This work was supported by a Wellcome Trust Clinical PhD fellowship (ECAT).

1. Fabrication of Parylene Patterns on SiO2: Process Flow (See Figure 1)
<ol>
	<li>Design desired parylene-C configuration using a layout editor software package, capable of reading/writing CIF (Caltech Intermediate Form) or GDS-II (Graphic Database System-II) files. CIF and GDS-II are industry standard file formats for integrated circuit artwork layout.</li>
	<li>Commission photo mask manufacture to an appropriate microelectronics facility, or make in-house if facilities exist.</li>
	<li>Oxidize a silicon wafer in an atmospheric horizontal furnace (H2 1.88 SLM and O2 1.25 SLM) at 950 &#176;C for 40 min to produce a 200 nm SiO2 layer (confirm thickness with a small spot spectroscopic reflectometer).</li>
	<li>Prime the oxidized wafer with a silane adhesion promoter. Now deposit parylene-C at 22 &#176;C at a rate of 1.298 nm/mg of dimer using a vacuum deposition system specifically designed to deposit parylene. A 100 nm thick parylene-C coating was used for all examples shown below.</li>
	<li>Next deposit hexamethyldisilazane (HMDS) adhesion promoter on the parylene-coated wafer using a suitable photo-resist coating system.</li>
	<li>Now spin the wafer at 4,000 rpm for 30 sec whilst applying positive photo-resist (resulting in a theoretical thickness of 1 &#956;m), using the same photoresist coating system as above.</li>
	<li>Soft bake the wafer for 60 sec at 90 &#176;C.</li>
	<li>Insert both the wafer and premanufactured photo mask into a mask aligner.</li>
	<li>Expose the photoresist-coated wafer with a UV negative representation of the desired parylene-C configuration.</li>
	<li>Bake exposed wafer for 60 sec at 110 &#176;C.</li>
	<li>Remove all exposed photo-resist from the wafer by developing in an appropriate developer solution.</li>
	<li>Etch off the unprotected parylene. Use an oxygen plasma etch system (at a 50 mTorr chamber pressure, 49 sccm O2, 100 W RF power at 13.56 MHz, and an etch rate of 100 nm/min) to reveal the underlying SiO2.</li>
	<li>Dice the wafer using an appropriate dicing saw (spindle speed 30,000 rpm, feed speed 7 mm/sec).</li>
	<li>Rinse chips in deionized H2O and blow dry with nitrogen.</li>
	<li>Store chips (indefinitely) in dust free boxes until required.</li>
</ol>
2. Chip Cleaning and Activation: Protocol
<ol>
	<li>Remove residual photoresist from chips by washing in acetone for 10 sec.</li>
	<li>Rinse in deionized distilled H2O 3x.</li>
	<li>Make up fresh piranha acid (a 5:3 ratio of 30% hydrogen peroxide and 98% sulfuric acid). 
	CAUTION: Prepare piranha acid with great care. It is an extremely powerful oxidizer, is strongly acidic, and mixing hydrogen peroxide with sulfuric acid is an exothermic reaction. Perform this stage in an acid fume hood. Allow 2 min&#160;to pass after mixing the piranha acid but use within 20 min.</li>
	<li>Clean and etch chips by immersion in piranha acid for 10 min.</li>
	<li>Rinse chips 3x in deionized H2O and transfer to a sterile culture dish.</li>
	<li>Now activate chips for cell patterning. For example, add two chips per well to a 6-well plate and then add 2 ml of fetal bovine serum so as to fully immerse all chips. Perform this, and all subsequent cell culture stages, under sterile conditions in a laminar flow tissue culture hood.</li>
	<li>Incubate chips in serum for 3-12 hr&#160;at 37 &#176;C. 
	NOTE: Steps 2.4-2.7 should be performed sequentially and without delay between steps. If serum activation is delayed by &#8805;24 hr&#160;after piranha-treatment, cell patterning is impaired due to reduced cell repulsion from SiO2 regions. 
	Alternative activation solutions containing specific cell adhesion proteins can be used in place of fetal bovine serum. Such solutions alter the cell-adhesion characteristics of the two contrasting substrates (see representative results for an example).</li>
</ol>
3. Plating Cell Lines On-chip: Protocol
<ol>
	<li>Remove chips from their activation solution and wash once for 10 sec in Hank&#39;s Balanced Salt Solution.</li>
	<li>Place chip in a culture well and plate chosen cell type as a suspension in its usual growth media. Optimum cell plating density depends both on cell type and geometric pattern of parylene-C on-chip. A density of 5 x 104 cells/ml is a sensible starting point.</li>
	<li>Imaging is tailored to the underlying motivation for cell patterning but live cell behavior can easily be assessed using a dissecting microscope and a digital camera with a suitable relay lens.</li>
</ol>

