Abstract:
The present invention details the design and operation of a miniaturized device array in which a range of simultaneous mechanical forces are produced by a single external pressure source. The invention is primarily embodied in a microfabricated device arrays designed to rapidly probe biological cell response to various combinations of mechanical, chemical and extra-cellular matrix parameters in a high-throughput fashion.

Description:
PRIORITY INFORMATION 
     This application claims priority from U.S. Provisional Patent Application No. 60/976,069 filed Sep. 28, 2007. 
    
    
     FIELD OF THE INVENTION 
     The present invention relates to the field of microsystems and devices. The present invention in particular relates to an apparatus comprising an array of devices for applying strain to a material and methods of using said apparatus. The present invention includes applications in the fields of biomedical engineering. 
     BACKGROUND OF THE INVENTION 
     High-throughput screening (HTS) is a method used in life science research and the biopharmaceutical industry for drug discovery, toxicology testing, and functional genomics. Typically, HTS is used to rapidly determine the physiological response of groups of cells to various combinations and quantities of biologically active chemical compounds and biomaterials surrounding the cell. 
     Cellular activity is also influenced by applied mechanical stimulation, which has been shown to have a strong impact on biological function in certain types of cells (McBeath, et al.,  Dev. Cell  2004; Wang &amp; Thampatty,  Biomech Model Mechanobiol  2006; Saha et al.,  J Cell Physiol,  2006). Existing experimental techniques are unable to adequately characterize cellular response to varying degrees of mechanical stimulation with a high accuracy in a high-throughput manner. These limitations have prevented systematic investigations into the effects of mechanical stimuli on cell behaviour and hindered discovery of new control strategies for cell-based therapies. 
     Furthermore, despite the demonstrated individual importance of mechanical forces; chemical cues; and the composition and structure of surrounding biomaterials in regulated cellular function, the lack of HTS techniques for mechanical factors precludes the ability to effectively study combinations of these various parameters. This patent application discloses a system designed to meet this need for rapidly probing either single cells or colonies of cells. 
     Existing low-throughput experimental techniques in this field make use of three main mechanical loading schemes to probe cellular response: compressive loading, deformation of the substrate to which cells adhere, and fluid flow-induced shear stresses. U.S. Pat. No. 6,048,723 discloses a flexible bottom culture plate for applying mechanical loads to cell culture; U.S. Pat. No. 6,218,178 discloses the loading assembly for the plates of U.S. Pat. No. 6,048,723; U.S. Pat. No. 6,645,759 discloses a device for growing cells in culture under shear stress and/or strain; and U.S. Pat. No. 6,037,141 discloses a system for culturing cells under compression conditions. However, the systems described the cited US patents are all low-throughput, applying a single strain across at most, six experimental locations. This drawback significantly impacts the time required to perform such studies. It also precludes the ability to perform combinatorial manipulation of chemical and mechanical parameters, as can be performed in our disclosed invention. 
     Moreover, there are two modes of cell culture: two-dimensional culture on a flat surface, and three-dimensional culture within a porous biomaterial. Each of these culture techniques and loading scenarios provide insight into the inner workings of the cell, but typically require radically different experimental setups. 
     Microsystems are engineered systems with critical structural or functional features of micrometers, where the microfabricated component of the system typically ranges in size from millimeters to centimeters. They have such advantages as low cost, small size, minimal reagent consumption and fast response time. Because of the reduced system footprint, a dense array of functional sub-units is possible, and as such they are ideal for developing array based HTS systems. Similarities between system feature sizes and the size of a cell make this technology suitable for developing HTS systems for single- or multi-cell biology. Advances in microfabrication have enabled the rapid development of complex, elastomeric, monolithic polymer structures with well-defined features with a resolution of micrometers. To provide an example, these techniques—termed Multilayer Soft Lithography (MSL)—have been used to develop a fully controllable microfluidic network, actuated by a number of 2-state valves (Unger et al., “Monolithic microfabricated valves and pumps by multilayer soft lithography,” Science, vol. 288, pp. 113-6, Apr. 7 2000; and U.S. Pat. No. 6,793,753). 
     In view of the foregoing, an improved apparatus, system and method for HTS applications is desirable. 
     The disclosed invention introduces new aspects in MSL device development, including the use of mechanical solid elements in an all-polymer device, and the application of a single pressure load to obtain a range of mechanical activity. 
     SUMMARY OF THE INVENTION 
     The present invention relates to an apparatus for applying mechanical forces of varying magnitudes to a material and methods of using said apparatus. 
