Name: control of cell geometry through infrared laser assisted micropatterning
Uploaded: 2021-07-10T15:00:00
Description: Watch this Scientific Journal Video about Control of Cell Geometry through Infrared Laser Assisted Micropatterning at JoVE.com

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.

1. Coverslip preprocessing
<ol>
	<li>Prepare squeaky-clean coverslips as described in Waterman-Storer, 199825.</li>
	<li>Prepare 1% (3-aminopropyl)trimethoxysilane (APTMS) solution and incubate the coverslips in the solution for 10 min with gentle agitation. Make sure that coverslips move freely in the solution.</li>
	<li>Wash coverslips twice with dH2O for 5 min each.</li>
	<li>Prepare 0.5% glutaraldehyde (GA) solution and incubate the coverslips in the solution for 30 min on a shake...

<ol>
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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 ...

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Bioengineering

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