COOPER-WHITE, Justin, John (Lot 2, 914 Upper Brookfield RoadUpper Brookfield, Queensland 4069, AU)
DORAN, Michael, Robert (Unit 21 Hardgrave Road, West End, Queensland 4101, AU)
COOPER-WHITE, Justin, John (Lot 2, 914 Upper Brookfield RoadUpper Brookfield, Queensland 4069, AU)
Claims
1. A device for assessing cellular response including a first transport layer and a second transport layer for carrying, supplying or removing a flow of at least one cell metabolite, a cell housing layer having at least one cell chamber for cultivating cells, the cell housing layer enabling the at least one cell metabolite to transfer from at least the first transport layer to at least the cell chamber, and means to measure at least one of the physical process parameters of the fluid flowing through the at least the first transport layer.
2. The device of claim 1 , wherein the cell chamber of the cell housing layer is separated from the first transport and second transport layer by a first and second membrane respectively permeable to the at least one metabolite
3. The device of claim 2, wherein the first transport layer includes a fluid passage contacting the membrane between the chamber of the cell housing layer and the first transport layer.
4 The device of claim 2, wherein the second transport layer includes a second fluid passage contacting the second membrane between the cell chamber of the cell housing layer and the second transport layer.
5. The device of claim 1 , wherein the cell housing layer further includes at least one access channel communicating with the cell chamber, the at least one channel being capable of providing a flow of cell metabolites or cells to or from the cell within the cell chamber.
6. The device of claim 1 wherein the height of the cell chamber is in the range of 10 μm to 1000 μm.
7. The device of claim 1 , wherein the height of the cell chamber is in the range of 10μm to 200 μm.
8. The device of claim 1 wherein the cell housing layer includes a plurality of cell chambers.
9. The device of claim 8 wherein the cell chambers are arranged in a two dimensional array, with the cell chambers aligned across the cell housing layer and parallel to at least the first transport layer.
10. The device of claim 1 further including a probe able to assess the metabolite concentration across the cell housing layer.
11. A microfluidic device for cultivating cells including:
a first transport layer for carrying, supplying or removing a flow of fluid containing at least one cell metabolite and a second transport layer for carrying, supplying or removing a flow of fluid containing at least one cell metabolite separated by a cell housing layer, the cell housing layer having at least one cell chamber for cultivating cells, the cell housing layer being permeable to the at least one cell metabolite required by the cell; and means to measure the physical process parameters of the fluid flowing through the at least the first transport layer wherein the dimension across the cell chamber is in the range of 10 μm to 1000 μm.
!2. The device of claim 12, wherein the dimension across the cell chamber is in the range of 10 μm to 200 μm.
13 The device of claim 1 or 11 , wherein the cell housing layer is contiguous with the ceil chambers.
14. The device of claim 11 , wherein the membrane or cell housing layer separating the second transport layer and the cell chamber is permeable to the at least one cell metabolite within a flow passage within the second transport layer. 15 The device of claim 1 or 12 wherein the measured physical process parameters of the fluid flowing in the transport layer or layers are at least one variable selected from the group consisting of pressure, temperature, pH, oxygen concentration and carbon dioxide concentration.
16 The device of claim 15 wherein the variables are measurable in real time and the device includes a means to control the measured variables in real time.
17. The device of claim 1 or 11 , wherein the interior of the chamber of the housing layer comprises a 2 dimensional support surface or a 3 dimensional scaffold for supporting cells.
18. The device of 1 or 11 , further including one or more heating elements positioned in the transport layer or layers, the cell housing layer or within the permeable membrane.
19. The device of claim 1 or 11 , wherein the cell chamber includes an elongated channel.
20. The device of claim 19 wherein the elongated channel includes a plurality of parallel elongated sections aligned parallel to the cell housing layer which travel across the cell housing layer.
21. The device of claim 19 or 20 wherein the elongated channel further includes a plurality of parallel elongated sections aligned perpendicular to the cell housing layer which travel across the cell housing layer
22. The device of claim 1 or 11 , wherein the interior of the chamber of the housing layer includes a 2 dimensional support surface or a 3 dimensional scaffold for supporting cells.
23. A method of controlling process variables within a cell chamber to create a microenvironment in the cell chamber of predetermined conditions including the steps of providing a device having a permeable cell housing layer between at least one side of the cell chamber, and at least one transport layer of cell metabolites,
providing a flow of at least one cell metabolite through the at least one transport layer to permeate through the permeable cell housing layer into the cell chamber,
monitoring the process variables within the flow of cell metabolites in the at least one transport layer, and/or the cell housing layer and
determining the process variables within the cell chamber based on the known characteristics of the permeable cell housing layer and the mass transfer gradients of the nutrients established across the cell housing layer.
24. The method of claim 23 wherein, the device includes a first and second transport layer for carrying, supplying or removing a flow of at least one cell metabolite, a cell housing layer having at least one cell chamber for cultivating cells, the cell housing layer being between the first and second transport layer, the method including the step of establishing a metabolite transport gradient across between the first and second transport layers and assessing the response of the cells in the cell chambers to the metabolite concentration.
25. The method of claim 24 wherein the cell housing layer is provided with a plurality of cell chambers, the cell chambers being arranged in an array of cell chambers across the cell housing layer, the metabolite concentration gradient being established across the row of cell chambers which span the cell housing layer.
26. The method of claim 25 wherein each column of cells in the array has substantially the same metabolite concentration.
27. The method of claim 24 or 25 wherein at least one of the first or second transport layer is a flow passage.
28. The method of claim 23 wherein one of the first or second transport layers is a controlled environment.
29. The method of claim 23 or 24 wherein at least the two sides of the cell housing layer include permeable membranes to separate the cell chambers from the at least one transport layer.
30. The method of claim 23 or 24 further including the step of locating one or more cells or a tissue culture on a surface or scaffold positioned within the one or more cell chambers and evaluating the response of the cells to the established conditions.
31. The method of claim 30 wherein the predetermined conditions are the physical characteristics of the surface.
32. The method of claim 23 or 24 further including the steps of
establishing known microenvironmental conditions in a cell chamber, and
varying at least one of the environmental condition in a known manner to monitor and assess the effect that varying the at least one known environmental conditions has on one or more cells in the cell chamber.
33. The method of claim 23 or 24 further including the step of establishing known microenvironment conditions in a cell chamber, and
monitoring the effect of the cell on the condition being monitored
34. A method of assessing the effect of surface conditions on the growth of one or more cells including the steps of introducing or creating in a cell chamber a microenvironment having predetermined environmental conditions at, or in proximity to the surface of one or more cells and monitoring and assessing the effect of the surface condition on the growth of such cells.
35. The method of claim 34 wherein the predetermined micro-environmental conditions being assessed include parameters relating to the physical characteristics of the surface of the cell chamber or a 3dimensional scaffold in the cell chamber. |
Microbioreactor
Field of the invention
This invention relates to a device for controlling the microenvironment around one or more cells and/or tissue and a method of assessing the effects of parameters in a microenvironment on the cells and/or tissue.
