Cytoskeletal-mediated tension modulates the directed self-assembly of microtissues

The ability of cells to self-assemble into a microtissue such as a spheroid has been attributed mainly to intercellular cohesiveness achieved by the binding of surface membrane proteins such as cadherins. However, highly dynamic and complex cytoskeletal rearrangements are coordinated with these binding events and therefore are likely to participate in self-assembly. By inhibiting such cytoskeletal activity, Y-27632 has been used to prevent and treat fibrotic disease. Here, we used the Rho kinase inhibitor to investigate the role that cellular contraction plays in self-assembly. Normal human fibroblasts (NHFs), Reuber-H35 hepatoma cells (H35s), and mixes of the two (hybrid) were treated with drug during directed self-assembly of microtissues in nonadhesive, micromolded hydrogels. The kinetics of self-assembly of both constrained and unconstrained NHF microtissues were dramatically slowed by the drug, and inhibition was dose responsive and reversible. Although sorting of NHFs and H35s occurred normally in the presence of drug, Y-27632-treated NHFs sorted to the outside of a spheroid when mixed with untreated NHFs. When mixed with H35s in trough micromolds, NHFs could drive spheroid formation from the core of the hybrid microtissues even in small numbers relative to H35s (1:19). These findings demonstrate that cellular contraction controls the kinetics of self-assembly and suggests that NHFs within the core of a microtissue can transmit contractile forces through heterotypic bonds with H35s. The control of directed self-assembly using fibroblasts and contraction inhibitors may be useful for in vitro tissue engineering as well as represent an in vitro model for fibrotic disease.