Manipulating cell-substrate interactions through matrix protein deposition from Physical confinement signals regulate the organization of stem cells in three dimensions

To confirm that collagen is responsible for the cell-substrate adhesion on PDMS channels, cells were seeded without collagen and incubated for 48hrs. Whether on the (a) 100 µm channel or the (b) flat substrate, no cells had successfully adhered (Scale bar = 100 µm and applies to (b) as well). (c) In an attempt to reverse the spheroidal geometry observed in the channels, we increased cell-substrate adhesion by depositing fibronectin in addition to collagen. Increased cell substrate adhesion appeared to have no effect of embryonic stem cells growth, as the aggregate still formed a highly three dimensional geometry. Interestingly, the increased substrate interaction promoted cell adhesion at the top of the channels; however, these cells formed typical two dimensional flat aggregates.To ascertain whether the observed changes in geometry are the result of a higher cell density, cells were seeded to match the density of 100 µm channels (~450 cells/mm2). Shown are cells at (d) 12 hrs and (e) 48 hours after seeding, displaying a typical island shaped morphology. Scale bar = 100 µm.