<ol>
	<li>Zhi, Z. 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=A+Versatile+Gold+Surface+Approach+for+Fabrication+and+Interrogation+of+Glycoarrays.">A Versatile Gold Surface Approach for Fabrication and Interrogation of Glycoarrays.</a> ChemBioChem. 9, 1568-1575 (2009).</li><li>Michelini, E., Roda, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Staying+alive:+new+perspectives+on+cell+immobilization+for+biosensing+purposes.">Staying alive: new perspectives on cell immobilization for biosensing purposes.</a> Anal. Bioanal. Chem. 402 (5), 1785-1797 (2012).</li><li>Franks, W., Tosatti, S., Heer, F., Seif, P., Textor, M., Hierlemann, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterned+cell+adhesion+by+self-assembled+structures+for+use+with+a+CMOS+cell-based+biosensor.">Patterned cell adhesion by self-assembled structures for use with a CMOS cell-based biosensor.</a> Biosens. Bioelectron. 22 (7), 1426-1433 (2007).</li><li>Bacakova, L., Filova, E., Parizek, M., Ruml, T., Svorcik, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+cell+adhesion,+proliferation+and+differentiation+on+materials+designed+for+body+implants.">Modulation of cell adhesion, proliferation and differentiation on materials designed for body implants.</a> Biotechnol. Adv. 29 (6), 739-767 (2011).</li><li>Bacakova, L., Svorcik, V., Kimura, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cell colonization control by physical and chemical modification of materials. In: Cell growth process: new research. , 5-56 (2008).</li><li>Sanjana, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+fast+flexible+ink-jet+printing+method+for+patterning+dissociated+neurons+in+culture.">A fast flexible ink-jet printing method for patterning dissociated neurons in culture.</a> J. Neurosci. Methods. 136, 151-163 (2004).</li><li>Brittain, S., Paul, K., Zhao, X. -. M., Whitesides, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soft+lithography+and+micro-+fabrication.">Soft lithography and micro- fabrication.</a> Phys. World. 11, 31-36 (1998).</li><li>Maher, M., Pine, J., Wright, J., Tai, Y. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+neurochip:+a+new+multielectrode+device+for+stimulating+and+recording+from+cultured+neurons.">The neurochip: a new multielectrode device for stimulating and recording from cultured neurons.</a> J. Neurosci. Methods. 87 (1), 45-56 (1999).</li><li>Martinoia, S., Bove, M., Tedesco, M., Margesin, B., Grattarola, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+micro-+fluidic+system+for+patterning+populations+of+neurons+on+silicon+micro-+machined+substrates.">A simple micro- fluidic system for patterning populations of neurons on silicon micro- machined substrates.</a> J. Neurosci. Methods. 87 (1), 35-44 (1999).</li><li>Zeck, G., Fromherz, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+neuroelectronic+interfacing+with+synaptically+connected+snail+neurons+immobilized+on+a+semiconductor+chip.">Noninvasive neuroelectronic interfacing with synaptically connected snail neurons immobilized on a semiconductor chip.</a> Proc. Natl. Acad. Sci. U.S.A. 98 (18), 10457-10462 (2001).</li><li>Michel, R., 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=Selective+molecular+assembly+patterning:+a+new+approach+to+micro-+and+nanochemical+patterning+of+surfaces+for+biological+applications.">Selective molecular assembly patterning: a new approach to micro- and nanochemical patterning of surfaces for biological applications.</a> Langmuir. 18 (8), 3281-3287 (2002).</li><li>Rajalingam, B., Selvarasah, S., Dokmeci, M., Khademhosseini, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+static+and+dynamic+patterned+cocultures+using+microfabricated+parylene-C+stencils.">Generation of static and dynamic patterned cocultures using microfabricated parylene-C stencils.</a> Lab Chip. 7, 1272-1279 (2007).</li><li>Wright, D., Rajalingam, B., Karp, J., Selvarah, S., Ling, Y., Yeh, J., Langer, R., Dokmeci, M., Khademhosseini, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reusable,+reversibly+sealable+parylene+membranes+for+cell+and+protein+patterning.">Reusable, reversibly sealable parylene membranes for cell and protein patterning.</a> J. Biomed. Mater. Res. A. 85 (2), 530-538 (2008).</li><li>Delivopoulos, E., Murray, A. F., MacLeod, N. K., Curtis, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guided+growth+of+neurons+and+glia+using+microfabricated+patterns+of+parylene-C+on+a+SiO2+background.">Guided growth of neurons and glia using microfabricated patterns of parylene-C on a SiO2 background.</a> Biomaterials. 30, 2048-2058 (2009).</li><li>Delivopoulos, E., Murray, A. F., Curtis, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+parylene-C+photooxidation+on+serum-assisted+glial+and+neuronal+patterning.">Effects of parylene-C photooxidation on serum-assisted glial and neuronal patterning.</a> J. Biomed. Mater. Res. A. 94, 47-58 (2010).</li><li>Delivopoulos, E., Murray, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+adhesion+and+growth+of+long+term+glial+and+neuronal+cultures+on+parylene-C.">Controlled adhesion and growth of long term glial and neuronal cultures on parylene-C.</a> PLoS One. 6 (9), (2011).</li><li>Hughes, M. A., Bunting, A., Cameron, K., Murray, A. F., Shipston, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulating+patterned+adhesion+and+repulsion+of+HEK+293+cells+on+micro-engineered+parylene-C/SiO2+substrates.">Modulating patterned adhesion and repulsion of HEK 293 cells on micro-engineered parylene-C/SiO2 substrates.</a> J. Biomed. Mat. Res. Mater. A. 101 (2), 349-357 (2013).</li><li>Unsworth, C. P., Graham, E. S., Delivopoulos, E., Dragunow, M., Murray, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=First+human+hNT+neurons+patterned+on+parylene-C/silicon+dioxide+substrates:+Combining+an+accessible+cell+line+and+robust+patterning+technology+for+the+study+of+the+pathological+adult+human+brain.">First human hNT neurons patterned on parylene-C/silicon dioxide substrates: Combining an accessible cell line and robust patterning technology for the study of the pathological adult human brain.</a> J. Neurosci. Methods. 194, 154-157 (2010).</li></ol>

