments, the ECM protein comprises heparan sulfate. Heparan sulfate is a linear polysaccharide found in all animal tissues. It typically occurs as a proteoglycan in which two or three heparan sulfate chains are attached in close proximity to cell surface or ECM proteins. In some embodiments, the ECM protein comprises chondroitin sulfate.

Chondroitin sulfate is a sulfated glycosaminoglycan comprised of a chain of alternating sugars (e.g. , N-acetylgalactosamine) and is typically found attached to proteins as part of a proteoglycan. In some embodiments, the ECM protein comprises keratan sulfate. Keratan sulfate typically has a variable sulfate content and does not contain uronic acid. Generally, karatan sulfates are found in the cornea, cartilage, bones, and horns of animals.

In some embodiments, at least a portion of a wall of the microfluidic device is coated with a fibrillar ECM protein, which refers to a group of proteins present in an extracellular matrix as fibrous proteins, that typically provide structural support to resident cells. In some embodiments, the ECM protein is a collagen. Typically, collagen is exocytosed as a precursor form (e.g., procollagen), which is cleaved by a protease , thereby allowing extracellular assembly. In some embodiments, the collagen is a fibrillar collagen. For example, the collagen may be a type I collagen, at type II collagen, a type III collagen, a type V collagen, or a type XI collagen. In some embodiments, the collagen is a facit collagen. For example the collagen may be a type IX collagen, a type XII collagen, or a type XIV collagen. In some embodiments, the collagen is a short chain collagen. For example the collagen may be a type VIII collagen, or a type X collagen. In some embodiments, the collagen is a basement membrane collagen. For example, the collagen may be a type IV collagen. It should be appreciated that additional types of collagen may be used, for example type VI collagen, type VII collagen, and type XIII collagen, which are within the scope of this disclosure. In some embodiments, the protein is an elastin, which is a type of protein involved in modulating an elastic property of a tissue.

In some embodiments, at least a portion of a wall of the microfluidic device is coated with a fibronectin or a variant thereof. In general, fibronectins are proteins that connect cells with collagen fibers in the ECM. Typically, fibronectins bind collagen and cell-surface integrins, which may facilitate cell movement. In some embodiments, the fibronectin comprises the amino acid sequence as set forth in (SEQ ID NO: 1). In some embodiments, the fibronectin comprises a tag. In some embodiments, the fibronectin comprises a fluorescent tag. In some embodiments, the fibronectin comprises a fluorescent tag. In some embodiments, the fibronectin comprises a FITC tag.

Fibronectin:

MLGGPGPGLLLLLA VLS LGT A VPS AG AS KS RRQ AQQIVQPQS PLT VS QS KPGC YDNG KHYQINQQWERTYLGSALVCTCYGGSRGFNCESKPEPEETCFDKYTGNTYRVGDTY ERPKDSMIWDCTCIGAGRGRISCTIANRCHEGGQSYKIGDTWRRPHETGGYMLECVC LGNGKGEWTCKPIAEKCFDQAAGTSYVVGETWEKPYQGWMMVDCTCLGEGSGRIT CTSRNRCNDQDTRTSYRIGDTWSKKDNRGNLLQCICTGNGRGEWKCERHTSLQTTS AGSGSFTDVRTAIYQPQPHPQPPPYGHCVTDSGVVYSVGMQWLKTQGNKQMLCTC LGNGVSCQETAVTQTYGGNSNGEPCVLPFTYNGKTFYSCTTEGRQDGHLWCSTTSN YEQDQKYSFCTDHTVLVQTRGGNSNGALCHFPFLYNNHNYTDCTSEGRRDNMKWC GTTQNYDADQKFGFCPMAAHEEICTTNEGVMYRIGDQWDKQHDMGHMMRCTCVG NGRGEWTCVAYSQLRDQCIVDGITYNVNDTFHKRHEEGHMLNCTCFGQGRGRWKC DPVDQCQDSETRTFYQIGDSWEKYLQGVRYQCYCYGRGIGEWACQPLQTYPDTSGP VQ VIITETPS QPNS HPIQWS APES S HIS KYILRWKPKNS PDRWKE ATIPGHLNS YTIKGL RPG V V YEGQLIS VQH YGQRE VTRFDFTTTS TS P A VTS NT VTGETTPLS P V V ATS ES VT ΕΓΤ AS S F V VS W VS AS DT VS GFR VE YELS EEGDEPQ YLDLPS T ATS VNIPDLLPGRKYT VNVYEISEEGEQNLILSTSQTTAPDAPPDPTVDQVDDTSIVVRWSRPRAPITGYRIVYS PS VEGS S TELNLPET ANS VTLS DLQPG VQ YNITIY A VEENQES TP VFIQQETTG VPRS D KVPPPRDLQFVEVTDVKITIMWTPPESPVTGYRVDVIPVNLPGEHGQRLPVSRNTFAE VTGLSPGVTYHFKVFAVNQGRESKPLTAQQATKLDAPTNLQFINETDTTVIVTWTPP RARIVG YRLT VGLTRGGQPKQ YN VGP A AS Q YPLRNLQPGS E Y A VS LV A VKGNQQS P RVTGVFTTLQPLGSIPHYNTEVTETTIVITWTPAPRIGFKLGVRPSQGGEAPREVTSES GSIVVSGLTPGVEYVYTISVLRDGQERDAPIVKKVVTPLSPPTNLHLEANPDTGVLTV S WERS TTPDITG YRITTTPTNGQQG YS LEE V VH ADQS S CTFENLS PGLE YN VS V YT VK DDKESVPISDTIIPEVPQLTDLSFVDITDSSIGLRWTPLNSSTIIGYRITVVAAGEGIPI FE DFVDSSVGYYTVTGLEPGIDYDISVITLINGGESAPTTLTQQTAVPPPTDLRFTNVGPD TMRVT W APPS S IELTNLLVR YS P VKNEED V AELS IS PS DN A V VLTNLLPGTE YLVS VS S V YEQHES IPLRGRQKT ALDS PS GIDFS DIT ANS FT VHWIAPR ATITG YRIRHHPENMG GRPREDR VPPS RNS ITLTNLNPGTE Y V VS IV ALNS KEES LPLVGQQS TVS D VPRDLE VI AATPTSLLISWDAPAVTVRYYRITYGETGGSSPVQEFTVPGSKSTATISGLKPGVDYTI TVYA VTGRGDSPAS S KPVS INYRTEIDKPS QMQVTD VQDNSIS VRWLPS S SPVTGYR VTTAPKNGPGPSKTKTVGPDQTEMTIEGLQPTVEYVVSVYAQNQNGESQPLVQTAV TNIDRPKGLAFTDVDVDSIKIAWESPQGQVSRYRVTYSSPEDGIHELFPAPDGEEETA ELQGLRPGSEYTVSVVALHDDMESQPLIGTQSTTIPAPTNLKFTQVTPTSLTAQWTAP NVQLTGYRVRVTPKEKTGPMKEINLAPDSSSVVVSGLMVATKYEVSVYALKDTLTS RPAQGVVTTLENVSPPRRARVTDATETTITISWRTKTETITGFQVDAIPANGQTPIQRT IRPD VRS YTITGLQPGTD YKIHLYTLNDN ARS S P V VID AS T AID APS NLRFLATTPNS L LVSWQPPRARITGYIIKYEKPGSPPREVVPRPRPGVTEATITGLEPGTEYTIQVIALKNN QKSEPLIGRKKTDELPQLVTLPHPNLHGPEILDVPSTVQKTPFITNPGYDTGNGIQLPG TS GQQPS LGQQMIFEEHGFRRTTPPTTATPVRHRPRPYPPNVNEEIQIGHVPRGD VDH HLYPHVVGLNPNASTGQEALSQTTISWTPFQESSEYIISCHPVGIDEEPLQFRVPGTSA SATLTGLTRGATYNIIVEAVKDQQRQKVREEVVTVGNSVDQGLSQPTDDSCFDPYTV SHYAIGEEWERLSDSGFKLSCQCLGFGSGHFRCDSSKWCHDNGVNYKIGEKWDRQG ENGQMMSCTCLGNGKGEFKCDPHEATC YDDGKTYHVGEQWQKEYLGAICSCTCFG GQRGWRCDNCRRPGAEPGNEGSTAHSYNQYSQRYHQRTNTNVNCPIECFMPLDVQ ADREDSRE (SEQ ID NO: 1)

In some embodiments, at least a portion of a wall of the microfluidic device is coated with laminin or a variant thereof. In general, laminins are proteins found in the basal laminae of animals. Typically, laminins form networks of web-like structures that resist tensile forces in the basal lamina. In some embodiments, the laminin comprises the amino acid sequence as set forth in (SEQ ID NOs: 2-4). In some embodiments, the laminin comprises a tag. In some embodiments, the laminin comprises a fluorescent tag. In some embodiments, the laminin comprises a fluorescent tag. In some embodiments, the laminin comprises a rhodamine tag.

Laminin >spIP191371LAMAl_MOUSE Laminin subunit alpha- 1 OS=Mus

musculusGN=LamalPE=lSV=l:

MRGS GTG A ALLVLLAS VLW VT VRS QQRGLFP AILNLATN AHIS AN ATC GEKGPEMF CKLVEHVPGRPVRHAQCRVCDGNSTNPRERHPISHAIDGTNNWWQSPSIQNGREYH WVTVTLDLRQVFQVAYIIIKAANAPRPGNWILERSVDGVKFKPWQYYAVSDTECLT RYKITPRRGPPT YRADNE VICTS Y YS KLVPLEHGEIHTS LINGRPS ADDPS PQLLEFTS A RYIRLRLQRIRTLNADLMTLSHRDLRDLDPIVTRRYYYSIKDISVGGMCICYGHASSC PWDEEAKQLQCQCEHNTCGESCDRCCPGYHQQPWRPGTISSGNECEECNCHNKAK DCYYDSSVAKERRSLNTAGQYSGGGVCVNCSQNTTGINCETCIDQYYRPHKVSPYD DHPCRPCNCDPVGSLSSVCIKDDRHADLANGKWPGQCPCRKGYAGDKCDRCQFGY RGFPNCIPCDCRTVGSLNEDPCffiPCLCKKNVEGKNCDRCKPGFYNLKERNPEGCSEC FCFGVS GVCDSLTWS IS QVTNMS GWLVTDLMSTNKIRS QQD VLGGHRQIS INNT A V MQRLTSTYYWAAPEAYLGNKLTAFGGFLKYTVSYDIPVETVDSDLMSHADIIIKGNG LTISTRAEGLSLQPYEEYFNVVRLVPENFRDFNTRREIDRDQLMTVLANVTHLLIRAN YNSAKMALYRLDSVSLDIASPNAIDLAVAADVEHCECPQGYTGTSCEACLPGYYRV DGILFGGICQPCECHGHASECDIHGICSVCTHNTTGDHCEQCLPGFYGTPSRGTPGDC QPCACPLSIDSNNFSPTCHLTDGEEVVCDQCAPGYSGSWCERCADGYYGNPTVPGGT CVPCNCSGNVDPLEAGHCDSVTGECLKCLWNTDGAHCERCADGFYGDAVTAKNCR ACDCHENGSLSGVCHLETGLCDCKPHVTGQQCDQCLSGYYGLDTGLGCVPCNCSVE GSVSDNCTEEGQCHCGPGVSGKQCDRCSHGFYAFQDGGCTPCDCAHTQNNCDPAS GECLCPPHTQGLKCEECEEAYWGLDPEQGCQACNCSAVGSTSAQCDVLSGHCPCKK GFGGQSCHQCSLGYRSFPDCVPCGCDLRGTLPDTCDLEQGLCSCSEDSGTCSCKENV VGPQCS KC Q AGTF ALRGDNPQGC S PCFCFGLS QLC S ELEGY VRTLITLAS D QPLLH V VSQSNLKGTIEGVHFQPPDTLLDAEAVRQHIYAEPFYWRLPKQFQGDQLLAYGGKL QYSVAFYSTLGTGTSNYEPQVLIKGGRARKHVIYMDAPAPENGVRQDYEVQMKEEF WKYFNS VS EKH VTHS DFMS VLS NID YILIKAS YGQGLQQS RIANIS ME VGRKA VELP AEGEAALLLELCVCPPGTAGHSCQDCAPGYYREKLPESGGRGPRPLLAPCVPCNCNN HSDVCDPETGKCLSCRDHTSGDHCELCASGYYGKVTGLPGDCTPCTCPHHPPFSFSP TCVVEGDSDFRCNACLPGYEGQYCERCSAGYHGNPRAAGGSCQTCDCNPQGSVHS DCDRASGQCVCKPGATGLHCEKCLPRHILMESDCVSCDDDCVGPLLNDLDSVGDAV LSLNLTGVSPAPYGILENLENTTKYFQRYLIKENAKKIRAEIQLEGIAEQTENLQKELT RVLARHQKVNAEMERTSNGTQALATFIEQLHANIKEITEKVATLNQTARKDFQPPVS ALQSMHQNISSLLGLIKERNFTEMQQNATLELKAAKDLLSRIQKRFQKPQEKLKALK EANSLLSNHSEKLQAAEELLKEAGSKTQESNLLLLLVKANLKEEFQEKKLRVQEEQN VTSELIAKGREWVDAAGTHTAAAQDTLTQLEHHRDELLLWARKIRSHVDDLVMQM S KRR ARDLVHR AEQH AS ELQS R AG ALDRDLEN VRN VS LN ATS A AH VHS NIQTLTEE AEMLAADAHKTANKTDLISESLASRGKAVLQRSSRFLKESVGTRRKQQGITMKLDEL KNLTSQFQESVDNITKQANDSLAMLRESPGGMREKGRKARELAAAANESAVKTLED VLALSLRVFNTSEDLSRVNATVQETNDLLHNSTMTTLLAGRKMKDMEMQANLLLD RLKPLKTLEENLS RNLS EIKLLIS RARKQ A AS IKV A VS ADRDCIRA YQPQTS S TN YNTL ILN VKTQEPDNLLF YLGS S S S S DFLA VEMRRGKV AFLWDLGS GS TRLEFPE VS INNNR WHS IYITRFGNMGS LS VKE AS A AENPP VRTS KS PGPS KVLDINNS TLMF VGGLGGQIK KSPA VKVTHFKGCMGE AFLNGKS IGLWNYIEREGKCNGCFGS S QNEDS SFHFDGS G YAMVEKTLRPTVTQIVILFSTFSPNGLLFYLASNGTKDFLSIELVRGRVKVMVDLGSG PLTLMTDRRYNNGTWYKIAFQRNRKQGLLAVFDAYDTSDKETKQGETPGAASDLN RLEKDLIYVGGLPHSKAVRKGVSSRSYVGCIKNLEISRSTFDLLRNSYGVRKGCALEP IQS VS FLRGG Y VEMPPKS LS PES S LLATF ATKNS S GILLV ALGKD AEE AGG AQ AH VPF FSIMLLEGRIEVHVNSGDGTSLRKALLHAPTGSYSDGQEHSISLVRNRRVITIQVDENS PVEMKLGPLTEGKTIDISNLYIGGLPEDKATPMLKMRTSFHGCIKNVVLDAQLLDFTH ATGSEQVELDTCLLAEEPMQSLHREHGELPPEPPTLPQPELCAVDTAPGYVAGAHQF GLS QNS HLVLPLNQS D VRKRLQ VQLS IRTF AS S GLIY Y V AHQNQMD Y ATLQLQEGRL HFMFDLGKGRTK VS HP ALLS DGKWHT VKTE YIKRKAFMT VDGQES PS VT V VGN AT TLDVERKLYLGGLPSHYRARNIGTITHSIPACIGEIMVNGQQLDKDRPLSASAVDRCY VVAQEGTFFEGS GYA ALVKEGYKVRLDLNITLEFRTTS KNGVLLGIS S AKVD AIGLEI VDGKVLFHVNNGAGRITATYQPRAARALCDGKWHTLQAHKSKHRIVLTVDGNSVR AES PHTHS TS ADTNDPIY VGG YP AHIKQNCLS S R AS FRGC VRNLRLS RGS Q VQS LDLS RAFDLQGVFPHSCPGPEP (SEQ ID NO: 2)

Laminin>spIP024691LAM131_MOUSELamininsubunitbetalOS=Mu smusculusGN=LamblP E=1SV=3:

MGLLQVFAFGVLALWGTRVCAQEPEFSYGCAEGSCYPATGDLLIGRAQKLSVTSTC GLHKPEPYCIVSHLQEDKKCFICDSRDPYHETLNPDSHLIENVVTTFAPNRLKIWWQS ENGVENVTIQLDLEAEFHFTHLIMTFKTFRPAAMLIERSSDFGKAWGVYRYFAYDCE SSFPGISTGPMKKVDDIICDSRYSDIEPSTEGEVIFRALDPAFKIEDPYSPRIQNLLKIT N LRIKFVKLHTLGDNLLDSRMEIREKYYYAVYDMVVRGNCFCYGHASECAPVDGVN EEVEGMVHGHCMCRHNTKGLNCELCMDFYHDLPWRPAEGRNSNACKKCNCNEHS SSCHFDMAVFLATGNVSGGVCDNCQHNTMGRNCEQCKPFYFQHPERDIRDPNLCEP CTCDPAGSENGGICDGYTDFSVGLIAGQCRCKLHVEGERCDVCKEGFYDLSAEDPY GCKSCACNPLGTIPGGNPCDSETGYCYCKRLVTGQRCDQCLPQHWGLSNDLDGCRP CDCDLGGALNNSCSEDSGQCSCLPHMIGRQCNEVESGYYFTTLDHYIYEAEEANLGP GVIVVERQYIQDRIPSWTGPGFVRVPEGAYLEFFIDNIPYSMEYEILIRYEPQLPDHWE KAVITVQRPGKIPASSRCGNTVPDDDNQVVSLSPGSRYVVLPRPVCFEKGMNYTVRL ELPQYTASGSDVESPYTFIDSLVLMPYCKSLDIFTVGGSGDGEVTNSAWETFQRYRCL ENSRSVVKTPMTDVCRNIIFSISALIHQTGLACECDPQGSLSSVCDPNGGQCQCRPNV VGRTCNRCAPGTFGFGPNGCKPCDCHLQGSASAFCDAITGQCHCFQGIYARQCDRCL PGYWGFPSCQPCQCNGHALDCDTVTGECLSCQDYTTGHNCERCLAGYYGDPIIGSG DHCRPCPCPDGPDSGRQFARSCYQDPVTLQLACVCDPGYIGSRCDDCASGFFGNPSD FGGSCQPCQCHHNIDTTDPEACDKETGRCLKCLYHTEGDHCQLCQYGYYGDALRQD CRKCVCNYLGTVKEHCNGSDCHCDKATGQCSCLPNVIGQNCDRCAPNTWQLASGT GCGPCNCNAAHSFGPSCNEFTGQCQCMPGFGGRTCSECQELFWGDPDVECRACDCD PRGIETPQCDQSTGQCVCVEGVEGPRCDKCTRGYSGVFPDCTPCHQCFALWDAIIGE LTNRTHKFLEKAKALKISGVIGPYRETVDSVEKKVNEIKDILAQSPAAEPLKNIGILFE EAEKLTKDVTEKMAQVEVKLTDTASQSNSTAGELGALQAEAESLDKTVKELAEQLE FIKNSDIQGALDSITKYFQMSLEAEKRVNASTTDPNSTVEQSALTRDRVEDLMLERES PFKEQQEEQARLLDELAGKLQSLDLSAVAQMTCGTPPGADCSESECGGPNCRTDEG EKKC GGPGCGGLVT V AHS A WQKAMDFDRD VLS ALAE VEQLS KM VS E AKVR ADE A KQNAQDVLLKTNATKEKVDKSNEDLRNLIKQIRNFLTEDSADLDSIEAVANEVLKME MPSTPQQLQNLTEDIRERVETLSQVEVILQQSAADIARAELLLEEAKRASKSATDVKV TADMVKEALEEAEKAQVAAEKAIKQADEDIQGTQNLLTSIESETAASEETLTNASQRI SKLERNVEELKRKAAQNSGEAEYIEKVVYSVKQNADDVKKTLDGELDEKYKKVESL IAQKTEESADARRKAELLQNEAKTLLAQANSKLQLLEDLERKYEDNQKYLEDKAQE LVRLEGE VRS LLKDIS EKV A V YS TCL (SEQ ID NO: 3)

