High-throughput durotaxis assay plate

Abstract

A method of preparing a hydrogel array comprising a multi-well plate includes preparing a polyacrylamide (PA) hydrogel with alternating stiffnesses. Preparing the PA hydrogel includes preparing a polymer solution, plasma treating the multi-well plate, attaching a patterned mask to a plate containing the polymer solution, the plate comprising a plurality of wells, and treating the polymer solution with UV light to form the PA hydrogel, the PA hydrogel having first regions with a first stiffness and second regions with a second stiffness, the first stiffness being greater than the second stiffness.

Description

HIGH-THROUGHPUT DUROTAXIS ASSAY PLATECROSS-REFERENCE TO RELATED APPLICATIONS[00011 This application claims the benefit of and priority to U.S. Application No.63 / 640,797, filed on April 30, 2024, and U.S. Application No. 63 / 646,456, filed on May 13, 2024, the entire disclosures of which are hereby incorporated by reference herein.STATEMENT OF GOVERNMENT SUPPORT

[0002] This invention was made with government support under HL160507 and NS116802, awarded by the National Institutes of Health. The government has certain rights in the invention.TECHNICAL FIELD

[0003] The present disclosure relates generally to the field of hydrogel assay preparation. Specifically, the present disclosure relates to preparing hydrogel arrays and assessing cell substrate interactions using the hydrogel compositions and transparent mask.BACKGROUND

[0004] Durotaxis is a type of cell migration, where cells migrate based on rigidity gradients arising from various structural properties of a substrate or other extracellular matrix. Two- dimensional durotaxis models can be used in a variety of applications including, but not limited to, small-molecule screenings.SUMMARY OF THE INVENTION

[0005] One aspect of the present disclosure relates to a method of preparing a hydrogel array comprising a multi-well plate. The method includes preparing a polyacrylamide (PA) hydrogel. Preparing the PA hydrogel includes preparing a polymer solution, plasma treating the multi-well plate, attaching a patterned mask to a plate containing the polymer solution, the plate comprising a plurality of wells, and treating the polymer solution with UV light to form the PA hydrogel. The PA hydrogel has a plurality of first regions with a first stiffnessand a plurality of second regions with a second stiffness, the first stiffness being greater than the second stiffness.

[0006] In various embodiments, the plate is a 96-well plate. In various implementations, a height of the plurality of first regions is the same as a height of the plurality of second regions. In some implementations, the plurality of first regions and the plurality of second regions are positioned in an alternating arrangement so as to form a striated configuration. In other implementations, a first width of each of the plurality of first regions is approximately 620 microns. In yet other implementations, a second width of each of the plurality of second regions is approximately 1.2 millimeters. In various implementations, treating the polymer solution with UV light includes providing UV light to the bottom surface of the plate so light radiates upward through the plate (i.e., to react with a photoinitiator molecule in the PA solution and cause gelation). In some implementations, the patterned mask comprises mylar. In other implementations, the PA hydrogel comprises between 3% and 8% acrylamide and about 0.48% Bis-acrylamide. In yet other implementations, the method also includes rinsing the plurality of wells with phosphate buffered saline after treating the polymer with UV light. In various implementations, attaching the patterned mask to the plate includes depositing droplets of diH2O onto a bottom surface of the plate and coupling the patterned mask to the plate. In some implementations, a volume of the polymer solution within each of the plurality of wells is between about 27uL to 35uL.

[0007] Another aspect of the present disclosure relates to a method of screening a hydrogel array. The method includes preparing the hydrogel array within a plate having a plurality of wells, wherein the hydrogel array includes a plurality of first regions with a first stiffness, and a plurality of second regions with a second stiffness, the first stiffness being greater than the second stiffness, and wherein the plurality of first regions and the plurality of second regions are positioned in an alternating arrangement to form a striated configuration. The method also includes seeding the hydrogel array with a cell and at least one small-molecule and analyzing the hydrogel array (e.g., for cell location). Analyzing the hydrogel array includes measuring a surface stiffness (e.g., Young’s Modulus) observed across the surface of the hydrogel array using atomic force microscopy (AFM), obtaining a plurality of fluorescent cell images corresponding to the plate, and generating at least one heat map of corresponding to theplurality of wells, the at least one heat map illustrating a durotactic response of the plurality of cells and the at least one small-molecule.

[0008] In various implementations, the at least one small-molecule comprises traction force microscopy beads. In some implementations, the method includes storing the plate for a period of time less than 2 months. In other implementations, the method includes labeling the plurality of cells with a fluorescent probe. In yet other implementations, the first stiffness is between about 7kPa and 12kPa, and wherein the second stiffness is between about 2kPa and 7kPa. In various implementations, analyzing the hydrogel array includes measuring (e.g., imaging) the hydrogel array in a direction perpendicular to a lined pattern defined by the striated configuration. In some implementations, measuring the position of the plurality of cells in the presence or absence of small molecules within the plate across a plurality of time points. In other implementations, the plurality of time points includes at least a first time point and a second time point, the first time point being immediately after seeding.BRIEF DESCRIPTION OF THE DRAWINGS

[0009] The disclosure will become more fully understood from the following detailed description, taken in conjunction with the accompanying figures, wherein like reference numerals refer to like elements, in which:

[0010] FIG. 1 A is a schematic representation of a hydrogel well having alternating stiffness regions formed by a mylar film pattern, according to at least one embodiment.

[0011] FIG. IB is a schematic representation of a hydrogel well having alternating stiffness regions formed by a mylar film pattern, according to at least one embodiment.

[0012] FIG. 2 is a schematic representation of a patterned mylar film mask for a 96-well plate containing polyacrylamide hydrogels, according to at least one embodiment.

