Abstract:
A multi-well plate can be loaded with a range of compliant substrates. Commerically-available assays can be used to test cellular responses across a plate with shear modulus from 50 to 51200 Pascals. Cells can be grown in the plates, and can be manipulated and analyzed. Hydrogels can be attached to the bottom of a well. The plates can support the attachment and growth of different cell types and can be compatible with standard 96-well and 384-well plate assays. The mechanical properties of the hydrogels can be reproducible and stable to increase the shelf life of the substrate. The hydrogel can be compatible with growth of a variety of cell types, various attachment ligands such as collagen I, collagen IV, fibronectin, vitronectin, laminin, or RGD peptides and can be coupled to the gel surface.

Description:
CROSS-REFERENCE TO RELATED ACTIONS 
       [0001]    This application claims the benefit of U.S. Provisional Application No. 60/969,104, filed on Aug. 30, 2007. 
     
    
     GRANTS 
       [0002]    The government has certain rights to the present invention under contract NIH HL-82856, GM-073628. 
     
    
     BACKGROUND 
       [0003]    The physical environment of a living cell influences its ability to proliferate, metabolize, differentiate and remodel. Living cells specify lineage and express different phenotypic and physical states with extreme responsiveness to stiffness (i.e., shear modulus) of their underlying matrix. 
         [0004]    A general consensus is emerging among bioengineers and life scientists that rigid substrates may, in many cases, be inappropriate for assessing cell behaviors. Early efforts to more realistically mimic the tissue environment relied on three dimensional reconstituted biological components that form hydrogels, such as collagen, fibrin, and Matrigel. More recently, synthetic, self-assembling peptide gels (PuraMatrix), alginate sponges (AlgiMatrix), and hyaluronan-derived gels (Glycosan) have become commercially available. However, these methods can be prohibitively costly, technologically impractical, or incompatible with many cell-based high-throughput assays, which are designed with 2-D systems in mind. And while culture in three dimensions is appreciated, in general, these systems do not allow wide control of elasticity. Further, in many cases, cell responses to pharmaceutical compounds and biologics in vitro do not accurately predict in vivo functionality and toxicity. 
       SUMMARY 
       [0005]    In accordance with implementations of the invention, one or more of the following capabilities may be provided. A collection of wells with varying shear modulus (i.e., stiffness) can be provided. In an embodiment, a multi-well plate can be fabricated with a range of compliant substrates. Commerically-available or custom-made assays can be used to test cellular responses across a plate with stiffness ranging from 50 to 150,000 Pascals. For example, lung fibroblast proliferation and apoptosis which can be strongly dependent upon substrate shear modulus can be tested. Cells can be grown in the plates, and can be manipulated and analyzed in a manner consistent with conventional multi-well plates. 
         [0006]    In an embodiment, hydrogels can be affixed (i.e., firmly attached) to the bottom of a well (e.g., wells in 24, 96, 384 well plates). The firm attachment enables long-term cell cultures, as well as compatibility with some assay reagents that may cause the hydrogels to shrink and detach. The plates can support the attachment and growth of different cell types and can be compatible with standard multi-well plate assays. The mechanical properties of the hydrogels can be reproducible and stable to increase the shelf life of the substrate. The hydrogel can be compatible with growth of a variety of cell types, various attachment ligands such as collagen I, collagen IV, fibronectin, vitronectin, laminin, or RGD peptides and can be coupled to the gel surface. 
         [0007]    Multiple shear modulus gels, varying in orientation, can be casted and derivatized in a multi-well glass-bottom plate. Stiffness-dependent biology can be assessed in a high-throughput manner and subject to a standard multi-well plate assay, including but not limited to, cell proliferation, apoptosis, signaling events, and detection of soluble and insoluble factors. Cells grown in the multiple shear modulus plate can be fixed and immunologically stained, or isolated for gene expression and protein analysis. Attachment-dependent cell types can be conceivably studied, including fibroblasts, smooth muscle, endothelial, epithelial, tumor, osteoid, and neuronal. The plate can serve as a tool to direct the differentiation of adult or embryonic stem cells. 
