We acknowledge Austin Cyphersmith and Mayandi Sivaguru (Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana&#8211;Champaign) for assistance with microscopy and for generously accommodating screen and video capture at the microscopy core.

The authors declare that they have no competing financial interests.

In our experiments, we have found that the most common failures are related to the quality of fabricated arrays and poorly characterized response in the biological system of interest. We refer the reader to Table 1 for common failure modes in cell microarray experiments and associated troubleshooting steps. Regarding quality of arrays in particular, we recommend the following. Confirm the technical quality and robustness of arraying programs, parameters, and buffers using fluorescently labeled molecules such as rhodamine-conjugated dextran. Thoroughly clean pins either before or after arraying per the manufacturer&#39;s instructions and further visually check that the pin channels are clear of debris using a light microscope. Confirm arrayed biomolecule retention using general protein stains or immunolabeling. Note that biomolecules with a molecular weight below 70 kDa are frequently not retained in the hydrogel23,31. Validate arrayed biomolecule cell-functionality using multiple cell types. Note that only adherent cells are compatible with arrays; additionally, adhesion to arrays is dependent on both cell-specific properties (e.g., integrin expression profile) and the selected ECM proteins.
Due to limited space, we have not provided an extensive treatment of array design, layout, and fabrication here and refer the reader to previous works23,25. We generally use 100 spot subarrays (150 &#181;m spot diameter, 450 &#181;m center-to-center distance) composed of 10&#8211;20 unique biomolecule conditions (i.e., 5&#8211;10 spots/condition). The number of subarrays in one array varies depending on the number of biomolecule conditions of interest, which can be comfortably scaled up to 1,280 on one 25 &#215; 75 mm microscope slide (~ 6,400 spots in 64 subarrays)25,31. The parameters above will further vary depending on the pattern size of interest; pins capable of generating patterns from 75 &#8211; 450 &#181;m are readily available.
Array experiments are best complemented by validation of high-scoring arrayed conditions of interest using other culture formats, assay readouts, and biological model systems. Specifically, we recommend further validating effects of select arrayed conditions using bulk cultures in conjunction with standard molecular biology techniques (e.g., qRT-PCR, immunoblotting) or standard TFM. Genetic manipulation (e.g., knockdown or overexpression) of the factor of interest in an appropriate biological model system can also serve to confirm effects observed in arrays. In vivo animal models represent another means of validation and were recently used, for example, to confirm the central role of galectin-3 and galectin-8 in the lung cancer metastatic niche, as initially identified via cell microarray31,49.
A number of other methods have been used to probe microenvironmental regulation of cellular functions, including a variety of two-dimensional microfabricated systems18,50,51,52,53,54,55 and three-dimensional engineered biomaterial systems56,57,58,59,60,61. In comparison with other methods, the particular advantages of the cell microarray platform described here consist of: (1) throughput up to hundreds or thousands of different combinations of factors, enabling analysis of interaction effects; (2) accessible, automated imaging and analysis; (3) integration of both biochemical and biophysical readouts with controlled presentation of arrayed factors; (4) ability to vary substrate material properties; and (5) high-content single-cell analysis of cell fate and function.
In summary, the combination of cell microarrays with TFM on substrates of tunable substrate stiffness enables thorough characterization of both biochemical and biophysical cues. As presented here, this platform is generalizable and can be readily applied to a variety of adherent cell types and tissue contexts towards an improved understanding of combinatorial microenvironmental regulation of cell differentiation and mechanotransduction.

