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 purp...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 &#181;L aliquots and diluted to 20 &#181;g/&#181;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,<...

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

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

Plasma Lithography Surface Patterning for Creation of Cell Networks

Systematic manipulation of a cell microenvironment with micro- and nanoscale resolution is often required for deciphering various cellular and molecular phenomena. To address this requirement, we have developed a plasma lithography technique to manipulate the cellular microenvironment by creating a patterned surface with feature sizes ranging from 100 nm to millimeters. The goal of this technique is to be able to study, in a controlled way, the behaviors of individual cells as well as groups of cells and their interactions.
This plasma lithography method is based on selective modification of the surface chemistry on a substrate by means of shielding the contact of low-temperature plasma with a physical mold. This selective shielding leaves a chemical pattern which can guide cell attachment and movement. This pattern, or surface template, can then be used to create networks of cells whose structure can mimic that found in nature and produces a controllable environment for experimental investigations. The technique is well suited to studying biological phenomenon as it produces stable surface patterns on transparent polymeric substrates in a biocompatible manner. The surface patterns last for weeks to months and can thus guide interaction with cells for long time periods which facilitates the study of long-term cellular processes, such as differentiation and adaption. The modification to the surface is primarily chemical in nature and thus does not introduce topographical or physical interference for interpretation of results. It also does not involve any harsh or toxic substances to achieve patterning and is compatible for tissue culture. Furthermore, it can be applied to modify various types of polymeric substrates, which due to the ability to tune their properties are ideal for and are widely used in biological applications. The resolution achievable is also beneficial, as isolation of specific processes such as migration, adhesion, or binding allows for discrete, clear observations at the single to multicell level.
This method has been employed to form diverse networks of different cell types for investigations involving migration, signaling, tissue formation, and the behavior and interactions of neurons arraigned in a network.

A versatile plasma lithography technique has been developed to generate stable surface patterns for guiding cellular attachment. This technique can be applied to create cell networks including those that mimic natural tissues and has been used for studying several, distinct cell types.

Systematic manipulation of a cell microenvironment with micro- and nanoscale resolution is often required for deciphering various cellular and molecular ...

Soft Lithographic Functionalization and Patterning Oxide-free Silicon and Germanium

The development of hybrid electronic devices relies in large part on the integration of (bio)organic materials and inorganic semiconductors through a stable interface that permits efficient electron transport and protects underlying substrates from oxidative degradation. Group IV semiconductors can be effectively protected with highly-ordered self-assembled monolayers (SAMs) composed of simple alkyl chains that act as impervious barriers to both organic and aqueous solutions. Simple alkyl SAMs, however, are inert and not amenable to traditional patterning techniques. The motivation for immobilizing organic molecular systems on semiconductors is to impart new functionality to the surface that can provide optical, electronic, and mechanical function, as well as chemical and biological activity. 
Microcontact printing (&mu;CP) is a soft-lithographic technique for patterning SAMs on myriad surfaces.1-9 Despite its simplicity and versatility, the approach has been largely limited to noble metal surfaces and has not been well developed for pattern transfer to technologically important substrates such as oxide-free silicon and germanium. Furthermore, because this technique relies on the ink diffusion to transfer pattern from the elastomer to substrate, the resolution of such traditional printing is essentially limited to near 1 &mu;m.10-16
In contrast to traditional printing, inkless &mu;CP patterning relies on a specific reaction between a surface-immobilized substrate and a stamp-bound catalyst. Because the technique does not rely on diffusive SAM formation, it significantly expands the diversity of patternable surfaces. In addition, the inkless technique obviates the feature size limitations imposed by molecular diffusion, facilitating replication of very small (&lt;200 nm) features.17-23 However, up till now, inkless &mu;CP has been mainly used for patterning relatively disordered molecular systems, which do not protect underlying surfaces from degradation.
Here, we report a simple, reliable high-throughput method for patterning passivated silicon and germanium with reactive organic monolayers and demonstrate selective functionalization of the patterned substrates with both small molecules and proteins. The technique utilizes a preformed NHS-reactive bilayered system on oxide-free silicon and germanium. The NHS moiety is hydrolyzed in a pattern-specific manner with a sulfonic acid-modified acrylate stamp to produce chemically distinct patterns of NHS-activated and free carboxylic acids. A significant limitation to the resolution of many &mu;CP techniques is the use of PDMS material which lacks the mechanical rigidity necessary for high fidelity transfer. To alleviate this limitation we utilized a polyurethane acrylate polymer, a relatively rigid material that can be easily functionalized with different organic moieties. Our patterning approach completely protects both silicon and germanium from chemical oxidation, provides precise control over the shape and size of the patterned features, and gives ready access to chemically discriminated patterns that can be further functionalized with both organic and biological molecules. The approach is general and applicable to other technologically-relevant surfaces.