[0013] FIG. 3 A is a bar chart illustrating stiffness (i.e., Young’s Modulus) of designated “soft” and “stiff’ regions within a well, according to at least one embodiment.100141 FIGS. 3B-3F are flow diagrams illustrating methods related to preparation and assessment of high durotaxis assay plates, according to at least one embodiment.

[0015] FIG. 4 is a graphical illustration of cumulative heatmaps of cell density across wells containing 8% Acrylamide / 0.48% Bis-Acrylamide hydrogels, according to at least one embodiment.10016] FIG. 5 is a graphical illustration of enrichment ratios in cells in un-patterned wells containing 8% Acrylamide / 0.48% Bis-Acrylamide hydrogels, according to at least one embodiment.

[0017] FIG. 6 is a graphical illustration of enrichment ratios in cells in patterned wells containing 8% Acrylamide / 0.48% Bis-Acrylamide hydrogels, according to at least one embodiment.

[0018] FIG. 7 is a graphical illustration of cumulative heatmaps of cell density across wells containing 5% Acrylamide / 0.48% Bis-Acrylamide hydrogels, according to at least one embodiment.

[0019] FIG. 8 is a graphical illustration of enrichment ratios in cells in un-patterned wells containing 5% Acrylamide / 0.48% Bis-Acrylamide hydrogels, according to at least one embodiment.

[0020] FIG. 9 is a graphical illustration of enrichment ratios in cells in patterned wells containing 5% Acrylamide / 0.48% Bis-Acrylamide hydrogels, according to at least one embodiment.

[0021] FIG. 10 is a graphical illustration of cumulative heatmaps of cell density across wells containing 3% Acrylamide / 0.48% Bis-Acrylamide hydrogels, according to at least one embodiment.

[0022] FIG. 11 is a graphical illustration of enrichment ratios in cells in un-patterned wells containing 3% Acrylamide / 0.48% Bis-Acrylamide hydrogels, according to at least one embodiment.

[0023] FIG. 12 is a graphical illustration of enrichment ratios in cells in patterned wells containing 3% Acrylamide / 0.48% Bis-Acrylamide hydrogels, according to at least one embodiment.

[0024] FIG. 13 is a fluorescent image of cells within patterned wells 0 hours and 48 hours after seeding, according to at least one embodiment.

[0025] FIG. 14 is a graphical illustration of cumulative heatmaps of cell density across wells, according to at least one embodiment.

[0026] FIG. 15 is a graphical illustration of mean fold change in cell number after 4 weeks of storage, according to at least one embodiment.

[0027] FIG. 16 is a graphical illustration of mean fold change in cell number after 8 weeks of storage, according to at least one embodiment.DETAILED DESCRIPTION

[0028] Referring generally to the figures, a method for hydrogel array preparation and assessing cell substrate interactions using the hydrogel compositions is described. Generally, mechanically patterned stripes of stiff substrate on a soft background are created to encourage cells to migrate to the stiffer stripes in a process called durotaxis. In some embodiments, photo-initiated polyacrylamide hydrogels can be made within a 96-well plate. In some embodiments, a transparent mask can be used to create alternating stiff and soft polymer bands across the well that are approximately the same height. In various embodiments, cells can be seeded on top of the hydrogel and imaged after 1 hours, 24 hours, and 48 hours after seeding to assess location within the hydrogel.

[0029] In various embodiments, lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate (LAP) with 365 nm UV light can be used as the reaction initiator to polymerize polyacrylamide (PA) hydrogels. In some embodiments, an occlusive mask (e.g., transparent mylar film) can be used to restrict light to certain areas of the hydrogel, forming regions of varying stiffness across a surface of the hydrogel. In some embodiments, the mask includes a pattern with alternating opacity, which causes corresponding bands of alternating stiff and soft regions on the hydrogel surface in multiple polyacrylamide gel formations. In some embodiments, the alternating stiff and soft regions include two stiff bands across the top surface of the hydrogel (i.e., a cell contact surface) in multiple polyacrylamide gel formulations.

[0030] Although various methods have been used to prepare hydrogels for durotaxis studies, various implementations involve six-well plates, which can limit study scope. In various implementations, different sized plates (e.g., 12, 24, 48, 96, 384, or 1536-well plates) can be used. Use of the herein disclosed systems and methods can be used to generate high throughput, two-dimensional durotaxis models in 96-well plate formats. In various implementations, these models can be used for a variety of purposes, including, but not limited to anti -durotaxis small molecule screenings.

[0031] Referring to FIGS. 1A and IB, a schematic representation of a photo-initiated polyacrylamide hydrogel 103 (hereinafter “hydrogel”) within a well 100 is shown, according to at least one embodiment. In some embodiments, the hydrogel 103 is made within a 96-well plate. To form the hydrogel 103 for durotaxis study, a transparent mask can be used to create a striated configuration of alternating polymer bands across the well 100. For example, as shown in FIGS. 1 A and IB, the hydrogel 103 includes a first set of polymer bands 105 (“soft” polymer bands) arranged in an alternating configuration with a second set of polymer bands 110 (“stiff’ polymer bands) across the well 100, where the “stiff’ polymer bands 110 have a higher stiffness than the “soft” polymer bands 105. In various embodiments, a height (defined in a direction substantially parallel with a primary axis of the well 100) of the “stiff’ bands 110 and “soft” bands 105 is approximately the same. In some embodiments, a width (defined in a direction substantially perpendicular to the primary axis of the well 100) of the “stiff’ bands 110 is smaller than a width of the “soft” bands 105. In some embodiments, the width of the “stiff’ bands 110 is approximately 620 microns. In other embodiments, the width of the “soft” bands 105 is approximately 1.20 millimeters.

