Phenotypic and Functional Characterization of Long-Term Cryopreserved Human Adipose-derived Stem Cells

Abstract

Cryopreservation represents an effective technique to maintain the functional properties of human adipose-derived stem cells (ASCs) and allows pooling of cells via long-term storage for clinical applications, e.g., cell-based therapies. It is crucial to reduce freezing injury during the cryopreservation process by loading the ASCs with the optimum concentration of suitable cryoprotective agents (CPAs). In this study, human ASCs were preserved for 3 months in different combinations of CPAs, including 1) 0.25 M trehalose; 2) 5% dimethylsulfoxide (DMSO); 3) 10% DMSO; 4) 5% DMSO + 20% fetal bovine serum (FBS); 5) 10% DMSO + 20% FBS; 6) 10% DMSO + 90% FBS. Interestingly, even with a reduction of DMSO to 5% and without FBS, cryopreserved ASCs maintained high cell viability comparable with standard cryomedium (10% DMSO + 90% FBS), with normal cell phenotype and proliferation rate. Cryopreserved ASCs also maintained their differentiation capability (e.g., to adipocytes, osteocytes and chondrocytes) and showed an enhanced expression level of stemness markers (e.g., NANOG, OCT-4, SOX-2 and REX-1). Our findings suggest that 5% DMSO without FBS may be an ideal CPA for an efficient long-term cryopreservation of human ASCs. These results aid in establishing standardized xeno-free long-term cryopreservation of human ASCs for clinical applications.

Cryopreservation and its clinical applications

“…Like glycerol, DMSO acts by reducing the electrolyte concentration in the residual unfrozen solution in and around a cell at any given temperature. However, a decline in the survival rate and the induction of cell differentiation caused by DNA methylation and histone alteration have been reported 28, 29. These negative effects of DMSO in cryopreservation create some difficulties for its use in routine clinical applications.…”

Assessment of tumourigenic potential in long-term cryopreserved human adipose-derived stem cells

“…To date, cryopreservation, an efficient method of preserving the viability and functional properties (e.g. proliferation and differentiation potential) of hMSCs in the long term (Gonda et al ., ; Yong et al ., ), is an alternative method that allows pooling of hMSCs to obtain sufficient cells required for clinical applications. However, the biosafety profile of cryopreserved hMSCs has not yet been established.…”

The Relative Functionality of Freshly Isolated and Cryopreserved Human Adipose-Derived Stromal/Stem Cells

“…For clinical and research purposes, ASCs often need to be cryopreserved in liquid nitrogen in a manner that maintains their immunophenotype, proliferative and differentiation properties. While some published comparisons have reported that cryopreserved ASCs retain these features [Yong et al, 2015], some authors have noted a decrease in differentiation potential [James et al, 2011]. Cell surface antigens are used to distinguish cell types and it has been reported that ASCs retain their immunophenotype after cryopreservation [De Rosa et al, 2009].…”