Name: simple lithography-free single cell micropatterning using laser-cut stencils
Uploaded: 2020-04-03T13:00:00.000+00:00
Duration: 8 min 59 s
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 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 in photolithography and microfabrication techniques. The described method circumvents significant challenges associated with generating stamps using soft lithography and transferring proteins from stamp molds to substrates of interest involved in the microcontact printing technique. This protocol provides a workflow to 1) create custom-designed stencils with an area and shape of interest in a cost-effective manner; 2) fabricate a hydrogel substrate with the stiffness of interest; and 3) efficiently conjugate ECM proteins on the hydrogel substrate.
Traditional soft lithography methods have been used for patterning various shapes of ECM proteins including triangles and stars on substrates of different materials such as silicon-based materials and glass coverslips13,14. In this study, we only demonstrate rectangular patterns given the physiologically- and pathologically-relevant shape of cardiomyocytes. We believe stencil-based patterning of other complex shapes can work as long as the smallest feature of the shape does not go beyond the resolution (~10 &#181;m). With regards to substrate materials, we demonstrate the stencil-based patterning can be applied on hydrogel (Figure 2, Figure 3, Figure 4, Figure 5, Figure 6 and Figure 8) or silicon-based elastomer substrate (Figure 7). The presented method does not work on substrates with minimal adhesiveness to polyimide-materials such as tissue culture plastics and glass coverslips because the tight sealing between the polyimide-based stencil and these substrates is not achieved. To enable tight sealing, further surface modification is required. Lastly, we show that the method can be used to reliably produce cell-pair patterns on hydrogels with different stiffnesses (i.e., 10 kPa, 60 kPa) (Figure 8).
In the protocol, the most critical step is optimization of ECM protein concentration (protocol section 6). The goal of this step is to find the optimal concentration of ECM protein that is viscous enough to inhibit the protein solution to seep through the holes of the stencil, but not too concentrated, to avoid gelation (Figure 5). This stencil-based method does not restrict the type of ECM protein. Although viscous ECM protein solution is more likely to stay on the top of the stencil, a less viscous protein can also be used. One requirement is making sure that the surfaces of the stencil and PA hydrogel are dry enough to form a relative stable hydrophobic interface. In this demonstration, basement membrane matrix protein was used because it is viscous and suitable for culturing hiPSC-CMs. However, other ECM proteins can likely be used (e.g., fibronectin, gelatin, collagen, laminin).
In addition to optimization of the ECM protein solution for each given application, generating a proper seal between the stencil and the hydrogel surface is crucial to obtain a precise pattern with the ECM protein. Because excessive drying can lead to rupturing of the hydrogel, it is essential to not overdry the stencil. These steps (i.e., placing the stencil on the hydrogel as well as gently pealing it off afterwards) require some practice. Establishing an optimal handling procedure for each given condition will also avoid having the hydrogel stick to the stencils. With this optimization, submicron scale laser burns caused by flaws in the laser cutting will not substantially affect the technique in regards to hydrogel damage.
Another important step is to use the front of the stencil when placing the stencil on the hydrogel substrate. We found that use of each side has an effect on the substrate, presumably due to laser cutting (Figure 6). One recommendation when patterning cells in specific size or shape for the first time is to generate an optimization stencil. Stencil-design features do not necessarily match the final patterned cell sizes. We recommend generating an optimization stencil that contains a gradient of feature sizes. We prepared a table listing potential problems and possible solutions to help with troubleshooting (Table 1).
It is not required to use the same microfabrication company mentioned in this manuscript. The keys to precise generation of the stencil are the beam diameter of the laser and the thickness of the stencil film, in our case, polyimide (50 &#181;m). We believe a laser lab equipped with an appropriate laser could generate the stencil with a few steps, optimizing laser intensity, alignment, and other variables. Although we have only worked with one company, there are many available companies who perform precision laser cutting.
A limitation to this method is resolution. While photolithography can achieve nanoscale resolution, stencil-based patterning resolution is limited to the resolution of the laser-cutting of the polyimide stencils, which to date is usually on the scale of a few microns (~10 &#181;m). Nevertheless, given that the average cell size is over 10 &#181;m, the resolution is generally applicable to any single cell patterning.
In summary, this method provides versatile platforms for studying cell-ECM interactions, examining cell structure-function correlation, and many other applications. We believe this protocol will benefit many biomedical researchers and facilitate single cell studies.

