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Title:
MICROFLUIDIC DEVICE FOR SHEAR FLOW TESTING AND METHODS FOR USING THE SAME
Document Type and Number:
WIPO Patent Application WO/2017/142950
Kind Code:
A1
Abstract:
A microfluidic system comprising a base layer having a microchannel that links a fluid reservoir to an actuation port and at one resisting device positioned there between. The microchannel includes first and second base ports. The system includes a valve device including a lower layer including a first portion of a membrane-holding device and first and second channels generally aligned with the first and second base ports. The device further includes an upper layer including a second portion of the membrane-holding device and an indent coupled to an upper port. The indent is generally aligned with the second channel. The upper port is generally aligned with the first channel. The lower layer is positioned between the base and upper layers. The device further includes a flexible membrane between the lower and upper layers. A first end of the membrane is held over the second channel by the first and second portions of the membrane-holding device.

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Inventors:
NOVAK RICHARD (US)
MAYOR ELIZABETH (US)
INGBER DONALD ELLIOT (US)
BAHINSKI ANTHONY (US)
MASOUMI NAFISEH (US)
MAYER JOHN (US)
Application Number:
PCT/US2017/017980
Publication Date:
August 24, 2017
Filing Date:
February 15, 2017
Export Citation:
Click for automatic bibliography generation   Help
Assignee:
HARVARD COLLEGE (US)
THE CHILDREN'S MEDICAL CENTER CORP (US)
International Classes:
C12M3/02; B01L3/00; B81B3/00; C12M1/12; C12M3/00; C12N5/071; G01N33/50
Domestic Patent References:
WO2015138032A22015-09-17
WO2016004394A12016-01-07
Foreign References:
US20090281250A12009-11-12
US20040228770A12004-11-18
Attorney, Agent or Firm:
RESNICK, David, S. et al. (US)
Download PDF:
Claims:
CLAIMS

What is claimed is:

1. A microfiuidic system, comprising:

a base layer having a microchannel that links a fluid reservoir to an actuation port, the microchannel including at least one resisting device positioned between the reservoir and the actuation port, the microchannel also including first and second base ports positioned at opposing ends of the microchannel; and

a valve device including

a lower layer including a first portion of a membrane-holding device, the lower layer including first and second channels, the first and second channels being generally aligned with a respective one of the first and second base ports; and

an upper layer including a second portion of the membrane-holding device, the upper layer further including an indent coupled to an upper port, the indent being generally vertically aligned with the second channel, the upper port being generally vertically aligned with the first channel, the lower layer being positioned between the base layer and the upper layer, and

a flexible membrane positioned between the lower layer and the upper layer, a first end of the membrane being held in a generally stationary position over the second channel by the first and second portions of the membrane-holding device, the flexible membrane further including a working portion abutting a portion of the lower layer such that the membrane completely covers the second channel.

2. The microfiuidic system of claim 1, wherein at least one surface of the membrane has cells adhered thereto.

3. The microfiuidic system of claim 1, wherein at least one side of the first and second portions of the membrane-holding device has a generally concave shape.

4. The microfiuidic device of claim 3, wherein the generally concave shape is a generally semicircular shape.

5. The microfiuidic device of claim 1, wherein the second portion of the membrane- holding device is configured to mate with the first portion of the membrane-holding device.

6. The microfluidic system of claim 1, further comprising a fluid pump coupled to the actuation port, the fluid pump being configured to perfuse and withdraw fluid from the reservoir.

7. The microfluidic system of claim 6, wherein the fluid pump is configured to move the fluid in a first direction during perfusion through the resisting device into the fluid reservoir, and wherein the fluid pump is configured to move the fluid in a second direction during withdrawal to move the fluid from the reservoir and through each of the second base port, second channel, and upper opening, past the membrane, and through the first channel and first port.

8. The microfluidic device of claim 7, wherein the membrane is configured to seal the second channel when the fluid is being moved in the first direction.

9. The microfluidic system of claim 7, wherein the at least one resisting device is configured to inhibit flow of fluid through the at least one resisting device in the second direction during the withdrawal.

10. The microfluidic device of claim 7, wherein the membrane is configured to flex to form an opening between the working portion of the membrane and the abutted portion of the lower layer by the fluid flowing in the second direction through the second channel.

11. The microfluidic system of claim 7, wherein the fluid includes cells.

12. The microfluidic system of claim 11, wherein, when the fluid passes through the membrane, a plurality of the cells adhere to at least one surface thereof.

13. The microfluidic system of claim 6, wherein the fluid pump is selected from a syringe pump, a peristaltic pump, a compressor pump, a diaphragm pump, and a piston pump.

