Funding for this research was provided by the New Jersey Commission on Spinal Cord Research (NJCSCR) (to FHK), grant CSCR14IRG005 (to BLF), NIH grant R15NS087501 (to CHC), and the F.M. Kirby Foundation (to ETA).

NOTE: Please consult all relevant material safety data sheets (MSDS) before use. Some of the chemicals used in this protocol are toxic and carcinogenic. Please use all appropriate safety practices (fume hood, glovebox) and personal protective equipment (safety glasses, gloves, lab coat, full length pants, closed-toe shoes) when using toxic or acid/base materials.
1. Fabrication of Master Molds for Soft Lithography using Photolithography
<ol>
	<li>Draw the layout of the microchannel using a computer-aided design (CAD) drawing tool.</li>
	<li>Print the layout on a blank mask plate using laser mask writer.</li>
	<li>Rinse a 4 inch silicon wafer with acetone followed by isopropanol and dry with a nitrogen air gun to ensure that no solvent is left on the wafer.</li>
	<li>Dehydrate the wafer by baking it on a hot plate at 200 &#176;C for 5 min.</li>
	<li>Select a negative photoresist designed for the desired height of the microchannels. For example, use negative photoresist SU-8 50 to obtain microchannels with a height of 50 &#181;m.</li>
	<li>Allow the wafer to cool to room temperature, and then align the wafer on the chuck of a spin coater.</li>
	<li>Dispense 4 mL (1 mL/inch diameter of the wafer) of negative photoresist onto the center of the wafer.</li>
	<li>Spin coat the wafer with a two-step spin cycle using a spinner. First, apply a spread cycle of 500 rpm with an acceleration of 100 rpm/s for 10 s. Then apply a spin cycle of 2,000 rpm with an acceleration of 300 rpm/s2 for 30 s to obtain a 50 &#181;m coating of photoresist on a wafer.</li>
	<li>Soft bake the wafer in two steps on a hot plate according to the manufacturer&#39;s directions. For a 50 &#181;m coating of photoresist, first pre-bake the wafer at 65 &#176;C for 6 min and then immediately ramp up the hot plate temperature to post-bake the wafer at 95 &#176;C for 20 min.</li>
	<li>Allow the wafer to cool to room temperature and then load the wafer onto the lithography stage.</li>
	<li>Align the mask on the wafer using mask aligner and expose the wafer to UV light (as per the manufacturer&#39;s instructions) at an intensity of 1.5 mW/cm2 for 146 s to apply the total UV dose of 220 mJ/cm2 required for a 50 &#181;m thick layer of photoresist.</li>
	<li>Apply post exposure bake to the wafer in two steps on a hot plate. For a 50-&#181;m coating of photoresist, first pre-bake the wafer at 65 &#176;C for 1 min and immediately ramp up the hot plate temperature to post-bake it at 95 &#176;C for 5 min.</li>
	<li>Develop the wafer in by submerging the wafer in SU-8 developer and gently shaking until the features become clear on the wafer. Develop the wafer for ~6 min for the 50 &#181;m thick photoresist.</li>
	<li>Rinse off excess developer with isopropanol and ethanol, then dry the wafer with a nitrogen air gun.</li>
	<li>Hard bake the silicon wafer on a hot plate at 150 &#176;C for 15 min.</li>
	<li>Place the wafer in a desiccator with 25 &#181;L of the silanizing agent tridecafluoro-1,1,2,2-tetrahydrooctyl-1-trichlorosilane. 
	NOTE: The silanizing agent is corrosive, thus should be used in an acid/base fume hood, with appropriate personal protective equipment (safety glasses, gloves, lab coat, full length pants, closed-toe shoes).</li>
	<li>Apply the vacuum for 5 min and expose the wafer to the vapors of silane for 30 min without releasing the vacuum.</li>
	<li>Transfer the wafer into a suitably sized Petri dish.</li>
</ol>
2. Fabrication of Master Molds for Soft Lithography Using Adhesive Tape
<ol>
	<li>Draw the layout of the microchannel using a CAD drawing tool and print the drawing on white paper.</li>
	<li>Clean a glass slide large enough to accommodate the design with isopropanol and then dry the slide with an air gun.</li>
	<li>Attach adhesive tape onto the cleaned glass slide. Be careful not to trap any air bubbles.</li>
	<li>Tape the sides of the glass slide onto the paper with the design, with the tape side up.</li>
	<li>Use a scalpel to cut the tape on the glass slide using the paper design as a reference and then peel off tape from unwanted areas of the glass slide.</li>
	<li>Rinse the glass slide with isopropanol and then dry with an air gun. Bake the glass slide in an oven at 65 &#176;C for 30 min.</li>
	<li>Gently roll over the tape with a rubber roller to remove air bubbles and allow the slide to cool to room temperature.</li>
</ol>
3. Soft Lithography Fabrication of the PDMS Devices
<ol>
	<li>Mix PDMS elastomer and its curing agent in a ratio of 10:1 (w:w), stir the mixture vigorously, and then degas the mixture by placing it into a vacuum chamber until no air bubbles appear in the mixture.</li>
	<li>Pour the mixture onto a silanized silicon mold or adhesive tape mold in a petri dish to obtain a ~2 mm thick layer of PDMS and degas until all air bubbles disappear.</li>
	<li>Cure the PDMS in an oven at 65 &#176;C for 2 h.</li>
	<li>Use a scalpel to cut the PDMS layer at least 5 mm away from the features and then peel the cured PDMS off the mold using tweezer.</li>
	<li>Punch a single inlet hole with a 1 mm biopsy punch anywhere on the microchannel, preferably at the end of a network of microchannels as seen in Figure 2a and 4b.</li>
	<li>Clean the PDMS cast with adhesive tape to remove dust particles adsorbed onto the device. Sterilize the PDMS microchannel device (PDMS cast) by rinsing with a solution of 70% ethanol followed by sterile deionized (DI) water, and expose to UV for 30 min.</li>
	<li>Keep the sterile PDMS device in a sterile petri dish until use.</li>
</ol>
4. Substrate Patterning
<ol>
	<li>Place the sterile PDMS cast using tweezers onto a sterile glass coverslip or petri dish and apply gentle pressure using the tip of the tweezers. 
	NOTE: The PDMS cast makes a conformal but reversible seal with the glass coverslip or petri dish forming microchannels without use of any adhesive.</li>
	<li>Place a droplet (20 - 40 &#181;L) of the substrate solution on the inlet completely covering the inlet hole. Pattern Poly-D-Lysine (PDL) by placing a 20 &#181;L droplet of PDL in sodium tetraborate buffer on the inlet of the microchannels.</li>
	<li>Put the petri dish in a vacuum of ~254 mmHgA (equivalent to house vacuum in biological laboratories) for 10 min, and observe air coming out of the channel in the form of bubbles.</li>
	<li>Release the vacuum and observe the substrate solution flowing into the microchannel.</li>
	<li>Incubate the dish at the appropriate conditions for substrate adherence. See Tables 1 and 2 for incubation conditions (these vary depending on the substrate). For PDL patterning, incubate for 1 h at 37 &#176;C.</li>
	<li>Carefully peel off the PDMS stamp using sterile tweezers and wash the pattern on glass coverslip thrice with DI water.</li>
	<li>Add a 1% bovine serum albumin (BSA) solution to the petri dish to cover the dish or the glass coverslip and incubate overnight at 37 &#176;C. 
	NOTE: This will reduce non-specific adherence in uncoated regions.</li>
	<li>Aspirate off the 1% BSA solution, and the patterned substrate is now ready to use.</li>
</ol>
5. Indirect Patterning of Cells
<ol>
	<li>Prepare the cell suspension in serum-free culture medium. The cell type and cell density are listed in Table 1.</li>
	<li>Add the cell suspension to the patterned Petri dish and completely submerge the patterned region in cell suspension.</li>
	<li>Incubate the petri dish at 37 &#176;C in an incubator for 15 min to allow cells to attach to the patterned substrate.</li>
	<li>Aspirate the excess cell suspension from the Petri dish and wash the patterned cells three times with phosphate buffered saline (PBS) while gently shaking for 10 s to remove unattached cells.</li>
	<li>Add the appropriate culture medium to the patterned cell cultures and incubate the patterned cells at 37 &#176;C in a CO2 incubator.</li>
</ol>
6. Direct Patterning of Cells
NOTE: This technique is an alternative to the indirect cell patterning described in step 5. However, unlike in step 5, in this technique cells are patterned on tissue culture surfaces with or without substrate coating.
<ol>
	<li>Sterilize the microfluidic device by rinsing with a solution of 70% of ethanol followed by DI water.</li>
	<li>Soak the device overnight in a solution of 1% BSA to prevent the cell adhesion to the PDMS.</li>
	<li>Dry the device at room temperature and attach it to the bottom of the tissue culture-treated petri dish.</li>
	<li>Prepare the cell suspension in serum-free culture media. The cell type and cell density are listed in Table 2.</li>
	<li>Place a droplet (4 - 8 &#181;L) of the cell suspension, enough to fill the microchannels, on the inlet completely covering the inlet hole.</li>
	<li>Put the petri dish in a vacuum for 10 min and observe air coming out of the channel in the form of bubbles. Release the vacuum and observe the cell suspension flowing into the microchannel.</li>
	<li>Incubate the Petri dish in an incubator to promote cell adhesion as per the incubation conditions (depend on type of cell patterned) mentioned in Table 2. Add PBS to the petri dish submerging the PDMS device.</li>
	<li>Peel the PDMS carefully off the petri dish with tweezers and wash the pattern with PBS. Add cell culture media to the Petri dish and place the cell culture in the incubator.</li>
</ol>