The authors declare no competing financial interests.

Immersion of chips in piranha acid serves not only to remove any residual organic material but also etches the substrate surfaces. This is key to enabling effective activation with fetal bovine serum. Failure to do so prevents cell-patterning and profoundly alters cell behavior on-chip. There is no requirement to sterilize chips after cleaning with piranha acid. Indeed sterilization by UV exposure has been shown to undermine cell patterning in a dose-dependent fashion13. Care must be taken to wash off all residual photoresist after the photolithographic process. Persisting photoresist can act as an unwanted cyto-adhesive layer that overrides patterning dictated by parylene-C/SiO2 geometry. Acetone is effective when using the photolithographic process described above and with the reagents specified. However, other types of photoresist may require a different solvent.
To assess the impact and success of the different fabrication steps, the contact angle of the two contrasting substrates can be measured. Figure 2 illustrates the alterations that occur during the chip activation process. It is likely, however, that specific adhesive and repulsive protein components in serum ultimately enable the parylene-patterned chip to exert its respective cyto-adhesive or cyto-repulsive characteristics.
All representative results used chips with a parylene thickness of 100 nm, though we have successfully patterned using both thicker and thinner parylene layers. Importantly, this photolithographic etching technique allows much greater three-dimensional control of parylene configuration than that illustrated here. For example, using a combination of photomasks, it is possible to create parylene regions of mixed thickness. This opens the way to creating cell cultures with defined three-dimensional topography, going beyond simply dictating regions of cell adhesion/repulsion, potentially offering a means of integrating microfluidic channels into the construct.
As shown, however, this patterning platform is not universally effective across cell types. Different cell lines, with their varied cell adhesion molecule profiles, unsurprisingly behave differently when cultured on this platform. We have not yet identified the key components in serum, nor the complimentary cell-membrane receptors, which underpin this cell-patterning platform. Doing so in future promises to broaden its utility and specificity. For example, a &#8216;non-patterning&#8217; cell line could be genetically modified to express the requisite adhesion molecule and so promote patterning.