     In one aspect, the present invention is an apparatus for applying mechanical forces of varying magnitudes to a material characterized in that the apparatus comprises at least one array defining a surface and a plurality of actuation devices disposed thereon, each of said actuation devices having a structural configuration, said structural configuration including an opening; and a flexible membrane fixed to the surface and covering said opening, said membrane having an upper surface that permits attachment of the material thereto, wherein the array is structured to enable pressure or vacuum to be delivered to the plurality of actuation devices, and wherein the array is further structured to enable variation of said structural configuration from actuation device to actuation device such that the delivery of pressure or vacuum to the plurality of actuation devices results in application of varying magnitudes of mechanical force to the material by means of actuation of the flexible membrane covering said openings_based on the structural configuration thereof. 
     In one aspect the strain fields produced by the mechanical, forces on the material comprise non-uniform strain fields of varying magnitudes on the material. 
     In another aspect, the present invention is an apparatus for applying mechanical forces of varying magnitudes to a material comprising at least one array defining a surface and a plurality of actuation devices disposed thereon, each of said actuation devices having a structural configuration, said structural configuration including: (i) a base including a first opening, (ii) a flexible actuation membrane fixed to the base and covering said first opening, said actuation membrane having an upper surface; and (iii) an upper structure resting on said upper surface of the actuation membrane and including a second opening that opens on the surface; a moving member extending from the upper surface of the actuation membrane into the upper structure towards the second opening; a substrate membrane fixed to the surface and covering said second opening, said substrate membrane having an upper surface that permits attachment of the material thereto, wherein the array is structured to enable pressure or vacuum to be delivered to the plurality of actuation devices, and wherein the array is further structured to enable variation of said structural configuration from actuation device to actuation device such that the delivery of pressure or vacuum to the plurality of actuation devices results in application of varying magnitudes of mechanical force to the material by means of actuation of the actuation membrane covering said first openings based on the structural configuration thereof thereby moving said moving member to direct the mechanical force to the material. 
     In one aspect of the disclosed invention, the strain fields comprise various uniform strain fields of varying magnitudes on the material. 
     In yet another aspect of the invention is an apparatus for applying mechanical forces of varying magnitudes to a material characterized in that the apparatus comprises: at least one array defining a surface and a plurality of actuation devices disposed thereon, each of said actuation devices having a structural configuration, said structural configuration including an opening; a flexible membrane fixed to the surface and covering said opening, said membrane having an upper surface; a moving member extending from the upper side of the membrane and having a top that permits attachment of the material thereto; and a weight means, wherein the array is structured to enable pressure or vacuum to be delivered to the plurality of actuation devices, and wherein the array is further structured to enable variation of said structural configuration from actuation device to actuation device such that the delivery of pressure or vacuum to the plurality of actuation devices results in application of varying magnitudes of mechanical force to the material by means of actuation of the flexible membrane covering said openings based on the structural configuration thereof thereby moving said moving member to compress the material against the weight means. 
     In a further aspect of the present invention is a method of high-throughput screening responses of a material to mechanical forces of varying magnitudes, characterised in that the method comprises: providing an apparatus of the invention; delivering pressure or vacuum to the apparatus; and measuring the effect of said mechanical forces on the material. 
     Non-limiting advantages of the apparatus of the present invention include an apparatus that allows high-throughput screening and large out-of-plane actuation distances, which are difficult to achieve in a traditional low-throughput apparatus. Another advantage of the apparatus of the present invention comprises the capability of translating a single input pressure into mechanical forces of varying magnitudes. Yet another advantage of the present invention includes a single apparatus capable of delivering mechanical stimulation and chemical stimulation simultaneously to a material of interest. Yet another advantage of the present invention includes a single apparatus capable of delivering a number of mechanical loading schemes simultaneously to a material of interest. Other advantages of the present invention will become apparent in the description of this invention. 
    
    
     
       BRIEF DESCRIPTION OF DRAWINGS 
         FIG. 1A : illustrates a radial distribution network of channels to supply pressure or lubricant to individual units in the microfabricated array. 
         FIG. 1B : illustrates a branching distribution network of channels to supply pressure or lubricant to individual units in the microfabricated array. 
         FIG. 2 : top-down schematic of the non-uniform substrate strain embodiment of the invention, illustrating the varying size of the actuation cavities across the array. 
         FIG. 3 : cross-sectional view of non-uniform substrate strain embodiment of the invention, illustrating the actuation cavity, and polymeric thin film. 
         FIG. 4 : demonstrates the working principles of this embodiment of the invention. Increases in actuation cavity size create different vertical displacements. 
