This work was supported by Connaught Fund New Investigator Award to S.P., Canada Foundation for Innovation, NSERC Discovery Grant Program (grants RGPIN-2015-05114 and RGPIN-2020-05881), University of Manchester and University of Toronto Joint Research Fund, and University of Toronto XSeed Program. C.T. was supported by NSERC USRA fellowship.

<ol>
	<li>Harris, T. J. C., Sawyer, J. K., Peifer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+the+Cytoskeleton+Helps+Build+the+Embryonic+Body+Plan+Models+of+Morphogenesis+from+Drosophila.">How the Cytoskeleton Helps Build the Embryonic Body Plan Models of Morphogenesis from Drosophila.</a> Current Topics in Developmental Biology. 89, 55-85 (2009).</li><li>Keren, K., 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=Mechanism+of+shape+determination+in+motile+cells.">Mechanism of shape determination in motile cells.</a> Nature. 453 (7194), 475-480 (2008).</li><li>Castor, L. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+Division+by+Cell+Contact+and+Serum+Concentration+in+Cultures+of+3T3+Cells.">Control of Division by Cell Contact and Serum Concentration in Cultures of 3T3 Cells.</a> Experimental Cell Research. 68 (1), 17-24 (1971).</li><li>Jain, N., Iyer, K. V., Kumar, A., Shivashankar, G. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+geometric+constraints+induce+modular+gene-expression+patterns+via+redistribution+of+HDAC3+regulated+by+actomyosin+contractility.">Cell geometric constraints induce modular gene-expression patterns via redistribution of HDAC3 regulated by actomyosin contractility.</a> Proceedings of the National Academy of Sciences. 110 (28), 11349-11354 (2013).</li><li>Paluch, E., Heisenberg, C. -. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biology+and+Physics+of+Cell+Shape+Changes+in+Development.">Biology and Physics of Cell Shape Changes in Development.</a> Current Biology. 19 (17), 790-799 (2009).</li><li>Pollard, T. D., Borisy, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+Motility+Driven+by+Assembly+and+Disassembly+of+Actin+Filaments.">Cellular Motility Driven by Assembly and Disassembly of Actin Filaments.</a> Cell. 112 (4), 453-465 (2003).</li><li>Ridley, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+Migration:+Integrating+Signals+from+Front+to+Back.">Cell Migration: Integrating Signals from Front to Back.</a> Science. 302 (5651), 1704-1709 (2003).</li><li>Cramer, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+cell+rear+retraction+in+migrating+cells.">Mechanism of cell rear retraction in migrating cells.</a> Current Opinion in Cell Biology. 25 (5), 591-599 (2013).</li><li>Lee, J., Ishihara, A., Oxford, G., Johnson, B., Jacobson, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+cell+movement+is+mediated+by+stretch-activated+calcium+channels.">Regulation of cell movement is mediated by stretch-activated calcium channels.</a> Nature. 400 (6742), 382-386 (1999).</li><li>Leptin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gastrulation+Movements:+the+Logic+and+the+Nuts+and+Bolts.">Gastrulation Movements: the Logic and the Nuts and Bolts.</a> Developmental Cell. 8 (3), 305-320 (2005).</li><li>Chen, C. S., Mrksich, M., Huang, S., Whitesides, G. M., Ingber, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Geometric+Control+of+Cell+Life+and+Death.">Geometric Control of Cell Life and Death.</a> Science. 276 (5317), 1425-1428 (1997).</li><li>Whitesides, G. M., Ostuni, E., Takayama, S., Jiang, X., Ingber, D. E. <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+in+Biology+and+Biochemistry.">Soft Lithography in Biology and Biochemistry.</a> Annual Review of Biomedical Engineering. 3, 335-373 (2001).</li><li>Cirelli, R. A., Watson, G. P., Nalamasu, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Techniques+and+Processing:+Surface,+Micro-,+and+Nanoscale+Processing.">Techniques and Processing: Surface, Micro-, and Nanoscale Processing.</a> Encyclopedia of Materials: Science and Technology. , 6441-6448 (2001).</li><li>Stricker, J., Aratyn-Schaus, Y., Oakes, P. W., Gardel, 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=Spatiotemporal+Constraints+on+the+Force-Dependent+Growth+of+Focal+Adhesions.">Spatiotemporal Constraints on the Force-Dependent Growth of Focal Adhesions.</a> Biophysical Journal. 100 (12), 2883-2893 (2011).</li><li>Azioune, A., Storch, M., Bornens, M., Th&#233;ry, M., Piel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+and+rapid+process+for+single+cell+micro-patterning.">Simple and rapid process for single cell micro-patterning.</a> Lab on a Chip. 9 (11), 1640-1642 (2009).