This work was supported by postdoctoral fellowship from Stanford Child Health Research Institute (CHRI) and National Institute of Health (1F32HL142205-01) to S.L, the NIH Office of Director&#8217;s Pioneer Award (LM012179-03), the American Heart Association Established Investigator Award (17EIA33410923), the Stanford Cardiovascular Institute, the Hoffmann and Schroepfer Foundation, and the Stanford Division of Cardiovascular Medicine, Department of Medicine to S.M.W, awards from National Institute of Health (UG3 TR002588, P01 HL141084, R01 HL126527, R01 HL113006, R01 HL123968) and Tobacco-related Disease Research Program (TRDRP 27IR-0012) to J.C.W, the American Heart Association (AHA) Postdoctoral Fellowship Award (18POST34030106) to H.Y, and the Hengstberger fellowship to T.S. We thank Dr. Andrew Olsen from Stanford Neuroscience Microscopy Service on the support of confocal imaging of the micropatterned hiPSC-CM. We thank H.Y. for first stencil design, fabrication, single cell micropatterning of iPSC-CM on the polyacrylamide hydrogel coated coverslip, and preliminary confocal imaging of the sarcomere structure of single cell micropatterned iPSC-CMs.

1. Fabrication of negative pattern polyimide-based stencils
<ol>
	<li>Generate a pattern (Figure 2A) in .dxf format using computer-aided design software (e.g., AutoCAD, SolidWorks, Onshape, Adobe Illustrator).
	<ol>
		<li>Generate a circle (diameter = 22 mm) to delineate the border of the stencil.</li>
		<li>Draw a solid-filled shape or the pattern desired.</li>
		<li>Include a chiral letter (e.g., R) to mark the front side of the stencils. 
		NOTE: As an example, an array of squares and rectangles is generated to pattern iPSC-CMs at single-cell and cell-pair levels. The design files are available (see Supplemental Files). The microfabrication resolution limit is ~10 &#181;m.</li>
	</ol>
	</li>
	<li>Submit the design to a microfabrication company to laser-cut polyimide film and fabricate stencils (Figure 2B,C).</li>
</ol>
2. Preparation of sulfo-SANPAH aliquots
NOTE: The protocol is modified from Fischer et al.11.
<ol>
	<li>Use a moisture-resistant cardboard sample storage box (e.g., 100 well, 10 x 10 spaces for centrifuge tubes, dimensions: 13.4 cm x 13.4 cm x 5.1 cm). Label it &#34;name/date/sulfo-SANPAH 40 &#181;L/reconstituted with 1,200 &#181;L of PBS&#34;.</li>
	<li>Label 50 microcentrifuge tubes (polypropylene, 1.5 mL) with an &#39;S&#39; on the lid.</li>
	<li>Take 4 mL of anhydrous, extra dry dimethyl sulfoxide (DMSO) and sterile filter the solution using a membrane filter unit (pore size = 0.22 &#181;m) into a sterile 15 mL conical tube.</li>
	<li>Prepare a liquid nitrogen bath and cover with the lid to minimize the evaporation of the liquid nitrogen.</li>
	<li>Dissolve 50 mg of sulfo-SANPAH in 2 mL of the filtered DMSO.</li>
	<li>Vortex well to fully dissolve the sulfo-SANPAH in the DMSO.</li>
	<li>Distribute 40 &#181;L aliquots of the sulfo-SANPAH solution into sterile 1.5 mL tubes.</li>
	<li>Transfer the tubes into the box.</li>
	<li>Flash-freeze the tubes in liquid nitrogen for 5 min.</li>
	<li>Store the aliquots at -80 &#176;C. 
	NOTE: The aliquots can be used for up to 6 months without diminished efficiency. The stock sulfo-SANPAH concentration is 25 mg/mL or 50.77 mM.</li>
</ol>
3. Sterilization of tools
<ol>
	<li>Autoclave two forceps, 24 glass coverslips (22 x 22 mm, No. 1 or No. 1.5), one razor blade, and three lint-free absorbent wipes. 
	NOTE: To make 12 hydrogel constructs, 24 glass coverslips are needed. There should be two coverslips per construct. It is advisable to prepare some spare coverslips.</li>
	<li>Place two pieces of paraffin film (4 x 4 inch) in an ethanol resistant plastic box (e.g., a 1,000 &#181;L pipette tip box) and sterilize them in fresh 70% ethanol for at least 15 min.&#160;</li>
</ol>
4. Preparation of polyacrylamide hydrogel precursor solution
NOTE: The protocol is modified from Lee et al.12.
<ol>
	<li>Dissolve 125 mg of aminoethyl methacrylate (AEM) in 36.25 mL of deionized water in a 50 mL polypropylene centrifuge tube by vortexing.</li>
	<li>To prepare 50 mL of 10 kPa polyacrylamide precursor solution (8&#8211;0.15&#8211;15 mM), mix 10 mL of 40% acrylamide solution, 3.75 mL of 2% bis-acrylamide solution, and 36.25 mL of the AEM solution prepared in step 4.1 in a new 50 mL polypropylene centrifuge tube. 