         [0008]    The devices and methods described herein are useful to program or reprogram embryonic and/or adult stem cells, e.g., the latter being obtained from normal or tumor-derived tissue. The cells are cultured in the chamber and can be subjected to variations in stiffness of the substrate. Reprogrammed stem cells are identified by their behavior (e.g., movement or lack thereof), physical state of the cytosol, appearance, change in gene/protein expression, or elaboration of intracellular or secreted factors, among other parameters. The methods and imaging systems are used to monitor characteristics of cells and identify/screen for cells of a desired phenotype, e.g., cells at a desired state of differentiation or “sternness”, e.g., phenotype as a response to variations in changes of stiffness of culture substrate applied to a cell or plurality of cells. The methods include non-invasive, non-perturbing, automatable, and quantitative methods and are applied to the examination of cells such as adult or embryonic stem cells as well as differentiated cells of all phenotypes and to cells at various stages of differentiation. Viability, sternness, or plasticity of the cell, in response to the culture environment or physical stresses to which the cell or cells are exposed are monitored and quantified at various points during culture, as preserved/fixed, or in real time. 
         [0009]    In general, in an aspect, the invention provides a method for fabricating hydrogels with elastic properties covering a physiological range, including placing a first polymerizing solution into a well, covering the first polymerizing solution with a plate, such that the area of the plate is less than the area of the well, conjugating the first polymerizing solution with a ligand, placing a second polymerizing solution into the well, such that oxygen in the air substantially inhibits polymerization at the air-liquid interface; and detoxifying the well. 
         [0010]    Implementations of the invention may include one or more of the following features. The uniformity of ligand binding can be assessed with anti-ligand and anti-IgG-coated fluorescent beads. The second polymerizing solution can be distributed evenly by tapping the well. The second polymerizing solution can substantially cover a ring shaped area defined by the edge of the first polymerizing solution and the well. The second polymerizing solution can affix the first polymerizing solution to the well. 
         [0011]    In general, in another aspect, the invention provides an apparatus including a well, a dispensing system configured to deliver a polymerization solution to the well, a peg configured to be inserted into the well, and a glass plate disposed at the distal end of the peg, such that the area of the glass plate is less than the area of the well and is configured to contact the polymerization solution in the well. 
         [0012]    Implementations of the invention may include one or more of the following features. The well can be a well in a multi-well plate, and the peg can be a more than one peg, such that each of the pegs can be configured to be inserted into a corresponding well in the multi-well plate. A peg insertion system can be configured to move the peg into and out of the well. A second dispensing system can be configured to deliver a second polymerization solution into the well. The dispensing system can configured to deliver the second polymerization solution into the well. The well can be a 96 or 384 well plate. A well plate stage can be configured to move the well in at least one axis. A well tapper can be configured to tap the well. 
         [0013]    In general, in another aspect, the invention provides an apparatus including a multi-well plate, a first gel affixed in at least a first well in the plate, and a second gel affixed in at least a second well in the plate, such that the shear modulus of the first and second gels are different. 
         [0014]    Implementation of the invention may include one or more of the following features. A third gel can be disposed in at least a third well of the plate, such that the shear modulus of the first, second, and third gels are different from one another. The wells can be disposed on the plate in columns and the shear modulus of the gels in the wells in a column can be substantially the same. The multi-well plate can be a 96 well plate, and can include 12 columns of wells such that one column of wells is empty and 11 columns of wells contain gel, whereby the gels in a well of a column can have a substantially similar shear modulus to the gels in wells of the same column. 
         [0015]    In general, in another aspect, the invention provides an apparatus including a multi-well plate, a gel affixed in at least a first well in the plate, wherein the thickness of the gel is less than 1 millimeter. 
         [0016]    Implementations of the invention may include one or more of the following features. The thickness of the gel can be less than 100 microns. A second gel can be disposed in at least a second well in the plate, such that the thickness of the second gel is less than 1 millimeter, and the shear modulus of the first and second gels are different. The thickness of the second gel can be less than 100 microns. The gel can be derivatized with a heterobifunctional crosslinker and a ligand. 
         [0017]    Also within the invention is a method of simulating a physiological growth condition by contacting a cell with the device or apparatus. For example, the cell is a cardiovascular, gastrointestinal, kidney, genitourinary, musculoskeletal, nervous system, oral, breast, periodontal, or skin cell or progenitor thereof. The shear modulus of the cell culture substrate of the device is in the range of the tissue type to be evaluated. Moreover, cell behavior or response are induced by contact with substrates of varying stiffness. For example, the differentiation path of stem or progenitor cells is driven toward a certain phenotype based on the shear modulus of the substrate upon which the cells are cultured. 
     
