Multilayered fibroblasts constructed by accelerated cellular self-assembly and applications for regenerative medicine

Abstract

Regenerative medicine research requires animal experiments to evaluate treatment effects. According to the 3Rs prin­ciples, alternative models have been developed and utilized to evaluate the efficacy and safety of new products. Three-dimensional (3D) cell cultures have been recognized for their relevant structures and biological functions that are akin to native tissues. They can better represent in vivo conditions than two-dimensional (2D) cell cultures. Herein, we present a fast and simple technique for the construction of 3D dermal fibroblasts (3D-DFs) without exogenous scaffolds. The 3D-DFs can be obtained within 3 days by seeding DFs at a level that exceeds their confluent density and culturing them in the presence of ascorbic acid. The 3D-DFs have a compact, multilayer structure as revealed by histology and their collagen content is drastically increased compared to the monolayer. The 3D-DF-derived extracellular matrix can serve for 3D culturing of other cells. A gap closure assay was performed with the 3D-DFs to represent a 3D-wounded dermal model. Interestingly, the multilayered structure of the 3D-DFs could be regenerated after wounding even when cultured in the absence of ascorbic acid. Moreover, skin grafting using the 3D-DFs was demonstrated in vitro using wounded in vitro human full-thickness skin models. The 3D-DFs will be potentially useful for regenerative medicine and as tissue models for in vitro studies.