Laminin>5pn ^j 024681LAMCl_MOUSELamininsubunitgammalOS=MusmusculusGN= Lam clPE=lSV=2:

MTGGGRAALALQPRGRLWPLLAVLAAVAGCVRAAMDECADEGGRPQRCMPEFVN AAFNVTVVATNTCGTPPEEYCVQTGVTGVTKSCHLCDAGQQHLQHGAAFLTDYNN QADTTWWQSQTMLAGVQYPNSINLTLHLGKAFDITYVRLKFHTSRPESFAIYKRTRE DGPWIPYQYYSGSCENTYSKANRGFIRTGGDEQQALCTDEFSDISPLTGGNVAFSTLE GRPS A YNFDNS P VLQEW VT ATDIR VTLNRLNTFGDE VFNEPKVLKS Y Y Y AIS DF A VG GRCKCNGHASECVKNEFDKLMCNCKHNTYGVDCEKCLPFFNDRPWRRATAESASE SLPCDCNGRSQECYFDPELYRSTGHGGHCTNCRDNTDGAKCERCRENFFRLGNTEA CSPCHCSPVGSLSTQCDSYGRCSCKPGVMGDKCDRCQPGFHSLTEAGCRPCSCDLRG STDECNVETGRCVCKDNVEGFNCERCKPGFFNLESSNPKGCTPCFCFGHSSVCTNAV G YS V YDIS S TFQIDEDGWRVEQRDGS E AS LEWS S DRQDIA VIS DS YFPR YFIAP VKFL GNQ VLS YGQNLS FS FRVDRRDTRLS AEDLVLEG AGLR VS VPLIAQGNS YPS ETT VKYI FRLHE ATD YPWRP ALS PFEFQKLLNNLTS IKIRGT YS ERT AG YLDD VTLQS ARPGPG V PATWVESCTCPVGYGGQFCETCLPGYRRETPSLGPYSPCVLCTCNGHSETCDPETGV CDCRDNTAGPHCEKCSDGYYGDSTLGTSSDCQPCPCPGGSSCArVPKTKEVVCTHCP TGTAGKRCELCDDGYFGDPLGSNGPVRLCRPCQCNDNIDPNAVGNCNRLTGECLKC IYNTAGFYCDRCKEGFFGNPLAPNPADKCKACACNPYGTVQQQSSCNPVTGQCQCL PHVSGRDCGTCDPGYYNLQSGQGCERCDCHALGSTNGQCDIRTGQCECQPGITGQH CERCETNHFGFGPEGCKPCDCHHEGSLSLQCKDDGRCECREGFVGNRCDQCEENYF YNRSWPGCQECPACYRLVKDKAAEHRVKLQELESLIANLGTGDDMVTDQAFEDRL KEAEREVTDLLREAQEVKDVDQNLMDRLQRVNSSLHSQISRLQNIRNTIEETGILAER ARSRVESTEQLIEIASRELEKAKMAAANVSITQPESTGEPNNMTLLAEEARRLAERHK QEADDIVRVAKTANETSAEAYNLLLRTLAGENQTALEIEELNRKYEQAKNISQDLEK QAARVHEEAKRAGDKAVEIYASVAQLTPVDSEALENEANKIKKEAADLDRLIDQKL KDYEDLREDMRGKEHEVKNLLEKGKAEQQTADQLLARADAAKALAEEAAKKGRS TLQEANDILNNLKDFDRRVNDNKTAAEEALRRIPAINRTIAEANEKTREAQLALGNA AADATEAKNKAHEAERIASAVQKNATSTKADAERTFGEVTDLDNEVNGMLRQLEE AENELKRKQDD ADQDMMM AGM AS Q A AQE AELN ARKAKNS VS S LLS QLNNLLDQL GQLDTVDLNKLNEIEGSLNKAKDEMKASDLDRKVSDLESEARKQEAAIMDYNRDIA EIIKDIHNLEDIKKTLPTGCFNTPSIEKP (SEQ ID NO: 4)

The proteins useful herein also include variants or fragments thereof. As used herein, "variant", refers to a portion of a protein retaining at least one functional i.e. binding or interaction ability and/or therapeutic property thereof. The level or degree of which the property is retained may be reduced relative to the wild type protein but is typically the same or similar in kind. Generally, variants are overall very similar, and, in many regions, identical to the amino acid sequence of the protein described herein.

The variant proteins may comprise, or alternatively consist of, an amino acid sequence which is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%, identical to, for example, the amino acid sequence of a protein such as an ECM protein. Further polypeptides encompassed by the invention are polypeptides encoded by polynucleotides which hybridize to the complement of a nucleic acid molecule encoding a protein such as an ECM protein under stringent hybridization conditions (e.g., hybridization to filter bound DNA in 6.times. Sodium chloride/Sodium citrate (SSC) at about 45 degrees Celsius, followed by one or more washes in 0.2. times. SSC, 0.1% SDS at about 50-65 degrees Celsius), under highly stringent conditions (e.g., hybridization to filter bound DNA in 6.times. sodium chloride/Sodium citrate (SSC) at about 45 degrees Celsius, followed by one or more washes in 0.1. times. SSC, 0.2% SDS at about 68 degrees Celsius), or under other stringent hybridization conditions which are known to those of skill in the art (see, for example, Ausubel, F. M. et al., eds., 1989 Current protocol in Molecular Biology, Green publishing associates, Inc., and John Wiley & Sons Inc., New York, at pages 6.3.1-6.3.6 and 2.10.3).

By a polypeptide having an amino acid sequence at least, for example, 95%

"identical" to a query amino acid sequence, it is intended that the amino acid sequence of the subject polypeptide is identical to the query sequence except that the subject polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the query amino acid sequence. In other words, to obtain a polypeptide having an amino acid sequence at least 95% identical to a query amino acid sequence, up to 5% of the amino acid residues in the subject sequence may be inserted, deleted, or substituted with another amino acid. These alterations of the reference sequence may occur at the amino- or carboxy-terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.

As a practical matter, whether any particular polypeptide is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, the amino acid sequence of a protein such as an ECM protein, can be determined conventionally using known computer programs. A preferred method for determining the best overall match between a query sequence (a sequence of the present invention) and a subject sequence, also referred to as a global sequence alignment, can be determined using the FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245 (1990)). In a sequence alignment the query and subject sequences are either both nucleotide sequences or both amino acid sequences. The result of said global sequence alignment is expressed as percent identity. Preferred parameters used in a FASTDB amino acid alignment are: Matrix=PAM 0, k- tuple=2, Mismatch Penalty=l, Joining Penalty=20, Randomization Group Length=0, Cutoff Score=l, Window Size=sequence length, Gap Penalty=5, Gap Size Penalty=0.05, Window Size=500 or the length of the subject amino acid sequence, whichever is shorter.

If the subject sequence is shorter than the query sequence due to N- or C-terminal deletions, not because of internal deletions, a manual correction must be made to the results. This is because the FASTDB program does not account for N- and C-terminal truncations of the subject sequence when calculating global percent identity. For subject sequences truncated at the N- and C-termini, relative to the query sequence, the percent identity is corrected by calculating the number of residues of the query sequence that are N- and C- terminal of the subject sequence, which are not matched/aligned with a corresponding subject residue, as a percent of the total bases of the query sequence. Whether a residue is matched/aligned is determined by results of the FASTDB sequence alignment. This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score. This final percent identity score is what is used for the purposes of the present invention. Only residues to the N- and C-termini of the subject sequence, which are not matched/aligned with the query sequence, are considered for the purposes of manually adjusting the percent identity score. That is, only query residue positions outside the farthest N- and C-terminal residues of the subject sequence. Cell Surface Proteins

Any number of cell surface proteins, or variants thereof, may be used in accordance with this disclosure. In some embodiments, at least a portion of a wall of any of the microfluidic devices, provided herein, is coated with at least one cell surface protein. In some embodiments, the protein is a cell surface protein or a variant thereof. As used herein, a "cell surface protein" refers to a protein that is embedded in or spans a layer of a cell plasma membrane in one or more organisms. Generally, cell surface proteins are exposed to an external side of the cell membrane, for example by embedding in or spanning the plasma membrane of a cell, which separates the inside of a cell from the external environment. Any number of cell surface proteins may be used in accordance with this disclosure. In some embodiments, the protein is a cell adhesion molecule (CAM) or a variant thereof.

In some embodiments, the cell surface protein is a cell adhesion molecule (CAM) or variant thereof. The term "cell adhesion molecule" refers to a protein that is typically located on the cell surface that is involved in binding with other cells or with the extracellular matrix (ECM). Cell adhesion molecules typically include four protein families, the immunoglobulin superfamily (IgSF CAMs), the integrins, the cadherins, and the selectins. In some embodiments, the protein is an IgSF, an integrin, a cadherin, or a selectin. Cell adhesion molecules may be classified as calcium-independent CAMs or calcium-dependent CAMs. In some embodiments, the protein is a calcium-independent CAM. For example, the calcium- independent CAM may be an IgSF protein, or a lymphocyte homing receptor {e.g., CD34 or GLYCAM-1). Additional non-limiting examples of calcium-independent CAMs include N- CAM {e.g., Myelin protein zero), ICAM {e.g., ICAM1, or ICAM5), VCAM-1, PE-CAM Ll- CAM, or Nectin {e.g., PVRL1, PVRL2, or PVRL3). In some embodiments, the protein is in immunoglobulin {e.g., an antibody). Exemplary antibodies that may be used in accordance with this disclosure are described in more detail below. In some embodiments, the protein is a calcium-dependent CAM. For example, the calcium-dependent CAM may be a cadherin, a selectin, or an integrin. In some

embodiments, the protein is a cadherin. In some embodiments, the cadherin is a classical cadherin, for example CDH1, CDH2, or CDH3. In some embodiments, the cadherin is a desmosomal cadherin, for example desmoglein (e.g., DSG1, DSG2, DSG3, or DSG4), or desmocollin (e.g. , DSC 1, DSC2, or DSC3). In some embodiments, the cadherin is a protocadherin, for example PCDH1, or PCDH15. In some embodiments, the cadherin may be an unconventional cadherin, for example T-cadherin, CDH4, CDH5, CDH6, CDH8, CDH11, CDH12, CDH15, CDH16, CDH17, CDH9, or CDH10. However additional cadherins would be apparent to the skilled artisan and are within the scope of this disclosure.

In some embodiments, the protein is a selectin. Selectins are single-chain

transmembrane glycoproteins that share similar properties to C-type lectins due to a related amino terminus and calcium-dependent binding. Typically, selectins bind to sugar moieties and so are considered to be a type of lectin, cell adhesion proteins that bind sugar polymers. In some embodiments, the selecting is an E-selectin, an L-selectin, or a P-selectin. However, it should be appreciated that this list is not meant to be limiting.

In some embodiments, the protein is an integrin. Integrins are transmembrane receptors that typically bridge cell-cell and cell-extracellular matrix (ECM) interactions. For example, in some embodiments, the integrin is an αΐβΐ, α2β 1, α4β1, α5β 1, αόβ ΐ, αίβ2, αΜβ2, αΙ¾β3, ανβ3, ανβ5, ανβό, or a α6β4 integrin. Additional examples of integrins include, without limitation LFA- 1 (e.g., CDl la and CD18), Integrin alphaXbeta2 (e.g., CD 11c and CD 18) Macrophage- 1 antigen (e.g. , CD l ib and CD 18) VLA-4 (e.g., CD49d and CD29) and Glycoprotein Ilb/IIIa (e.g., ITGA2B and ITGB3). However, additional integrins would be apparent to the skilled artisan and are within the scope of this disclosure.

In some embodiments, the protein is a receptor tyrosine kinase, or a G-protein coupled receptor. In some embodiments, the receptor tyrosine kinase is a an ErbB family receptor (e.g., EGF receptor), insulin receptor, PDGF receptor, FGF receptor, VEGF receptor, HGF receptor, Trk receptor, Eph receptor, AXL receptor, LTK receptor, TIE receptor, ROR receptor, DDR receptor, RET receptor, KLG receptor, RYK receptor, or MuSK receptor. In some embodiments, the G-protein coupled receptor is a rhodopsin-like receptor, the secretin receptor, metabotropic glutamate/pheromone receptor, cyclic AMP receptor, frizzled/smoothened receptor, CXCR4, CCR5, or beta-adrenergic receptor. It should be appreciated that additional tyrosine kinase receptors and G-protein coupled receptors would be apparent to the skilled artisan and are within the scope of this disclosure. In some embodiments, the protein is a protein of a vessel wall (e.g., a blood vessel wall). For example, in some embodiments, the protein is selectin, vascular cell adhesion molecule- 1, alpha -V Beta-3 integrin, CD36, fibronectin, thrombospondin, Von Willebrand Factor, or laminin. However, additional proteins found on the walls of blood vessels are known and would be apparent to the skilled artisan.

Immunoglobulins

Any number of immunoglobulin proteins (e.g. antibodies), or variants thereof, may be used in accordance with this disclosure. In some embodiments, at least a portion of a wall of any of the microfluidic devices, provided herein, is coated with at least one antibody. As used herein, an "antibody" encompasses not only intact (e.g. , full-length) polyclonal or monoclonal antibodies, but also antigen-binding fragments thereof (such as Fab, Fab', F(ab')2, Fv), single chain (scFv), mutants thereof, fusion proteins comprising an antibody portion, humanized antibodies, chimeric antibodies, diabodies, linear antibodies, single chain antibodies, multispecific antibodies (e.g. , bispecific antibodies) and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site of the required specificity, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies. An antibody includes an antibody of any class, such as IgD, IgE, IgG, IgA, or IgM (or sub-class thereof), and the antibody need not be of any particular class. Depending on the antibody amino acid sequence of the constant domain of its heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g. , IgGl, IgG2, IgG3, IgG4, IgAl and IgA2. The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

Any of the antibodies, provided herein may be designed to bind to any antigen of interest, for example, and antigen on the surface of a cell. In some embodiments, the antibody binds to an antigen on the surface of a cell. In some embodiments, the antibody binds to an antigen on the surface of a specific cell type. For example, the antibody may be designed to bind an erythrocyte (i.e. , a red blood cell). In some embodiments, the antibody binds to an antigen on a differentiated cell. For example, in some embodiments, the antibody binds to an antigen on a blood or immune cell. In some embodiments, the antibody binds to an antigen on an erythrocyte (e.g., a red blood cell), a megakaryocyte (e.g. , a platelet precursor), a monocyte (e.g. , a white blood cell), a connective tissue macrophage, an epidermal

Langerhans cell, an osteoclast (e.g., in bone), a dendritic cell (e.g., in lymphoid tissues), a microglial cell (e.g., in central nervous system), a neutrophil granulocyte, an eosinophil granulocyte, a basophil granulocyte, a hybridoma cell, a mast cell, a helper T cell, a suppressor T cell, a cytotoxic T cell, a natural killer T cell, a B cell, or a reticulocyte.

In some embodiments, any of the antibodies provided herein may bind to an antigen on a cultured cell or a cell from a subject (e.g. , a human subject). In some embodiments, the antibody binds to an antigen on a mammalian cell, examples of mammalian cells include but are not limited to, a cell from a rodent, a mouse, a rat, a hamster, or a non-human primate. The target cell may also be from a human. The cell may be also from an established cell line (e.g., a 293T cell), or a primary cell cultured ex vivo (e.g., cells obtained from a subject and grown in culture). The antibody may bind to an antigen on a hematologic cell (e.g. , a hematopoietic stem cell, a leukocyte, a thrombocyte, or erythrocyte), or on a cell from a solid tissue, such as a liver cell, a kidney cell, a lung cell, a heart cell, a bone cell, a skin cell, a brain cell, or any other cell found in a subject. It should be appreciated that any of the antibodies provided herein may bind to an antigen on a cell in order to selectively bind to the cell of interest. It would be apparent to a person skilled in the art how to design an antibody that specifically binds to a particular cell type based, at least in part, on one or more unique antigens that are expressed on the cell.

In some embodiments, any of the antibodies provided herein may bind to an antigen on a stem cell, such as a pluripotent stem cell or a totipotent stem cell. Some non-limiting examples of antigens specific to pluripotent stem cells include Oct4 and Nanog, which were the first proteins identified as essential for both early embryo development and pluripotency maintenance in embryonic stem cells. Stem cell antigens are known in the art and have been described previously, for example in Nichols J, et al. "Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription factor Oct4.", Cell. 95:379-91, 1998; the contents of which are hereby incorporated by reference). In addition to Oct4, Sox2 and Nanog, many other pluripotent stem cell markers have been identified, including Sall4, Daxl, Essrb, Tbx3, Tell, Rifl, Nacl and Zfp281. See, for example, Loh Y, et al. "The Oct4 and Nanog transcription network regulates pluripotency in mouse embryonic stem cells.", Nat Genet. 38:431-40, 2006.

