The Potential of a Tailored Biomimetic Hydrogel for In Vitro Cell Culture Applications: Characterization and Biocompatibility

In this study, the Pluronic F127 with modified tripeptide Gly-Arg-Gly-Asp copolymer (hereafter defined as 3BE) hydrogel was evaluated in terms of its biocompatibility potentials. The fibroblasts (Swiss 3T3 cell line) and human hair follicles-derived mesenchymal stem cells (HFMSCs) were cultured in different concentrations of the 3BE hydrogel (0%, 0.05%, 0.1%, 0.25%, and 0.5%, respectively). The cell morphology and differentiation potential of HFMSCs were observed through optical microscopy, and the cell viability was investigated via Live/Dead Kit and Cell Counting Kit-8 assay. Analytical results showed that HFMSC can differentiate into adipogenic, chondrogenic, and osteogenic lineages. The HFMSC and Swiss 3T3 cells would properly assemble into a spherical shape as cultured with the 3BE hydrogel. Most importantly, cell viability could be maintained above 70%. The formation of spheroid structures of cells within this hydrogel is predicted to promote cell differentiation potentials of HFMSC that benefit in generating functional adipocytes, chondrocytes, and osteoblasts. Therefore, these findings demonstrate that the 3BE hydrogel has great potential as a three-dimensional cell culture scaffold for tissue engineering applications.

Dynamics of Actin Stress Fibers and Focal Adhesions during Slow Migration in Swiss 3T3 Fibroblasts: Intracellular Mechanism of Cell Turning

To understand the mechanism regulating the spontaneous change in polarity that leads to cell turning, we quantitatively analyzed the dynamics of focal adhesions (FAs) coupling with the self-assembling actin cytoskeletal structure in Swiss 3T3 fibroblasts. Fluorescent images were acquired from cells expressing GFP-actin and RFP-zyxin by laser confocal microscopy. On the basis of the maximum area, duration, and relocation distance of FAs extracted from the RFP-zyxin images, the cells could be divided into 3 regions: the front region, intermediate lateral region, and rear region. In the intermediate lateral region, FAs appeared close to the leading edge and were stabilized gradually as its area increased. Simultaneously, bundled actin stress fibers (SFs) were observed vertically from the positions of these FAs, and they connected to the other SFs parallel to the leading edge. Finally, these connecting SFs fused to form a single SF with matured FAs at both ends. This change in SF organization with cell retraction in the first cycle of migration followed by a newly formed protrusion in the next cycle is assumed to lead to cell turning in migrating Swiss 3T3 fibroblasts.