<ol>
	<li>Kane, R. S., Takayama, S., Ostuni, E., Ingber, D. 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=Patterning+proteins+and+cells+using+soft+lithography.">Patterning proteins and cells using soft lithography.</a> Biomaterials. 20 (23-24), 2363-2376 (1999).</li><li>Lin, R. Z., Ho, C. T., Liu, C. H., Chang, H. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dielectrophoresis+based-cell+patterning+for+tissue+engineering.">Dielectrophoresis based-cell patterning for tissue engineering.</a> Biotechnol J. 1 (9), 949-957 (2006).</li><li>Veiseh, M., Zareie, M. H., Zhang, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+Selective+Protein+Patterning+on+Gold-Silicon+Substrates+for+Biosensor+Applications.">Highly Selective Protein Patterning on Gold-Silicon Substrates for Biosensor Applications.</a> Langmuir. 18 (17), 6671-6678 (2002).</li><li>Kung, F., Wang, J., Perez-Castillejos, R., Townes-Anderson, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Position+along+the+nasal/temporal+plane+affects+synaptic+development+by+adult+photoreceptors,+revealed+by+micropatterning.">Position along the nasal/temporal plane affects synaptic development by adult photoreceptors, revealed by micropatterning.</a> Integr Biol. 7 (3), 313-323 (2015).</li><li>Dickinson, L. E., Lutgebaucks, C., Lewis, D. M., Gerecht, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterning+microscale+extracellular+matrices+to+study+endothelial+and+cancer+cell+interactions+in+vitro.">Patterning microscale extracellular matrices to study endothelial and cancer cell interactions in vitro.</a> Lab Chip. 12 (21), 4244-4248 (2012).</li><li>Khademhosseini, A., 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=Co-culture+of+human+embryonic+stem+cells+with+murine+embryonic+fibroblasts+on+microwell-patterned+substrates.">Co-culture of human embryonic stem cells with murine embryonic fibroblasts on microwell-patterned substrates.</a> Biomaterials. 27 (36), 5968-5977 (2006).</li><li>Bogdanowicz, D. R., Lu, H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studying+cell-cell+communication+in+co-culture.">Studying cell-cell communication in co-culture.</a> Biotechnol J. 8 (4), 395-396 (2013).</li><li>Choi, Y., Lee, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guided+cell+growth+through+surface+treatments.">Guided cell growth through surface treatments.</a> J of Mech Sci Technol. 19 (11), 2133-2137 (2005).</li><li>Hwang, I. -. 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=Efficient+Immobilization+and+Patterning+of+Biomolecules+on+Poly(ethylene+terephthalate)+Films+Functionalized+by+Ion+Irradiation+for+Biosensor+Applications.">Efficient Immobilization and Patterning of Biomolecules on Poly(ethylene terephthalate) Films Functionalized by Ion Irradiation for Biosensor Applications.</a> ACS Appl Mater Interf. 3 (7), 2235-2239 (2011).</li><li>Clark, P., Britland, S., Connolly, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+cone+guidance+and+neuron+morphology+on+micropatterned+laminin+surfaces.">Growth cone guidance and neuron morphology on micropatterned laminin surfaces.</a> J Cell Sci. 105 (1), 203-212 (1993).</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> J Cell Sci. 123 (24), 4201-4213 (2010).</li><li>Douvas, A., 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=Biocompatible+photolithographic+process+for+the+patterning+of+biomolecules.">Biocompatible photolithographic process for the patterning of biomolecules.</a> Biosens Bioelectron. 17 (4), 269-278 (2002).</li><li>Alom, R. S., Chen, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microcontact+printing:+A+tool+to+pattern.">Microcontact printing: A tool to pattern.</a> Soft Matter. 3 (2), 168-177 (2007).</li><li>Ess&#246;, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modifying+Polydimethylsiloxane+(PDMS)+surfaces.">Modifying Polydimethylsiloxane (PDMS) surfaces.</a> Institutionen f&#246;r biologi och kemiteknik. , (2007).</li><li>Zhou, J., Ellis, A. V., Voelcker, N. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+developments+in+PDMS+surface+modification+for+microfluidic+devices.">Recent developments in PDMS surface modification for microfluidic devices.</a> Electrophoresis. 31 (1), 2-16 (2010).</li><li>Folch, A., Jo, B. H., Hurtado, O., Beebe, D. J., Toner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfabricated+elastomeric+stencils+for+micropatterning+cell+cultures.">Microfabricated elastomeric stencils for micropatterning cell cultures.</a> J Biomed Mater Res. 52 (2), 346-353 (2000).</li><li>Yeo, W. S., Yousaf, M. N., 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=Dynamic+interfaces+between+cells+and+surfaces:+electroactive+substrates+that+sequentially+release+and+attach+cells.">Dynamic interfaces between cells and surfaces: electroactive substrates that sequentially release and attach cells.</a> J Am Chem Soc. 125 (49), 14994-14995 (2003).</li><li>Bhatia, S. N., Toner, M., Tompkins, R. G., Yarmush, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+adhesion+of+hepatocytes+on+patterned+surfaces.">Selective adhesion of hepatocytes on patterned surfaces.</a> Ann N Y Acad Sci. 745, 187-209 (1994).</li><li>Song, E., Kim, S. Y., Chun, T., Byun, H. -. J., Lee, Y. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collagen+scaffolds+derived+from+a+marine+source+and+their+biocompatibility.">Collagen scaffolds derived from a marine source and their biocompatibility.</a> Biomaterials. 27 (15), 2951-2961 (2006).</li><li>Yamato, M., Konno, C., Utsumi, M., Kikuchi, A., Okano, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermally+responsive+polymer-grafted+surfaces+facilitate+patterned+cell+seeding+and+co-culture.">Thermally responsive polymer-grafted surfaces facilitate patterned cell seeding and co-culture.