         FIG. 5 : Finite element simulations displaying radial and circumferential strains obtained across the radius of bulged films of different sizes. 
         FIG. 6 : Picture of sample microfabricated device for the substrate strain embodiment of the invention. 
         FIG. 7 : Image of the non-uniform substrate strain microsystem in the cell culture incubator, with associated peripheral devices (including pump, valves and controllers). 
         FIG. 8 : Graph presenting results for percentage of cells expressing alpha-smooth muscle actin across the mechanically active culture regions of the array. 
         FIG. 9 : illustrates the working principles of the uniform substrate strain embodiment of the invention. As pressure is applied to the actuation cavity beneath the support layer, the loading post is driven upwards into the culture membrane. 
         FIG. 10 : demonstrates the working principles of this embodiment of the invention. Increases in actuation cavity size create increases in vertical displacements of the loading post, for a given applied pressure. 
         FIG. 11A-F : Sequence of images from the finite element analyses performed for this embodiment of the device. 
         FIG. 12 : Graphical representation of the radial and circumferential strains obtained across the surface of the device, for different sizes of actuation cavity, for a circular loading post profile. 
         FIG. 13 : Finite element simulation results for a square loading post profile. 
         FIG. 14A-E : demonstrates variations in various segments of the material area. 
         FIG. 15A : illustrates the 5×5 array produced as an example of this embodiment of the invention. 
         FIG. 15B : top down view of the example array fabricated as an embodiment of this invention. 
         FIG. 15C : images of the device at rest and while actuated. 
         FIG. 16 : example of a larger array for the uniform substrate-strain embodiment of this invention. 
         FIG. 17 : illustrates the displacement of fluorescent beads on the surface of the membrane, used to calibrate the strains produced by the device. 
         FIG. 18 : Graph representing radial and circumferential strains results obtained from analysis of the fluorescent bead displacements. 
         FIG. 19 : Graph presenting results for the fluorescent bead calibration across the array. 
         FIG. 20 : Fluorescently stained image where Blue=Hoechst nuclear stain, and Red=BrdU stain for proliferating cells. 
         FIG. 21 : Graph presenting results for the fraction of proliferating cells across mechanically active culture regions of the array. 
         FIG. 22 : Schematic illustrating the incorporation of microfluidic channels to deliver and control available chemical factors. 
         FIG. 23A , B: illustrates the procedure by which hydrogels can be micropatterned onto the device, creating an array of three dimensional constructs. 
         FIG. 24A , B: Schematic illustrating compressive loading of the constructs. 
         FIG. 25 : Image demonstrating micropatterning of polyethylene glycol into an array of hydrogel cylinders. 
         FIG. 26 : Cross-sectional view of the hydrogel cylinders fabricated. 
         FIG. 27 : Schematic of peripheral setups for each embodiment of the invention. 
     
    
    
     In the drawings, one or more embodiments of the present invention are illustrated by way of example. It is to be expressly understood that the description and drawings are only for the purpose of illustration and as an aid to understanding, and are not intended as a definition of the limits of the present invention. 
     DETAILED DESCRIPTION OF THE INVENTION 
     The present invention is particularly useful for applying a range of mechanical forces to a material across length scales on the order of micrometers and millimeters. This novel actuation scheme is versatile, and can be used in several configurations and for various purposes. Examples related to uniform and non-uniform substrate-stretch and tissue construct deformation with multiple loading modes are outlined in this disclosed invention. The various embodiments of the invention can be used to apply: (a) non-uniform strain fields of varying magnitudes to a material sample of interest; (b) uniform strain fields of varying magnitudes to a material sample of interest; and (c) compressive stresses of varying magnitudes to a three-dimensional construct. 
     Commonalities between each of the embodiments will be described first, followed by details relevant to specific configurations. 
     “Material” as used herein should be understood to indicate any material of interest, including without limitation organic and inorganic materials, films, a combination of multiple substances into an aggregate mixture, cells, tissues, organs, cell cultures. 