Interactions between cells and surrounding microenvironmental factors mediate a large variety of biological processes throughout development, homeostasis, and disease pathogenesis1,2,3,4. These microenvironmental interactions include delivery of soluble factors to cells, cell&#8211;matrix binding, and cell&#8211;cell interactions via ligand&#8211;receptor binding. In addition to the above biochemical considerations, biophysical parameters, such as substrate mechanical properties (e.g., Young&#39;s modulus, porosity) and cell shape, and associated downstream mechanotransduction have increasingly gained recognition as key mediators of cell differentiation5,6,7,8,9,10. Signals resulting from these microenvironmental interactions serve as inputs to gene networks and signaling pathways. Moreover, these cell-intrinsic components also provide feedback to the microenvironment via secreted factors and matrix-degrading enzymes, completing a complex co-regulatory loop between cell-intrinsic genetic programs and cell-extrinsic microenvironmental factors5,11,12.
The use of engineered systems for the controlled presentation of microenvironmental factors has proven useful in a range of different contexts13,14,15. Microfabricated systems in particular have facilitated precise spatial patterning of proteins and cells as well as highly parallelized analysis via miniaturization13,16,17,18,19,20,21,22. Cell microarrays represent one such microfabricated system in which combinations of biomolecules are contact-printed onto an elastic polyacrylamide hydrogel substrate23,24,25. The inclusion of cell-adhesive components (namely matrix proteins) enables sustained cell adhesion and culture on microarrays, which is frequently followed by downstream analysis via immunocytochemistry and fluorescent reporters. Cell microarrays have been productively directed towards attaining an improved understanding of liver cell phenotype23,26, neural precursor differentiation27, mammary progenitor fate decisions28, embryonic stem cell maintenance/differentiation23,29,30, lung cancer metastasis31, and therapeutic response in melanoma32. We have recently demonstrated the use of cell microarrays for defining the role of extracellular matrix (ECM) protein composition in endoderm specification33, liver progenitor differentiation34,35, and lung tumor cell drug response36. In these works, we have focused on extending the combinatorial capabilities of the array platform and exploring the intersections of cell-intrinsic signaling with extracellular matrix composition and biomechanics. In addition, we have implemented biophysical readouts in this array platform to provide the ability to quantitatively characterize the role of cell contractility in differentiation processes35. To do so, we integrated traction force microscopy (TFM) with cell microarrays to enable high-throughput assessment of cell-generated traction. TFM is a widely utilized method to measure cell-generated traction forces and has provided significant insights regarding the coordination of single-cell and tissue-level function with the composition and biomechanics of the local microenvironment37,38,39,40. Thus, combining TFM with cell microarrays provides a high-throughput system for measuring key, physiologically relevant biophysical parameters.
The cell microarray platform described here consists of four sections: fabrication of polyacrylamide substrates, fabrication of arrays, cell culture and assay readout, and analysis of data. See Figure 1 for a schematic summary of the first three experimental sections; see Figure 2 for a schematic summary of the final section with a focus on analysis of immunofluorescence data. In order to adapt the cell microarray platform to studies of biomechanical cell&#8211;substrate interactions, we used polyacrylamide substrates of tunable Young&#39;s modulus but similar porosity, per Wen et al.41. To enable TFM measurements of forces exerted by cells on their substrate, we implemented a glass-bottom Petri dish format in addition to the thick glass microscope slides frequently utilized by other groups. Thus, this cell microarray platform is capable of parallel measurements of cell differentiation via immunofluorescence on microscope slides and cell-generated forces via TFM on separate glass-bottom dishes. We have also applied several improvements to the analytical approach commonly used with cell microarrays. Specifically, instead of parametric Z-scoring of overall island intensity, we measure single-cell intensity and apply quantile normalization in order to account for non-normal distributions and more accurately describe cellular behavior. We believe these improvements provide particular utility towards investigations of biological processes in which both biochemical and biophysical cues play significant, intersecting roles. Further, our analytical improvements enable the application of cell microarrays to studies of a range of cellular functions for which single-cell and population-level behavior diverge.