    
     
       BRIEF DESCRIPTION OF THE FIGURES 
         [0018]      FIG. 1  is a side view of an assembly for fabricating hydrogels. 
           [0019]      FIG. 2  is a flowchart and illustrative view of a method and apparatus for creating compliant substrates with two polymerization solutions. 
           [0020]      FIG. 3  are photographs of edge effects of a polymer surface. 
           [0021]      FIG. 4  is a flowchart and illustrative view of method and apparatus for creating compliant substrates with a single polymerization solution. 
           [0022]      FIG. 5  is an exemplary 96-well plate configured with a plurality of gels. 
           [0023]      FIG. 6  is an exemplary 384-well plate configured with a plurality of gels. 
           [0024]      FIG. 7  is a graph depicting human lung fibroblast cell growth in a 384-well plate containing hydrogels with a range of shear modulus values. 
           [0025]      FIG. 8  is a graph depicting apoptosis in human lung fibroblast grown in a 384-well plate containing hydrogels with a range of shear modulus values. 
           [0026]      FIG. 9  includes graphs comparing the cell growth of different cell types as a function of the shear modulus values of the hydrogels the cell are grown on. 
       
    
    
     DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS 
       [0027]    Cells grown on rigid plastic behave differently than the same cells residing in soft tissues. For example, rigid substrates can typically support high rates of proliferation, but also tend to attenuate cell differentiation, which can distort the normal function of cells in their native environment. Accordingly, the inventions described herein provide for growing cells on substrates that mimic the elasticity of soft tissues and offers an approach that will capture a fundamental aspect of the tissue environment while retaining the simplicity of in vitro systems. 
         [0028]    Embodiments of the invention provide techniques for fabricating hydrogels with elastic properties covering a broad, physiologically relevant range. Glass-bottomed multi-well plates can be used. Cells can be studied in an appropriate elastic environment. A multi-well plate can use synthetic matrix-coated hydrogels to span a physiological range of shear modulus values. For example, a 96-well plate, which is a generally used format for biological assays, can be used. The system can also be extended to other formats that are amendable to high-throughput screening (384-well plates) or cell cultivation (petri dish). In general, to replicate soft tissue elasticity, a polyacrylamide hydrogel is polymerized as a thin, optically transparent layer which is affixed to the bottom of each well. By controlling the number of crosslinks that interconnect the hydrogel network, the elasticity can be tuned over the range of typical soft tissues (heart, lung, kidney, liver, muscle, neural, etc.) from elastic modulus ˜20 Pascals (fat) to ˜100,000 Pascals (skeletal muscle). In comparison, prior art cell culture dishes made from polystrene plastic have a stiffness of ˜3,000,000,000 Pascals. 
         [0029]    Referring to  FIG. 1 , an assembly for fabricating hydrogels  10  is shown. The assembly  10  includes at least one peg  12 , a well  14 , a polymerization solution  16 , a glass well bottom  18 , and a glass plate  20 . The well  14  can be a multi-well configuration such as a 96-well assembly comprising a 12×8 matrix of wells in a plate (e.g., a Matrical 96-well assembly). The well  14  can also be a 6-well, 24-well, 384-well configurations. Generally, the well  14  can include standard multi-well plates used to study various biological endpoints under different interventions. The bottom of the well bottom  18  can be glass to allow for observation of cells placed within the well  14 . The well bottom  18  can be other material configured to inhibit oxygen from flowing into the polymerization solution  16 . In an embodiment, the polymerization solution  16  can be comprised of variable ratios of acrylamide:bis-acrlamide, and can be delivered into the well via a dispensing system (e.g., pipette, automated liquid dispensing system). The polymerization solution  16  can be polymer hydrogels such as polyalkylimide, poly(N-vinyl formamide), polyvinyl alcohol, poly(ethylene glycol). Also, polydimethylsiloxane, silicone, glycosaminoglycans, hyaluronic acid, chondroitin sulfate, polysaccharide, self-assembling peptides, collagen, gelatin, fibrin, methylcellulose, agarose. For a 384-well assembly, each well can typically receive 1-2 micro liters of polymerization solution  16 . A 96-well assembly typically will receive about 5 micro liters. The amount of polymerization solution can change based on the desired thickness of resulting gel. The delivery system can be a pipette or similar liquid dispensing system (e.g., BioTek Microplate Liquid dispensing system). The glass plate  20  can be hydrophobic glass, and is disposed at the distal end of the peg  12 . As an example, and not a limitation, a circular well  14 , the diameter of the glass plate  20  is less than the diameter of the well  14 . The well  14 , peg  12 , well bottom  18 , and glass plate  20  can be other shapes. 