Background of the invention
Tissue engineering offers the prospect of replacing missing or non functional body parts and organs with newly created tissue. The strategy most often used to generate these new tissues is the seeding and culture of antilogous cells within highly engineered biocompatible bioabsorbable polymeric scaffolds. The scaffolds are designed with the aims of mimicking the microenvironment created by the extracellular matrix which encases all cells in normal tissue as well as maintaining the required space for supporting new tissue growth. Tissue engineering involves four clearly defined steps:
(i) the formation of a three dimensional porous and/or fibrous scaffold of desirable porosity and morphology that mimics the critical supramolecular architectures of native extracellular matrix from suitable biocompatible (natural or synthetic) polymers;
(ii) the creation of functional surfaces throughout these three dimensional porous scaffolds with biochemical and topological properties that mimic the biofunctionality of the native extracellular matrix;
(iii) the seeding of living cells, ideally harvested from the patient into the functionalised scaffold; and
(iv) the engineering of tissue in vitro prior to transplantation.
Ideally the engineered scaffold is resorbed (biosorbable) or degraded (biodegradable) in situ, leaving only healthy, functional generated tissue. Steps (i) and (ii) are aimed at
creating the appropriate microenvironment for optimum cell physiology and steps (iii) and (iv) then provide the appropriate microenvironment to support cell viability, phenotype and tissue cultivation.
In this regard under in vivo conditions, none of the polymers currently under investigation for tissue engineering of natural or synthetic origin have been truly successful as scaffolds for tissue generation. In fact most known FDA approved biocompatible implants, whether they are drug delivery systems, breast implants or bone prostheses, induce a similar foreign body reaction (chronic low level inflammation and local macrophage activity) when implanted in vivo, resulting in the formation of an avascular, thick fibrous capsule (50-200 microns thick). This capsule effectively walls off the implant from the body preventing true integration of the implant and as a result the performance of many implanted devices is significantly impeded. However all of the same biomaterials can be shown to perform well in existing in vitro compatibility assays.
It is anticipated that the internal architecture, surface topography and surface chemistry of biomaterials intended to replicate biological systems will be complex multicomponent, multilayered, oriented, patterned and biofunctional. Such properties are to be expected if the scaffold used is to be integrated seamlessly with the host normal healing behaviour.
In assessing and producing integrated complex scaffolds, factors such as the cell- surface and cell-cell interactions control cell attachment, migration, proliferation, differentiation and functionality of newly formed cells within the 3D spaces of a scaffold.
Hence the effects of the physical characteristics of the scaffold such as pore size, pore size distribution, pore connectivity and tortuosity on cell regeneration and repair are expected to not only be cell specific and culture systems specific but also dependent on surface characteristics such as the topography, biochemical interaction and mobility of the scaffold. In order to determine whether a chosen artificial surface or porous or fibrous scaffold will support controlled cell expansion and de novo tissue regeneration ex vivo, an integrated systematic approach is needed which allows a number of these variables to be modified whilst maintaining others invariant allowing true assessment of the variables of interest.
The applicant is of the view that the absence of in vitro models which allow quantitative, real time assessment of the effect on cells of the microenvironment surrounding those cells whether in two dimensions or three dimensions are tools which are currently not available to scientists and engineers working in the tissue engineering industry. Such a model must be capable of providing direct insight into the effects of discrete differences in architecture, surface functionality and environmental conditions on the development of any specific cell type. Additionally, they must be able to provide quantitative data for the validation of mathematical models to allow for true predictability in future advanced scaffold designs and cell culture methods.
Existing models do not allow the complex microenvironment to be reliably reproduced and do not allow direct quantification of the cellular response to the microenvironment without significant post processing. Hence there is a need for tools to provide real time data which is critical for the creation of suitable scaffolds. Furthermore these new in vitro models will have to address two distinctly different dimensional scales of investigation, one applicable to the size of the pores within the scaffold and one to the length scale of the total scaffold. Such insights are especially important for the proposed application of stem cells in tissue engineering. These models must not only provide insights but must be robust, sterilisable, simple to use, easy to manufacture and compatible with existing cell biology analysis tools.
US patent application publication no. 2003/0186217 discloses a method and device for growing and/or treating cells. A cell culture plate is disclosed as having cells located at the base of a cell culture chamber and in one embodiment O 2 , air and nutrients to the cell culture chamber are supplied through membranes in the top and bottom of the chamber by pressure applied to those membranes. This reference is not capable of controlling the microenvironment around a cell and quantifying the cellular response to the microenvironment nor would it be possible to assess scaffold design variables in such a device.
Summary of the invention
According to one aspect of the invention there is provided a device for assessing cellular response including a first transport layer and a second transport layer for
carrying, supplying or removing a flow of at least one cell metabolite, a cell housing layer having at least one cell chamber for cultivating cells, the cell housing layer enabling the at least one cell metabolite to transfer from the first transport layer and/or second transport layer to at least the cell chamber, and means to measure at least one of the physical process parameters of the fluid flowing through the at least the first transport layer.
The cell chamber of the cell housing layer may be separated from the first transport and second transport layer by a first and second membrane respectively permeable to the at least one metabolite.
The term cell metabolites is intended to include molecules which are essential to cell homeostasis, survival and growth as well as cell type determinants. Such metabolites include oxygen/air and nutrient medium, growth factors, mitogens, cytokines and chemokines.
Within the context of the invention, the term 'transfer" is intended to cover transport mechanisms such as diffusion and/or convection.
The physical process parameters of the fluid are preferably at least one variable selected from the group including pressure, temperature, oxygen concentration, carbon dioxide concentration and pH. All of the above variables may be measurable in real time. The above apparatus may further include a means to control the measured variable or variables preferably in real time.
In a preferred form of this aspect of the invention, the first transport layer includes a fluid passage contacting the membrane between the chamber of the cell housing layer and the first transport layer. Additionally the second transport layer includes a second fluid passage contacting the second membrane between the cell chamber of the cell housing layer and the second transport layer.
The cell housing layer may further include at least one access channel communicating with the cell chamber. The at least one channel may be capable of further providing a flow of cell metabolites to the cell within the cell chamber. The access channel may
further provide a means by which waste products from the cell can be removed from the cell chamber and cell metabolites generally not capable of passing through the permeable membranes are supplied to the cell chamber. Additionally the access channel may be used for seeding of one or more cells into the cell chamber.
In this preferred form of the invention, the height of the cell chamber is sufficient to not physically interfere with the cell but sufficiently thin to allow predictable determination of the conditions around the cells in the cell chamber. The interior of the chamber of the housing layer comprises a 2 dimensional support surface or a 3 dimensional scaffold for supporting cells. With this construction, any flow within cell chamber and in particular in proximity to the cells may be kept in the laminar region.
In order to fulfil the requirements of the cell chamber being sufficiently thin to allow predictable determination of the conditions around or at the surface of the cells in the cell chamber, the surface or scaffold is located within the laminar flow regions of the cell chamber. It is anticipated that the height of the cell chamber would be in the range of 10 μm to 1000 μm (preferably less than 200 μm and most preferably less than 100 μm) in order to fulfil this requirement. In most circumstances, the cells on the surface or scaffold need to be no greater than 200 μm from the source of oxygen (membrane) to enable oxygen to diffuse to the cell through the culture at a sufficient rate to ensure survival of the cell. In order to provide a cell chamber which is sufficiently thin to satisfy the above requirements, it is preferable that the device is a micro fluidic device in which each of the layers of the device are produced from layers of materials commonly used in such reactor construction including without limitation silicon, glass, metal, polymer using known manufacturing techniques for producing micro fluidic devices.
