Slow Freezing Cryopreservation in Combination With Cryoprotectants Preserve Gingival Mesenchymal Stem Cells

Abstract

Introduction: Development of gingival mesenchymal stem cells (GMSCs) cryopreservation procedures is needed to conserve GMSC in stem cell-based therapy. This study aimed to evaluate slow freezing cryopreservation and cryoprotectants for GMSCs. Methods: Gingival tissue from extracted human third molars was separated from teeth. The minced-gingival tissues were plated in tissue culture dishes, added culture media, and incubated at 37o C in 5% CO2 . Morphology and flowcytometry analysis were determined on the fourth passage of gingival cells. GMSCs were separated into two groups of noncryopreserved-GMSCs (ncGMSCs) and cryopreserved-GMSCs (cGMSCs). The GMSCs were frozen by slow freezing with CPA in the following combinations: 1) 100% Cell Banker (CB-group), 2) 90% FBS+10% DMSO (FDs-group), 3) 90% FBS+10% DMEM (FD-group), and 4) 90% DMEM+10% DMSO (DDs-group). Trypan blue dye exclusion was used to assess the proliferation of ncGMSCs and cGMSCs. The Oil Red O, Alizarin Red, and Alcian Blue staining were used to determine their multipotencies. Results: Gingival cells and GMSCs showed fibroblastic-like morphology. They did not express hematopoietic cell markers of CD11b/CD19/CD34/CD45 and HLA-DR, but expressed more than 90% positive MSC surface markers of CD90, CD73, CD150, and CD44. The cGMSCs viability of FDs-group was 81% and 80% in -80o C and LN2 , respectively. There was no statistically significant difference (p>0.05) in proliferation and doubling time between ncGMSCs and cGMSCs. They had ability to develop into chondrogenic, osteogenic, and adipogenic differentiation. Conclusion: Slow freezing cryopreservation in combination with 90% FBS+10% DMSO retain the biological properties of GMSCs, and it can be developed to GMSCs banking.