Understanding mechanisms that dictate cell adhesion and patterning on synthetic materials is important for applications such as tissue engineering, drug discovery, and the fabrication of biosensors1-3. Many techniques are available and evolving, each taking advantage of the myriad biological, chemical, and physical factors that influence cell adhesion.
Here, we describe a cell-patterning technique that utilizes processes initially developed for microelectronic fabrication purposes. As such, the platform is well-placed to enable downstream integration of microelectronic technologies, such as MEAs, into the patterning platform.
The interface between a cell membrane and an adjacent material is bi-directional and complex. In vivo, extracellular matrix proteins provide structure and strength and impact upon cell behavior via interactions with cell adhesion receptors. Similarly, cells in vitro interact with synthetic substrates via absorbed layers of proteins4 whilst physico-chemical influences also modulate adhesion. For example, a polymer surface can be rendered more &#8220;wettable&#8221; (hydrophilic) by ions or ultraviolet light irradiation, or etching by treatment with acid or hydroxide5. Established methods for cell patterning take advantage of these and other cell adhesion mediators. Examples include inkjet printing6, microcontact stamping7, physical immobilization8, microfluidics9, real-time manipulation10, and selective molecular assembly patterning (SMAP)11. Each has specific benefits and limitations. A key driver in our work, however, is to integrate cell patterning with microelectromechanical systems (MEMS).
MEMS refer to extremely small mechanical devices driven by electricity. This overlaps with the nanoscale equivalent, nanoelectromechanical systems. This concept became practical only when semiconductor strategies enabled fabrication to take place at the microscale. Microfabrication techniques developed originally for semiconductor electronics have inadvertently been found useful for other uses such as cellular electrophysiology, for example. A key downstream aim is to combine such microelectronic technologies with a high fidelity cell patterning process (forming a bioMEMS device). Several existing and otherwise reliable and practical cell-patterning techniques are incompatible with this idea. For example, accurate alignment of any embedded microelectronics or biosensors is fundamental to their efficacy but is extremely difficult to achieve using a technique such as microcontact stamping.
To circumvent this problem, we are working on a SiO2-based patterning platform that uses photolithographically printed parylene-C. Photolithography involves transfer of geometric features from a mask to a substrate via UV illumination. A mask is designed using an appropriate computer-aided design program. Upon a glass plate, a thin layer of nontransparent chromium represents the desired geometric pattern (a feature resolution of 1-2 mm is possible). The substrate to be patterned is coated with a thin layer of photoresist (a UV-sensitive polymer). The coated polymer is then aligned and brought into close contact with the mask. A UV source is applied such that unprotected areas are irradiated and therefore become soluble and removable in the next development step, leaving a parylene-C representation of the mask pattern behind. This process originated during development of semiconductor devices. As such, silicon wafers are frequently used as a substrate. Photolithographic deposition of parylene-C on SiO2 is hence a straightforward and reliable process that routinely takes place in microelectronic cleanroom facilities.
Whilst parylene has several desirable bioengineering characteristics (chemically inert, non bio-degradable), a factor restricting its direct use in cell patterning is its innately poor cell adhesiveness, attributed in part to its extreme hydrophobicity. Nevertheless, parylene-C has previously been used indirectly for cell patterning, for example as a peel-away cellular template12,13. This approach is limited by poor resolution and requires multiple steps. The process described here instead utilizes an acid etch step, followed by serum incubation, to ensure that parylene-C regions become cell-adhesive, through a combination of reduction in hydrophobicity and serum protein binding.
The end result is a construct composed of two different substrates which, after biological activation, manifest respective cyto-adhesive or cyto-repulsive characteristics and so represents an effective cell pattering platform. Importantly, there is no need to introduce biological agents into the cleanroom facility as the patterned substrates can be stored indefinitely prior to use (whereupon they are activated using fetal bovine serum or another activation solution).
This parylene-C/SiO2 patterning platform is therefore a good candidate for a coalition with MEMS components, as the fabrication processes so closely mirror those used for microelectronic fabrication.

