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>
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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. 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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...

Watch this Scientific Journal Video about Simple Lithography-Free Single Cell Micropatterning using Laser-Cut Stencils at JoVE.com

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

Micro-masonry for 3D Additive Micromanufacturing

Transfer printing is a method to transfer solid micro/nanoscale materials (herein called &#8216;inks&#8217;) from a substrate where they are generated to a different substrate by utilizing elastomeric stamps. Transfer printing enables the integration of heterogeneous materials to fabricate unexampled structures or functional systems that are found in recent advanced devices such as flexible and stretchable solar cells and LED arrays. While transfer printing exhibits unique features in material assembly capability, the use of adhesive layers or the surface modification such as deposition of self-assembled monolayer (SAM) on substrates for enhancing printing processes hinders its wide adaptation in microassembly of microelectromechanical system (MEMS) structures and devices. To overcome this shortcoming, we developed an advanced mode of transfer printing which deterministically assembles individual microscale objects solely through controlling surface contact area without any surface alteration. The absence of an adhesive layer or other modification and the subsequent material bonding processes ensure not only mechanical bonding, but also thermal and electrical connection between assembled materials, which further opens various applications in adaptation in building unusual MEMS devices.

This paper introduces a 3D additive micromanufacturing strategy (termed &#8216;micro-masonry&#8217;) for the flexible fabrication of microelectromechanical system (MEMS) structures and devices. This approach involves transfer printing-based assembly of micro/nanoscale materials in conjunction with rapid thermal annealing-enabled material bonding techniques.

Transfer printing is a method to transfer solid micro/nanoscale materials (herein called &#8216;inks&#8217;) from a substrate where they are generated to ...

Engineering

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 ...

Creating Adhesive and Soluble Gradients for Imaging Cell Migration with Fluorescence Microscopy

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as growth factors. Here, we outline a method to create opposing gradients of adhesive and soluble cues in a microfluidic chamber, which is compatible with live cell imaging. A copolymer of poly-L-lysine and polyethylene glycol (PLL-PEG) is employed to passivate glass coverslips and prevent non-specific adsorption of biomolecules and cells. Next, microcontact printing or dip pen lithography are used to create tracks of streptavidin on the passivated surfaces to serve as anchoring points for the biotinylated peptide arginine-glycine-aspartic acid (RGD) as the adhesive cue. A microfluidic device is placed onto the modified surface and used to create the gradient of adhesive cues (100% RGD to 0% RGD) on the streptavidin tracks. Finally, the same microfluidic device is used to create a gradient of a chemoattractant such as fetal bovine serum (FBS), as the soluble cue in the opposite direction of the gradient of adhesive cues.

A method for the assembly of adhesive and soluble gradients in a microscopy chamber for live cell migration studies is described. The engineered environment combines antifouling surfaces and adhesive tracks with solution gradients and therefore allows one to determine the relative importance of guidance cues.

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as ...

Bioengineering

A Microfluidic Platform for High-throughput Single-cell Isolation and Culture

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet high-throughput, method to perform single-cell culture experiments. Here, we report a microfluidic chip-based strategy for high-efficiency single-cell isolation (~77%) and demonstrate its capability of performing long-term single-cell culture (up to 7 d) and cellular heterogeneity analysis using clonogenic assay. These applications were demonstrated with KT98 mouse neural stem cells, and A549 and MDA-MB-435 human cancer cells. High single-cell isolation efficiency and long-term culture capability are achieved by using different sizes of microwells on the top and bottom of the microfluidic channel. The small microwell array is designed for precisely isolating single-cells, and the large microwell array is used for single-cell clonal culture in the microfluidic chip. This microfluidic platform constitutes an attractive approach for single-cell culture applications, due to its flexibility of adjustable cell culture spaces for different culture strategies, without decreasing isolation efficiency.

Here, we present a protocol for isolating and culturing single cells with a microfluidic platform, which utilizes a new microwell design concept to allow for high-efficiency single cell isolation and long-term clonal culture.

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet ...

Expanding Nanopatterned Substrates Using Stitch Technique for Nanotopographical Modulation of Cell Behavior

Substrate nanotopography has been shown to be a potent modulator of cell phenotype and function. To dissect nanotopography modulation of cell behavior, a large area of nanopatterned substrate is desirable so that enough cells can be cultured on the nanotopography for subsequent biochemical and molecular biology analyses. However, current nanofabrication techniques have limitations to generate highly defined nanopatterns over a large area. Herein, we present a method to expand nanopatterned substrates from a small, highly defined nanopattern to a large area using stitch technique. The method combines multiple techniques, involving soft lithography to replicate poly(dimethylsiloxane) (PDMS) molds from a well-defined mold, stitch technique to assemble multiple PDMS molds to a single large mold, and nanoimprinting to generate a master mold on polystyrene (PS) substrates. With the PS master mold, we produce PDMS working substrates and demonstrate nanotopographical modulation of cell spreading. This method provides a simple, affordable yet versatile avenue to generate well-defined nanopatterns over large areas, and is potentially extended to create micro-/nanoscale devices with hybrid components.

A protocol for producing a large area of nanopatterned substrate from small nanopatterned molds for study of nanotopographical modulation of cell behavior is presented.

Substrate nanotopography has been shown to be a potent modulator of cell phenotype and function. To dissect nanotopography modulation of cell behavior, a ...