</a> Biomaterials. 23 (2), 561-567 (2002).</li><li>Takayama, 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=Patterning+cells+and+their+environments+using+multiple+laminar+fluid+flows+in+capillary+networks.">Patterning cells and their environments using multiple laminar fluid flows in capillary networks.</a> Proc Natl Acad Sci U S A. 96 (10), 5545-5548 (1999).</li><li>Kim, D. S., Lee, K. -. C., Kwon, T. H., Lee, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micro-channel+filling+flow+considering+surface+tension+effect.">Micro-channel filling flow considering surface tension effect.</a> J of Micromech Microeng. 12 (3), 236 (2002).</li><li>Kim, E., Xia, Y., 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=Micromolding+in+Capillaries:+Applications+in+Materials+Science.">Micromolding in Capillaries: Applications in Materials Science.</a> J Am Chem Soc. 118 (24), 5722-5731 (1996).</li><li>Kim, E., Xia, Y. N., 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=Polymer+Microstructures+Formed+by+Molding+in+Capillaries.">Polymer Microstructures Formed by Molding in Capillaries.</a> Nature. 376 (6541), 581-584 (1995).</li><li>Jeon, N. L., Choi, I. S., Xu, B., 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=Large-area+patterning+by+vacuum-assisted+micromolding.">Large-area patterning by vacuum-assisted micromolding.</a> Adv Mater. 11 (11), 946 (1999).</li><li>Shrirao, A. B., 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=System+and+method+for+novel+microfluidic+device.">System and method for novel microfluidic device.</a> US patent. , (2010).</li><li>Merkel, T. C., Bondar, V. I., Nagai, K., Freeman, B. D., Pinnau, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gas+sorption,+diffusion,+and+permeation+in+poly(dimethylsiloxane).">Gas sorption, diffusion, and permeation in poly(dimethylsiloxane).</a> J Polym Sci Part B Polym Phys. 38 (3), 415-434 (2000).</li><li>Shrirao, A. B., Hussain, A., Cho, C. H., Perez-Castillejos, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adhesive-tape+soft+lithography+for+patterning+mammalian+cells:+application+to+wound-healing+assays.">Adhesive-tape soft lithography for patterning mammalian cells: application to wound-healing assays.</a> Biotechniques. 53 (5), 315-318 (2012).</li><li>Shrirao, A. B., Perez-Castillejos, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chips+&amp;+tips:+simple+fabrication+of+microfluidic+devices+by+replicating+scotch-tape+masters.">Chips &amp; tips: simple fabrication of microfluidic devices by replicating scotch-tape masters.</a> Lab Chip. , (2010).</li><li>Anil, B. S., Frank, H. K., Derek, Y., Cheul, H. C., Ellen, T. -. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vacuum-assisted+fluid+flow+in+microchannels+to+pattern+substrates+and+cells.">Vacuum-assisted fluid flow in microchannels to pattern substrates and cells.</a> Biofabrication. 6 (3), 035016 (2014).</li><li>Yuen, P. K., Goral, V. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low-cost+rapid+prototyping+of+flexible+microfluidic+devices+using+a+desktop+digital+craft+cutter.">Low-cost rapid prototyping of flexible microfluidic devices using a desktop digital craft cutter.</a> Lab on a Chip. 10 (3), 384-387 (2010).</li><li>Wang, L., 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=Self-loading+and+cell+culture+in+one+layer+microfluidic+devices.">Self-loading and cell culture in one layer microfluidic devices.</a> Biomed Microdevices. 11 (3), 679-684 (2009).</li><li>Feng, 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=Survival+of+mammalian+cells+under+high+vacuum+condition+for+ion+bombardment.">Survival of mammalian cells under high vacuum condition for ion bombardment.</a> Cryobiology. 49 (3), 241-249 (2004).</li><li>Haubert, K., Drier, T., Beebe, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PDMS+bonding+by+means+of+a+portable,+low-cost+corona+system.">PDMS bonding by means of a portable, low-cost corona system.</a> Lab on a Chip. 6 (12), 1548-1549 (2006).</li><li>Fan, D. -. H., Yuan, S. -. W., Shen, Y. -. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+modification+with+BSA+blocking+based+on+in+situ+synthesized+gold+nanoparticles+in+poly+(dimethylsiloxane)+microchip.">Surface modification with BSA blocking based on in situ synthesized gold nanoparticles in poly (dimethylsiloxane) microchip.</a> Colloids Surf, B. 75 (2), 608-611 (2010).</li><li>Hideshima, S., Sato, R., Inoue, S., Kuroiwa, S., Osaka, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+tumor+marker+in+blood+serum+using+antibody-modified+field+effect+transistor+with+optimized+BSA+blocking.">Detection of tumor marker in blood serum using antibody-modified field effect transistor with optimized BSA blocking.</a> Sens Actuator B-Chem. 161 (1), 146-150 (2012).</li><li>Zheng, C., 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=High-throughput+immunoassay+through+in-channel+microfluidic+patterning.">High-throughput immunoassay through in-channel microfluidic patterning.</a> Lab on a Chip. 12 (14), 2487-2490 (2012).</li><li>MacLeish, P., Barnstable, C., Townes-Anderson, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+a+monoclonal+antibody+as+a+substrate+for+mature+neurons+in+vitro.">Use of a monoclonal antibody as a substrate for mature neurons in vitro.</a> Procs Nat Acad of Sci. 80 (22), 7014-7018 (1983).</li><li>Suchodolskis, A., 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=Elastic+properties+of+chemically+modified+baker's+yeast+cells+studied+by+AFM.">Elastic properties of chemically modified baker's yeast cells studied by AFM.</a> Surf Interface Anal. 43 (13), 1636-1640 (2011).</li></ol>