	NOTE: The final concentration of precursor solution is 8% acrylamide, 0.15% bis-acrylamide, and 15 mM AEM, which was measured to have ~10 kPa stiffness by atomic force microscopy. According to the tissue applications of interest, the stiffness of the hydrogel can be altered by changing the ratio of the 40% acrylamide to the 2% bis-acrylamide solution. For example, 8% acrylamide&#8211;0.08% bis-acrylamide solution yields 3 kPa hydrogels; 8% acrylamide&#8211;0.48% bis-acrylamide solution yields 38 kPa hydrogels; 8% acrylamide&#8211;0.48% bis-acrylamide solution yields 60 kPa hydrogels. It is advisable to confirm the stiffness of hydrogels made from the precursor solutions with altered ratios.</li>
</ol>
5. Preparation of photoinitiator solution
<ol>
	<li>For 1 mL of 5% photoinitiator solution, first dissolve 50 mg of 2-hydroxy-4&#8217;-(2-hydroxyethoxy)-2-methylpropiophenone powder in 700 &#181;L of 100% ethanol in a 1.5 mL polypropylene centrifuge tube with lid. 
	NOTE: 2-Hydroxy-4&#8217;-(2-hydroxyethoxy)-2-methylpropiophenone is not soluble in water. Make sure to fully dissolve the powder in ethanol by vortexing.</li>
	<li>After completely dissolving the 2-hydroxy-4&#8217;-(2-hydroxyethoxy)-2-methylpropiophenone in ethanol by vortexing, add 300 &#181;L of phosphate-buffered saline (PBS) for a final volume of 1 mL. 
	NOTE: The final concentration of 2-hydroxy-4&#8217;-(2-hydroxyethoxy)-2-methylpropiophenone solution is 5%. The foiled 5% photoinitiator solution is good for 4 weeks at 4 &#176;C.</li>
</ol>
6. Preparation of basement membrane matrix protein solution
NOTE: Basement membrane matrix (Table of Materials) is a temperature-sensitive material. Make sure to work on ice with chilled pipette tips and tubes as well as ice-cold solutions. A new stock of basement membrane matrix should be thawed slowly on ice at 4 &#176;C overnight.
<ol>
	<li>Prepare 200 &#181;L of basement membrane matrix protein solution per hydrogel construct. For 12 constructs, 2.4 mL of basement membrane matrix protein solution are needed. Prepare 10% extra solution (e.g., 2.6 mL).</li>
	<li>For every new batch/lot of basement membrane matrix protein solution, optimize the dilution ratio. For the first time, test dilution ratios of 1:10, 1:20, 1:40 to find the dilution ratio that yields the best qualities for cell patterning. 
	NOTE: If the basement membrane matrix protein solution is too viscous, it will form a gel on top of the stencil, and it is more likely to peel off when removing the stencil. If the protein solution is too fluid, it will penetrate through the pattern holes of the stencils, which will result in poor quality patterning.</li>
	<li>Dilute basement membrane matrix protein stock solution on ice with ice-cold Dulbecco&#8217;s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12) media or 1x PBS to the optimized dilution ratio above. For example, dilute 120 &#181;L of the stock solution in 2.4 mL of DMEM/F12 media (1:20) and keep on ice for later use.</li>
</ol>
7. Hydrogel fabrication
<ol>
	<li>Place a UV bench lamp (365 nm, 4 mW/cm2) in a biological safety hood.</li>
	<li>Place the sterilized paraffin films from step 3.2 and dry completely under sterile conditions. If the paraffin films are not completely dry, use a vacuum aspiration system to remove any residual liquid. Place six autoclaved coverslips from step 3.1 on each paraffin film using the autoclaved forceps.</li>
	<li>Prepare UV-crosslinkable polyacrylamide precursor solution by diluting 5% photoinitiator solution (step 5.2) with polyacrylamide precursor solution (step 4.2) in a 50 mL polypropylene centrifuge tube. For 5 mL of UV-crosslinkable polyacrylamide precursor solution, add 50 &#181;L of 5% photoinitiator solution to 5 mL of polyacrylamide precursor solution.</li>
	<li>Filter the precursor solution from step 7.3 using a 50 mL filter unit with a 0.22 &#181;m pore size for sterilization. 
	NOTE: Always prepare fresh precursor solution.</li>
	<li>Dispense 200 &#181;L of polyacrylamide precursor solution from step 7.4 on each 22 x 22 mm glass cover slip in the paraffin filmed Petri dish and carefully cover with another 22 x 22 mm glass coverslip on top to sandwich the solution in between (Figure 3A). 
	NOTE: Paraffin film is used to avoid spilling and to confine the precursor solution in between the coverslips.</li>
	<li>Place the coverslip-containing Petri dish from step 7.5 under the UV bench lamp and photopolymerize the polyacrylamide hydrogels for 5 min (Figure 3B). 
	NOTE:&#160;If the surface is non-white, the UV absorbance of surface reduces the photocrosslinking efficiency. The coverage of white paper on the non-white surface is important to minimize the UV intensity loss.&#160;To protect from UV radiation, cover the lamp with aluminum foil and wear UV protection goggles.</li>
	<li>Carefully detach one cover slip off from the other using a razor blade by leverage action (Figure 3C). 
	NOTE: The hydrogel will usually stick to the top coverslip. Pay extra attention when detaching the coverslip from the soft hydrogel, because it is likely to break.</li>