</li><li>Chang, F., Atilgan, E., Burgess, D., Minc, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manipulating+Cell+Shape+by+Placing+Cells+into+Microfabricated+Chambers.">Manipulating Cell Shape by Placing Cells into Microfabricated Chambers.</a> Methods in Molecular Biology. 1136, 281-290 (2014).</li><li>Takeuchi, S., DiLuzio, W. R., Weibel, D. 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=Controlling+the+Shape+of+Filamentous+Cells+of+Escherichia+Coli.">Controlling the Shape of Filamentous Cells of Escherichia Coli.</a> Nano Letters. 5 (9), 1819-1823 (2005).</li><li>Minc, N., Boudaoud, A., Chang, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+Forces+of+Fission+Yeast+Growth.">Mechanical Forces of Fission Yeast Growth.</a> Current Biology. 19 (13), 1096-1101 (2009).</li><li>Durand-Smet, P., Spelman, T. A., Meyerowitz, E. M., J&#246;nsson, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytoskeletal+organization+in+isolated+plant+cells+under+geometry+control.">Cytoskeletal organization in isolated plant cells under geometry control.</a> Proceedings of the National Academy of Sciences. 117 (29), 17399-17408 (2020).</li><li>Haske, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=65+nm+feature+sizes+using+visible+wavelength+3-D+multiphoton+lithography.pdf.">65 nm feature sizes using visible wavelength 3-D multiphoton lithography.pdf.</a> Optics Express. 15 (6), 3426-3436 (2007).</li><li>Song, J., Michas, C., Chen, C. S., White, A. E., Grinstaff, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+Simple+to+Architecturally+Complex+Hydrogel+Scaffolds+for+Cell+and+Tissue+Engineering+Applications:+Opportunities+Presented+by+Two-Photon+Polymerization.">From Simple to Architecturally Complex Hydrogel Scaffolds for Cell and Tissue Engineering Applications: Opportunities Presented by Two-Photon Polymerization.</a> Advanced Healthcare Materials. 9 (1), 1901217 (2020).</li><li>Torgersen, J., Qin, X., Li, Z., Ovsianikov, A., Liska, R., Stampfl, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogels+for+Two-Photon+Polymerization:+A+Toolbox+for+Mimicking+the+Extracellular+Matrix.">Hydrogels for Two-Photon Polymerization: A Toolbox for Mimicking the Extracellular Matrix.</a> Advanced Functional Materials. 23 (36), 4542-4554 (2013).</li><li>Doyle, A. D., Wang, F. W., Matsumoto, K., Yamada, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One-dimensional+topography+underlies+three-dimensional+fibrillar+cell+migration.">One-dimensional topography underlies three-dimensional fibrillar cell migration.</a> The Journal of Cell Biology. 184 (4), 481-490 (2009).</li><li>Doyle, A. D. <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+Micropatterned+Substrates+Using+Micro+Photopatterning.">Generation of Micropatterned Substrates Using Micro Photopatterning.</a> Current Protocols in Cell Biology. 45 (1), 1-35 (2009).</li><li>Waterman-Storer, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtubule/Organelle+Motility+Assays.">Microtubule/Organelle Motility Assays.</a> Current Protocols in Cell Biology. 00 (1), 1-21 (1998).</li><li>Inou&#233;, S., Spring, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Video Microscopy: The Fundamentals. , (1997).</li><li>Schneider, I. C., Hays, C. K., Waterman, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidermal+Growth+Factor-induced+Contraction+Regulates+Paxillin+Phosphorylation+to+Temporally+Separate+Traction+Generation+from+De-adhesion.">Epidermal Growth Factor-induced Contraction Regulates Paxillin Phosphorylation to Temporally Separate Traction Generation from De-adhesion.</a> Molecular Biology of the Cell. 20 (13), 3155-3167 (2009).</li><li>Helmchen, F., Denk, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deep+tissue+two-photon+microscopy.">Deep tissue two-photon microscopy.</a> Nature Methods. 2 (12), 932-940 (2005).</li><li>Xing, J., Cao, Y., Yu, Y., Li, H., Song, Z., Yu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Vitro+Micropatterned+Human+Pluripotent+Stem+Cell+Test+(&#181;P-hPST)+for+Morphometric-Based+Teratogen+Screening.">In Vitro Micropatterned Human Pluripotent Stem Cell Test (&#181;P-hPST) for Morphometric-Based Teratogen Screening.</a> Scientific Reports. 7 (1), 8491 (2017).</li><li>Ankam, S., Teo, B. K., Kukumberg, M., Yim, E. K. <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+screening+to+investigate+the+interaction+of+stem+cells+with+their+extracellular+microenvironment.">High throughput screening to investigate the interaction of stem cells with their extracellular microenvironment.</a> Organogenesis. 9 (3), 0 (2013).</li></ol>

The authors disclose no conflict of interests.