<table><tbody><tr><td>0.2 &amp;micro;m syringe filter</td><td>Pall Corporation</td><td>4433</td><td>Match with appropriately-sized Luer lock plastic syringes.</td></tr><tr><td>100 &amp;times; penicillin&amp;ndash;streptomycin solution</td><td>Fisher Scientific</td><td>SV30010</td><td></td></tr><tr><td>22 &amp;times; 60 mm coverglasses</td><td>Electron Microscopy Sciences</td><td>63765</td><td></td></tr><tr><td>3-(trimethoxysilyl)propyl methacrylate (3-TPM)</td><td>Sigma-Aldrich</td><td>440159</td><td>Store under inert gas per manufacturer&#039;s instructions. Exposure of 3-TPM to air could compromise silanization of glass substrates. CAUTION: 3-TPM is a combustible liquid. Keep away from heat, sparks, open flames, and hot surfaces and use only in a chemical fume hood.</td></tr><tr><td>3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate hydrate (CHAPS)</td><td>Sigma-Aldrich</td><td>C3023</td><td></td></tr><tr><td>35 mm glass-bottom Petri dishes</td><td>Cell E&amp;amp;G</td><td>GBD00002-200</td><td>13 mm well consisting of #1.5 coverglass. Enables TFM and live-cell imaging.</td></tr><tr><td>384-well polypropylene V-bottom microplate, non-sterile</td><td>USA Scientific</td><td>1823-8400</td><td></td></tr><tr><td>6-well polystyrene microplates</td><td>Fisher Scientific</td><td>08-772-1B</td><td>35 mm glass-bottom Petri dishes fit into wells of microplate, easing array fabrication.</td></tr><tr><td>Acetone</td><td>Sigma-Aldrich</td><td>179973</td><td></td></tr><tr><td>Acrylamide</td><td>Sigma-Aldrich</td><td>A3553</td><td>CAUTION: Exposure to acrylamide can result in acute toxicity and irritation. Wear protective gloves, clothing, and eye protection.</td></tr><tr><td>Collagen I, rat tail</td><td>EMD Millipore</td><td>08-115MI</td><td></td></tr><tr><td>Collagen III, human</td><td>EMD Millipore</td><td>CC054</td><td></td></tr><tr><td>Collagen IV, human</td><td>EMD Millipore</td><td>CC076</td><td></td></tr><tr><td>Crosslinker, 365 nm</td><td>UVP</td><td>CL-1000</td><td></td></tr><tr><td>Dextran, rhodamine B-conjugated, 70 kDa</td><td>ThermoFisher Scientific</td><td>D1841</td><td>Used as a marker for array location.</td></tr><tr><td>Dimethyl sulfoxide</td><td>Fisher Scientific</td><td>BP231</td><td></td></tr><tr><td>Dulbecco&#039;s phosphate-buffered saline (PBS)</td><td>Fisher Scientific (HyClone)</td><td>SH3001302</td><td></td></tr><tr><td>Ethyl alcohol</td><td>Decon Labs</td><td>2701</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid (EDTA)</td><td>Sigma-Aldrich</td><td>ED</td><td></td></tr><tr><td>Fc-recombinant DLL1, mouse</td><td>R&amp;amp;D Systems</td><td>5026-DL-050</td><td></td></tr><tr><td>Fc-recombinant DLL4, mouse</td><td>AdipoGen</td><td>AG-40A-0145-C050</td><td></td></tr><tr><td>Fc-recombinant JAG1, rat</td><td>R&amp;amp;D Systems</td><td>599-JG-100</td><td></td></tr><tr><td>Fibronectin, human</td><td>Sigma-Aldrich</td><td>F2006</td><td></td></tr><tr><td>Fluorescent microscope, inverted</td><td>Zeiss</td><td>Axiovert 200M</td><td>Ensure microscope is equipped with a robotic stage for both automated fluorescent imaging and TFM. Environmental control (&lt;em&gt;i.e&lt;/em&gt;., 37 &amp;deg;C and 5% CO&lt;sub&gt;2&lt;/sub&gt;) is highly advisable for TFM.</td></tr><tr><td>Fluoromount G with DAPI</td><td>SouthernBiotech</td><td>0100-20</td><td></td></tr><tr><td>Glacial acetic acid</td><td>Sigma-Aldrich</td><td>695092</td><td>CAUTION: Acetic acid is flammable and corrosive. Wear protective gloves, clothing, and eye protection.</td></tr><tr><td>Glycerol</td><td>Sigma-Aldrich</td><td>M6145</td><td></td></tr><tr><td>Irgacure 2959</td><td>BASF Corporation</td><td>55047962</td><td></td></tr><tr><td>Laminin, mouse</td><td>EMD Millipore</td><td>CC095</td><td></td></tr><tr><td>Methanol</td><td>Sigma-Aldrich</td><td>179957</td><td></td></tr><tr><td>Microarray scanner</td><td>GenePix</td><td>4000B</td><td>Fluorophores must be Cy3- or Cy5-compatible.</td></tr><tr><td>Microarrayer</td><td>Digilab</td><td>OmniGrid Micro</td><td>Other microarrayers of similar or greater capability can readily be substituted.</td></tr><tr><td>Microscope slides, 25 &amp;times; 75 mm</td><td>Sigma-Aldrich</td><td>CLS294775X25</td><td>~0.9 &amp;ndash; 1.1 mm thickness.</td></tr><tr><td>&lt;em&gt;N,N&amp;prime;&lt;/em&gt;-Methylenebisacrylamide (bisacrylamide)</td><td>Sigma-Aldrich</td><td>M7279</td><td>CAUTION: Exposure to acrylamide can result in acute toxicity and irritation. Wear protective gloves, clothing, and eye protection.</td></tr><tr><td>Paraformaldehyde (PFA), 16% v/v</td><td>Electron Microscopy Sciences</td><td>RT15710</td><td>Prepare PFA fresh (do not store) for optimal fixation. CAUTION: Exposure to PFA can result in acute toxicity and can also irritate or corrode skin on contact. Wear protective gloves, clothing, and eye protection and use only in a chemical fume hood.</td></tr><tr><td>Protein A/G, recombinant</td><td>ThermoFisher Scientific</td><td>21186</td><td></td></tr><tr><td>Pyrex drying tray, 2,000&amp;nbsp;mL</td><td>Fisher Scientific</td><td>15-242B</td><td></td></tr><tr><td>Rectangular 4-chambered culture dish</td><td>Fisher Scientific (Nunc)</td><td>12-565-495</td><td>For cell culture on arrayed microscope slides.</td></tr><tr><td>Sodium acetate</td><td>Sigma-Aldrich</td><td>S2889</td><td></td></tr><tr><td>Sodium hydroxide</td><td>Sigma-Aldrich</td><td>415413</td><td>CAUTION: NaOH is highly caustic and can cause severe skin burns and eye damage. Wear protective gloves, clothing, and eye protection.</td></tr><tr><td>Stealth pin for arraying</td><td>ArrayIt</td><td>SMP3</td><td>Clean pins after each array run using the instructions of the manufacturer. Produces 150 micron domains; purchase other pin sizes (75&amp;ndash;450 microns) as suited to your particular application.</td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>X100</td><td></td></tr></tbody></table>