In another preferred form of this aspect of the invention, the cell housing layer includes a plurality of cell chambers. The cell chambers are preferably circular to maximise the cell chamber volume relative to its surface area, thereby enabling rapid equilibration of cell metabolite(s) within the cell chamber. The cell chambers are preferably arranged in a two dimensional array, with the cell chambers aligned across the cell housing layer and parallel to the first transport layer and/or second transport layer. Once a metabolite gradient has been established over the cell housing layer, each column of cell
chambers which are parallel to the first transport layer and/or second transport layer will have the same metabolite concentration. A metabolite concentration gradient is established across the row of cell chambers which span the cell housing layer, thereby, enabling different cell chamber environments (eg. scaffold architecture or culture medium) to be evaluated against varying metabolite levels. Further, as each chamber cell will have a relatively small metabolite concentration gradient, the migration of cells in response to metabolite concentration changes (eg. cytokines, such as vascular endothelial growth factor) may be conveniently assessed.
The device may further include a probe able to assess the metabolite concentration across the cell housing layer. Preferably the probe includes a spectrometer with a fibre optic component which may extend across the cell housing layer. The probe may be used to assess the metabolite concentration across each column of cell chambers, parallel to the first transport layer. It may also be maintained above the cell chambers to monitor, detect and record any disturbances in metabolite concentration over time.
To increase the metabolite concentration gradient across the cell housing layer, the cell housing layer is preferably contiguous with the cell chambers. The transport of the metabolite(s) across a fluid permeable solid cell housing layer extending across the cell housing layer enables a \/ery steep, yet stable metabolite concentration gradient to be established thereover.
In another aspect of the invention, there is provided a micro fluidic device for cultivating cells including:
a first transport layer for carrying, supplying or removing a flow of fluid containing at least one cell metabolite and a second transport layer for carrying, supplying or removing a flow of fluid containing at least one cell metabolite separated by a cell housing layer, the cell housing layer having at least one cell chamber for cultivating cells, the cell housing layer being permeable to the at least one cell metabolite required by the cell; and
means to measure the physical process parameters of the fluid flowing through the transport layer wherein the dimension across the cell chamber is in the range of 10 μm to 1000 μm, preferably less than 200 μm.
In this aspect of the invention, the cell housing layer may further include at least one channel communicating with the cell chamber. The at least one access channel is preferably capable of further providing a flow of cell metabolites to the cell within the cell chamber. The access channel may further be used to seed one or more cells into the cell chamber.
This aspect of the invention is provided with a second transport layer adjacent the cell housing layer, the second transport layer and the cell chamber being separated by a membrane (cell housing layer) permeable to the at least one cell metabolite within a flow passage within the second transport layer. Preferably the monitored physical process parameters of the fluid flowing in the first and second transport layer are variables selected from the group including pressure, temperature, pH, oxygen concentration and carbon dioxide concentration and other process variables useful for controlling conditions within the cell housing layer. All of the above variables are measurable in real time. The above apparatus may further include a means to control the measured variables preferably in real time.
By measuring the physical process parameters of the fluid flowing in the transport layer and knowing the permeability properties of the cell housing layer separating the fluid flow passage of the transport layer and cell chamber, the flux of cell metabolites into the cell chamber side of the membrane can be determined. Furthermore, due to the physical dimensions of the cell chamber and the positioning of the cells in the cell chamber, flow of fluid in the cell chamber is substantially laminar enabling a more predictable and precise determination of flux of cell metabolites from the membrane surface to the surface of the cells. Thus by controlling the process parameters within the transport passage, the conditions or process variables within the cell microenvironment can be individually controlled. Alternatively process parameters such as temperature can be monitored and/or controlled by one or more heating elements positioned in the transport layer or layers, the cell housing layer or within the permeable membrane.
While laminar conditions are preferred and easiest to monitor, if turbulent flow conditions are to be studied then a turbulent flow regime can be established. Under turbulent flow conditions, the mass transfer relationships are more complex and a greater level of real time monitoring may be required to effect the same level of control of the microenvironment within the cell chamber.
The cell chamber may include an elongated channel. The elongated channel preferably includes a plurality of parallel elongated sections (aligned parallel to the cell housing layer) which travel across the cell housing layer. These elongated channel sections align to specific metabolite concentrations and thus enables the optimum metabolite concentration to be determined over a relatively short distance. The width of the channels is preferably between 10μm to 100μm and more preferably between 20μm and 40μm. The distance between the parallel elongated sections is preferably 10μm to 100μm and more preferably between 20μm and 40μm.
The elongated channel may further include a plurality of parallel elongated sections (aligned perpendicular to the cell housing layer) which travel across the cell housing layer. These elongated channel sections may enable cells to migrate towards an metabolite source, such as oxygen if the cells are capable of aerotaxis. The width of the channels is preferably between 10μm to 100μm and more preferably between 20μm and 40μm. The distance between the parallel elongated sections is preferably 10μm to 100μm and more preferably between 20μm and 40μm.
The arrangement of an array of cell chambers with parallel elongated sections aligned both parallel and perpendicular to the cell housing layer enables the study of the effects of different metabolite concentrations on cell proliferation and migration simultaneously.
In a further aspect of the invention, there is provided a method of controlling process variables within a cell chamber including the steps of providing a device having a permeable cell housing layer between at least one side of the cell chamber, and a flow passage of cell metabolites, providing a flow of at least one cell metabolite through the flow passage to permeate through the permeable cell housing layer into the cell chamber, monitoring the process variables within the flow of cell metabolites in the flow
passage, and/or the cell housing layer and determining the process variables within the cell chamber based on the known characteristics of the permeable cell housing layer and the mass transfer gradients of the nutrients established across the cell housing layer.
The preferred form of the invention requires at least the two sides of the cell housing layer to comprise permeable membranes to separate the cell chambers from the flow passages of nutrients .
The above aspect of the invention further provides the step of locating one or more cells or a tissue culture on a surface or scaffold positioned within the one or more cell chambers. The above method of controlling the conditions within the cell chamber enables the conditions at the surface of the cell to be controlled. The process parameters are preferably monitored in real time and the measured variable controlled by feed back control preferably in real time
In a further aspect of the invention, there is provided a method of assessing the effect of surface conditions on the growth of one or more cells including the steps of introducing or creating in a cell chamber a microenvironment having predetermined environmental conditions at, or in proximity to the surface of one or more cells and monitoring and assessing the effect of the surface condition on the growth of such cells.
The predetermined micro-environmental conditions being assessed may be parameters including the physical characteristics of the surface such as surface chemistry and topography.
In this aspect, the method may further provide for recreating the known environmental conditions in a cell chamber, varying at least one of the environmental conditions in a known manner to monitor and assess the effect of the varying environmental conditions on one or more cells.