         [0030]    In operation, the peg  12  can be lowered and raised within the well  14  such that the glass plate  20  can contact the polymerization solution  16 . For example, the glass plate  20  can be disposed within 50 microns of the well bottom  18  such that the polymerization solution  16  is compressed to a uniform thickness across the diameter of the well  14 . The glass plate  20 , however, need not be a uniform surface. The plate  20  can include positive and negative designs (e.g., bumps, dimples, holes) to create creases and other irregularities in the surface of the polymerization solution  16 . The well  14  and peg  12  can be coupled to linear and rotary actuators such that they can be moved independently, for example, to present the well  14  to a dispensing system, to place the well  14  under the peg  12 , and to move the bottom of the well  18  and the glass plate  20  closer together. 
         [0031]    Referring to  FIG. 2 , with further reference to  FIG. 1 , a method and apparatus for creating compliant substrates  100  is shown. The process  100 , however, is exemplary only and not limiting. The process  100  may be altered, e.g., by having stages added, removed, or rearranged. 
         [0032]    At stage  102 , a polymerization solution  16  is placed into a well. As an example and not a limitation, each well of a 384-well tray contains approximately 1-2 μl of a polymer hydrogel. For example, an acrylamide/bis-acrylamide mixture can be used. Different concentrations of the acrylamide/bis-acrylamide mixture can be used to produce gels of different shear modulus, for example, ranging from 20 to 100,000 Pa. Other polymer hydrogels such as polyalkylimide, poly(N-vinyl formamide), polyvinyl alcohol, poly(ethylene glycol), polydimethylsiloxane, silicone, glycosaminoglycans, hyaluronic acid, chondroitin sulfate, polysaccharide, self-assembling peptides, collagen, gelatin, fibrin, methylcellulose, and agarose can be used. The shear modulus of the gels in each well  14  need not cover a range. In an embodiment, a multi-well tray can include gels with a standard shear modulus (e.g., 5000, 10,000, 17,000 Pa) in each well. 
         [0033]    At stage  104 , the relative position between the peg  12  and the well  14  is changed such that the glass plate  20  contacts the polymerization solution  16 . As an example, and not a limitation, the polymerization solution  16  is compressed to thickness of less than 50 microns. The glass plate  20  remains in contact with the polymerization solution  16  for approximately 5-10 minutes. Thicker gels may require longer set-up time. 
         [0034]    At stage  106 , the polymerization solution  16  may not completely polymerize at the wall of the well  14  due to oxygen inhibition. In general, polymerization relies on a free radical generating system and hence can be susceptible to other factors. For example, oxygen is a free radical trap, and surfaces which absorb or are permeable to oxygen will inhibit polymerization, which can result in localized gel distortions or unpolymerized gel. Accordingly, as the peg  12  and glass plate  20  are removed at stage  108 , a non-polymerized area  16   a  may exist. Referring to Photo A in  FIG. 3 , the non-uniform edge of the polymerization solution  16  is shown in relation to the gaps  16   a  and the well wall  14 . 
         [0035]    At stage  110 , the polymerization solution  16  (i.e., gel) is functionalized via conjugation with a ligand. The surface ligand  22  is generally added to improve cell adhesion to the gel  16 . For example, the gel  16  surfaces are treated with a heterobifunctional crosslinker (e.g., sulfo-SANPAH), which can covalently link a desired ligand to the gel. Other heterobifunctional crosslinkers such as amine-reactive photoreactive (e.g., NHS/nitrophenyl azide crosslinkers: ANB-NOS, sulfo-SBED), amine-carboxyl reactive, amine-sulfhydryl reactive, and acrylic acid esters, can be used. Exemplary ligands include extracellular matrix (ECM) proteins such as collagen (type I collagen), laminin, fibronectin or combinations thereof. Other cell attachment ligands/promoters such as collagen (type III, IV), vitronectin, poly-d-lysine, cyclic and linear RGD peptides, peptide sequences, and negatively or positively charged groups, can