3D Culture of MSCs on a Gelatin Microsphere in a Dynamic Culture System Enhances Chondrogenesis

Abstract

Recent advancement in cartilage tissue engineering has explored the potential of 3D culture to mimic the in vivo environment of human cartilaginous tissue. Three-dimensional culture using microspheres was described to play a role in driving the differentiation of mesenchymal stem cells to chondrocyte lineage. However, factors such as mechanical agitation on cell chondrogenesis during culture on the microspheres has yet to be elucidated. In this study, we compared the 2D and 3D culture of bone-marrow-derived mesenchymal stem cells (BMSCs) on gelatin microspheres (GMs) in terms of MSC stemness properties, immune-phenotype, multilineage differentiation properties, and proliferation rate. Then, to study the effect of mechanical agitation on chondrogenic differentiation in 3D culture, we cultured BMSCs on GM (BMSCs-GM) in either static or dynamic bioreactor system with two different mediums, i.e., F12: DMEM (1:1) + 10% FBS (FD) and chondrogenic induction medium (CIM). Our results show that BMSCs attached to the GM surface and remained viable in 3D culture. BMSCs-GM proliferated faster and displayed higher stemness properties than BMSCs on a tissue culture plate (BMSCs-TCP). GMs also enhanced the efficiency of in-vitro chondrogenesis of BMSCs, especially in a dynamic culture with higher cell proliferation, RNA expression, and protein expression compared to that in a static culture. To conclude, our results indicate that the 3D culture of BMSCs on gelatin microsphere was superior to 2D culture on a standard tissue culture plate. Furthermore, culturing BMSCs on GM in dynamic culture conditions enhanced their chondrogenic differentiation.