The above method may further comprise the step of monitoring the effect of the cell on the condition being monitored.
In a further aspect of the invention, there is provided a method of assessing a scaffold including the steps of introducing a scaffold into the cell chamber of a device for cultivating cells, establishing a predetermined microenvironment with the cell chamber, seeding one or more cells onto the scaffold and monitoring and assessing the effect the scaffold and microenvironment have on the behaviour of the one or more cells.
The features of the scaffold being assessed may be parameters including the physical characteristics of the scaffold such as the nature of the scaffold and properties of the scaffold material, the pore size, pore distribution, pore connectivity and tortuosity.
The method according to this aspect of the invention may further include the step of varying at least one of the microenvironment conditions in a known manner to monitor and access the effect of varying the environmental condition on the one or more cells.
Description of the drawings
Figures 1(a)-1(g) are plan views of the components of an embodiment of the invention with Figures 1(b) and 1(d),
Figure 2 is a sectional view of the embodiment of Figures 1 (a)-1 (g),
Figure 3 is a second embodiment of the invention,
Figure 4 is a sectional view of a third embodiment of the invention showing the control circuit layout,
Figure 5 is a sectional view of the embodiment of Figure 4,
Figure 6 is a fourth embodiment a cell housing layer for incorporation into the invention,
Figures 7 and 8 are further alternative embodiments of fluid transport layers for incorporation into the invention,
Figures 9, 11-15 are plan views of alternative designs of surface and scaffold for incorporation into the cell chamber in the apparatus of the invention, Figure 10 is a plan view of a grid sensor pattern for use with the cell chamber in the apparatus of the invention,
Figure 16 is a plan view of an optional mask which may be applied to the membrane separating the cell housing layer from the first and/or second transport layer/s,
Figures 17, 18 and 19 illustrate possible micro heater configurations which could be used in the present invention,
Figure 20 is a schematic diagram of a device of the present invention,
Figure 21 is a perspective view of the device of Figure 20,
Figure 22 is a graph of the oxygen concentration gradient over the cell housing layer of the device of Figure 21 under the conditions described in Example 1 ,
Figure 23 is a graph of the Fibroblast 3T3 fold expansion under discreet oxygen concentrations as described in Example 1 ,
Figure 24 is a schematic diagram of a microbioreactor of the present invention constructed from an integral piece of PDMS material,
Figure 25 is a schematic diagram of the microreactor of Figure 24 further including a glass plate and polycarbonate shell,
Figure 26 is a graph of the oxygen concentration gradient across the cell housing layer for the three by three well array of Figure 25,
Figure 27 is a graph of the oxygen concentration gradient across the cell housing layer for the two by three well array of Figure 25,
Figure 28 is a graph of the estimated oxygen profile through a microbioreactor of Figure 25 maintained in an anaerobic atmosphere,
Figure 29 is a graph of the cell expansion data for Experiment 1 (Example 2),
Figure 30 is a schematic diagram of an embodiment of the invention used in Experiment 3;
Figure 31 is a schematic diagram of an embodiment of the invention used in Experiment 4;
Figure 32 is a plan view of serpentine channels with imposed oxygen gradient of a microfluidic microbioreactor of the present invention, and
Figure 33 is a schematic diagram of the cell housing layer with a serpentine cell chamber as shown in figure 32;
Figure 34 is a side view of a microbioreactor similar to Figure 32;.
Figure 35 is a photograph showing cell expansion in a serpentine cell chamber between and oxygen gradient of 0% to 20%vol; and
Figure 36 shows the layers of the device used in this experiment 5.
Detailed description of the embodiments
As shown in Figures 1(a)-1(g) and Figure 2 the microbioreactor 1 includes a cell housing layer 2 between a first transport layer 3 and preferably a second transport layer 4. A membrane 7 permeable to at least one cell metabolite is provided between the cell housing layer 2 and the first transport layer 3 and when present a membrane 6 is present between the cells housing layer 2 and the second transport layer 4.
In order for the cell to be supplied with the metabolites, the membrane material is selected and produced at a thickness to allow these metabolites to permeate from the
transport layers 3, 4 into the cell housing layer 2. Furthermore to allow easy analysis of the cell behaviour, it is preferable that the transport layers and the membranes have similar light transmission properties in the wavelengths at which analysis is to take place. For example, if analytical techniques involving light confocal microscopy are used then the transport layer 3, 4 and membrane should be transparent. A suitable membrane material for such an analytical technique is PDMS. Similarly, if light fluorescent microscopy or Micro CT analytical techniques are used then the transport layers and membranes should allow transmission of the relevant wavelengths.
The cell housing layer 2 further includes a cell chamber 5 within which cells introduced via a well or port within the cell chamber (which may contain either a surface or a scaffold) are able to grow. The height of the cell chamber may be altered to closely mimic the in vivo microenvironment and/or enable the control and/or monitoring of the conditions. The cell housing layer may also be provided with an auxiliary passage 8 for the delivery of cell metabolites to the cell and may be used for removal of waste products from the cell. This auxiliary passage is not intended to provide the majority of the cell metabolites to the cell and while laminar conditions are generally maintained through the cell chamber, turbulent conditions can be induced if required.
The cell chamber 5 is shown as having a functional surface 15. The surface 15 may be hyaluronic acid/chitsan layer which has been added or layered on the interior of the cell chamber. The functional surface provides a surface upon which the cells may be seeded and/or cultivated on thus separating them from interacting with the membrane. The functional layer may also serve as a secondary membrane offering increased molecular selectivity over the membrane bordering the cell housing layer 2. The functional surface layer is preferably formed of multiple oriented layers up to 20 μm thick. For example, a hyaluronic acid/acid layer of 20 μm would consist of approximately 130 layers. Alternatively the layered functional surface is made up of alternating polycationic substance and polyanionic substance layers.
The first transport layer 3 is provided with a passage 9 through which fluid is able to pass. The passage is preferably open adjacent to the cell chamber and separated from the cell chamber by the permeable membrane. In this way, mass transfer gradients
established between the first transport layer 3, second transport layer 4 and the cell chamber 5 enable material to transfer from the transport layer or layers to the cell chamber 5. Furthermore, the flux of material transferring across the permeable membrane can be accurately determined.
The passage 9 through the first transport layer 3 and corresponding passage 10 in the second transport layer 4 is provided with inlets 11 , 13 and outlets 12, 14 respectively, to enable the flow of fluid containing cell metabolites through the passages 9, 10.
The positioning of the inlet and outlet into the fluid passages 9 and 10 will depend on the proximity of other equipment to the apparatus. For example, in one embodiment a plurality of microbioreactors of the present invention may be stacked into a multi reactor column. In this embodiment, the respective inlets 11 1 , 13 1 and outlets 12 1 , 14 1 are in through the side of the transport layers as shown in Figure 3.
Alternatively, in a further embodiment a number of microbioreactors may be placed side by side, an inlet 11 , 13" and outlet 12, 14" arrangement shown in Figure 5 could be used optional with multiple reactors 20, 21 with inlets 22 and outlets 23 on a single cell housing layer 24 (Figure 6).