[0032] FIG. 2 is a schematic representation of a patterned transparent mask (e.g., mylar film mask) 220 for a 96-well plate (i.e., such that the plate includes 96 wells, each of which being the same or equivalent to the well 100). The mask 220 includes a plurality of mask wells 200 containing patterned regions 203, each corresponding to a well 100 containing the hydrogel 103. In some embodiments, the transparent mask 220 can be used to restrict UV light exposure to specific areas of each hydrogel 103 (i.e., within each well 100) such that, due to the resulting different degrees of polymerization, the hydrogel 103 is formed with alternating regions having different stiffnesses. Accordingly, each patterned region 203 includes a firstset of bands 210 alternated by the second set of bands 205, which cause the corresponding first set of bands 105 and second set of bands 110 on each hydrogel 103. In various embodiments, each of the hydrogels 103 can be a polyacrylamide (PA) hydrogel. In some embodiments, the hydrogels 103 can be formed using lithium phenyl(2,4,6- trimethylbenzoyl)phosphinate (LAP) treated with 365nm UV light to facilitate polymerization.

[0033] In some embodiments, the alternating regions can be characterized as being “stiff’ (i.e., the areas exposed to more UV light) or “soft” (i.e., the areas exposed to less UV light), as shown in FIG. 2. For example, as shown in FIG. 2 and as described above, each patterned region 203 includes a first set of opaque bands 210 arranged in an alternating configuration with a second set of transparent bands 205 across the patterned region 203, where the resulting top-surface of the hydrogel 103 has “soft” polymer bands 105 alternating with “stiff’ polymer bands 110 100.

[0034] In various embodiments, a width, thickness, and / or opacity of each of the first set of bands 210 and second set of bands 205 correspond to a respective width, thickness, and / or stiffness of the bands 110 and bands 105 on each hydrogel 103. For example, a larger width of the bands 210 and / or 205 corresponds to a larger width of the respective bands 110 and and / or 105. Similarly, a greater thickness and / or opacity of the first set of bands 210 and the second set of bands 205 corresponds to a lower stiffness of the respective bands 110 and 105. In various embodiments, a height (defined in a direction substantially parallel with a primary axis of the well 100) of the “stiff’ bands 110 and “soft” bands 105 is approximately the same. In some embodiments, a width (defined in a direction perpendicular to the primary axis of the well 100) of the “stiff’ bands 110 is smaller than a width of the “soft” bands 105. In some embodiments, the width of the pattern of “stiff’ bands 110 is approximately 620 microns. In other embodiments, the width of the “soft” bands 105 is approximately 1.20 millimeters.

[0035] In various embodiments, the transparent mask 220 to restrict UV light exposure to specific areas of the hydrogel 103 is a mylar film. In some embodiments, the transparent mask 220 results in a pattern having two “stiff’ bands 110 across a top contact surface (i.e., the cell contact surface) of the hydrogel 103 in each well 100, where each of the “stiff’ bands110 is alternated with a “soft” band 105. In various embodiments, a pattern of striations across the top contact surface of the hydrogel 103 can include any number of “stiff’ bands 110 and “soft” bands 105. In various embodiments, the pattern shown in FIG. 2 is produced via FineLine imaging. As illustrated in FIG. 2, the regions / bands 205 indicated in white are substantially clear (corresponding to “stiff’ bands 110) and regions / bands 210 indicated with black are semi-opaque (corresponding to “soft” bands 105). In various embodiments, centers of each well 100 (i.e., and thus centers of each mask well 200) are spaced approximately 9 millimeters apart, with approximately 600 micron-thick bands of clear space (i.e., bands 205) interspersed by approximately 1.20-millimeter bands of opaque space (i.e., bands 210).

[0036] In various embodiments, the PA hydrogel 103 can have a first formulation of 3% Acrylamide / 0.48% Bis-Acrylamide, a second formulation of 5% Acrylamide / 0.48% Bis- Acrylamide, a third formulation of 8% Acrylamide / 0.48% Bis-Acrylamide, or any other suitable formulation known in the art. In various embodiments, the “stiff’ bands (i.e., patterned) 110 and the “soft” bands (i.e., “un-patterned”) 105 within the PA hydrogel 103 have stiffness ranging as shown in FIG. 3 A, which illustrates stiffness values for PA hydrogels 103 for each of the first formulation, the second formulation, and third formulation. The stiffness ranges for each formulation are further shown in Table 1 below. The difference between mathematical means of the “stiff’ bands 110 and the “soft” bands 105 is calculated by subtracting the mean of the “soft” bands 105 from the mean of the “stiff’ bands 110.Table 1Polyacrylamide Hydrogel Synthesis

[0037] In various implementations, durotactic responses for each PA hydrogel 103 can be determined using a multi method 300 disclosed herein and shown in FIG. 3B. At 305, the PA pre-polymer solution can be prepared as desired. In various embodiments, at 310, a lOmLmethacrylate solution can be prepared for each 96-well plate (including 96 wells 100, each structured to contain the hydrogel 103) to be treated in a fume hood. In some embodiments, the methacrylate solution can include lOmL Ethanol, 300uL 10% Acetic Acid, and 50uL Methacrylate. The PA solutions can be prepared according to the formulations shown in Table 2 below.Table 2

[0038] To prepare the solutions, a 96-well glass bottom plate (i.e., a plate similar or equivalent to the plate 220) can be plasma treated for 30 seconds with O2. In some implementations, the plate can be immediately removed once the interlocks are released, moved to the fume hood, and added the methacrylate solution dropwise to the center of the well until the glass is just covered. Afterward, the plate can be washed with the methacrylate solution at 315. In some implementations, the plate can be incubated at room temperature (i.e., 20-22 °C) for about 5 min before pipetting out methacrylate solution and rinsed with 200uL ethanol. The plate can be air dried upside down, wherein the plate can be sitting crossangled across the plate lid, for about 5 minutes.