The results above demonstrate that the described IR laser assisted micropatterning (microphotopatterning) protocol provides reproducible adherent patterns of various shapes that enables the manipulation of cell shape and cytoskeletal architecture. Although numerous micropatterning methods have been developed both prior to and after the debut of microphotopatterning, this method possesses several advantages. First, it does not require specialized equipment and cleanrooms that are usually only found within Engineering depa...

Cell shape is a key determinant of fundamental biological processes such as tissue morphogenesis1, cell migration2, cell proliferation3, and gene expression4. Changes in cell shape are driven by an intricate balance between dynamic rearrangements of the cytoskeleton that deforms the plasma membrane and extrinsic factors such as external forces exerted on the cell and the geometry of cell-cell and cell-matrix adhesions5. Migrating mesenchymal cells, for instance, polymerize a dense actin network at the leading edge that pushes the plasma membrane forward and creates a wide lamellipodia6, while actomyosin contractility retracts the cell&#39;s narrow trailing edge to detach the cell from its current position7,8. Disrupting signaling events that give rise to such specialized cytoskeletal structures perturbs shape and polarity and slows cell migration9. In addition, epithelial sheet bending during gastrulation requires actomyosin-based apical constriction that causes cells and their neighbors to become wedge-shaped10. Although these studies highlight the importance of cell shape, the inherent heterogeneity in cell shape has encumbered efforts to identify mechanisms that connect morphology to function.
To this end, numerous approaches to manipulate cell shape have been developed over the past three decades. These approaches achieve their goal by either constraining the cell with a three-dimensional mold or controlling cellular adhesion geometry through patterned deposition of extracellular matrix (ECM) proteins onto an antifouling surface, a technique termed micropatterning11. Here we will review a number of techniques that have gained popularity throughout the years.
Originally pioneered as an approach for microelectronic applications, soft lithography-based microcontact printing has unequivocally become a cult favorite12. A master wafer is first fabricated by selectively exposing areas of a photoresist-coated silicon substrate to photoirradiation, leaving behind a patterned surface13. An elastomer, such as PDMS, is then poured onto the master wafer to generate a soft &#34;stamp&#34; that transfers ECM proteins to a desired substrate11,14. Once fabricated, a master wafer can be used to cast many PDMS stamps that give rise to highly reproducible micropatterns12. However, the patterns cannot be readily adjusted due to the lengthy photolithography process. This process also requires highly specialized&#160;equipment and cleanrooms that are not typically available in Biology departments.
More recently, direct printing using deep UV&#160;has been reported to circumvent limitations posed by traditional lithography-based approaches. Deep UV light is directed through a photomask to selective areas of a glass coverslip coated with poly-L-lysine-grafted-polyethylene glycol. Chemical groups exposed to deep UV are photoconverted without the use of photosensitive linkers to enable binding of ECM proteins15. The lack of photosensitive linkers enables patterned coverslips to remain stable at room temperature for over seven months15. This method avoids the use of cleanrooms and photolithography equipment and requires less specialized training. However, the requirement for photomasks still poses a substantial hurdle for experiments that require readily available changes in patterns.
In addition to methods that manipulate cell geometry through controlled deposition of ECM proteins on a 2D surface, other seek to control cell shape by confining cells in 3D microstructures. Many studies have adapted the soft lithography-based approach described above to generate 3D, rather than 2D, PDMS chambers to investigate shape-dependent biological processes in embryos, bacteria, yeast and plants16,17,18,19. Two-photon polymerization (2PP) has also taken the lead as a microfabrication technique that can create complex 3D hydrogel scaffolds with nanometer resolution20. 2PP relies on the principles of two-photon adsorption, where two photons delivered in femtosecond&#160;pulses are absorbed simultaneously by a molecule - photoinitiator in this case - that enables local polymerization of photopolymers21. This technique has been heavily employed to print 3D scaffolds that mimic the native ECM structures of human tissue and has been shown to induce low photochemical damage to cells22.