In some embodiments, any of the antibodies provided herein may bind to an antigen on a tumor or cancer cell. In some embodiments, any of the antibodies provided herein may bind to an antigen that is unique to a tumor or cancer cell, for example, as compared to a non- tumor or non-cancer (e.g., normal) cell. In some embodiments, the protein is a protein that binds a tumor associated or tumor specific antigen. In some embodiments, the protein is an antibody that binds a tumor associated or tumor specific antigen. Some non-limiting examples of tumor antigens include, CA19-9, c-met, PD-1, CTLA-4, ALK, AFP, EGFR,

Estrogen receptor (ER), Progesterone receptor (PR), HER2/neu, KIT, B-RAF, S lOO, MAGE, Thyroglobulin, MUC-1, and PSMA. However, immunoglobulins that bind other tumor antigens would be apparent to the skilled artisan and are within the scope of this disclosure. For example, tumor specific antigens have been described in Bigbee W., et al. "Tumor markers and immunodiagnosis.", Cancer Medicine . 6th ed. Hamilton, Ontario, Canada: BC Decker Inc., 2003.; Andriole G, et al. "Mortality results from a randomized prostate-cancer screening trial.", New England Journal of Medicine, 360(13): 1310-1319, 2009.; Schroder FH, et al. "Screening and prostate-cancer mortality in a randomized European study." New England Journal of Medicine, 360(13): 1320-1328, 2009.; Buys SS, et al. "Effect of screening on ovarian cancer mortality: the Prostate, Lung, Colorectal and Ovarian (PLCO) Cancer Screening Randomized Controlled Trial.", JAMA, 305(22):2295-2303, 2011.;

Cramer DW et al. "Ovarian cancer biomarker performance in prostate, lung, colorectal, and ovarian cancer screening trial specimens." Cancer Prevention Research, 4(3):365-374, 2011.; Roy DM, et al. "Candidate prognostic markers in breast cancer: focus on extracellular proteases and their inhibitors.", Breast Cancer. Jul 3;6:81-91, 2014.; Tykodi SS. et al. "PD-1 as an emerging therapeutic target in renal cell carcinoma: current evidence." Onco Targets Ther. Jul 25;7: 1349-59, 2014.; and Weinberg RA. The Biology of Cancer, Garland Science, Taylor & Francis Group LLC, New York, NY,2007.; the entire contents of each are incorporated herein by reference).

Producing Antibodies

Any of the antibodies used in accordance with this disclosure, for example antibodies that are coated onto at least a portion of a wall of any of the microfluidic devices provided herein, may be produced by any suitable method. Numerous methods may be used for obtaining antibodies, or antigen binding fragments thereof, of the disclosure. For example, antibodies can be produced using recombinant DNA methods. Monoclonal antibodies may also be produced by generation of hybridomas (see e.g., Kohler and Milstein (1975) Nature, 256: 495-499) in accordance with known methods. Hybridomas formed in this manner are then screened using standard methods, such as enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (e.g. , OCTET or BIACORE) analysis, to identify one or more hybridomas that produce an antibody that specifically binds to a specified antigen. Any form of the specified antigen may be used as the immunogen, e.g. , recombinant antigen, naturally occurring forms, any variants or fragments thereof, as well as antigenic peptide thereof (e.g. , any of the epitopes described herein as a linear epitope or within a scaffold as a

conformational epitope). One exemplary method of making antibodies includes screening protein expression libraries that express antibodies or fragments thereof (e.g. , scFv), e.g. , phage or ribosome display libraries. Phage display is described, for example, in Ladner et al., U.S. Pat. No. 5,223,409; Smith (1985) Science 228: 1315-1317; Clackson et al. (1991) Nature, 352: 624-628; Marks et al. (1991) J. Mol. Biol., 222: 581-597W092/18619; WO 91/17271 ; WO 92/20791 ; WO 92/15679; WO 93/01288; WO 92/01047; WO 92/09690; and WO 90/02809.

In addition to the use of display libraries, the specified antigen (e.g. , a blood cell antigen) can be used to immunize a non-human animal, e.g. , a rodent, e.g. , a mouse, hamster, or rat. In one embodiment, the non-human animal is a mouse.

In another embodiment, a monoclonal antibody is obtained from the non-human animal, and then modified, e.g. , chimeric, using suitable recombinant DNA techniques. A variety of approaches for making chimeric antibodies have been described. See e.g. ,

Morrison et al., Proc. Natl. Acad. Sci. U.S.A. 81 :6851, 1985; Takeda et al., Nature 314:452, 1985, Cabilly et al., U.S. Pat. No. 4,816,567; Boss et al., U.S. Pat. No.

4,816,397; Tanaguchi et al., European Patent Publication EP171496; European Patent Publication 0173494, United Kingdom Patent GB 2177096B.

For additional antibody production techniques, see Antibodies: A Laboratory Manual, eds. Harlow et al., Cold Spring Harbor Laboratory, 1988. The present disclosure is not necessarily limited to any particular source, method of production, or other special characteristics of an antibody.

Protein variants

It should be appreciated that the proteins provided herein may be from any organism. For example the protein may be a naturally-occurring protein from an animal. In some embodiments, the protein is a naturally-occurring protein from a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, cattle, a primate, a pig, or a fish. However, proteins from other organisms would be apparent to the skilled artisan and are within the scope of this disclosure. In some aspects, the disclosure provides microfluidic devices that are coated with cells. In some embodiments, at least a portion of a wall of any of the microfluidic devices, provided herein, is coated with at least one cell. The cell may be attached to a wall of the device covalently or non-covalently. In some embodiments, any of the proteins provided herein are comprised in a cell. In some embodiments, the cell is a cultured cell (e.g., from a cell line). In other embodiments, the cell is a primary cell, from a subject, which may, or may not be taken directly from a subject, or may be cultured after being obtained from the subject.

The one or more walls of any of the microfluidic devices provided herein may be coated with one or more proteins, which may be naturally-occurring (e.g. , naturally produced by a cell), or non-naturally occurring, meaning that the protein does not occur in nature. In some embodiments, a non-naturally occurring protein may comprise a naturally-occurring protein. For example, a naturally occurring human fibronectin protein fused to a protein tag (e.g. a FLAG tag) represents one non-naturally occurring protein that comprises a naturally occurring protein. It should be appreciated that the disclosure provides proteins that are variants of or homologous to any naturally-occurring protein. For example any of the naturally-occurring proteins provided. In some embodiments, the protein is homologous to any of the proteins provided herein.

As used herein, the term "homology" refers to the overall relatedness between polypeptides. In some embodiments, proteins are considered to be "homologous" to one another if their sequences are at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical. In some embodiments, proteins are considered to be "homologous" to one another if their sequences are at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% similar. The term "homologous" necessarily refers to a comparison between at least two sequences (e.g., two amino acid sequences).

Devices

Devices containing a microfluidic channel further contain a first wall adjacent to the microfluidic channel, where at least a portion of the first wall is coated with at least one protein and a second wall adjacent to the microfluidic channel, where at least a portion of the second wall comprises a gas permeable membrane or film. As used herein, "adjacent to" refers to a physical proximity to the channel such that at least a portion of a wall (e.g., a first wall or a second wall) and at least a portion of the channel are in physical contact or are separated by a space that contains the gas. "Adjacent to" could mean that the wall defines a surface of the channel. Adjacent to could also mean that the wall defines an inner surface and/or outer surface of the microfluidic device. For example the microfluidic channel may have a top surface, bottom surface, side surface or end surface that contacts and/or contains a fluid that is flowed through one or more of the microfluidic channels.

The gas permeable portion of a wall (e.g., a second wall), which can be, for example a gas permeable membrane or film e.g., polydimethylsiloxane (PDMS), permits the control of the level of a gas in the microfluidic device. In some embodiments, the gas permeable film has a thickness ranging from 5 μιη to 500 μιη. In some embodiments, the gas permeable film has a thickness ranging from 5 μιη to 20 μιη, from 5 μιη to 50 μιη, from 5 μιη to 100 μιη, from 5 μηι to 150 μιη, from 5 μιη to 200 μιη, from 5 μιη to 250 μιη, from 5 μιη to 300 μιη, from 5 μηι to 400 μιη, from 5 μιη to 500 μιη, from 50 μιη to 100 μιη, from 50 μιη to 150 μιη, from 50 μιη to 200 μιη, from 50 μιη to 300 μιη, from 50 μιη to 400 μιη, from 50 μιη to 500 μηι, from 100 μιη to 200 μιη, from 100 μιη to 300 μιη, from 100 μιη to 400 μιη, from 100 μηι to 500 μιη, from 200 μιη to 300 μιη, from 200 μιη to 400 μιη, from 200 μιη to 500 μιη, from 300 μιη to 400 μιη, from 300 μιη to 500 μιη or from 400 μιη to 500 μιη. As one specific example, the gas permeable film has a thickness of aboutl50 μιη. It should be appreciated that the gas permeable membrane or film may make up an entire wall (e.g., a second wall) or a portion of a wall (e.g., a second wall) of the microfluidic channel.

In some embodiments the gas permeable membrane makes up from 1% to 100% of the surface area of a wall (e.g., the second wall) of the device. In some embodiments, the gas permeable membrane makes up from 1% to 5%, from 1% to 10%, from 1% to 20%, from 1% to 30%, from 1% to 50%, from 1% to 60%, from 1% to 80%, from 1% to 100%, from 5% to 10%, from 5% to 20%, from 5% to 30%, from 5% to 50%, from 5% to 60%, from 5% to

80%, from 5% to 100%, from 20% to 30%, from 20% to 50%, from 20% to 60%, from 20% to 80%, from 20% to 100%, from 30% to 50%, from 30% to 60%, from 30% to 80%, from 30% to 100%, from 50% to 60%, from 50% to 80%, from 50% to 100%, or from 80% to 100% of a wall (e.g., a second wall) of the microfluidic device. It should be appreciated that one or more walls of the microfluidic device may have at least a portion of a wall (e.g., a second wall) that is made of a gas permeable membrane or film.

The gas permeable membrane or film may be permeable to any number of gases that are supplied to the gas permeable membrane or film. For example the membrane or film may be permeable to gasses including but not limited to oxygen, nitrogen, carbon dioxide, nitric oxide, carbon monoxide, nitrous oxide, nitrogen dioxide and/or methane. In a particular embodiment, the membrane or film is permeable to oxygen. The gas permeable membrane or film may be constructed of any suitable material that is permeable to any of the gases, described herein. For example the gas permeable membrane or film may be made of a material including but not limited to polydimethylsiloxane (PDMS), hydroxypropyl cellulose (HPC), hydroxypropyl methylcellulose (HPMC), cellulose triacetate (CTA), or poly(methyl methacrylate) (PMMA). Other gas permeable membranes or films are known in the art, such as those disclosed in Budd et al. (Peter M. Budd and Neil B. McKeown, Highly permeable polymers for gas separation membranes, Polym. Chem., 2010, 1, 63-68; the entire contents of which are hereby incorporated by reference). In a specific embodiment, the gas permeable film is made of PDMS.

In some embodiments, the gas permeable film or membrane comprises PDMS (Dow Corning Sylgard elastomer 184). In some embodiments, Sylgard 184 silicone elastomer is supplied as a two-part liquid component kit, a pre-polymer base (part A) and a crosslinking curing agent (part B), that when mixed together is curable at both room temperature (RT = 25 °C) and elevated temperatures (RT > T > 200 °C). In some embodiments, the PDMS membrane or film can be produced by mixing different ratios of the pre-polymer base with the crosslinking curing agent. It should be appreciated that mixing the pre-polymer base with the crosslinking curing agent can be done at different ratios to control the properties of the PDMS membrane or film. In some embodiments, the PDMS pre-polymer base is mixed with the crosslinking curing agent at a ratio (PDMS pre-polymer : crosslinking curing agent) from 1 : 1 to 20: 1. In some embodiments, the PDMS pre-polymer base is mixed with the crosslinking curing agent at a ratio (PDMS pre-polymer : crosslinking curing agent) from 1 : 1 to 2: 1, from 1 : 1 to 4: 1, from 1 : 1 to 6: 1, from 1 : 1 to 8: 1, from 1 : 1 to 10: 1, from 1 : 1 to 12: 1, from 1 : 1 to 14: 1, from 1 : 1 to 18: 1, or from 1 : 1 to 20: 1. In some embodiments, the PDMS pre-polymer base is mixed with the crosslinking curing agent at a ratio (PDMS pre-polymer : crosslinking curing agent) of 5: 1, 10: 1, 15: 1 or 20: 1. It should be appreciated that the ratio may be modified to control one or more properties of the PDMS membrane or film produced.

In some aspects, a portion of a wall (e.g. , a first wall), or an entire wall (e.g. , a first wall) that is coated with at least one protein (e.g., fibronectin) may permit or facilitate binding of a cell to a portion of the wall that is coated with a protein. In some embodiments, at least a portion of a wall (e.g. , a first wall) may be coated with at least one protein, for example, from 1% to 100% of the surface area of a wall (e.g. , a first wall) may be coated with at least one protein. In some embodiments, from 1% to 5%, from 1% to 10%, from 1% to 20%, from 1% to 30%, from 1% to 50%, from 1% to 60%, from 1% to 80%, from 1% to 100%, from 5% to 10%, from 5% to 20%, from 5% to 30%, from 5% to 50%, from 5% to 60%, from 5% to 80%, from 5% to 100%, from 20% to 30%, from 20% to 50%, from 20% to 60%, from 20% to 80%, from 20% to 100%, from 30% to 50%, from 30% to 60%, from 30% to 80%, from 30% to 100%, from 50% to 60%, from 50% to 80%, from 50% to 100%, or from 80% to 100% of a wall (e.g., a first wall) of the microfluidic device is coated with at least one protein. It should be appreciated that one or more walls of the microfluidic device may be coated with at least one protein.

The walls may be coated with any suitable amount of protein, for example, an amount sufficient to permit or facilitate binding of a cell to a wall of the device. In some

embodiments, a wall of the device may be coated with a given amount of protein in a given surface area. For example, a protein may be densely coated or thinly coated onto a wall of the device. In some embodiments, a wall or a portion of a wall is coated with from 0.0^g/in to 50 μg/in of protein. In some embodiments, a wall or a portion of a wall is coated with

2 2 2 2 2 2 from 0.0^g/in to 0.1 μg/in , from 0.0^g/in to 1 μg/in , from 0.0^g/in to 2 μg/in , from

2 2 2 2 2 2

0.0^g/in to 0.3 μg/in , from 0.0^g/in to 4 μg/in , from 0.0^g/in to 5 μg/in , from

2 2 2 2 2 2

0.0^g/in to 10 μg/in , from 0.0^g/in to 20 μg/in , from 0.0^g/in to 30 μg/in , or from

2 2

0.0^g/in to 40 μg/in , of a protein. However, it should be appreciated that additional amounts of protein may be used in accordance with this disclosure and the examples provided herein are not meant to be limiting.

In some embodiments, the proteins provided herein may be coated onto a wall of the device using any suitable method, for example a method that would be apparent to the skilled artisan. In some embodiments, the protein is bound to the surface of a wall. In some embodiments, the protein is non-covalently bound to the surface of a wall. In some embodiments, the protein is covalently bound to the surface of a wall. The protein may be bound directly to the wall, or indirectly to the wall, e.g., via a linker. The term "linker," as used herein, refers to a chemical group or a molecule linking two molecules or moieties, e.g., a wall (e.g., a coated wall) to a protein, such as laminin. Typically, the linker is positioned between, or flanked by, two groups, molecules, or other moieties and connected to each one via a covalent bond, thus connecting the two. In some embodiments, the linker is an amino acid or a plurality of amino acids (e.g., a peptide or protein). In some embodiments, the linker is an organic molecule, group, polymer, or chemical moiety. In some embodiments, the linker is 5- 100 amino acids in length, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 30-35, 35-40, 40-45, 45-50, 50-60, 60- 70, 70-80, 80-90, 90-100, 100-150, or 150-200 amino acids in length. Longer or shorter linkers are also contemplated.

Any of the devices, described herein, may also contain a gas channel. This gas channel may be used to supply a gas to the gas permeable membrane or film of the device in order to regulate the gas content of the fluid in the device. The gas channel may encase the gas permeable membrane or film on a wall of the microfluidic channel such that gas exchange can occur between the gas in the gas channel and the fluid in the microfluidic channel through the gas permeable membrane or film. An exemplary microfluidic device with a gas channel encasing a gas permeable layer is shown in Figs. 1 and 1 1 - 16. The gas channel is separated from the microfluidic channel by a gas permeable membrane to allow gas exchange between the gas channel and a fluid in the microfluidic device. The gas channel may be any size or shape suitable for supplying a gas to the gas permeable membrane or film of the microfluidic device. In some embodiments, the gas channel is between 10 μιη and 10mm in height. In one specific embodiment, the gas channel is 100 μιη in height. It should also be appreciated that any of the microfluidic devices may comprise one or more gas channels to deliver one or more gasses to any portion of the microfluidic device with a gas permeable membrane or film.

In some embodiments, the gas channel comprises at least one inlet (e.g. for gas to enter the gas channel) and/or at least one outlet (e.g., for gas to exit or flow out of). A gas or gas mixture may be supplied to the inlet of the gas channel from one or more tanks containing the gas or gas mixture. In some non-limiting embodiments, the gas supplied to the gas channel is oxygen, nitrogen, carbon dioxide, nitric oxide, carbon monoxide, nitrous oxide, nitrogen dioxide, methane, or any combination thereof. In some embodiments the gas supplied to the gas channel contains oxygen. In some embodiments the gas supplied contains between 1% and 100 % oxygen. In some embodiments the gas supplied contains from 1% to 2%, from 1% to 3%, from 1% to 5%, from 1% to 10%, from 1% to 20%, from 1% to 40%, from 1% to 60%, from 1% to 80%, from 1% to 100%, from 5% to 10%, from 5% to 20%, from 5% to 40%, from 5% to 60%, from 5% to 80%, from 5% to 100%, from 20% to 40%, from 20% to 60%, from 20% to 80%, from 20% to 100%, from 40% to 60%, from 40% to 80%, from 40% to 100%, from 60% to 80%, from 60% to 100% or from 80% to 100% oxygen. In some embodiments the gas supplied to the gas channel contains about 2%, about 5%, or about 20% oxygen. As used herein, the term "about," or "approximately" as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In certain embodiments, the term "approximately" or "about" refers to a range of values that fall within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (for example, when such number would exceed 100% of a possible value). In some embodiments the gas supplied to the gas channel contains about 20% oxygen, 5% carbon dioxide and about 75% nitrogen. In other embodiments the gas supplied to the gas channel contains about 5% oxygen, 5% carbon dioxide and about 90% nitrogen. In yet other embodiments the gas supplied to the gas channel contains about 2% oxygen, 5% carbon dioxide and about 93% nitrogen. The gas or gas mixture may be supplied to one or more inlets of one or more gas channels using any suitable means, such as tubing or hoses.

