High Throughput Traction Force Microscopy Using PDMS Reveals Dose-Dependent Effects of Transforming Growth Factor-β on the Epithelial-to-Mesenchymal Transition

J Vis Exp. 2019 Jun 1:(148):10.3791/59364. doi: 10.3791/59364.

Abstract

Cellular contractility is essential in diverse aspects of biology, driving processes that range from motility and division, to tissue contraction and mechanical stability, and represents a core element of multi-cellular animal life. In adherent cells, acto-myosin contraction is seen in traction forces that cells exert on their substrate. Dysregulation of cellular contractility appears in a myriad of pathologies, making contractility a promising target in diverse diagnostic approaches using biophysics as a metric. Moreover, novel therapeutic strategies can be based on correcting the apparent malfunction of cell contractility. These applications, however, require direct quantification of these forces. We have developed silicone elastomer-based traction force microscopy (TFM) in a parallelized multi-well format. Our use of a silicone rubber, specifically polydimethylsiloxane (PDMS), rather than the commonly employed hydrogel polyacrylamide (PAA) enables us to make robust and inert substrates with indefinite shelf-lives requiring no specialized storage conditions. Unlike pillar-PDMS based approaches that have a modulus in the GPa range, the PDMS used here is very compliant, ranging from approximately 0.4 kPa to 100 kPa. We create a high-throughput platform for TFM by partitioning these large monolithic substrates spatially into biochemically independent wells, creating a multi-well platform for traction force screening that is compatible with existing multi-well systems. In this manuscript, we use this multi-well traction force system to examine the Epithelial to Mesenchymal Transition (EMT); we induce EMT in NMuMG cells by exposing them to TGF-β, and to quantify the biophysical changes during EMT. We measure the contractility as a function of concentration and duration of TGF-β exposure. Our findings here demonstrate the utility of parallelized TFM in the context of disease biophysics.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Video-Audio Media

MeSH terms

  • Animals
  • Cell Movement*
  • Cells, Cultured
  • Dimethylpolysiloxanes / chemistry*
  • Epithelial-Mesenchymal Transition*
  • Female
  • Mammary Glands, Animal / drug effects
  • Mammary Glands, Animal / pathology*
  • Mice
  • Microscopy, Atomic Force / methods*
  • Transforming Growth Factor beta / pharmacology*

Substances

  • Dimethylpolysiloxanes
  • Transforming Growth Factor beta
  • baysilon