be used. As an example, and not a limitation, the surface of the gel  16  can be activated by adding approximately a solution containing 1 mM sulfosuccinimidyl-6-(4-azido-2-nitrophenylamino) hexanoate dissolved in 200 mM HEPES. The dishes can be exposed to ultraviolet light for 5 minutes, washed twice with 0.1M HEPES solution, washed once with PBS, coated with type I Collagen solution (0.1 mg/ml) and stored overnight at 4° C. On the following day, the gel  16  can be washed, hydrated with a serum free media solution and stored in an incubator at 37° C. and 5% CO 2 . A 96-well plate, for example, can be loaded with plurality of polymerization solutions  16  in different wells. The wells can be covered by an array of pegs  14  and plates  20  and cured simultaneously to produce gels  16  of uniform thickness and various shear moduli (e.g., 100, 200, 400, 800, 1600, 3200, 6400, 12800, 25600, and 51200 Pa). 
         [0036]    At stage  112  a second polymerization solution  26  can be added via a delivery system  24 . The delivery system  24  can be a pipette, or similar liquid dispensing system. The second polymerization solution  26  can be used to overcome the edge effect caused by oxygen inhibition (i.e.,  16   a ). In general, the second polymerization solution  26  is a softer polyacrylamide (i.e., a relatively elastic polymer) which can adhere to the well. 
         [0037]    At stage  114 , a meniscus can be formed by tapping the well  14  to distribute the second polymerization solution  26  evenly. The tapping can be performed by a mechanical tapper or manually. At stage  116 , oxygen in the air inhibits polymerization at the interface between the air and the second polymerization solution  26 . That is, polymerization occurs only at the gel/well edge, where the depth of the meniscus is the greatest. At stage  118 , a uniform circular boarder is formed around the well  14 . The ring of second polymerization solution  26  assists in affixing (i.e., holding in place) the functionalized gel  16  in the well. Also, the second polymerization solution  26  can be non-functionalized (and soft) which can minimize cell attached an growth on the ring  26 . Referring to Photo B of  FIG. 3 , a photograph of a uniform edge of the first polymerization solution  16  as outlined by the second polymerization solution  26  is shown. 
         [0038]    Acrylamide, and other polymer solutions, can be highly toxic to cells, so it is generally necessary to include a detoxification process. At stage  120 , glutathione  32  can be added to detoxify free, unpolymerized acrylamide. For example, a solution of glutathione  32  can be dispensed into the well  14  and incubated for several hours prior to seeding the well  14  with cells. 
         [0039]    In another embodiment, an assay may include cells that are particularly sensitive to the second polymer solution used in process  100 , and the cleaning at stage  120  may be insufficient to detoxify the well  14  to a required level. Referring to  FIG. 4 , with further reference to  FIGS. 1 and 2 , a method and apparatus for creating compliant substrates  200  with a single polymer solution is shown. The process  200 , however, is exemplary only and not limiting. The process  200  may be altered, e.g., by having stages added, removed, or rearranged. 
         [0040]    The method and apparatus  200  can include a multi-well pin block  201  comprising a plurality of pin assemblies  210  operably connected to a plurality of glass plates  20 . The dimensions of each of the glass plates a slightly less than the dimensions of each of the wells  14  on a multi-well plate, such that a glass plate  20  can be disposed within a well  14 . In an embodiment, the multi-well plate can be a 96-well plate from Matrical, Inc., that uses a non-cytotoxic adhesive to bind the glass bottom  18  to well. Typically, the glass thickness is compatible with fluorescence-based assays, as well as high power and confocal microscopy (i.e., at thickness of about 170 microns). 
         [0041]    At stage  220 , a multi-well pin block  201  can be assembled and cleaned. The pin block glass  20  can be treated with a hydrophobicizing agent (e.g., Surfasil, Pierce), and cleaned thoroughly with methanol prior to gel casting. In an embodiment, the thickness of the gel can be controlled by placing a spacer on each of the four corners of the plate  20 . 
         [0042]    At stage  222 , each of the wells  14  of a multi-well plate can be treated with 50 micro-liters of an aqueous solution of silane to ensure the subsequently added polymer solution will be affixed to the well. In general, the well  14  is treated with agent which can be a substituted propyl-trimethoxysilane of the formula: 
         [0000]    
       