[0039] At 320, the mask can be attached to the plate. In various implementations, approximately l-2uL droplets of diELO can be deposited onto a bottom surface of the plate (e.g., at each comer and at the center of the plate) to attach the mask (i.e., mylar) to the bottom of plate (i.e., the surface of the plate opposite the well openings). This ensures that the mask is aligned with each of the wells (i.e., each similar or equivalent to the wells 100) and adheres to the plate when the plate is flipped over.

[0040] At 325, gel / polymer solution can then be added to each well (e.g., 27uL per well) of desired polyacrylamide stiffness range. In some embodiments, 2.592mL of polymer solution is needed per 96-well plate. During this process, about 2.592 mL of pre-made acrylamide gel solution is mixed with 25.92uL of a visible light photoinitiator (e.g., LAP) (5 weight percent, final weight percent of 0.05%), and a small molecule (e.g., traction force microscopy beads) if desired (1 : 100 dilution, 25.92uL). The plate can be centrifuged at 400rpm for 30 seconds.

[0041] After gel addition and centrifugation, the plate can be removed gently from the centrifuge, and immediately moved to a UV light source at 330, where the light source should be radiating upwards through the gel (i.e., the UV light can be provided at a bottom surface of the plate). In various embodiments, the light is UV at 350nm light (e.g., with a minimum intensity of 40mW / cm2) for 120 seconds. The mask (e.g., mylar) can be removed from the bottom of the plate at 335. At 340, the wells can be gently rinsed with 200uL phosphate buffered saline (PBS) and then shaken at about 50rpm for 5 min. At 340, the PBS can be gently aspirated out of each well, and the plate can be dried / sterilized (uncovered) in a sterile biological safety cabinet (BSC) hood for a period of time. In various implementations, the period of time is approximately 1 hour. In various embodiments, the gel thickness can be less than or equal to 0.8mm.ECM Treatment of Gels

[0042] After the plate is dried / sterilized at 340 of the method 300, extracellular matrix (ECM) treatment of the PA gels can commence using a method 400, which is shown in FIG. 3C. At 405, lOOuL / well of sulfo-SANPAH solution in 50mM HEPES solution is prepared to a final dilution of 0.2mg / mL. Afterward, PBS within the wells can be aspirated gently, and the sulfo- SANPAH solution added to each well. At 410, each well is irradiated for a periodof time until solution turns brown using 350nm light. In various implementations, the period of time is approximately 10 minutes. At 415, the wells can then be washed with PBS. In various implementations, the wells are washed with PBS three times for five minutes. Subsequently at 420, the ECM and PBS can be mixed on ice, after which lOOuL of protein is added to each well and incubated for 1 hour at 37°C. In various embodiments, the protein is collagen type 1 (rat tail) (25 ug / mL). While protein attachment to the hydrogel is occurring, at 420, a T-75 flask (or other suitable vessel) containing the desired cell line with lOmL PBS can be rinsed, aspirated, and 4mL Accutase added at 425. This can then be incubated for 4 minutes at 37°C.

[0043] Subsequently at 430, a volume of media (e.g., 8mL) containing at least 10% fetal bovine serum (FBS) can be added to the flask, and cells can be lifted via gentle pipetting from the flask. The solution can then be collected and centrifuged for a period of time. In various implementations, the solution can be centrifuged at about 1500 rpm for about 5 minutes. Afterward, at 435 the cell pellet can be resuspended in serum free media at a concentration of l*106cells / mL. In various embodiments, the serum free media is Dulbecco’s Modified Eagle Medium (DMEM) or Roswell Park Memorial Institute 1640 medium (RPMI). Afterward, at 440 an amount (e.g., luL) of a fluorescent probe (e.g., CellTracker Green CMFDA) per 106cells (ImL) can be added to label the cells, where the CellTracker should be at a final concentration of lOuM. The mixture can be incubated for 25 minutes at 37°C.

[0044] Following incubation, at 445 the cells can be spun at 1500 rpm for 5 minutes, or until the cell pellet is noticeable. While cells spin down, the 96-well plate can be removed from the incubator and the ECM solution gently removed from wells. The wells can be washed once with 200uL sterile PBS, waiting to remove PBS until just before adding cell suspension. This can be set aside in the BSC. In an operation 450, media can be aspirated from the cell pellet, and the pellet can be resuspended in culture media to the desired cell concentration (using 200uL media per well). Wells can then be seeded with cells at 455. In various embodiments, wells seeded with NIH 3T3 cells can include about 2000-8000 cells / well (i.e., 6,600-27,000 cells / cm2). In various embodiments, wells seeded with IMR-90 cells can include about 6000 cells / well (i.e., 19,870 cells / cm2). Prior to seeding, the PBS should be removed, and 200uLcell suspension added the desired seeding density. In various embodiments, the desired seeding density can be approximately 9000 cells / well (i.e., density of 30,000 cells / cm2). The cells can then be immediately imaged for a time (t) at 460. In various embodiments, the cells can be imaged at the 0-hour timepoint and repeated at 24 hours and 48 hours after seeding.Atomic Force Microscopy (AFM) of Polymer Stiffness

[0045] Imaging of the cells within the wells can be carried out using a method 500, which is shown in FIG. 3D. To image the cells, polymer solutions can be prepared according to the formulations outlined in Table 1. Initially at 505, #0 cover glass slides can be prepared with plasma cleaner and methacrylate solution (e.g., such as described above in relation to method 300) and adhered (e.g., using diH2O) onto the mylar film. At 510, corresponding wells can then be traced using a hydrophobic pen onto the #0 glass surface. Subsequently at 515, LAP and a small molecule (e.g., TFM beads) can be added at the above final concentrations to a small volume of the pre-polymer solution, and 27uL of polymer solution can be immediately pipetted into the center of the traced well. In some embodiments, the solution can be spread across the full traced interior of the well.