<table border="0" cellpadding="0" cellspacing="0" width="1784">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Layout editor software package&#160;</td>
			<td style="width:572px;">e.g. CleWin 5.0 from WieWeb, http://www.wieweb.com/ns6/index.html</td>
			<td style="width:215px;"></td>
			<td style="width:615px;">Capable of reading/writing CIF or GDS-II files. Used to create parylene design for photo mask manufacture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Bespoke photo mask</td>
			<td colspan="2">e.g. Compugraphics International Ltd, Glenrothes, Scotland, www.compugraphics-photomasks.com</td>
			<td>Either fabricate in-house of facilities exist or commision</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">3&#34; Silicon wafers</td>
			<td>e.g. Siltronix, Archamps, France, http://www.siltronix.com</td>
			<td style="width:215px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Atmospheric horizontal furnace</td>
			<td style="width:572px;">e.g. Sandvik, http://www.mrlind.com</td>
			<td style="width:215px;"></td>
			<td>For oxidising silicon wafer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Small spot spectroscopic reflectometer</td>
			<td>e.g. Nanometrics NanoSpec 3000 reflectometer, www.nanometrics.com/</td>
			<td style="width:215px;"></td>
			<td>To measure depth of silicon dioxide layer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Silane adhesion promoter</td>
			<td>e.g. Merck Silane A174 adhesion promoter. Merck Chemicals, www.merck-chemicals.de/</td>
			<td style="width:215px;">1076730050</td>
			<td>Pre-applied to wafer to encourage parylene deposition</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Parylene-C&#160;</td>
			<td style="width:572px;">e.g. Ultra Electronics, www.ultra-cems.com</td>
			<td style="width:215px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SCS Labcoter 2 deposition Unit, Model PDS2010</td>
			<td style="width:572px;">SCS equipment, Surrye, UK, www.scscoatings.com/</td>
			<td style="width:215px;">Model PDS2010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hexamethyldisilazane (HMDS) adhesion promoter</td>
			<td style="width:572px;">e.g. SpiChem, www.2spi.com</td>
			<td style="width:215px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:383px;">Automated track system for dispensing photoresist on wafers.</td>
			<td>e.g SVG (silicon Valley Group) 3 inch photo-resist track,&#160;</td>
			<td style="width:215px;"></td>
			<td>Automated track system for dispensing photoresist on wafers. A prime oven bakes the wafer and dispenses the adhesion promoter, HMDS. A combination spinner dispenses photoresist.&#160; Pre-bake oven cures the resist.&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Photo-resist: Rohm &#38; Haas</td>
			<td style="width:572px;">Rohm &#38; Haas, www.rohmhaas.com/</td>
			<td style="width:215px;">&#160;SPR350-1.2 positive photo-resist</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Phot-mask aligner</td>
			<td>e.g. Suss Microtech MA/BA8 mask aligner, www.suss.com</td>
			<td style="width:215px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microchem MF-26A developer</td>
			<td>Microchem MF-26A developer, www.microchem.com</td>
			<td style="width:215px;"></td>
			<td>Removes exposed reogions of photoresist</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Plasma etch system</td>
			<td>e.g. JLS RIE80 etch system, JLS Designs, www.jlsdesigns.co.uk</td>
			<td style="width:215px;"></td>
			<td>Removes exposed regions of parylene</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Wafer dicing saw</td>
			<td>e.g. DISCO DAD 680 Dicing Saw, DISCO Corporation, Japan, www.disco.co.jp</td>
			<td style="width:215px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Acetone&#160;</td>
			<td style="width:572px;">e.g. Fisher Scientific, www.fishersci.com/</td>
			<td style="width:215px;">A929-4&#160;</td>
			<td>To wash off residual photoresist</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">30% Hydrogen Peroxide&#160;</td>
			<td style="width:572px;">e.g. Sigma-Aldrich, www.sigmaaldrich.com</td>
			<td style="width:215px;">H1009</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">98% Sulphuric Acid</td>
			<td style="width:572px;">e.g. Sigma-Aldrich, www.sigmaaldrich.com</td>
			<td style="width:215px;">435589</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal Bovine Serum&#160;</td>
			<td style="width:572px;">Gibco-Invitrogen, www.invitrogen.com</td>
			<td style="width:215px;">10437</td>
			<td>Standard chip activation.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hank&#39;s Balanced Salt Solution</td>
			<td style="width:572px;">Gibco-Invitrogen, www.invitrogen.com</td>
			<td style="width:215px;">14170</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