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 &amp;times; L 22 mm &amp;times; 22 mm</td><td>Sigma</td><td>CLS285522</td><td></td></tr><tr><td>Irgacure (2-Hydroxy-4&amp;prime;-(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 &amp;micro;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 back side of the stencil is used, the size of the matrix protein islands increases due to the direction of the laser cutting (Figure 6A,B). While the width of the rectangular patterns did not significantly change (Figure 6C), the height significantly increased when using the back side of the stencil, leading to a reduction in the intended aspect ratio (Figure 6D,E). We applied stencil-based patterning to silicon elastomer substrates (Figure 7). The patterned cardiomyocytes on silicon elastomer substrates were visualized by immunocytochemistry of cardiac troponin T at low magnification (Figure 7A) and sarcomeric protein &#945;-actinin at high magnification (Figure 7B). The stencil-based patterning could also be applied to pattern two cardiomyocytes side-by-side on hydrogels with different stiffnesses. As a demonstration, we show the cardiomyocyte patterning on physiologically-relevant stiffness (Figure 8A) and pathologically-relevant stiffness (Figure 8B).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60888/60888fig01.jpg" /> 
Figure 1: Schematic of three gold-standard micropatterning strategies.&#160;(A) Photolithography. (B) Microcontact printing. (C) Microfluidic patterning. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60888/60888fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60888/60888fig02.jpg" /> 
Figure 2: Workflow of designing stencils.&#160;(A) The computer-assisted design of the stencil. (B) A representative photograph of a laser-cut polyimide stencil. (C) A representative microscopic image of a stencil. The yellow area represents the polyimide stencil, while the light blue area represents an array of laser-cut holes. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60888/60888fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60888/60888fig03.jpg" /> 
Figure 3: Photographs of hydrogel fabrication steps.&#160;(A) Polyacrylamide hydrogel fabrication by sandwiching the precursor solution in between two coverslips. (B) UV photo-crosslinking of polyacrylamide hydrogels under the UV bench lamp. The UV lamp is covered with aluminum foil for user protection. (C) The photo-crosslinked hydrogel is retrieved by detaching a coverslip on one side. (D) The hydrogel construct is in contact with sulfo-SANPAH solution to modify the hydrogel for ECM protein incorporation. Before UV activation, the sulfo-SANPAH is orange. (E) After UV activation, sulfo-SANPAH turns brown, indicating that it is activated and incorporated onto the hydrogel surface. (F) After dabbing the hydrogel with sterile lint-free wipes to remove excess water, the stencil is placed on the hydrogel. (G) Basement membrane matrix protein solution is pipetted over the stencil-hydrogel constructs. (H) If the stencil-hydrogel contact is successfully tight, the basement membrane matrix protein solution will stay on top and will not seep through. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60888/60888fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/60888/60888fig04.jpg" /> 
Figure 4: Micropatterning of basement membrane matrix proteins and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) with different rectangular shapes.&#160;(A) Microscopic images of stencils containing laser-cut rectangles with different aspect ratios (1:1, 4:1, 11:1). (B) Representative images of immunostained matrix protein islands on hydrogel substrates. (C) Representative images of hiPSC-CMs stained for nuclei (cyan) and cardiac troponin T (magenta). Scale bar = 20 &#181;m for panels B and C. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60888/60888fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/60888/60888fig05.jpg" /> 