[0046] At 520, 350nm UV light (i.e., minimum intensity 40mW / cm2) can be turned on for 120 seconds, followed by removal of the cover glass from mylar film and submersion in PBS for 5 minutes. At 525, the slide can be removed from PBS and dried under UV light for 1 hour. The slide can be resubmerged in PBS for a minimum of 1 hour before taking AFM measurements. At 530, the AFM stage can be prepared, and small dots of an adhesive (e.g., superglue) can be deposited along the edges of the #0 glass slide to adhere the #0 glass slide to a thicker microscope slide (i.e., with the polymer facing up). At 535, a known scale (e.g., a 200um-marked transparency) can be adhered under the gel, or the x-y stage can be calibrated to a known scale. At 540, beginning at one end of the gel, imaging can proceed across the hydrogel perpendicular to the lined pattern (i.e., the pattern of bands 105 and bands 110 within the well 100), measuring deflection (i.e., of the AFM cantilever probe) at known time intervals and fitting the measured deflection data to a calibrated Hertz model. In various implementations, deflection measurement is carried out using atomic force microscopy (AFM). Measured deflection can then be used to determine the stiffness at various pointsacross the surface of the hydrogel 103 withing each well 100 of the 96-well plate. In some embodiments, the stiffness is the Young’s Modulus.Image Acquisition and Analysis

[0047] In various implementations, image acquisition and analysis can be carried out using a method 600, as shown in FIG. 3E. In various embodiments, imaging can be performed on a Keyence BZ-X800 All-in-One Fluorescent Microscope. Before imaging, at 605, a multi-point capture can be set up, with the center of each well (i.e., each well 200 within the plate 220) added as a Registered Area, and Stitching enabled for a 3x3 image (i.e., 8.696mm x 6.522mm area). At 610, the 96-well plate (i.e., the plate 220) can be placed inside a temperature- controlled chamber. For example, the plate can be placed inside a Tokai Hit live-cell chamber with 5% CO2 and 37°C temperature control during imaging.

[0048] At 615, using the single-channel image capture setting, the BZ-X filter cube (excitation: 470 / 40nm, emission: 525 / 50nm) can preview a subset of random wells from (e.g., 3 wells of the 96 wells within the plate) to establish a z-stack range, with a known pitch (e.g., 99.0 pm). Generally, 2-3 z-layers can be sufficient to encapsulate variations across the plate. In some embodiments, z-layers can be recorded for future timepoints (e.g., 24 hr., 48 hr., etc.). At 620, excitation levels can be set for predetermined time points. For example, at time zero (i.e., immediately after seeding), an excitation of l / 50s can be used with black balance and increased for future timepoints as necessary. Standard resolution sensitivity can be used. Once an appropriate z-stack range and excitation are chosen, capture can start at 625.Captures can take approximately 20-30 minutes per plate.10049] After the imaging is complete, images can be stitched using BZ-X800 software at 630. The GCI file can be opened to the “Stich” setting and stitched using the “Full Focus / Sectioning Image” option to create a maximum projection of each 9x9x3 image stack. Images can be saved as compressed TIFs labeled by well and timepoint.ROI Definition

[0050] Processing of the images collected using the method 600 can be carried out using method 700, which is shown in FIG. 3F. To threshold the images, cells can be identified andthe coordinates exported a CSV format at 705. In some embodiments, this can be accomplished using one or more algorithms to facilitate calibrating image scale, image rotation, image thresholding, and exporting of the cell coordinates. For example, the (Fiji Is Just) ImageJ Macro Script, KeyenceAnalysis can be used to calibrate the image scale, rotate the image capture 90 degrees to align with the horizontal pattern format, threshold the image to identify particles within an applicable cell / cell group area, and export the x-y coordinates of identified ROIs to a CSV file.

[0051] At 710, user input can be used to select one or more image-processing parameters. In various implementations, the image-processing parameters can include the area of the image to be cropped around the bounds of the well, validation of the image threshold, and entry of the well number (e.g., from the image file title) corresponding to a particular well within the plate. In various implementations, each time the script is used, it can be adjusted for “dir,” “OutPatb,” and “ResultsPath” locations. The timepoints (i.e., “tp#”), group (i.e., “group#,” the path that the original stitched images are in), the template (i.e., for re-naming the cropped images and resulting CSV files with necessary identifying information), and the size bounds of cells (i.e., “loSize” and “hiSize”) can be adjusted based on the experiment parameters. The script can iterate through all images in an image grouping, including timepoints and software (e.g., Keyence) subfolders created. A CSV for each well at each timepoint can be created in the designated results folder at 720, as well as a copy of the thresholded and selected ROI image.Image Visualization

[0052] After each stitched image (e.g., Keyence image) has been run through the image processing software (e.g., ImageJ script, KeyenceAnalysis. ijm), the resulting CSV files can be grouped by condition, normalized, and graphed at 725. For example, the CSV files can be grouped using MATLAB. In various implementations, individual CSVs sharing a group name (e.g., ‘*_gel_0hr_3p_-IMR90.csv’) can be read into a cell array for each timepoint. As each data set is read, a custom user-defined function (UDF) can be used to plot the x-y data points at 730. For example, the x-y data points can be plotted in a scatterplot, in a histogram binned by the y-axis, and / or a 3D histogram showing the cell count per bin region in the x-ycoordinate system. Additionally or alternatively the x-y data can be used to create a heatmap for the cell distribution across each well. Each of these images can be optionally saved using the UDF. In various implementations, each well within the 96-well plate can be divided into a nxn grid, where non-zero whole number. In some implementations, n can be 30. Accordingly, in such embodiments, each bin region can represent a 200 pm x 200 pm area. In various implementations, any ROI with a center reported within the designated bin region can be counted so that the “cell count per bin region” corresponds to a number of ROIs within that specific bin region. Accordingly, discretization of each hydrogel surface into definitive regions can be carried out.

