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. © The Authors, 2019.