The gas or gas mixture may be delivered to the gas channel continuously such that the gas enters the inlet of the gas channel and exits from the outlet of the gas channel. This ensures that the gas or gas mixture in the channel remains consistent as gas exchanges across the gas permeable membrane. As used herein, consistent means that the level of, or % composition of a gas in a given space (e.g., a channel) does not vary by a large amount. In some embodiments consistent means that the level of, or % composition of a gas entering the gas channel does not vary by more than 1%, 2%, 3%, 4%, 5%, 8% or 10% before exiting the gas channel. The gas or gas mixture may be flowed through the gas channel at any suitable rate. The gas in the gas channel may regulated at a specific pressure. In some embodiments the pressure of the gas in the gas channel is from lpsi to lOpsi. In a specific embodiment, the pressure of the gas in the gas channel is regulated to be about 5psi.

The device may be configured such that the gas or gas mixture, supplied to one or more gas inlets of the device, can be switched to a different gas or gas mixture. This enables the device to dynamically control the gas content of a fluid in the microfluidic channel. For example, a fluid containing cells flowing through one or more microfluidic channels of the device can be exposed to a gas with high oxygen content (e.g., 20% oxygen) for a given time through the gas channel. As the fluid containing cells flows through the one or more microfluidic channels, a different gas may be supplied to the same gas channel or a different gas channel. For example the gas delivered to the gas channel can be switched to a gas with low oxygen content (e.g., 2% oxygen). This allows for the dynamic

observation/measurement of cell parameters in response to dynamically changing gas conditions. For example, a fluid containing red blood cells is flowed through the microfluidic device where the gas delivered to the gas channel contains about 20% oxygen, about 5% carbon dioxide and about 75% nitrogen. One or more measurements, for example a morphological measurement (e.g., cell sickling) or a mechanical measurement (e.g., cell stretching) can be made as the cells flow through the microfluidic device under high oxygen content. The gas delivered to the gas channel can then be switched to a gas having a low oxygen content (e.g., about 2% oxygen, about5% carbon dioxide and about 75% nitrogen) to regulate the oxygen content of the fluid containing red blood cells. One or more additional measurements may be taken over time to dynamically observe/measure one or more cell parameters in response to low oxygen conditions. For example, cell sickling time, or adhesiveness may be determined for a given cell sample when oxygen levels decrease. It should be appreciated that the device may be used to measure a cell-scale parameter in response to any gas or gas mixture and is not limited to the examples provided herein.

Devices containing a microfluidic channel can further contain a substantially planar transparent wall that defines a wall of a microfluidic channel. This substantially planar transparent wall, which can be, for example, glass or plastic, permits observation into the microfluidic channel by microscopy so that at least one measurement of each cell that passes through one of the microfluidic channels can be obtained. In one example, the transparent wall has a thickness of 0.05 mm to 2 mm. In some cases, the transparent wall may be a microscope cover slip, or similar component. Microscope coverslips are widely available in several standard thicknesses that are identified by numbers, as follows: No. 0 - 0.085 to 0.13 mm thick, No. 1 - 0.13 to 0.16 mm thick, No. 1.5 - 0.16 to 0.19 mm thick, No. 2 - 0.19 to 0.23 mm thick, No. 3 - 0.25 to 0.35 mm thick, No. 4 - 0.43 to 0.64 mm thick, any one of which may be used as a transparent wall, depending on the device, microscope, cell size, and cell detection strategy.

Any of the microfluidic channels of the present disclosure may have a height, for example from a top wall to a bottom wall, ranging from 0.5 μιη to 100 μιη. The microfluidic channel of any of the devices provided herein may have a height in a range of 0.5 μιη to 100 μιη, 0.1 μπι to 100 μιη, 1 μιη to 50 μιη, 1 μιη to 50 μιη, 10 μιη to 40 μιη, 5 μιη to 15 μιη, 0.1 μιη to 5 μιη, or 2 μιη to 5 μιη. The microfluidic channel may have a height of up to 0.5 μιη, 1 μιη, 1.5 μιη, 2.0 μιη, 2.5 μιη, 3.0 μιη, 3.5 μιη, 4.0 μιη, 4.5 μιη, 5.0 μιη, 5.5 μιη, 6.0 μιη, 6.5 μιη, 7.0 μιη, 7.5 μιη, 8.0 μιη, 8.5 μιη, 9.0 μιη, 9.5 μιη, 10 μιη, 20 μιη, 30 μιη, 40 μιη, 50 μιη, 75 μιη, 100 μιη, or more. In a specific embodiment, the microfluidic channel has a height of 15 μιη, or about 15 μιη. It should be appreciated that the height of the microfluidic channel can be any suitable height, which may be based on the intended use of the microfluidic device. For example, the height of the device may be larger than a cell to allow the cell to flow through the device. As another example, the height of the device may be slightly larger than a cell that is introduced into the device so that it can be squeezed between the top wall and the bottom wall, where one of the walls is a membrane or film. A force (e.g., pressure) can be exerted on the membrane to narrow the distance between the top wall and bottom wall, thereby squeezing the cell. Accordingly, in some embodiments, the height of the microfluidic channel is from Ο.ΟΙμιη to 100 μιη larger than a dimension (e.g. , diameter) of a cell in the device. In some embodiments, the microfluidic channel is from Ο.ΟΙμιη to 0.1 μιη, from Ο.ΟΙμιη to 1 μιη, from Ο.ΟΙμιη to 10 μιη, from Ο.ΟΙμιη to 20 μιη, from Ο.ΟΙμιη to 50 μιη, from Ο.ΟΙμιη to 80 μιη, from Ο. ΐμιη to 1 μιη, from Ο. ΐμιη to 10 μιη, from Ο. ΐμιη to 20 μιη, from Ο. ΐμιη to 50 μιη, from Ο. ΐμιη to 80 μιη, from Ιμιη to 10 μιη, from Ιμιη to 20 μιη, from Ιμιη to 50 μιη, from Ιμιη to 80 μιη, from 10 μιη to 20 μιιι, from 10 μιη to 50 μιη, from 10 μπι to 80 μιη, from 20 μιη to 50 μπι, from 20 μιη to 80 μπι, or from 20 μιη to 80 μιη larger than a dimension (e.g. , diameter) of a cell in the device.

Any of the microfluidic channels of the present disclosure may have a width, for example from a first side wall to a second side wall, ranging from 0.01 mm to 5 mm. The microfluidic channel of any of the devices provided herein may have a width in a range of 0.01 mm to 4 mm, 0.1 mm to 3 mm, 0.1 mm to 2 mm, 0.2 mm to 2 mm, 0.5 mm to 2 mm, 0.5 mm to 1.5 mm, 0.8 mm to 1.5 mm, or 1 mm to 1.4 mm. In some embodiments, the microfluidic channel may have a width of up to 0.01 mm, 0.05 mm, 0.2 mm, 0.4 mm, 0.6 mm, 0.8 mm, 1 mm, 1.2 mm, 1.3 mm, 1.4 mm, 1.5 mm, 1.6 mm, 1.7 mm, 1.8 mm, 1.9 mm, 2.0 mm, 2.2 mm, 2.4 mm, 2.8 mm, 3 mm, 3.5 mm, 4 mm, 4.5 mm, 6 mm, 6.5 mm, 7 mm, or more. In a specific embodiment, the microfluidic channel has a width of 1.3 mm, or about 1.3 mm.

Any of the microfluidic channels of the present disclosure may have a length, for example from a first end wall to a second end wall, or from a first inlet to a second inlet, ranging from 0.1 mm to 20 mm. The microfluidic channel of any of the devices provided herein may have a length in a range of 0.1 mm to 19 mm, 0.1 mm to 18 mm, 0.1 mm to 15 mm, 0.2 mm to 14 mm, 0.2 mm to 12 mm, 0.5 mm to 10 mm, 0.5 mm to 5 mm, or 1 mm to 5 mm. In some embodiments, the microfluidic channel may have a length of up to 0.01 mm, 0.05 mm, 0.2 mm, 0.4 mm, 0.6 mm, 0.8 mm, 1 mm, 1.2 mm, 1.3 mm, 1.4 mm, 1.5 mm, 1.6 mm, 1.7 mm, 1.8 mm, 1.9 mm, 2.0 mm, 2.2 mm, 2.4 mm, 2.8 mm, 3 mm, 3.5 mm, 4 mm, 4.5 mm, 6 mm, 6.5 mm, 7 mm, or more. In a specific embodiment, the microfluidic channel has a length of 3 mm, or about 3 mm.

The device described above can further contain a reservoir fluidically connected with the one or more microfluidic channels, and a pump that perfuses fluid from the reservoir through the one or more microfluidic channels, and optionally, a microscope arranged to permit observation within the one or more microfluidic channels. The reservoir may contain cells suspended in a fluid. The fluidics connecting the reservoir to the microfluidic channel may include one or more filters to prevent the passage of unwanted or undesirable components into the microfluidic channels.

The device may be designed and configured to create a pressure gradient from the channel inlet to the channel outlet of 0.05 Pa/μιη, 0.1 Pa/μιη, 0.15 Pa/μιη, 0.2 Pa/μιη, 0.25 Pa/μιη, 0.3 Pa/μιη, 0.35 Pa/μιη, 0.4 Pa/μιη, 0.45 Pa/μιη, 0.5 Pa/μιη, 0.55 Pa/μιη, 0.6 Pa/μιη, 0.65 Pa/μιη, 0.7 Pa/μιη, 0.75 Pa/μιη, 0.8 Pa/μιη, 0.85 Pa/μιη, 0.9 Pa/μιη, 0.95 Pa/μιη, 1 Pa/μιη, 2 Pa/μιη, 3 Pa/μιη, 4 Pa/μιη, 5 Pa/μιη, 10 Pa/μιη, or more. In other embodiments, the device may be designed and configured to create a pressure gradient from the channel inlet to the channel outlet of about 0.05 Pa/μιη, 0.1 Pa/μιη, 0.15 Pa/μιη, 0.2 Pa/μιη, 0.25 Pa/μιη, 0.3 Pa/μιη, 0.35 Pa/μιη, 0.4 Pa/μιη, 0.45 Pa/μιη, 0.5 Pa/μιη, 0.55 Pa/μιη, 0.6 Pa/μιη, 0.65 Pa/μιη, 0.7 Pa/μιη, 0.75 Pa/μιη, 0.8 Pa/μιη, 0.85 Pa/μιη, 0.9 Pa/μιη, 0.95 Pa/μιη, 1 Pa/μιη, 2 Pa/μιη, 3 Pa/μm, 4 Pa/μm, 5 Pa/μιη, 10 Pa/μm, or more.

It should be appreciated that the pressure gradient may be expressed in other units, for example in mbar. In some embodiments, The device may be designed and configured to create a pressure gradient from the channel inlet to the channel outlet from 50 mbar to 500 mbar, for example 60 mbar, 80 mbar, 100 mbar, 150 mbar, 200 mbar, 250 mbar, 300 mbar, 350 mbar, 400 mbar, 450 mbar, or 500 mbar. In some embodiments, the device may be designed and configured to create a pressure gradient from the channel inlet to the channel outlet of about 60 mbar, 80 mbar, 100 mbar, 150 mbar, 200 mbar, 250 mbar, 300 mbar, 350 mbar, 400 mbar, 450 mbar, or 500 mbar.

The device may be designed and configured to create a pressure gradient from a channel inlet to a channel outlet in a range of 0.05 Pa/μιη to 0.1 Pa/μιη, 0.1 Pa/μιη to 0.3 Pa/μιη, 0.1 Pa/μιη to 0.5 Pa/μιη, 0.1 Pa/μιη to 0.8 Pa/μιη, 0.5 Pa/μιη to 1 Pa/μιη, 1 Pa/μιη to 10 Pa/μιη, for example. The pressure gradient may be linear or non-linear.

The device may be designed and configured to create a pressure (gauge pressure) in the channel of up to 5 Pa, 10 Pa, 20 Pa, 30 Pa, 40 Pa, 50 Pa, 100 Pa, 200 Pa, 300 Pa, 400 Pa, 500 Pa, 600 Pa, 700 Pa, 800 Pa, 900 Pa, 1 kPa, 2 kPa, 5 kPa, 10 kPa or more. The device may be designed and configured to create a pressure (gauge pressure) in the channel in a range of 50 Pa to 200 Pa, 100 Pa to 500 Pa, 100 Pa to 800 Pa, 100 Pa to 1 kPa, 500 Pa to 5 kPa, or 500 Pa to 10 kPa.

The device may be designed and configured to create an average fluid velocity within the channel of up to 1 μητ/s, 2 μιη/s, 5 μιη/s, 10 μιη/s, 20 μιη/s, 50 μιη/s, 100 μιη/s, or more. The device may be designed and configured to create an average fluid velocity within the channel in a range of 1 μητ/8 to 5 μιη/s, 1 μιη/s to 10 μιη/s, 1 μιη/s to 20 μιη/s, 1 μιη/s to 50 μιη/s, 10 μιη/s to 100 μιη/s, or 10 μιη/s to 200 μιη/s, for example.

The device may be configured to pulse a fluid through the channel of any of the microfluidic devices. In some embodiments, pulsing the fluid allows the gas content of the fluid within the chamber (e.g., under conditions of hypoxia) to be maintained under high pressure or flow rates. For example, at continuous high flow rates (e.g., high pressures) the fluid being regulated to be hypoxic may increase in oxygen concentration. In some embodiments, the device is configured so that the pulsing may be controlled. For example, the amplitude, duty cycle, and period, duration of the pulse may be controlled.

The device may be designed and configured to have a channel cross-sectional area, perpendicular to the flow direction, of 0.1 mm ² , 0.5 mm ² , 10 mm ² , 20 mm ² , 30 mm ² , 40 mm ² , 50 mm ² , 60 mm ² , 70 mm ² , 80 mm ² , 90 mm ² , 100 mm ² , 150 mm ² , 200 mm ² , 300 mm ² , 400 mm ² , 500 mm ² , 600 mm ² , 700 mm ² , 800 mm ² , 900 mm ² , or 1000 mm ² .

The device may be designed and configured to produce any of a variety of different shear rates (e.g., up to 1000 s ^"1 ). For example, the device may be designed and configured to produce a shear rate in a range of 10 s ^"1 to 50 s ^"1 , 10 s ^"1 to 100 s ^"1 , 50 s ^"1 to 200 s ^"1 , 100 s ^"1 to 200 s ^"1 , 100 s ^"1 to 500 s ^"1 , 50 s ^"1 to 500 s ^"1 , or 50 s ^"1 or 1000 s ^"1 .

Alternatively or additionally, the device described herein further contains a heat transfer element, which can maintain the fluid at a predetermined temperature (e.g. , a physiologically relevant temperature (e.g., a temperature that would be found in vivo in a healthy or diseased subject or one with a particular condition as provided herein), such as 30 °C to 45 °C, preferably 37 °C, 40 °C or 41 °C).

In some embodiments, non-microfluidic devices are provided. In some embodiments, the non-microfluidic device is AFM, optical tweezers, micropipette, magnetic twisting cytometer, cytoindenter, microindenter, nanoindenter, microplate stretcher, microfabricated post array detector, micropipette aspirator, substrate stretcher, shear flow detector, diffraction phase microscope, or tomographic phase microscope.

Non-limiting examples of the microfluidic devices and systems provided herein are shown in Figs. 1, 11- 16 and 20. It should be appreciated that the examples provided herein are not to be construed as limiting and are included for the purpose of providing exemplary embodiments of the devices and systems described herein. In some embodiments, The microfluidic device 1 includes a microfluidic channel 10 comprising at least one inlet 5 and one outlet 15, each connected to a reservoir (inlet reservoir 20, outlet reservoir 140). An example of one embodiment of a microfluidic device is shown in Fig. 1. Other exemplary schematic representations of the device design can be found in FIGs. 11-16 and 20. In some embodiments, the microfluidic channel comprises a first wall 110 wherein at least a portion of this wall is coated with at least one protein and a second wall 120 adjacent to the microfluidic channel, wherein at least a portion of the second wall 120 comprises a gas permeable membrane or film 130. The device may include a pump 25 that perfuses fluid from a reservoir 20. In some embodiments, the reservoir 20 further comprises a filter. In some embodiments, a single syringe 150 is used as a source of back pressure (FIG. 15). A flow sensor 80 and/or pressure regulator 90 may connect the reservoir 20 to the microfluidic channel 10. The device can accommodate a microscope 30 arranged to permit observation within the microfluidic channel 10. The device may further comprise a heat transfer element 40 to regulate temperature within the microfluidic channel. The device may comprise a gas channel 50, which contacts at least a portion of the gas permeable membrane or film 130. A gas flow sensor or regulator 70 may connect the gas channel 50 to the gas source 60. The device system may also comprise a computer 100 in electronic communication with the microscope 30, heat transfer element 40, gas flow sensor or regulator 70, the pressure regulator 90, the fluid flow sensor 80, and/or the pump 25.

Methods

Methods of Measuring Individual Cell Properties

Described herein are devices and methods for assessing cell properties under controlled gas environments. Accordingly, a microfluidics-based model was developed to quantify cell-level processes modulating the pathophysiology of disease {e.g., sickle cell disease (SCD), spherocytosis, ovalocytosis, alpha thalassemia, beta thalassemia, delta thalassemia, malaria and anemia). This in vitro model enabled quantitative investigations of the kinetics of cell processes and transformations such as cell sickling, unsickling and cell rheology. Examples of the use of the devices of the invention are included in the Examples below. These results support the use of the microfluidic platform described as a unique and quantitative approach to assess the kinetic, rheological and hematological factors involved in vaso-occlusive events associated with disease and to develop alternative diagnostic tools of disease severity. Such insights may also lead to a better understanding of the pathogenic basis and mechanism of drug response in disease.

In some cases, the methods may be carried out in a high throughput manner. In some aspects, methods are provided that are useful for diagnosing, assessing, characterizing, evaluating, and/or predicting disease based on transit characteristics of cells, e.g. , red blood cells, platelets, cancer cells, and tissues, e.g. , blood in microfluidic devices. In one aspect, the present disclosure includes a high throughput method of measuring a morphological and/or mechanical property of an individual cell under controlled gas conditions including: flowing a fluid comprising a plurality of cells through a channel comprising a wall, wherein at least a portion of the wall is coated with at least one protein, obtaining at least one measurement of a cell in the fluid; and regulating a level of gas in the fluid. It should be appreciated that the protein may be any protein provided herein.

In some embodiments, the method measures morphological properties, including cell shape, such as round, disk shaped, biconcave, oblong, or sickle shaped. The method may be used to assess normal or abnormal cell textures, including smooth, coarse, or spiky textures.

Furthermore, the method may measure a fraction of cells with an abnormal shape or texture by examining the delay time of recovery from an abnormal shape change. In one embodiments, the cell shape change is between sickling and unsickling.