From the known response of variables to the mass transfer gradients and the properties of the permeable membrane, the flux of material transported from the transport layers across the membrane interface to the growth cell layer can be reliably predicted and determined. Due to the laminar flow conditions within the cell chamber, the transfer of these materials from the membrane interface to the surface of the cell is a linear distance related relationship. Therefore the concentration of cell metabolites at the cell surface can be reliably predicted, determined and included within associated process control methodologies. Other variables such as temperature may be more accurately measured directly in order to define the environmental state within the cell.
The process variables in the first transport layer are monitored to establish the nature of the mass transfer gradients from the transport layers 3, 4 to the cell chamber 5. These variables may be selected from the group of variables including pressure within the fluid
transport passage 9, 10, the pH, oxygen and carbon dioxide concentration of the fluid as well as the concentration of other cell metabolites entering and leaving the entrance 11 , 13 and exit 12, 14 ports of the transport layers 3, 4. Temperature may also be measured in the transport layer although as mentioned above, it is preferable to measure temperature of the cell housing layer directly. These conditions are monitored during operation of the device of the invention. Additionally the process variables of any fluid entering within, and leaving the auxiliary passage is monitored to enable a mass balance to be established around the entire vessel and thereby provide information on the cell consumption of cell metabolites and also the effect in which predetermined changes in the microenvironment have on the cells within the cell housing layer.
These monitoring systems further enable levels of material entering the system to be set or varied in a known manner over a predetermined period of time. Once these material levels have been varied in a known manner, the effect of these and other variables on the cells and the response of the cell can be monitored.
In order to effect real time measurement and control of process parameters, a more complex integrated measurement and control apparatus as shown in Figure 4 can be used. By using direct measurement of process parameters such as O 2 , CO 2 , glucose, temperature etc, the concentration within the cell chamber can be directly measured with micro fluidic optic fibre sensors using fluorescent lead technology at multiple sensor locations. The sensors may be arranged as a grid of sensors (Figure 10) providing local measurements on a range of process variables over the surface of the cell chamber or membrane. The microenvironment within cell chambers can be controlled directly through a central environmental controller controlling the flow of fluid media into the transport layer. For convenience only one master controller 20 is shown controlling the flow of media through inlet 13". In response to measurements taken at positions 21, 22, 23 and 24 within the cell chamber 5, a slave controller feeds information back to a master controller which compares the readings against set points for the variables being measured. Variations from the set points results in changes to the flow rates through the inlet. Changes in the flow rate into inlet 13 11 are then fed back to the slave controller which resets the set points until the next measurement cycle.
Figures 17, 18 and 19 show possible micro heater configurations which could be used in the present invention. In the embodiment of Figure 17, the micro heater 51 is located in the cell housing layer 50 surrounding the cell chamber. In Figure 17, a two chambered layer is shown.
In the embodiment of Figure 18, the micro heater 53 is located in the membrane layer above and/or below the cell chamber and in Figure 19, micro heaters 54, 55 are positioned in the transport layers 56 and 57 respectively.
In order to monitor and assess the effect of temperature on the cells, a temperature sensor or sensors may be incorporated into the control apparatus. The feedback from the control sensor may be used to directly control heating elements located in close proximity to or embedded in the transport layer or layers, membrane or cell chamber layer. This enables localised heating to be provided to the cell chamber. The heating elements may be any available micro heater design.
By being able to monitor the concentration of materials and process variables in the microenvironment of the cell(s), the effect of variations in environmental conditions on the cells can be determined for different cell types. Furthermore the microenvironment around the cell can be reliably reproduced.
During operation of the microbioreactor of the present invention, cells are introduced via a port (not shown) on to a surface or into a well positioned within the cell chamber. The conditions for cultivation in the cell are set by a combination of establishing the required variables in the fluid flowing through the transport layers and the auxiliary passage of the cell housing layer as well as other surface related conditions on which the cells exist. Any one of a number of conditions may then be varied in a known manner to monitor the effect of the variable change on the condition of the cells.
For example, a method of determining the effect of one or more microenvironmental conditions on the cell can be performed. In this example the cell chamber includes highly programmable pores emanating from the central or off centre where cells are introduced. Properties or conditions of the pores are known and their effect on the cell
can be observed. As the other microenvironmental conditions of the cell are controlled and known, the effect of changes to the properties or conditions of the pores is directly observable. The properties or conditions of pores which are variable and hence can be found to have an observable effect are the size, length, shape, tortuosity, surface biochemistry and topology and pore interconnectivity. The pore sizes and characteristics may be representative of structures found in nature or those in manufactured rigid and hydrogel based scaffolds. The characteristics and features of surface biochemistry include the inclusion of growth factors, growth factor complexes, peptidomimetics, oligopeptides, protein segments, proteins (glycoproteins etc), glycosamines (e.g. GAG, HA), carbohydrates or mixtures thereof in constant concentrations or concentration gradients on the surface via various surface treatment techniques such as wet chemistry, block copolymer templating, plasma templating, and plasma polymerisation. These surface treatment techniques may be applied to create surface structures with controlled functionality and topology at defined locations throughout the highly defined pores.
In other methods of using the microbioreactor of the present invention, a number of variations and configurations of the cell chamber can be established within a single microbioreactor in order to observe the actual effect of selected surfaces and/or scaffold materials on the cells. In such a method, for example, a radial disc (Figure 9) of selected surfaces and/or scaffold materials 31 around the disc with the cells initially centrally located can be used to observe the preference of certain types of cell behaviour to different surface or scaffold characteristics. The differing selected surfaces can be arranged as sectors of a radial disc or may be arranged as concentric layers and these differing selected surfaces may incorporate different growth factor complexes and/or extracellular matrix molecules with specific distributions at specific locations. In this example, the cells would be inoculated into the centre of the scaffold but they will be distributed at specific locations throughout the scaffold to ascertain the most effective inoculation technique for functional tissue growth.
In the embodiments shown in Figures 11-15, different scaffold architectures can be designed to provide the effect of different conditions of surface chemistry and pore size distribution and tortuosity (Figures 11 , 12, 13, 15).
Alternatively, where a soft scaffold is being investigated such as different types of hydrogel, the cell chamber can be filled with the scaffold (Figure 14) and then seeded with cells. The growth of the cell and the effect of various parameters as the cell creates pores in the hydrogel can be monitored and the suitability of such a scaffold assessed.
Another method of providing or designing microenvironments within the cell chamber is to use baffles, tortuous pathways or structures within the first and second transport layers. An example of two possible arrangements is shown in Figures 7 and 8 although a variety of arrangements could be conceived depending on the microenvironment and concentrating gradients to be constructed within the cell chamber. In the flow path of Figure 7, the concentration of cell metabolites closest to the inlet 33 would be at its greatest. Hence by providing a larger pathway through the transport layer to outlet 34, a greater lateral concentration gradient of cell metabolites in the cell chamber can be created.
In the flow path shown in Figure 8, the fluid flow spirals from inlet 35 in towards a centre outlet 36. Such a flow construction would establish a concentration gradient of metabolites which is greater at the outer regions reducing into the centre of the cell chamber.