a high-throughput cell microarray platform for correlative analysis of cell differentiation and traction forces

Microfabricated cellular microarrays, which consist of contact-printed combinations of biomolecules on an elastic hydrogel surface, provide a tightly controlled, high-throughput engineered system for measuring the impact of arrayed biochemical signals on cell differentiation. Recent efforts using cell microarrays have demonstrated their utility for combinatorial studies in which many microenvironmental factors are presented in parallel. However, these efforts have focused primarily on investigating the effects of biochemical cues on cell responses. Here, we present a cell microarray platform with tunable material properties for evaluating both cell differentiation by immunofluorescence and biomechanical cell&#8211;substrate interactions by traction force microscopy. To do so, we have developed two different formats utilizing polyacrylamide hydrogels of varying Young&#39;s modulus fabricated on either microscope slides or glass-bottom Petri dishes. We provide best practices and troubleshooting for the fabrication of microarrays on these hydrogel substrates, the subsequent cell culture on microarrays, and the acquisition of data. This platform is well-suited for use in investigations of biological processes for which both biochemical (e.g., extracellular matrix composition) and biophysical (e.g., substrate stiffness) cues may play significant, intersecting roles.

Using this platform, we investigated the role of both biochemical and biophysical cues in the fate specification of liver progenitors34,35. Protein A/G-conjugated Notch ligands showed improved retention and clustering in the polyacrylamide hydrogel (Figure 3A) and were furthermore capable of driving differentiation of liver progenitors towards a bile duct cell fate (Figure 3B). Using single-cell analysis, we quantified the response to the Notch ligands for the ECM proteins collagen I, collagen III, collagen IV, fibronectin, and laminin (Figure 3C), finding that the response of liver progenitors to the ligand depends also on the ECM context. Last, we utilized shRNA knockdown to generate liver progenitors without the ligands Dll1 and Jag1. The response to the arrayed Notch ligand varied depending on the presence of either ligand, confirming that the responsiveness to the cell-extrinsic ligand is also a function of the cell-intrinsic ligand expression (Figure 3D). Further, we observed a distinct subpopulation of double-positive (ALB+/OPN+) cells in the Dll1 knockdown (Figure 3D). Together, these representative results show: (1) the combinatorial capabilities of the array format, as exemplified by the pairing of multiple arrayed ECM proteins and Notch ligands with the knockdown of individual ligands; (2) the functionality of not only arrayed ECM proteins but also arrayed cell&#8211;cell ligand via Protein A/G-mediated conjugation; and (3) the implementation of our single-cell analysis and its ability to discern unique subpopulations.
We also observed that the differentiation of liver progenitors is dependent on both the substrate stiffness and the ECM composition (Figure 4A), specifically finding that collagen IV is supportive of differentiation on both soft and stiff substrates while fibronectin only supports differentiation on stiff substrates (Figure 4B). Representative heat maps of TFM measurements suggested that sustained traction stress at low substrate stiffness on collagen IV promoted differentiation into bile duct cells (Figure 4C), a finding confirmed by average root-mean-square values (Figure 4D). Together, these representative results show: (1) the successful integration of TFM with cell microarrays on substrates with a tunable stiffness to assess both the cell phenotype and the traction stress; (2) the coordination of the liver progenitor cell fate with both the matrix composition and the substrate stiffness; and (3) the implementation of our TFM analysis and typical traction stress profiles in cell microarrays.
<img alt="Figure 1" src="/files/ftp_upload/55362/55362fig1.jpg" /> 
Figure 1: Overview Schematic Showing the First Three Experimental Sections. In Section 1, glass substrates are cleaned and silanized to facilitate the fabrication of polyacrylamide hydrogels. In Section 2, the biomolecule combinations of interest are prepared in a 384-well source microplate. A robotic arrayer is then loaded with clean pins, the source microplate, and the polyacrylamide hydrogels and initialized, fabricating arrays on the hydrogels. In Section 3, cells are seeded onto the arrayed domains and allowed to adhere, after which the culture protocol of interest is performed. At the endpoint, cells are either fixed for immunocytochemistry/immunofluorescence or analyzed using TFM. Scale bars are 75 &#181;m. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55362/55362fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/55362/55362fig2.jpg" /> 
Figure 2: Processing and Analysis of Immunofluorescence Data from Arrays. (A) Tiled, composite 32-bit RGB images are first binned and then split into individual 8-bit channels. Using a combination of arrayed fluorescent markers and cell islands, three corners of the array are identified to allow for automated orientation and gridding of the arrays. (B) Single-cell data is generated for each channel of the input arrays. In order to account for experimental drift, quantile normalization is applied by biological replicate, producing a single shared distribution across all replicates. Quantile normalized data is subsequently plotted and interpreted via calculation of ensemble measurements (e.g., cells/island, mean intensity, percentage cells positive for a label) or direct analysis of single-cell distributions. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55362/55362fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/55362/55362fig3.jpg" /> 