In some embodiments, the cells are bound to the portion of the wall coated with at least one protein, and in other embodiments, the cells are not bound to the portion of the wall coated with at least one protein. The measurement may be used to determine a proportion of cells having an abnormal shape and/or texture at a certain temperature, flow rate and/or gas concentration. The property may be a mechanical property, such as adhesiveness. For example, the method includes measuring the adhesiveness of the sample to the portion of the wall coated with at least one protein. Cells may slide, roll, or tumble along the portion of the wall that is coated with at least on protein, and the device may measure the number and/or fraction of cells that bind to the portion of the wall coated with at least one protein. Other examples of measurements contemplated include: the rate at which the cells bind to the portion of the wall coated with at least one protein, the speed that the cells move along the portion of the wall coated with at least one protein, and the number and/or fraction of cells that detach from a portion of the wall that is coated with at least one protein. Each measurement can be conducted at a certain temperature, flow rate, and/or gas concentration and average rates, speeds, and distances can be determined from the measurements.

Furthermore, the measurement may be used to determine the number of and/or fraction of cells that detach from the wall that is coated with at least one protein at a certain temperature, flow rate, and/or gas concentration.

The method also may include contacting the cell with the wall that is coated with at least one protein while fluid flows through the channel. In a further embodiment, the flow of the fluid through the channel may be stopped. The cell may contact the wall that is coated with at least one protein when the fluid is stopped, and the fluid may pass through the channel after it had been stopped.

Furthermore, mechanical properties, such as deformability, may be examined. The cell may be bound to a fixed position on the portion of the wall coated with at least one protein, where its deformability may be measured. Deformability can be measured as the distance that a cell stretches or the ratio of the length versus width of a cell, The

measurement can be used to determine the amount, or average amount, a cell deforms under certain temperatures, flow rates, and/or gas concentrations. However, additional indices may be used in the assessment of cell deformability. For example, cell location, cell adhesion sites, cell area (projected), and cell shape parameters, such as elongation ratio, maximum Feret diameter ratio, and Convex Hull etc. However, additional indices for assessing cell deformability would be apparent to the skilled artisan and are within the scope of this disclosure.

Some aspects of the disclosure provide devices and methods for measuring the fragility of one or more objects (e.g., cells). In some embodiments, the fragility of cells (e.g., red blood cells) is determined. In some embodiments, the mechanical fragility of one or more cells is determined. In some embodiments, the mechanical fragility index of one or more cells (e.g., a red blood cells) is determined. The mechanical fragility index (MFI) is an in vitro measurement of the extent of RBC sublethal injury. Sublethal injury might constitute a component of the RBC storage lesion. Methods for determining the MFI of cells have been described in Raval JS, et al., "The use of the mechanical fragility test in evaluating sublethal RBC injury during storage." Vox sanguinis 2010; 99(4):325 D331; the entire contents of which are incorporated herein by reference. It should be appreciated that both lethal and sub- lethal conditions may be assessed. For example, by determining (1) an amount of

compressive stress (force/unit area); when the cells are compressed with the membrane their surface area increases; (2) applied load vs time curve; (3) applied compressive stress vs time curve; and (4) applied compressive force vs area increase.

In some embodiments, the cells are from a subject. Such cells can be obtained from a blood sample, and may comprise red blood cells, white blood cells, stem cells or epithelial cells. In a particular embodiment, the cells comprise one or more tumor cells.

In some embodiments, the gas channel comprises at least one inlet and/or at least one outlet. A gas or gas mixture may be supplied to the inlet of the gas channel from one or more tanks containing the gas or gas mixture. In some non-limiting embodiments, the gas supplied to the gas channel is oxygen, nitrogen, carbon dioxide, nitric oxide, carbon monoxide, nitrous oxide, nitrogen dioxide, methane, or any combination thereof. In some embodiments the gas supplied to the gas channel contains oxygen. In some embodiments the gas supplied contains between 1% and 100 % oxygen. In some embodiments the gas supplied contains from 1% to 2%, from 1% to 3%, from 1% to 5%, from 1% to 10%, from 1% to 20%, from 1% to 40%, from 1% to 60%, from 1% to 80%, from 1% to 100%, from 5% to 10%, from 5% to 20%, from 5% to 40%, from 5% to 60%, from 5% to 80%, from 5% to 100%, from 20% to 40%, from 20% to 60%, from 20% to 80%, from 20% to 100%, from 40% to 60%, from 40% to 80%, from 40% to 100%, from 60% to 80%, from 60% to 100% or from 80% to 100% oxygen. In some embodiments the gas supplied to the gas channel contains about 2%, about 5%, or about 20% oxygen. As used herein, the term "about," or "approximately" as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In certain embodiments, the term "approximately" or "about" refers to a range of values that fall within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (for example, when such number would exceed 100% of a possible value). In some embodiments the gas supplied to the gas channel contains about 20% oxygen, 5% carbon dioxide and about 75% nitrogen. In other embodiments the gas supplied to the gas channel contains about 5% oxygen, 5% carbon dioxide and about 90% nitrogen. In yet other embodiments the gas supplied to the gas channel contains about 2% oxygen, 5% carbon dioxide and about 93% nitrogen. The gas or gas mixture may be supplied to one or more inlets of one or more gas channels using any suitable means, such as tubing or hoses.

In some embodiments, the level of gas in the fluid is regulated to be at a concentration of less than 5%. In other embodiments, the level of the gas in the fluid is regulated to be at a concentration from 5% to 10%, 5% to 15%, 5% to 20%, 20% to 25%, 20% to 30%, 20% to 35%, 20% to 40%, 40% to 45%, 40% to 50%, 40% to 55%, 40% to 60%, or to a level greater than 60%. In some embodiments, the level of gas in the fluid is regulated to be at a concentration of about 5%, about 4%, about 3%, or about 2%.

In some embodiments, the property is measured at two or more different gas concentrations. The gas concentration may be increased and/or decreased, and the property may be measured as a function of time and as a function of gas concentration.

Any appropriate condition or disease of a subject may be evaluated using the methods herein, typically provided that a test agent may be obtained from the subject that has a material property (e.g. , deformability, shear modulus, viscosity, Young's modulus, etc.) that is indicative of the condition or disease. The condition or disease to be detected may be, for example, a fetal cell condition, HPV infection, or a hematological disorder, such as sickle cell disease, sickle cell trait (SCT), spherocytosis, ovalocytosis, alpha thalassemia, beta thalassemia, delta thalassemia, malaria, anemia, diabetes, leukemia, hematological cancer, infectious mononucleosis, HIV, malaria, leishmaniasis, babesiosis, monoclonal gammopathy of undetermined significance or multiple myeloma. Examples of hematological cancer include, but are not limited to, Hodgkin's disease, Non-Hodgkin's lymphoma, Burkitt's lymphoma, anaplastic large cell lymphoma, splenic marginal zone lymphoma, hepatosplenic T-cell lymphoma, angioimmunoblastic T-cell lymphoma (AILT), multiple myeloma, Waldenstrom macroglobulinemia, plasmacytoma, acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), B cell CLL, acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), T-cell prolymphocytic leukemia (T-PLL), B-cell prolymphocytic leukemia (B-PLL), chronic neutrophilic leukemia (CNL), hairy cell leukemia (HCL), T-cell large granular lymphocyte leukemia (T-LGL) and aggressive NK-cell leukemia. In one embodiment, the cells are from a subject having or suspected of having sickle cell disease. The foregoing diseases or conditions are not intended to be limiting. It should thus be appreciated that other appropriate diseases or conditions may be evaluated using the methods disclosed herein.

In some embodiments, the fluid comprising the cells is flowed at a predetermined flow rate. The device may be designed and configured to create a flow rate in a range of about 0.01 μί/ηιίη to 0.1 μί/ηιίη, 0.1 μί/ηιίη to 1 μί/ηιίη , 0.1 μΙ7ιηίη to 10 μί/ηιίη, 0.1 μΙ7ιηίη to 20 μί/ηιίη, 0.1 μυηιίη to 30 μΙ7ιηίη, 0.1 to 40 μΙ7ιηίη, 0.1 μΙ7ιηίη to 50 μυηιίη, 0.1 μΙ7ιηίη to 60 μυηιίη, 0.1 μυηιίη to 70 μΙ7ιηίη, 0.1 μΙ7ιηίη to 80 μυηιίη, 0.1 μΙ7ιηίη to 90 μυηιίη, or 0.1 μυηιίη to 100 μΙ7ιηίη.

In some embodiments, the fluid comprising the cells is flowed at a predetermined pressure gradient. The device may be designed and configured to create a pressure gradient from the channel inlet to the channel outlet of 0.01 Pa/μιη, 0.05 Pa/μιη, 0.1 Pa/μιη, 0.15 Pa/μιη, 0.2 Pa/μιη, 0.25 Pa/μιη, 0.3 Pa/μιη, 0.35 Pa/μιη, 0.4 Pa/μιη, 0.45 Pa/μιη, 0.5 Pa/μιη, 0.55 Pa/μιη, 0.6 Pa/μιη, 0.65 Pa/μιη, 0.7 Pa/μιη, 0.75 Pa/μιη, 0.8 Pa/μιη, 0.85 Pa/μιη, 0.9 Pa/μιη, 0.95 Pa/μιη, 1 Pa/μιη, 2 Pa/μιη, 3 Pa/μιη, 4 Pa/μιη, 5 Pa/μιη, 10 Pa/μιη, or more.

The device may be designed and configured to create a pressure gradient from the channel inlet to the channel outlet in a range of 0.01 Pa/μιη to 0.05 Pa/μιη, 0.05 Pa/μιη to 0.1 Pa/μιη, 0.1 Pa/μιη to 0.3 Pa/μιη, 0.1 Pa/μιη to 0.5 Pa/μιη, 0.1 Pa/μιη to 0.8 Pa/μιη, 0.1 Pa/μιη to 2 Pa/μηι, 0.1 Pa/μηι to 5 Pa/μηι, 0.5 Pa/μηι to 1 Pa/μηι, 1 Pa/μηι to 10 Pa/μηι, for example. The pressure gradient may be ceased, continuous, or not continuous.

In some embodiments, the methods provide regulating the pressure of a gas or fluid in the gas channel of any of the devices provided herein. It should be appreciated that, as one example, pressurizing the gas channel may exert a force on the membrane of any of the devices provided herein to narrow the microfluidic channel, thereby squeezing one or more objects (e.g., cells) in the microfluidic channel. In some embodiments, the gas or fluid is supplied to the gas channel via one or more inlets or outlets of the gas channel. In some embodiments, the gas or fluid is at a pressure from 0.1 psi to 100 psi in the gas channel. In some embodiments, the gas or fluid is at a pressure from 0.1 psi to 1 psi, from 0.1 psi to 2 psi, from 0.1 psi to 5 psi, from 0.1 psi to 10 psi, from 0.1 psi to 20 psi, from 0.1 psi to 40 psi, from 0.1 psi to 60 psi, from 0.1 psi to 80 psi, from 1 psi to 2 psi, from 1 psi to 5 psi, from 1 psi to 10 psi, from 1 psi to 20 psi, from 1 psi to 40 psi, from 1 psi to 60 psi, from 1 psi to 80 psi, from 2 psi to 5 psi, from 2 psi to 10 psi, from 2 psi to 20 psi, from 2 psi to 40 psi, from 2 psi to 60 psi, from 2 psi to 80 psi, from 5 psi to 10 psi, from 5 psi to 20 psi, from 5 psi to 40 psi, from 5 psi to 60 psi, from 5 psi to 80 psi, from 10 psi to 20 psi, from 10 psi to 40 psi, from 10 psi to 60 psi, from 10 psi to 80 psi, from 20 psi to 40 psi, from 20 psi to 60 psi, from 20 psi to 80 psi, from 40 psi to 60 psi, from 40 psi to 80 psi, or from 60 psi to 80 psi in the gas channel.

In some embodiments, the methods provide regulating the pressure of a gas or fluid in the gas channel of any of the devices provided herein. In some embodiments, the pressure of the gas or fluid is increased. In some embodiments, the pressure of the gas or fluid is decreased. In some embodiments, the pressure of the gas or fluid is increased at a rate from 1 psi/min to 100 psi/min. In some embodiments, the pressure of the gas or fluid is increased at a rate from 1 psi/min to 5 psi/min, from 1 psi/min to 10 psi/min, from 1 psi/min to 15 psi/min, from 1 psi/min to 20 psi/min, from 1 psi/min to 40 psi/min, from 1 psi/min to 60 psi/min, from 1 psi/min to 80 psi/min, from 5 psi/min to 10 psi/min, from 5 psi/min to 15 psi/min, from 5 psi/min to 20 psi/min, from 5 psi/min to 40 psi/min, from 5 psi/min to 60 psi/min, from 5 psi/min to 80 psi/min, from 10 psi/min to 15 psi/min, from 10 psi/min to 20 psi/min, from 10 psi/min to 40 psi/min, from 10 psi/min to 60 psi/min, from 10 psi/min to 80 psi/min, from 20 psi/min to 40 psi/min, from 20 psi/min to 60 psi/min, from 20 psi/min to 80 psi/min, from 40 psi/min to 60 psi/min, from 40 psi/min to 80 psi/min, or from 60 psi/min to 80 psi/min. In some embodiments, the pressure of the gas or fluid is decreased at a rate from 1 psi/min to 100 psi/min. In some embodiments, the pressure of the gas or fluid is decreased at a rate from 1 psi/min to 5 psi/min, from 1 psi/min to 10 psi/min, from 1 psi/min to 15 psi/min, from 1 psi/min to 20 psi/min, from 1 psi/min to 40 psi/min, from 1 psi/min to 60 psi/min, from 1 psi/min to 80 psi/min, from 5 psi/min to 10 psi/min, from 5 psi/min to 15 psi/min, from 5 psi/min to 20 psi/min, from 5 psi/min to 40 psi/min, from 5 psi/min to 60 psi/min, from 5 psi/min to 80 psi/min, from 10 psi/min to 15 psi/min, from 10 psi/min to 20 psi/min, from 10 psi/min to 40 psi/min, from 10 psi/min to 60 psi/min, from 10 psi/min to 80 psi/min, from 20 psi/min to 40 psi/min, from 20 psi/min to 60 psi/min, from 20 psi/min to 80 psi/min, from 40 psi/min to 60 psi/min, from 40 psi/min to 80 psi/min, or from 60 psi/min to 80 psi/min.

The device may be designed and configured to measure the designated property after one or more reoxygenation (ReOxy) and/or deoxygenation (DeOxy) cycles. Methods are provided herein for evaluating, characterizing, and/or assessing mechanical, morphological, kinetic, rheological or hematological properties of cells under controlled gas conditions. In particular, methods are provided for measuring, evaluating and/or characterizing dynamic mechanical responses of biological cells, e.g. , red blood cells, white blood cells,

reticulocytes, platelets, etc. The methods typically involve obtaining measurements of cell deformability, cell velocity and cell shape. Measurements of cell deformability often involve an assessment of the transit time of one or more cells through one or more constrictions within a fluid channel of a microfluidic device, or an assessment of another parameter indicative of a resistance to deformation In further aspects, methods are provided that are useful for measuring changes in cell properties or characteristics in response to changes in the concentration of one or more gasses. As one example, the transit characteristics of a red blood cell through one or more constrictions of a microfluidic device are measured at high oxygen content (e.g., 20% oxygen) and low oxygen content (e.g., 2% oxygen).

Some aspects of the disclosure relate to determining cell properties in response to repetitive or cyclical changes in the concentration of one or more gases (e.g., alternating between relatively high and low concentrations of a gas in a fluid). In some aspects, methods are provided that are useful for measuring changes in cell properties or characteristics in response to one or more cycles of a gas concentration. For example, in some embodiments one or more changes in cell properties or characteristics are measured in response to one or more cycles of an oxygen, a nitrogen, a carbon dioxide, a carbon monoxide, a nitric oxide, a nitrous oxide, a nitrogen dioxide, or a methane gas concentration. However, it should be appreciated that cell properties or characteristics may be determined in response to one or more cycles of any suitable gas concentration. In some embodiments, one or more changes in cell properties or characteristics are measured in response to one or more changes in oxygen concentration.

A cycle of a gas concentration refers to a change from a relatively high gas concentration (e.g. , 20% oxygen) to a relatively low gas concentration (e.g. , 2% oxygen) and back to a relatively high gas concentration. A cycle of a gas concentration also refers to a change from a relatively low gas concentration (e.g. , 2% oxygen) to a relatively high gas concentration (e.g. , 20% oxygen) and back to a relatively low gas concentration. In some embodiments a cycle of a gas concentration refers to a change from a relatively high oxygen concentration to a relatively low oxygen concentration and back to a relatively high oxygen concentration, referred to herein as a deoxygenation (DeOxy) cycle. In some embodiments a cycle of a gas concentration refers to a change from a relatively low oxygen concentration to a relatively high oxygen concentration and back to a relatively low oxygen concentration, referred to herein as a reoxygenation (ReOxy) cycle. In some embodiments a change from a relatively high gas concentration (e.g. , of oxygen) to a relatively low gas concentration refers to a decrease in gas concentration of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or at least 100% in a gas or fluid. In some embodiments a change from a relatively low gas concentration (e.g. , of oxygen) to a relatively high gas

concentration refers to an increase in gas concentration of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100% in a gas or fluid.

Some aspects of the disclosure relate to determining cell properties in response to one or more cycles of a gas concentration. In some embodiments one or more cell properties are determined after being exposed to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, or at least 1000 cycles of a gas concentration. In some

embodiments, one or more cell properties are determined after being exposed to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, or at least 1000

deoxygenation (DeOxy) cycles. In some embodiments, one or more cell properties are determined after being exposed to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, or at least 1000 reoxygenation (ReOxy) cycles. In some embodiments, the cycles of a gas concentration provided herein may be performed for any suitable duration of time, which may depend on, among other factors, the intended purpose or the nature of the cells (e.g., healthy or diseased cells). In some embodiments, the duration of two or more consecutive cycles are the same. For example, in some embodiments, two or more consecutive cycles may be 360 seconds long. In some embodiments, the length of two or more consecutive cycles are different. For example a first cycle may be 360 seconds long and a second cycle may be 400 seconds long. In some embodiments, the length of two or more consecutive cycles may be increased. In some embodiments, the length of two or more consecutive cycles may be decreased. In some embodiments a cycle is from 5seconds (5s) to 1 hour (lh) long. However, it should be appreciated that a cycle may be any suitable duration and any exemplary cycle durations provided herein are not intended to be limiting. In some embodiments, a cycle is from 5s to 20s, from 5s to 100s, from 5s to 200s, from 5s to 400s, from 5s to 600s, from 5s to 1000s, from 5s to 20min, from 5s to 30min, from 5s to 40min, from 5s to 50min, from 100s to 200s, from 100s to 400s, from 100s to 600s, from 100s to 1000s, from 100s to 20min, from 100s to 30min, from 100s to 40min, from 100s to 50min from 100s to lh, from 200s to 400s, from 200s to 600s, from 2s to 1000s, from 200s to 20min, from 200s to 30min, from 200s to 40min, from 200s to 50min from 200s to lh, from 400s to 600s, from 400s to 1000s, from 400s to 20min, from 400s to 30min, from 400s to 40min, from 400s to 50min, or from 400s to lh in duration.