                 
         
             
             
         
       
     
         [0000]    where 
       R is NR 1 R 2  or OR 3    
       [0043]    R 1  and R 2  are independently hydrogen or C 1 -C 6  alkyl;
 
R 3  is alkyl, alkenyl, alkynyl or acyl (e.g., C(O)R 4 , where R4 is hydrogen, alkyl, alkenyl, or alkynyl).
 
       Certain Compounds Include 
       [0044]    
       
                 
         
             
             
         
       
     
         [0045]    For example, for polyacrylamide hydrogels, a solution of approximately 0.004-0.4% 3-methacryloxypropyltrimethoxysilane can be placed in the well  14  for approximately 10 minutes. At stage  224 , each of the wells  14  is rinsed in distilled water and dried (e.g., the entire multi-well plate can be placed distilled water and air or blown dry). The treated glass surface  18  reacts with subsequently added polyacrylamide to from a covalent, long-lasting bond which affixes the polyacrylamide to the well. 
         [0046]    In an embodiment, at stage  222 , each of the wells of a multi-well plate can be treated with 3-aminopropyltrimethoxysilane. For example, for polyacrylamide hydrogels, a well can be coated with a solution of approximately 97% 3-aminopropyltrimethoxysilane for approximately 10 minutes. At stage  224 , each of the wells is rinsed in distilled water and dried (e.g., the entire multi-well plate can be placed in distilled water and air or blown dry). The treated glass surface is then coated with 0.5% glutaraldehyde in water for 30 minutes and then rinsed and dried. The fully functionalized glass reacts with subsequently added polyacrylamide to form a covalent, long-lasting bond which affixes the polyacrylamide to the well. 
         [0047]    At stage  226 , a small amount (e.g., approximately 5 microliters for each well in a 96-well tray) of polymerization solution  16  is added to the bottom of each well  14 . As an example, and not a limitation, the polymerization solution  16  can be mixture of acrylamide and bisacrylamide. Other polymerization solutions as discussed above with the respect to the process  100  may also be used. The ratio of acrylamide and bisacrylamide can vary based on the desired stiffness of the completed hydrogel. The mixture may also contain 0.15% TEMED, 0.075% ammonium persulfate, and 1 mM sodium bisulfite (which acts as an oxygen scavenger). 
         [0048]    At stage  228 , the multi-pin array block  201  is aligned over the multi-well tray such that the each well  14  receives a pin and glass assembly  210 ,  20 . In an embodiment, the multi-pin array block can be constructed with a second multi-well tray, where each well is affixed to a rod and glass plate assembly  210 ,  20 , such that the ends of the rods that are affixed to the glass plate extend out of the second multi-well tray. In operation, the second multi-well tray can be inverted and aligned over the multi-well tray that contains the polymerization solution  16 , such that the glass plates  20  can be lowered into the wells  14 . 
         [0049]    At stage  230 , the glass plate  20  is pressed firmly against the polymerization solution  16  and the glass bottom  18 . As an example, and not a limitation, the thickness of the solution  16  (i.e., gel) after compression can be less than 100 microns (e.g., 10, 25, 50, 70, 80 microns). Depending on the assay, the thickness of the gel  16  can also be larger (e.g., 0.3, 0.5, 0.75, 1.0 mm). The volume of polymer solution added can impact the thickness of the resulting gel. The glass plate  20  remains in position until the solution  16  is completely polymerized (e.g., approximately 10-15 minutes, but longer time may be required for thicker gels). In an embodiment, the multi-well tray and pin-array assemblies can be placed in an anoxic chamber. For example, an nitrogen environment. 
         [0050]    At stage  232 , the glass plate  20  is removed from the well  14 . Typically, the plate  20  is removed by lifting while applying a lateral force such that plate  20  peels off of the gel  16 . Once the plate  20  is removed from each of the wells  14 , approximately 100 micro liters of distilled water  212  is added to the well to allow the gel  16  to hydrate. 
         [0051]    At stage  234 , the distilled water  212  is removed and the gel  16  is derivatized with a heterobifunctional crosslinker, and then a ligand. In an embodiment, 35 micro liters of 0/5 mg/ml Sulfo-SANPAH (N-Sulfosuccinimidyl-6-[4′-azido-2′-nitrophenylamino]hexanoate) in 50 mM HEPES buffer is added to each well  14  (i.e., for a well in a 96-well tray). The wells  14  can then be exposed to UV light for approximately 5 minutes. After exposure to the UV light, the sulfo-SANPAH solution can be removed, and the gels  16  can be rinsed with 100 micro liters of 50 mM HEPES. 100 micro liters of 100 ug/ml collagen I (pepsin-solubilized) in PBS can then be added to each well, and then incubated from approximately 4 hours at room temperature. The collagen I solution is then removed from the wells. Other crosslinkers and ligands as discussed above in relation to the process  100  can be used. In an embodiment, the polymerization solution  16  can be derivatized prior to being added to the well  14 . 