14. The microfluidic system of claim 1, wherein at least one of the second channel or the upper port is positioned at an angle relative to the membrane.

15. The microfluidic system of claim 1, wherein the membrane is positioned at an angle relative to the base layer.

16. The microfluidic system of claim 1, further comprising a glass slide generally adjacent a surface of the bottom layer generally opposing the lower layer.

17. The microfluidic device of claim 1, wherein the at least one resisting device is a fluidic resistor.

18. The microfluidic device of claim 1, wherein the at least one resisting device is a valve.

19. The microfluidic device of claim 1, wherein the membrane includes electrospun material, cast material, woven material, three-dimensional printed material, explanted material, or any combination thereof.

20. The microfluidic device of claim 1, wherein the membrane is formed of material of bacterial origin, plant origin, fungal origin, mammalian origin, synthesized origin, or any combination thereof.

21. The microfluidic device of claim 1, further including a microscope, the microscope configured to provide real-time imaging.

22. The microfluidic system of claim 1, further comprising one or more sensors, the one or more sensors being configured to test flow, electrical, protein, nucleic acid, other cell components, or any combination thereof.

23. A microfluidic device for simulating a heart valve, comprising:

an elastic membrane that is capable of receiving living cells, the elastic membrane including a working portion that inhibits fluid flow in a first direction and permits fluid flow in a second direction;

a first layer located above the elastic membrane; and

a second layer located below the elastic membrane, the first layer and the second layer clamping the elastic membrane outside of the working portion.

24. The microfluidic device of claim 23, wherein the first layer and the second layer define a fluid inlet and a fluid outlet.

25. The microfluidic device of claim 24, wherein at least one of the location and angle of the fluid inlet relative to the elastic membrane is preselected to create a desired angle of impingement flow against a lower surface of the membrane.

26. The microfluidic device of claim 24, wherein at least one of the location and angle of the fluid outlet relative to the elastic membrane is preselected to create a desired fluid outlet angle.

27. The microfluidic device of claim 23, wherein the fluid flow in the second direction includes flow along a lower surface of the working portion of the membrane.

28. The microfluidic device of claim 23, wherein deflection of the membrane is inhibited by a portion of the first layer.

29. The microfluidic device of claim 23, wherein deflection of the membrane is inhibited by a portion of the second layer.

30. The microfluidic device of claim 23, wherein at least one end of the working portion of the membrane is curved.

31. A microfluidic system for simulating a heart valve, comprising:

a fluid pump capable of moving fluid in an outward direction and an inward direction; a first fluid path coupled to the fluid pump;

a second fluid path coupled to the fluid pump;

a fluid reservoir that is fluidically coupled to the fluid pump channel the first fluid path and the second fluid path;

a valve device located within the second fluid path, the valve device including a membrane that is capable of receiving living cells, an upper layer located above the membrane, and a lower layer located below the membrane, the upper layer and the lower layer clamping the membrane in a region outside of a deflectable working portion, the working portion closing the second fluid path in response to the outward direction of fluid flow from the fluid pump such that fluid flows from the fluid pump to the fluid reservoir channel along the first fluid path, the working portion deflecting in response to the inward direction of fluid flow to the fluid pump such that fluid flows from the fluid reservoir to the fluid pump channel along the second fluid path.

Description:
MICROFLUIDIC DEVICE FOR SHEAR FLOW TESTING AND METHODS FOR

USING THE SAME

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 62/296,448, filed February 17, 2016, the contents of which are incorporated herein by reference in its entirety.

GOVERNMENT SUPPORT

[0002] This invention was made with government support under grant number HHSF223201310079 awarded by the Defense Advanced Research Projects Agency (DARPA). The government has certain rights in the invention.

TECHNICAL FIELD

[0003] The present invention relates to cell culture systems and fluidic systems. More specifically, the invention relates to a microfluidic pulsatile bidirectional flow platform for tissue maturation and analysis.

BACKGROUND

[0004] Microfluidic and/or mesofluidic devices (hereinafter referred to as microfluidic devices) allow for various types of experimentation to be performed on various types of cells contained within the devices. The experimentation often requires the flow of fluid across the cells, which mimics the flow of fluid in vivo. With respect to certain cells (e.g., heart cells), the flow of fluid is bidirectional. The amount of fluid that passes through the microfluidic devices generally ranges from microliters to milliliters.