The debut of microphotopatterning 10 years ago gave researchers the opportunity to fabricate micropatterns while avoiding inaccessible and specialized equipment. Microphotopatterning creates patterns on the micron scale by thermally removing selective regions of poly-vinyl alcohol (PVA) coated on activated glass surfaces using an infrared&#160;laser23,24. ECM proteins that attach only the underlying glass surface and not PVA then serve as biochemical cues to enable controlled spreading dynamics and cell shape. This method also offers superior flexibility since patterns can be readily changed in real time. Here, we provide a step-by-step protocol of microphotopatterning by using a commercial multi-photon imaging system. The described protocol is designed for rapid and automated fabrication of large patterns. We demonstrated that these patterns efficiently control cell shape by constraining the geometry of cell-ECM adhesions. Finally, we demonstrate that the described patterning technique modulates the organization and dynamics of the actin cytoskeleton.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>(3-Aminopropyl)trimethoxysilane</td>
			<td>Aldrich</td>
			<td>281778</td>
			<td></td>
		</tr>
		<tr>
			<td>10 cm Cell Culture Dish</td>
			<td>VWR</td>
			<td>10062-880</td>
			<td>Polysterene, TC treated, vented</td>
		</tr>
		<tr>
			<td>25X Apo LWD Water Dipping Objective</td>
			<td>Nikon</td>
			<td>MRD77225</td>
			<td></td>
		</tr>
		<tr>
			<td>3.5 cm Cell Culture Dish</td>
			<td>VWR</td>
			<td>10861-586</td>
			<td>Polysterene, TC treated, vented</td>
		</tr>
		<tr>
			<td>4&#39;,6-Diamidino-2-Phenylindole (DAPI)</td>
			<td>Thermo</td>
			<td>62248</td>
			<td>1mg/mL dihydrochloride solution</td>
		</tr>
		<tr>
			<td>Bovine Serine Albumin</td>
			<td>BioShop</td>
			<td>ALB005</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate-Buffered Saline</td>
			<td>Wisent</td>
			<td>311-425-CL</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanolamine</td>
			<td>Sigma-Aldrich</td>
			<td>E9508</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibronectin</td>
			<td>Sigma-Aldrich</td>
			<td>FC010</td>
			<td>1mg/mL in pH 7.5 buffer</td>
		</tr>
		<tr>
			<td>Fibronectin Antibody</td>
			<td>BD</td>
			<td>610077</td>
			<td>Mouse</td>
		</tr>
		<tr>
			<td>Fiji</td>
			<td>ImageJ</td>
			<td></td>
			<td>Version 1.53c</td>
		</tr>
		<tr>
			<td>Fluorescent Phalloidin</td>
			<td>Invitrogen</td>
			<td>A12380</td>
			<td>568nm</td>
		</tr>
		<tr>
			<td>Glass Coverslip</td>
			<td>VWR</td>
			<td>16004-302</td>
			<td>22 &#215; 22 mm</td>
		</tr>
		<tr>
			<td>Glutaraldehyde</td>
			<td>Electron Microscopy Sciences</td>
			<td>16220</td>
			<td>25% aqueous solution</td>
		</tr>
		<tr>
			<td>Hydrochloric Acid</td>
			<td>Caledon</td>
			<td>6025-1-29</td>
			<td>37% aqueous solution</td>
		</tr>
		<tr>
			<td>IR Laser</td>
			<td>Coherent</td>
			<td>Chameleon Vision</td>
			<td></td>
		</tr>
		<tr>
			<td>Minimal Essential Medium &#945;</td>
			<td>Gibco</td>
			<td>12561-056</td>
			<td></td>
		</tr>
		<tr>
			<td>Mounting Medium</td>
			<td>Sigma</td>
			<td>F4680</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse Secondary Antibody</td>
			<td>Cell Signaling Technology</td>
			<td>4408S</td>
			<td>Goat, 488nm</td>
		</tr>
		<tr>
			<td>Multi-Photon Microscope</td>
			<td>Nikon</td>
			<td>A1R MP+</td>
			<td></td>
		</tr>
		<tr>
			<td>Myosin Light Chain Antibody</td>
			<td>Cell Signaling Technology</td>
			<td>3672S</td>
			<td>Rabbit</td>
		</tr>
		<tr>
			<td>NIS Elements</td>
			<td>Nikon</td>
			<td></td>
			<td>Version 5.21.03</td>
		</tr>
		<tr>
			<td>Nitric Acid</td>
			<td>Caledon</td>
			<td>7525-1-29</td>
			<td>70% aqueous solution</td>
		</tr>
		<tr>
			<td>Photoshop</td>
			<td>Adobe</td>
			<td></td>
			<td>Version 21.2.1</td>
		</tr>
		<tr>
			<td>Pluronic F-127</td>
			<td>Sigma</td>
			<td>P2443</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>Poly(vinyl alchohol)</td>
			<td>Aldrich</td>
			<td>341584</td>
			<td>MW 89000-98000, 98% hydrolyzed</td>
		</tr>
		<tr>
			<td>Rabbit Secondary Antibody</td>
			<td>Cell Signaling Technology</td>
			<td>4412S</td>
			<td>Goat, 488nm</td>
		</tr>
		<tr>
			<td>Shaker</td>
			<td>VWR</td>
			<td>10127-876</td>
			<td>Alsoknown as analog rocker</td>
		</tr>
		<tr>
			<td>Sodium Borohydride</td>
			<td>Aldrich</td>
			<td>452882</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>Sodium Hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td>S8045</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Phosphate Dibasic</td>
			<td>Sigma</td>
			<td>S5136</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>Sodium Phosphate Monobasic</td>
			<td>Sigma</td>
			<td>S5011</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>Spyder</td>
			<td>Anaconda</td>
			<td>4.1.4</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Wisent</td>
			<td>325-042-CL</td>
			<td>0.05% aqueous solution with 0.53mM EDTA</td>
		</tr>
	</tbody>
</table>

