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

Abstract

Background: Adipose-derived stem cells (ADSCs) promote tissue regeneration and repair. Cryoprotective agents (CPAs) protect cells from cryodamage during cryopreservation. Safe and efficient cryopreservation of ADSCs is critical for cell-based therapy in clinical applications. However, most CPAs are used at toxic concentrations, limiting their clinical application. Objective: The aim of this study is to develop a non-toxic xeno-free novel CPA aiming at achieving high-efficiency and low-risk ADSC cryopreservation.Methods: We explored different concentrations of trehalose (0.3 M, 0.6 M, 1.0 M, and 1.25 M) and glycerol (10%, 20%, and 30% v/v) for optimization and evaluated and compared the outcomes of ADSCs cryopreservation between a combination of trehalose and glycerol and the commonly used CPA DMSO (10%) + FBS (90%). All samples were slowly frozen and stored in liquid nitrogen for 30 days. The effectiveness was evaluated by the viability, proliferation, migration and multi-potential differentiation of the ADSCs after thawing. Results: Compared with the groups treated with individual reagents, the 1.0 M Tre + 20% Gly group showed significantly higher efficiency in preserving ADSC activities after thawing, with better outcomes in both cell viability and proliferation capacity. Compared with the 10% DMSO + 90% FBS treatment, the ADSCs preserved in 1.0 M Tre + 20% Gly showed similar cell viability, surface markers and multi-potential differentiation but a significantly higher migration capability. The results indicated that cell function preservation can be improved by 1.0 M Tre + 20% Gly. Conclusions: The 1.0 M Tre + 20% Gly treatment preserved ADSCs with a higher migration capability than 10% DMSO + 90% FBS and with viability higher than that with trehalose or glycerol alone but similar to that with 10% DMSO + 90% FBS and fresh cells. Moreover, the new CPA achieves stemness and multi-potential differentiation similar to those in fresh cells. Our results demonstrate that 1.0 M Tre + 20% Gly can more efficiently cryopreserve ADSCs and is a non-toxic CPA that may be suitable for clinical applications.

The Effect of Glycerol as a Cryoprotective Agent in the Cryopreservation of Adipose Tissue

“…Several studies have reported that using DMSO or DMSO + FBS as CPAs was effective in protecting adipose tissue cell survival and retention of the adipose tissue volume compared with the absence of CPA 9,[21][22][23][24] . However, the use of DMSO and FBS has the risk of toxicity and zoonotic infection 25 . When these agents are applied in tissue cryopreservation, especially in adipose tissue that is grafted into the human body in a large volume, toxicity may become a major concern [26][27][28] .…”