Figure 3: Notch Ligand Presentation Mediates Liver Progenitor Differentiation. (A) Fc-recombinant Notch ligands Jagged-1 (JAG1) and Delta-like 1 (DLL1) exhibited improved retention and clustering when arrayed with Protein A/G. Scale bar is 50 &#181;m. (B) Liver progenitors differentiated into bile duct cells upon presentation with Notch ligand. 4&#39;,6-Diamidino-2-phenylindole (DAPI) is a nuclear label, albumin (ALB) is a hepatic cell marker, and osteopontin (OPN) is a bile duct cell marker. Scale bar is 150 &#181;m. (C) Quantification of percentage of cells positive for OPN for the Notch ligands JAG1, DLL1, and Delta-like 4 (DLL4) on the ECM proteins collagen I, collagen III, collagen IV, fibronectin, and laminin. Student&#39;s t-tests were performed against control IgG for each arrayed Notch ligand within each ECM protein with P-values indicated for P&#60;0.05 (*). (D) Imaging cytometry of ALB and OPN for cells on collagen III presented with the Notch ligands JAG1, DLL1, and DLL4. Liver progenitors without the Notch ligands Dll1 and Jag1 (i.e., shDll1 and shJag1) were generated using shRNA knockdown. Data in (C) presented as mean &#177; s.e.m. This figure has been modified from Kaylan et al.34. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55362/55362fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/55362/55362fig4.jpg" /> 
Figure 4: Matrix Composition and Substrate Stiffness Coordinate Liver Progenitor Differentiation. (A) Liver progenitor differentiation to bile duct cells is dependent on both ECM composition and substrate stiffness. DAPI is a nuclear label, ALB is a hepatic cell marker, and OPN is a bile duct cell marker. (B) Quantification of percentage of cells positive for OPN on substrates of Young&#39;s modulus 30 kPa, 13 kPa, and 4 kPa for collagen I (C1), collagen IV (C4), fibronectin (FN), and all two-way combinations of those ECM proteins. (C) Cell traction stress is dependent on both substrate stiffness and ECM composition. (D) Quantification of root-mean-square values of traction stress on substrates of Young&#39;s modulus 30 kPa and 4 kPa for collagen I (C1), collagen IV (C4), fibronectin (FN), and all two-way combinations of those ECM proteins. In (B) and (D), data were presented as mean &#177; s.e.m and Student&#39;s t-tests were performed against 30 kPa for each ECM combination with P-values indicated for P&#60; 0.05 (*), P&#60; 0.01 (**), and P&#60; 0.001 (***). Scale bars are 50 &#181;m. This figure has been modified from Kourouklis et al.35. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55362/55362fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td fo:width="1.2in">Section</td>
			<td fo:width="1.5in">Problem</td>
			<td fo:width="1.5in">Potential Causes</td>
			<td>Solution</td>
		</tr>
		<tr>
			<td rowspan="3">1. Fabrication of Polyacrylamide Substrate.</td>
			<td>Coverglass cannot be removed from hydrogel.</td>
			<td>Overpolymerization.</td>
			<td>Reduce polymerization time to &#60;10 minutes (4 W/m2). Check that UV crosslinker output is within expected range.</td>
		</tr>
		<tr>
			<td>Poor polyacrylamide hydrogel polymerization.</td>
			<td>Underpolymerization.</td>
			<td>Increase polymerization time to &#62;10 minutes (4 W/m2).&#160; Check that UV crosslinker output is within expected range.</td>
		</tr>
		<tr>
			<td>Polyacrylamide hydrogels are damaged after removal of coverglass.</td>
			<td>Soft polyacrylamide hydrogels are easy to damage.&#160;</td>
			<td>We observe decreasing hydrogel fabrication yield (~50%) for the softest (i.e., 4 kPa) hydrogels in particular. Handle hydrogels gently and increase starting numbers to attain desired yield.</td>
		</tr>
		<tr>
			<td rowspan="2">2. Fabrication of Arrays.</td>
			<td rowspan="2">Poor or inconsistent spot morphology.</td>
			<td>Inconsistent humidifier function.</td>
			<td>Check that humidifier and rheometer a functional throughout each print run and maintain 65% RH.</td>
		</tr>
		<tr>
			<td>Pins stuck in printhead or clogged.</td>
			<td>Clean the printhead to allow for free pin movement. Clean pins thoroughly before or after each print run to remove aggregates from pin channels.</td>
		</tr>
		<tr>
			<td rowspan="8">3. Cell Culture and Assay Execution.</td>
			<td rowspan="2">Cell detachment or death on arrays after initial attachment.</td>
			<td>Overseeding and excessive proliferation.</td>
			<td>Reduce initial seeding density and time. Use &#34;maintenance&#34; or &#34;differentiation&#34; media during array culture to reduce cell proliferation.</td>
		</tr>
		<tr>
			<td>Release of toxic acrylamide monomer from hydrogel.</td>
			<td>Soak hydrogels in dH2O for at least 3 d to allow for diffusion/release of acrylamide monomer and reduce cell toxicity.</td>
		</tr>
		<tr>
			<td rowspan="4">Cells don&#39;t attach to arrays.</td>
			<td>Underseeding.</td>
			<td>Increase initial seeding density and time. Use a more strongly adherent cell type.</td>
		</tr>
		<tr>
			<td>Poor deposition of matrix or biomolecule condition.</td>
			<td>Clean pins of particles and aggregates, confirm printing parameters, and evaluate spotting of fluorescent markers, e.g., rhodamine-conjugated dextran.</td>
		</tr>
		<tr>
			<td>Specificity of cell&#8211;matrix interactions.</td>
			<td>Different cell types adhere specifically to some but not other ECM proteins. Test multiple different ECM proteins with your cells.</td>
		</tr>
		<tr>
			<td>Suboptimal array storage after fabrication.</td>
			<td>We recommend storing fabricated arrays overnight at 65% RH and room temperature, in part to avoid phase changes during freezing. Cell adhesion is sensitive to both humidity, temperature, and storage time; make sure these parameters are consistent/optimized for your experiments.</td>
		</tr>
		<tr>
			<td rowspan="2">Detachment of hydrogel from glass substrate during cell culture.</td>
			<td>Poor slide cleaning and silanization.</td>
			<td>Replace working solutions for slide cleaning and silanization.</td>
		</tr>
		<tr>
			<td>Overdehydrated hydrogel.</td>
			<td>Don&#39;t leave hydrogels dehydrating on a hot plate for longer than 15&#8211;30 min.</td>
		</tr>
		<tr>
			<td>4. Analysis of Data.</td>
			<td>High variability between replicate spots and slides.</td>
			<td>Variability in array fabrication.</td>
			<td>Check that pins and printhead are clean. Confirm humidifier function. Visualize and quantify spot and array quality using fluorescent markers. Store arrays as recommended above.&#160;</td>
		</tr>
	</tbody>
</table>
Table 1: Troubleshooting.