	<li>Fill a disposable polypropylene reservoir with PBS. Transfer the hydrogel-coverslip composites&#160;in a PBS-containing reservoir to rinse the hydrogels in PBS for 5 min. After rinsing, place the hydrogel construct back on the paraffin film in the Petri dish.</li>
</ol>
8. Protein conjugation
<ol>
	<li>Prepare an ice bucket and precool PBS. 
	NOTE: For six hydrogels, 1,200 &#181;L of cold PBS is needed.</li>
	<li>Take out the sulfo-SANPAH aliquot (1 vial = 6 gels). 
	NOTE: If necessary, the amount of sulfo-SANPAH used can be reduced after optimization.</li>
	<li>Add 1,200 &#181;L of cold PBS into a vial of sulfo-SANPAH aliquot and mix by pipetting up and down.</li>
	<li>Dispense 200 &#181;L of sulfo-SANPAH on the paraffin film in the Petri dish and cover with the hydrogel-coverslip composite with hydrogel contacting sulfo-SANPAH (Figure 3D). 
	NOTE: Sulfo-SANPAH is very susceptible to water due to hydrolysis. Freshly thaw from -80 &#176;C right before usage. Do not reuse or refreeze any leftovers.</li>
	<li>Expose the hydrogel to the UV lamp at 365 nm, 4 mW/cm2 for 5 min to activate sulfo-SANPAH. 
	NOTE: After exposure, the sulfo-SANPAH will change from orange to brown (Figure 3E). If the hydrogel turns brown, it indicates successful activation of the phenyl azide group of sulfo-SANPAH and incorporation of sulfo-SANPAH onto the hydrogel. Directly proceed with steps 8.6&#8722;8.9 within a maximum of 10 min, because the activity of sulfo-SANPAH will decline.</li>
	<li>Replenish a disposable polypropylene reservoir with fresh PBS. After sulfo-SANPAH activation, transfer the activated hydrogels and quickly rinse the hydrogels in a PBS reservoir to remove unbound sulfo-SANPAH. 
	NOTE: A long wash can result in low protein conjugation efficiency.</li>
	<li>After a quick rinse, place the hydrogel-attached coverslips on the paraffin film in the Petri dishes and carefully dab the hydrogels with sterile lint-free wipes. 
	NOTE: The remaining PBS on top of the hydrogel will result in leakage of the basement membrane matrix protein solution through the stencil, and drying too extensively will lead to rupture of the hydrogel upon removal of the stencil due to too high adherence to the stencil.</li>
	<li>Place the stencil on top of the hydrogels and dab with autoclaved lint-free wipes to ensure tight sealing between the stencil and the hydrogel (Figure 3F).</li>
	<li>Carefully dispense 200 &#181;L of the diluted basement membrane matrix protein solution prepared from step 6.3 (Figure 3G) on top. Leave overnight in the incubator (37 &#176;C). 
	NOTE: When the stencil-hydrogel contact is tight and the basement membrane matrix protein solution concentration is optimal, the basement membrane matrix protein solution will be sequestered and remain on top of the stencil (Figure 3H). Incubation time can be reduced upon optimization. Theoretically, it can be reduced down to 30 min&#8211;2 h.</li>
	<li>The next day, transfer the hydrogels to a 6 well plate and fully immerse them in PBS. 
	NOTE: It is recommended to peel off the stencil with PBS immersion over the coverslip to prevent tearing and sticking of the hydrogel to the stencil.</li>
	<li>Carefully remove the stencil using autoclaved tweezers without tearing the hydrogel. Resuspend well with DMEM, return the patterned hydrogels to the incubator, and leave them overnight to test for any contamination.</li>
	<li>If the media remains clear, the hydrogels are ready to be used for cell plating. Optimize the cell plating density and incubation time to allow adhesion of the cells for respective applications. 
	NOTE: For single cell patterning of hiPSC-CMs, seed and incubate overnight. The following cell numbers are recommended for seeding: 30,000, 60,000, and 90,000 cells/well. Seeding too many cells leads to more than one cell per pattern. A cell strainer is recommended to strain out non-single cells before cell seeding.</li>
	<li>The next day, observe cell attachment and spreading, and change the media to get rid of detached cells.</li>
</ol>
9. Cleaning of the used stencils
<ol>
	<li>To remove residual matrix proteins on the used stencils, submerge the stencils in enzyme solution (Table of Materials) for 10 min at 37 &#176;C. 
	NOTE: The enzyme should be selected depending on the matrix proteins used. For collagen-rich matrix protein solution, use collagenase. A 10&#8211;15 min ultrasonication in enzyme solution is also recommended.</li>
	<li>Aspirate the solution and replenish with 10% bleach. Incubate for 10 min at room temperature.</li>
	<li>Rinse 3x with PBS.</li>
	<li>Store in 70% ethanol at 4 &#176;C until use.</li>
</ol>