[0005] Mimicking structural and mechanical characteristics of native tissues has been the focus of tissue engineered heart valve (TEHV) design for more than a decade. Modern biomaterials, however, generally have substantial limitations associated therewith. The goal is to create a bio-compatible scaffold with optimum degradation and mechanics and appropriate chemistry for cell channelbility and tissue formation. The aim is to precisely mimic, for example, the tri-layered structure of heart valve leaflets, fibrous layers of fibrosa and ventricularis, and the middle hydrogel-like layer of spongeosa by including cell encapsulation and fibrous-hydrogel layer assembles.

[0006] The bidirectional flow of fluid through microfluidic devices is difficult to achieve with existing equipment. Moreover, existing equipment generally cannot provide such bidirectional flow at controllable flow rates at the small microliter and/or milliliter volumes required to mimic flow in vivo.

[0007] Microfluidic devices for testing experimental membrane (e.g., tricuspid heart valve) materials should provide flow actuation that mimics the flow in a human. They should also provide the ability to study cell seeding under flow onto a variety of materials and structures, tissue maturation under mechanical forces and fluidic shear, and pre-seeded cells in gels inside the membrane materials.

[0008] In existing systems, life-size valves are typically used and tested in a bioreactor setup. While these systems allow for some in vitro testing, it is generally difficult to perform such testing due to the volume of media required. Additionally, testing multiple conditions with several replicates is very difficult or nearly impossible using existing systems. Furthermore, existing systems typically prevent imaging of the tissue until it is removed after culture.

[0009] The below-described devices, methods, and systems address many of the disadvantages associated with the current art by providing a device that has the ability to test multiple parameters quickly and easily at a significantly smaller scale than in existing systems and devices.

SUMMARY

[0010] According to aspects of the present disclosure, a microfluidic system comprises a base layer having a microchannel that links a fluid reservoir to an actuation port. The microchannel includes at least one resisting device positioned between the reservoir and the actuation port. The microchannel also includes first and second base ports positioned at opposing ends of the microchannel. The microfluidic system includes a valve device including a lower layer including a first portion of a membrane-holding device. The lower layer includes first and second channels. The first and second channels are generally aligned with a respective one of the first and second base ports. The valve device further includes an upper layer including a second portion of the membrane-holding device. The upper layer further includes an indent coupled to an upper port. The indent is generally vertically aligned with the second channel. The upper port is generally vertically aligned with the first channel. The lower layer is positioned between the base layer and the upper layer. The valve device further includes a flexible membrane positioned between the lower layer and the upper layer. A first end of the membrane is held in a generally stationary position over the second channel by the first and second portions of the membrane-holding device. The flexible membrane further includes a working portion abutting a portion of the lower layer such that the membrane completely covers the second channel.

[0011] According to additional aspects of the present disclosure, a microfluidic device for simulating a heart valve comprises an elastic membrane that is capable of receiving living cells. The elastic membrane includes a working portion that inhibits fluid flow in a first direction and permits fluid flow in a second direction. The device further includes a first layer located above the elastic membrane and a second layer located below the elastic membrane. The first layer and the second layer clamp the elastic membrane outside of the working portion.

[0012] According to a further aspect of the present disclosure, a microfluidic system for simulating a heart valve comprises a fluid pump capable of moving fluid in an outward direction and an inward direction. The system further includes a first fluid path coupled to the fluid pump and a second fluid path coupled to the fluid pump. The system further includes a fluid reservoir that is fluidically coupled to the fluid pump channel the first fluid path and the second fluid path. The system further includes a valve device located within the second fluid path. The valve device includes a membrane that is capable of receiving living cells, an upper layer located above the membrane, and a lower layer located below the membrane. The upper layer and the lower layer clamp the membrane in a region outside of a deflectable working portion. The working portion closes the second fluid path in response to the outward direction of fluid flow from the fluid pump such that fluid flows from the fluid pump to the fluid reservoir channel along the first fluid path. The working portion deflects in response to the inward direction of fluid flow to the fluid pump such that fluid flows from the fluid reservoir to the fluid pump channel along the second fluid path.

[0013] These and other capabilities of the inventions, along with the inventions themselves, will be more fully understood after a review of the following figures, detailed description, and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] While the invention is susceptible to various modifications and alternative forms, specific embodiments have been shown by way of example in the drawings and will be described in detail herein. It should be understood, however, that the invention is not intended to be limited to the particular forms disclosed. Rather, the invention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the invention as defined by the appended claims. [0015] FIG. 1 illustrates a top view of a microfluidic device with a valve device, in accord with some aspects of the present concepts.

[0016] FIG. 2 is a cross-section of the microfluidic device taken along line B-B of FIG. 1, in accord with some aspects of the present concepts.

[0017] FIG. 3 is an exploded view of the microfluidic device of FIGs. 1 and 2.

[0018] FIGs. 4A-4F illustrates non-limiting examples of membrane-holding devices that may be used with the embodiments described herein.

