Xeno-free plant-derived hydrolysate-based freezing of human embryonic stem cells

Human embryonic stem cells (hESCs) are one of the most interesting cell types for tissue engineering and cell therapy. The large-scale banking of hESCs for research and future clinical application requires economic, defined, and xeno-free cryopreservation protocols. In this study, the possibility to substitute knockout serum replacement (KO-SR) in the cryopreservation process with vegetal and synthetic hydrolysates was investigated. To our knowledge, the use of hydrolysates in hESC cryopreservation has not been yet explored. Initially, 3 different hydrolysates (Ultrapep Soy, Hypep 4601 and EX-CELL(®) CD Hydrolysate Fusion) were tested in the cryopreservation solution. A concentration of 8 mg/mL EX-CELL CD Hydrolysate Fusion in the cryopreservation solution leads to the highest recovery ratio; thus, this solution was selected for additional cryopreservation experiments. To ensure reproducibility of the results, 3 hESC lines (HS181, H9, and BG01) were used. The hESCs were collected prefreezing by application of collagenase IV and cell dissociation solution. Experiments showed that it was feasible to substitute the KO-SR in both the cryopreservation solution as the thawing solution. The obtained recovery ratios were comparable to those obtained with KO-SR (no statistical significant difference; Student's t-test, P<0.05). Further optimization protocols showed a doubling of the obtained recovery ratio after addition of Rock-inhibitor Y-27632 post-thawing. The expansion profile and pluripotency analysis revealed no changes in normal hESC behavior. We conclude that the application of vegetal or synthetic hydrolysates is suitable for xeno-free hESC cryopreservation.