A Versatile Method of Patterning Proteins and Cells

Substrate and cell patterning techniques are widely used in cell biology to study cell-to-cell and cell-to-substrate interactions. Conventional patterning techniques work well only with simple shapes, small areas and selected bio-materials. This article describes a method to distribute cell suspensions as well as substrate solutions into complex, long, closed (dead-end) polydimethylsiloxane (PDMS) microchannels using negative pressure. This method enables researchers to pattern multiple substrates including fibronectin, collagen, antibodies (Sal-1), poly-D-lysine (PDL), and laminin. Patterning of substrates allows one to indirectly pattern a variety of cells. We have tested C2C12 myoblasts, the PC12 neuronal cell line, embryonic rat cortical neurons, and amphibian retinal neurons. In addition, we demonstrate that this technique can directly pattern fibroblasts in microfluidic channels via brief application of a low vacuum on cell suspensions. The low vacuum does not significantly decrease cell viability as shown by cell viability assays. Modifications are discussed for application of the method to different cell and substrate types. This technique allows researchers to pattern cells and proteins in specific patterns without the need for exotic materials or equipment and can be done in any laboratory with a vacuum.

This report describes a simple, easy to perform technique, using low pressure vacuum, to fill microfluidic channels with cells and substrates for biological research.

Substrate and cell patterning techniques are widely used in cell biology to study cell-to-cell and cell-to-substrate interactions. Conventional patterning ...

Simple, Affordable, and Modular Patterning of Cells using DNA

The relative positioning of cells is a key feature of the microenvironment that organizes cell-cell interactions. To study the interactions between cells of the same or different type, micropatterning techniques have proved useful. DNA Programmed Assembly of Cells (DPAC) is a micropatterning technique that targets the adhesion of cells to a substrate or other cells using DNA hybridization. The most basic operations in DPAC begin with decorating cell membranes with lipid-modified oligonucleotides, then flowing them over a substrate that has been patterned with complementary DNA sequences. Cells adhere selectively to the substrate only where they find a complementary DNA sequence. Non-adherent cells are washed away, revealing a pattern of adherent cells. Additional operations include further rounds of cell-substrate or cell-cell adhesion, as well as transferring the patterns formed by DPAC to an embedding hydrogel for long-term culture. Previously, methods for patterning oligonucleotides on surfaces and decorating cells with DNA sequences required specialized equipment and custom DNA synthesis, respectively. We report an updated version of the protocol, utilizing an inexpensive benchtop photolithography setup and commercially available cholesterol modified oligonucleotides (CMOs) deployed using a modular format. CMO-labeled cells adhere with high efficiency to DNA-patterned substrates. This approach can be used to pattern multiple cell types at once with high precision and to create arrays of microtissues embedded within an extracellular matrix. Advantages of this method include its high resolution, ability to embed cells into a three-dimensional microenvironment without disrupting the micropattern, and flexibility in patterning any cell type.

Here we present a protocol to micropattern cells at single-cell resolution using DNA-programmed adhesion. This protocol uses a benchtop photolithography platform to create patterns of DNA oligonucleotides on a glass slide and then labels cell membranes with commercially available complementary oligonucleotides. Hybridization of the oligos results in programmed cell adhesion.

The relative positioning of cells is a key feature of the microenvironment that organizes cell-cell interactions. To study the interactions between cells ...

Control of Cell Geometry through Infrared Laser Assisted Micropatterning

Micropatterning is an established technique in the cell biology community used to study connections between the morphology and function of cellular compartments while circumventing complications arising from natural cell-to-cell variations. To standardize cell shape, cells are either confined in 3D molds or controlled for adhesive geometry through adhesive islands. However, traditional micropatterning techniques based on photolithography and deep UV etching heavily depend on clean rooms or specialized equipment. Here we present an infrared laser assisted micropatterning technique (microphotopatterning) modified from Doyle et al. that can be conveniently set up with commercially available imaging systems. In this protocol, we use a Nikon A1R MP+ imaging system to generate micropatterns with micron precision through an infrared (IR) laser that ablates preset regions on poly-vinyl alcohol coated coverslips. We employ a custom script to enable automated pattern fabrication with high efficiency and accuracy in systems not equipped with a hardware autofocus. We show that this IR laser assisted micropatterning (microphotopatterning) protocol results in defined patterns to which cells attach exclusively and take on the desired shape. Furthermore, data from a large number of cells can be averaged due to the standardization of cell shape. Patterns generated with this protocol, combined with high resolution imaging and quantitative analysis, can be used for relatively high throughput screens to identify molecular players mediating the link between form and function.

The protocol presented here enables automated fabrication of micropatterns that standardizes cell shape to study cytoskeletal structures within mammalian cells. This user-friendly technique can be set up with commercially available imaging systems and does not require specialized equipment inaccessible to standard cell biology laboratories.

Micropatterning is an established technique in the cell biology community used to study connections between the morphology and function of cellular ...

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 ...

Microfabrication via Photolithography

The fabrication of BioMEM&#160;devices is often done using a microfabrication technique called photolithography. This widely used method utilizes light to transfer a pattern onto a silicon wafer, and provides the basis for the fabrication of many types of BioMEM&#160;devices.
This video presents the photolithography technique, shows how the process is performed in the cleanroom, and introduces some applications of the process.

Microfabrication of BioMEM Devices via Photolithography

The fabrication of BioMEM&#160;devices is often done using a microfabrication technique called photolithography. This widely used method utilizes light to ...