Patent Publication Number: US-2013230911-A1

Title: Porous structure with independently controlled surface patterns

Description:
CROSS-REFERENCE TO RELATED PATENT APPLICATIONS 
     This application claims priority to Provisional U.S. Patent Application 61/606,087, filed Mar. 2, 2012, which is incorporated herein by reference in its entirety. 
    
    
     BACKGROUND OF THE DISCLOSURE 
     Reabsorptive transport in vivo occurs through natural barriers, formed by a single layer of polarized epithelial cells supported by a basement membrane (BM) which governs the transport. Solutes and molecules cross the epithelial barrier by transcellular or paracellular pathways to the interstitial space and surrounding blood vessels, resulting in reabsorption of essential water and solutes. Common examples of reabsorptive or absorptive barriers in the body include those of the respiratory, gastrointestinal, and urinary tracts. Fluid and solute transport across these barriers make them particularly susceptible to injury by circulating toxins, pathogenic antibodies or certain drugs. 
     SUMMARY OF THE DISCLOSURE 
     According to one aspect of the disclosure, a cell culture support device includes a first and second polymer layer, each with a flow chamber defined therethrough. Additionally, the cell culture support device includes a surface (also referred to as a cross channel interface) between the first polymer layer and the second polymer layer. The surface separates the first flow chamber from the second flow chamber. The surface includes a plurality of pores configured to allow communication and transport between the flow chambers. Furthermore, the surface includes a pattern independent of the geometry of the plurality of pores. 
     In some implementations, the surface is a membrane and the flow chambers are configured to be cellular chambers. In some implementations, the top layer of the device is configured to allow imaging of the surface in some implementations. 
     In certain implementations, the pattern is a topographic pattern and/or a chemical pattern. The pattern can be selected to enhance the growth of specific cell types. In some implementations, more than one pattern is formed on the face of the surface and/or flow chambers. 
     In other implementations, the pores are selected to produce a specific type of interaction between the flow chambers. In some implementations, the size of the pores is selected to prevent cell migration between the flow chambers but to allow cell nutrients and cell signaling analytes to migrate between the flow chambers. The size of the pores is between about 3 μm and about 15 μm in some implementations. In other implementations, the pattern and/or the geometry of the pores is selected to elicit a particular arrangement, function, shape, or density of cellular growth. 
     In yet other implementations, at least one of the polymer layers and/or the surface includes a biodegradable polymer. In some implementations, the pattern is selected to influence a degradation rate of the surface and/or to facilitate cellular attachment to particular locations within the cell culture support device. 
     According to another aspect of the disclosure, a method for fabricating a cell culture support device includes forming a first flow chamber in a first polymer layer, and forming a second flow chamber in a second polymer layer. Additionally, pores of a specific size are formed through a surface. A selected pattern is then applied to the surface. The selection of the pattern is independent from the selection of the pore size and position. The forming is done such that the formation of the pattern on the surface preserves the plurality of pores. Additionally, the method includes coupling the first polymer layer and the second polymer layer such that the surface separates the first flow chamber from the second flow chamber. 
     In some implementations, cells are seeded into at least one of the flow chambers. In certain implementations, the pores are formed such that they have a specific pore density along the surface. In other implementations, the pattern is a topographic pattern and/or a chemical pattern selected responsive to the type of cells to be grown on the surface. 
     In some implementations, the method also includes selecting and forming additional patterns onto the surface and or walls of the flow chamber. The additional patterns can be the same as, or different than, the initially selected pattern. In some implementations, the patterns are selected to elicit a particular arrangement, function, shape, or density of cells grown on the surface. 
     In yet other implementations, the polymer layers and/or surface include a biodegradable polymer. The pattern is selected to influence a degradation rate of the surface in some implementations. 
     According to yet another aspect of the disclosure, a cell culture support system includes a first and second polymer layer each with flow chambers defined therethrough and a surface separating the flow chamber of the first polymer layer from the flow chamber of the second polymer layer. The surface includes a plurality of pores configured to allow communication and transport between the flow chambers. Additionally, at least one face of the surface is patterned. The pattern is independent of the geometry of the pores and preserves the size of the pores when formed. The system also includes an imager configured to image a face of the surface. 
     In certain implementations, the system includes a means for coupling the surface between the first and second polymer layers, a flow meter configured to measure flow through at least one of the flow chambers, a pressure sensor configured to measure the pressure at an inlet and/or an outlet of the flow chambers, a fluid pump configured to flow fluid through the flow chambers, and macro-molecule injector coupled to an inlet of the flow chambers. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       The skilled artisan will understand that the figures, described herein, are for illustration purposes only. It is to be understood that in some instances various aspects of the described implementations may be shown exaggerated or enlarged to facilitate an understanding of the described implementations. In the drawings, like reference characters generally refer to like features, functionally similar and/or structurally similar elements throughout the various drawings. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating the principles of the teachings. The drawings are not intended to limit the scope of the present teachings in any way. The system and method may be better understood from the following illustrative description with reference to the following drawings in which: 
         FIG. 1  is a block diagram of an example system in which a cell culture support device is employed. 