control of cell geometry through infrared laser assisted micropatterning

Micropatterning is an established technique in the cell biology community used to study connections between the morphology and function of cellular compartments while circumventing complications arising from natural cell-to-cell variations. To standardize cell shape, cells are either confined in 3D molds or controlled for adhesive geometry through adhesive islands. However, traditional micropatterning techniques based on photolithography and deep UV etching heavily depend on clean rooms or specialized equipment. Here we present an infrared laser assisted micropatterning technique (microphotopatterning) modified from Doyle et al. that can be conveniently set up with commercially available imaging systems. In this protocol, we use a Nikon A1R MP+ imaging system to generate micropatterns with micron precision through an infrared (IR) laser that ablates preset regions on poly-vinyl alcohol coated coverslips. We employ a custom script to enable automated pattern fabrication with high efficiency and accuracy in systems not equipped with a hardware autofocus. We show that this IR laser assisted micropatterning (microphotopatterning) protocol results in defined patterns to which cells attach exclusively and take on the desired shape. Furthermore, data from a large number of cells can be averaged due to the standardization of cell shape. Patterns generated with this protocol, combined with high resolution imaging and quantitative analysis, can be used for relatively high throughput screens to identify molecular players mediating the link between form and function.

The quality of the experimental data obtained through&#160;micropatterning is largely dependent on the quality of the patterns. To determine the quality of patterns generated with the method above, we first used reflectance microscopy to assess the shape and size of the photo ablated areas of the coverslip. We found that each individual pattern looked very similar to the ablation mask and displayed clear boarders and a surface that reflected light uniformly (Figure 2B). A variety of shapes a...

Watch this Scientific Journal Video about Control of Cell Geometry through Infrared Laser Assisted Micropatterning at JoVE.com

Control of Cell Geometry through Infrared Laser Assisted Micropatterning

The protocol presented here enables automated fabrication of micropatterns that standardizes cell shape to study cytoskeletal structures within mammalian cells. This user-friendly technique can be set up with commercially available imaging systems and does not require specialized equipment inaccessible to standard cell biology laboratories.

Micropatterning is an established technique in the cell biology community used to study connections between the morphology and function of cellular ...

control-cell-geometry-through-infrared-laser-assisted

Research

JoVE Journal

Bioengineering

3.3K Views.  University of Toronto. The protocol presented here enables automated fabrication of micropatterns that standardizes cell shape to study cytoskeletal structures within mammalian cells. This user-friendly technique can be set up with commercially available imaging systems and does not require specialized equipment inaccessible to standard cell biology laboratories. 

Video: Control of Cell Geometry through Infrared Laser Assisted Micropatterning

Generation of Aligned Functional Myocardial Tissue Through Microcontact Printing

Advanced heart failure represents a major unmet clinical challenge, arising from the loss of viable and/or fully functional cardiac muscle cells. Despite optimum drug therapy, heart failure represents a leading cause of mortality and morbidity in the developed world. A major challenge in drug development is the identification of cellular assays that accurately recapitulate normal and diseased human myocardial physiology in vitro. Likewise, the major challenges in regenerative cardiac biology revolve around the identification and isolation of patient-specific cardiac progenitors in clinically relevant quantities. These cells have to then be assembled into functional tissue that resembles the native heart tissue architecture. Microcontact printing allows for the creation of precise micropatterned protein shapes that resemble structural organization of the heart, thus providing geometric cues to control cell adhesion spatially. Herein we describe our approach for the isolation of highly purified myocardial cells from pluripotent stem cells differentiating in vitro, the generation of cell growth surfaces micropatterned with extracellular matrix proteins, and the assembly of the stem cell-derived cardiac muscle cells into anisotropic myocardial tissue.

The generation of aligned myocardial tissue is a key requirement for adapting the recent advances in stem cell biology to clinically useful purposes. Herein we describe a microcontact printing approach for the precise control of cell shape and function. Using highly purified populations of embryonic stem cell derived cardiac progenitors, we then generate anisotropic functional myocardial tissue.

Advanced heart failure represents a major unmet clinical challenge,  arising from the loss of viable and/or fully functional cardiac muscle  cells. ...

