Fibroblast elongation and dendritic extensions in constrained versus unconstrained microtissues

Cytoskeletal tension is fundamental to many biological processes, including germ layer sorting during embryogenesis [Krieg et al., 2008]. In vitro, such tension influences cell sorting in self-assembled, 3D microtissues and can be of sufficient magnitude to cause complex-shaped microtissue failure [Dean et al., 2007]. To examine the process of failure under cell-derived tension, we subjected normal human fibroblasts (NHFs) to directed self-assembly [Dean et al., 2007] in micro-molds designed to yield self-constraining microtissues. As cells contracted in this assay, the constrained microtissues narrowed, thinned and ultimately failed at their midpoints. By adding small numbers of GFP+ cells, changes in cell movement and morphology were assessed and compared to those of unconstrained microtissues. We found that cells formed numerous dendritic extensions within an hour of self-assembly and retracted these extensions as they elongated up to 30 times their initial diameter ( approximately 600 microm) just prior to failure. Surprisingly, significant coordination in cell motility was observed over large distances within microtissues. Pharmacologic interventions showed that failure was myosin II and Rho kinase dependent and inhibition of failure resulted in shorter cells with greater numbers of extensions. These findings further our understanding of cellular self-assembly and introduce the use of GFP+ cells with directed self-assembly as a scaffold-free analogue to fibroblast-populated collagen gels (FPCGs).