         FIG. 2  is a solid model of an example cell culture support device, as can be employed in the system of  FIG. 1 . 
         FIG. 3  is a cross sectional view of an example cell culture support device, in which the flow chambers are seeded with cells. 
         FIGS. 4A-4C  are a series of solid models illustrating example cross channel interface pore topographies. 
         FIG. 5  is a flow chart of an example method for manufacturing a cell culture support device similar to the device of  FIG. 2 . 
         FIG. 6  is a flow chart of an example method for using a cell culture support device in a system similar to the system of  FIG. 1 . 
         FIG. 7A  is a cross sectional schematic of an example cell culture support device. 
         FIG. 7B  is an image of a cell culture support device manufactured based on the schematic shown in  FIG. 7A . 
         FIGS. 7C-7E  are a series of scanning electron micrographs, at various magnifications, of the cell culture support device shown in  FIG. 7B . 
         FIGS. 8A-8E  are a series of scanning electron micrographs showing topographies of various example cross channel interfaces. 
         FIG. 9A  is a plot illustrating the relationship between the change in pore diameter and hot-embossing dwell time. 
         FIG. 9B  is a plot illustrating how pore diameter is affected by cross channel interface topography when hot embossed. 
         FIG. 9C  is a series of scanning electron micrographs of example cross channel interfaces with various topographies. 
         FIG. 9D  is a plot illustrating how pore elongation is affected by cross channel interface topography and pore diameter. 
         FIG. 10A  is a brightfield microscopy image of a cell culture support device&#39;s flow chamber seeded with HK-2 cells. 
         FIG. 10B  is a brightfield microscopy image of the same flow chamber show in  FIG. 10A , viewed under higher magnification. 
         FIG. 10C  is a series of confocal microscopy images of the cells seeded in the flow chamber shown in  FIG. 10A . 
         FIG. 10D  is a brightfield microscopy image of a cell culture support device&#39;s flow chamber seeded with primary renal proximal tubule epithelial cells. 
         FIG. 10E  is a brightfield microscopy image of the same flow chamber shown in  FIG. 10D  viewed under higher magnification. 
         FIG. 10F  is a series of confocal microscopy images of the cells seeded in the flow chamber shown in  FIG. 10D . 
         FIG. 11A  is a scanning electron micrograph of an example cross channel interface prior to cellular seeding. 
         FIG. 11B  is a scanning electron micrograph of an example cross channel interface after a cellular mat has formed on the cross channel interface. 
     
    
    
     DETAILED DESCRIPTION 
     The various concepts introduced above and discussed in greater detail below may be implemented in any of numerous ways, as the described concepts are not limited to any particular manner of implementation. Examples of specific implementations and applications are provided primarily for illustrative purposes. 
     Models of absorptive barriers in the respiratory, gastrointestinal, and urinary tracts would offer a platform to better understand the biology and function of reabsorptive barriers, to interrogate underlying disease mechanisms affecting those barriers, and to provide rapid screening of drugs for toxic effects to and excretion by organs containing those barriers. In particular, since the kidney is susceptible to drug toxicity and governs excretion of drugs, its renal epithelial structures provide valuable test cases for in vitro models of reabsorptive barriers. Disclosed herein is a systems and methods for the manufacture and use of such barriers in vitro. In some implementations, the systems and methods disclosed herein are used as a medical device to assist organ function. 
       FIG. 1  illustrates a cell culture support system  100 . The system  100  includes at least one cell culture support device  101 . The system  100  also includes at least one pump  103  that pumps fluid from a first fluid reservoir  102  into an inlet of the cell culture support device  101 . As the fluid passes from the pump  103  to the cell culture support device  101 , it passes through a flow meter  104  and past a molecule injector  105 . Upon exiting the cell culture support device  101  the fluid passes by a fluid sampler  108  and through a second flow meter  109  and is then deposited into a second fluid reservoir  110 . A pressure sensor  106  measures the pressure at, or near, the inlet and the outlet of the cell culture support device  101 . Additionally, the system  100  includes an imager  107 , which is used to view cells within the cell culture support device  101 . 
     As discussed above, the system  100  includes a number of components to support the cell culture support device  101 . The pump  103  drives fluid from the first fluid reservoir  102  through the cell culture support device  101 . In some implementations, the pump  103  is a peristaltic pump or a syringe pump. In implementations using a syringe pump, the fluid reservoir  102  is the barrel of a syringe. The pump  103  controls the fluid flowing through the cell culture support device  101 . For example, the pump can control the fluid&#39;s flow rate and the duration of the flow through the cell culture support device  101 . In some implementations, the flow is continuous and in other implementations the flow is pulsatile. The pump  103  can be configured to control the shear stress the fluid exerts on cells within the cell culture support device  101 . The fluids passed through the cell culture support device  101  can include, but are not limited to, cell culture medium, cell nutrients, reagents, test agents, buffer fluids, reactant fluids, fixing agents, stains, simulated and real biological fluids such as blood filtrate, whole blood, blood serum, blood plasma, urine, dilute urine. 