     The major structural components of the apparatus may be made, for example, from polydimethylsiloxane (PDMS, Sylgard 184, DOW CORNING™). The apparatus of the invention may be fabricated using principles of multilayer soft lithography (MSL) in several layers, each layer is formed by casting the liquid prepolymer onto a negative relief mold. The layers are then aligned and bonded to create complex multilevel structures. The integration of other membrane types is also permitted through the use of uncured PDMS as an adhesive layer. Through such bonding techniques, when the material to be tested include cell cultures, membranes of other polymers, including but not limited to polyurethane, polyacrylamide, or a custom-designed polymer membrane can be used as a substrate for the cell culture. The inventors have successfully demonstrated this technique to integrate alternative materials into the PDMS fabrication process with membranes of polydimethylsiloxane and polyurethane. Although not necessary for the utility of the present invention, limiting these membranes to optically transparent materials enables the use of inverted microscopy, a standard tool in biology labs used to visually examine cells and to observe fluorescent reporters and reagents. The transparent feature is advantageous because it enables spatially and temporally heterogeneous cell responses to be visually detected, which is not possible if an assay only measures the end-point response of the entire population, as is typical with HTS. 
     The apparatus of the present invention allows for large out-of-plane actuation distances, which are difficult to achieve in a traditional microdevice. Although the magnitude of strain fields can be varied by changing the pressure applied, this would require several external pump systems, to obtain a variety of strain magnitudes in one device. The present inventors have solved the problem of requiring several external pump systems by providing a mechanical design solution. In order to apply a range of mechanical forces across the microfabricated array in each of the embodiments, variations in geometry are employed. A single external pressure and vacuum source is connected to the apparatus of the invention, which by means of a network of microfabricated channels delivers pressure or vacuum to each of the individual units (also known as “actuation devices”) in the array.  FIG. 1A  and  FIG. 1B  illustrate examples of such pressure delivery channel network  22 . Variations in geometric dimensions of individual units  10  in the array are used to vary the amount of mechanical force generated by that actuation device  10 . The mechanisms for generating the types of mechanical forces are outlined in the following embodiments. 
     In one aspect the present invention is an apparatus for applying mechanical forces of varying magnitudes to a material characterized in that the apparatus comprises: at least one array defining a surface and a plurality of actuation devices disposed thereon, each of said actuation devices having a structural configuration, said structural configuration including an opening; and a flexible membrane fixed to the surface and covering said opening, said membrane having an upper surface that permits attachment of the material thereto, wherein the array is structured to enable pressure or vacuum to be delivered to the plurality of actuation devices, and wherein the array is further structured to enable variation of said structural configuration from actuation device to actuation device such that the delivery of pressure or vacuum to the plurality of actuation devices results in application of varying magnitudes of mechanical force to the material by means of actuation of the flexible membrane covering said openings_based on the structural configuration thereof. 
     A top-down schematic of the array  1  is shown in  FIG. 2 . A cross-sectional view of a single actuation device  10  in the array  1  is provided in  FIG. 3 , showing the single unit  10  in the array  1  at rest. The actuation device  10  comprises a structural configuration including an opening  9 . The actuation device  10  includes an actuation cavity  18  including a cavity wall  12  having a thickness  13  and a bottom wall  14 . A flexible membrane  20  is fixed to the array surface (not shown) and covering the opening  9 . The flexible membrane  20  has an upper surface  21  that permits attachment of a material of interest. In one aspect the flexible membrane  20  comprises a thin polymer film. Applying a positive or negative pressure within the actuation cavity  18  bows the thin film  20  upwards or downwards.  FIG. 4  illustrates the use of variation in geometry to provide different non-uniform strain fields. The left side of the figure shows cross-sectional views of actuation devices with two dimensions of the actuation cavity  18 . A pair of dimensions are used merely for illustration purposes. The right side of the figure shows the effect of varying actuation cavity  18  geometry on the bending applied to the thin polymeric film  20  under the same positive pressure. By decreasing the thickness  13  of the cavity wall  12  the unsupported membrane  20  over the actuation cavity  18  increases, the stiffness of that membrane  20  decreases, and the membrane  20  is bowed further.  FIG. 5  shows finite element simulation results for various actuation geometries of a circular unit. The results show non-uniform strain fields in the radial and circumferential directions of the polymeric film  20 . The finite element analysis shown in  FIG. 5  simulates a circular membrane being deformed by a pressure applied beneath it. These simulations were conducted using the same applied pressure for circular membranes of various dimensions, ranging from diameters of 500 μm to 1.6 mm in 100 μm increments. A three-quarter view of a representative simulation is provided to better illustrate the function of this embodiment. Radial and circumferential strains across the surface of the membrane are presented in graphical form. The results indicate non-uniform strain fields across the surface of the membrane, with unequal radial and circumferential components of the applied strains. 
