Microfabricated Post-Array-Detectors (mPADs): an Approach to Isolate Mechanical Forces

Summary

In this video, we demonstrate how to fabricate and utilize microfabricated post array detectors (mPADs) to assess modulations of cellular contractility.

Abstract

In this video, we will present our approach to measure cellular traction forces using a microfabricated array of posts. Traction forces are generated through myosin-actin interactions and play an important role in our physiology. During development, they enable cells to move from one location to the next in order to form the early structures of tissue. Traction forces help in the healing processes. They are necessary for the proper closure of wounds or the migration and crawling of leukocytes through our body. These same forces can be detrimental to our health in the case of cancer metastasis or vascular growth towards a tumor. The most common method by which to study cells in vitro has been to use a glass or polystyrene dish. However, the rigidity of the substrates makes it impossible to physically measure cell traction forces, and there are relatively few methods to study traction forces. Our lab has developed a technique to overcome these limitations. The method is based on a vertical array of flexible cantilevers, the stiffness and size scale of which are such that individual cells spread across many cantilevers and deflect them in the process. The pillars we use are 3 μm in diameter, 10 μm tall, and are configured in a regular array with 9 μm center-to-center spacing. But these physical dimensions can be readily varied to accommodate a variety of studies. We start with a silicon master, but the final posts are made out of silicone rubber called poly (dimethyl siloxane), or PDMS. We can measure the deflections under a microscope and calculate the magnitude and direction of traction forces required to produce the observed deflections. We call these substrates microfabricated post-array-detectors, or mPADs. Here, we will show you how we fabricate and use the mPADs to assess modulations of cellular contractility.

Protocol

Silicon Master Mold FabricationLithographyMaterials: 75 mm Silicon wafers SU-8 2 Negative Photoresist (Microchem, Boston, MA) N2 Method: Dehydrate Si wafer at 120 °C for 2 hours. UV ozone treat surface for 7 minutes to remove organics. N2 blow surface to remove particulates. Apply SU-8 2 to cover 70% of surface (~10 ml for 75 mm wafer). Spin at 2000 rpm for 20 seconds with 300 rpm/s a…