<ol>
	<li>Burridge, P. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemically+Defined+and+Small+Molecule-Based+Generation+of+Human+Cardiomyocytes.">Chemically Defined and Small Molecule-Based Generation of Human Cardiomyocytes.</a> Nature Methods. 11 (8), 855-860 (2014).</li><li>Sayed, N., Liu, C., Wu, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translation+of+Human-Induced+Pluripotent+Stem+Cells:+From+Clinical+Trial+in+a+Dish+to+Precision+Medicine.">Translation of Human-Induced Pluripotent Stem Cells: From Clinical Trial in a Dish to Precision Medicine.</a> Journal of the American College of Cardiology. 67 (18), 2161-2176 (2016).</li><li>Ribeiro, A. J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contractility+of+single+cardiomyocytes+differentiated+from+pluripotent+stem+cells+depends+on+physiological+shape+and+substrate+stiffness.">Contractility of single cardiomyocytes differentiated from pluripotent stem cells depends on physiological shape and substrate stiffness.</a> Proceedings of the National Academy of Sciences. 112 (41), 12705-12710 (2015).</li><li>Kumar, A., Biebuyck, H. A., Whitesides, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterning+Self-Assembled+Monolayers:+Applications+in+Materials+Science.">Patterning Self-Assembled Monolayers: Applications in Materials Science.</a> Langmuir. 10 (5), 1498-1511 (1994).</li><li>Wilbur, J. L., Kumar, A., Kim, E., Whitesides, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfabrication+by+microcontact+printing+of+self-assembled+monolayers.">Microfabrication by microcontact printing of self-assembled monolayers.</a> Advanced Materials. 6 (7-8), 600-604 (1994).</li><li>Th&#233;ry, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micropatterning+as+a+tool+to+decipher+cell+morphogenesis+and+functions.">Micropatterning as a tool to decipher cell morphogenesis and functions.</a> Journal of Cell Science. 123 (24), 4201-4213 (2010).</li><li>Toepke, M. W., Beebe, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PDMS+absorption+of+small+molecules+and+consequences+in+microfluidic+applications.">PDMS absorption of small molecules and consequences in microfluidic applications.</a> Lab on a Chip. 6 (12), 1484-1486 (2006).</li><li>Seeger, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Premature+Termination+Codon+Mutation+in+MYBPC3+Causes+Hypertrophic+Cardiomyopathy+via+Chronic+Activation+of+Nonsense-Mediated+Decay.">A Premature Termination Codon Mutation in MYBPC3 Causes Hypertrophic Cardiomyopathy via Chronic Activation of Nonsense-Mediated Decay.</a> Circulation. 139 (6), 799-811 (2019).</li><li>Lee, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+PDGF+pathway+links+LMNA+mutation+to+dilated+cardiomyopathy.">Activation of PDGF pathway links LMNA mutation to dilated cardiomyopathy.</a> Nature. 572 (7769), 335-340 (2019).</li><li>Wu, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modelling+diastolic+dysfunction+in+induced+pluripotent+stem+cell-derived+cardiomyocytes+from+hypertrophic+cardiomyopathy+patients.">Modelling diastolic dysfunction in induced pluripotent stem cell-derived cardiomyocytes from hypertrophic cardiomyopathy patients.</a> European Heart Journal. 40 (45), 3685-3695 (2019).</li><li>Fischer, R. S., Myers, K. A., Gardel, M. L., Waterman, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stiffness-controlled+three-dimensional+extracellular+matrices+for+high-resolution+imaging+of+cell+behavior.">Stiffness-controlled three-dimensional extracellular matrices for high-resolution imaging of cell behavior.</a> Nature Protocols. 7 (11), 2056-2066 (2012).</li><li>Lee, S., Stanton, A. E., Tong, X., Yang, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogels+with+enhanced+protein+conjugation+efficiency+reveal+stiffness-induced+YAP+localization+in+stem+cells+depends+on+biochemical+cues.">Hydrogels with enhanced protein conjugation efficiency reveal stiffness-induced YAP localization in stem cells depends on biochemical cues.</a> Biomaterials. 202, 26-34 (2019).</li><li>Th&#233;ry, M., P&#233;pin, A., Dressaire, E., Chen, Y., Bornens, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+distribution+of+stress+fibres+in+response+to+the+geometry+of+the+adhesive+environment.">Cell distribution of stress fibres in response to the geometry of the adhesive environment.</a> Cell Motility. 63 (6), 341-355 (2006).</li><li>Kilian, K. A., Bugarija, B., Lahn, B. T., Mrksich, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Geometric+cues+for+directing+the+differentiation+of+mesenchymal+stem+cells.">Geometric cues for directing the differentiation of mesenchymal stem cells.</a> Proceedings of the National Academy of Sciences. 107 (11), 4872-4877 (2010).</li></ol>

