Efficient cryopreservation of human mesenchymal stem cells using silkworm hemolymph-derived proteins

Cryopreservation methods for human mesenchymal stem cells (hMSCs) typically depend on the presence of fetal bovine serum (FBS) with dimethyl sulphoxide (DMSO), which is not appropriate for therapeutic applications. In our previous study, we found that storage protein 2 (SP2), a natural material derived from silkworm hemolymph, has an inhibitory effect on the generation of reactive oxygen species (ROS). In this study, we used SP2 as an alternative to establish an effective, low-DMSO and FBS-free cryopreservation agent for the cryostorage of hMSCs. We investigated the cell viability and stem cell characterization of umbilical cord-derived MSCs in different freezing media through the freezing and thawing process. We also evaluated the efficacy of cryostorage using these media over 1 week and 1 year. When the cell characteristics (cell viability and stemness) were analysed after thawing, those obtained using 5 mg/ml SP2 were comparable to those obtained using a freezing medium with FBS. The stable cell viability and characteristics were shown even after 1 year of cryopreservation. In addition, when the cells were differentiated into adipocytes and osteocytes, we confirmed that the differentiation behaviours of the thawed cells were well maintained. The positive results could be also obtained when SP2 was applied to other MSCs. The results clearly indicate that SP2 could be used as an alternative to FBS for a freezing medium with reduced DMSO. Copyright (c) 2016 John Wiley & Sons, Ltd.