Watch this Scientific Journal Video about A High-throughput Cell Microarray Platform for Correlative Analysis of Cell Differentiation and Traction Forces at JoVE.com

A High-throughput Cell Microarray Platform for Correlative Analysis of Cell Differentiation and Traction Forces

Cell differentiation is regulated by a host of microenvironmental factors, including both matrix composition and substrate material properties. We describe here a technique utilizing cell microarrays in conjunction with traction force microscopy to evaluate both cell differentiation and biomechanical cell&#8211;substrate interactions as a function of microenvironmental context.

Microfabricated cellular microarrays, which consist of contact-printed combinations of biomolecules on an elastic hydrogel surface, provide a tightly ...

a-high-throughput-cell-microarray-platform-for-correlative-analysis

Nanyang Technological University

Research

JoVE Journal

Bioengineering

9.6K Views.  University of Illinois at Urbana–Champaign. Cell differentiation is regulated by a host of microenvironmental factors, including both matrix composition and substrate material properties. We describe here a technique utilizing cell microarrays in conjunction with traction force microscopy to evaluate both cell differentiation and biomechanical cell–substrate interactions as a function of microenvironmental context.

Video: A High-throughput Cell Microarray Platform for Correlative Analysis of Cell Differentiation and Traction Forces

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In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or ...

A Versatile Automated Platform for Micro-scale Cell Stimulation Experiments

Study of cells in culture (in vitro analysis) has provided important insight into complex biological systems. Conventional methods and equipment for in vitro analysis are well suited to study of large numbers of cells (&ge;105) in milliliter-scale volumes (&ge;0.1 ml). However, there are many instances in which it is necessary or desirable to scale down culture size to reduce consumption of the cells of interest and/or reagents required for their culture, stimulation, or processing. Unfortunately, conventional approaches do not support precise and reproducible manipulation of micro-scale cultures, and the microfluidics-based automated systems currently available are too complex and specialized for routine use by most laboratories. To address this problem, we have developed a simple and versatile technology platform for automated culture, stimulation, and recovery of small populations of cells (100 - 2,000 cells) in micro-scale volumes (1 - 20 &mu;l). The platform consists of a set of fibronectin-coated microcapillaries ("cell perfusion chambers"), within which micro-scale cultures are established, maintained, and stimulated; a digital microfluidics (DMF) device outfitted with "transfer" microcapillaries ("central hub"), which routes cells and reagents to and from the perfusion chambers; a high-precision syringe pump, which powers transport of materials between the perfusion chambers and the central hub; and an electronic interface that provides control over transport of materials, which is coordinated and automated via pre-determined scripts. As an example, we used the platform to facilitate study of transcriptional responses elicited in immune cells upon challenge with bacteria. Use of the platform enabled us to reduce consumption of cells and reagents, minimize experiment-to-experiment variability, and re-direct hands-on labor. Given the advantages that it confers, as well as its accessibility and versatility, our platform should find use in a wide variety of laboratories and applications, and prove especially useful in facilitating analysis of cells and stimuli that are available in only limited quantities.

We have developed an automated cell culture and interrogation platform for micro-scale cell stimulation experiments. The platform offers simple, versatile, and precise control in cultivating and stimulating small populations of cells, and recovering lysates for molecular analyses. The platform is well suited to studies that use precious cells and/or reagents.

Study of cells in culture (in vitro analysis) has provided important insight into complex biological systems. Conventional methods and equipment for in ...

Easy and Accurate Mechano-profiling on Micropost Arrays

Cell culture substrates with integrated flexible microposts enable a user to study the mechanical interactions between cells and their immediate surroundings. Particularly, cell-substrate interactions are the main interest. Today micropost arrays are a well-characterized and established method with a broad range of applications that have been published over the last decade. However, there seems to be a reservation among biologists to adapt the technique due to the lengthy and challenging process of micropost manufacture along with the lack of easily approachable software for analyzing images of cells interacting with microposts. 
The force read-out from microposts is surprisingly easy. A micropost acts like a spring with the cell ideally attached at its tip. Depending on size a cell applies force from its cytoskeleton through one or multiple focal adhesion points to the micropost, thus deflecting the micropost. The amount of deflection correlates directly to the applied force in direction and in magnitude. The number of microposts covered by a cell and the post deflection patterns are characteristic and allow determination of values like force per post and many biologically relevant parameters that allow &#8220;mechano-profiling&#8221; of cell phenotypes.
A convenient method for mechano-profiling is described here combining the first generation of ready-to-use commercially available microposts with an in-house developed software package that is now accessible to all researchers. As a demonstration of typical application, single images of bone cancer cells were taken in bright-field microscopy for mechano-profiling of cell line models of metastasis. This combination of commercial traction force sensors and open source software for analysis allows for the first time a rapid implementation of the micropost array technique into routine lab work done by non-expert users. Furthermore, a robust and streamlined analysis process enables a user to analyze a large number of micropost images in a highly time-efficient manner.

Described are protocols for quantifying mechanical interactions between adherent cells and microstructured substrates. These interactions are closely linked to essential cell behaviors including migration, proliferation, differentiation, and apoptosis. The protocols present an open-source image analysis software called MechProfiler, which enables determination of involved forces for each micropost.

Cell culture substrates with integrated flexible microposts enable a user to study the mechanical interactions between cells and their immediate ...

High Throughput Characterization of Adult Stem Cells Engineered for Delivery of Therapeutic Factors for Neuroprotective Strategies

Mesenchymal stem cells (MSCs) derived from bone marrow are a powerful cellular resource and have been used in numerous studies as potential candidates to develop strategies for treating a variety of diseases. The purpose of this study was to develop and characterize MSCs as cellular vehicles engineered for delivery of therapeutic factors as part of a neuroprotective strategy for rescuing the damaged or diseased nervous system. In this study we used mouse MSCs that were genetically modified using lentiviral vectors, which encoded brain-derived neurotrophic factor (BDNF) or glial cell-derived neurotrophic factor (GDNF), together with green fluorescent protein (GFP). 
Before proceeding with in vivo transplant studies it was important to characterize the engineered cells to determine whether or not the genetic modification altered aspects of normal cell behavior. Different culture substrates were examined for their ability to support cell adhesion, proliferation, survival, and cell migration of the four subpopulations of engineered MSCs. High content screening (HCS) was conducted and image analysis performed. 
Substrates examined included: poly-L-lysine, fibronectin, collagen type I, laminin, entactin-collagen IV-laminin (ECL). Ki67 immunolabeling was used to investigate cell proliferation and Propidium Iodide staining was used to investigate cell viability. Time-lapse imaging was conducted using a transmitted light/environmental chamber system on the high content screening system.
Our results demonstrated that the different subpopulations of the genetically modified MSCs displayed similar behaviors that were in general comparable to that of the original, non-modified MSCs. The influence of different culture substrates on cell growth and cell migration was not dramatically different between groups comparing the different MSC subtypes, as well as culture substrates. 
This study provides an experimental strategy to rapidly characterize engineered stem cells and their behaviors before their application in long-term in vivo transplant studies for nervous system rescue and repair.