The authors have nothing to disclose.

We describe a lithography-free stencil-based micropatterning method that enables effective patterning of adherent cells. In this protocol, we demonstrate patterning of hiPSC-CMs in different length-to-width ratios by micropatterning basement membrane matrix protein islands on polyacrylamide hydrogels with physiologically- or pathologically-relevant tissue stiffnesses or silicon-based elastomer substrates. This method is relatively simple and highly accessible to any researchers, including those who have little background...

The advent of hiPSCs and the subsequent development of protocols for their directed differentiation into different cell types have made it possible to study development and disease at a molecular and patient-specific level, specifically using patient-derived iPSC cardiomyocytes (iPSC-CMs) to model cardiomyopathies1,2. However, a major limitation to studying development and physiology using the iPSC system and other in vitro models is the absence of a structured microenvironment. In situ, cells are subjected to the constraints of the extracellular matrix (ECM), as well as neighboring cells. The particular biochemical composition and stiffness of these microenvironments dictate the spatial distribution of cells as well as factors available for engaging in cell adhesion. This, in turn, influences intracellular signaling pathways, gene expression, and cell fate determination. For example, micropatterned iPSC-CM in an adult-like rod shape has a significantly better contractile ability, calcium flow, mitochondrial organization, electrophysiology, and transverse-tubule formation3. Thus, the properties of the microenvironment are integral in the regulation of cellular functions.
Previous micropatterning techniques heavily relied on photolithography (Figure 1A). In this technique, a layer of photosensitive polymer, or photoresist, is spun on a flat substrate from solution to form a thin film about 1 &#956;m thick. Next, ultraviolet (UV) light is applied onto the photoresist through a mask containing the desired pattern. Exposure to ultraviolet (UV) light chemically alters the photoresist by modifying its solubility in its respective developer solution, transferring the desired pattern from the mask onto the substrate. Many micropatterning methods incorporate photolithography, as it confers nanometer to micrometer-level control over the design of the cell patterns. However, the spinning of the photoresist is highly sensitive to impurities, because the smallest dust particles will disrupt the spreading of the solution into a thin film. Photolithography must therefore be carried out in uncontaminated facilities, which are costly to maintain and require special expertise to utilize. In addition, the chemicals used in photolithography are often toxic to cells and can denature important biomolecules. Thus, photolithography poses significant obstacles to the fabrication of micropatterns for convenient biological applications.
In 1994, Whitesides and colleagues4 overcame some of the challenges associated with photolithography by pioneering a collection of techniques called soft lithography. In soft lithography, a microstructured surface made with polydimethylsiloxane (PDMS), a transparent, rubber-like material, is used to generate a pattern of ECM proteins4. Common soft lithographic techniques include microcontact printing and microfluidic patterning. In microcontact printing, currently the most popular soft lithographic method, a PDMS stamp coated with ECM proteins transfers the material onto a surface at the areas contacted by the stamp (Figure 1B). In microfluidic patterning, microstructures are designed on a PDMS surface such that when the stamp is pressed to a substrate, a network of microchannels, through which fluids can be delivered to desired areas, is created (Figure 1C)5. Soft lithography offers several benefits over photolithography. Once a master wafer is microfabricated, the PDMS stamps can easily be replicated without further employment of clean-room facilities. In addition, the absence of organic solvents in the process of soft lithography allows for utilization of polymeric materials such as polystyrene, typically used in cell culture. Finally, micropatterning using soft lithographic methods is not restricted to flat surfaces. Thus, soft lithography increases the accessibility and functionality of micropattern fabrication over photolithography6. However, soft lithography has significant drawbacks. For example, an initial etching step, using photolithography, is still required to microfabricate the stamp. In addition, micropatterning using a PDMS stamp is subject to variations in the quality of protein transfer onto the substrate6. Avoiding these discrepancies requires optimization and consistency in the pressure applied to the PDMS stamp during protein transfer, otherwise deformation and distortion of the feature sizes of the PDMS molds can occur6. There is also a major concern of repeatedly using the PDMS due to small molecule absorption7.
To avoid using soft photolithography and PDMS stamps, we describe a stencil-based, lithography-free single cell micropatterning method that overcomes many of the obstacles associated with photolithography and soft lithography. In this method, a polyacrylamide hydrogel is used as a substrate for stencil-based ECM protein incorporation, allowing for selective plating of single hiPSC-CMs. This technique is highly compatible with polymeric materials used in classic cell culture conditions. Moreover, with proper cleaning and maintenance, the stencils are reusable and resistant to degradation and protein absorption during the microfabrication process. Finally, the patterning process is technically robust, inexpensive, customizable, and accessible to those with no specialized bioengineering skills. This stencil-based micropatterning technique has been broadly utilized in our recent publications modeling varied cardiomyopathies8,9,10.

<table>
	<tbody>
		<tr>
			<td>2-Aminoethyl methacrylate hydrochloride (powder)</td>
			<td>Sigma-Aldrich</td>
			<td>516155</td>
			<td></td>
		</tr>
		<tr>
			<td>Acrylamide solution 40% (solution)</td>
			<td>Sigma-Aldrich</td>
			<td>A-4058-100mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Bench UV lamp 365 nm</td>
			<td>UVP</td>
			<td>UVP 95-0042-07, XX-15L</td>
			<td></td>
		</tr>
		<tr>
			<td>BioFlex culture plate</td>
			<td>FLEXCELL INTERNATIONAL CORPORATION, Burlington, NC</td>
			<td></td>
			<td>6-well plate with silicon elastomer substrate</td>
		</tr>
		<tr>
			<td>Bis-acrylamide solution 2% (solution)</td>
			<td>Sigma-Aldrich</td>
			<td>M1533-25mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning cover glasses square, No. 2, W &#215; L 22 mm &#215; 22 mm</td>
			<td>Sigma</td>
			<td>CLS285522</td>
			<td></td>
		</tr>
		<tr>
			<td>Irgacure (2-Hydroxy-4&#8242;-(2-hydroxyethoxy)-2-methylpropiophenone) (powder)</td>
			<td>Sigma-Aldrich</td>
			<td>410896</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Corning</td>
			<td>356231</td>
			<td>basement membrane matrix protein solution</td>
		</tr>
		<tr>
			<td>Methyl sulfoxide, 99.7+%, Extra Dry, AcroSeal, ACROS Organics</td>
			<td>Acros Organics</td>
			<td>326881000</td>
			<td></td>
		</tr>
		<tr>
			<td>Millex (13mm) filter unit with Durapore Membrane</td>
			<td>Millipore</td>
			<td>SLGV013SL</td>
			<td></td>
		</tr>
		<tr>
			<td>Millipore 50mL Steriflip (0.22 &#181;m)</td>
			<td>Fisher Scientific</td>
			<td>SCGP00525</td>
			<td></td>
		</tr>
		<tr>
			<td>Stencils</td>
			<td>Potomac</td>
			<td>custom design</td>
			<td></td>
		</tr>
		<tr>
			<td>Sulfo-SANPAH</td>
			<td>ThermoFisher Scientific</td>
			<td>22589</td>
			<td></td>
		</tr>
		<tr>
			<td>TrypLE Select 10x</td>
			<td>ThermoFisher Scientific</td>
			<td>A1217702</td>
			<td>Enzyme used for stencil cleaning</td>
		</tr>
	</tbody>
</table>

simple lithography-free single cell micropatterning using laser-cut stencils

Micropatterning techniques have been widely used in cell biology to study effects of controlling cell shape and size on cell fate determination at single cell resolution. Current state-of-the-art single cell micropatterning techniques involve soft lithography and micro-contact printing, which is a powerful technology, but requires trained engineering skills and certain facility support in microfabrication. These limitations require a more accessible technique. Here, we describe a simple alternative lithography-free method: stencil-based single cell patterning. We provide step-by-step procedures including stencil design, polyacrylamide hydrogel fabrication, stencil-based protein incorporation, and cell plating and culture. This simple method can be used to pattern an array of as many as 2,000 cells. We demonstrate the patterning of cardiomyocytes derived from single human induced pluripotent stem cells (hiPSC) with distinct cell shapes, from a 1:1 square to a 7:1 adult cardiomyocyte-like rectangle. This stencil-based single cell patterning is lithography-free, technically robust, convenient, inexpensive, and most importantly accessible to those with a limited bioengineering background.