[0019] FIGs. 5 A-5D illustrate non-limiting cross-sectional views along line B-B of FIG. 1 of fluid inlets relative to the membrane of various embodiments of valve devices described herein.

[0020] FIGs. 6A-6D illustrate cross-sectional views along line B-B of FIG. 1 of valve devices according to various non-limiting embodiments showing various indents formed in the upper layer.

[0021] FIGs. 6E-6G are cross-sectional views of upper layers of valve devices described herein illustrating exemplary shape profiles for the indent formed therein.

[0022] FIGs. 7A-7D illustrate non-limiting cross-sectional views along line B-B of FIG.

1 of fluid outlets relative to the membrane of various embodiments of valve devices described herein.

[0023] FIG. 8 illustrates a system for introducing bi-directional fluid flow through a microfluidic device, in accord with some aspects of the present disclosure.

[0024] FIG. 9A is cross-sectional view along line B-B in FIG. 1, depicting a membrane in closed position according to some embodiments.

[0025] FIG. 9B is a cross-sectional view along line B-B in FIG. 1, depicting the membrane in an open position according to some embodiments.

[0026] FIG. 10A is a cross-sectional view of the valve device of FIG. 9A along line 10a- 10a.

[0027] FIG. 10B is a cross-sectional view of the valve device of FIG. 9B along line 10b- 10b.

DETAILED DESCRIPTION

[0028] While the inventions are susceptible of embodiment in many different forms, there is shown in the drawings and will herein be described in detail preferred embodiments of the inventions with the understanding that the present disclosure is to be considered as an exemplification of the principles of the inventions and is not intended to limit the broad aspects of the inventions to the embodiments illustrated.

[0029] The functionality of cells and tissue types (and even organs) can be implemented in one or more microfluidic devices or "chips" that enable researchers to study these cells and tissue types outside of the body while mimicking much of the stimuli and environment to which the tissue is exposed in vivo. It can also be desirable to implement these microfluidic devices into interconnected components that can simulate groups of organs or tissue systems. In some embodiments, the microfluidic devices are easily inserted and removed from an underlying fluidic system that connects to the devices in order to vary the simulated in vivo conditions and organ systems. More details on such microfluidic devices and chips can be found in, for example, U.S. Patent No. 8,647,861, which is incorporated herein by reference in its entirety.

[0030] The present disclosure relates to systems that incorporate material/tissue that acts as a valve when exposed to a pulsatile, bidirectional flow of medium. FIGs. 1-3 illustrate one type of a microfluidic device 10, in accord to some aspects disclosed herein. Referring to FIGs. 1-3, the device 10 includes a base layer 12 and a valve device 13. The valve device 13 includes a lower layer 14, a flexible, elastic membrane 15, and an upper layer 16.

[0031] Each of the base layer 12, lower layer 14, and upper layer 16 may made of a polymeric material, such as polydimethysyloxane (PDMS), poly(methyl methacrylate) (PMMA), polycarbonate, cyclic olefin copolymer (COP), cyclic olefin polymer (COC), polyurethane, polystyrene, styrene-butadiene-styrene (SBS), poly(styrene-ethylene/butylene- styrene) (SEBS) block copolymers, other thermoplastic or thermoset polymer(s), glass, ceramic, combinations thereof, or the like.

[0032] The base layer 12 includes a microchannel 20 that links a fluid reservoir 22 to an actuation port 24. The microchannel 20 also includes first and second base ports 30, 32 positioned at opposing ends of the microchannel 20. The microchannel includes a first fluid path 25 extending from the actuation port 24 toward the second base port 32 (in the direction of Arrow C) and a second fluid path 27 extending from the actuation port 24 toward the first base port 30. The dimensions of the microchannel 20 may range from about 10 microns wide/deep to more than 2 mm wide/deep. The dimensions may depend upon factors such as, but not limited to, the desired flow rate of the fluid media through the microchannel 20 and other needs.

[0033] The microchannel 20 includes at least one resisting device 26 positioned in the first flow path 25 between the reservoir 22 and the actuation port 24. The size of the resisting device 26 may vary depending, for example, on the desired backflow rate through the resisting device 26.

[0034] In the illustrated embodiments, the resisting device 26 is a fluidic resistor. It is contemplated, however, that the resisting device may be a valve, such as a passive valve, an active valve, a pneumatically activated valve, a mechanical valve, or other suitable type of valve. The valve may be controlled externally and in sync with the bidirectional flow. The resisting device 26 may also include a second membrane, which may or may not be formed from the same material as the membrane 15. Including a second membrane could allow for two tests to be conducted per device 10. For example, the second membrane could be used to test various heart valve pathologies.