The photolithographic process of patterning SiO2 with parylene-C is illustrated in Figure 1. Once prepared, activation of chips in fetal bovine serum enables a wide range of cell types to be patterned in culture. Our group has successfully patterned primary murine hippocampal cells14-16, the HEK 293 cell line17, the human neuron-like teratocarcinoma (hNT) cell line18, primary murine cerebellar granule cells, and primary human glioma-derived stem-like cells.
Figure 3 illustrates robust patterning of HEK 293 cells on a parylene pattern consisting of circular nodes with &#8216;cross-hair&#8217; extensions. Chip activation in this example was with fetal bovine serum. By contrast, Figure 4 illustrates the potential to augment the patterning platform by using alternative activation solutions. Using a solution of bovine serum albumin (3 mg/ml) and fibronectin (1 &#956;g/ml) in HBSS, the previous patterning precept has been inverted.
Figure 5 illustrates a different cell type (a primary human-derived stem-like cell line derived from a high grade glioma). Here, the geometry of the underlying pattern impacts cell behavior, with the pattern shown in Figure 4A&#160;promoting cell process growth along thin parylene-C tracks as shown in Figures 4B&#160;and 4C.
Some cell types do not pattern when using the established fetal bovine serum activation protocol. Figure 6 illustrates 3T3 L1 cells growing to confluence with no discernable cyto-repulsive or cyto-adhesive difference between parylene-C and SiO2 regions.
<img alt="Figure 1" fo:content-width="5in" fo:src="/files/ftp_upload/50929/50929fig1highres.jpg" src="/files/ftp_upload/50929/50929fig1.jpg" /> 
Figure 1.&#160;Flow diagram illustrating the process for fabrication of parylene-C patterns on SiO2.
<img alt="Figure 2" fo:content-width="5in" fo:src="/files/ftp_upload/50929/50929fig2highres.jpg" src="/files/ftp_upload/50929/50929fig2.jpg" /> 
Figure 2.&#160;Flow diagram illustrating the changes in contact angle for patterned parylene-C and SiO2 domains during chip activation steps.
<img alt="Figure 3" fo:content-width="5in" fo:src="/files/ftp_upload/50929/50929fig3highres.jpg" src="/files/ftp_upload/50929/50929fig3.jpg" /> 
Figure 3.&#160;Live cell imaging of HEK 293 cells cultured on parylene-C/SiO2 after three days in vitro. Chips incubated for 3&#160;hr in fetal bovine serum after which cells were plated in suspension at a concentration of 5 x 104 cells/ml. Parylene-C promotes cell adhesion whilst bare SiO2 repels cells. <a href="https://www.jove.com/files/ftp_upload/50929/50929fig3highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" fo:content-width="5in" fo:src="/files/ftp_upload/50929/50929fig4highres.jpg" src="/files/ftp_upload/50929/50929fig4.jpg" /> 
Figure 4.&#160;Live cell imaging of HEK 293 cells cultured on parylene-C/SiO2 after three days in vitro. Chips activated for 3&#160;hr&#160;in different rationalized activation solutions: A: fetal bovine serum, B: vitronectin (1 &#956;g/ml) in HBSS, C: Bovine serum albumin (3 mg/ml) + vitronectin (1 &#956;g/ml) in HBSS, D: Bovine serum albumin (3 mg/ml) + fibronectin (1 &#956;g/ml) in HBSS. Cells plated were in suspension at a density of 5 x 104 cells/ml. Note how different treatment of the chip has resulted in reversal of the previous patterning dogma, with SiO2 now adhesive and parylene-C repulsive. Parylene-C node diameter 250 &#956;m, adapted from Hughes et al.17 <a href="https://www.jove.com/files/ftp_upload/50929/50929fig4highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" fo:content-width="5in" fo:src="/files/ftp_upload/50929/50929fig5highres.jpg" src="/files/ftp_upload/50929/50929fig5.jpg" /> 
Figure 5.&#160;Immunofluorescence images of primary human glioma-derived stem-like cells grown on various patterns of parylene-C on SiO2. A: schematic illustrating the reticular parylene design shown in B and C. B: Fluorescence micrograph of fixed cells (after 4&#160;days in vitro) stained for glial fibrillary acidic protein (GFAP). C: Light micrograph of live cells on same chip. D: Fluorescence image illustrating GFAP-stained cells on a different parylene design. E: Reflectance image of the node and spoke parylene design imaged in D. <a href="https://www.jove.com/files/ftp_upload/50929/50929fig5highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" fo:content-width="5in" fo:src="/files/ftp_upload/50929/50929fig6highres.jpg" src="/files/ftp_upload/50929/50929fig6.jpg" /> 
Figure 6.&#160;Live cell imaging of 3T3 L1 cells cultured on parylene-C/SiO2 after four days in vitro. Chips activated for 3&#160;hr&#160;in fetal bovine serum after which cells were plated in suspension (3 x 104 cells/ml). In this instance, the platform does not enable patterning, with cells becoming equally confluent on parylene-C and SiO2 regions. <a href="https://www.jove.com/files/ftp_upload/50929/50929fig6highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates at JoVE.com

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 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 ...

cell-patterning-on-photolithographically-defined-parylene-c-sio2

Research

JoVE Journal

Bioengineering

13.2K ビュー.  The University of Edinburgh. このプロトコルは、SiO 2のセルパターニングのための微細加工と互換性のある方法を記載している。事前に定義されたパリレン-Cの設計は、フォトリソグラフィによってSiO 2のウェハ上に印刷されています。血清（または他の活性化溶液）とのインキュベーション後の細胞を特異的に接着し（との適合性に応じて成長する）はSiO 2領...

ビデオ: 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...

photopatterning proteins and cells in aqueous environment using tio2 photocatalysis

Photopatterning Proteins and Cells in Aqueous Environment Using TiO2 Photocatalysis

We describe a protocol for modifying cell affinity of a scaffold surface in aqueous environment. The method takes advantage of titanium dioxide photocatalysis to decompose organic film in the photo-irradiated region. We show that it can be used to create microdomains of scaffolding proteins, both ex situ and in...