     In some implementations, the above agents and/or other molecules are added to the fluid flowing into the cell culture support device  101  by the molecule injector  105 . In certain implementations, the molecule injector  105  is a second syringe pump. In other implementations, continuous delivery of nutrients by the fluid creates favorable conditions for long term cell culture within the cell culture support device  101 . The system  100  also includes a fluid sampler  108 . The fluid sampler  108  is positioned near the outlet of the cell culture support device  101 . In some implementations, the fluid sampler  108  is configured to siphon off a small amount of the fluid exiting the cell culture support device  101 . The collected fluid may be tested for specific molecular markers, reagents, or other such molecules. 
     The system  100  further includes a flow meter  104  near the inlet of the cell culture support device  101 , a flow meter  109  near the outlet of the cell culture support device  101 , and a pressure sensor  106 . The pressure sensor  106  measures the pressure of the fluid as it enters and exits the cell culture support device  101 . In certain implementations, the measurements made by the flow meters  104  and  109  and the pressure sensor  106  are used to calculate the shear stress imparted on cells within the cell culture support device  101 . 
     The imager  107  is used to observe cells within the cell culture support device  101 . In some implementations, the cells are imaged while fluid is flowing through the cell culture support device  101 . In other implementations, at the end of an experiment fixing fluid is passed through the cell culture support device  101  and the cells are imaged upon completion of experimentation. In other implementations, the imager  107  is configured to monitor the integrity of the cross channel interface. For example, the imager  107  can be configured to measure the degree to which the cross channel interface  202  has degraded. 
       FIG. 2  is a solid model illustrating the cell culture support device  101  in greater detail. As illustrated, the cell culture support device  101  is a multi-layered polymer device. The cell culture support device  101  includes a first polymer layer  203  with a first flow chamber  206  defined therethrough, and a second polymer layer  201  has a second flow chamber  205  defined therethrough. The first polymer layer  203  of the cell culture support device  101  also includes a cross channel interface  202 . In some other implementations, the cross channel interface  202  is an additional polymer layer that is coupled between the first polymer layer  203  and second polymer layer  201 . In certain implementations, the cross channel interface  202  is a membrane made of a thermoplastic, such as polystyrene, polycarbonate, polyimide, polysulfone, polyethersulfone; biodegradable polyesters, such as polycaprolactone (PCL); soft elastomers, such as polyglycerol sebacate (PGS); or other polymers such as polydimethylsiloxane (PDMS) and poly(N-isopropylacrylamide). In yet other implementations, the cross channel interface  202  is made from silicon, glass, or silicon nitride. The cross channel interface  202  is manufactured, in some implementations, through processing methods such as track-etching, electro-spinning, microfabrication, micromolding, gel deposition, phase separation, particle leaching, and solvent leaching. In yet other implementations, the cross channel interface  202  is a multilayered membrane that includes several layers of material. For example, the material can be a structural backing, a skin layer, a porous layer, a layer that serves as a permeable spacer, or allows lateral flow. 
     As discussed above, in certain implementations, the interior of the cell culture support device  101  is imaged with the imager  107 . Accordingly, in some implementations, the roof  204  of the first flow chamber  206  is configured to allow for visual inspection of the first flow chamber  206 , cross channel interface  202 , and/or second flow chamber  205 . In other implementations, the roof  204  is a polymer layer manufactured out of a material similar to, or the same as, the polymer layers. In certain implementations, the cell culture support device  101  includes more than one flow chamber within a polymer layer. Additionally, in some implementations, the cell culture support device  101  includes more than two polymer layers with flow chambers. For example, the cell culture support device  101  can include three or more polymer layers each separated from one another by a different cross channel interface  202 . The polymer layers can include, but are not limited to, a thermoplastic, such as polystyrene, polycarbonate, polyimide; biodegradable polyesters, such as polycaprolactone (PCL); soft elastomers, such as polyglycerol sebacate (PGS); or other polymers such as polydimethylsiloxane (PDMS) and poly(N-isopropylacrylamide). In certain implementations, the polymer material is selected for its ability to be micro-machined and support cell growth. In some implementations, the length, width, and height of the flow chambers are selected to mimic kidney structures. In other implementations, the height of a flow chamber is between about 10 μm and about 100 μm, the width is between about 250 μm and about 2 mm, and the length is between about 5 mm and about 10 mm. 
     Discussed in greater detail in relation to FIGS.  3  and  4 A- 4 C, but briefly, the cross channel interface  202  enables communication between the first flow chamber  206  and the second flow chamber  205 . A plurality of pores  207  are defined through the cross channel interface  202  and at least one face of the cross channel interface  202  includes a topography that is independent of the pores  207 . A cross channel interface  202  with pores  207  that are created independent of the topography generates a porous membrane that facilitates basement membrane (BM)-like architecture and enables better control of experimental variables. In some implementations, the topography is selected such that it has a specific effect on the pores  207 . For example, the topography can be selected such that it alters the pore share or closes the pores in a specific area of the cross channel interface  202  or reduces the size of the pores  207  by a specific size. 
