Cellular Mechanotransduction via Microfabricated Post Arrays

Substrate-based biophysical cues, which interact with cells through mechanotransductive pathways, influence many biological processes and cellular behaviors. By leveraging microfabrication techniques, this work aims to build biophysical stimuli into cellular substrates through discrete microtopographic features to study cellular responses. Here, uniform and gradient arrays of circular microposts have been geometrically tuned to change the apparent rigidity of a substrate and the placement of available cellular attachment sites. Three areas of cellular interaction with these micropost array substrates have been investigated: (i) single cell motility, (ii) maintenance and inhibition of collective cell behavior, and (iii) reprogramming and differentiation processes of induced pluripotent stem cells (iPSCs)./nSingle cell migration was induced through gradients in substrate stiffness and spacing of available attachment sites - phenomena known as durotaxis and herein referred to as spatiotaxis, respectively. Unidirectional micropost arrays gradients were designed with increasing stiffnesses at low and high gradient strengths of 0.5 nN/μm and 7.5 nN/μm, respectively. On these surfaces, bovine aortic endothelial cells (BAECs) were found to preferentially migrate toward the direction of increasing micropost stiffness. In 18-hour studies, with more than 12 single cells in each case, BAECs had average displacements of 26.5 ± 8.7 μm and 41.9 ± 14.7 μm for the low and high gradient strengths, respectively. Furthermore, BAECs were found to migrate in favor of the direction of decreasing interpost spacing over the direction of increasing stiffness in the prototype micropost arrays, demonstrating that spatial cues can dominate stiffness cues in the migratory response of cells./nThe maintenance and inhibition of collective cell behavior was studied through changes in substrate stiffness and spacing via uniform and gradient micropost arrays with stiffnesses in the range of 24-106 nN/μm. BAEC collectives directly cultured on these surfaces exhibited area contraction or expansion, which corresponded to maintenance and inhibition of group behavior on soft and stiff substrates, respectively. The micropost mechanical stiffness required for collective-to-single cell transitions was characterized as 30 ± 6 nN/μm. Effects of spacing on collective cell behavior were also explored, and results showed that BAEC collectives were unable to maintain group behavior with favorable stiffness cues, demonstrating again the significance of micropost spacing./nThe effects of microtopography on the reprogramming and differentiation of iPSCs was investigated through uniform micropost arrays of varying radii and heights with mouse ear fibroblasts (mERFs) on micropost arrays with stiffnesses in the range of 24-2900 nN/μm. Preliminary results have shown that microtopography had influence over the formation of iPSC colonies and the number of colonies that exhibited beating. Specifically, beating colonies were observed as early as 10 days after the infection of mERFs on micropost surfaces, suggesting that microtopography might direct the differentiation of iPSCs. Characterizations of changes in mERF morphology, expression of nuclear structural proteins, and intracellular localization of proteins that regulate gene expression provide evidence for possible mechanisms responsible for the effects of microtopography on the reprogramming and differentiation processes of iPSCs.

Advisor: Lin, Liwei