Selective Cell Elimination from Mixed 3D Culture Using a Near Infrared Photoimmunotherapy Technique

Recent developments in tissue engineering offer innovative solutions for many diseases. For example, tissue engineering using induced pluripotent stem cell (iPS) emerged as a new method in regenerative medicine. Although this tissue regeneration is promising, contamination with unwanted cells during tissue cultures is a major concern. Moreover, there is a safety concern regarding tumorigenicity after transplantation. Therefore, there is an urgent need for eliminating specific cells without damaging other cells that need to be protected, especially in established tissue. Here, we present a method for specific cell elimination from a mixed 3D cell culture in vitro with near infrared photoimmunotherapy (NIR-PIT) without damaging non-targeted cells. This technique enables the elimination of specific cells from mixed cell cultures or tissues.

Eliminating specific cells without damaging other cells is extremely difficult, especially in established tissue, yet there is an urgent need for a cell elimination method in the tissue engineering field. Here, we present a method for specific cell elimination from a mixed 3D cell culture using near infrared photoimmunotherapy (NIR-PIT).

Recent developments in tissue engineering offer innovative solutions for many diseases. For example, tissue engineering using induced pluripotent stem ...

Whole-body Mass Spectrometry Imaging by Infrared Matrix-assisted Laser Desorption Electrospray Ionization (IR-MALDESI)

Ambient ionization sources for mass spectrometry (MS) have been the subject of much interest in the past decade. Matrix-assisted laser desorption electrospray ionization (MALDESI) is an example of such methods, where features of matrix-assisted laser desorption/ionization (MALDI) (e.g., pulsed nature of desorption) and electrospray ionization (ESI) (e.g., soft-ionization) are combined. One of the major advantages of MALDESI is its inherent versatility. In MALDESI experiments, an ultraviolet (UV) or infrared (IR) laser can be used to resonantly excite an endogenous or exogenous matrix. The choice of matrix is not analyte dependent, and depends solely on the laser wavelength used for excitation. In IR-MALDESI experiments, a thin layer of ice is deposited on the sample surface as an energy-absorbing matrix. The IR-MALDESI source geometry has been optimized using statistical design of experiments (DOE) for analysis of liquid samples as well as biological tissue specimens. Furthermore, a robust IR-MALDESI imaging source has been developed, where a tunable mid-IR laser is synchronized with a computer controlled XY translational stage and a high resolving power mass spectrometer. A custom graphical user interface (GUI) allows user selection of the repetition rate of the laser, number of shots per voxel, step-size of the sample stage, and the delay between the desorption and scan events for the source. IR-MALDESI has been used in variety of applications such as forensic analysis of fibers and dyes and MSI of biological tissue sections. Distribution of different analytes ranging from endogenous metabolites to exogenous xenobiotics within tissue sections can be measured and quantified using this technique. The protocol presented in this manuscript describes major steps necessary for IR-MALDESI MSI of whole-body tissue sections.

Whole-body Mass Spectrometry Imaging by Infrared Matrix-assisted Laser Desorption Electrospray Ionization IR-MALDESI

A mass spectrometry imaging (MSI) source operated at atmospheric pressure was developed by coupling mid-infrared laser desorption and electrospray post-ionization. Exogenous ice matrix was used as the energy-absorbing matrix to facilitate resonant desorption of tissue-related material. This manuscript provides a step-by-step protocol for performing IR-MALDESI MSI of whole-body neonatal mouse.

Ambient ionization sources for mass spectrometry (MS) have been the subject of much interest in the past decade. Matrix-assisted laser desorption ...

Micropatterning and Assembly of 3D Microvessels

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass based substrates, and hydrogel-based tube formation assays. These assays, while informative, do not recapitulate lumen geometry, proper extracellular matrix, and multi-cellular proximity, which play key roles in modulating vascular function. This manuscript describes an injection molding method to generate engineered vessels with diameters on the order of 100 &#181;m. Microvessels are fabricated by seeding endothelial cells in a microfluidic channel embedded within a native type I collagen hydrogel. By incorporating parenchymal cells within the collagen matrix prior to channel formation, specific tissue microenvironments can be modeled and studied. Additional modulations of hydrodynamic properties and media composition allow for control of complex vascular function within the desired microenvironment. This platform allows for the study of perivascular cell recruitment, blood-endothelium interactions, flow response, and tissue-microvascular interactions. Engineered microvessels offer the ability to isolate the influence from individual components of a vascular niche and precisely control its chemical, mechanical, and biological properties to study vascular biology in both health and disease.

This manuscript presents an injection molding method to engineer microvessels that recapitulate physiological properties of endothelium. The microfluidic-based process creates patent 3D vascular networks with tailorable conditions, such as flow, cellular composition, geometry, and biochemical gradients. The fabrication process and examples of potential applications are described.

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass ...