In order to remove cells from the microbioreactor without disassembly of the device, the cells in the cell chamber 5 are dosed with a digestive proteinase such as trypsin through the inlet access port of the cell chamber to detach the cells from the scaffold. This allows the cells to be removed through the access passage or other port designed in the cell housing layer for that purpose.
In a further method of designing microenvironments within the cell chamber, an optional masking layer 40 adjacent or on the membrane layer is provided. The masking layer effectively allows for the passage of cell metabolites through a predetermined region of the membrane. An example of such a masking layer 40 is shown in figure 16 where only a thin annulus 41 of membrane is available for the transfer of cell metabolites into the cell chamber. The masking layer 40 may be applied directly to the membrane layer 40 or provided on a separate membrane layer having the same or different permeability
properties to the first membrane layer. It would be appreciated that the masking layer could be shaped and sized so that only a point source is available for the transfer of cell metabolites into the cell chamber.
The microbioreactor of the present invention may be manufactured using microfabrication technology which is know to those in the art and preferably would be sterilisable, optically clear and include sensors for environmental monitoring and hence control of the process variables. The microbioreactor of the present invention can be readily assembled and disassembled, for post hoc histological tissue processing. Any surface or scaffold material can be assessed in the cell chamber of the present invention as described above.
The microbioreactors of the present invention allow for systematic investigations into the effects of surface and scaffold structure and function on cell culture and tissue growth and allow for the development of reliable wet control manufacturing techniques for programmed microbioreactors.
The relevance of oxygen concentration in the study of disease, embryonic development, aging, wound repair and tissue engineering is becoming increasingly obvious. While conventional tissue culture is generally performed at atmospheric oxygen concentration (20%), physiological oxygen concentrations range from 0-5. These two facts require the development of culture/cell devices which allow cell cultures to be observed and manipulated over this range and further discrete desired concentrations anywhere from 0-21%. However, due to the challenge of establishing and maintaining a range of oxygen environments, conventional devices are limited in that they rely on models to predict local oxygen concentrations and do not allow for the real time assessment of the actual cell culture environment.
Example 1
The generation of an array of oxygen concentrations within a single cell culture device for the purpose of research diagnostics.
Microbioreactor design
This example relates to a device (microbioreactor) of the present invention designed to provide a tool to study the response of mammalian cells to various oxygen concentrations in otherwise identical culture conditions, including maintenance of constant CO 2 concentrations.
To study the effects of oxygen concentration on cell behaviour in real time, the microbioreactor is:
(i) able to allow for individual cultures at a range of oxygen tensions;
(ii) optically clear for real time assessment;
(iii) able to maintain uniform culture conditions, and
(iv) able to have the ability to measure the local oxygen concentration at any time throughout the experiment.
The device generates and maintains an oxygen gradient over an array of discreet tissue culture micro wells. An oxygen gradient is maintained in the gas phase of the device by flowing gases having differing oxygen composition through the ports at opposing ends of the device. Figure 20 illustrates how this diffusion gradient is maintained within the device while Figure 21 provides a perspective drawing of the actual device.
Gas functioning either as an oxygen source 60 (represented by shaded arrows) or oxygen sink 62 (represented by unshaded arrows) diffuses through a manifold 64, containing a microporous membrane intended to minimize convection, into the cell chamber. The oxygen gradient achieves rapid equilibrium within the gas layer of a culture/cell chamber. The actual oxygen content within the gas phase can be measured above any row of wells (cell chambers) 66 during operation without disturbing the gradient through use of the oxygen probe 68. The culture medium contained within the micro wells equilibrates rapidly due to the small volume relative to surface area.
An alternative approach to studying the response of mammalian cells to various oxygen concentrations would be to generate a gradient by diffusion in a liquid phase. This is not feasible on a reasonable time scale, thus devices which do use a liquid phase gradient rely on convection. In a convective system measurements of the local oxygen concentration are more challenging making control of the gradient limited and often the measurement process disrupts the gradient.
The ability to assess the oxygen concentration in the gas phase at any time over the length of the gradient, as well as data log the gradient throughout the culture, is unique to this reactor and is necessary to enable representative cbmparison of results obtained in different laboratories. Figures 20 and 21 show that the oxygen concentration in the gas phase can be measured at any time along the length of the chamber using a fiber optic oxygen probe 68 which slides freely in the axial direction. The semi rigid aluminium casing on the probe keeps it at a constant vertical position above the wells and also keeps it from contacting the culture medium. The probe 68 accesses the culture environment though a narrow stainless steel tube on the same end in which the gas providing the oxygen source is fed. This placement mitigates any concern over the quality of the gas seal at the probe access point.
When a gradient is established the precise oxygen concentration over each row of wells 66 is measured and recorded. Having established the culture conditions throughout the device, the probe 68 is locked in place over a central well and data logged to detect any disturbances in the gradient over the culture period.
The aim of the culture well design of this embodiment is to maximize the medium surface area to volume ratio for rapid equilibrium with the gas phase and to minimize medium depth such that oxygen tension at the cell layer most closely reflects the concentration in the gas phase. This aim was achieved by fabricating the base of the wells from glass and the walls from polydimethylsiloxane (PDMS) (Sylgard 184, Dow Corning Corporation, Ml, USA). The glass base provides a suitable substrate for cell adhesion, while the PDMS repels culture medium, resulting in an inverted drop geometry which maximizes the surface area to volume ratio. Well dimensions are 4 mm diameter by 2 mm deep. This diameter was selected as the entire well could be
observed in the 4x field of view on a light microscope. The well array occupies an area 5 cm by 2 cm while the total chamber length is 6 cm. The head space in the chamber above the wells is 1.5 mm, allowing for gas diffusion and space for the oxygen probe.
The glass plate 70 on which the wells are situated can simply be lifted in and out of the polycarbonate shell 72 in which the oxygen gradient is maintained. This provides flexibility in that multiple cultures can be initiated in separate well arrays and transferred into the gradient device for evaluation under different gradient conditions, thus facilitating the rapid analysis of many cultures.
Sterility of the wells and polycarbonate shell can be achieved through autoclaving as all materials are heat stable. Gas enters the device through stainless steel tubes which feed into ports 74 and 76 shown in Figure 21. Before entering the gradient chamber, the gases pass through 0.22 μm polycarbonate membranes (Polycarbonate lsopore membrane filters, 0.22 μm, Millipore, Australia) situated under the gas manifolds 64.
The filter/manifold design ensures sterility while minimizing convective mass transport within the chamber.
The gas which provides either the oxygen source or sink preferably contains a constant concentration of 5% carbon dioxide such that a CO2/PH gradient is not formed along the length of the device. The flow rate of pressurized gas mixes into the device are measured using conventional rotameters and controlled manually through needle valves. Prior to entering the device, gasses were humidified by bubbling through water in order to mitigate evaporative effects on the cultures.
Experimental design
It is known from the literature that mammalian cells generally suffer hypoxia at oxygen concentrations less than 1.5%, 1% being mild hypoxia, 0.05% moderate hypoxia, ≤0.02% as extreme hypoxia, and anoxia at 0%. In this example, the device is used to study cell expansion under oxygen concentrations spanning from 0-2 %, which includes both the hypoxic and anoxic regions. This range was further subdivided into two separate experiment defined as Culture A (0-1% oxygen) and Culture B (1-2% oxygen).
