Growth and Differentiation of Human Dental Pulp Stem Cells Maintained in Fetal Bovine Serum, Human Serum and Serum-free/Xeno-free Culture Media

Abstract

Introduction: Dental pulp stem cells (DPSCs) are an accessible cell source with therapeutic applicability in regeneration of damaged tissues. Current techniques for expansion of DPSCs require the use of Fetal Bovine Serum (FBS). However, animal-derived reagents stage safety issues in clinical therapy. By expanding DPSCs in serumfree/ xenofree medium (SF/XF-M) or in medium containing human serum (HS-M), the problems can be eliminated. Therefore, the aim of our study was to identify suitable cell culture media alternatives for DPSCs. Methods: We studied the isolation, proliferation, morphology, cell surface markers (CD29, CD44, CD90, CD105, CD31, CD45 and CD146), stemness markers expression (Oct3/4, Sox2, Nanog and SSEA-4) and in vitro multilineage differentiation of DPSCs in HS-M or SF/XF-M in comparison to FBS-M. Results: DPSCs expressed the cell surface and stemness markers in all studied conditions. The proliferation analysis of cells cultured in different HS concentrations revealed that cells isolated in 20% HS-M and passaged in 10% or 15% HS-M supported the cell growth. Direct isolation of cells in SF/XF-M did not support cell proliferation. Therefore, cells cultured in 20% HS-M were used for further SF/XF-M studies. However, proliferation of DPSCs was significantly lower in SF/XF-M when compared with cells cultured in FBS-M and HS-M. In addition, proliferation of DPSCs in SF/XF-M could be enhanced by addition of 1% HS in cell culture medium. There were differences in osteogenic, chondrogenic and adipogenic differentiation efficacy between cells cultured in FBS, HS and SF/XF differentation media. More pronounced adipogenic and osteogenic differentiation was observed in HS differentiation medium, however, in FBS-M cultured cells more effective chondrogenic differentiation was detected. Conclusions: Our results indicate that HS is a suitable alternative to FBS for the expansion of DPSCs. The composition of SF/XF-M needs to be further optimized in terms of cell expandability and differentiation efficiency to reach clinical applicability.