In some embodiments, the duration of time that a gas is at a relatively high

concentration, within a cycle, may vary. In some embodiments, the duration of time that a gas is at a relatively low concentration ,within a cycle, may vary. In some embodiments, the duration of time at which a gas is at a relatively high concentration and the duration of time at which a gas is at a relatively low concentration, within a cycle, is the same. In some embodiments, the duration of time at which a gas is at a relatively high concentration and the duration of time at which a gas is at a relatively low concentration, within a cycle, is different. In some embodiments, the duration of time at which a gas is at a relatively high concentration is greater than the duration of time at which a gas is at a relatively low concentration, within a cycle. In some embodiments, the duration of time at which a gas is at a relatively high concentration is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, 500%, 1000%, or at least 5000% greater than the duration of time at which a gas is at a relatively low concentration, within a cycle. In some embodiments, the duration of time at which a gas is at a relatively high concentration, within a cycle, is from Is to 20s, from Is to 100s, from Is to 200s, from Is to 400s, from Is to 600s, from Is to 1000s, from Is to 20min, from Is to 30min, from Is to 40min, from Is to 50min, from Is to lh, from 100s to 200s, from 100s to 400s, from 100s to 600s, from 100s to 1000s, from 100s to 20min, from 100s to 30min, from 100s to 40min, from 100s to 50min from 100s to lh, from 200s to 400s, from 200s to 600s, from 2s to 1000s, from 200s to 20min, from 200s to 30min, from 200s to 40min, from 200s to 50min from 200s to lh, from 400s to 600s, from 400s to 1000s, from 400s to 20min, from 400s to 30min, from 400s to 40min, from 400s to 50min, or from 400s to lh in duration. In some embodiments, the duration of time at which a gas is at a relatively high concentration is less than the duration of time at which a gas is at a relatively low concentration, within a cycle. In some embodiments, the duration of time at which a gas is at a relatively high concentration is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, 500%, 1000%, or at least 5000% less than the duration of time at which a gas is at a relatively low concentration, within a cycle. In some embodiments, the duration of time at which a gas is at a relatively low concentration, within a cycle, is from Is to 20s, from Is to 100s, from Is to 200s, from Is to 400s, from Is to 600s, from Is to 1000s, from Is to 20min, from Is to 30min, from Is to 40min, from Is to 50min, from Is to lh, from 100s to 200s, from 100s to 400s, from 100s to 600s, from 100s to 1000s, from 100s to 20min, from 100s to 30min, from 100s to 40min, from 100s to 50min from 100s to lh, from 200s to 400s, from 200s to 600s, from 2s to 1000s, from 200s to 20min, from 200s to 30min, from 200s to 40min, from 200s to 50min from 200s to lh, from 400s to 600s, from 400s to 1000s, from 400s to 20min, from 400s to 30min, from 400s to 40min, from 400s to 50min, or from 400s to lh in duration.

Alternatively or additionally, the device described herein further contains a heat transfer element, which can maintain the fluid at a predetermined temperature (e.g., a physiologically relevant temperature (e.g., a temperature that would be found in vivo in a healthy or diseased subject or one with a particular condition as provided herein), such as 30 °C to 45 °C, preferably 37 °C, 40 °C or 41 °C). In some embodiments, the fluid comprising the cells is flowed at a predetermined temperature, preferably a physiological temperature.

In some embodiments, the method includes a protein, such as a cell surface protein or extracellular matrix (ECM) protein. The cell surface protein may be a cell adhesion molecule, such as an integrin, a cadherin, a selectin, a receptor tyrosine kinase, or a G-protein coupled receptor. The ECM protein, in some embodiments, may be collagen, elastin, laminin, or fibronectin. The protein may comprise an antibody.

In further embodiments, the fluid comprising a plurality of cells is flowed through the device of the present disclosure.

Monitoring Efficacy of Therapeutic Agents and Testing Candidate Therapeutic Agents

Methods are also provided for testing candidate therapeutic agents for treating a condition or disease in a subject. The methods typically involve: (a) perfusing a fluid comprising one or more cells from the subject through the any of the microfluidic devices, described herein, where the level of one or more gases is regulated, (b) administering one or more compounds to the fluid of (a), or wherein the fluid comprises the one or more compounds; (c) determining a property of one or more of the cells; and (d) comparing the property to an appropriate standard, wherein the results of the comparison are indicative of the status of the condition or disease in the subject.

The two or more compounds may be administered to the fluid sequentially or simultaneously. An effective therapeutic agent may be identified based on the comparison in (d). The cells may be from a subject, and the effective therapeutic agent may be administered to the subject. The compounds may be from a library of compounds, and in some

embodiments, are candidate therapeutic agents.

In some embodiments, this method may be used to identify candidate therapeutic agents that improve blood flow in subjects with circulation problems such as sickle cell disease, leg ulcers, pain from diabetic neuropathy, eye and ear disorders, and altitude sickness. Similarly for subjects with aggregation or clotting disorders of cells or insufficient delivery of essential chemicals such as oxygen to the brain in subjects with strokes from blood clots.

Method for Analyzing Condition or Disease in a Subject

In some embodiments, a method for analyzing, diagnosing, detecting, or determining the severity of a condition or disease in a subject, includes (a) perfusing a fluid comprising one or more cells from the subject through the any of the microfluidic devices, described herein, where the level of one or more gases is regulated, (b) determining a property of one or more of the cells; and (c) comparing the property to an appropriate standard, wherein the results of the comparison are indicative of the status of the condition or disease in the subject. The results of the comparison are typically indicative of the status of the condition or disease in the subject. In one example, the device is a microfluidic channel with a gas permeable membrane or film. In another example, the device is a microfluidic channel with a gas permeable membrane or film and a gas channel. The deformable object, in this example, typically has a mechanical property, the value of which is indicative of the presence of sickle cell disease. In one example, the method is used to determine the severity of the disease based on differences in mechanical properties. In another example, the method is used to predict the likelihood that a subject will undergo vaso-occlusion crisis based on differences in mechanical properties. In such methods, the methods may be performed under different regulated gas conditions.

In other examples, the property may be adhesiveness and/or detachment. Further, certain methods of the invention provide for measurement of cytoadhesive properties of a cell population, in combination with or separate from measurement of the deformability of the cell population. The combination of determining cytoadhesive properties and the

deformative properties of a cell population, particularly a cell population containing a plurality of different cell types (e.g. , red blood cells and white blood cells), may be used to generate a "Health Signature" that comprises an array of properties that can be tracked in a subject over a period of time. Such a Health Signature may facilitate effective monitoring of a subject's health over time. Such monitoring may lead to an early detection of potential acute or chronic infection, or other disease, disorder, fitness, or condition. In some cases, further, knowledge of the overall rheology of a material, along with either the deformative or cytoadhesive property of a cell, allows the determination of the other property.

An "appropriate standard" is a parameter, value or level indicative of a known outcome, status or result (e.g. , a known disease or condition status). An appropriate standard can be determined (e.g. , determined in parallel with a test measurement) or can be preexisting (e.g. , a historical value, etc.). The parameter, value or level may be, for example, a transit characteristic (e.g. , transit time), a value representative of a mechanical property, a value representative of a rheological property, etc. For example, an appropriate standard may be the transit characteristic of a test agent obtained from a subject known to have a disease, or a subject identified as being disease-free. In the former case, a lack of a difference between the transit characteristic and the appropriate standard may be indicative of a subject having a disease or condition. Whereas in the latter case, the presence of a difference between the transit characteristic and the appropriate standard may be indicative of a subject having a disease or condition. The appropriate standard can be a mechanical property or rheological property of a cell obtained from a subject who is identified as not having the condition or disease or can be a mechanical property or rheological property of a cell obtained from a subject who is identified as having the condition or disease.

The magnitude of a difference between a parameter, level or value and an appropriate standard that is indicative of known outcome, status or result may vary. For example, a significant difference that indicates a known outcome, status or result may be detected when the level of a parameter, level or value is at least 1%, at least 5%, at least 10%, at least 25%, at least 50%, at least 100%, at least 250%, at least 500%, or at least 1000% higher, or lower, than the appropriate standard. Similarly, a significant difference may be detected when a parameter, level or value is at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 100-fold, or more higher, or lower, than the level of the appropriate standard. Significant differences may be identified by using an appropriate statistical test. Tests for statistical significance are well known in the art and are exemplified in Applied Statistics for Engineers and Scientists by Petruccelli, Chen and Nandram Reprint Ed. Prentice Hall (1999).

Any appropriate condition or disease of a subject may be evaluated using the methods herein, typically provided that a test agent may be obtained from the subject that has a material property (e.g. , deformability, shear modulus, viscosity, Young's modulus, etc.) that is indicative of the condition or disease. The condition or disease to be detected may be, for example, a fetal cell condition, HPV infection, or a hematological disorder, such as sickle cell disease, sickle cell trait (SCT), spherocytosis, ovalocytosis, alpha thalassemia, beta thalassemia, delta thalassemia, malaria, anemia, diabetes, leukemia, hematological cancer, infectious mononucleosis, HIV, malaria, leishmaniasis, babesiosis, monoclonal gammopathy of undetermined significance or multiple myeloma. Examples of hematological cancer include, but are not limited to, Hodgkin's disease, Non-Hodgkin's lymphoma, Burkitt's lymphoma, anaplastic large cell lymphoma, splenic marginal zone lymphoma, hepatosplenic T-cell lymphoma, angioimmunoblastic T-cell lymphoma (AILT), multiple myeloma, Waldenstrom macroglobulinemia, plasmacytoma, acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), B cell CLL, acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), T-cell prolymphocytic leukemia (T-PLL), B-cell prolymphocytic leukemia (B-PLL), chronic neutrophilic leukemia (CNL), hairy cell leukemia (HCL), T-cell large granular lymphocyte leukemia (T-LGL) and aggressive NK-cell leukemia. The foregoing diseases or conditions are not intended to be limiting. It should thus be appreciated that other appropriate diseases or conditions may be evaluated using the methods disclosed herein.

In some embodiments, the appropriate standard is the value of a transit characteristic for a test agent at a regulated gas concentration that has been contacted with a control therapeutic agent (e.g., hydroxyurea or 5 -hydroxy methy If urfural). Typically, a control therapeutic agent is a molecule that has a known effect on deformability of a test agent and that is effective for treating the condition or disease. Thus, comparing the transit

characteristic of a candidate therapeutic agent with that of a control therapeutic agent provides a basis for identifying candidate therapeutic agents that are likely to be useful for treating the disease or condition. For example, a candidate therapeutic agent that results in the same or a similar value for a particular transit characteristic as that of a control therapeutic agent that is known to be effective for treating the disease or condition is likely to be an agent that is also effective for treating the disease or condition.

Typically the therapeutic agent or candidate therapeutic agent is a small molecule or pharmaceutical agent. "Small molecule" refers to organic compounds, whether naturally- occurring or artificially created (e.g., via chemical synthesis) that have relatively low molecular weight and that are not proteins, polypeptides, or nucleic acids. Small molecules are typically not polymers with repeating units. In certain embodiments, a small molecule has a molecular weight of less than about 1500 g/mol. In certain embodiments, the molecular weight of the polymer is less than about 1000 g/mol. Also, small molecules typically have multiple carbon-carbon bonds and may have multiple stereocenters and functional groups.

"Pharmaceutical agent," also referred to as a "drug," is used herein to refer to an agent that is administered to a subject to treat a disease, disorder, or other clinically recognized condition that is harmful to the subject, or for prophylactic purposes, and has a clinically significant effect on the body to treat or prevent the disease, disorder, or condition.

Therapeutic agents include, without limitation, agents listed in the United States

Pharmacopeia (USP), Goodman and Gilman 's The Pharmacological Basis of Therapeutics, th

10 Ed., McGraw Hill, 2001; Katzung, B. (ed.) Basic and Clinical Pharmacology, McGraw- Hill/ Appleton & Lange; 8th edition (September 21, 2000); Physician 's Desk Reference

th

(Thomson Publishing), and/or The Merck Manual of Diagnosis and Therapy, 17 ed. (1999), th

or the 18 ed (2006) following its publication, Mark H. Beers and Robert Berkow (eds.),

th

Merck Publishing Group, or, in the case of animals, The Merck Veterinary Manual, 9 ed., Kahn, C.A. (ed.), Merck Publishing Group , 2005. Method for Monitoring and/or Determining Effectiveness of Therapeutic Agent

The methods herein also provide for monitoring and/or determining the effectiveness of a therapeutic agent. One method for monitoring the effectiveness of a therapeutic agent for treating a disease or condition in a subject includes (a) perfusing a fluid comprising one or more cells from the subject through the microfluidic device described above; (b) determining a property of one or more of the cells; (c) treating the subject with the therapeutic agent; and (d) repeating steps (a) and (b) at least once wherein a difference in the property of one or more cells is indicative of the effectiveness of the therapeutic agent.

Determining the effectiveness of a therapeutic is also contemplated. One such method includes (a) obtaining a biological sample from a subject comprising a cell; (b) perfusing a fluid comprising one or more cells from the subject through the microfluidic device described above; (c) determining a property of one or more of the cells; (d) contacting the biological sample comprising a cell with the therapeutic; (e) perfusing a fluid comprising the product of (d) through the microfluidic device; (f) determining a property of one or more of the cells from (e); and (g) comparing the property of one or more cells from (c) with the property of one or more cells from (f), wherein the results of the comparison are indicative of the effectiveness of the therapeutic.

Such methods may be used for a therapeutic to treat sickle cell disease, for example. In some embodiments, the therapeutic is hydroxyurea (HU) or 5-hydroxymethylfurfural (Aes-103).

Real-Time Method for Cell Morphological Kinetic Quantification

It should be appreciated that methods provided herein allow for the real-time observation of changes in cell morphology in response to various levels of gas. A method includes (a) perfusing a fluid comprising one or more blood cells through a microfluidic described above, wherein the fluid has a first level of gas; (b) determining a property of one or more of the cells from (a); (c) perfusing a fluid comprising one or more cells through the microfluidic device described above; wherein the fluid has a second level of gas that is different from the first level; (d) determining a property of one or more of the cells from (c); and (e) quantifying the cell morphological kinetics of the cells from (b) and (d).

The method may be used to examine cell sickling and/or unsickling kinetics, such as cell sickling in response to low oxygen concentrations. The methods provided also allow for the real-time observation of reoxygenation induced cell shape recovery. For example the transition of sickled cells into normal disk shaped red blood cells. In other embodiments, a morphological characteristic, such as a cell shape change (e.g., sickling, a sphericity change, and aspect ratio change or a texture change), of one or more cells is measured at one or more gas concentrations. For example a morphological characteristic of one or more red blood cells from a subject is measured at a low oxygen concentration (e.g., 2% oxygen). Upon increase of the oxygen concentration (e.g., 20% oxygen), another transient characteristic of one or more cells can be measured. The measurements of cell morphology of the cells may be used to determine the fraction of abnormally shaped (e.g., sickled) cells. It should be appreciated that methods provided herein allow for the real-time observation of hypoxia- induced changes in cell morphology. For example, cell sickling in response to low oxygen concentrations. The methods provided also allow for the real-time observation of reoxygenation induced cell shape recovery. For example the transition of sickled cells, cells with a rough texture, or spiky cells into normal disk shaped red blood cells. Accordingly, the methods, described herein, allow for the simultaneous measurement of cell shape changes over time and cell transit characteristics in response to changes in gas concentration, for example, cell sickling delay time and sickled fraction can be simultaneously measured in real-time in response to decreased oxygen concentration.

The methods, described herein, may be used to determine the fraction of obstructed cells, the fraction of cells with an abnormal shape and/or texture, the capillary obstruction ratio, the delay time of an abnormal cell shape change, and/or the delay time of recovering from an abnormal cell shape change.

EXAMPLES

In order that the invention described herein may be more fully understood, the following examples are set forth. The examples described in this application are offered to illustrate the devices, compounds, pharmaceutical compositions, and methods provided herein and are not to be construed in any way as limiting their scope. Example 1: Microfluidic-based multiplex Cell Assay for Drug Compound Testing

The optically transparent microfluidic-based polymeric platform utilizes a combination of geometric, physical, chemical and biological means to quantify cell biophysical properties in vitro to mimic the in vivo microenvironment. Effective integration of the pathological processes within a single system offers a) the quantitative investigation of microvascular occlusion, thrombosis and other hematologic diseases and b) In silico cell modeling development. Hence, the platform may be used as a vehicle for both drug testing experimentation and for the manifestation of underlying microvascular-related mechanisms.

The microenvironment of the cells is mimicked through precise shear stress modulation imposed by liquid flow profile, ambient gas partial pressure modulation

(diffusion rate control), gas mixture, osmolality control (constant or modulated osmolality), biophysical interaction between the cell/cell and/or cell/protein coated microchannel walls.

For a given test drug compound/sample, the following assays can be performed sequentially with "on-demand" cell microenvironment modulation: a) identification and classification of the cell shape/type population, b) cell (specific binding) attachment assay; and/or actively control the rectangular microchannel hydraulic diameter geometry by manipulation of free standing polymeric membranes (% degree of constriction), c) adherent cell deformability cytometer d) cell detachment. The assays may all be performed under hypoxia and normoxia conditions:

A) Identification and classification of the cell shape/type population: Image processing.

Image snapshots of the cell population for statistical analysis under multiple region of interests; under hypoxia and normoxia conditions (Fig. 17).

B) Cell (specific binding) attachment assay: Microchannel walls are coated with proteins or cells. Adherent cell enumeration assay under specific flow profile (liquid shear rate range), protein type; under hypoxia and normoxia conditions (Figs. 3A-3B, Fig. 7).

C) Adherent cell deformability cytometer (or adhesion-assisted deformability

cytometry): Application and release of a shear stress range to effectively measure the cell shear modulus and viscosity via a dynamic shape recovery process; under hypoxia and normoxia conditions. (Figs. 8A-8B, Fig. 9, Figs. 10A-10B, and Table 1) Tethering formation and breakup as a function of shear rate and hypoxia (Fig. 5).

D) Cell detachment assay: Suitable laminar flow profile (pulsatile, continuous, sinus etc.) to modulate the shear stress on the adherent cells and quantify detachment over shear rate range, protein type; under hypoxia and normoxia conditions. (Figs. 4A-4B, Figs. 6A-6B) Cell ( specific binding ) attachment assay

Oxygenated and deoxygenated conditions were controlled using gas mixtures as described above prior to initiation of attachment assay. Attachment was quantified as percentage of cells remaining attached after low flow washing per mm . Multiple fields of view were observed.