     To demonstrate the applicability of the first embodiment of the present invention,  FIG. 6  depicts an apparatus  5  of the present invention having circular actuation cavities  18 , however other obvious patterns may be used ( FIG. 6 ). In the apparatus of  FIG. 6 , the circular actuation cavities  18  are used to provide strain fields similar to those shown in the finite element simulations of  FIG. 5 . However, various strain fields can also be generated by changing the shape of the actuation cavity  18 . This requires no change to the actual manufacturing process of the apparatus, and can be achieved by modifying the template used to build the apparatus. The sample apparatus  5  of  FIG. 6  shows six isolated, identical groups, each of which contains twenty mechanically active actuation sites  10 . By applying a single positive pressure to the entire apparatus  5  (not shown), the channel network  22  delivers the pressure from a source of pressure to each of the mechanically active actuation sites  10  in all the isolated groups, bowing the membranes, the out-of-plane displacement of which is commensurate with the actuation cavity geometry. In this example, polyurethane films are bonded to the surface of the PDMS device. 
     In one non-limiting example the experimental setup of  FIG. 6  is being used to apply a range of non-uniform strain fields to adherent biological cells cultured on the surface of the array  1 . The setup shown in a cell culture incubator is shown in  FIG. 7 . The well  7  shown in the apparatus  5  is used to hold cell culture media, which allows the array  1  in the apparatus  5  to respond to a specific set of chemical factors in the culture media. In this specific example extra-cellular matrix (ECM) proteins are deposited by adsorption on the surface of the polyhrethane films—these ECM proteins can include but are not limited to collagen (Types I-IV), fibronectin, laminin, vinculin and heparin. Each array  1  in each apparatus  5  can be patterned with a different ECM protein at different concentrations. A further extension to this example would be achieved by employing well-established techniques for protein patterning, such as those described in R. S. Kane et al., “Patterning proteins and cells using soft lithography,”  Biomaterials , vol. 20, pp. 2363-2376, December 1999, which can be used to deposit on each actuation site  10  in the array  1  various types and concentrations of proteins. This will allow control over extra-cellular matrix composition for individual bioreactor units. 
     Specific to this particular experiment, subcultured porcine aortic valvular interstitial cells (PAVICs) isolated from pig heart valve leaflets were seeded on the surface of the array and allowed to attach and spread without mechanical stimulation. This was achieved using standard cell culture techniques. Initial experiments involved applying a cyclic mechanical deformation to the film upon which the cells were attached, over a period of two days. Analysis of the effects of mechanical stimulation involve staining the cells for the presence of a-smooth muscle actin (αSMA), a mechanosensitive cytoskeletal protein. Fluorescent imaging and analysis yielded results shown in  FIG. 8  for the percentage of cells expressing αSMA from the total population on each experimental unit. This experiment demonstrates the practicality of collecting data on biological cell response through fluorescent imaging techniques. Various other combinations of ECM proteins, culture media composition and mechanical stimulation to tease out differences in biological activity in response to varying non-uniform strain fields may be studied with the use of the apparatus of the present invention. 
     The apparatus of the present invention is useful to probe all adherent cell types, including but not limited to heterogeneous cell populations, stem cells, progenitor cells, primary isolates, and cell lines, which has broad scope for use in experiments in biomedical research. Possible applications include determining the effects of various external stimuli in combination with non-uniform cyclic mechanical strain on cells, including but not limited to levels of drug uptake, efficacy of gene therapy, receptor formation, cytokine production, proliferation, apoptosis, structural reorganization, morphology, gene and protein expression, and differentiation on a large number of cell types from various model organisms. 
     The apparatus of the present invention could also be used to applying varying non-uniform strains to native tissue samples, cells encapsulated in a thin membrane, or as a material testing unit for thin polymer films. This last application is of particular relevance to the materials science community, looking for novel experimental methods to test mechanical properties of thin films, membranes and biological tissue samples, which have been shown to have different properties than when in their bulk forms. Previously patented techniques include mechanical characterization through laser excitation (U.S. Pat. No. 5,672,830); microindentation using piezoelectric positioners (U.S. Pat. No. 5,553,486). These techniques are serial in nature, and cannot collect data quickly. A more recent attempt to create a high-throughput system has been patented (U.S. Pat. No. 6,772,642), in which an array of samples is tested by a positionable force generator. However, data collection is still serial. 
     This potential setup has the advantage of higher throughput over current attempts—a series of data for responses to a range of mechanical forces is collected simultaneously. In one aspect a sample of the material of interest, such as a thin membrane of the material or biological tissue to be studied is bonded by an adhesive agent to the surface of an array and suspended over a series of actuation devices with increasing radii. By applying a controlled positive pressure and determining the vertical displacement of the membrane, the stiffness and Young&#39;s modulus of the film can be determined. Increasing the pressure to breakage determines ultimate stress properties of the material. Continuous cycling of the pressure source determines fatigue, elasticity and plasticity. Because of the device throughput, a great deal of data for various stresses can be obtained simultaneously. 