     In some implementations, the pores  207  of the cross channel interface  202  are generated by track-etching. Track-etching creates highly uniform pores  207 . The pore sizes range between about 3 μm and 15 μm wide. The cross channel interface  202  is between about 6 μm and 30 μm thick. 
     As indicated above, at least one face of the cross channel interface  202  is patterned with a selected topography. In certain implementations, at least one wall of the first flow chamber  206  and/or second flow chamber  205  is also patterned with a selected topography. In some implementations, the patterned faces (e.g. a cross channel interface  202  face and a first flow chamber  206  wall) are patterned with a different topographies. In yet other implementations, different sections of the same face are patterned with different topographies. 
       FIG. 3  is a cross-sectional view of a cell culture support device  300  similar to the cell culture support device  101  of  FIG. 2 . The cell culture support device  300  includes a first polymer layer in which a first flow chamber  206  is defined, and a second polymer layer  201  in which a second flow chamber  205  is defined. A cross channel interface  202  forms the roof of the second flow chamber  205  and the floor of the first flow chamber  206 . A top polymer layer  204  forms the roof of the first flow chamber  206 . As previously discussed, the cross channel interface  202  includes a plurality of pores  207 . As illustrated, the cell culture support device  300  includes a first plurality of cells  302  seeded in the first flow chamber  206  and a second plurality of cells  303  seeded in the second flow chamber  205 . In some implementations, cells are only seeded into one of the flow chambers  206  and  205 . As illustrated by the flow arrows  301 , fluid communication occurs through the pores  207 . The fluid communication can include, but is not limited to, the passive transport of macromolecules, nutrients, and test agents. In some implementations, the size of the pores  207  allows for cells to migrate across the cross channel interface  202  and in other implementations the size and/or shape of the pores  207  inhibits trans-chamber migration of cells but allows for the migration of nutrients and cellular signaling analytes between the chambers. The pores  207  can be dispersed randomly or in an ordered fashion throughout the length of the cross channel interface  202 , and in some implementations, the pores  207  are limited to specific regions of the cross channel interface  202 . In some implementations, the arrangement, shape, and size is referred to as the pore geometry. 
       FIG. 4A-4C  are solid models illustrating topographical patterns that can be applied to a cross channel interface. For illustrative purposes the cross channel interfaces includes three pores  207 ; however, the is no requirement for similar pore spacing, alignment, or concentrations. The topography of a cross channel interface  202  is selected based on the sells that are to be seeded into the cell culture support device  101 , and can be selected to effect a particular arrangement, function, shape and/or density of cells. In some implementations, the cross channel interface  202  is manufactured from a biodegradable polymer. In some of these implementations, the surface pattern is selected to facilitate and/or control the degradation of the cross channel interface  202 . In certain implementations, the topographies are selected and manufactured such that they degrade at a specific rate or when exposed to a specific chemical agent. For example, the patterning topography and cross channel interface  202  may be configured such that the cross channel interface  202  completely dissolves once a cellular mat has grown on the cross channel interface  202 . Thus, in such an implementation, after cross channel interface  202  degradation, the first polymer layer  203  and second polymer layer  201  are separated only by the cellular mat. 
       FIG. 4A  illustrates a portion of cross channel interface  410  that is smooth and does not contain a topographical pattern.  FIG. 4B  illustrates a cross channel interface  420  with a ridge and groove pattern. The ridge and groove pattern can be aligned perpendicular to, parallel with, or angled to the flow of fluid through a flow chamber. In other implementations, the ridges are between about 0.5 μm and about 1.0 μm wide, have a pitch between about 1.0 μm and about 2 μm, and are between about 0.5 μm and 1.0 μm tall. In some implementations, the dimensions and spacing of the ridges is constant along the entirety of a patterned surface. In other implementations, one or more of the ridge parameters is varied along the length of the patterned surface. For example, the spacing between the ridges may start at 1.0 μm and transition to a 2.0 μm spacing at the flow chamber&#39;s outlet. In certain implementations, the ridges and/or grooves are rounded. 
       FIG. 4C  illustrates a cross channel interface  430  with a pit pattern. As illustrated, the pits are cylinderical, but in other implementations the pits can be rectangular, square, frustroconical, conical, and/or hemispherical. Additionally, the above pit patterns can be inversed to create posts. The pits and posts can be patterned in a regular, ordered fashion or randomly. For example, in the regular, ordered fashion, hemispherical posts may be aligned in ordered rows with 1.0 μm between each post of a row and 1.5 μm between each row. In some implementations, the cross channel interface  202  topographies includes a plurality of the above described topographies. 