Static and dynamic attachment was studied using functionalized microfluidic devices. For static attachment assays, cell suspensions were loaded into the fluid channel of BSA passivated protein functionalized devices. After loading, cells were incubated at 37°C without flow for 10-15 min. Following incubation, very low flow (0.1-1 μΐ/min) was introduced for 15 s to quantify how many cells had indeed attached. For dynamic attachment assays, a similar protocol was followed. However, instead of static incubation for 10-15 min at 37°C, cells were perfused continuously at very low flow rates (0.1-1 μΐ/min), to simulate the vasoocclusion microenvironment. Cell detachment assay

A detachment assay was conducted following the static or dynamic adhesion incubation period described above. Using the Elveflow flow sensor to provide real-time information on flow rates, the flow rate was increased for specified durations until all cells had detached. The flow rates at which cells detached indicated the strength of the adhesive bond between the cell and the functionalized protein. Detachment was quantified as percentage of cells detached after specified flow rate per mm . Multiple fields of view were observed.

Deformability properties of attached sickle RBCs was also attained from detachment studies using elongation index measurements. The ratio of minor to major axes (length) of attached RBCs was used to determine deformability. Length of axes was measured in ImageJ (NIH).

Results

To recapitulate the vasculature microenvironment present in vaso-occlusions, a microfluidic device was designed with comparable geometry to post-capillary venules, the site often associated with vasoocclusions in the sickle cell patients. Devices included cell channels with 15 μιη height, 1.3 mm width and 3 mm length dimensions. This width and length were selected to enable imaging of higher number of cells and prevent entrance effects, respectively. The channels were casted in the silicone elastomer

polydimethylsiloxane (PDMS), a well-established polymer often used in microfluidic device fabrication offering advantages such as low cost, reproducibility, mechanical properties similar to vasculature and advantageous optical properties for imaging studies (the material is optically clear).

Previous microfluidic-based studies have utilized PDMS designs to study cell adhesion ^1- . These studies, however, have failed to address the critical hypoxic feature of the microenvironment in obstructed vessels. The design described herein provides gas control of the cell channel through an additional gas channel and a previously described gas-permeable thin (150 μηι) PDMS membrane ⁴ . Such a design offers the benefits of precise dynamic control of gas mixture, gas pressure and diffusion rate into the cell channel. To mimic the hypoxic microenvironment, a gas mixture of 2% 0 ₂ , 5% C0 ₂ , 93% N ₂ was used while normoxic conditions were mimicked using a 20% 0 ₂ , 5% C0 ₂ , 75% N ₂ gas mixture. The gas pressure was maintained to below 300 mbar for all studies.

To study adhesion in these microfluidic devices, the cell channel was functionalized (e.g., coated ) with proteins known to be relevant for adherent sickle RBCs. Specifically, the extra-cellular matrix proteins fibronectin and laminin were chosen as both have been well characterized in promoting sickle RBC adhesion to the endothelium. Fibronectin binds to sickle RBCs through α4β 1 integrins while laminin interacts with the basal cell adhesion molecule/Lutheran protein (B-CAM/LU) receptor ⁵ ' ⁶ . In addition, fluorescently tagged versions of these proteins were selected to aid in imaging and measurement of surface concentration prior to and during the course of the studies. To ensure robust protein functionalization, APTMS gas vapor deposition was used followed by protein

functionalization. Through imaging and protein measurement studies, the presence of proteins on the surface of the microfluidic device was demonstrated (Figs. 2A-2C). Protein concentration could be tailored to match the desired range (Figs. 2A-2C). Imaging of devices following adhesion assays confirmed protein presence even after exposure to increasing flow rates (data not shown). Finally, use of a flow sensor allowed real-time measurement of the flow rate and gas pressure during adhesion studies. To this end, flow rates consistent to what are found in microcirculation could be provided. Furthermore, since hematocrit of cell suspensions were known and flow rate was known, shear stress could also be calculated.

The above features of the design were exploited to systematically characterize how cell density, hypoxia, extracellular matrix proteins, and concentration of proteins affected cell adhesion. A series of assays were designed to qualitatively and quantitatively assess the adhesive properties of sickle RBCs, namely attachment and strength of adhesive bond. By varying the flow rate, the deformability of adherent cells was also characterized. Finally, dynamically changing the oxygen content in the microenvironment under fluid flow allowed observation of how the focal adhesion sites differ for stiff sickled cells as compared to oxygenated flexible cells.

Exemplary device and system designs are summarized in Figs. 1 and 11-16.

Effect of sickle cell density on adhesion to fibronectin

The heterogeneity of the sickle RBC population in terms of morphology and density add a level of complexity to the events leading up to vaso-occlusion. Indeed, several studies have demonstrated higher levels of adhesion in lighter density and irregularly shaped discoid cells and lower adhesion for high density irreversibly sickled cells to endothelial cells 7—10. A low flow adhesion scheme under oxygenated conditions was utilized in the fibronectin (50 μg/ml) functionalized microfluidic devices to study the role density played in adhesion. Shear rate may be calculated and related to a physiological flow rate. To separate the sickle blood in to lighter and denser fractions, a density gradient fractionation technique was used, which has been previously published. Fractions trapped within four bands were collected (Figs. 3A-3B) and with these performed adhesion assays where sickle RBCs were incubated under low flow and oxygenated conditions in fibronectin functionalized microfluidic devices for 15 min. Results confirmed adhesion to fibronectin of all cells with greater adhesion for lighter density fractions (discocytes) confirming findings found in literature for sickle RBC -endothelial cell adhesion. Control microfluidic devices with bovine serum albumin blocked surfaces confirmed adhesion to fibronectin was not non-specific (data not shown). Ostensibly, it seems that morphologic abnormalities leading to minimal contact between protein and sickle RBC surface may cause the decreased adhesion as described by Kaul et al, although future studies may be necessary to confirm the effect of morphology on adhesion ¹⁰ .

In at least one instance, cells in fractions exhibited various behaviors prior to attachment.

Some cells slid on the protein functionalized surface, decreased sliding velocity and stopped altogether. Other cells tumbled and bonded immediately to a specific location. Some cells attached and then quickly detached. Sliding, rolling, and tumbling were observed in all fractions.

Protein-specific adhesion of sickle RBCs

Several studies suggest inflammatory conditions, increased levels of interleukins for example, are associated with higher exposure of ECM proteins such as fibronectin and laminin. For example, interleukin-6 has been shown to induce greater amounts of fibronectin synthesis by monocytes and hepatocytes ⁵ . Increased laminin exposure has also been shown under high concentrations of interleukin- 1 and tumor necrosis factor due to endothelial cell retraction ⁶ . Moreover, inflammation is not uncommon in sickle cell disease patients. Thus, delineating the role each protein plays in adhesion can help unravel the more complicated vasocclusive cascade. To this end, a series of static adhesion studies under oxygenated conditions were performed. Patient samples exhibited various levels of adhesion potential to fibronectin and laminin (50 μg/ml). In general, it seems adhesion was higher to fibronectin than laminin in most patient samples studied, although further studies to provide additional confirmation may be performed (data not shown). Once a patient- specific protein profile was observed, the strength of bonding for these proteins was characterized. Inflammatory status information, plasma protein levels may also be measured to determine their effects on cells, which may play a role in adhesion. The strength of binding was measured by increasing the fluid flow rate using stepwise increments after 15 min of static incubation under oxygenated conditions. As shown by the shift in the distribution of cell detachment rates in Figs. 4A-4B, sickle RBCs in this patient, exhibited stronger bonding to fibronectin than laminin.

Interestingly, point adhesion in deformable adherent sickle RBCs was observed under increasing fluid flow when studying both proteins. Identification and classification of cell shapes under oxygenated and deoxygenated conditions is included in Fig. 17. Tethering of the RBC membrane, which occurs when the lipid bilayer detaches from the cytoskeleton was also observed during increasing fluid flow often times for deformable sickle RBCs (Fig. 5). Further characterization of tethering in unique sickle RBC types can provide additional information on adhesion strength and dynamics of membrane detachment for individual proteins ¹¹ . Nondeformable adherent sickle RBCs exhibited multiple adhesion points and tethering but seemingly shorter tether lengths before detachment. Previous studies have observed tethering of adherent sickle RBCs to endothelial cells upon detachment but not characterized the phenomenon in detail 5 ' 7 ' 8. Deformability of adherent sickle RBCs was measured by measuring the ratio of major to minor axis length initially and before detachment and calculated the difference (Figs. 8A-8B, Table 1). Irreversibly sickled cells appear to have minimal change in elongation ratio from initiation to the point of detachment confirming their rigidity. In addition, relaxation time or the time necessary to return back to the original shape also informs on the deformability, specifically the viscosity properties of each cell (Fig. 9). As these results show, the platform can characterize both the adhesive and mechanical properties of sickle RBCs, offering an added benefit. Future elongation ratio and relaxation time analyses of larger number of adherent cells is necessary to understand deformability properties of cell types. Deoxygenation conditions were pursued with the closed loop gravitational potential driven setup but were unable to maintain the deoxygenated microenvironment after ~9ul/min flow rate. For this reason, an alternate system design was pursued. One way to address this issue is to pulse the fluid through the device, which allows the fluid within the device to maintain a deoxygenated microenvironment under high pressures or flow rates.

Table 1. Change in elongation ratio summary for adherent sickle RBCs (fibronectin).

Shape Time (s) Sheaf rate Elongation Ratio {Initial} Elongation Ratio (Detachment) % Change

Discocyte 12.1 below 285 1.04 1.28 23.67

Discocyte 36.3 =570 1.13 1 37 21.44

54 i I ^' M 3 i? a? 33,35

Elongated 59.2 ΪΪ40 2.10 2.04 -3.21

*>,' _· J y0 22,5¾

Elongated 78.7 1652 2.00 2.62 31,14

- ^: : ^: : ^: : ^: : ^: : ^: : ^: : ^: ¾ϊ>ϊϊ¾: ^: : ^: : ^: : ^: : ^: : ^: :

irreversibly Sickled Cell 8L2 1652 2,82 3.36 19,02

^: :: _: ^: :: _: ^: :: _: ^: :: _: ^: :: _: ^: :: _: ^: :: _: ^: :¾¾¾ϊέίΜ¾: _: ^: :: _: ^: :: _: ^: :: _: ^: :: _: ^: :: _: ^: :: _:

Elongated Ϊ0Ϊ.2 2279 2 ^' .24 2.95 31.56

Irreversibly Sickled Cell 106.9 2279 2.68 3.10 15,72

tir.ovwciiA.- *tfitW t:¾ ¾ ¾i ¾ 1 ) A

Elongated 155.5 4615 2.24 2.67 19.06

Effect of hypoxia on adhesion

As noted, characterization of sickle RBC adhesion under deoxygenated conditions in microfluidic devices has not yet been reported. The ability to tailor this aspect of the microenvironment is a major improvement to existing microfluidic device designs. Such a device that better resembles the in vivo microenvironment of vasoocclusions in sickle cell patients holds great promise for drug therapy studies.

Initially, adhesion studies were conducted where sickle RBCs were incubated statically with fibronectin (100 μg/ml) under oxygenated or deoxygenated conditions and then detached using stepwise increments. Using higher magnification, visual differences were observed in response to increasing shear stress for adherent cells in oxygenated and deoxygenated conditions. In general, adherent deoxygenated sickle cells appeared stiffer and failed to change shape while oxygenated cells appeared more flexible (Fig. 10). In addition, a single focal adhesion point appeared to dominate for the oxygenated condition as noted previously whereas multiple adhesion points were visible for adherent cells under deoxygenation. The detachment rate revealed differences in strength of binding under oxygenated and deoxygenated conditions (Figs. 6A-6B). As shown, 80% of the original fibronectin-adherent sickle cell population were still bound at 4000 mbar of pressure in oxygenated conditions whereas less than 20% were bound at the equivalent pressure in deoxygenated conditions. This demonstrated a much stronger bonding under oxygenated conditions, which could be a result of increased contact area available for binding.

In terms of quantification of adhesion percentage, interestingly, a close to 10-fold increase in adhesion to fibronectin was observed under deoxygenated conditions as compared to oxygenated conditions (Fig. 7). However, as Figs. 6A-6B show, the majority of the adherent cells under deoxygenation were removed with low flow suggesting these cells are not as problematic in a vasoocclusive environment. These findings support the hypothesis that morphologic abnormalities of sickle cells inhibit close contact with endothelial cells, preventing adhesion 7—10. To eliminate any bias associated with devices or cell loading, a similar experiment was performed where sickle RBCs were incubated under deoxygenated and oxygenated conditions sequentially under flow for 5 min each. These studies were performed with both fibronectin and laminin and observed a greater amount of adhesion under deoxygenated conditions (data not shown). Furthermore, a dose-related increase in adhesion to fibronectin was also observed under conditions of static incubation and deoxygenation (Fig. 7). These results have implications for inflammatory conditions in sickle cell disease patients where ECM protein concentrations may be high. Cycling of oxygenation and deoxygenation conditions

One of the powerful features of the device design is the ability to dynamically change the oxygenation in the microenvironment and observe the sickle RBCs response real-time. Some preliminary studies exploring the effect of cycling on adhesion and deformability of the adherent cells were performed. It was first observed that sickling and unsickling under deoxygenated and oxygenated conditions, respectively, did not affect the attachment properties of the cells, at least for 1-5 oxy-deoxy cycles that were conducted. Furthermore, mechanical properties of attached deformable sickle RBCs were not affected after 5 cycles of switching, i.e. cells did not stiffen and continued to appear flexible if initially flexible. Most likely much greater cycle numbers are necessary to affect mechanical properties of the sickle RBCs.

Discussion of Data

Characterizing the role adhesion plays in a deoxygenated microenvironment may help in understanding the vasoocclusive cascade of events and possibly aid in predicting when these painful crises occur. The ability to precisely tune the microenvironment with respect to geometry, oxygen content, proteins present and fluid flow in an in vitro device offers tremendous potential for patient-specific therapeutic applications.

Examples of the types of cell specific adhesion dysfunction that may be detected by the methods, devices, and systems provided herein are numerous. For example, (A) reduced tether elongation in adherent deoxygenated, stiffer sickle cells is visible using continuous flow and push/pull systems, (B) adherent deoxygenated sickle cells exhibit reduced elongation and rigid behavior as compared to adherent oxygenated sickle cells in response to increased continuous flow and pressure amplitude of push/pull system, (C) deoxygenated sickle cells exhibit increased adhesion as compared to oxygenated sickle cells in both continuous flow and push/pull systems, (D) fibronectin exhibits stronger adhesive bonding to sickle cells than laminin at identical concentrations, evident through increased shear rate necessary for detachment. Increased adhesion to fibronectin as compared to laminin is also observed in sickle cells for identical concentrations tested (using both systems), (E) sickle cells from patients on hydroxyurea exhibit reduced adhesion to fibronectin and laminin in comparison to off hydroxyurea patients in both continuous flow and push/pull systems, (F) adherent nondeformable sickle cells in oxygenated and deoxygenated environments can require higher flow rates and increased pressure in the continuous flow and push/pull system to detach.

Methods

Blood specimens

Blood was drawn into ethylenediaminetetraacetic acid (EDTA) coated "purple-top" vacutainers from homozygous SS sickle cell disease patients at Brigham and Women's Hospital, Boston, MA during routine patient care and from homozygous SS sickle cell disease patients at University of Pittsburgh Medical Center, Pittsburgh, PA in agreement with approved research protocols 2006P000066 and PRO08110422, respectively. Specimens used for this study were restricted to patients that were not in crisis and patients with transfusions earlier than 30 days prior to the time of study. Both on hydroxyurea treatment and off hydroxyurea treatment samples were used. Prior to attachment or detachment assays, blood was centrifuged at 821 g for 5 minutes and buffy coat and plasma were removed. Packed red blood cells (pRBCs) were then washed and centrifuged twice with D-PBS (GE Healthcare, Pittsburgh, PA). The pRBC pellet was resuspended in RPMI-1640 (Thermofisher Scientific, Pittsburgh, PA) with 1% (w/v) bovine serum albumin (BSA) (EMD Millipore, Billerica, MA) at a concentration of 2-3 μΐ pRBC per 200 μΐ of medium solution. Samples were stored at 4°C until use and tested within 1-3 days after collection.

Density -gradient fractionation

Sickle red blood cells were fractionated into four fractions of increasing density using methods described (Du PNAS 2015). In short, solutions of increasing density (1.081, 1.091, 1.100 and 1.111 g/ml) were made using Optiprep density gradient medium (Sigma Aldrich, St. Louis, MO) and D-PBS. 2.5 ml volumes of solutions were then layered such that the densest fraction (1.111 g/ml) was on the bottom of the gradient. Washed pRBCs were resuspended in D-PBS to reach a 70-80% hematocrit and layered on top of the gradient. Cells were then centrifuged for 30 minutes at 821 g and 21°C. Cells between density fractions were removed carefully and washed twice for 5 minutes at 821 g and 21°C. Fractions were then resuspended in the RPMI solution described above. Microfluidic device fabrication

Devices for adhesion study were fabricated using standard photolithography and polydimethylsiloxane (PDMS) methods. The microfluidic platform consisted of a two layer device with a fluidic channel for cells and a gas channel to allow control of the amount of oxygen within the microenvironment. Device design was similar to the cell sickling device as it has been described in Du et al. (Du PNAS 2015). Channels were separated by a 150 μιη thick PDMS gas permeable membrane. The amount of oxygen and its diffusion rate into the fluidic channel was regulated by the gas and gas pressure in the gas channel and thickness of the PDMS membrane. Hypoxic conditions were created by using a 2% 0 ₂ , 5% C0 ₂ , 93% N ₂ gas mixture while normoxic conditions were mimicked using a 20% 0 ₂ , 5% C0 ₂ , 75% N ₂ gas mixture. Both fluidic and gas channels were 3 mm in length and 1.326 mm in width. The height of the fluidic and gas channel were 15 μιη and 100 μιη, respectively. Standard photolithography techniques were used to create SU-8 masters on 6 in silicon wafers. To aid in removal of PDMS from masters, wafers were passivated using tridecafluoro-1,1,2,2- tetrahydrooctyl)-l-trichlorosilane for 2 h under vacuum. A polymer to curing agent ratio of 10 to 1 (w/w) of Sylgard® 184 silicone elastomer (Dow Corning, Auburn, MI) was mixed, and poured onto wafers after all bubbles from mixture had been removed. PDMS was cured for at least 2 h at 80°C before removal. For the membrane layer, mixed PDMS was spincoated onto the wafer to attain a thickness of 150 μιη and then cured as described. An inlet and outlet were created in both gas and cell channels using a 1.5 mm diameter biopsy punch, 1.2 mm away from the middle. Both channels were bonded to cleaned cover glasses using plasma treatment.