       FIGS. 9 through 13  depict the mechanical principles of the second embodiment of the present invention. With reference to  FIG. 9 , each actuation device  110  comprises: a base  120  including a first opening  121  and comprising a first actuation cavity  126  including a cavity wall  122  having a thickness  123  and a bottom wall  124 . The said cavity wall  122  has an upper end  128  configured to fix a flexible actuation membrane  130  that covers the opening  121 , said actuation membrane  130  having an upper side  132 ; an upper structure  140  comprising a side wall  142  that ends on the array surface  144 , a second opening  146  and a second cavity  148 . A substrate membrane  220  is fixed to the surface  144  and covers the second opening  146 . The substrate membrane  220  is configured to support a material of interest  222  across the opening  146 ; a post  160  extending from the upper side  132  of the actuation membrane  130  into the second cavity  148  towards the second opening  146 . 
     As a non-limiting example,  FIG. 9  demonstrates the principle for applying a substrate-induced deformation to adherent cells in a two-dimensional culture. The upper diagram illustrates the cross-sectional view of a single unit of the actuation device  110  at rest. The actuation membrane  130  then bows upwards, driving the post  160  up into the substrate membrane  220 , atop which adherent cells  222  are cultured. The culture membrane  220  then slips and stretches over the raised loading post  160 , creating a uniform strain field, the features of which depends on the geometry of the loading post  160 . Examples include but are not limited to: a circular post, which will create a cylindrical equibiaxial strain field; a square post which will create an equibiaxial strain field; and a rectangular post which will create an anisotropic biaxial strain field, approaching uniaxial strains. As in the previous embodiment, this embodiment of the system can be used to probe all adherent cell types, including but not limited to heterogeneous cell populations, stem cells, progenitor cells, primary isolates, and cell lines. 
     The use of varying geometry to change mechanical forces applied is demonstrated in  FIG. 10 , which uses the substrate-stretch embodiment of this invention as a descriptive aid. The right half of the image depicts a cross-sectional view of the actuation device  110  with no external positive pressure applied (at rest). The device  110  of lower quadrant of  FIG. 10  features a much larger size actuation cavity  126  than the upper quadrant of  FIG. 10 . As the size of the unsupported actuation membrane  130  increases, the stiffness of the membrane  130  decreases, and the post  160  is vertically displaced further. In this way, by varying the lateral geometry of the system, the vertical actuation distance is varied for a fixed applied pressure, and hence the strains generated in the culture membrane  220  are also varied. The magnitudes of generated strain fields are limited by the material&#39;s ultimate elastic strain: preliminary finite element simulations indicate a range from 0-5% strain for our specific initial design—however, based on experimental results, the design can be realistically adjusted to provide mechanical stimulation ranging from 0 to approximately 20% strain. 
     To better illustrate the mechanical actuation of the system, a sequence of images taken from a finite element simulation have been included. The simulation depicts a circular loading post as an example, in a substrate-stretch configuration. The simulation assumes frictionless interaction between the post and the membrane. The images shown in  FIG. 11A-F  show how the post is driven up into the membrane, causing stretch to occur. Note that this sequence is not indicative of any specific elapsed time. The quantitative results for this simulation are shown in  FIG. 12 . The radial and circumferential strains along the membrane surface are graphically displayed. This exercise confirms an equibiaxial stretch for this particular situation, and indicates a region of uniform strain within the radius of the loading post. Two-dimensional simulations were also performed for a square post geometry: results for a section of square post geometries indicate similarly uniform results, and are shown in  FIG. 13 . 
     The fabrication process for this second embodiment of the device  110  may be based on known standard processes of multilayer soft lithography. No claims of novelty are made on these techniques. Essentially, a negative relief mold is created for each layer of the device, again by standard processes. Two examples are provided for illustration and not, limitation. The first is the use of Microchem&#39;s SU-8 negative photoresist to pattern molds of various thicknesses. Alternatively, silicon micromachining in a silicon-on-insulator wafer can be used with Deep Reactive Ion Etching to create molds with vertical side walls and flat bottoms. A liquid prepolymer (for example, PDMS) is poured over the mold and temperature-cured. The PDMS can then be peeled off the mold and it retains the microscale features. PDMS can also be spin coated on a second mold, resulting in a very thin patterned film. Alternatively, the technique developed by Jo et al., (B. H. Jo, L. M. Van Lerberghe, K. M. Motsegood, and D. J. Beebe, “Three-dimensional micro-channel fabrication in polydimethylsiloxane (PDMS) elastomer,”  Journal of Microelectromechanical Systems , vol. 9, pp. 76-81, March 2000.) can be used in which the liquid polymer layer is squeezed in a mechanical clamp, creating a very thin film. These films are then aligned using a micromanipulator and bonded together by treating the surfaces with a corona discharge unit. 