Fabrication of stencils containing an array of squares or rectangles has been demonstrated (Figure 4A). Following this protocol, we obtained patterned matrix protein islands (Figure 4B and Figure 5A) and cells (Figure 4C). Suboptimal matrix protein solution concentration led to suboptimal patterning (Figure 5B). It is critical to use the front side of the stencil. If the ba...

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Simple Lithography-Free Single Cell Micropatterning using Laser-Cut Stencils

This protocol introduces a lithography-free micropatterning method that is simple and accessible to those with a limited bioengineering background. This method utilizes customized laser-cut stencils to micropattern extracellular matrix proteins in a shape of interest for modulating cell morphologies. The procedure for micropatterning is demonstrated using induced pluripotent stem cell derived cardiomyocytes.

Micropatterning techniques have been widely used in cell biology to study effects of controlling cell shape and size on cell fate determination at single ...

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Research

JoVE Journal

Developmental Biology

7.4K Views.  Stanford University School of Medicine. This protocol introduces a lithography-free micropatterning method that is simple and accessible to those with a limited bioengineering background. This method utilizes customized laser-cut stencils to micropattern extracellular matrix proteins in a shape of interest for modulating cell morphologies. The procedure for micropatterning is demonstrated using induced pluripotent stem cell derived cardiomyocytes.

Video: Simple Lithography-Free Single Cell Micropatterning using Laser-Cut Stencils

Epigenetic Conversion as a Safe and Simple Method to Obtain Insulin-secreting Cells from Adult Skin Fibroblasts

Regenerative medicine requires new, fully functional cells that are delivered to patients in order to repair degenerated or damaged tissues. When such cells are not readily available, they can be obtained using different approaches that include, among the many, reprogramming and trans-differentiation, with advantages and limitations that are specific of the different techniques. Here a new strategy for the conversion of an adult mature fibroblast into an insulin-secreting cell, arbitrarily designated as epigenetic converted cells (EpiCC), is described. The method has been developed, based on the increasing understanding of the mechanisms controlling epigenetic regulation of cell fate and differentiation. In particular, the first step uses an epigenetic modifier, namely 5-aza-cytidine, to drive adult cells into a "highly permissive" state. It then takes advantage of this brief and reversible window of epigenetic plasticity, to re-address cells toward a different lineage. The approach is designated "epigenetic cell conversion". It is a simple and robust way to obtain an efficient, controlled and stable cellular inter-lineage switch. Since the protocol does not involve the use of any gene transfection, it is free of viral vectors and does not involve a stable pluripotent state, it is highly promising for translational medicine applications.

Here, a new method that allows the conversion of adult skin fibroblasts into insulin-secreting cells is presented. This technique is based on epigenetic conversion, does not involve the use of retroviral vectors nor the acquisition of a stable pluripotent state. It is therefore highly promising for translational medicine applications.

Regenerative medicine requires new, fully functional cells that are delivered to patients in order to repair degenerated or damaged tissues. When such ...

Stencil Micropatterning of Human Pluripotent Stem Cells for Probing Spatial Organization of Differentiation Fates

Human pluripotent stem cells (hPSCs), including embryonic stem cells and induced pluripotent stem cells, have the intrinsic ability to differentiate into all three germ layers. This makes them an attractive cell source for regenerative medicine and experimental modeling of normal and diseased organogenesis. However, the differentiation of hPSCs in vitro is heterogeneous and spatially disordered. Cell micropatterning technologies potentially offer the means to spatially control stem cell microenvironments and organize the resultant differentiation fates. Micropatterning hPSCs needs to take into account the stringent requirements for hPSC survival and maintenance. Here, we describe stencil micropatterning as a method that is highly compatible with hPSCs. hPSC micropatterns are specified by the geometries of the cell stencil through-holes, which physically confine the locations where hPSCs can access and attach to the underlying extracellular matrix-coated substrate. Due to this mode of operation, there is greater flexibility to use substrates that can adequately support hPSCs as compared to other cell micropatterning methods. We also highlight critical steps for the successful generation of hPSC micropatterns. As an example, we demonstrate that stencil micropatterning of hPSCs can be used to modulate spatial polarization of cell-cell and cell-matrix adhesions, which in turn determines mesoendoderm differentiation patterns. This simple and robust method to micropattern hPSCs widens the prospects of establishing experimental models to investigate tissue organization and patterning during early embryonic development.

Human pluripotent stem cells (hPSCs) have the intrinsic ability to differentiate and self-organize into distinct tissue patterns; although this requires the presentation of spatial environmental gradients. We present stencil micropatterning as a simple and robust method to generate biochemical and mechanical gradients for controlling hPSC differentiation patterns.

Human pluripotent stem cells (hPSCs), including embryonic stem cells and induced pluripotent stem cells, have the intrinsic ability to differentiate into ...