Predicting Gene Silencing Through the Spatiotemporal Control of siRNA Release from Photo-responsive Polymeric Nanocarriers

New materials and methods are needed to better control the binding vs. release of nucleic acids for a wide range of applications that require the precise regulation of gene activity. In particular, novel stimuli-responsive materials with improved spatiotemporal control over gene expression would unlock translatable platforms in drug discovery and regenerative medicine technologies. Furthermore, an enhanced ability to control nucleic acid release from materials would enable the development of streamlined methods to predict nanocarrier efficacy a priori, leading to expedited screening of delivery vehicles. Herein, we present a protocol for predicting gene silencing efficiencies and achieving spatiotemporal control over gene expression through a modular photo-responsive nanocarrier system. Small interfering RNA (siRNA) is complexed with mPEG-b-poly(5-(3-(amino)propoxy)-2-nitrobenzyl methacrylate) (mPEG-b-P(APNBMA)) polymers to form stable nanocarriers that can be controlled with light to facilitate tunable, on/off siRNA release. We outline two complementary assays employing fluorescence correlation spectroscopy and gel electrophoresis for the accurate quantification of siRNA release from solutions mimicking intracellular environments. Information gained from these assays was incorporated into a simple RNA interference (RNAi) kinetic model to predict the dynamic silencing responses to various photo-stimulus conditions. In turn, these optimized irradiation conditions allowed refinement of a new protocol for spatiotemporally controlling gene silencing. This method can generate cellular patterns in gene expression with cell-to-cell resolution and no detectable off-target effects. Taken together, our approach offers an easy-to-use method for predicting dynamic changes in gene expression and precisely controlling siRNA activity in space and time. This set of assays can be readily adapted to test a wide variety of other stimuli-responsive systems in order to address key challenges pertinent to a multitude of applications in biomedical research and medicine.

We present a novel method that uses photo-responsive block copolymers for more efficient spatiotemporal control of gene silencing with no detectable off-target effects. Additionally, changes in gene expression can be predicted using straightforward siRNA release assays and simple kinetic modeling.

New materials and methods are needed to better control the binding vs. release of nucleic acids for a wide range of applications that require the precise ...

Initial 3D Cell Cluster Control in a Hybrid Gel Cube Device for Repeatable Pattern Formations

The importance of in vitro 3D cultures is considerably emphasized in cell/tissue culture. However, the lack of experimental repeatability is one of its restrictions. Producing few repeatable results of pattern formation deteriorates the analysis of the mechanisms underlying the self-organization. Reducing variation in initial culture conditions, such as the cell density and distribution in the extracellular matrix (ECM), is crucial to enhance the repeatability of a 3D culture. In this article, we demonstrate a simple but robust procedure for controlling the initial cell cluster shape in a 3D extracellular matrix to obtain highly repeatable pattern formations. A micromold with a desired shape was fabricated by using photolithography or a machining process, and it formed a 3D pocket in the ECM contained in a hybrid gel cube (HGC). Highly concentrated cells were then injected in the pocket so that the cell cluster shape matched with the fabricated mold shape. The employed HGC allowed multi-directional scanning by its rotation, which enabled high-resolution imaging and the capture of the entire tissue structure even though a low-magnification lens was used. Normal human bronchial epithelial cells were used to demonstrate the methodology.&#160;

We present a procedure for controlling the initial cell cluster shape in a 3D extracellular matrix to obtain a repeatable pattern formation. A cubic device containing two different hydrogels is employed to achieve multi-directional imaging for tissue pattern formation.

The importance of in vitro 3D cultures is considerably emphasized in cell/tissue culture. However, the lack of experimental repeatability is one of its ...

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

Human embryonic stem cells demonstrate a unique ability to respond to morphogens in vitro by self-organizing patterns of cell fate specification that correspond to primary germ layer formation during embryogenesis. Thus, these cells represent a powerful tool with which to examine the mechanisms that drive early human development. We have developed a method to culture human embryonic stem cells in confined colonies on compliant substrates that provides control over both the geometry of the colonies and their mechanical environment in order to recapitulate the physical parameters that underlie embryogenesis. The key feature of this method is the ability to generate polyacrylamide hydrogels with defined patterns of extracellular matrix ligand at the surface to promote cell attachment. This is achieved by fabricating stencils with the desired geometric patterns, using these stencils to create patterns of extracellular matrix ligand on glass coverslips, and transferring these patterns to polyacrylamide hydrogels during polymerization. This method is also compatible with traction force microscopy, allowing the user to measure and map the distribution of cell-generated forces within the confined colonies. In combination with standard biochemical assays, these measurements can be used to examine the role mechanical cues play in fate specification and morphogenesis during early human development.

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 specification.

