The Combination of Trehalose and Glycerin: An Effective and Non-Toxic Recipe for Clinical Cryopreservation of Human Adipose-Derived Stem Cells

Abstract

Background: Adipose-derived stem cells (ADSCs) promote tissue regeneration and repair. Cryoprotective agents (CPA) protect cells from cryodamage in the process of cryopreservation. Safe and efficient cryopreservation of ADSCs is critical in the clinical application of cell-based therapy. However, most CPAs contain toxic concentrations limiting the possibility of their clinical application. Objective: The aim of this study is to develop a non-toxic xeno-free CPA for ADSCs to achieve high-efficiency and low-risk cryopreservation. Methods: We explored the most efficient concentrations in different concentrations of trehalose (0.3M, 0.6M, 1.0M, and 1.25M) and glycerol (10%, 20%, 30% v/v); then evaluated the outcome of the combination of trehalose and glycerol in ADSC cryopreservation, compared to the commonly used CPA, DMSO (10%) + FBS (90%). All samples were slowly freezed and stored in liquid nitrox for 30 days. The effectiveness was evaluated by the cell viability, proliferation, migration and multi-potential differentiation of ADSCs after thawing. Results: Compared to the CPAs with single reagent, 1.0M Tre + 20%Gly group showed significantly higher efficiency in preserving ADSCs activities after thawing, with better outcome in both cell viability and proliferating capacity. Compared to 10%DMSO+90%FBS, ADSCs preserved in 1.0M Tre + 20%Gly group showed similar cell viability, surface markers and multi-potential differentiation but significantly higher migration capability, indicating that better cell function preservation can be achieved by 1.0M Tre + 20%Gly. Conclusions: 1.0M Tre + 20%Gly can preserve ADSCs with high migration capability and cell viability compared to 10%DMSO+90%FBS and maintain similar stemness and multi-potential differentiation as fresh cells. Our results demonstrate that 1.0M Tre + 20%Gly can achieve highly efficient cryopreservation of ADSCs and is suitable for clinical applications.