Here we describe a simple method for patterning oxide-free silicon and germanium with reactive organic monolayers and demonstrate functionalization of the patterned substrates with small molecules and proteins. The approach completely protects surfaces from chemical oxidation, provides precise control over feature morphology, and provides ready access to chemically discriminated patterns.

The development of hybrid electronic devices relies in large part on the integration of (bio)organic materials and inorganic semiconductors through a ...

Visualization of Cortex Organization and Dynamics in Microorganisms, using Total Internal Reflection Fluorescence Microscopy

TIRF microscopy has emerged as a powerful imaging technology to study spatio-temporal dynamics of fluorescent molecules in vitro and in living cells1. The optical phenomenon of total internal reflection occurs when light passes from a medium with high refractive index into a medium with low refractive index at an angle larger than a characteristic critical angle (i.e. closer to being parallel with the boundary). Although all light is reflected back under such conditions, an evanescent wave is created that propagates across and along the boundary, which decays exponentially with distance, and only penetrates sample areas that are 100-200 nm near the interface2. In addition to providing superior axial resolution, the reduced excitation of out of focus fluorophores creates a very high signal to noise ratios and minimizes damaging effects of photobleaching2,3. Being a widefield technique, TIRF also allows faster image acquisition than most scanning based confocal setups.
At first glance, the low penetration depth of TIRF seems to be incompatible with imaging of bacterial and fungal cells, which are often surrounded by thick cell walls. On the contrary, we have found that the cell walls of yeast and bacterial cells actually improve the usability of TIRF and increase the range of observable structures4-8. Many cellular processes can therefore be directly accessed by TIRF in small, single-cell microorganisms, which often offer powerful genetic manipulation techniques. This allows us to perform in vivo biochemistry experiments, where kinetics of protein interactions and activities can be directly assessed in living cells.
We describe here the individual steps required to obtain high quality TIRF images for Saccharomyces cerevisiae or Bacillus subtilis cells. We point out various problems that can affect TIRF visualization of fluorescent probes in cells and illustrate the procedure with several application examples. Finally, we demonstrate how TIRF images can be further improved using established image restoration techniques.

Total Internal Reflection Fluorescence (TIRF) microscopy is a powerful approach to observe structures close to the cell surface at high contrast and temporal resolution. We demonstrate how TIRF can be employed to study protein dynamics at the cortex of cell wall-enclosed bacterial and fungal cells.

TIRF microscopy has emerged as  a powerful imaging technology to study spatio-temporal dynamics of fluorescent molecules  in vitro and in living cells1. ...

A Versatile Automated Platform for Micro-scale Cell Stimulation Experiments

Study of cells in culture (in vitro analysis) has provided important insight into complex biological systems. Conventional methods and equipment for in vitro analysis are well suited to study of large numbers of cells (&ge;105) in milliliter-scale volumes (&ge;0.1 ml). However, there are many instances in which it is necessary or desirable to scale down culture size to reduce consumption of the cells of interest and/or reagents required for their culture, stimulation, or processing. Unfortunately, conventional approaches do not support precise and reproducible manipulation of micro-scale cultures, and the microfluidics-based automated systems currently available are too complex and specialized for routine use by most laboratories. To address this problem, we have developed a simple and versatile technology platform for automated culture, stimulation, and recovery of small populations of cells (100 - 2,000 cells) in micro-scale volumes (1 - 20 &mu;l). The platform consists of a set of fibronectin-coated microcapillaries ("cell perfusion chambers"), within which micro-scale cultures are established, maintained, and stimulated; a digital microfluidics (DMF) device outfitted with "transfer" microcapillaries ("central hub"), which routes cells and reagents to and from the perfusion chambers; a high-precision syringe pump, which powers transport of materials between the perfusion chambers and the central hub; and an electronic interface that provides control over transport of materials, which is coordinated and automated via pre-determined scripts. As an example, we used the platform to facilitate study of transcriptional responses elicited in immune cells upon challenge with bacteria. Use of the platform enabled us to reduce consumption of cells and reagents, minimize experiment-to-experiment variability, and re-direct hands-on labor. Given the advantages that it confers, as well as its accessibility and versatility, our platform should find use in a wide variety of laboratories and applications, and prove especially useful in facilitating analysis of cells and stimuli that are available in only limited quantities.

