Stretching Micropatterned Cells on a PDMS Membrane

Summary

This manuscript presents a technique to apply or release forces on adherent cells or tissues using unidirectional stretching.

Abstract

Mechanical forces exerted on cells and/or tissues play a major role in numerous processes. We have developed a device to stretch cells plated on a PolyDiMethylSiloxane (PDMS) membrane, compatible with imaging. This technique is reproducible and versatile. The PDMS membrane can be micropatterned in order to confine cells or tissues to a specific geometry. The first step is to print micropatterns onto the PDMS membrane with a deep UV technique. The PDMS membrane is then mounted on a mechanical stretcher. A chamber is bound on top of the membrane with biocompatible grease to allow gliding during the stretch. The cells are seeded and allowed to spread for several hours on the micropatterns. The sample can be stretched and unstretched multiple times with the use of a micrometric screw. It takes less than a minute to apply the stretch to its full extent (around 30%). The technique presented here does not include a motorized device, which is necessary for applying repeated stretch cycles quickly and/or computer controlled stretching, but this can be implemented. Stretching of cells or tissue can be of interest for questions related to cell forces, cell response to mechanical stress or tissue morphogenesis. This video presentation will show how to avoid typical problems that might arise when doing this type of seemingly simple experiment.

Introduction

The cells composing a tissue in higher organisms are subject to mechanical tensions and stretching forces coming either from the external environment or from surrounding cells^1,2. Cells must adapt to and resist these forces in order to maintain tissue integrity. Such forces are also important for tissues morphogenesis during development^3,4. Applying mechanical forces on cultured cells is a way to mimic what might happen in a tissue, but with a quantitative and independent control of cell shape and cell deformation^5,6. For this, several techniques might be used. One can press on the cells (the whole cell or part of it), for example usin....

Protocol

1. Passivation of the PDMS

1. Cut a piece of PDMS approximately 35 mm x 20 mm from a pre made sheet (for instance, GelPak, as listed in the table of materials).
2. Remove the top and the bottom protective layers of plastic (if necessary) and use tweezers to place the PDMS in a plastic (not cell culture treated) Petri dish.
3. Wash the PDMS with 70% ethanol for 5 min on a rotator at 30 oscillations/min.
4. Dry the surface by flowing air on it.
5. Illuminate with deep UV (λ = 180 nm) for 5 min at a distance from the UV bulbs of about 5 cm (see Materials sheet for lamp reference; parameters will vary for different lamps).

Results

The technique presented in this video protocol allowed the application of forces on the retraction fibers of mitotic mammalian cells. Indeed, during cell division, at the mitotic stage, mammalian cells retract to take the shape of a sphere and leave behind thin actin cables surrounded by membrane which are attached to the substrate. These cables (retraction fibers), are the memory of the cell geometry before going into division. Making micropatterns with deep UV through a photomask on PDMS thin film (Figure 1

Discussion

Although this technique has been used numerous times and is thoroughly tested, there are several critical steps that can lead to a failed experiment.

About the PDMS:

For this work, GelPak, a commercially available thin PDMS sheet, was used. Alternatively PDMS sheets can be cast directly from PDMS mix. We recommend using GelPak because it is more reproducible, and is less likely to break compared to custom made PDMS.

About the device:

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
GelPak	GelPak	PF-60-X4	Different thickness/stickiness are available. One alternative could be to cast your PDMS yourself.
Silicon grease	GE Bayer Silicones	Baysilone-Paste	This one is biocompatible
Stretching device	GREM mécanique	Stretcher 2011
EDC (N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride)	Sigma	3450	Stable 6 months at -20 °C
NHS (N-Hydroxysulfosuccinimide sodium salt)	Sigma	56485	Protect from humidity
Pll-g-peg (PLL(20)-g[3.5]-PEG(2) 20 mg)	SurfaceSolutions (Zurich)
Synthetic Quartz photomask	Toppan		Take standard binary photomask in Quartz
Fibronectin from bovine plasma	Sigma	F1141