[0053] After each well is read at 730, a custom UDF can be used to combine well replicates into a same coordinates (i.e., same x-y group) at 735. In various implementations, each plate includes a first subset of wells without a patterned hydrogel (i.e., un-patterned wells) and a second subset of wells with the patterned hydrogel (i.e., patterned wells). Accordingly, in carrying out at 735, it is possible to group the “Control” (i.e., un-patterned wells) into one variable per timepoint, and the “Patterned” wells into a separate variable (i.e., separate from the Control). These cumulative variables can then plotted as a heatmap (e.g., using the hist3() function of MATLAB), and the resulting data frame containing a square matrix defined by the number of bins, which was used in further analysis described below. This data can be normalized at 740, and the total number of cells or the percent of cells in each area at a given timepoint can also be compared.

[0054] To create an unbiased sample of the cell density in each area, two UDFs can be created to separate the areas of each well based on user-defined parameters, a random subset of elements can be chosen from both the stiff area and the soft area, and an enrichment ratio of the number of stiff area cell density to the soft area cell density can be generated at 745. Using one or more computational operators (e.g., MATLAB’s randperm and numel functions), which selects a random set of non-repeating integers within a specified range, an equal number of cell densities can be selected from each region at 750. Accordingly, the mean of the resulting subsets from the “stiff’ and “soft” regions can be reported, and the “mean stiff’ cell density can be divided by the “mean soft” cell density at each timepoint to report the enrichment ratio of cell density in the stiff region. It should be noted that theseenrichment densities correspond to the cumulative wells in each group normalized by the number of wells included to compare the patterned wells (n=84) to the un-patterned group (n=12). Alternatively, random cell densities can be collected for each individual well and the enrichment ratio of soft to stiff densities calculated, then collectively plotted with each well as a replicate value for each condition. Error bars for these values can be plotted as the standard deviation of the dataset. Other optional functions for presenting the data can be included and annotated.In various embodiments, the methods 600 and / or 700 can be carried out by at least one controller. In various embodiments, the at least one controller is configured to include a processor and a non-transitory computer readable medium (e.g., a memory device) having computer-readable instructions stored thereon that, when executed by the processor, cause the at least one controller to carry out one or more operations. In various embodiments, the at least one controller is a computing device (e.g., a microcomputer, microcontroller, or microprocessor). In other embodiments, the at least one controller is configured as part of a data cloud computing system configured to receive commands from a user control device and / or remote computing device.Results(0055] In various embodiments, upon formation of the PA hydrogels, durotactic responses can be measured for each formulation. FIGS. 4-12 show durotactic responses to surface stiffness (i.e., Young’s Modulus) over time for the various PA hydrogels.

[0056] When carried out, the method / assay described above resulted in migration of cells from a homogeneous distribution at time zero to stiffer regions of the well. First, the stiffness of hydrogels created using the photomask was measured using AFM. The difference between the stiff and soft regions of hydrogels was measured to be between approximately 3.5kPa to 5.1kPa across the surface of the hydrogel. Polymers made with 8% acrylamide had a stiffness (i.e., Young’s Modulus) in the “stiff’ regions (i.e., bands 110) of approximately 12kPa, and a stiffness in the “soft” regions (i.e., bands 105) of approximately 7kPa. Polymers made with 5% acrylamide had a stiffness in the “stiff’ regions (i.e., bands 110)of approximately 9kPa, and a stiffness in the “stiff’ regions (i.e., bands 105) of approximately 6kPa. Hydrogels madewith 3% acrylamide had a stiff modulus of approximately 7kPa, and a soft modulus of approximately 2kPa.

[0057] In some embodiments, when seeded with NIH 3T3 fibroblasts, cells showed a durotactic response to the surface stiffness over time, such as shown in FIG. 4-9. In various implementations, immediately after seeding, cells can be distributed homogenously across the surface of both the un-pattemed and patterned wells. Using this method, as shown in FIGS. 4- 6 (illustrating the first formulation) after 24 hours (see, e.g., sections A-B and D-E of FIG. 4), cells on the patterned wells were seen to be preferentially clustered within the stiff areas of the hydrogels but were generally homogenously distributed on the control wells. This pattern continued at the 48-hour timepoint, as appreciated from sections C and F of FIG. 4.Furthermore, when an equal number of areas were compared from the stiff and soft regions, it was observed that there was a high enrichment ratio of cell density in the stiff areas at 24 and 48 hours, as appreciated from FIG. 5-6. This pattern was observed in both 5% acrylamide and 8% acrylamide polymers, as illustrated in FIGS. 7-9, which show durotactic responses for the second formulation.[0058| In other implementations, PA hydrogels according to the third formulation can be seeded with IMR90 fibroblasts. As appreciated from FIGS. 10-12, which illustrate durotactic responses for the third formulation, cell migration trends are less pronounced than those of the NH4-3T3 fibroblast responses (i.e., of the first and second formulations shown in FIGS. 4- 9). Furthermore, as shown in the figures, the patterned wells show an increase in the enrichment ratio of the cell density in the stiff areas relative to the soft areas across 24 and 48 hours.