Figure 5: The ECM protein concentration plays an essential role in generating proper patterns.&#160;(A) Representative image of an optimal concentration with very homogenous distribution of basement membrane matrix proteins in the single patterns. (B) Suboptimal matrix protein patterns due to low concentration, which caused leakage of matrix protein outside the patterns and low amounts of matrix proteins within the patterns as determined by wheat germ agglutinin staining. Scale bar = 20 &#181;m for panels A and B. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60888/60888fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/60888/60888fig06.jpg" /> 
Figure 6: Stencil side effect.&#160;(A) Basement membrane matrix protein patterns when the front side of the stencil was used. (B) Basement membrane matrix protein patterns when the back side of the stencil was used. Quantification of (C) width, (D) height, (E) aspect ratio of rectangular basement membrane matrix protein islands. Graph is plotted mean &#177; SEM. Statistics are calculated by unpaired student T test. n.s. = not significant. **** = p &#60; 0.0001. Scale bar = 20 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60888/60888fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/60888/60888fig07.jpg" /> 
Figure 7: Stencil-based patterning on silicon elastomer substrate.&#160;(A) A representative image of single cell patterned cardiomyocytes on silicon elastomer substrates stained by the cardiac marker, cardiac troponin T (cTnT), at low magnification. Scale bar = 300 &#181;m. (B) A representative single-cell-patterned cardiomyocyte stained by sarcomeric protein &#945;-actinin at high magnification. Scale bar = 10 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60888/60888fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/60888/60888fig08.jpg" /> 
Figure 8: Micropatterning of hiPSC-CMs on polyacrylamide hydrogels with different stiffnesses.&#160;(A) 10 kPa, (B) 60 kPa. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60888/60888fig08large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Step</td>
			<td>Problem observed</td>
			<td>Possible reason</td>
			<td>Solution</td>
		</tr>
		<tr>
			<td>1.1</td>
			<td>The pattern size is bigger than expected</td>
			<td>Stencil is flipped</td>
			<td>Include a chiral letter/shape in the stencil design to indicate the front- and back-side of stencils</td>
		</tr>
		<tr>
			<td rowspan="2">8.1</td>
			<td rowspan="2">Hydrogel fractures when peeling stencils</td>
			<td rowspan="2">Hydrogel sticks to stencils</td>
			<td>Cleaning of stencils</td>
		</tr>
		<tr>
			<td>Do not dry hydrogel too much before putting stencils</td>
		</tr>
		<tr>
			<td rowspan="3">8.13</td>
			<td rowspan="3">Cell attachment is suboptimal</td>
			<td>Degraded sulfo-SANPAH</td>
			<td>Use new sulfo-SANPAH</td>
		</tr>
		<tr>
			<td>Inefficient protein incorporation</td>
			<td>Prepare new ECM protein solution</td>
		</tr>
		<tr>
			<td>Old ECM protein solution</td>
			<td></td>
		</tr>
		<tr>
			<td>8.13</td>
			<td>Cell patterning is suboptimal</td>
			<td>Basement membrane matrix protein solution spills over</td>
			<td>Perform immunostaining using fluorophore-conjugated anti mouse IgG antibody or Wheat germ agglutinine to examine the distribution of basement membrane matrix protein solution on hydrogel</td>
		</tr>
		<tr>
			<td rowspan="2">8.13</td>
			<td rowspan="2">More than one cell on one slot</td>
			<td>Not in single cell suspension</td>
			<td>Use 40&#8722;70 &#181;m cell strainer to have single cell suspension</td>
		</tr>
		<tr>
			<td>Seeding density not optimal</td>
			<td>Optimize the seeding density from 30 k to 90 k per mL</td>
		</tr>
	</tbody>
</table>
Table 1: Troubleshooting guidelines. Frequent problems, possible reasons, and potential solutions are discussed.
Supplemental Files.&#160;&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60888/Supplemental_Files.zip" target="_blank">Please click here to download files.</a>

