Long-term cryopreservation of human mesenchymal stem cells using carboxylated poly-l-lysine without the addition of proteins or dimethyl sulfoxide.

Human bone marrow-derived mesenchymal stem cells (hBMSCs) are known for their potential to undergo mesodermal differentiation into many cell types, including osteocytes, adipocytes, and chondrocytes. Therefore, hBMSCs can be used for a variety of regenerative medicine therapies, in fact, hBMC-derived osteocytes have already been used in bone reconstruction. This study discusses the viability and the differentiation properties of hBMSCs that have been cryopreserved in the absence of proteins or dimethyl sulfoxide (DMSO) by using a novel polyampholyte cryoprotective agent (CPA). This CPA is based on carboxylated poly-l-lysine (COOH-PLL) and it was produced by a reaction between ε-poly-l-lysine and succinic anhydride. (1)H-NMR and two-dimensional correlation ((1)H-(13)C HSQC) spectroscopy revealed that COOH-PLL did not have a special structure in solution. The hBMSCs can be cryopreserved for 24 months at -80 °C by using a 7.5% (w/w) cryopreserving solution of COOH-PLL, which introduces carboxyl groups that result in > 90% cell viability after thawing. Furthermore, the cryopreserved hBMSCs fully retained both their proliferative capacity as well as their potential for osteogenic, adipogenic, and chondrogenic differentiation. Confocal laser-scanning microscopy showed that the polyampholyte CPA did not penetrate the cell membrane; rather, it attached to the membrane during cryopreservation. These results indicate that the cryoprotective mechanisms of COOH-PLL might differ from those of currently used small molecule CPAs. These results also suggest that using COOH-PLL as a CPA for hBMSC preservation can eliminate the use of proteins and DMSO, which would be safer if these cells were used for cell transplantation or regenerative medicine.