Fetal bovine serum-free cryopreservation methods for clinical banking of human adipose-derived stem cells

The use of fetal bovine serum (FBS) as a cryopreservation supplement is not suitable for the banking of mesenchymal stem cells (MSCs) due to the risk of transmission of disease as well as xenogeneic immune reactions in the transplanted host. Here, we investigated if human serum albumin (HSA), human serum (HS), or knockout serum replacement (KSR) can replace FBS for the cryopreservation of MSCs. In addition, we examined the characteristics of MSCs after multiple rounds of cryopreservation. Human adipose-derived stem cells (ASCs) cryopreserved with three FBS replacements, 9% HSA, 90% HS, or 90% KSR, in combination with 10% dimethyl sulfoxide (Me2SO) maintained stem cell properties including growth, immunophenotypes, gene expression patterns, and the potential to differentiate into adipogenic, osteogenic, and chondrogenic lineages, similar to ASCs frozen with FBS. Moreover, the immunophenotype, gene expression, and differentiation capabilities of ASCs were not altered by up to four freeze-thaw cycles. However, the performance of three or four freeze-thaw cycles significantly reduced the proliferation ability of ASCs, as indicated by the longer population doubling time and reduced colony-forming unit-fibroblast frequency. Together, our results suggest that HSA, HS, or KSR can replace FBS for the cryopreservation of ASCs, without altering their stemness, and should be processed with no more than two freeze-thaw cycles for clinical approaches.