[0059] As appreciated from FIGS. 4-12, there is evidence of fibroblast durotaxis across multiple cell types within 48 hours after seeding hydrogels formed with stiff and soft bands as described above. Additionally, fibroblasts exhibit durotaxis across multiple polyacrylamide polymer compositions (i.e., the first, second, and third formulations) in a 96- well plate. Furthermore, the methods described herein provide a method for quantifying cell location and bulk durotaxis across each well. Overall, this protocol establishes a method forpreparing hydrogels in 96-well plates, seeding fibroblasts on top of the hydrogels, and quantifying cell durotaxis in a high-throughput manner.

[0060] In various embodiments, the method described above can be extended to include small-molecule agonists / antagonists against durotaxis. For example, Blebbistatin, a myosin II inhibitor, could be used to treat cells seeded in this system versus an untreated control. The comparative cell density in stiff and soft areas could then be compared at multiple timepoints using the analysis methods shown in this presentation, with the expectation that blebbistatin would reduce the enrichment ratio of cells in the stiff region versus the soft region because inhibition of cell contraction would impair cells from migrating to the stiffer regions of the PA hydrogel. In various implementations, dimethyl sulfoxide (DMSO) is used as the vehicle control for small molecule additions in which small molecules are reconstituted in DMSO. In some implementations, lOuM lysophosphatidic acid (LPA) can be used to enhance / maintain durotaxis, and 20uM of a Rho-associated protein kinase (ROCK) inhibitor (e.g., Y27632) can be used to inhibit durotaxis. Accordingly, the ratio of cells quantified in the stiff versus soft area will increase with LPA treatment at non-zero timepoints, whereas treatment with Y27632 will result in a comparable ratio of cells within the stiff versus soft region to that of an un-pattemed control well.

[0061] In various implementations, storage time of high throughput durotaxis plates can be increased by adapting various aspects of one or more of the methods 300-500. In various implementations, fresh 3-(Trimethoxysilyl)propyl methacrylate can be used. This is a moisture-sensitive chemical that degrades over time, which can be replaced (i.e., solution can be replaced with a fresh bottle of reagent) to improve gel adhesion over time relative to implementations in which similar replacements were not made. In various implementations, manufacturer guidelines for storage conditions, time, and flushing during reagent removal with inert gas to extend shelf life can be followed. In addition, or as an alternative to using fresh 3 -(Trimethoxy silyl)propyl methacrylate, the volume of pre-polymer solution added to each well can be increased from 27uL to 35uL (i.e., at 515 of the method 500). Increasing pre-polymer solution volume and / or using fresh 3-(Trimethoxysilyl)propyl methacrylate can improve hydrogel coverage and long-term gel adhesion at weekly timepoints as compared to hydrogel prepared without these adjustments, as shown in FIGS. 13-15.(0062] FIG. 13 illustrates NIH-3T3 cells labeled with CellTracker Green CMFDA immediately after seeding (0 hours) and 48 hours after seeding. The cells were prepared according to the methods 300-500, with 3-(Trimethoxysilyl)propyl methacrylate added regularly and using a volume of 27 uL (left column) or 35 uL (right column) pre-polymer solution. The cells were stored at 4°C for 4 weeks before treating with Sulfo-Sanpah and ECM-coating with Collagen I RT (i.e., in a manner similar to or equivalent to 405 of the method 400). FIG. 13 includes representative images of 2 wells at two timepoints and includes a scale bar of lOOOum. It should be noted that although volumes greater than 35uL can be used and result in durotactic behavior, using a volume of 35uL showed the most consistent and largest stiff area per well after 8 weeks of storage, as illustrated in FIG. 15. In various implementations, multiple polymer volumes can be added to regulate the degree of the polymer stiffness gradient within the hydrogel / well.

[0063] FIG. 14 illustrates heatmaps for 3 wells of NIH-3T3 cells labeled with CellTracker Green CMFDA. As noted above, the plates were stored for 4 weeks at 4°C before coating with collagen and seeding with cells. As shown in FIG. 15, which shows mean fold change of the number of cells in binned areas designated as “stiff’ or “soft” after 4 weeks of plate storage, increasing the volume of pre-polymer solution to 35 uL resulted in greater change in number of cells (significance of p-value <0.05 shown for a two-way ANOVA with mean comparison between conditions per timepoint). As shown in FIG. 16, which shows mean fold change of the number of cells in binned areas designated as “stiff’ or “soft” after 8 weeks of plate storage, increasing the volume of pre-polymer solution to 35 uL resulted in greater change in number of cells (significance of p-value <0.05 shown for a two-way ANOVA with mean comparison between conditions per timepoint).100641 In various implementations, after the polymer has been made, washed with PBS to remove excess LAP / unpolymerized polymer, and dried for 1 hour (i.e., 515-535 of the method 500), excess PBS can be left on the gel surface (i.e., not removed). A thin layer of PBS that did not fully evaporate during the UV sterilization can then be observed, and no further PBS can be added or removed before wrapping the plate with Parafilm and storing it at 4 degrees Celsius. In various implementations, the wrapping and storing can be carried out as part of 535 and / or 540. After sealing the lid to the plate with parafilm, the plate can bestored flat at about 4 degrees Celsius (e.g., in a temperature-controlled chamber) for up to two months. During storage, a small layer of PBS covering the gel surface for the entirety of storage time can be maintained. In various implementations, if the gel surface is directly visible, a volume of sterile PBS can be added. For example, 20-40uL of sterile PBS can be added. After the desired storage time, the plate can be removed from the 4°C environment and allowed to warm to room temperature for ~30 minutes before proceeding with Sulfo- SANPAH treatment and ECM coating (i.e., in a manner similar or equivalent to 405). It should be noted that although the methods described herein relate to storage times of less than two months, preliminary data suggests the plate may be stable for longer periods of time (i.e., more than two months of storage).