       FIG. 5  is a flow chart of a method  500  for manufacturing a cell culture support device, such as that of system  100 . The method  500  includes forming a first flow chamber in a first polymer layer (step  501 ), and also forming a second flow chamber in a second polymer layer (step  502 ). Additionally, the method  500  includes forming pores in a cross channel interface (step  503 ). A topographical pattern is selected (step  504 ) and formed into the cross channel interface (step  505 ). In some implementations, the cross channel interface is coupled between the first polymer layer and the second polymer layer (step  506 ). 
     As set forth above, a first flow chamber is manufactured in a first polymer layer (step  501 ) and a second flow chamber is manufactured in a second polymer layer (step  502 ). The flow chambers can be manufactured by photolithographic techniques, injection molding, direct micromachining, deep RIE etching, hot embossing, or any combinations thereof. In some implementations, as illustrated in  FIG. 2 , the cross channel interface  202  is a component of the first polymer layer  203 . In other implementations, the cross channel interface  202  is a separate component that is separately manufactured and subsequently coupled between the first and second polymer layers. 
     The method  500  of manufacturing a cell culture support device continues with the formation of pores in the cross channel interface (step  503 ). In some implementations, the pores  207  of the cross channel interface  202  are manufactured by leaching micro-particles from the cross channel interface  202 , phase separation micro-molding, track etching, or any combination thereof. In certain implementations, the cross channel interface  202  is obtained prefabricated with pores  207 . 
     The method  500  continues with the selection of a topographical pattern to apply to the cross channel interface (step  504 ) and then the application of the selected pattern to the cross channel interface (step  505 ). As discussed above, the selection of the pattern can be dependent on the cells to be grown on the cross channel interface  202  and/or the desired arrangement, function, shape, and density of the seeded cells. Responsive to selecting the topographical pattern, the pattern is applied to the cross channel interface  202 . 
     The cross channel interface  202 , and any wall of a flow chamber to be patterned, can be patterned with hot-embossing. Hot-embossing can be accomplished as a two step molding process. First, a silicon mold is fabricated using photolithography and reactive ion etching. The first step of the process generates a positive of the selected pattern. Next, a negative is created from the positive. The negative is formed by electroforming nickel to the positive mold. The electroforming is accomplished by applying a voltage difference between a nickel source and the silicon mold. This causes the nickel to flow into the silicon mold. In some implementations, prior to embossing, the patterned face of the nickel mold is soaked in a 1 mM solution of hexadecanethiol (HDT), which forms a self-assembled monolayer (SAM) to decrease surface energy to aid in subsequent polymer release. 
     The second step of the hot-embossing method includes placing the cross channel interface  202  in contact with the topographically patterned face of the nickel mold. The mold and cross channel interface  202  are sandwiched between two Kapton polyimide films and silicone rubber sheets to decrease sticking and add compliance. The stack is then placed in a uniformly heated, temperature- and pressure-controlled automatic hydraulic press. A light load is applied to the stack while the temperature is set to about 150° C. The load is applied for a specified dwell time before being cooled to 60° C. under constant pressure. Upon cooling, the newly patterned membrane is released from the nickel mold and analyzed for changes in pore size and geometry. The combination of the dwell time, pressure and temperature are selected such that the topographical pattern is fully created in the cross channel interface  202 , but does not cause polymer material to flow into the pores  207 . In some implementations, the dwell time was selected to be between about 10 and about 20 minutes, under a pressure of between about 700 kPa and about 850 kPa, and at a temperature of about 125° C. to about 175° C. For example, a dwell time of 15 minutes at 820 kPa and 150° C. preserves pore architecture. In other implementations, alternative embossing parameters and processes may be employed. 
     In some implementations, the method  500  of manufacturing a cell culture support device includes coupling the cross channel interface between the first and second polymer layers (step  506 ). As discussed above, in some implementations, the cross channel interface  202  is a component of the first or second polymer layers, and therefore, in these implementations, the first polymer layer  203  would be directly completed to the second polymer layer  201 . In other implementations, the cross channel interface  202  is coupled between the first polymer layer and the second polymer layer. In certain implementations, the components of the cell culture support device  101  are reversibly coupled to one another. For example, a clamp can be used to couple the components together during an experiment and allow for the cross channel interface  202  to be removed after an experiment for further analysis. 
       FIG. 6  is a flow chart of a method  600  for using a cell culture support device in a system similar to the system  100 . The method  600  begins with the provisioning of a cell culture support device (step  601 ). Then, cells are seeded into at least one flow chamber of the cell culture support device (step  602 ). The method  600  continues with the flowing of fluid through the flow chambers of the cell culture support device (step  603 ) and the injection of a molecule into the inlet of at least one of the flow chambers (step  604 ). Responsive to flowing fluid through the flow chambers, at least one flow parameter is measured (step  605 ) and the concentration of the injected molecule is measured at an outlet of at least one flow chamber (step  606 ). 
