Dynamics of the Self-Assembly of Complex Cellular Aggregates on Micromolded Nonadhesive Hydrogels

The process by which cells self-assemble to form three-dimensional (3D) structures is central to morphogenesis and development of living tissues and hence is of growing interest to the field of tissue engineering. Using rapid prototyping technology we made micromolded nonadhesive hydrogels to study the dynamics of self-assembly in a low-shear environment with simple spherical geometries as well as more complex geometries such as a toroid. Aggregate size, shape, and composition were easily controlled; aggregates were easily retrieved; and the dynamics of the assembly process were readily observed by time-lapse microscopy. When two cell types, normal human fibroblasts (NHFs) and human umbilical vein endothelial cells (HUVECs), were seeded together, they self-segregated into multilayered spherical microtissues with a core of NHFs enveloped by a layer of HUVECs. Surprisingly, when a single cell suspension of NHFs was added to 7-day-old HUVEC spheroids, the HUVEC spheroid reorganized such that NHFs occupied the center and HUVECs coated the outside, demonstrating that self-assembly is not terminal and that spheroids are fluid structures that retain the ability to reassemble. We also showed that cells can self-assemble to form a complex toroid shape, and we observed several phenomena indicating that cellular contraction and tension play a significant role in the assembly process of complex shapes.