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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Bioengineering

Control of Cell Geometry through Infrared Laser Assisted Micropatterning

Published: July 10th, 2021

DOI:

10.3791/62492

1Department of Cell and Systems Biology, 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.

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.

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 forw....

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1. Coverslip preprocessing

  1. Prepare squeaky-clean coverslips as described in Waterman-Storer, 199825.
  2. 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.
  3. Wash coverslips twice with dH2O for 5 min each.
  4. Prepare 0.5% glutaraldehyde (GA) solution and incubate the coverslips in the solution for 30 min on a shake.......

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The quality of the experimental data obtained through 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.......

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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.......

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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.

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

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