[0035] The lower layer 14 of the valve device 13 is positioned between the base layer 12 and the upper layer 16, under the membrane 15. The lower layer 14 includes a first portion 36 of a membrane-holding device 38. The lower layer 14 further includes first and second channels 40, 42. The first and second channels 40, 42 are generally aligned with a respective one of the first and second base ports 30, 32 (see, e.g., FIG. 2) to form a fluid inlet and a fluid outlet such that fluid may pass from the second base ports 32 of the base layer 12 through the second channel 42 of the lower layer 14 to the upper layer 16 and from the upper layer 16 through the first channel 40 of the lower layer 14 into the first base port 30 of the base layer 12. The lower layer 14 provides a valve "seat" 43 on which the membrane 15 may be positioned to seal fluid flow, as discussed in more detail below.

[0036] The upper layer 16 includes an indent 50 coupled to an upper port 52 (see FIG. 2). The indent 50 is a hollowed-out portion of the surface of the upper layer 16 that contacts into which fluid flowing through the expanded membrane 15 travels. The indent 50 is generally vertically aligned with the second channel 42. The upper port 52 is generally vertically aligned with the first channel 40 and the first base port 30.

[0037] The upper layer 16 further includes a second portion 46 of the membrane-holding device 38. The second portion 46 of the membrane-holding device 38 is configured to be coupled to and/or to mate with the first portion 36 of the membrane-holding device 38 positioned on the lower layer 14. Each of the first and second portions 36, 46 of the membrane-holding device 38 may include a ridged surface adapted to contact and clamp the membrane 15. For example, the first and second portions 36, 46 of the membrane-holding device 38 may include interlocking teeth that clamp down on the membrane 15 to hold it in place. In another embodiment, the membrane 15 is bonded to each of the first and second portions 36, 46 of the membrane-holding device 38. [0038] As shown in FIGs. 1-3, at least one side of the first and second portions 36, 46 of the membrane-holding device 38 has a generally curved, concave shape. The membrane- holding device 38 contacts the membrane 15 such that the sides of the membrane 15 are generally supported (e.g., do not fold or collapse) but still allows for free motion of a working (expandable) portion 54b of the membrane 15 in a flapping motion (see FIGs. 4A-4F). Put another way, the working portion 54b of the membrane 15 is still able to flex and expand upon contact with medium or fluid flowing generally upwardly through the second channel 42 (as discussed below). The size and shape of membrane-holding device 38 dictate the size and shape of the working portion 54b of the membrane 15.

[0039] FIGs. 4A-4F show various exemplary shapes and sizes of the working portion 54b of a membrane 15 according to various embodiments, with a stationary (e.g., clamped) portion of the membrane 15 being sandwiched between the membrane-holding device 38 shown in cross hatching. As shown in FIGs. 4A-4F, the generally concave shape of the membrane-holding device 38 may include a semicircular shape, a semi-elliptical shape, a generally parabolic shape, a U-shape, or the like. Such shapes generally mimic the in vivo geometry of a tricuspid heart valve and decouple the stiffness of the membrane material under test from its ability to function.

[0040] The flexible membrane 15 is positioned generally on the valve seat 43 of the lower layer 14 between the lower layer 14 and the upper layer 16 of the valve device 13. The stationary portion 54a of the membrane 15 is held in a place over the second channel 42 of the lower layer 14 by the first and second portions 36, 46 of the membrane-holding device 38. The working portion 54b of the membrane 15 abuts a portion 56 of the lower layer 14 such that the membrane 15 completely covers and is configured to form a seal over the second channel 42 by the fluid flowing in the direction of Arrow B (see FIG. 9A) from the upper port 52. The membrane 15 is further configured to flex to form an opening between the working portion 54b of the membrane 15 and the abutted portion 56 of the lower layer 14 by the fluid flowing in the direction of Arrow A through the second channel 42 (see FIG. 9B).

[0041] The amount of overlap and the deflection angle between the working portion 54b of the membrane 15 and the abutted portion 56 of the lower layer 14 may be varied to achieve a desired effect, as shown in FIGs. 5A-5D. Specifically, FIGs. 5A-5D are cross-sectional views along line B-B of FIG. 1 showing fluid inlets being modified relative to the membrane 15. Specifically, the size and shape of the fluid inlet (e.g., second channel 42) may be modified. Such modifications result in different types of fluid flow and different angles of impingement flow against the lower surface of the membrane 15. [0042] The membrane 15 may be formed of any suitable material including, but not limited to an electrospun material, cast material, woven material, three-dimensional printed material, explanted material, or any combination thereof. The membrane 15 may be formed of material of bacterial origin, plant origin, fungal origin, mammalian origin, synthesized origin, or any combination thereof. In one embodiment, the membrane 15 is made of a cured PDMS. The use of an optically clear material such as PDMS allows for live imaging of experimental membrane tissue during an experiment. The position of the membrane 15 relative to the microfluidic circuit also facilitates the ease of observation of the experimental material.