We have developed an automated cell culture and interrogation platform for micro-scale cell stimulation experiments. The platform offers simple, versatile, and precise control in cultivating and stimulating small populations of cells, and recovering lysates for molecular analyses. The platform is well suited to studies that use precious cells and/or reagents.

Study of cells in culture (in vitro analysis) has provided important insight into complex biological systems. Conventional methods and equipment for in ...

A Microfluidic Chip for the Versatile Chemical Analysis of Single Cells

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this purpose, the cells are passively trapped in the middle of a microchamber. Upon activation of the control layer, the cell is isolated from the surrounding volume in a small chamber. The surrounding volume can then be exchanged without affecting the isolated cell. However, upon short opening and closing of the chamber, the solution in the chamber can be replaced within a few hundred milliseconds. Due to the reversibility of the chambers, the cells can be exposed to different solutions sequentially in a highly controllable fashion, e.g. for incubation, washing, and finally, cell lysis. The tightly sealed microchambers enable the retention of the lysate, minimize and control the dilution after cell lysis. Since lysis and analysis occur at the same location, high sensitivity is retained because no further dilution or loss of the analytes occurs during transport. The microchamber design therefore enables the reliable and reproducible analysis of very small copy numbers of intracellular molecules (attomoles, zeptomoles) released from individual cells. Furthermore, many microchambers can be arranged in an array format, allowing the analysis of many cells at once, given that suitable optical instruments are used for monitoring. We have already used the platform for proof-of-concept studies to analyze intracellular proteins, enzymes, cofactors and second messengers in either relative or absolute quantifiable manner.

In this article we present a microfluidic chip for single cell analysis. It allows the quantification of intracellular proteins, enzymes, cofactors, and second messengers by means of fluorescent assays or immunoassays.&#160;

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this ...

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 ...

Delivery of Proteins, Peptides or Cell-impermeable Small Molecules into Live Cells by Incubation with the Endosomolytic Reagent dfTAT

Macromolecular delivery strategies typically utilize the endocytic pathway as a route of cellular entry. However, endosomal entrapment severely limits the efficiency with which macromolecules penetrate the cytosolic space of cells. Recently, we have circumvented this problem by identifying the reagent dfTAT, a disulfide bond dimer of the peptide TAT labeled with the fluorophore tetramethylrhodamine. We have generated a fluorescently labeled dimer of the prototypical cell-penetrating peptide (CPP) TAT, dfTAT, which penetrates live cells and reaches the cytosolic space of cells with a particularly high efficiency. Cytosolic delivery of dfTAT is achieved in multiple cell lines, including primary cells. Moreover, delivery does not noticeably impact cell viability, proliferation or gene expression. dfTAT can deliver small molecules, peptides, antibodies, biologically active enzymes and a transcription factor. In this report, we describe the protocols involved in dfTAT synthesis and cellular delivery. The manuscript describes how to control the amount of protein delivered to the cytosolic space of cells by varying the amount of protein administered extracellularly. Finally, the current limitations of this new technology and steps involved in validating delivery are discussed. The described protocols should be extremely useful for cell-based assays as well as for the ex vivo manipulation and reprogramming of cells.

We describe how to deliver proteins and cell-impermeable small molecules into cultured mammalian cells by a simple co-incubation protocol with a reagent that causes endocytic organelles to become leaky.

Macromolecular delivery strategies typically utilize the endocytic pathway as a route of cellular entry. However, endosomal entrapment severely limits the ...