A Simple Method to Identify Kinases That Regulate Embryonic Stem Cell Pluripotency by High-throughput Inhibitor Screening

Embryonic stem cells (ESCs) can self-renew or differentiate into all cell types, a phenomenon known as pluripotency. Distinct pluripotent states have been described, termed &#34;na&#239;ve&#34; and &#34;primed&#34; pluripotency. The mechanisms that control na&#239;ve-primed transition are poorly understood. In particular, we remain poorly informed about protein kinases that specify na&#239;ve and primed pluripotent states, despite increasing availability of high-quality tool compounds to probe kinase function. Here, we describe a scalable platform to perform targeted small molecule screens for kinase regulators of the na&#239;ve-primed pluripotent transition in mouse ESCs. This approach utilizes simple cell culture conditions and standard reagents, materials and equipment to uncover and validate kinase inhibitors with hitherto unappreciated effects on pluripotency. We discuss potential applications for this technology, including screening of other small molecule collections such as increasingly sophisticated kinase inhibitors and emerging libraries of epigenetic tool compounds.

Here, we present a quantitative and scalable protocol to perform targeted small molecule screens for kinase regulators of the na&#239;ve-primed pluripotent transition.

Embryonic stem cells (ESCs) can self-renew or differentiate into all cell types, a phenomenon known as pluripotency. Distinct pluripotent states have been ...

High-resolution Episcopic Microscopy (HREM) - Simple and Robust Protocols for Processing and Visualizing Organic Materials

We provide simple protocols for generating digital volume data with the high-resolution episcopic microscopy (HREM) method. HREM is capable of imaging organic materials with volumes up to 5 x 5 x 7 mm3 in typical numeric resolutions between 1 x 1 x 1 and 5 x 5 x 5 &#181;m3. Specimens are embedded in methacrylate resin and sectioned on a microtome. After each section an image of the block surface is captured with a digital video camera that sits on the phototube connected to the compound microscope head. The optical axis passes through a green fluorescent protein (GFP) filter cube and is aligned with a position, at which the bock holder arm comes to rest after each section. In this way, a series of inherently aligned digital images, displaying subsequent block surfaces are produced. Loading such an image series in three-dimensional (3D) visualization software facilitates the immediate conversion to digital volume data, which permit virtual sectioning in various orthogonal and oblique planes and the creation of volume and surface rendered computer models. We present three simple, tissue specific protocols for processing various groups of organic specimens, including mouse, chick, quail, frog and zebra fish embryos, human biopsy material, uncoated paper and skin replacement material.

High-resolution Episcopic Microscopy HREM - Simple and Robust Protocols for Processing and Visualizing Organic Materials

We provide simple and robust protocols for processing biopsy material of various species, embryos of biomedical model organisms and samples of other organic tissues in order to permit digital volume data generation with the high-resolution episcopic microscopy method.

We provide simple protocols for generating digital volume data with the high-resolution episcopic microscopy (HREM) method. HREM is capable of imaging ...

Clonal Analysis of Embryonic Hematopoietic Stem Cell Precursors Using Single Cell Index Sorting Combined with Endothelial Cell Niche Co-culture

The ability to study hematopoietic stem cell (HSC) genesis during embryonic development has been limited by the rarity of HSC precursors in the early embryo and the lack of assays that functionally identify the long-term multilineage engraftment potential of individual putative HSC precursors. Here, we describe methodology that enables the isolation and characterization of functionally validated HSC precursors at the single cell level. First, we utilize index sorting to catalog the precise phenotypic parameter of each individually sorted cell, using a combination of phenotypic markers to enrich for HSC precursors with additional markers for experimental analysis. Second, each index-sorted cell is co-cultured with vascular niche stroma from the aorta-gonad-mesonephros (AGM) region, which supports the maturation of non-engrafting HSC precursors to functional HSC with multilineage, long-term engraftment potential in transplantation assays. This methodology enables correlation of phenotypic properties of clonal hemogenic precursors with their functional engraftment potential or other properties such as transcriptional profile, providing a means for the detailed analysis of HSC precursor development at the single cell level.

Here we present methodology for the clonal analysis of hematopoietic stem cell precursors during murine embryonic development. We combine index sorting of single cells from the embryonic aorta-gonad-mesonephros region with endothelial cell co-culture and transplantation to characterize the phenotypic properties and engraftment potential of single hematopoietic precursors.

The ability to study hematopoietic stem cell (HSC) genesis during embryonic development has been limited by the rarity of HSC precursors in the early ...

Single-cell Photoconversion in Living Intact Zebrafish

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause disease when disrupted. Scientists have developed clever techniques to investigate characteristics and natural dynamics of these cells within intact tissue by expressing fluorescent proteins in subsets of cells. However, at times, experiments require more selected visualization of cells within the tissue, sometimes at the single-cell or population-of-cells manner. To achieve this and visualize single cells within a population of cells, scientists have utilized single-cell photoconversion of fluorescent proteins. To demonstrate this technique, we show here how to direct UV light to an Eos-expressing cell of interest in an intact, living zebrafish. We then image those photoconverted Eos+ cells 24 h later to determine how they changed in the tissue. We describe two techniques: single cell photoconversion and photoconversions of populations of cell. These techniques can be used to visualize cell-cell interactions, cell-fate and differentiation, and cell migrations, making it a technique that is applicable in numerous biological questions.

Here, we present a protocol to show how cell photoconversion is achieved through UV exposure to specific areas expressing the fluorescent protein, Eos, in living animals.

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause ...

Single-cell Transcriptomic Analyses of Mouse Pancreatic Endocrine Cells

Pancreatic endocrine cells, which are clustered in islets, regulate blood glucose stability and energy metabolism. The distinct cell types in islets, including insulin-secreting &#946; cells, are differentiated from common endocrine progenitors during the embryonic stage. Immature endocrine cells expand via cell proliferation and mature during a long postnatal developmental period. However, the mechanisms underlying these processes are not clearly defined. Single-cell RNA-sequencing is a promising approach for the characterization of distinct cell populations and tracing cell lineage differentiation pathways. Here, we describe a method for the single-cell RNA-sequencing of isolated pancreatic &#946; cells from embryonic, neonatal and postnatal pancreases.