[0043] The membrane 15 may include any suitable dimensions. The thickness of the membrane 15 is generally in the range of about 10 microns to about 500 microns. More specifically, the thickness may range from about 100 microns to about 300 microns. The size of the membrane 15 may range from about 1 mm to more than 10 mm. In one example, the membrane has a length ranging from about 8 mm to about 12 mm (e.g., about 10 mm) and a width ranging from about 8 mm to about 12 mm (e.g., about 10 mm).

[0044] In some embodiments, the valve seat 43 of the lower layer 14 - and the membrane 15 positioned thereon - is positioned at an angle relative to the fluid inlet and/or the fluid outlet. In one non-limiting embodiment, the valve seat 43 is tilted at an angle relative to the base layer 12, the second channel 42, or the like. Such positioning generally modifies the deflection angle of the membrane 15 and exposes the membrane 15 to various different amounts and directions of fluid shear flow. Thus, a range of deflection angles and the corresponding effects on the membrane 15 and device 10 may be tested.

[0045] As shown in FIGs. 6A-6D, the height and/or shape of the upper layer 16 and/or the indent 50 formed therein may be varied, for example, to restrict how high the membrane 15 may expand. For example, the height of the indent 50 may be smaller to provide a "ceiling" beyond which the membrane 15 may not expand so as to physically prevent further deflection (and, hence, fluid flow) of the membrane 15 during operation. As shown, the indent 50 may have a flat, planar undersurface for contacting the membrane 15, a curved undersurface for better matching the curved shape of the membrane 15 in operation, or some combination thereof. FIGs. 6E-6G illustrate exemplary cross-sectional views of the upper layer 16 where the indent 50 formed therein has various shape profiles. In other embodiments (not shown), the height of the upper layer 16 and/or the indent 50 may be greater than the expansion potential of the membrane 15 so as not to interfere with or inhibit the expansion of the membrane 15. [0046] It is contemplated that the position of the fluid outlet relative to the membrane 15 may be varied, as shown in FIGs. 7A-7D, to study the effects thereof. Specifically, FIGs. 7A-7D illustrate schematic cross-sectional views through the lower layer 14 and the upper layer 16 showing various types of the fluid outlet locations to encourage certain types of flow around the membrane 15.

[0047] The device 10 may also include a slide 18 coupled to the base 12, as shown in FIGs. 1-3. The slide 18 is generally adjacent a surface of the base 12 opposing the lower layer 14. The slide 18 may, for example, be made of glass (e.g., a standard 25 mm x 75 mm glass slide) or other optically clear material(s) that permits viewing of the fluids, media, particulates, etc. as they move through the device 10.

[0048] It is contemplated that living cells and/or reagents can be introduced into the medium or fluid passed through the device 10 to the membrane 15. Specifically, when cell- containing fluid passes through the membrane 15, a plurality of cells may attach/colonize to at least one surface of the membrane 15, thereby allowing testing of cell attachment/colonization and/or growth.

[0049] The device 10 is advantageous because it is configured to simulate a biological function, as would be experienced in vivo within organs, tissues, cells, or the like. For example, as discussed above, the membrane 15 is generally capable of stretching and expanding as fluid travels in the direction of Arrow A to simulate the physiological effects commonly experienced by heart valves. The membrane 15 is designed to have an elasticity corresponding with a desired function. For example, the membrane 15 may have an elasticity that corresponds with that of a tricuspid heart valve.

[0050] To provide the desired flow of fluid through the device 20, the systems described herein may include a fluid pump 70, as shown, for example, in FIG. 8. The fluid pump 70 may be, for example, a syringe pump, a peristaltic pump, a compressor pump, a diaphragm pump, a piston pump, or the like. In the illustrated embodiment of FIG. 8, the fluid pump 70 is a syringe. The syringe may be gas tight and can deliver fluid according to precise and accurate measurements based on the displaced volume within the syringe. That is, the syringe can include moveable plungers and fixed barrels, with the movable plungers being supported within the barrels. In other embodiments, the fluid pump 70 can be replaced with a mechanical actuator, a pneumatic actuator, or the like, depending on the requirements of the system.