This study describes an experimental platform to rapidly characterize engineered stem cells and their behaviors before their application in long-term in vivo transplant studies for nervous system rescue and repair.

Mesenchymal stem cells (MSCs) derived from bone marrow are a powerful cellular resource and have been used in numerous studies as potential candidates to ...

Medicine

High Throughput Traction Force Microscopy Using PDMS Reveals Dose-Dependent Effects of Transforming Growth Factor-&#946; on the Epithelial-to-Mesenchymal Transition

Cellular contractility is essential in diverse aspects of biology, driving processes that range from motility and division, to tissue contraction and mechanical stability, and represents a core element of multi-cellular animal life. In adherent cells, acto-myosin contraction is seen in traction forces that cells exert on their substrate. Dysregulation of cellular contractility appears in a myriad of pathologies, making contractility a promising target in diverse diagnostic approaches using biophysics as a metric. Moreover, novel therapeutic strategies can be based on correcting the apparent malfunction of cell contractility. These applications,&#160;however, require direct quantification of these forces.
We have developed silicone elastomer-based traction force microscopy (TFM) in a parallelized multi-well format. Our use of a silicone rubber, specifically polydimethylsiloxane (PDMS), rather than the commonly employed hydrogel polyacrylamide (PAA) enables us to make robust and inert substrates with indefinite shelf-lives requiring no specialized storage conditions. Unlike pillar-PDMS based approaches that have a modulus in the GPa range, the PDMS used here is very compliant, ranging from approximately 0.4 kPa to 100 kPa. We create a high-throughput platform for TFM by partitioning these large monolithic substrates spatially into biochemically independent wells, creating a multi-well platform for traction force screening that is compatible with existing multi-well systems.
In this manuscript, we use this multi-well traction force system to examine the Epithelial to Mesenchymal Transition (EMT); we induce EMT in NMuMG cells by exposing them to TGF-&#946;, and to quantify the biophysical changes during EMT. We measure the contractility as a function of concentration and duration of TGF-&#946; exposure. Our findings here demonstrate the utility of parallelized TFM in the context of disease biophysics.

We present a high throughput traction force assay fabricated with silicone rubber (PDMS). This novel assay is suitable for studying physical changes in cell contractility during various biological and biomedical processes and diseases. We demonstrate this method's utility by measuring a TGF-&#946; dependent increase in contractility during the epithelial-to-mesenchymal transition.

Cellular contractility is essential in diverse aspects of biology, driving processes that range from motility and division, to tissue contraction and ...

Fabrication and Implementation of a Reference-Free Traction Force Microscopy Platform

Quantifying cell-induced material deformation provides useful information concerning how cells sense and respond to the physical properties of their microenvironment. While many approaches exist for measuring cell-induced material strain, here we provide a methodology for monitoring strain with sub-micron resolution in a reference-free manner. Using a two-photon activated photolithographic patterning process, we demonstrate how to generate mechanically and bio-actively tunable synthetic substrates containing embedded arrays of fluorescent fiducial markers to easily measure three-dimensional (3D) material deformation profiles in response to surface tractions. Using these substrates, cell tension profiles can be mapped using a single 3D image stack of a cell of interest. Our goal with this methodology is to make traction force microscopy a more accessible and easier to implement tool for researchers studying cellular mechanotransduction processes, especially newcomers to the field.

This protocol provides instructions for implementing multiphoton lithography to fabricate three-dimensional arrays of fluorescent fiducial markers embedded in poly(ethylene glycol)-based hydrogels for use as reference-free, traction force microscopy platforms. Using these instructions, measurement of 3D material strain and calculation of cellular tractions is simplified to promote high-throughput traction force measurements.

Quantifying cell-induced material deformation provides useful information concerning how cells sense and respond to the physical properties of their ...

Traction Microscopy Integrated with Microfluidics for Chemotactic Collective Migration

Cells change migration patterns in response to chemical stimuli, including the gradients of the stimuli. Cellular migration in the direction of a chemical gradient, known as chemotaxis, plays an important role in development, the immune response, wound healing, and cancer metastasis. While chemotaxis modulates the migration of single cells as well as collections of cells in vivo, in vitro research focuses on single-cell chemotaxis, partly due to the lack of the proper experimental tools. To fill that gap, described here is a unique experimental system that combines microfluidics and micropatterning to demonstrate the effects of chemical gradients on collective cell migration. Furthermore, traction microscopy and monolayer stress microscopy are incorporated into the system to characterize changes in cellular force on the substrate as well as between neighboring cells. As proof-of-concept, the migration of micropatterned circular islands of Madin-Darby canine kidney (MDCK) cells is tested under a gradient of hepatocyte growth factor (HGF), a known scattering factor. It is found that cells located near the higher concentration of HGF migrate faster than those on the opposite side within a cell island. Within the same island, cellular traction is similar on both sides, but intercellular stress is much lower on the side of higher HGF concentration. This novel experimental system can provide new opportunities to studying the mechanics of chemotactic migration by cellular collectives.