Protein functionalization and characterization

To functionalize desired proteins to surface of channel, (3-Aminopropyl)- trimethoxysilane (APTMS) was deposited using gas phase deposition for 2 hours at room temperature on plasma-treated PDMS devices and coverslips. After silanization and bonding, devices were incubated with FITC conjugated fibronectin (Cytoskeleton, Denver, CO) or rhodamine conjugated laminin (Cytoskeleton, Denver, CO) in PBS at desired concentration for 1 h at room temperature. Fluorescently labeled proteins were chosen to aid in

visualization of protein. Fibronectin was selected for adhesion studies as its presence on the vessel lining and promotion of sickle RBC binding to the endothelium via a4bl integrins have been well-documented (Kasschau et al Blood 1996). Laminin is another ECM protein, known to bind to the basal cell adhesion molecule/Lutheran protein (B-CAM/LU) receptor which is overexpressed on sickle RBCs (Zen et al AJH 2004). Gently washed devices were passivated with a 3% (w/v) BSA (EMD Millipore, Billerica, MA) in PBS solution over night at 4°C and used within 24-48 h. Washing and blocking of devices were performed with a low flow rate using a syringe pump to minimize disturbance to functionalized protein.

To ensure a homogeneous distribution of protein on the channel surface and protein concentration consistency in the functionalization protocol each time, devices were qualitatively and quantitatively assessed prior to adhesion testing. Devices were observed in brightfield and fluorescence modes using an Olympus 1X71 inverted microscope. Filter cubes for FITC and rhodamine were used as needed. Images of the protein functionalized channel, specifically the gas and fluidic channel overlap, were acquired using a lOx objective lens under predefined settings. Fluorescence intensity was then compared using ImageJ (NIH, Bethesda, MD). For some devices, functionalized protein was quantified using Pierce Coomassie Plus (Bradford) Assay (ThermoFisher Scientific, Pittsburgh, PA) as per manufacturer's instructions. In brief, protein-functionalized devices (that had not been blocked with BSA) were washed twice with PBS. Original protein solutions along with washes were incubated with Coomassie Plus Reagent as per manufacturer's instructions.

Absorbance of solutions was observed under 595 nm filter using a spectrophotometer. BSA solutions of concentrations ranging from 0 to 25 μg/ml were used as controls.

Experimental setup For the LoC device: a mass production amenable technology was used to fabricate optically transparent Lab-on-a-Chip polymeric microfluidic devices that feature PDMS permeable membrane. This setup is advantageous due to: easy of processability, gas permeability, easy adhesion and bonding to different substrates, biocompatibility,

transparency, low cost processing for device fabrication.

Microfluidic devices were imaged using Zeiss Axiovert 200 and Olympus 1X71 inverted microscopes. To improve image contrast of RBCs, a 414/446-nm band-bass filter (Semrock, Rochester, NY) was used. A Hitachi HAL100 camera (Tarrytown, NY) and— were used for image acquisition for the Zeiss and Olympus microscopes, respectively. All testing was performed at 37°C using a heating incubator (ibidi heating system, ibidi USA, Madison, WI). The height of liquid in columns connected to the device reservoirs via Tygon tubing (0.02 in inner diameter x 0.06 in outer diameter) was used to regulate the flow. A system using pressurized air to control fluid flow was also used in specified experiments. Hypoxic and normoxic conditions were mimicked using gas mixtures of 2% oxygen, 5% carbon dioxide, 93% nitrogen and 20% oxygen, 5% carbon dioxide, 75% nitrogen, respectively. Gas mixtures were connected to the gas channel in microfluidic devices using Tygon tubing (0.02 in inner diameter x 0.06 in outer diameter). Gas mixtures then diffused through the PDMS permeable membrane to provide the desired environment to cell channel. An Elveflow flow sensor (Paris, France) was connected in-line with the tubing controlling fluid flow and gas flow to provide a real-time measurement of flow rate and gas pressure and ensure consistency. Flow rates of 0.1-1 μΐ/min were used for dynamic infusion studies and detachment rates up to 80 μΐ/min were used for detachment studies. Gas pressure was maintained at 300 mbar for both hypoxic and normoxic conditions. For studies where pressurized air was used to regulate fluid flow, amplitude, period, and duty cycle of the pulse was varied using an Elveflow computer interface. IC Capture 6.7 (Charlotte, NC), CellSens Standard (Olympus), and Oovoo software were used to acquire images and videos for analysis.

Example 1 References.

1. Alapan, Y., Little, J. A. & Gurkan, U. A. Heterogeneous red blood cell adhesion and deformability in sickle cell disease. Sci. Rep. 4, 7173 (2014).

2. Tsai, M. et al. In vitro modeling of the microvascular occlusion and thrombosis that occur in hematologic diseases using microfluidic technology. . Clin. Invest. 122, 408-418 (2012). 3. Antia, M., Herricks, T. & Rathod, P. K. Microfluidic modeling of cell-cell interactions in malaria pathogenesis. PLoS Pathog. 3, e99 (2007).

4. Du, E., Diez-Silva, M., Kato, G. J., Dao, M. & Suresh, S. Kinetics of sickle cell

biorheology and implications for painful vasoocclusive crisis. Proc. Natl. Acad. Sci. U. S. A. 112, 1422-1427 (2015).

5. Kasschau, M. R., Barabino, G. A., Bridges, K. R. & Golan, D. E. Adhesion of sickle neutrophils and erythrocytes to fibronectin. Blood 87, 771-780 (1996).

6. Zen, Q. et al. B-CAM/LU expression and the role of B-CAM/LU activation in binding of low- and high-density red cells to laminin in sickle cell disease. Am. J. Hematol. 75, 63- 72 (2004).

7. Mohandas, N. & Evans, E. Sickle erythrocyte adherence to vascular endothelium.

Morphologic correlates and the requirement for divalent cations and collagen-binding plasma proteins. J. Clin. Invest. 76, 1605-1612 (1985).

8. Mohandas, N. & Evans, E. Adherence of sickle erythrocytes to vascular endothelial cells: requirement for both cell membrane changes and plasma factors. Blood 64, 282-287

(1984).

9. Stone, P. C, Stuart, J. & Nash, G. B. Effects of density and of dehydration of sickle cells on their adhesion to cultured endothelial cells. Am. J. Hematol. 52, 135-143 (1996).

10. Kaul, D. K., Chen, D. & Zhan, J. Adhesion of sickle cells to vascular endothelium is critically dependent on changes in density and shape of the cells. Blood 83, 3006-3017

(1994).

11. Borghi, N. & Brochard-Wyart, F. Tether extrusion from red blood cells: integral proteins unbinding from cytoskeleton. Biophys. J. 93, 1369-1379 (2007).

The entire contents of each of references 1-11 are incorporated herein by reference.

Example 2: Microfluidic-based assay for testing mechanical fragility.

There is need for a fast and robust method for assessing the quality of stored blood before transfusion, which can depend on the duration of storage, storage conditions, and gender of the donor [12]. RBC mechanical fragility (MF) test has been suggested to provide such a measure [13, 14]. However, there is no individual cell based high-throughput fragility test available so far, and the existing methods normally need large amounts (3-20 mL) of blood [13, 15].

Described herein is a robust, individual-cell based microfluidic mechanical fragility (MF) test, requiring only 100 μΐ _^ blood for each test. The MF assay provided herein could also be used to evaluate the effect of new interventions (e.g. improved blood storage preservation solutions) intended to mitigate the susceptibility of RBCs to sub-hemolytic injury during storage.

Various approaches have been used to test the mechanical fragility (MF) of RBCs (i.e., susceptibility to damage by mechanical stress), which are typically based on the detection of the degree of hemolysis under constant or varying mechanical stress [13, 16] .

The resultant degree of hemolysis following a standardized shearing period is usually expressed as the "mechanical fragility index" (MFI) [12, 15], reflecting the mechanical stability of RBCs. Hence, the MFI is an in vitro measurement of the extent of RBCs' "sub- lethal" or "sub-hemolytic" injury. Changes in MFI reflect the damage to RBCs [12, 17], caused by mechanical forces which are not high enough to result in immediate hemolysis but do induce cellular alterations, including morphological changes [18, 19] and/or impaired deformability [20, 21] . Mechanical fragility (MF) profiles may provide a powerful and versatile tool as a metric of RBC quality.

Methods provided herein utilize a poly(dimethylsiloxane) (PDMS) thin elastomeric membrane in direct contact with the RBCs surface via compressive loading up to the point of cell lysing. This process is monitored and recorded (compression versus cell response) hence compressive loading-unloading curves can also reflect the sub-hemolytic RBC damage. For validation purposes, the assay can be designed to handle 20- 100 cells per test; however due to inherent advantages of microfluidic processability utilizing automated RBC positioning and fluidic multiplexing, it can be extended with further engineering development for hemolytic screening of 2,000- 10,000 cells in less than an hour.

As compared to existing RBC fragility tests, several key advantages of the methods and devices described herein are provided below:

1) individual cell profiling (& high throughput potential),

2) requiring only a small blood volume (-100 μΐ _^ instead of 3-20 mL),

3) capable of dynamically altering gas mixture microenvironment, and

4) capable of testing both hemolytic & sub-hemolytic conditions.

It should be appreciated that devices described herein involve mass production- amenable technology to fabricate optically transparent Lab-on-a-Chip polymeric microfluidic devices that feature a flexible poly(dimethylsiloxane) (PDMS) permeable membrane; acting as a mechanical actuator for compressive loading-unloading. This platform is advantageous due to, at least in part: ease of process ability (multilayer soft lithography), gas permeability, biocompatibility, optical transparency and low-cost processing for translational device fabrication. Consequently, the proposed platform uniquely utilizes a combination of geometric, physical, chemical and biological means to quantify ex vivo red blood cell fragility {i.e., susceptibility to hemolysis) at single-cell level via application of compressive load.

Development and characterization of a micro -hemolytic device, using hydraulic pressure loading.

Fabrication of a free-standing PDMS membrane with suitable geometry (thickness & surface area) and composition (related to elasticity) for mechanical fragility (MF) profiling up to 100 RBCs per test. Results have shown that a mechanical fragility (MF) test is feasible using the devices provided herein. A dual-layer microfluidic-test-bed with arbitrary membrane geometry and elasticity was fabricated and used pneumatic (gas) compressive loading/unloading to deflect the PDMS membrane until it comes to direct contact with the RBCs; and then we increased further the external pressure with an approximate rate of ~ 6 psi/min. Hence, "gas pressure-induced hemolysis" was performed and cell-specific lysis events as a function of the increasing gas pressure were recorded. See Figures 18 and 19.

Device Operation

Characterization of PDMS membrane deflection for compressive loading; pressure and flow control.

As mentioned previously the micro-hemolytic device is based on the deflection of a PDMS membrane that imposes compressive load in direct-contact with the RBCs. The membrane is free-standing within a dual-layer microchannel construction. A schematic representation of an exemplary microfluidic {e.g., micro-hemolytic) device is shown in

Figure 20. The dual-layer micro-hemolytic device comprises a "flow channel" in which there is flow of a cell {e.g., red blood cell (RBC)) suspension; and a "control microchannel," which may also be referred to as a "gas channel", for pressure regulation. Both channels may be connected with in-line pressure and flow sensors (with feedback loop) for precise PDMS membrane deflection position under compressive loading. Within the "control channel" pressurized gas or liquid can be delivered via a high precision pneumatic pump. Furthermore, both pressure and vacuum control may be regulated in the control channel for precise membrane placement and initialization prior to each MF test; to minimize fabrication device dependent discrepancies. Membrane deflection can be directly observed via the change in channel height filled with fluorescein solution. Fluorescence intensity change can be measured and correlated with the applied pressure under compressive loading/unloading (while monitoring the liquid pressure differential in the flow channel).

The RBC microenvironment and positioning within the micro-hemolytic device region of interest (ROI) can be tuned independently by: precise RBC x-positioning imposed by the liquid flow profile, ambient gas partial pressure modulation (diffusion rate control), gas mixture, temperature and PDMS membrane geometry and elasticity. Furthermore, the pneumatic pumps that control both the control and the flow channels, are fully programmable and can be integrated with the microscope's motorized stage and camera for fully automatic operation, improving assay reproducibility and robustness.

Head-to-head assay validation against established fragility tests in stored blood and sickle cell blood.

With operational device validation described above, healthy or diseased blood samples using the methods and devices provided herein were performed as provided below. a) Prior to compressive load cycling: Identification and classification of cell shapes/type via an in-house built video processing algorithm. RBCs projected surface area and shape factor population statistics recording as well as individual cell position mapping at the ROI. b) Application of compressive load (with incompressible fluid): Continuously monitoring and recording of individual cell's projected surface area as a function of the external applied pressure and individual cell lysing events.

c) Analyses of all the individual cell lysing events: Compilation of the mechanical fragility (MF) profile, and correlation of the cell lysing events with the cell parameters (shape/type) recorded in a).

Hemolytic screening under different or varying gas microenvironment, with gas pressure loading.

Development and characterization of a micro-hemolytic device, using gas for compressive loading control. Similar to the description provided above, we can and perform operational device validation of the micro-hemolytic device with gas loading. In brief, the assay can be carried out sequentially as follows:

a) prior to compressive load cycling: identification and classification of cell shapes/type, b) application of compressive load (with pressurized gas) and c) analyses of all the individual cell lysing events: compilation of the mechanical fragility (MF) profile, and correlation of the cell lysing events with the cell parameters (shape/type) recorded in a). DeOxy gas pressure-induced hemolysis.

Cells may be lysed under varying gas conditions. For example, Figure 21 shows a RBC under the same experimental conditions as described for Figs. 18 and 19, but under hypoxic (DeOxy) conditions(gas mixture of 2% 0 ₂ , 5% C0 ₂ , 93% N ₂ ). Panels a-j show high speed imaging snapshots of RBC lysis over time (Fig. 21). Abrupt sickled RBC membrane rupture and consequent release of visible clusters of polymerized HbS and hemolysis products under steady state deoxygenation are shown.

Example 2 References.

[12] Raval JS, Waters JH, Seltsam A, Scharberg EA, Richter E, Daly AR, Kameneva MV, Yazer MH. The use of the mechanical fragility test in evaluating sublethal RBC injury during storage. Vox sanguinis 2010; 99(4):325 D331.

[13] Ziegler LA, Olia SE, Kameneva MV. Red Blood Cell Mechanical Fragility Test for Clinical Research Applications. Artificial organs 2016.

[14] Tarasev M, Chakraborty S, Alfano K. RBC mechanical fragility as a direct blood quality metric to supplement storage time. Military medicine 2015; 180(3 Suppl): 150D 157.

[15] Gu L, Smith WA, Chatzimavroudis GP. Mechanical Fragility Calibration of Red Blood Cells. ASAIO Journal 2005; 51(3): 194D201.

[16] Nevaril CG, Lynch EC, Alfrey CP, Jr., Heliums JD. Erythrocyte damage and destruction induced by shearing stress. The Journal of laboratory and clinical medicine 1968; 71(5):784D790.

[17] V. KM, Antaki JF. Mechanical trauma to blood, edited by: Baskurt OK, Hardeman MR, Rampling MW,

Meiselman HJ. Handbook of Hemorheology and Hemodynamics. Amsterdam, Berlin, Oxford, Tokyo, Washington DC: IOS Press, 2007. p.206D227.

[18] Orear EA, Udden MM, Farmer JA, Mclntire LV, Lynch EC. Increased

Intracellular Calcium and Decreased Deformability of Erythrocytes from Prosthetic Heart□ Valve Patients. Clin Hemorheol 1984; 4(5):461 D471.

[19] Reinhart WH. Peculiar red cell shapes: Fahraeus Lecture 2011. Clinical hemorheology and microcirculation 2011; 49(1□ 4): 11 D27. [20] Kameneva MV, Undar A, Antaki JF, Watach MJ, Calhoon JH, Borovetz HS.

Decrease in red blood cell deformability caused by hypothermia, hemodilution, and

mechanical stress: factors related to cardiopulmonary bypass. ASAIO J 1999; 45(4):307 D

310.

[21] Kameneva MV, Marad PF, Brugger JM, Repko BM, Wang JH, Moran J,

Borovetz HS. In vitro evaluation of hemolysis and sublethal blood trauma in a novel

subcutaneous vascular access system for hemodialysis. ASAIO J 2002; 48(1):34D38.

The entire contents of each of references 12-21 are incorporated herein by reference.

Example 3: Assessing adhesion and sickle cell polymerization simultaneously under

hypoxia.

The methods and devices provided herein provide the opportunity to study for the first time, simultaneous adhesion and sickle cell polymerization under hypoxia in cells, for

example in the following cells:

a. any shape mature sickle red blood cells (Fig. 24)

b. reticulocytes (young sickle red blood cells) (Fig. 25)

c. irreversible sickle red blood cells. (Fig. 22; cell "m")

Assessment of different cells, such as cells described above in a, b, and c, were

performed and results are shown in Figures 22-25.

Sickle cell attachment phenomena observed under steady state hypoxia

a. HbS polymer fiber growth outwards of the cell: In very young cells (reticulocytes) there is apparent reorganization of the polymer content that grows even outwards of the cell and on the fibronectin (FN)-coated surface (reticulocyte in Fig. 25); polymer HbS polymer fibers growth can be monitored as well as apparent adherent contact area change (up to 15%) (Fig. 25) b. Cell membrane change at the contact line: In light-density, highly-deformable cells, where cell membrane is evidently separated from the bulk of the intracellular polymerized content when cell membrane attaches to the surface cell contact line appears spiky (small menisci while adherent) (Fig. 22; cells "g", "h", "e" and "j")

c. Increase in attachment points while adherent: In some cases, initially cells adhere only in one attachment point (focal adhesion) and develop over time more attachment points (Fig. 23) d. Apparent reorganization of intracellular polymerized content across various cell types while adherent (Fig. 24 cell "i" ).

OTHER EMBODIMENTS In the claims articles such as "a," "an," and "the" may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include "or" between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention includes embodiments in which more than one or all of the group members are present in, employed in or otherwise relevant to a given product or process.

Furthermore, the invention encompasses all variations, combinations, and

permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Where elements are presented as lists, e.g., in Markush group format, each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should it be understood that, in general, where the invention, or aspects of the invention, is/are referred to as comprising particular elements and/or features, certain embodiments of the invention or aspects of the invention consist, or consist essentially of, such elements and/or features. For purposes of simplicity, those embodiments have not been specifically set forth in haec verba herein. It is also noted that the terms "comprising" and "containing" are intended to be open and permits the inclusion of additional elements or steps. Where ranges are given, endpoints are included. Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or sub-range within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

This application refers to various issued patents, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference. If there is a conflict between any of the incorporated references and the instant specification, the specification shall control. In addition, any particular embodiment of the present invention that falls within the prior art may be explicitly excluded from any one or more of the claims. Because such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the invention can be excluded from any claim, for any reason, whether or not related to the existence of prior art.

Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments described herein. The scope of the present embodiments described herein is not intended to be limited to the above Description, but rather is as set forth in the appended claims. Those of ordinary skill in the art will appreciate that various changes and modifications to this description may be made without departing from the spirit or scope of the present invention, as defined in the following claims.