Engineering Substrates for the Study of Cell Mechanical Interactions

Edición de la obra Engineering Substrates for the Study of Cell Mechanical Interactions

This thesis describes the effect of geometries with controlled radius of curvature on cellular behavior, a novel approach to measure and map protrusive forces in cells and the application of fabrication techniques like wet etching and microcontact printing to answer fundamental biological problems. In order to isolate the effects of curvature from other factors, we have developed a technique to create features with nominally identical dimensions but varying radius of curvature. Using these substrates, we analyzed the effect of curvature on cell morphology. Cell area and aspect ratio were examined on various substrates, and immunostaining of focal adhesions, stress fibers and microtubules were used to show the effect of curvature on these cytoskeleton components. We show that feature curvature has an effect on both cell morphology and cytoskeleton organization. Using this technique, it may be possible to engineer precise geometries that can lead to better design of scaffolds and biomaterials for tissue engineering. The motivation behind measuring cellular forces was two fold, first to measure the protrusive forces locally and with spatial resolution and second to measure them at the same time as traction forces. This is important because cell motility is a result of forces generated within a cell and various biological processes like cancer metastasis, wound healing and immune response are a result of cell motility. Thus, measuring theses forces precisely and simultaneously will help us designing and develop devices that can have application in cancer diagnostics and wound healing therapies. The magnitude of protrusive force measured was 1.0 nN and traction force was computed to be 2.7 nN. Furthermore we also estimated the number of actin filaments per micron square which agree with previously reported values thus confirming the accuracy of this method. The approach presented here is the first study to simultaneously measure the protrusive and traction forces in cells. In chapter 4, I describe in detail the fabrication process for making high aspect ratio grooves and ridges by wet etching Silicon using boiling potassium hydroxide. The etched substrates were used as imprint masters and were faithfully replicated and molded in a silicone elastomer. Next the substrates were plasma fluorinated and used to form elastomer stamps for microcontact printing and other applications requiring easy mold release. In chapter 5, I have used microcontact printing to fabricate substrates to help us understand plasma membrane dynamics. The role of plasma membrane (PM) area as a critical factor during cell motility is poorly understood, mainly due to an inability to precisely follow PM area dynamics. To address this fundamental question, we developed static and dynamic assays to follow PM area changes during fibroblast spreading.