Human embryonic stem cells demonstrate a unique ability to respond to morphogens in vitro by self-organizing patterns of cell fate specification that ...

Synthesis of Near-Infrared Emitting Gold Nanoclusters for Biological Applications

Over the past decade, fluorescent gold nanoclusters (AuNCs) have witnessed growing popularity in biological applications and enormous efforts have been devoted to their development. In this protocol, a recently developed, facile method for preparation of water soluble, biocompatible, and colloidally stable near-infrared emitting AuNCs have been described in detail. This room-temperature, bottom-up chemical synthesis provides easily functionalizable AuNCs capped with thioctic acid and thiol-modified polyethylene glycol in aqueous solution. The synthetic approach requires neither organic solvents or additional ligand exchange nor extensive knowledge of synthetic chemistry to reproduce. The resulting AuNCs offer free surface carboxylic acids, which can be functionalized with various biological molecules bearing a free amine group without adversely affecting the photoluminescent properties of the AuNCs. A quick, reliable procedure for flow cytometric quantification and confocal microscopic imaging of AuNC uptake by HeLa cells also been described. Due to the large Stokes shift, proper setting of filters in flow cytometry and confocal microscopy is necessary for efficient detection of near-infrared photoluminescence of AuNCs.

A reliable and easily reproducible method for preparation of functionalizable, near-infrared emitting photoluminescent gold nanoclusters and their direct detection inside HeLa cells by flow cytometry and confocal laser scanning microscopy is described.

Over the past decade, fluorescent gold nanoclusters (AuNCs) have witnessed growing popularity in biological applications and enormous efforts have been ...

Mitigation of Blood Borne Cell Attachment to Metal Implants through CD47-Derived Peptide Immobilization

The key complications associated with bare metal stents and drug eluting stents are in-stent restenosis and late stent thrombosis, respectively. Thus, improving the biocompatibility of metal stents remains a significant challenge. The goal of this protocol is to describe a robust technique of metal surface modification by biologically active peptides to increase biocompatibility of blood contacting medical implants, including endovascular stents. CD47 is an immunological species-specific marker of self and has anti-inflammatory properties. Studies have shown that a 22 amino acid peptide corresponding to the Ig domain of CD47 in the extracellular region (pepCD47), has anti-inflammatory properties like the full-length protein. In vivo studies in rats, and ex vivo studies in rabbit and human blood experimental systems from our lab have demonstrated that pepCD47 immobilization on metals improves their biocompatibility by preventing inflammatory cell attachment and activation. This paper describes the step-by step protocol for the functionalization of metal surfaces and peptide attachment. The metal surfaces are modified using polyallylamine bisphosphate with latent thiol groups (PABT) followed by deprotection of thiols and amplification of thiol-reactive sites via reaction with polyethyleneimine installed with pyridyldithio groups (PEI-PDT). Finally, pepCD47, incorporating terminal cysteine residues connected to the core peptide sequence through a dual 8-amino-3,6-dioxa-octanoyl spacer, are attached to the metal surface via disulfide bonds. This methodology of peptide attachment to metal surface is efficient and relatively inexpensive and thus can be applied to improve biocompatibility of several metallic biomaterials.

Presented here is a protocol for appending peptide CD47 (pepCD47) to metal stents using polybisphosphonate chemistry. Functionalization of metal stents using pepCD47 prevents the attachment and activation of inflammatory cells thus improving their biocompatibility.

The key complications associated with bare metal stents and drug eluting stents are in-stent restenosis and late stent thrombosis, respectively. Thus, ...

Dual Raster-Scanning Photoacoustic Small-Animal Imager for Vascular Visualization

Imaging of vascular networks on small animals has played an important role in basic biomedical research. Photoacoustic imaging technology has great potential for application in the imageology of small animals. The wide-field photoacoustic imaging of small animals can provide images with high spatiotemporal resolution, deep penetration, and multiple contrasts. Also, the real-time photoacoustic imaging system is desirable to observe the hemodynamic activities of small-animal vasculature, which can be used to research the dynamic monitoring of small-animal physiological features. Here, a dual-raster-scanning photoacoustic imager is presented, featuring a switchable double-mode imaging function. The wide-field imaging is driven by a two-dimensional motorized translation stage, while the real-time imaging is realized with galvanometers. By setting different parameters and imaging modes, in vivo visualization of small-animal vascular network can be performed. The real-time imaging can be used to observe pulse change and blood flow change of drug-induced, etc. The wide-field imaging can be used to track the growth change of tumor vasculature. These are easy to be adopted in various areas of basic biomedicine research.

A dual raster-scanning photoacoustic imager was designed, which integrated wide-field imaging and real-time imaging.

Imaging of vascular networks on small animals has played an important role in basic biomedical research. Photoacoustic imaging technology has great ...