     As set forth above, the method  600  begins by providing a cell culture support device (step  601 ), such as the cell culture support device  101  of  FIG. 2 . Next, cells are seeded into the cell culture support device (step  602 ). In some implementations, the cell culture support device  101  is provided pre-assembled, and in other implementations, the cell culture support device  101  is provided unassembled. For example, cells can be seeded onto the cross channel interface  202  prior to assembly of the cell culture support device  101 . The cell seeded cross channel interface  202  can then be cultured in an incubator until the cells reach a maturity level appropriate for experimentation. In other implementations, cells are injected into the cell culture support device  101  with a syringe and allowed to adhere to the cross channel interface  202  and/or other surfaces of the flow chambers. In certain implementations, the cell culture support device  101  is sterilized prior to cellular seeding. For example, the cell culture support device  101  can be sterilized with ethylene oxide and then rinsed with 70% ethanol. Also prior to cellular seeding, in certain implementations, the cross channel interface  202  and/or remaining components of the cell culture support device  101  are coated with an agent, such as an agent to inhibit or encourage cellular growth. For example, surfaces of the cell culture support device  101  exposed to cells can be coated with an extracellular matrix, collagen IV, collagen I, laminin, fibronectin, agrin, nephrin, or similar proteins, Arginine-Glycine-Aspartic acid or similar peptides, or adhesive motifs. 
     The method  600  continues with the flowing of fluid through the cell culture support device (step  603 ). As described above in relation to  FIG. 1 , fluid flow through the chambers of the cell culture support device  101  is controlled with a pump  103 . In some implementations, fluid is only flowed through one of the flow chambers. For example, a first chamber can act as a cellular well without perfusion, such that the cells within the camber are not exposed to shear stress caused by flowing fluid. In this example, nutrients or other agents can be transported to the cellular well through the pores  207  in a cross channel interface  202  that separate the cellular well from flowing fluid in a flow chamber beneath the cellular well. 
     The method  600  also includes injecting a molecule into the inlet of at least one flow chamber (step  604 ). The molecule can be a cell culture medium, cell nutrient, reagent, test agent, buffer fluid, reactant fluid, fixing agent, and/or stain. In some implementations, the injection of the molecule and/or the pump  103  is computer controlled so that a specific flow rate and molecule concentration is achieved within the cell culture support device  101 . Example molecules to be injected can include, but are not limited to, water, sodium, potassium, chlorine and other ions; urea creatinine, and other metabolic products; oxygen, carbon dioxide, nitrogen, and other gases; macromolecules of defined molecular weights such as inulin, ficoll, dextran, albumin and other proteins; pharmaceutical agents and their metabolically-modified forms; toxins; cells and subcellular biological components such as platelets and microparticles; large particles of solids include micro and nano particles; lipid and other vesicles either synthetic or naturally-derived; bubbles or other gas-phase particles. 
     Responsive to flowing fluid through the cell culture support device, at least one flow parameter is measured (step  605 ). The measurement of the flow parameter can include parameters that are either directly measured, such as fluid flow rate and fluid pressure, derived measurements, such as shear stress measurements. In certain implementations, the measurements are made at the inlet, outlet and/or within the cell culture support device. In other implementations, cross channel permeability is measured. For example, hydraulic permeability, which measures the flux of a chemical, molecule or agent through a membrane at a given transmembrane pressure, can measured. In certain implementations, the transmembrane pressure is measured by direct measurements or by deriving the measurement based on pressure levels at channel inputs and outputs. The fluid flow rate can be quantified by measuring filling of a vessel of known size, measuring mass of inputs/outputs over time, flow visualization techniques, particle image velocimetry, and techniques using tracer elements or contrast agents. 
     Additionally, the method  600  includes measuring the concentration of the injected molecule at an outlet of at least one of the flow chambers (step  606 ). In some implementations, transport through the cross channel interface  202  (and in some implementations, the layer of cells seeded on the cross channel interface  202 ) is measured by injecting a molecule into an inlet of a first flow channel and then measuring the concentration of the molecule at the outlet of a second flow channel. 
     In some implementations, the transport of specific species across the membrane is analyzed by evaluating the concentrations of solutes, particles and other components of fluids in a cellular flow chamber, at the inlet of a cellular flow chamber, and/or at the outlet of a cellular flow chamber. In certain implementations, the concentration of the molecules is measured with a sensor, a molecule selective dye, a soluble nanosensor, a molecular label, such as a radioactive label or tracer. In some implementations, the evaluation of concentration and flow can be configured such that a sieving coefficient of a molecule or component is quantified for the device, cross channel interface  202 , and/or the membrane-cell construct. The sieving coefficient is the concentration of a specific analyte in the fluid passing through the membrane divided by the concentration of that same analyte in the fluid being fed to the membrane. The sieving coefficient can reflect the selectivity of a porous membrane. 