The authors declare no competing financial interests.

While conventional photolithography is a well-established technique for the creation of molds for soft lithography, the equipment, materials, and skills necessary to use conventional photolithography are not readily available to most laboratories. For laboratories without access to these resources, we have presented adhesive tape fabrication as a method of creating molds with relatively simple features for microfluidic devices. This method allows any laboratory to create and utilize microfluidic devices for research purposes with readily available tools. The adhesive tape method may be improved with a low-cost desktop vinyl cutter31. Desktop vinyl cutters may increase reproducibility, resolution, as well as layout complexity. Each of these options, conventional photolithography, desktop cutters, or adhesive tape fabrication have different capabilities and limitations and researchers must carefully consider which method would be sufficient for their needs.
In cured PDMS, the long polymeric chains of dimethyl siloxane form a lattice, nanoporous structure which creates empty regions that can be filled with air molecules27. This gas permeable property is key to our method of filling microfluidic channels. When placed into a low pressure environment, air molecules are removed from the PDMS bulk material as well as the microchannels themselves25. When the PDMS is then exposed to atmospheric pressure, the PDMS bulk material and microchannels retain a negative pressure for some time25,30. This negative pressure draws liquid into the microchannels automatically filling the microfluidic channels. This negative pressure continues to draw liquid into the channel until the bulk PDMS equilibrates with atmospheric pressure.
This vacuum-assisted technique allows for patterning of specific proteins and substrates onto a growth substrate. In this study, we were able to pattern several different substrates and cell types both directly and indirectly (Table 1 and 2). However, the experimenter may need to test different incubation times, cell densities, or seeding media for their particular cell type. These variables likely relate to the innate ability of either the cell or substrate to adhere to the surface it is being placed upon. For example, here C2C12 cells required the use of serum-free media for the seeding media before culturing the cells in DMEM with 2% horse serum. Additionally, conditions may vary based upon whether a glass coverslip or plastic petri dish is being patterned. Here, both worked well to allow for specific patterning of various cell types.
One of the initial concerns we had while utilizing this technique was how exposure to vacuum might affect cell viability. This study and previous studies, however, should alleviate those concerns as we have shown that vacuum has little to no effect on a variety of cells30. Wang et al. previously utilized degassed irreversibly sealed microfluidic chambers to load HeLa cells32. Other studies have used vacuum-assisted cell seeding for a variety of cell types including human stem cells, adherent and non-adherent cells, and human-hamster hybrid cell line (AL) cells to load into microfluidic channels33. In addition, other researchers have subjected cells to much greater vacuum pressures in comparison to this current study with little to no discernible effect on the cells33. Bubbles formed during the removal of air from the microchannel tend to congregate on the surface of the droplet of the suspension at the inlet. Often these bubbles do not rupture due to the surface tension of the suspension. We have not observed a noticeable decrease in cell viability due to bubbles. In addition, the experiment with Calcein-AM does not suggest a significant decrease in cell viability. Because of this, we have not thoroughly examined cell death specifically due to bubbles.
Previous attempts to pattern substrates or cells were often plagued by problems such as the formation of air bubbles in the microfluidic devices. The formation of air bubbles made it difficult to inject liquids easily and efficiently without the use of equipment or materials that are not available in most laboratories. For example, previous methods utilized the use of plasma treatment or corona treatment to decrease the hydrophobicity of PDMS microchannels15,34. While effective, plasma and corona treaters are not readily available in most laboratories. In this protocol, we demonstrate the ability to pattern cells and substrates simply using common laboratory vacuums. Using the adhesive tape fabrication technique, the methodology can be utilized to create PDMS microfluidic channels to pattern substrates or cells in nearly any laboratory.
In order to pattern substrates or cells with the vacuum patterning protocol, several steps are critical for successful patterning. First, to fabricate the adhesive tape master, the adhesive tape must be completely attached to the glass slide and free of air bubbles. Air bubbles weaken the bond between the tape and the glass slide and may lead to the tape being peeled off when cured PDMS is peeled off the adhesive tape mold. In addition, bubbles will distort the geometry of the microfluidic channel causing the surface of the channels to be non-planar. To make the PDMS cast from SU-8 mold, the SU-8 mold must be silanized before casting with PDMS. This step is critical in preventing the permanent bonding of PDMS to the SU-8 mold after curing of PDMS. Prior to conformal sealing of the PDMS microfluidic channel to glass coverslips or plastic petri dishes, the PDMS must be cleaned of all dust particles. These particles may prevent the formation of a conformal seal between PDMS and glass and thus prevent the injection of cells or substrates into the microfluidic channel. As stated in the above protocol, cleaning the PDMS channels with adhesive tape is critical prior to adhering the microchannel to glass coverslips or plastic petri dishes. Finally, after placing the device into the vacuum chamber, the vacuum must be gently released to prevent displacement of the droplet of substrate or cell solution from the inlet hole.
If no fluid is observed flowing into the microfluidic channel, there may be a leak in the microfluidic channel which would prevent liquid from flowing into the channel. Remove the PDMS stamp, dry and clean it and then repeat step 4, 5, or 6 of the technique. If the solution does not flow into the microchannel after release of vacuum, ensure that the solution is completely covering the entrance of the microchannel. This can be done by pipetting solution up and down several times while putting it in the inlet hole. If the substrate, after injecting into the microchannel, does not attach to the glass, evaluate and determine the optimal incubation conditions necessary for the substrate used. If removing the PDMS cast from the glass surface or plastic petri dish causes the patterned cells or substrates to be destroyed, use a thinner PDMS cast, submerge the PDMS cast in PBS or media, and slowly and gently peel off the PDMS cast from the glass or plastic surface with tweezers.
Finally, BSA may be critical in decreasing nonspecific adhesion of cells to either the unpatterned glass or plastic surface. BSA is typically used as a blocking reagent in western blotting, immunostaining, and other molecular biology techniques. Other researchers have also utilized it for indirect or direct patterning protocols to decrease nonspecific cell adhesion35,36,37. We found that incubation of the substrate with BSA was necessary for most cell types except for salamander retinal cells. This may be because of the more specialized nature of the substrate (an antibody called Sal-1) that was used as well as the general lack of cell adhesion by this cell type38. In our hands, BSA also did not noticeably decrease the adherence of cells to the patterned areas and mainly served to decrease nonspecific adherence.
While this method is designed to be versatile and flexible in its application to different substrate and cell types, there are several limitations to this protocol and the compatible cells and substrate. Because of the very nature of patterning cells onto a surface, the cell types that this technique can be applied to are limited to those which are amenable to any patterning protocol such as those cells that are able to survive with more limited cell to cell contact as well as those that can survive at lower cell seeding concentrations. For example, one potential application is the patterning of yeast cells for atomic force microscopy (AFM)39. While we have tested many different cell substrates, other substrates may not work with this particular technique such as those that form three-dimensional gels. At this time, we have not examined if gels properly form and stay on the surface of the glass coverslip or plastic petri dish after patterning. This technique is capable of patterning complex shapes and large areas. However, it cannot pattern an array of individual separated shapes without interconnecting microchannels. In the absence of interconnected microchannels, each individual shape would require its own inlet making the technique cumbersome. While we did not notice any non-uniformity in patterning protein, the distribution of cells patterned inside the microchannel may be non-uniform. This non-uniformity may be due to the random distribution of cells in cell suspensions, geometry and dimensions of the microchannels, and viscosity of the cell suspension.
In the future, this technique may be applied to other types of substrates and cells. For example, three-dimensional gels such as basement membrane matrix or collagen scaffolds may potentially be patterned and formed using PDMS microfluidic channels. By injecting a hydrogel into the microfluidic device, the hydrogel may take on the shape of the microfluidic device and be able to form complex patterns without the use of 3-D printers or other technologies. These would allow for rapid creation of structured scaffolds with readily available tools and microfluidic devices.