         [0052]    At stage  236 , the wells  14  and gel  16  is sterilized. In an embodiment, 200 micro liters of PBS is added to each well  14 . The wells  14  are then sterilized for approximately 2 hours under UV light  216 . The PBS is removed, and cells can be seeded in approximately 200 micro liters of media. 
         [0053]    Referring to  FIG. 5 , with further reference to  FIGS. 2 and 4 , in an embodiment, a 96-well plate can be configured so the shear modulus of the elastic gel  16  in each of the wells  14  varies columnwise. For example, the well columns can be loaded with gels  16  of increasing shear modulus in ascending order (i.e., 50, 100, 200, 400, 800, 1600, 3200, 6400, 12800, 25600 and 51200 Pa respectively). Further, a non-derivatized gels can serve as a background control for each shear modulus value. The rows of the plate can include different test cells and blanks as required for a test protocol. In an embodiment, the shear modulus values of the gels throughout a multi-well plate can be substantially similar. For example, for an assay in which shear modulus is consistent in the wells, and cell types vary. 
         [0054]    Referring to  FIG. 6 , with further reference to  FIGS. 2 and 4 , in an embodiment, a 384-well plate can be configured so the elastic modulus and the shear modulus of the elastic gel  16  in each of the wells  14  varies columnwise. For example, the gel  16  can have an elastic modulus of 150, 300, 600, 1200, 2400, 4800, 9600, 19200, 38400, 76800, and 153600 Pa, and a shear modulus of 50, 100, 200, 400, 800, 1600, 3200, 6400, 12800, 25600 and 51200 Pa. 
         [0055]    Referring to  FIG. 7 , with further reference to  FIGS. 2 ,  4  and  6 , a graph depicting human lung fibroblast cell growth in a 384-well plate containing hydrogels  16  with a range of shear moduls values is shown. The graph includes an x-axis  302  representing the shear modulus of the gels which the cells were grown. The scale of the x-axis  302  is logarithmic and ranges from 10 to 1,000,000 Pascal (Pa). The y-axis of the graph  304  represents the fold change in cell number (i.e., 2-fold, 3-fold) and has a range from 0 to 6-fold. Four groups of data are plotted on the graph  300 , including 0% serum  306 , 0.1% serum  308 , 1% serum  310 , and 10% serum  312 . The results plotted on the graph  300  are based on adult human lung fibroblasts (CCL-151) that were seeded in 384-well plates containing gels of different shear modulus values, and exposed to the indicated serum concentrations for a period of 48 hours. The results demonstrate that the number of cells increased at intermediate shear modulus, peaking at approximately 800 Pa. The right-most data points  314  represents cells grown on a rigid glass substrate. 
         [0056]    Referring to  FIG. 8 , with further reference to  FIGS. 2 ,  4  and  6 , a graph  320  depicting apoptosis in human lung fibroblast grown in a 384-well plate containing hydrogels  16  with a range of shear modulus values is shown. The graph includes an x-axis  302  representing the shear modulus of the gels which the cells were grown. The scale of the x-axis  302  is logarithmic and ranges from 10 to 1,000,000 Pascal (Pa). The y-axis of the graph  322  represents the relative caspase 3/7 activity per cell and has a range from 0 to 0.035. Four groups of data are plotted on the graph  300 , including 0% serum  306 , 0.1% serum  308 , 1% serum  310 , and 10% serum  312 . The results plotted on the graph  320  are based on adult human lung fibroblasts (CCL-151) that were seeded in 384-well plates containing gels of different shear modulus values, and exposed to the indicated serum concentrations for a period of 48 hours. The results demonstrate that caspase 3/7 activity, a marker of apoptosis, is elevated in cells grown on softer gels. The effect is potentiated when cells are grown in serum-free media. The right-most data points  314  represents cells grown on a rigid glass substrate. 