[0065] Notwithstanding the embodiments described above in FIGS. 1 - 16, various modifications and inclusions to those embodiments are contemplated and considered within the scope of the present disclosure.

[0066] While this specification contains specific implementation details, these should not be construed as limitations on the scope of what may be claimed but rather as descriptions of features specific to particular implementations. Certain features described in this specification in the context of separate implementations can also be implemented in combination in a single implementation. Conversely, various features described in the context of a single implementation can also be implemented in multiple implementations separately or in any suitable subcombination. Moreover, although features may be described as acting in certain combinations and even initially claimed as such, one or more features from a claimed combination can, in some cases, be excised from the combination, and the claimed combination may be directed to a subcombination or variation of a subcombination.

[0067] As utilized herein, the terms “substantially,” “generally,” “approximately,” and similar terms are intended to have a broad meaning in harmony with the common and accepted usage by those of ordinary skill in the art to which the subject matter of this disclosure pertains. It should be understood by those of skill in the art who review this disclosure that these terms are intended to allow a description of certain features described and claimed without restricting the scope of these features to the precise numerical rangesprovided. Accordingly, these terms should be interpreted as indicating that insubstantial or inconsequential modifications or alterations of the subject matter described and claimed are considered to be within the scope of the appended claims.

[0068] The term “coupled” and the like, as used herein, mean the joining of two components directly or indirectly to one another. Such joining may be stationary (e.g., permanent) or moveable (e.g., removable or releasable). Such joining may be achieved with the two components or the two components and any additional intermediate components being integrally formed as a single unitary body with one another, with the two components, or with the two components and any additional intermediate components being attached to one another.

[0069] It is important to note that the construction and arrangement of the various systems shown in the various example implementations is illustrative only and not restrictive in character. All changes and modifications that come within the spirit and / or scope of the described implementations are desired to be protected. It should be understood that some features may not be necessary, and implementations lacking the various features may be contemplated as within the scope of the disclosure, the scope being defined by the claims that follow. When the language “a portion” is used, the item can include a portion and / or the entire item unless specifically stated to the contrary.

[0070] It should be noted that the term “exemplary” as used herein to describe various embodiments is intended to indicate that such embodiments are possible examples, representations, and / or illustrations of possible embodiments (and such term is not intended to connote that such embodiments are necessarily extraordinary or superlative examples).

Claims

WHAT IS CLAIMED IS:

1. A method of preparing a hydrogel array comprising a multi-well plate, the method comprising: preparing a polyacrylamide (PA) hydrogel, wherein preparing the PA hydrogel comprises: preparing a polymer solution, plasma treating the multi-well plate; attaching a patterned mask to a plate containing the polymer solution, the plate comprising a plurality of wells; and treating the polymer solution with UV light to form the PA hydrogel, the PA hydrogel having a plurality of first regions with a first stiffness and a plurality of second regions with a second stiffness, the first stiffness being greater than the second stiffness.

2. The method of claim 1, wherein the plate is a 96-well plate.

3. The method of claim 1, wherein a height of the plurality of first regions is the same as a height of the plurality of second regions.

4. The method of claim 1, wherein the plurality of first regions and the plurality of second regions are positioned in an alternating arrangement so as to form a striated configuration.

5. The method of claim 4, wherein a first width of each of the plurality of first regions is approximately 620 microns.

6. The method of claim 4, wherein a second width of each of the plurality of second regions is approximately 1.2 millimeters.

7. The method of claim 1, wherein treating the polymer solution with UV light includes providing UV light to a bottom surface of the plate so light radiates upward through the plate.

8. The method of claim 1, wherein the patterned mask comprises mylar.

9. The method of claim 1, wherein the PA hydrogel comprises between 3% and 8% acrylamide and about 0.48% Bis-acrylamide.

10. The method of claim 1, further comprising rinsing the plurality of wells with phosphate buffered saline after treating the polymer with UV light.

11. The method of claim 1, wherein attaching the patterned mask to the plate comprises depositing droplets of diffcO onto a bottom surface of the plate and coupling the patterned mask to the plate.

12. The method of claim 1, wherein a volume of the polymer solution within each of the plurality of wells is between about 27uL to 35uL.

13. A method of screening a hydrogel array, the method comprising: preparing the hydrogel array within a plate having a plurality of wells, wherein the hydrogel array comprises: a plurality of first regions with a first stiffness; and a plurality of second regions with a second stiffness, the first stiffness being greater than the second stiffness; wherein the plurality of first regions and the plurality of second regions are positioned in an alternating arrangement to form a striated configuration; seeding the hydrogel array with a plurality of cells and at least one small-molecule; and analyzing the hydrogel array, wherein analyzing the hydrogel array comprises: measuring a surface stiffness across the hydrogel array using atomic force microscopy (AFM); obtaining a plurality of images corresponding to the plate; and generating at least one heat map of corresponding to the plurality of wells, the at least one heat map illustrating a durotactic response of the plurality of cells and the at least one small-molecule.

14. The method of claim 13, wherein the at least one small-molecule comprises traction force microscopy beads.

15. The method of claim 13, further comprising storing the plate for a period of time less than 2 months.

16. The method of claim 13, further comprising labeling the plurality of cells with a fluorescent probe.

17. The method of claim 13, wherein the first stiffness is between about 7kPa and 12kPa, and wherein the second stiffness is between about 2kPa and 7kPa.

18. The method of claim 13, wherein analyzing the hydrogel array comprises imaging the hydrogel array in a direction perpendicular to a lined pattern defined by the striated configuration.

19. The method of claim 13, wherein measuring the deflection of the plurality of cells and the at least one small-molecule within the plate comprises measuring the deflection across a plurality of time points.

20. The method of claim 19, wherein the plurality of time points includes at least a first time point and a second time point, the first time point being immediately after seeding.

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