In tissue engineering and biosensing, the ability to control the spatial organization of proteins and cells on a &#181;m scale, has become increasingly important over the last four decades1,2,3. Precise spatial organization of proteins and cells has allowed researchers to examine the interaction between cells and substrates containing similar or different types of cells, to guide cell growth, and to immobilize biomolecules for the fabrication of biosensors4,5,6,7,8,9.
Current methods of patterning proteins include photopatterning and microcontact printing. Photopatterning utilizes light sensitive material which is crosslinked upon exposure to ultra violet (UV) light. UV light directed at a photomask (consisting of transparent areas with darker regions to prevent UV light transmission) causes crosslinking in specific regions which can then be used for subsequent attachment of biomaterials or cells10,11. While this scheme is very accurate and allows for precise control of the topography of the culture surface, it is limited to UV-sensitive biomolecules that can be patterned by UV radiation12. Microcontact printing is another popular method of patterning specific proteins13,14. In this method, a poly-dimethyl siloxane (PDMS) stamp is treated with a variety of surface modification reagents before being soaked in a solution of the chosen biomolecular substrate. It is then gently pressed onto a glass coverslip or other surface thus &#34;stamping&#34; the biomolecule onto the culture surface. However, stamping is limited to the type of material that can be transferred as well as the wettability of biomolecules to the surface of the PDMS stamp15.
Direct patterning of cells can be more difficult and relies on complex methods such as switchable substrates, stencil based methods, or patterning with specific cell adhesion molecules16,17. These methods are limited in their ability to pattern cells due to the lack of compatible cell adhesion substrates, incompatibility of the process to work with sensitive biological cells and constraints, inconsistency in reproducing the patterning, and complexity of the procedure. For example, with switchable substrates, custom substrates need to be designed for every cell type, to switch their adherence to specific cell types without degradation upon exposure to the UV light and heat used in process17,18,19,20. Stencil based patterning methods are versatile in their ability to pattern cells; however, it is difficult to manufacture PDMS stencils at the appropriate thicknesses for use16,21. Direct injection of cells into PDMS microfluidic channels have some advantages such as: 1) ease in fabrication of microfluidic channels and 2) suitability for many different cells and substrates. However, the prevalent issue of air bubble capture during the injection process due to the hydrophobicity of PDMS without the use of plasma cleaning, or other methods to decrease air bubbles, makes it difficult to consistently create patterned cells on glass or plastic surfaces21.
This work expands upon capillary micromolding22,23,24,25,26 and reports a method to inject protein and cell suspensions into microchannels. The method used here demonstrates the patterning of substrates and both direct and indirect patterning of specific cell types. This technique overcomes the high hydrophobicity of PDMS and eliminates the presence of bubbles during injection of either substrates or cells by taking advantage of the gas permeability of PDMS27. This paper demonstrates the use of the technique with several different substrates and cell types. The article also highlights the fabrication of molds for soft lithography using conventional photolithography as well as a simple and low-cost adhesive tape method useful in resource limited settings28,29.