     One aspect of novelty is introduced into the fabrication process. When bonding the culture membrane to the first three layers of the device, in order to prevent the partially cured culture membrane from bonding to the post, a vacuum is applied to the actuation membrane, sucking the posts away from the culture membrane. The membrane is then bonded, and cured while the actuation membrane is under vacuum. Low viscosity oil is heated to further reduce viscosity and then flushed between the post and the actuation membrane, providing a lubricating layer, and preventing excessive friction. When the vacuum is released the loading post returns to its original position, unattached to the culture membrane, and lubricated by the oil. 
     To illustrate the range of other design considerations encompassed by the present invention, a number of modifications have been made to the design of the device  110 , and shown in  FIG. 14A-E . In  FIG. 14A , a different material is used for one of the structural layers of the device  110 .  FIG. 14B  shows a modified structural configuration in which structural means or notches  150  extend from surface  144  of the array are used to limit vertical movement of the actuation membrane  130 .  FIG. 14C  incorporates the use of a ‘lip’  162  on the loading post  160  to reduce the friction between the post and the culture membrane  220 , by reducing the total area of contact.  FIG. 14D  demonstrates one of the possibilities of actuation cavity geometry  170  achieved through a different fabrication process.  FIG. 14E  shows a post  164  profile that is different in the vertical as well as the planar directions. 
     To demonstrate the practicality and feasibility of such a system, a sample 5×5 array of individual units was constructed ( FIG. 15A ). Actuation of the structure is demonstrated in  FIGS. 15B  and C. Arrays with larger number of units can easily be fabricated ( FIG. 16 ), but for demonstration and initial experimentation purposes, a 5×5 array was used. This array has been successfully constructed with a polydimethylsiloxane culture layer, or with a polyurethane culture layer, using bonding techniques discussed previously. The density of the experimental units is equivalent to that of a 1536-well plate, which can provide a 256-fold increase over currently available substrate-stretching equipment. 
     In order to calibrate the strains exerted by the device, fluorescent beads, 1 micron in diameter were deposited on the surface, and imaged in a standard fluorescent microscope. The array was then actuated, and the locations of the fluorescent beads tracked, using standard image analysis techniques. The radial and circumferential strains across the surface of the membrane were then extracted from the raw displacement data, and the results plotted in  FIGS. 18A  and B. The graphs indicate uniform strains across the surface, and an equibiaxial condition (equal radial and circumferential strains). Deviations from uniformity can be attributed to errors in measurement of the fluorescent bead positions. The nominal strain values for the radial and circumferential axes were then tabulated for each of the differently-sized units in the array. The results for a polyurethane membrane are shown in  FIG. 19 , and indicate an increasing strain level across the array, as demonstrated by simulation. 
     The specific application of the particular experimental setup constructed is to provide uniform substrate strains as mechanical stimuli to determine the effects on biological cells grown on the culture membrane surface. As in the first embodiment of this invention, a PDMS well is used to hold cell culture media, to control the chemical stimuli seen by the cells, and to deposit ECM proteins prior to seeding the adherent cells. Also as in the first embodiment, standard techniques can also be used to pattern ECM protein type and concentration on individual units of the array. For a demonstration experiment, a mesenchymal stem cell line (C3H10T1/2) was seeded onto a polyurethane membrane, and subjected to cyclic 1 Hz strains ranging from 0 to 8% in 2% increments. The BrdU stain for proliferating cells was then used to determine the fraction of total cells that were proliferating, for each of the mechanically active regions (sample image shown in  FIG. 20 ). Obtaining fluorescent images can require purging the oil lubrication channels if the oil autofluoresces at a specific excitation wavelength. This can be done with a soap and water solution. The results, shown in  FIG. 21 , demonstrate the practicality of using fluorescent analysis techniques to obtain data from cells cultured on the apparatus of the present invention. 