     EXAMPLES 
     I. Topographic Pattering 
       FIG. 7A-7E  illustrates a series of images, at different magnification, of a cell culture support device manufactured using the above described hot-embossing method. In this example, the cross channel interface was manufactured as an independent topographically-patterned membrane assembled into a cell culture support device.  FIG. 7A  is a schematic of the overall cross-sectional architecture of the cell culture support device. As in the cell culture support device  101  of  FIG. 2 , the schematic illustrates a top cell chamber defined in a first polymer layer and a bottom cell chamber defined in a second polymer layer. A cross channel interface is coupled between the first polymer layer and second polymer layer, and a cover slide provides the roof for the top cell chamber.  FIG. 7B  illustrates an assembled cell culture support device, of which  FIGS. 7C-7E  provide greater detail. 
       FIG. 7C  is a scanning electron micrograph illustrating the cross section of the device in  FIG. 7B . The micrograph shows the porous nature of the cross channel interface separating the top and bottom chambers.  FIGS. 7D and 7E  are scanning electron micrographs magnifying the inserts of  FIG. 7C .  FIGS. 7D and 7E  illustrate the well-defined groove topography coexisting with the porous architecture. 
     II. Topographic Examples 
       FIGS. 8A-E  are a series of scanning electron micrographs showing example pattern topographies created with the hot-embossing method described above on cross channel interfaces with 8 μm diameter pores.  FIG. 8A  is an image of a cross channel interface  202  including three pores and a smooth topography.  FIGS. 8B-8D  illustrate the ridge and groove topography discussed in relation to  FIGS. 4A-4C .  FIGS. 8B-8D  also illustrate the effect of ridge width on topography. The width of the ridges in  FIG. 8B  is 0.5 μm, 0.75 μm in  FIG. 8C , and 1.0 μm  FIG. 8D . Similarly,  FIG. 8E  illustrates a cross channel interface with evenly spaced 1.0 μm pits. In  FIGS. 8B-8E , the topographical features are 0.75 μm deep. 
     III. Hot-Embossing Parameters 
     As discussed above, the embossing of the topographical pattern onto the cross channel interface are done in a controlled manner as to not alter the pore architecture. To determine the appropriate embossing parameters, a series of cross channel interfaces were hot-embossed under a constant load of 820 kPa at 150° C. For the trials the dwell time ranged from 10 to 30 minutes.  FIG. 9A  is a graph illustrating how pore diameter (y axis) changes with dwell time (x axis). The plot in  FIG. 9A  shows that nominal pore size did not significantly change for dwell times less than 15 minutes when compared to the pre-embossed cross channel interface. However, as dwell time increased past 20 minutes, pore diameter significantly decreased when compared to pre-embossing diameters. 
       FIG. 9B  is a series of bar charts that illustrate how different topographies affect pore diameter during the embossing process. For this experiment, cross channel interfaces were created to have 3 μm, 5 μm, 8 μm, or 12 μm diameter pores. The diameter of the pores on each cross channel interface were measured and averaged to serve as controls for the experiment. The cross channel interfaces were then hot-embossed with a dwell time of 20 minutes under 820 kPa at 150° C. The resulting pore diameters were measured and averaged. 
       FIG. 9B  shows that embossing reduces pore diameter for membranes with large pores, such as the 8 μm and the 12 μm diameter pores, but does not reduce pore diameter for pores with small pore diameters, such as the 3 μm and the 5 μm diameter pores. The 8 μm and 12 μm pore cross channel interfaces showed a decrease in pore diameter over all topographies after embossing. Also, while a pore diameter of 12 μm was desired for the fourth group (the 12 μm group), the actual pore diameter was measured to be closer to 10 μm. Thus, the decrease in pore diameter caused by the hot-embossing process in the 12 μm cross channel interface, although statistically significant, was smaller than the difference between the desired control pore diameter and the actual measured 12 μm pore diameter. 
     Some decrease in pore diameter is expected after embossing due to the flow of the polymer under high temperature and pressure. Pore deformation was independent of pattern type. A dwell time of 10-15 minutes provided a good balance of pattern transfer onto the cross channel interfaces without significantly changing pore diameter. 
     In some experiments, pores were not perfect circles and the elongation of the pores was exacerbated during the hot-embossing process.  FIG. 9C  is a series of scanning electron micrographs illustrating the differences in elongation of 3-12 μm pores embossed with a ridge and groove topography (a 10 Grat topography corresponds to 1.0 μm wide ridges spaced 2.0 μm apart).  FIG. 9D  is a bar chart, with the same groupings represented in  FIG. 9B , and illustrates the amount of elongation, represented as a fraction, induced by hot-embossing with a dwell time of 20 minutes under 820 kPa at 150° C. The average pore in the non-embossed control cross channel interfaces exhibited a slightly elongated shape with a fraction of elongation ranging from 0.16 for 12 μm pores to 0.34 for 3 μm pores.  FIG. 9D  shows how the fraction of elongation for pores after embossing was dependent on initial pore diameter and in some cases, the topography. Cross channel interfaces with smaller pore diameters, i.e. 3 μm and 5 μm, yielded significantly higher fractions of pore elongation when compared to larger pore sizes. Cross channel interfaces with a pore size of 3 μm exhibited pores with an average elongation fraction of almost 0.5 when embossed with the 10 Grat pattern, a 49% increase from the control. On average, elongation of the 3 μm pores increased by 37% across all linear patterns. For larger pore sizes, i.e. 8 μm and 12 μm, the change in pore elongation became insignificant across most topographies when compared to the control pore geometry. The elongation of 8 μm pores increased by an average of only 15.3% among linear patterns. Cross channel interfaces with 12 μm pores had the lowest fraction of elongation, with an average elongation of 0.23 among linear patterns. Topographical features in the form of 1 μm pits had the least affect on pore elongation for small and large pore sized membranes, with a range from 0.37 for 3 μm pores to 0.22 for 12 μm pores. 