We describe a method for the isolation of endocrine cells from embryonic, neonatal and postnatal pancreases followed by single-cell RNA sequencing. This method allows analyses of pancreatic endocrine lineage development, cell heterogeneity and transcriptomic dynamics.

Pancreatic endocrine cells, which are clustered in islets, regulate blood glucose stability and energy metabolism. The distinct cell types in islets, ...

Three-dimensional Angiogenesis Assay System using Co-culture Spheroids Formed by Endothelial Colony Forming Cells and Mesenchymal Stem Cells

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more than 50 different pathological conditions, such as rheumatoid arthritis, oculopathy, cardiovascular diseases, and tumor metastasis. During angiogenesis drug development, it is crucial to use in vitro assay systems with appropriate cell types and proper conditions to reflect the physiologic angiogenesis process. To overcome limitations of current in vitro angiogenesis assay systems using mainly endothelial cells, we developed a 3-dimensional (3D) co-culture spheroid sprouting assay system. Co-culture spheroids were produced by two human vascular cell precursors, endothelial colony forming cells (ECFCs) and mesenchymal stem cells (MSCs) with a ratio of 5 to 1. ECFCs+MSCs spheroids were embedded into type I collagen matrix to mimic the in vivo extracellular environment. A real-time cell recorder was utilized to continuously observe the progression of angiogenic sprouting from spheroids for 24 h. Live cell fluorescent labeling technique was also applied to tract the localization of each cell type during sprout formation. Angiogenic potential was quantified by counting the number of sprouts and measuring the cumulative length of sprouts generated from the individual spheroids. Five randomly-selected spheroids were analyzed per experimental group. Comparison experiments demonstrated that ECFCs+MSCs spheroids showed greater sprout number and cumulative sprout length compared with ECFCs-only spheroids. Bevacizumab, an FDA-approved angiogenesis inhibitor, was tested with the newly-developed co-culture spheroid assay system to verify its potential to screen anti-angiogenic drugs. The IC50 value for ECFCs+MSCs spheroids compared to the ECFCs-only spheroids was closer to the effective plasma concentration of bevacizumab obtained from the xenograft tumor mouse model. The present study suggests that the 3D ECFCs+MSCs spheroid angiogenesis assay system is relevant to physiologic angiogenesis, and can predict an effective plasma concentration in advance of animal experiments.

Three-dimensional co-culture spheroid angiogenesis assay system is designed to mimic the physiologic angiogenesis. Co-culture spheroids are formed by two human vascular cell precursors, ECFCs and MSCs, and embedded in collagen gel. The new system is effective for evaluating angiogenic modulators, and provides more relevant information to the in vivo study.

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more ...

Efficient Neural Differentiation using Single-Cell Culture of Human Embryonic Stem Cells

In vitro differentiation of human embryonic stem cells (hESCs) has transformed the ability to study human development on both biological and molecular levels and provided cells for use in regenerative applications. Standard approaches for hESC culture using colony type culture to maintain undifferentiated hESCs and embryoid body (EB) and rosette formation for differentiation into different germ layers are inefficient and time-consuming. Presented here is a single-cell culture method using hESCs instead of a colony-type culture. This method allows maintenance of the characteristic features of undifferentiated hESCs, including expression of hESC markers at levels comparable to colony type hESCs. In addition, the protocol presents an efficient method for neural progenitor cell (NPC) generation from single-cell type hESCs that produces NPCs within 1 week. These cells highly express several NPC marker genes and can differentiate into various neural cell types, including dopaminergic neurons and astrocytes. This single-cell culture system for hESCs will be useful in investigating the molecular mechanisms of these processes, studies of certain diseases, and drug discovery screens.

Presented here is a protocol for the generation of a single-cell culture of human embryonic stem cells and their subsequent differentiation into neural progenitor cells. The protocol is simple, robust, scalable, and suitable for drug screening and regenerative medicine applications.

In vitro differentiation of human embryonic stem cells (hESCs) has transformed the ability to study human development on both biological and molecular ...

Single Cell and Nuclei Extraction from Human Brain Organoids

Generation and Downstream Analysis of Single-Cell and Single-Nuclei Transcriptomes in Brain Organoids

Over the past decade, single-cell transcriptomics has significantly evolved and become a standard laboratory method for simultaneous analysis of gene expression profiles of individual cells, allowing the capture of cellular diversity. In order to overcome limitations posed by difficult-to-isolate cell types, an alternative approach aiming at recovering single nuclei instead of intact cells can be utilized for sequencing, making transcriptome profiling of individual cells universally applicable. These techniques have become a cornerstone in the study of brain organoids, establishing them as models of the developing human brain. Leveraging the potential of single-cell and single-nucleus transcriptomics in brain organoid research, this protocol presents a step-by-step guide encompassing key procedures such as organoid dissociation, single-cell or nuclei isolation, library preparation&#160;and sequencing. By implementing these alternative approaches, researchers can obtain high-quality datasets, enabling the identification of neuronal and non-neuronal cell types, gene expression profiles, and cell lineage trajectories. This facilitates comprehensive investigations into cellular processes and molecular mechanisms shaping brain development.

Author Spotlight: Integrating Organoid Models with Single-Cell and Spatial Transcriptomics Technologies

Here, we introduce a comprehensive protocol for the generation and downstream analysis of human brain organoids using single-cell and single-nucleus RNA sequencing.

Over the past decade, single-cell transcriptomics has significantly evolved and become a standard laboratory method for simultaneous analysis of gene ...