Fabrication of Spheroids with Uniform Size by Self-Assembly of a Micro-Scaled Cell Sheet (μCS): The Effect of Cell Contraction on Spheroid Formation.

Cell spheroid culture can be an effective approach for providing an engineered microenvironment similar to an in vivo environment. Our group had recently developed a method for harvesting uniformly sized multicellular spheroids via self-assembly of micro-scaled cell sheets (μCSs) induced by the expansion of temperature-sensitive hydrogels. However, the μCS assembly process was not fully understood. In this study, we investigated the effects of cell number, pattern shape, and contractile force of cells on spheroid formation from micropatterned (width of square pattern from 100-300 μm) hydrogels. We used human dermal fibroblasts (HDFBs) as a model cell type and cultured them for 24 and 72 h. The self-assembly of μCSs cultured on square micropatterns for 72 h rapidly occurred within 4 min after reducing the temperature from 37 to 4 °C. In addition, the size distribution of spheroids was narrower with μCSs from a 72 h culture. Treatment with a ROCK1 inhibitor disrupted cytoskeletal actin fibers and the corresponding μCSs were not detached from the hydrogel. The assembly of the μCS was also affected by the micropattern shape, and the spheroid harvest efficiency was decreased to 60% when using a circular micropattern, which was explained by the stress direction on the circular versus square micropattern upon hydrogel expansion. Therefore, we confirmed that the factors controlling cell-cell interactions are important for spheroid formation using micropatterned hydrogel systems. Finally, the μCSs with dual layers of HDFBs labeled with DiD and DiO dyes resulted in the formation of spheroids with discretely localized colors within the core and shell, respectively, which suggests an outside-in assembly of detached μCSs. In consideration of these complex environmental factors, our system could be utilized in various applications as a three-dimensional culture system to fabricate cell spheroids.