<table><tbody><tr><td>CorelDRAW X4 CAD Drawing Tools</td><td>Corel Corporation, Canada</td><td>X4 Version 14.0.0.701</td><td>CAD tool used to draw the layout of the microfluidic device</td></tr><tr><td>Laser Printer HP</td><td>Hewlett Packard, CA</td><td>1739629</td><td>Used to print the layout of microfluidic device for adhesive tape technique</td></tr><tr><td>Bel-Art Dessicator</td><td>Fisher Scientific, MA</td><td>08-594-16B</td><td>Used to degass the PDMS mixture</td></tr><tr><td>Adhesive Scotch Tape</td><td>3M Product, MN</td><td>Tape 600</td><td>Used to fabricate adhesive tape Master</td></tr><tr><td>PDMS Sylgard 184</td><td>Dow Corning, MI</td><td>1064291</td><td>Casting polymer</td></tr><tr><td>Petri Dish</td><td>Fisher Scientific, MA</td><td>08-772-23</td><td>Used to keep the mold to cast with PDMS</td></tr><tr><td>Stainless steel Scalpel (#3) with blade (# 11)</td><td>Feather Safety Razor Co. Ltd. Japan</td><td>2976#11</td><td>Used to cut the PDMS</td></tr><tr><td>Tweezers</td><td>Ted Pella, CA</td><td>5627-07</td><td>Used to handle the PDMS cast during peeling</td></tr><tr><td>Glass slides</td><td>Fisher Scientific, MA</td><td>12-546-2</td><td>Used as surface to pattern the Substrate</td></tr><tr><td>Glass slides</td><td>Fisher Scientific, MA</td><td>12-544-4</td><td>Used as surface to pattern the Substrate</td></tr><tr><td>Rubber Roller</td><td>Dick Blick Art Materials, IL</td><td>40104-1004</td><td>Used to attach adhesive tape on glass without trapping air bubbles</td></tr><tr><td>Laser Mask Writer</td><td>Heidelberg Instruments, Germany</td><td>DWL66fs</td><td>Used to fabricate quartz mask used in photolithography fabrication process</td></tr><tr><td>EVG Mask Aligner (Photolithography UV exposure tool)</td><td>EV Group, Germany</td><td>EVG 620T(B)</td><td>Used to expose the photoresist to UV light</td></tr><tr><td>Spin Coater Headway</td><td>Headway Research Inc, TX</td><td>PWM32-PS-CB15PL</td><td>Used to spin coat the photoresist on silicon wafer</td></tr><tr><td>Photoresists SU-8 50</td><td>MicroChem, MA</td><td>Y131269</td><td>Negative photoresist used for mold fabrication</td></tr><tr><td>SU-8 Devloper</td><td>MicroChem, MA</td><td>Y020100</td><td>Photoresist developer</td></tr><tr><td>Tridecafluoro-1,1,2,2-Tetrahydrooctyl-1-Trichlorosilane</td><td>UCT Specialties, PA</td><td>T2492-KG</td><td>Coat mold to avoid PDMS adhesion</td></tr><tr><td>Isopropanol</td><td>Sigma-Aldrich, MO</td><td>190764</td><td>Cleaning Solvent</td></tr><tr><td>Ethanol</td><td>Sigma-Aldrich, MO</td><td>24102</td><td>Sterilization Solvent</td></tr><tr><td>Poly-D-Lysine hydrobromide (PDL)</td><td>Sigma-Aldrich, MO</td><td>P0899-10MG</td><td>PDL solution is made at 0.1 mg/mL in Sodium Tetraborate Buffer</td></tr><tr><td>Laminin</td><td>Sigma-Aldrich, MO</td><td>L2020</td><td>Laminin aliquoted into 10 &amp;micro;L aliquots and diluted to 20 &amp;micro;g/&amp;micro;L in PBS prior to use</td></tr><tr><td>BSA</td><td>Fisher Scientific, MA</td><td>BP1605100</td><td>Cell culture</td></tr><tr><td>C2C12 Myoblast cell lline</td><td>ATCC, VA</td><td>CRL-1722</td><td>Used to demonstrate C2C12 patterning</td></tr><tr><td>PC12 Cell Line</td><td>ATCC, VA</td><td>CRL-1721</td><td>Used to demonstrate PC12 patterning</td></tr><tr><td>Collagen type 1, rat tail</td><td>BD Biosciences</td><td>40236</td><td>Cell culture</td></tr><tr><td>DMEM</td><td>GIBCO, MA</td><td>11965-084</td><td>Cell culture</td></tr><tr><td>Horse Serum, heat inactivated</td><td>Fisher Scientific, MA</td><td>26050-070</td><td>Cell culture</td></tr><tr><td>Phalloidin-tetramethylrhodamine B isothiocyanate (TRITC)</td><td>Sigma-Aldrich, MO</td><td>P1951</td><td>To label cells</td></tr><tr><td>Calcein-AM live dead cell Assay kit</td><td>Invitrogen, MA</td><td>L-3224</td><td>Cell viability Assay</td></tr><tr><td>Biopsy Hole Punch</td><td>Ted Pella, CA</td><td>15110-10</td><td>Punched hole in PDMS</td></tr></tbody></table>