Collective cell migration in development, wound healing, and cancer metastasis is often guided by the gradients of growth factors or signaling molecules. Described here is an experimental system combining traction microscopy with a microfluidic system and a demonstration of how to quantify the mechanics of collective migration under biochemical gradient.

Cells change migration patterns in response to chemical stimuli, including the gradients of the stimuli. Cellular migration in the direction of a chemical ...

Simplified, High-throughput Analysis of Single-cell Contractility using Micropatterned Elastomers

Cellular contractile force generation is a fundamental trait shared by virtually all cells. These contractile forces are crucial to proper development, function at both the cellular and tissue levels,and regulate the mechanical systems in the body. Numerous biological processes are force-dependent, including motility, adhesion, and division of single-cells, as well as contraction and relaxation of organs such as the heart, bladder, lungs, intestines, and uterus. Given its importance in maintaining proper physiological function, cellular contractility can also drive disease processes when exaggerated or disrupted. Asthma, hypertension, preterm labor, fibrotic scarring, and underactive bladder are all examples of mechanically driven disease processes that could potentially be alleviated with proper control of cellular contractile force. Here, we present a comprehensive protocol for utilizing a novel microplate-based contractility assay technology known as fluorescently labeled elastomeric contractible surfaces (FLECS), that provides simplified and intuitive analysis of single-cell contractility in a massively scaled manner. Herein, we provide a step-wise protocol for obtaining two six-point dose-response curves describing the effects of two contractile inhibitors on the contraction of primary human bladder smooth muscle cells in a simple procedure utilizing just a single FLECS assay microplate, to demonstrate proper technique to users of the method. Using FLECS Technology, all researchers with basic biological laboratories and fluorescent microscopy systems gain access to studying this fundamental but difficult-to-quantify functional cell phenotype, effectively lowering the entry barrier into the field of force biology and phenotypic screening of contractile cell force.

This work presents a flexible protocol for utilizing fluorescently labeled elastomeric contractible surfaces (FLECS) Technology in microwell format for simplified, hands-off quantification of single-cell contractile forces based on visualized displacements of fluorescent protein micropatterns.

Cellular contractile force generation is a fundamental trait shared by virtually all cells. These contractile forces are crucial to proper development, ...

Pattern Generation for Micropattern Traction Microscopy

Micropattern traction microscopy allows control of the shape of single cells and cell clusters. Furthermore, the ability to pattern at the micrometer length scale allows the use of these patterned contact zones for the measurement of traction forces, as each micropatterned dot allows for the formation of a single focal adhesion that then deforms the soft, underlying hydrogel. This approach has been used for a wide range of cell types, including endothelial cells, smooth muscle cells, fibroblasts, platelets, and epithelial cells. 
This review describes the evolution of techniques that allow the printing of extracellular matrix proteins onto polyacrylamide hydrogels in a regular array of dots of prespecified size and spacing. As micrometer-scale patterns are difficult to directly print onto soft substrates, patterns are first generated on rigid glass coverslips that are then used to transfer the pattern to the hydrogel during gelation. First, the original microcontact printing approach to generate arrays of small dots on the coverslip is described. A second step that removes most of the pattern to leave islands of small dots is required to control the shapes of cells and cell clusters on such arrays of patterned dots. 
Next, an evolution of this approach that allows for the generation of islands of dots using a single subtractive patterning step is described. This approach is greatly simplified for the user but has the disadvantage of a decreased lifetime for the master mold needed to make the patterns. Finally, the computational approaches that have been developed for the analysis of images of displaced dots and subsequent cell-generated traction fields are described, and updated versions of these analysis packages are provided.

We describe improvements to a standard method for measuring cellular traction forces, based on microcontact printing with a single subtractive patterning step of dot arrays of extracellular matrix proteins on soft hydrogels. This method allows for simpler and more consistent fabrication of island patterns, essential for controlling cell cluster shape.

Micropattern traction microscopy allows control of the shape of single cells and cell clusters. Furthermore, the ability to pattern at the micrometer ...

Single Cell Durotaxis Assay for Assessing Mechanical Control of Cellular Movement and Related Signaling Events

Durotaxis is the process by which cells sense and respond to gradients of tension. In order to study this process in vitro, the stiffness of the substrate underlying a cell must be manipulated. While hydrogels with graded stiffness and long-term migration assays have proven useful in durotaxis studies, immediate, acute responses to local changes in substrate tension allow focused study of individual cell movements and subcellular signaling events. To repeatably test the ability of cells to sense and respond to the underlying substrate stiffness, a modified method for application of acute gradients of increased tension to individual cells cultured on deformable hydrogels is used which allows for real time manipulation of the strength and direction of stiffness gradients imparted upon cells in question. Additionally, by fine tuning the details and parameters of the assay, such as the shape and dimensions of the micropipette or the relative position, placement, and direction of the applied gradient, the assay can be optimized for the study of any mechanically sensitive cell type and system. These parameters can be altered to reliably change the applied stimulus and expand the functionality and versatility of the assay. This method allows examination of both long term durotactic movement as well as more immediate changes in cellular signaling and morphological dynamics in response to changing stiffness.

Mechanical forces are important for controlling cell migration. This protocol demonstrates the use of elastic hydrogels that can be deformed using a glass micropipette and a micromanipulator to stimulate cells with a local stiffness gradient to elicit changes in cell structure and migration.

Durotaxis is the process by which cells sense and respond to gradients of tension. In order to study this process in vitro, the stiffness of the substrate ...