Rapid Subtractive Patterning of Live Cell Layers with a Microfluidic Probe

The microfluidic probe (MFP) facilitates performing local chemistry on biological substrates by confining nanoliter volumes of liquids. Using one particular implementation of the MFP, the hierarchical hydrodynamic flow confinement (hHFC), multiple liquids are simultaneously brought in contact with a substrate. Local chemical action and liquid shaping using the hHFC, is exploited to create cell patterns by locally lysing and removing cells. By utilizing the scanning ability of the MFP, user-defined patterns of cell monolayers are created. This protocol enables rapid, real-time and spatially controlled cell patterning, which can allow selective cell-cell and cell-matrix interaction studies.

We present a protocol to perform subtractive patterning of live cell monolayers on a surface. This is achieved by local and selective lysis of adherent cells using a microfluidic probe (MFP). The cell lysate retrieved from local regions can be used for downstream analysis, enabling molecular profiling studies.

The microfluidic probe (MFP) facilitates performing local chemistry on biological substrates by confining nanoliter volumes of liquids. Using one ...

Light-mediated Formation and Patterning of Hydrogels for Cell Culture Applications

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In particular, light-mediated click reactions, such as photoinitiated thiol&#8722;ene and thiol&#8722;yne reactions, afford spatiotemporal control over material properties and allow the design of systems with a high degree of user-directed property control. Fabrication and modification of hydrogel-based biomaterials using the precision afforded by light and the versatility offered by these thiol&#8722;X photoclick chemistries are of growing interest, particularly for the culture of cells within well-defined, biomimetic microenvironments. Here, we describe methods for the photoencapsulation of cells and subsequent photopatterning of biochemical cues within hydrogel matrices using versatile and modular building blocks polymerized by a thiol&#8722;ene photoclick reaction. Specifically, an approach is presented for constructing hydrogels from allyloxycarbonyl (Alloc)-functionalized peptide crosslinks and pendant peptide moieties and thiol-functionalized poly(ethylene glycol) (PEG) that rapidly polymerize in the presence of lithium acylphosphinate photoinitiator and cytocompatible doses of long wavelength ultraviolet (UV) light. Facile techniques to visualize photopatterning and quantify the concentration of peptides added are described. Additionally, methods are established for encapsulating cells, specifically human mesenchymal stem cells, and determining their viability and activity. While the formation and initial patterning of thiol-alloc hydrogels are shown here, these techniques broadly may be applied to a number of other light and radical-initiated material systems (e.g., thiol-norbornene, thiol-acrylate) to generate patterned substrates.

We describe a sequential process for light-mediated formation and subsequent biochemical patterning of synthetic hydrogel matrices for three-dimensional cell culture applications. The construction and modification of hydrogels with cytocompatible photoclick chemistry is demonstrated. Additionally, facile techniques to quantify and observe patterns and determine cell viability within these hydrogels are presented.

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In ...

Patterning Bioactive Proteins or Peptides on Hydrogel Using Photochemistry for Biological Applications

There are many biological stimuli that can influence cell behavior and stem cell differentiation. General cell culture approaches rely on soluble factors within the medium to control cell behavior. However, soluble additions cannot mimic certain signaling motifs, such as matrix-bound growth factors, cell-cell signaling, and spatial biochemical cues, which are common influences on cells. Furthermore, biophysical properties of the matrix, such as substrate stiffness, play important roles in cell fate, which is not easily manipulated using conventional cell culturing practices. In this method, we describe a straightforward protocol to provide patterned bioactive proteins on synthetic polyethylene glycol (PEG) hydrogels using photochemistry. This platform allows for the independent control of substrate stiffness and spatial biochemical cues. These hydrogels can achieve a large range of physiologically relevant stiffness values. Additionally, the surfaces of these hydrogels can be photopatterned with bioactive peptides or proteins via thiol-ene click chemistry reactions. These methods have been optimized to retain protein function after surface immobilization. This is a versatile protocol that can be applied to any protein or peptide of interest to create a variety of patterns. Finally, cells seeded onto the surfaces of these bioactive hydrogels can be monitored over time as they respond to spatially specific signals.

In this method, we use photopolymerization and click chemistry techniques to create protein or peptide patterns on the surface of polyethylene glycol (PEG) hydrogels, providing immobilized bioactive signals for the study of cellular responses in vitro.

There are many biological stimuli that can influence cell behavior and stem cell differentiation. General cell culture approaches rely on soluble factors ...