     IV. Cells Proliferate on and Respond to Porous Membrane Topography 
     A cell culture support device, similar to that of the device in  FIGS. 7A-7E , consisting of two channels separated by the patterned porous cross channel interface (also referred to as a membrane) served as a platform to characterize the response of renal epithelial cells cultured on the patterned porous membrane. The device&#39;s performance was evaluated in three ways. First, HK-2 response to unpatterned and patterned membranes outside the device was characterized by analyzing cellular alignment. Second, the cell culture support device was evaluated by scanning electron microscopy and optical microscopy to verify channel geometry and alignment. Third, immunofluorescent techniques were used to label markers indicative of a reabsorptive epithelial layer for cells cultured in the cell culture support device. 
     During the experiments, HK-2 cells and renal proximal tubule epithelial cells (RPTECs) proliferated from initial seeding to confluency within the cell culture support device over approximately 4 days. A uniform initial seeding density and appropriate culture time yielded complete confluency of both HK-2 and RPTECs over the cell culture support device channel area of 1.25 mm 2  shown respectively in the brightfield composites in  FIG. 10A  and  FIG. 10D , with higher magnification views provided in  FIGS. 10B and 10F . HK-2 and RPTEC monolayers expressed paxillin (FIGS.  10 C(i) and  10 F(i)), a typical epithelial marker of focal adhesions; ZO-tight junction complexes (FIGS.  10 C(ii) and  10 F(ii)); and acetylated tubulin, an indicator of primary cilia and cytoplasmic microtubules (FIGS.  10 C(iii) and  10 F(iii)). Paxillin expression in HK-2 samples signified focal adhesions that were less discrete with a weaker signal than RPTEC samples. Both HK-2s and RPTECs expressed ZO-1 in distinct borders outlining the perimeter of cells, indicating initial formation of a tight-junction-based sealed epithelial barrier. Acetylated tubulin morphology differed between the HK-2s and RPTECs. The HK-2s showed somewhat distributed cytoplasmic microtubules, but distinct primary cilia were not expressed on the apical surface. The RPTECs expressed acetlylated tubulin in a single punctuate spot on the apical surface of each cell, indicating formation of a primary cilia. 
     Formation of the complete, confluent cellular monolayer within the channel layer allows interrogation of the layer for permeability, a requisite for a reabsorptive barrier. As the layer is confluent, the cell culture support device allows fluidic and/or electrical access to any point on the flow chamber and thus the direct measurement of cellular transport. Therefore, the cell culture support device allows for the quantification of reabsorptive properties. Formation of ZO-1 junctions indicate an epithelial barrier capable of active transport. The cellular junctions can be improved by conditioning the cells with mechanical and/or other stimuli. The HK-2 cells formed a more mature monolayer due to its longer culture time, causing subtle differences in paxillin expression. Focal adhesions in highly developed monolayers are not discrete and tend to have a weaker signal, which was seen in the HK-2 samples as compared to the RPTECs. The lack of primary cilia in HK-2 cells was not abnormal. Primary cilia may not be fully expressed in HK-2 populations if their formation is not enhanced by serum starvation or shear stress. Cytoplasmic tubulin was more prevalent compared with RPTEC, with signs of a microtubule-organizing center that may nucleate cilia development. The presence of primary cilia in the RPTECs indicated the cells will be responsive to mechanical stimuli, such as shear stress, as the cilia can serve to transduce mechanical signals to chemical activity. Continuous flow of nutrient rich fluid through the cell culture support device delivers nutrients to the cell populations, thereby creating more favorable conditions for long term cell culture in a small channel volume while simultaneously mimicking the filtrate flow seen by proximal tubule cells in vivo. Finally, as shown by the scanning electron micrographs of  FIGS. 11A and 11B , the cells block the pores of the membrane.  FIG. 11A  is a scanning electron micrograph of a membrane prior to cellular seeding and  FIG. 11B  is a membrane showing that the seeded cells block the pores of the membrane.  FIG. 11B  indicates transport across the membrane-cell layer construct can be limited to transcellular transport if paracellular transport is limited through tight junction and other cell-cell junction formation. With the ability to stimulate cells mechanically; interrogate cells with imaging, electrical, and fluidic means; and support growth of an epithelial layer expressing indications of a mechanically-responsive reabsorptive barrier, the patterned porous membrane of the cell culture support device allows the quantification reabsorptive barrier function.