The required flow rates of gases were first determined. The source gas for both experimental conditions was composed of 3% O 2 , 5% CO 2 in N 2 , while sink gas was 5% CO 2 in N 2 . The gradients generated within the chamber and the specific oxygen concentration over each well is shown in Figure 22. The sink gas flow rate for both cultures was maintained at 10 ml/minute, while the source gas flow rate was set at 4 and 10 ml/minute for cultures A and B respectively.
The cells selected for this example were NIH mouse fibroblast 3T3's. Culture slides containing the well array were sterilized and seeded with 20 μl droplets containing 1000 cells. Culture medium was DMEM (GIBCO, Grand Island, NY) supplemented with 10% FBS (Serum Supreme, BioWhittaker, Walkersville, MD). Cells cultures were established in a standard 5% CO 2 incubator prior to being subjected to an oxygen gradient. Photos were taken at time zero to demonstrate that all wells contained similar numbers of cells and to determine the density of the starting cell population.
The doubling time of the NIH 3T3 fibroblast is roughly 22 hours and thus this time was selected as the duration for which the culture would be subjected to the oxygen gradient. After being cultured under the imposed gradient conditions of Culture A & B, cell numbers were determined by counting those cells attached in 16 randomly selected 1 mm 2 regions. Cell viability was assessed through evaluation of morphological features and through the use of live/dead stain Trypan blue.
Cell numbers at time zero and at 22 hours were determined by the manual counting of cells attached in randomly selected regions. Fold expansion was determined by dividing the number of cells per unit area at time 22 hours by those at time 0 hours.
Figure 23 shows that the full expansion potential of NIH 3T3 fibroblasts is achieved at oxygen concentrations greater than 1%. While less than 1% oxygen inhibits normal rates of expansion, loss of adherent cell populations does not occur until concentrations of less than 0.5% oxygen (less than 1-fold expansion or maintenance of the population). Generally these cells, which are no longer adhering to the substrate, have compromised viability, however in this example the viability of any floating cells was not assessed.
Example 2
For the purposes of studying cell migration a series of devices, within the scope of the present invention, were constructed. The oxygen gradient was maintained though a PDMS membrane rather than through the gas phase as demonstrated in Example 1. In this example the oxygen gradient is maintained in the gas phase which limits the maximum slope of the gradient. Utilising a gas permeable solid, such as PDMS, a very steep yet stable oxygen gradient is maintained over the cell housing layer.
Microbioreactor design
Figures 24 and 25 illustrate one of the designs which we have utilised to study cell migration. In this embodiment, the device contains a three by three array 80 and a two by three 82 array of culture wells. The chambers 84 flanking the well array function as either an oxygen sink or as an oxygen source. The oxygen concentration in each well can be estimated through a steady state model as shown in Figure 26 for the three by three well array and in Figure 27 for the two by three well array. In this model the source gas contains 3% oxygen and the sink gas contains 0% oxygen. Both source and sink gas contain 5% CO 2 thus eliminating a CO 2 /pH gradient. Figures 24 and 25 show the microbioreactor in an inverted position. In this position the culture wells are loaded. Once loaded with cells and medium the glass plate is placed over the wells and the entire device is inverted such that the cells settle and attach on the glass.
Experiment 1
The work described in this experiment utilized a microbioreactor with the basic design described in the previous section and shown in Figures 24 and 25. To ensure that there was no ingress of the outside atmosphere, the entire device was enclosed in a vessel having a 95% N 2 /5% CO 2 atmosphere. NIH 3T3 fibroblasts were subjected to gradient conditions for 24 hours, where the source gas contained 3% oxygen and the sink gas contained 0% oxygen. The model provided in Figure 24 cannot be applied to this modified device, with the introduction of an anaerobic atmosphere resulting in the far
steeper gradient profile(Figure 28). . In this experiment only data from rows 1, 3 and 5 is available.
Cell culture medium is DMEM (GIBCO, Grand Island, NY) supplemented with 10% FBS
(Serum Supreme, BioWhittaker, Walkersville, MD). NIH 3T3 fibroblasts suspended at 10,000 cells per ml in culture medium are introduced into individual culture wells of the device shown in Figure 1. Cultures are stabilized for 24 hours in a 20% O 2 , 5% CO 2 atmosphere. A gradient is imposed across the device for a 24 hour period by a source gas containing 3% O 2 , 5% CO 2 and sink gas containing 0% O 2 , 5% CO 2 in nitrogen.
Pictures of the cells in each well were taken before and after the introduction of the gradient. Cells numbers in each well were counted from the photo images and these numbers used to determine the rate of cell expansion in each well.
Cell expansion data is provided in Table 1 and graphically in Figure 29. The cell expansion data shown demonstrates that NIH 3T3 fibroblasts proliferate in oxygen environments greater than 1%, while cell death occurs in environment having less than 0.5% oxygen (Note that fold expansion (FE) = 1 implies constant cell numbers; FE > 1 implies an increase in cell number; FE < 1 implies cell death). It is likely that no migration is observed in this experiment due to the fact that the gradient was sustained for only 24 hours. In the following experiment an oxygen gradient is sustained for 72 hours.
Table 1. Cell expansion data for Experiment 1.
Experiment 2
The aim of experiment 2 is to investigate the potential for NIH Fibroblast 3T3s to migrate towards an oxygen source a gradient was imposed across the culture wells in a device similar to that shown in Figure 25. In this experiment, NIH fibroblast 3T3 cultures were given 24 hours in a standard incubator (20% O2, 5% CO 2 ) to stabilize. A gradient was imposed on the culture over a 72 hour period ranging from 0.6% to 1.2% oxygen across the wells.
Cell culture medium is DMEM (GIBCO 1 Grand Island, NY) supplemented with 10% FBS (Serum Supreme, BioWhittaker, Walkersville, MD). NIH 3T3 fibroblasts suspended at 20,000 cells per ml in culture medium were loaded into individual culture wells 86. Cultures were stabilized for 24 hours in a 20% O 2 , 5% CO 2 atmosphere. A gradient was imposed across the device for a 72 hour period using a source gas containing 5% O 2 , 5% CO 2 and sink gas containing 0% O 2 , 5% CO 2 in nitrogen. Pictures of the cells in each well were taken before and after subjecting the cultures to the gradient.
From comparing the cell distribution at time zero and at 72 hours, it was seen that the 3T3's do migrate from a region of hypoxia towards an oxygen source. Migration is limited by a confluent layer of 3T3s which appear to remain stationary and not to migrate further towards the oxygen source. This results in the maximal density of cells at roughly 0.8% oxygen. Cells density in this region is so great that a bilayer begins to form. However, contact inhibition prevents further migration or proliferation at this point This result is in agreement with our previous observations presented in Experiment 1 and Example 1.
In this model the hypoxic environment is one that contains less than 1% oxygen. A 0.5% oxygen environment appears sufficient for cell survival and 1% for proliferation (see Example 1). It is likely that the aerotaxis phenomenon is terminated in environments having oxygen levels sufficient for normal cell function (or at roughly ~1%). This proposition is supported by the observation that the cell migration front appears to be terminated at roughly 0.8% oxygen.
