Cells in multicellular organisms are exposed to a range of forces that would kill unicellular lifeforms. To cope, these cells have evolved a mechanically-sensitive signaling pathway called mechanotransduction. Mechanotransduction can direct lineage differentiation by detecting changes in environmental viscoelasticity. It has been shown that stiffness differs within and between tissues, resulting in a range of mechanical signals sensed by cells. Current tools to study cellular mechanotransduction either apply mechanical forces to cells grown in 2D, where cells adopt shapes different to their 3D shape in vivo, or ignore viscosity completely. Here, we apply dynamic control of polymer hydrogel scaffold viscoelasticity using macromolecular crowding (MMC), and observe the effects this dynamic viscoelasticity has on cell cultures. So-called crowding agents “crowd” polymer hydrogels by taking up space in the hydrogel matrix, restricting structural conformations, thereby stiffening the network.

For the purpose of this study, alginate and Matrigel, with pore sizes of about 5 nm and 2 μm, respectively, are ideal hydrogels. At these pore sizes, small crowding agents, such as poly(ethylene glycol) (PEG), may easily penetrate the polymer matrices, taking up network space without other kinds of interactions (see presentation by Scott, et al.). By dynamically changing the PEG concentration of the solution in which alginate hydrogels are incubated, results on 2D substrates have shown that such changes in viscoelasticity affect both cell adhesion and the cytoskeleton, without any negative effect on cell vitality or proliferation. While several studies have demonstrated that MMC could affect biological equilibria, we demonstrate with control experiments that crowding alone does not account for the observed changes in cell morphology. Our aim is to apply this methodology in a 3D environment, mimicking the complexity of an in vivo organism. This technique holds promise for disease modeling (i.e. cancer), and simulating the self-organization of tissues during morphogenesis. In particular, they can serve as effective tools for studying the effects of external and internal mechanical forces and their interactions with molecular signaling pathways.