[0051] The amount of fluid drawn in and pushed out of the device 10 by the fluid pump 70 can depend on several factors. One non-limiting factor that controls the amount of moved fluid is the size of the fluid pump 70 (e.g., the syringe), both with respect to length and diameter.

[0052] The fluid pump 70 generally has a controllable volume and frequency and may be programmed to cycle between withdrawal and perfuse steps. The fluid pump 70 is designed to cyclically push a medium or fluid into the device 10 and subsequently pull the medium or fluid out of the device 10. The flow of fluid during the withdrawal and perfuse steps generally mimics the flow of blood through a valve (e.g., the tricuspid valve) of a human heart during a heartbeat cycle. The volume and rate of the fluid moving through the device 10 can be accurately and precisely controlled based on controlling the fluid pump 70. The frequency of perfusion/withdrawal cycles, the volume of fluid, and the like may be varied accordingly. With respect to the device 10 described above, and by way of an example, the fluid pump 70 can be connected to and in fluid communication with the device 10 by way of the actuation port 24.

[0053] During perfusion, fluid is flowed from the fluid pump 70 in an outward direction to the reservoir 22. Referring to FIG. 3, fluid from the fluid pump 70 moving in the direction of Arrow B travels into the first base port 30, up through the first channel 40, into the upper port 52 and, subsequently, the indent 50, down toward the membrane 15. The fluid travelling in the direction of Arrow B applies a downward force on the membrane 15, thereby causing it to seal the valve seat 43 of the lower layer 14 around the second channel 42, thereby inhibiting or preventing the flow of fluid therethrough (see FIG. 9A). Because the fluid is, thus, effectively stopped from passing through the membrane 15, fluid passing from the fluid pump 70 through the actuation port 24 instead moves in the direction of Arrow C, passing through the resistor device 26 and into the reservoir 22. FIG. 1 OA is a schematic illustration of a cross-section through the working portion 54b of the valve device 13 along line 10a- 10a in FIG. 9A during perfusion.

[0054] The fluid pump 70 then reverses the fluid flow to generate a bi-directional flow. Specifically, during the withdrawal cycle, the fluid pump 70 pulls fluid by creating suction through the actuation port 24. Fluid is generally pulled in the direction of Arrow A from the reservoir 22, into the second base port 32, and toward the second channel 42. The fluid flow (and/or suction caused by the fluid pump 70) to the membrane 15 during withdrawal pushes on the membrane 15, causing it to expand and form an opening between the working portion 54b of the membrane 15 and the abutted portion 56 of the lower layer 14 (see FIG. 9B). As such, fluid may flow from the reservoir 22, through the second channel 42 and the opening, into the indent 50 of the upper layer 16, through the upper port 52, down through the first channel 40, into the first base port 30, and, finally, back into the fluid pump 70. FIG. 10B is a schematic illustration of a cross-section through the working portion 54b of the valve device 13 along line 10b- 10b in FIG. 9B during withdrawal.

[0055] During withdrawal, fluid may also flow from the reservoir 22 back toward the fluid pump 70 in an inward direction in the direction of Arrow D (see FIG. 3). However, the resisting device 26 generally inhibits or prevents such backflow of the fluid from the reservoir 22. The amount of resistance provided by the resisting device 26 can be used to tailor the amount of backflow (e.g., to mimic heart valve failure or damage). The combination of the membrane 15 and resisting device 26 allows for changes in the flow direction to actuate the membrane 15 with a single inlet/outlet (i.e., actuation port 24) to the fluid pump 70.

[0056] The fluid pump may include a motor, which can be controlled by a controller, as described in U.S. Patent Application Serial No. 62/141,560, which is fully incorporated by reference herein. Bioreactor control software may implement algorithms to operate the mechanical components of the system (e.g., pumps, resisting devices, etc.) in a way that allows delivery of well-controlled physiologic stresses to the engineered construct in the microfluidic channel 20.

[0057] By way of example, and without limitation, an operator of the fluid pump 70 may program various rhythms and volumes for the flow of fluid into and out of the device 10. The rhythm can be a continuous or intermittent oscillation of fluid into and out of the device 10. The volume of fluid for each cycle of drawing fluid into and out of the device 10 can be the same volume of fluid or a different volume of fluid. Various profiles of the controller can operate the fluid pump 70 to mimic various patterns of heartbeats in humans. By way of example and without limitation, one profile can mimic the heartbeat of a human during rest. The fluid pump may be controlled such that the period of the actuating the syringe and the volume of fluid moved by the syringe generally mimics the period and volume of a human heartbeat during rest. Another profile can be of heartbeats during exercise or exertion, such as a shorter period and/or larger volumes of fluid. Thus, the fluid pump 70 may generate different shear stress levels on the cells lining the membrane 15 within the device 10.