         [0057]    Referring to  FIG. 9 , with further reference to  FIGS. 2 ,  4  and  6 , graphs  330 ,  332 ,  334 ,  336 ,  338 ,  340  comparing the cell growth of different cell types as a function of the stiffness of the hydrogels  16  the cell are grown on are shown. Each of the graphs includes an x-axis representing the shear modulus of the gels which the cells were grown. The scale of the x-axis is logarithmic and ranges from 10 to 1,000,000 Pascal (Pa). The y-axis of the graphs represents the fold change in cell number (i.e., 2-fold, 3-fold) and have a range from 0 to 10-fold. The cells were seeded at a density of 100 cells/sq mm in 384-well plates and cultured for 48 hours in media containing 10% serum. The cells include fetal lung fibroblast  330 , adult lung fibroblast  332 , normal human lung fibroblast  334 , dermal fibroblast  336 , myoblast  338  and Madin-Darby canine kidney  340 . The results indicate that the change in cell number is based on shear modulus and the cell type. 
         [0058]    A compliant surface multi-well culture plate can be considered an upgrade to conventional 2-D cell cultures. In general, the compliant surface multi-well culture plate can meet the a needs for more realistic cell culture. For example, researchers have demonstrated that mesenchymal stem cell differentiation along different lineages can be accomplished simply by tuning extracellular stiffness. The compliant surface multi-well culture plate technology can be used to screen for a differentiation stiffness, and once identified, would serve as the substrate for all future studies. Further, mechanical and biochemical factors can be assessed in a combinatorial fashion to create a powerful discovery matrix. 
       Simulation of Physiological Conditions 
       [0059]    Most drug screens, particularly those for cancer, are performed with cells plated on rigid multi-well plates. There a number of chemotherapeutic drugs, however, which fail to inhibit proliferation on rigid plates, but succeed on soft substrates; there are examples of the reverse as well. In general, the knowledge of what mechanical properties (i.e., elastic modulus) will evoke a desired cell phenotype can be of tremendous value to tissue engineers in their design of 3-D scaffolds for tissue engineering. 
         [0060]    The compliant surface multi-well culture plate can also be used for disease-based research. In general, there is distinct stiffening (or softening) of tissue that can occur in a number of diseases, such as sclerodoma, atherosclerosis, emphysema, and fibrosis of the lung, liver and kidney. The compliant surface multi-well culture plate technology opens up the field to the assessment of stiffness-dependent cell behaviors at a level of detail that is not currently possible. For example, simulation of cell growth/behavior to contact with fat tissue is carried out by growing cells on a surface characterized by a shear modulus of fat (approximately 10 Pascal). Different tissue types are characterized by different stiffness, e.g., normal brain tissue has a shear modulus of approximately 200 Pascal. Cell growth/behavior also differs relative to the disease state of a given tissue, e.g., the shear modulus of normal mammary tissue is approximately 100 Pascal, whereas that of breast tumor tissue is approximately 2000 Pascal. Similarly, normal liver tissue has a shear modulus of approximately 300 Pascal compared to fibrotic liver tissue, which is characterized by a shear modulus of approximately 800 Pascal. Growth, signal transduction, gene or protein expression/secretion, as well as other physiologic parameters are altered in response to contact with different substrate stiffness and evaluated in response to contact with substrates characterized by mechanical properties that simulate different tissue types or disease states. 
         [0061]    Differentiation of stem cells is influenced by mechanical properties of the culture substrate. For example, stem cells, e.g., mesenchymal stem cells, cultured on a substrate with a shear modulus of approximately 300 Pascal induces differentiation to cells of a neurogenic phenotype. Stem cells exposed to an intermediate stiffness, e.g., 3000 Pascal, are induced to differentiate into myogenic tissue. In another example, stem cells grown on a substrate characterized by a relative stiff shear modulus (e.g., 10-15,000 Pascal) are induced to differentiate into cells with an osteogenic phenotype. Thus, substrates of varying stiffness described herein and devices containing those substrates are also useful to influence or drive differentiation of stem or progenitor cells toward one phenotype compared to a different phenotype based on contact with the mechanical properties of the 
         [0062]    Further, in phenotype-targeted cell culture, the general rigidity of plastic is an extreme condition that can induce cell phenotypes that may be undesirable, or not prevalent in native environments. The compliant surface multi-well culture plate technology can be particularly suited for the culture of neurons, which exhibit increased branching and growth on soft substrates. 
         [0063]    While the description above refers to the invention, the description may include more than one invention.