     Although the apparatus of the present invention has been used for particular applications, the description of such is not intended to limit the scope of this invention. Theoretically, any culture membrane material that can be processed into a thin film can be used on the device. Any adherent cell type can be used, and because of the 1536-well plate format, currently available robotic dispensing is capable of controlling the chemical environment for individual units within the array. Furthermore, a microfluidic network can be incorporated (as in  FIG. 22 ) to deliver precise quantities and combinations of chemicals to individual cell locations. Provided the chemicals are in liquid form, they can be distributed to each individual bioreactor. Examples of such chemical stimulation can include but are not limited to growth factors, hormones, cytokines, dissolved gases, and bioactive molecules. With this configuration, the bioreactor array can combinatorially probe cellular response to various mechanical strains, chemical cues, and extra-cellular matrix compositions. Controlling the flow rate of chemicals in the microfluidic channel also allows control over shear stresses exerted on the cells. Variations in shape of the loading post can create different strain fields in the culture membrane. 
     With reference to  FIGS. 22 through 26  in a third embodiment, the present invention discloses a modification to the structure of the device  310 , which allows compressive strains to be applied to a three-dimensional construct. This embodiment makes use of the lower portion of the above described embodiments—the culture membrane is removed entirely and the supporting structure for the culture membrane can optionally be removed. Three-dimensional constructs can then be fabricated with standard materials, including but not limited to natural or synthetic hydrogels, porous polymeric scaffolds, other tissue engineering scaffolds, biomaterials for cell encapsulation, native tissue or a custom-designed material. These constructs can be patterned and seeded with cells, using standard techniques (such as in Liu &amp; Bhatia: “Three-dimensional Photopatterning of Hydrogels containing Living Cells”,  Biomedical Microdevices,  4, p 257, 2004). 
       FIGS. 23A-B  illustrate the process by which a patterned hydrogel is photopolymerized atop the loading posts: a cell suspension and prepolymer mixture is flushed between the loading posts and a chemically functionalized glass slide. A mask is then aligned and placed atop the glass slide. UV light is then used to photopolymerize the array over the loading posts.  FIG. 24  demonstrates compressive loading of the structure. Compressive loading is achieved using the same mechanism as in the previous embodiment: a pressure-actuated loading post atop an actuation cavity of varying dimensions. The loading posts squeeze the constructs against a holding means, which in this case comprises the functionalized glass slide. 
     This embodiment of the system can be used to study both adherent and non-adherent cell types. Non-adherent cell types would necessitate the use of an encapsulating polymer as the construct. Obtaining this setup is achievable by a number of methods—an alternative method is provided here: A cell suspension in pre-polymer solution can be prepared and patterned onto a glass substrate. The patterned constructs are then aligned with the array and spaced by means of a gasket. The setup is then mounted in a light clamp with appropriately flexible spacers. A positive pressure applied to the actuation cavity will bow the post upwards, compressing the construct ( FIG. 24 ). Cycling the pressure will result in a dynamic, high-throughput compressive bioreactor array. 
     To demonstrate the practicality of this approach, an array of cells encapsulated within photopolymerizable polyethylene-glycol (PEG) constructs are photopatterned on a chemically functionalized glass slide using a standard masking technique: The unpolymerized solution is then washed away, and the results, shown in  FIG. 25  demonstrate the formation of large arrays of micropatterned cylinders.  FIG. 26  illustrates a side view of one of the constructs. These arrays are then aligned with the device and clamped into place using a suitable supporting structure, for dynamic mechanical compressive stimulation. 
     The following numbers are reasonable estimates. The strain ranges for compressive testing are estimated to range between 0 and 80%—the upper end is limited by the porosity and stiffness of the construct, and the lower limit would depend on the size of the desired construct. Tensile testing can be expected to generate strains between 0 and 200%, based on finite element results for the distention of the loading post, and the minimum size of a desirable construct. 
     An idealized peripheral setup and integrated system for each of the embodiments described above is outlined in  FIG. 27 , which includes a computer controlled pump with a pressure sensor and valve to form a closed-loop control system for accurately applying dynamic pressures to the reactor array. The computer also controls chemical feed rates through a multichannel peristaltic or syringe pump, which provide nutrients to the cells, and can also be used to apply shear forces to each cell. The required control algorithms are simple and readily available in the public domain. The entire array is mounted in a ‘live cell’ imaging chamber, on an automated motion stage. The live-cell imaging system allows the cells to survive under a microscope for an extended period of time, and the motion stage will allow the microscope to take pictures of each unit at various time intervals. All this data can then be time-stamped and catalogued, and saved on the computer for subsequent automated or manual analysis.