Experiment 3
Driving the gradient generation through use of an anaerobic sachet (as is done when performing anaerobic microbiology culture systems) simplifies use of the oxygen gradient device. A glass Petri dish was modified by drilling a hole through the base of the dish providing access to the outside atmosphere. This hole is sealed by a channel which extends parallel to the well array functioning to provide oxygen equally to each column of cell chambers. This is shown in figure 30.
Experiment 4
A model was generated for cell migration towards a point source. In order to generate data to substantiate the model, a simple point source culture chamber was developed
(not shown). This chamber included 5 tissue culture treated polystyrene wells, each with a 300 μm hole in the centre. On the bottom of the well array, there was a 100 μm
PDMS membrane which provides both gas exchange and a barrier from foreign pathogens. The headspace above the wells was sealed off with the exception of an inlet and exit feed for a controlled atmosphere. When the headspace in the vessel was replaced with an atmosphere containing 5% CO2 in nitrogen, the single hole in the middle of each well became the only oxygen source (point source).
Cell culture medium is DMEM (GIBCO, Grand Island, NY) supplemented with 10% FBS (Serum Supreme, BioWhittaker, Walkersville, MD). In this experiment A7r5 cells were utilized (A7r5 is a smooth muscle cell line derived from rat aorta) (Kimes and Brandt 1976). Each well is equivalent to that in a 24 well plate. Cells were seeded at 20,000 cells per ml and cultures permitted to establish for 24 hours in a standard 20% O 2 , 5% CO 2 incubator. Each well contained 0.5 ml medium. For the next 48 hours the headspace in the chamber was exchanged with humidified 5% CO 2 in nitrogen at a flow rate of 2-3 ml/minute. Pictures were taken at 24 and 48 hours.
From the photographs, (not shown), it was clear that there was a tendency in all cases for greater cell densities near the oxygen point source, and that this tendency increases with time. In all 5 cases it is clearly seen that cell density in the area immediately
around the point source is confluent while the areas away from the point source are not. In order to determine if migration or proliferation is the cause of the density distribution, a device as shown in figure 33 can be used in conjunction with the use of a fluorescent cell tracker and time-lapse microscopy is proposed.
The modified point source design shown in Figure 31 is equivalent to the device used in Experiment 4 above. Essentially the membrane filter is replaced by a PDMS membrane in Figure 31. With this modification (Figure 31), it is possible to reduce the thickness of the cell layer allowing greater control of the oxygen environment by rapid purging and the elimination of convection.
Experiment s
Figure 36 shows the layers of a three layer device to explore NIH 3T3 fibroblast morphology used in this experiment. The figure shows the layers of the device. From left to right the layers are: (1) glass to isolate from external atmosphere, (2) fluid layer containing specifically conditioned fluid through the left and right chamber, (3) a polydimethylsiloxane (PDMS) gas permeable membrane, (4) cell culture array or cell layer, (5) a cuprophan or nutrient permeable membrane, (6) a medium layer, and (7) glass to isolate the external environment.
NIH 3T3 fibroblasts were subjected to the following environments.
(a) Gel scaffolds comprising all collagen diluted in DMEM supplemented with 10% foetal calf serum (FBS) and penicillin and streptomycin
• 2 mg/ml collagen I
• 1 mg/ml collagen I
(b) Surfaces (all protein ligands diluted in DMEM supplemented with 10% foetal calf serum (FBS) and penicillin and streptomycin)
• Collagen I (133 μg/ml)
• Fibronectin (10 μg/ml)
• FBS (20%)
The behaviour of the fibroblast were contrasted between 20% and 5% oxygen environments.
The NIH 3T3 fibroblasts were cultured for 48 hours in the device of figure 36 at the oxygen levels defined above and on the above specified gel scaffolds or surfaces. Cells were loaded into cell layer at 10 5 cells/ml.
Cells in the collagen I gels remained rounded and bunched at 20% oxygen. At 5% oxygen they appeared to have either found there way to the membrane which enabled better spreading or perhaps were more viable. Typically a fibroblast would be able to spread on a 2mg/ml collagen gel. Throughout the study, it appears that cells maintained at 5% oxygen were better able to spread, a characteristic that correlates with viability.
Cells on the surface do not spread well and nor do the cells with the fibronectin surface coating. Only the surface of 20% serum demonstrate good spreading. Likely the Cuprophan membrane does not bind some ligands as well as others. Again, we saw that the cells spread more in the 5% oxygen environment than in the 20% oxygen environment. Likely this again is a reflection of greater viability in response to high levels of oxygen. This is also observed in other microbioreactor systems according to the invention which deliver oxygen to within close proximity (hundreds of microns) of the cell microenvironment.
Bioreactor design utilising microfluidics
The design (Figures 32, 33 and 34) incorporates an array of micro channels formed using PDMS fused onto a glass substrate. The serpentine channels double back on themselves first in an arrangement parallel to the gas membrane (cell housing layer) where the gradient is maintained and then perpendicularly. The serpentine channels have an oxygen gradient superimposed above the cell chamber (layer 2), as oxygen diffuses between the first and second transport layers (layer 3 and 1). The cell housing
layer forms a contiguous and permeable connection between the first and second transport layers and the cell chamber to enable a stable oxygen gradient to be imposed across the cell chamber.
The aerobic transport layer (layer 3) and anaerobic layer (layer 1) are preferably supplied an aerobic and anaerobic gas supply respectively, which contains 5% carbon dioxide to maintain the pH level of the system.
An enlarged view of the channels parallel to the cell housing layer is shown in Figure 32 and 33. As the channel loops back through the cell housing layer many oxygen concentration may be tested simultaneously. Ideally the cell housing layer thickness is minimal as this facilitates rapid equilibrium. With soft lithography it is possible to fabricate channels 25 μm in width and 25 μm spacing between channels. Using this thickness and spacing it is possible to place 20 channels beneath a 1 mm thick membrane. Such an arrangement allows for one channel for each percent oxygen concentration from 20% to 1 %. One of the other strengths of this design is that controls are maintained in both the source gas (first transport layer) and the sink gas (second transport layer).
In the embodiment illustrated in Figure 34, the cell chamber (cell layer) is confined to the region between the first and second transport layers, in which a metabolite concentration gradient exists. However, as illustrated in Figure 32 and 33, the cell chambers may extend into each of the transport layers. This configuration provides control environments in each of the fully defined atmospheres.
The array of channels running perpendicular to the membrane is used to study cell migration. Their orientation allows cells to migrate towards an oxygen source if the test cells are capable of aerotaxis. The strength in this design is that the channel length spans a very short distance resulting in a steep concentration gradient. This design is integrated with a temperature control mechanism allowing for real-time microscopy of migrating cells. The length scale of the device enables the visualisation of many channels within the same field of view on a microscope.
Figure 35 is a diagram of cell expansion in a channel network shown in figure 32 with the aerobic conditions represented as 20% oxygen on the right of the diagram and 0% oxygen or anaerobic conditions on the left of the diagram.
It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the invention.
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