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 ...

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

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

7.4K Views.  Stanford University School of Medicine. We beschrijven een op stencil gebaseerde lithografie-vrije eencellige micropattern methode die veel obstakels in verband met fotolithografie en zachte lithografie overwint. De stencils zijn herbruikbaar en het proces is goedkoop en aanpasbaar. Deze methode opende een nieuwe weg voor niet-ingenieur onderzoekers om celvorm en gebied-geïnduceerde mechanotransductie in verschillende fysiologische en pathologische contexten te bestuderen.Bovendien, dit protocol maakt het mogelijk voor micr...

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

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

Bioengineering

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 ...

Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain central aspects of cellular physiology. To easily combine cell patterning with Multi-Electrode Arrays (MEAs) and silicon-based &#8216;lab on a chip&#8217; technologies, a microfabrication-compatible protocol is required. We describe a method that utilizes deposition of the polymer parylene-C on SiO2&#160;wafers. Photolithography enables accurate and reliable patterning of parylene-C at micron-level resolution. Subsequent activation by immersion in fetal bovine serum (or another specific activation solution) results in a substrate in which cultured cells adhere to, or are repulsed by, parylene or SiO2 regions respectively. This technique has allowed patterning of a broad range of cell types (including primary murine hippocampal cells, HEK 293 cell line, human neuron-like teratocarcinoma cell line, primary murine cerebellar granule cells, and primary human glioma-derived stem-like cells). Interestingly, however, the platform is not universal; reflecting the importance of cell-specific adhesion molecules. This cell patterning process is cost effective, reliable, and importantly can be incorporated into standard microfabrication (chip manufacturing) protocols, paving the way for integration of microelectronic technology.

Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

This protocol describes a microfabrication-compatible method for cell patterning on SiO2. A predefined parylene-C design is photolithographically printed on SiO2 wafers. Following incubation with serum (or other activation solution) cells adhere specifically to (and grow according to the conformity of) underlying parylene-C, whilst being repulsed by SiO2 regions.

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain ...

Microcontact Printing of Proteins for Cell Biology

The ability to pattern proteins and other biomolecules onto substrates is important for capturing the spatial complexity of the extracellular environment. Development of microcontact printing by the Whitesides group (<a href="http://gmwgroup.harvard.edu/">http://gmwgroup.harvard.edu/</a>) in the mid-1990s revolutionalized this field by making microelectronics/microfabrication techniques accessible to laboratories focused on the life sciences. Initial implementations of this method used polydimethylsiloxane (PDMS) stamps to create patterns of functionalized chemicals on material surfaces1. Since then, a range of innovative approaches have been developed to pattern other molecules, including proteins2. This video demonstrates the basic process of creating PDMS stamps and uses them to pattern proteins, as these steps are difficult to accurately express in words. We focus on patterning the extracellular matrix protein fibronectin onto glass coverslips as a specific example of patterning.
An important component of the microcontact printing process is a topological master, from which the stamps are cast; the raised and lowered regions of the master are mirrored into the stamp and define the final pattern. Typically, a master consists of a silicon wafer coated with photoresist and then patterned by photolithography, as is done here. Creation of masters containing a specific pattern requires specialized equipment, and is best approached in consultation with a fabrication center or facility. However, almost any substrate with topology can be used as a master, such as plastic diffraction gratings (see Reagents for one example), and such serendipitous masters provide readily available, simple patterns. This protocol begins at the point of having a master in hand.

Microcontact printing is used extensively to pattern proteins and other molecules on material surfaces. We demonstrate the basic steps of this process, stamping patterns of fibronectin onto glass.

The ability to pattern proteins and other biomolecules onto substrates is important for capturing the spatial complexity of the extracellular environment. ...

Biology

Automated Robotic Dispensing Technique for Surface Guidance and Bioprinting of Cells

This manuscript describes the introduction of cell guidance features followed by the direct delivery of cells to these features in a hydrogel bioink using an automated robotic dispensing system. The particular bioink was selected as it allows cells to sediment towards and sense the features. The dispensing system bioprints viable cells in hydrogel bioinks using a backpressure assisted print head. However, by replacing the print head with a sharpened stylus or scalpel, the dispensing system can also be employed to create topographical cues through surface etching. The stylus movement can be programmed in steps of 10 &#181;m in the X, Y and Z directions. The patterned grooves were able to orientate mesenchymal stem cells, influencing them to adopt an elongated morphology in alignment with the grooves&#39; direction. The patterning could be designed using plotting software in straight lines, concentric circles, and sinusoidal waves. In a subsequent procedure, fibroblasts and mesenchymal stem cells were suspended in a 2% gelatin bioink, for bioprinting in a backpressure driven extrusion printhead. The cell bearing bioink was then printed using the same programmed coordinates used for the etching. The bioprinted cells were able to sense and react to the etched features as demonstrated by their elongated orientation along the direction of the etched grooves.

This protocol describes a bioprinting methodology using an automated robotic depositing system that incorporates etched topographical guidance cues with the precision deposition of a cell bearing hydrogel bioink. The printed cells are directly delivered to the etched features and are able to sense and orientate with them.

This manuscript describes the introduction of cell guidance features followed by the direct delivery of cells to these features in a hydrogel bioink using ...

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 ...

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

Samenvatting

Meer video's verkennen

Hoofdstukken in deze video