Photopatterning Proteins and Cells in Aqueous Environment Using TiO2 Photocatalysis

patterning the geometry of human embryonic stem cell colonies on compliant substrates to control tissue-level mechanics

Patterning the Geometry of Human Embryonic Stem Cell Colonies on Compliant Substrates to Control Tissue-Level Mechanics

Extracellular matrix ligands can be patterned onto polyacrylamide hydrogels to enable the culture of human embryonic stem cells in confined colonies on compliant substrates. This method can be combined with traction force microscopy and biochemical assays to examine the interplay between tissue geometry, cell-generated forces, and fate...

creating defined gaseous environments to study the effects of hypoxia on c. elegans

Creating Defined Gaseous Environments to Study the Effects of Hypoxia on C. elegans

This paper details how to use continuous-flow hypoxia chambers to generate atmospheres with defined concentrations of O2 to understand biological responses to decreased O2. This system is easy to setup and maintain, and flexible enough to suit a wide range of O2 concentrations and model...

Creating Defined Gaseous Environments to Study the Effects of Hypoxia on C. elegans

the effect of interfacial chemical bonding in tio2-sio2 composites on their photocatalytic nox abatement performance

The Effect of Interfacial Chemical Bonding in TiO2-SiO2 Composites on Their Photocatalytic NOx Abatement Performance

The focus of the present work is to establish means to generate and quantify levels of Ti-O-Si linkages and to correlate these with the photocatalytic properties of the supported...

The Effect of Interfacial Chemical Bonding in TiO2-SiO2 Composites on Their Photocatalytic NOx Abatement Performance

plasma lithography surface patterning for creation of cell networks

Plasma Lithography Surface Patterning for Creation of Cell Networks

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...

cell patterning using magnetic-archimedes strategy

Author Spotlight: Magnetic-Based Cell Patterning Method for High-Throughput Biomedical Applications 

This protocol describes an ink-free, label-free, substrate-independent, high-throughput cell patterning method based on the Magnetic Archimedes...

Cell Patterning Using Magnetic-Archimedes Strategy

patterning cells on optically transparent indium tin oxide electrodes

Patterning Cells on Optically Transparent Indium Tin Oxide Electrodes

Non-fouling PEG silane monolayer was desorbed from individually addressable ITO electrodes on glass by application of a reductive potential.   Electrochemical stripping of PEG-silane layer from ITO microelectrodes allowed for cell adhesion to take place in a spatially defined fashion, with cellular patterns corresponding closely to electrode...

rapid subtractive patterning of live cell layers with a microfluidic probe

Rapid Subtractive Patterning of Live Cell Layers with a Microfluidic Probe

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...

cell co-culture patterning using aqueous two-phase systems

Cell Co-culture Patterning Using Aqueous Two-phase Systems

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...

antifouling self-assembled monolayers on microelectrodes for patterning biomolecules

Antifouling Self-assembled Monolayers on Microelectrodes for Patterning Biomolecules

We present a procedure for forming a poly(ethylene glycol) self-assembled monolayer (PEG-SAM) on a silicon substrate with gold microelectrodes. The PEG-SAM is formed in a single step and prevents biofouling on silicon and gold surfaces. Electrophoresis is then used for patterning biomolecules down to the...

patterning bioactive proteins or peptides on hydrogel using photochemistry for biological applications

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

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...

growth of gold dendritic nanoforests on titanium nitride-coated silicon substrates

Growth of Gold Dendritic Nanoforests on Titanium Nitride-coated Silicon Substrates

This study presents a feasible procedure for synthesizing gold dendritic nanoforests on titanium nitride/silicon substrates. The thickness of gold dendritic nanoforests increases linearly within 15 min of a synthesis...

proof-of-concept for gas-entrapping membranes derived from water-loving sio2/si/sio2 wafers for green desalination

Proof-of-Concept for Gas-Entrapping Membranes Derived from Water-Loving SiO2/Si/SiO2 Wafers for Green Desalination

Presented here is a stepwise protocol for realizing gas-entrapping membranes (GEMs) from SiO2/Si wafers using integrated circuit microfabrication technology. When silica-GEMs are immersed in water, the intrusion of water is prevented, despite the water-loving composition of...

Proof-of-Concept for Gas-Entrapping Membranes Derived from Water-Loving SiO2/Si/SiO2 Wafers for Green Desalination

creating two-dimensional patterned substrates for protein and cell confinement

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

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...