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 method allows the patterning of proteins and indirect patterning of cells using dead-end microfluidic channels with dimensions as small as 10 &#181;m and equipment available in almost all biological laboratories once the master mold is made. This technique can be utilized with PDMS microfluidic channels created using traditional soft photolithography, or with PDMS microfluidic channels created with adhesive tape fabrication (Figure 1)28,29. We have previously demonstrated the use of this procedure in indirect patterning of neuronal cells for evaluation of axon guidance in photoreceptors4,30, and have now demonstrated its use in several additional cell types of varying origins. Embryonic rat cortical neurons patterned on the poly-D-Lysine (PDL) and laminin surfaces demonstrate general adherence to the patterned areas and growth along the PDL and laminin stripes (Figure 3A). C2C12 myoblast cell lines also adhere to the patterned area and demonstrate fusion into multinucleated myotubes (Figure 3B). We have also adapted this technique for direct cell patterning as seen with fibroblasts in Figure 4. As previously reported, this direct cell patterning method showed no adverse effect on cell survival30. The variables that lead to successful direct or indirect patterning of stated cell types are listed in Table 1 and 2. Thus in the future, researchers may utilize this technique to pattern cells and discover phenomena related to their specific patterning.
<img alt="Figure 1" src="/files/ftp_upload/55513/55513fig1.jpg" /> 
Figure 1: Fabrication of Mold by Soft Lithography. Left: Photolithography process. SU-8 is spin coated onto a silicon wafer, exposed to UV radiation, and developed to remove excess SU-8. Right: Adhesive tape fabrication process. Adhesive tape is attached to a clean glass slide, cut with a scalpel, and excess tape is carefully removed. PDMS cast is then fabricated from each mold using soft lithography. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55513/55513fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/55513/55513fig2.jpg" /> 
Figure 2: Example of a PDMS Device for Substrate Patterning. (a) PDMS microfluidic cast with inlet used for microfluidic patterning of substrates. Inlet was punched out at one end of microchannels. (b) Microfluidic channel as seen under phase contrast microscopy. Microfluidic channels are 15 mm in length, 20 &#181;m wide, and separated by 200 &#181;m. Figure 2 is reproduced with permission from IOP Publishing. All rights reserved30. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55513/55513fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/55513/55513fig3.jpg" /> 
Figure 3: Indirect Cell Patterning. (a) Vacuum assisted protein patterning process. PDMS microfluidic channel device is attached to a petri dish, substrate solution is loaded into inlet, vacuum is briefly applied to chamber and released, PDMS device is removed, pattern is washed, and ready to use. (b) Embryonic cortical rat neurons seeded onto a coverslip with a patterned PDL and laminin substrate. (c) C2C12 myoblast cell lines grown on patterned PDL and laminin. Figures 3a and 3c are reproduced with permission from IOP Publishing. All rights reserved30. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55513/55513fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/55513/55513fig4.jpg" /> 
Figure 4: Direct Cell Patterning. (a-c) Fibroblasts patterned via direct cell patterning. (a) Microfluidic channel device fabricated using adhesive tape lithography. (b) Microfluidic channels after filling with fibroblast cell suspension using vacuum assisted patterning technique. (c) Fibroblasts after removal of microfluidic device. (d) Calcein-AM staining of fibroblasts after microfluidic patterning. (e) Phase contrast image of fibroblasts after microfluidic patterning. (f-g) Fibroblasts stained with phalloidin-TRITC (red) and DAPI (blue). Figure 4 is reproduced with permission from IOP Publishing. All rights reserved30. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55513/55513fig4large.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>Indirect Cell Patterning</td>
			<td>Substrate</td>
			<td>Protein Incubation Conditions</td>
			<td>Cell Density</td>
			<td>Cell Adhesion Duration</td>
		</tr>
		<tr>
			<td>PC12</td>
			<td>Collagen-1</td>
			<td>1 h at 50 &#176;C</td>
			<td>5 x 106/ mL</td>
			<td>1 h at 37 &#176;C</td>
		</tr>
		<tr>
			<td>Fibroblast</td>
			<td>Collagen-1</td>
			<td>1 h at 50 &#176;C</td>
			<td>5 x 106 / mL</td>
			<td>1 h at 37 &#176;C</td>
		</tr>
		<tr>
			<td>C2C12</td>
			<td>PDL + Laminin</td>
			<td>1 h at 37 &#176;C, wash PBS, overnight at 37 &#176;C</td>
			<td>2 x 106 / mL</td>
			<td>15 min at 37 &#176;C</td>
		</tr>
		<tr>
			<td>Embryonic Rat Cortical Neurons</td>
			<td>PDL + Laminin</td>
			<td>1 h at 37 &#176;C, wash PBS, overnight at 37 &#176;C</td>
			<td>2 x 106 / mL</td>
			<td>15 min at 37 &#176;C</td>
		</tr>
		<tr>
			<td>Salamander Retinal Neurons</td>
			<td>Sal-1</td>
			<td>overnight at 10 &#176;C</td>
			<td>N/A</td>
			<td>1 h at 10 &#176;C</td>
		</tr>
	</tbody>
</table>
Table 1: Indirect Cell Patterning Conditions. List of incubation conditions such as substrate type, time, temperature, and cell density for typical cell types. Researchers should optimize the incubation conditions for their own model substrates and cell type. Sal-1 is the antibody substrate used to grow Salamander retinal neurons.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Direct Cell Patterning</td>
			<td>Substrate</td>
			<td>Cell Density</td>
			<td>Cell Adhesion time</td>
		</tr>
		<tr>
			<td>PC12</td>
			<td>Collagen-1</td>
			<td>6 x 106 / mL</td>
			<td>1 h at 37 &#176;C</td>
		</tr>
		<tr>
			<td>Fibroblast</td>
			<td>Collagen-1</td>
			<td>6 x 106 / mL</td>
			<td>1 h at 37 &#176;C</td>
		</tr>
		<tr>
			<td>C2C12</td>
			<td>PDL + Laminin</td>
			<td>2 x 106 / mL</td>
			<td>15 min at 37 &#176;C</td>
		</tr>
		<tr>
			<td>Embryonic Rat Cortical Neurons</td>
			<td>PDL + Laminin</td>
			<td>2 x 106 / mL</td>
			<td>15 min at 37 &#176;C</td>
		</tr>
		<tr>
			<td>Salamander Retinal Neurons</td>
			<td>Sal-1</td>
			<td>N/A</td>
			<td>1 h at 10 &#176;C</td>
		</tr>
	</tbody>
</table>
Table 2: Direct Cell Patterning Conditions. List of incubation conditions such as temperature, time, and cell density for typical cell types. Researchers should optimize the incubation conditions for their own model cell type.

Watch this Scientific Journal Video about A Versatile Method of Patterning Proteins and Cells at JoVE.com

A Versatile Method of Patterning Proteins and Cells

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 ...

a-versatile-method-of-patterning-proteins-and-cells

Nanyang Technological University

Research

JoVE Journal

Bioengineering

9.3K Views.  Rutgers University. This report describes a simple, easy to perform technique, using low pressure vacuum, to fill microfluidic channels with cells and substrates for biological research.

