Pattern Generation for Micropattern Traction Microscopy

Summary

We describe improvements to a standard method for measuring cellular traction forces, based on microcontact printing with a single subtractive patterning step of dot arrays of extracellular matrix proteins on soft hydrogels. This method allows for simpler and more consistent fabrication of island patterns, essential for controlling cell cluster shape.

Abstract

Micropattern traction microscopy allows control of the shape of single cells and cell clusters. Furthermore, the ability to pattern at the micrometer length scale allows the use of these patterned contact zones for the measurement of traction forces, as each micropatterned dot allows for the formation of a single focal adhesion that then deforms the soft, underlying hydrogel. This approach has been used for a wide range of cell types, including endothelial cells, smooth muscle cells, fibroblasts, platelets, and epithelial cells.

This review describes the evolution of techniques that allow the printing of extracellular matrix proteins onto polyacrylamide hydrogels in a regular array of dots of prespecified size and spacing. As micrometer-scale patterns are difficult to directly print onto soft substrates, patterns are first generated on rigid glass coverslips that are then used to transfer the pattern to the hydrogel during gelation. First, the original microcontact printing approach to generate arrays of small dots on the coverslip is described. A second step that removes most of the pattern to leave islands of small dots is required to control the shapes of cells and cell clusters on such arrays of patterned dots.

Next, an evolution of this approach that allows for the generation of islands of dots using a single subtractive patterning step is described. This approach is greatly simplified for the user but has the disadvantage of a decreased lifetime for the master mold needed to make the patterns. Finally, the computational approaches that have been developed for the analysis of images of displaced dots and subsequent cell-generated traction fields are described, and updated versions of these analysis packages are provided.

Introduction

Most cell phenotypes exert traction forces on their environment. These traction forces are generated by a cell's contractile cytoskeleton, which is a network of actin and myosin, and other filamentous biopolymers and crosslinking proteins^1,2,3,4. Forces generated within the cell can be transmitted to the extracellular environment or adjacent cells, primarily via transmembrane proteins such as integrins and cadherins, respectively^5,6. How a cell spreads or contracts-and the magnit....

Protocol

1. Creation of silicone masters

NOTE: Most of the process of the design, creation, and troubleshooting of silicon masters for the repeated molding of PDMS stamps has been covered previously²¹, so only key differences in this new approach will be described here.

1. Create the design for the photomask using AutoCAD or similar design software. Coat one side of the photomask, a thin piece of glass, with a thin layer of chrome to control UV light sc.......

Discussion

An improved method of indirectly patterning PAA hydrogels is described in this paper. This approach builds on methods that have been used previousely^{20,35,36,37,38,39,40,41,42}. The primary change is that PDMS stamps are no.......

Materials

Name	Company	Catalog Number	Comments
(3-aminopropyl)trimethoxysilane	Sigma Aldrich	#281778
1.5 mL Microcentrifuge tube	Fisher Scientific	#05-408-129
15 mL conical tube	Fisher Scientific	#05-539-12
4 x 4 in 0.060 Quartz LR Chrome Photomask	Advance Reproductions Corporation	N/A	Custom-designed mask
6 Well Plates	Fisher Scientific	#07-200-83
Acetone	Fisher Scientific	#A18P-4
Acrylamide Solution, 40%	Sigma Aldrich	#A4058
AlexaFluor 488	Thermo Fisher	#A20000
Aminonium Persulfate	Fisher Scientific	#BP179-25
Bisacrylamide	Fisher Scientific	#PR-V3141
Ethanol	Greenfield Global	#111000200C1GL
Glass Coverslips, 25 mm round	Fisher Scientifc	#12-545-102
Glass Coverslips, 30 mm round	Warner	#64-1499
Hamamatsu ORCA-R2 Camera	Hamamatsu	#C10600-10B
Human Plasma	Valley Biomedical	#HP1051P	Used to isolate fibronectin
Hydrochloric Acid, 1.0 N	Millipore Sigma	#1.09057
ImageJ	Wayne Rasband	#1.53n
Interchangeable Coverslip Dish Set	Bioptechs	#190310-35
Kim Wipes	Fisher Scientific	#06-666-11C
Mask Alinger	Karl Suss	#MA6
Matlab 2021	Mathworks	#R2021a
MetaMorph Basic	Molecular Devices	#v7.7.1.0
N-hydroxysuccinimide ester	Sigma Aldrich	#130672-5G
NucBlue Live Cell Stain	Thermo Fisher	#R37605
Olympus IX2-ZDC Inverted Microscope	Olympus	#IX81
PD-10 Desalting Columns	GE Healthcare	#52-1308-00
Photoresist Spinner Hood	Headway Research	#PWM32
Plasma Cleaner	Harrick	#PDC-001
Plasma Etcher	TePla	#M4L
Prior Lumen 200Pro Light Source	Prior Scientific	#L200
Silicon Wafers, 100 mm	University Wafer	#809
SU-8 2005	Kayaku Advanced Materials Inc.	#NC9463827
SU-8 Developer	Kayaku Advanced Materials Inc.	#NC9901158
Sylgard 184 Silicone Elastomer	Essex Brownell	#DC-184-1.1
Tetramethylethylenediamine	Fisher Scientific	#BP150-20
Trichloro(1H,1H,2H,2H-perfluorooctyl)silane	Sigma Aldrich	#448931
UAPON-40XW340 Objective	Olympus	#N2709300
UV Flood Exposure	Newport	#69910
Wafer Carrier Tray, 110 x 11 mm	Ted Pella, Inc.	#19395-40