Practical and safe method of cryopreservation for clinical application of human adipose-derived mesenchymal stem cells without a programmable freezer or serum

BackgroundAdipose-derived mesenchymal stem cells (ADSCs) have emerged as a promising therapeutic modality for cellular therapy because of their rapid proliferation and potent cellular activity compared to conventional bone marrow-derived mesenchymal stem cells (MSCs). Cosmetic lipoaspirates provide an easily obtainable source of ADSCs. Cryopreservation facilitates their clinical application due to increased transportability and pooling of sufficient numbers of cells. However, proper cryopreservation techniques have not been established yet.MethodsWe evaluated the post-thaw viability and ADSC functions after cryopreservation with three cryoprotectants (serum containing 10% dimethylsulfoxide (DMSO), serum-free: CP-1TM, DMSO-free: SCB-DFTM) at two temperature (−80°C, −150°C) and two cell densities: (1 × 106, 7 × 106cells/mL) for up to 18 months using cryovials. After determining optimal conditions, we also tested if large quantities of ADSCs remained viable after 18 months of cryopreservation in a 100-mL cryobag. Rate-controlled freezing methods or liquid nitrogen storage were not exploited.ResultsADSCs cryopreserved in serum containing 10% DMSO or CP-1TMat −150°C and 7 × 106cells/mL were most viable (>85%) after 18 months without perturbation of MSC functions. Even suboptimal conditions (−80°C, 1 × 106cells/mL, no DMSO) assured >80% viability when stored for up to 9 months. Large quantities of ADSCs in a cryobag were properly cryopreserved.ConclusionsA programmable freezer or liquid nitrogen storage is not necessary. CP-1TMis preferable in terms of side effects. Simplified cryopreservation methods (−80°C and no DMSO) can be used for up to 9 months, resulting in reduced infusion toxicities and lower costs.