[0058] Shear flow can be adjusted in intensity by changing fluid volumetric flow rate or geometry. For example, a low shear may be applied to initially seed the cells from the fluid onto the membrane 15, and a higher shear may be applied to adhere more cells thereon. Shear flow rate may also be adjusted in angle/directionality (e.g., by adjusting the deflection angles, as discussed above) to test the effect of shear properties on cells, material structure, combinations thereof, or the like.

[0059] As discussed above, various factors associated with the fluid pump 70 may be controlled including the duration of each aspect of the cycle (intake duration and output duration) and the volume of fluid that is moved through the microfluidic channel 20 within each cycle. Furthermore, the temperature of the fluids used in the cycles and the content of the fluid in the cycles can be controlled. The adjustable parameters of the devices can mimic systemic circulation on a wide range of operating points (e.g., aortic or pulmonary valves, arterial or venous systemic circulation, peripheral circulation, and the like - from fetal to adult).

[0060] The above described system is one embodiment of a system for testing the effects of fluid flow on cells seeding on and/or lining the membrane 15 of the device 10. Cellular interactions may be tested with new biomaterials in a dynamic environment stimulated by both valve movement and changes in flow. For example, the systems and devices described herein may be used to test heart valve materials and implantation characteristics such as cell seeding and/or degradation of the material as well as material failure testing of artificial, acellular valves.

[0061] The systems and devices described herein may also or alternatively be used to test shear-based delivery of compounds or to test efficacy of clot-busting drugs by incorporating a clot in an artery. The systems and devices may also be used to test biomechanical effects on a range of tissues, including HSC development, bone marrow cell migration, muscle cells, combinations thereof, or the like. The systems and devices may also be used for bacterial and/or other non-mammalian or mammalian cell bioreactors to avoid sedimentation or to provide biomechanical cues. The systems and devices may also be used to test components of robotic systems in various fluids under a range of stresses including, but not limited to, robot wings in turbulence, fins under water, or the like.

[0062] The system and/or components thereof described herein may be housed within a chamber. According to some embodiments, the chamber can be an incubator that allows for control of the conditions inside of the chamber. By way of example, and without limitation, the chamber can be operated at a set temperature, such as 37 °C, which mimics the internal temperature of the human body. The chamber can also be operated at other conditions that mimic the internal conditions of a human body. By operating the device under conditions that mimic the internal conditions of a human body, such as providing a hydrostatic pressure similar to the systolic blood flow pressure at the position of PV or AV (about 30mmHg, about 85 mmHg), the system may be operated at conditions that mimic conditions in vivo.

[0063] The systems and devices described herein generally allow for high throughput testing of conditions or materials due to the small scale of the microfluidic devices and the ability for multiple tests to be multiplexed into a single incubator. They are scalable to allow for ease of replicates and multi-condition experiments in a reasonable time and space (e.g., one experiment per standard incubator, which could include, for example, about 8 to about 16 tests or devices). The compact devices described herein have a low medium volume, but are generally large enough to support cell culture for several days. The systems and devices described herein generally enable testing of various membrane materials less than about 500 microns in thickness. Additionally, the systems and devices are amenable to real-time imaging or simple imaging without terminating experiments and provide simple ways for adding cells or reagents to the medium.

[0064] According to the embodiments described herein, by perfusing and withdrawing fluid through the device 10, the device 10 generally mimics a human heart. As such, the device 10 may be used to study the effects of various factors on a heart valve and/or to assist in creating improved artificial valves. The systems described herein allow for various different applications to test the effects of fluid, device construction, and/or agents on the device 10. By way of example, and without limitation, such applications include optimizing media/fluid, analyzing the effects of fluid flow on cell seeding onto the membrane 15, analyzing the effect of mechanical conditioning on cell growth, tissue formation and expressed markers, studying the mechanical stress of delivering fluid in the system, and mimicking different heartbeat patterns (e.g., exercise, rest, or the like).

[0065] It is contemplated that the system described herein may include other components. For example, the system may include a (automated) microscope configured to provide realtime imaging. Additionally or alternatively, the system may include one or more sensors configured to test flow, electrical components, protein, nucleic acid, other cell components, or any combination thereof.

[0066] While the present invention has been described with reference to one or more particular embodiments, those skilled in the art will recognize that many changes may be made thereto without departing from the spirit and scope of the present invention. Each of these embodiments and obvious variations thereof is contemplated as falling within the spirit and scope of the invention. It is also contemplated that additional embodiments according to aspects of the present invention may combine any number of features from any of the embodiments described herein.