Video: A Versatile Method of Patterning Proteins and Cells

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 ...

Cell Co-culture Patterning Using Aqueous Two-phase Systems

Cell patterning technologies that are fast, easy to use and affordable will be required for the future development of high throughput cell assays, platforms for studying cell-cell interactions and tissue engineered systems. This detailed protocol describes a method for generating co-cultures of cells using biocompatible solutions of dextran (DEX) and polyethylene glycol (PEG) that phase-separate when combined above threshold concentrations. Cells can be patterned in a variety of configurations using this method. Cell exclusion patterning can be performed by printing droplets of DEX on a substrate and covering them with a solution of PEG containing cells. The interfacial tension formed between the two polymer solutions causes cells to fall around the outside of the DEX droplet and form a circular clearing that can be used for migration assays. Cell islands can be patterned by dispensing a cell-rich DEX phase into a PEG solution or by covering the DEX droplet with a solution of PEG. Co-cultures can be formed directly by combining cell exclusion with DEX island patterning. These methods are compatible with a variety of liquid handling approaches, including manual micropipetting, and can be used with virtually any adherent cell type.

Aqueous two-phase systems were used to simultaneously pattern multiple populations of cells. This fast and easy method for cell patterning takes advantage of the phase separation of aqueous solutions of dextran and polyethylene glycol and the interfacial tension that exists between the two polymer solutions.

Cell patterning  technologies that are fast, easy to use and affordable will be required for the  future development of high throughput cell assays, ...

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

Mapping the Emergent Spatial Organization of Mammalian Cells using Micropatterns and Quantitative Imaging

A fundamental goal in biology is to understand how patterns emerge during development. Several groups have shown that patterning can be achieved in vitro when stem cells are spatially confined onto micropatterns, thus setting up experimental models which offer unique opportunities to identify, in vitro, the fundamental principles of biological organisation.
Here we describe our own implementation of the methodology. We adapted a photo-patterning technique to reduce the need for specialized equipment to make it easier to establish the method in a standard cell biology laboratory. We also developed a free, open-source and easy to install image analysis framework in order to precisely measure the preferential positioning of sub-populations of cells within colonies of standard shapes and sizes. This method makes it possible to reveal the existence of patterning events even in seemingly disorganized populations of cells. The technique provides quantitative insights and can be used to decouple influences of the environment (e.g., physical cues or endogenous signaling), on a given patterning process.

The method presented here uses micropatterning together with quantitative imaging to reveal spatial organization within mammalian cultures. The technique is easy to establish in a standard cell biology laboratory and offers a tractable system to study patterning in vitro.

A fundamental goal in biology is to understand how patterns emerge during development. Several groups have shown that patterning can be achieved in vitro ...

Developmental Biology

Micropatterning Transmission Electron Microscopy Grids to Direct Cell Positioning within Whole-Cell Cryo-Electron Tomography Workflows

Whole-cell cryo-electron tomography (cryo-ET) is a powerful technology that is used to produce nanometer-level resolution structures of macromolecules present in the cellular context and preserved in a near-native frozen-hydrated state. However, there are challenges associated with culturing and/or adhering cells onto TEM grids in a manner that is suitable for tomography while retaining the cells in their physiological state. Here, a detailed step-by-step protocol is presented on the use of micropatterning to direct and promote eukaryotic cell growth on TEM grids. During micropatterning, cell growth is directed by depositing extra-cellular matrix (ECM) proteins within specified patterns and positions on the foil of the TEM grid while the other areas remain coated with an anti-fouling layer. Flexibility in the choice of surface coating and pattern design makes micropatterning broadly applicable for a wide range of cell types. Micropatterning is useful for studies of structures within individual cells as well as more complex experimental systems such as host-pathogen interactions or differentiated multi-cellular communities. Micropatterning may also be integrated into many downstream whole-cell cryo-ET workflows, including correlative light and electron microscopy (cryo-CLEM) and focused-ion beam milling (cryo-FIB).

The goal of this protocol is to direct cell adhesion and growth to targeted areas of grids for cryo-electron microscopy. This is achieved by applying an anti-fouling layer that is ablated in user-specified patterns followed by deposition of extra-cellular matrix proteins in the patterned areas prior to cell seeding.

Whole-cell cryo-electron tomography (cryo-ET) is a powerful technology that is used to produce nanometer-level resolution structures of macromolecules ...

Biochemistry

Preparation of Tunable Extracellular Matrix Microenvironments to Evaluate Schwann Cell Phenotype Specification

Traumatic peripheral nervous system (PNS) injuries currently lack suitable treatments to regain full functional recovery. Schwann cells (SCs), as the major glial cells of the PNS, play a vital role in promoting PNS regeneration by dedifferentiating into a regenerative cell phenotype following injury. However, the dedifferentiated state of SCs is challenging to maintain through the time-period needed for regeneration and is impacted by changes in the surrounding extracellular matrix (ECM). Therefore, determining the complex interplay between SCs and differing ECM to provide cues of regenerative potential of SCs is essential. To address this, a strategy was created where different ECM proteins were adsorbed onto a tunable polydimethylsiloxane (PDMS) substrate which provided a platform where stiffness and protein composition can be modulated. SCs were seeded onto the tunable substrates and critical cellular functions representing the dynamics of SC phenotype were measured. To illustrate the interplay between SC protein expression and cellular morphology, differing seeding densities of SCs in addition to individual microcontact printed cellular patterns were utilized and characterized by immunofluorescence staining and western blot. Results showed that cells with a smaller spreading area and higher extent of cellular elongation promoted higher levels of SC regenerative phenotypic markers. This methodology not only begins to unravel the significant relationship between the ECM and cellular function of SCs, but also provides guidelines for the future optimization of biomaterials in peripheral nerve repair.

This methodology aims to illustrate the mechanisms by which extracellular matrix cues such as substrate stiffness, protein composition and cell morphology regulate Schwann cell (SC) phenotype.

Traumatic peripheral nervous system (PNS) injuries currently lack suitable treatments to regain full functional recovery. Schwann cells (SCs), as the ...

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 ...

A Versatile Method of Patterning Proteins and Cells

Samenvatting

Meer video's verkennen

Hoofdstukken in deze video