A Novel Ink-Free, Label-Free Magnetic-Archimedes Cell Patterning Method

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reactive vapor deposition of conjugated polymer films on arbitrary substrates

Reactive Vapor Deposition of Conjugated Polymer Films on Arbitrary Substrates

This paper presents a protocol for reactive vapor deposition of poly(3,4-ethylenedioxythiophene), poly(3,4-propylenedioxythiophene), and poly(thieno[3,2-b]thiophene) films on glass slides and rough substrates, such as textiles and paper.

micropunching lithography for generating micro- and submicron-patterns on polymer substrates

Micropunching Lithography for Generating Micro- and Submicron-patterns on Polymer Substrates

A micropunching lithography approach is developed to generate micro- and submicron-patterns on top, sidewall and bottom surfaces of polymer substrates. It overcomes the obstacles of patterning conducting polymers and generating sidewall patterns. This method allows rapid fabrication of multiple features and is free of aggressive...

ultrahigh density array of vertically aligned small-molecular organic nanowires on arbitrary substrates

Ultrahigh Density Array of Vertically Aligned Small-molecular Organic Nanowires on Arbitrary Substrates

We report a simple method for fabricating an ultrahigh density array of vertically ordered small-molecular organic nanowires. This method allows for synthesis of complex heterostructured hybrid nanowire geometries, which can be inexpensively grown on arbitrary substrates. These structures have potential applications in organic electronics, optoelectronics, chemical sensing, photovoltaics and...

light-mediated formation and patterning of hydrogels for cell culture applications

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

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.

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 ...

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 ...

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. ...

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, ...

Using Adhesive Patterning to Construct 3D Paper Microfluidic Devices

We demonstrate the use of patterned aerosol adhesives to construct both planar and nonplanar 3D paper microfluidic devices. By spraying an aerosol adhesive through a metal stencil, the overall amount of adhesive used in assembling paper microfluidic devices can be significantly reduced. We show on a simple 4-layer planar paper microfluidic device that the optimal adhesive application technique and device construction style depends heavily on desired performance characteristics. By moderately increasing the overall area of a device, it is possible to dramatically decrease the wicking time and increase device success rates while also reducing the amount of adhesive required to keep the device together. Such adhesive application also causes the adhesive to form semi-permanent bonds instead of permanent bonds between paper layers, enabling single-use devices to be non-destructively disassembled after use. Nonplanar 3D origami devices also benefit from the semi-permanent bonds during folding, as it reduces the likelihood that unrelated faces may accidently stick together. Like planar devices, nonplanar structures see reduced wicking times with patterned adhesive application vs uniformly applied adhesive.

We demonstrate the use of patterned aerosol adhesives to construct 3D paper microfluidic devices. This method of adhesive application forms semi-permanent bonds between layers, enabling single-use devices to be non-destructively disassembled after use and to ease folding complex nonplanar structures.

We demonstrate the use of patterned aerosol adhesives to construct both planar and nonplanar 3D paper microfluidic devices. By spraying an aerosol ...

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 ...

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 ...

A Versatile Method of Patterning Proteins and Cells

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

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

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

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 ...

Evaluation of Keratinocyte Proliferation on Two- and Three-dimensional Type I Collagen Substrates

Type I collagen, useful as a substrate for cell culture, exists in two forms: the two-dimensional, non-fibrous form and three-dimensional, fibril form. Both forms can be prepared with the same type I collagen. In general, the non-fibrous form promotes cell adhesion and proliferation. The fibril form (gels) provides more physiological conditions in many types of cells; therefore, gel culture is useful for examining physiological behaviors of cells, such as drug efficacy.
Researchers can select the appropriate form according to the purpose of its use. For example, in the case of keratinocytes, on-gel culture has been used as a wound healing model. FEPE1L-8, a keratinocyte cell line cultured on the non-fibrous form of type I collagen, promote cell adhesion. Notably, keratinocyte proliferation is slower on the fibril form than the non-fibrous form. Protocols for the preparation of type I collagen for cell culture are simple and have wide applications depending on the experimental needs.

Here, we present a protocol for the preparation of two different forms of culture substrates utilizing type I collagen. Depending on how collagen is handled, collagen molecules either maintain two-dimensional, non-fibrous form or reassemble into three-dimensional, fibril form. Cell proliferation on type I collagen is drastically affected by fibril formation.

Type I collagen, useful as a substrate for cell culture, exists in two forms: the two-dimensional, non-fibrous form and three-dimensional, fibril form. ...

Cell Patterning on Photolithographically Defined Parylene-C: SiO2 Substrates

Cell Patterning on Photolithographically Defined Parylene-C: SiO₂ Substrates