Name: simple lithography-free single cell micropatterning using laser-cut stencils
Uploaded: 2020-04-03T13:00:00
Description: Watch this Scientific Journal Video about Simple Lithography-Free Single Cell Micropatterning using Laser-Cut Stencils at JoVE.com

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 ...

<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. 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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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