Cryopreservation of monkey stem cells: Development of safe and efficacious protocols

Cryopreservation is a procedure to preserve living cells and tissues without loss of viability using ultra low temperatures. Since there is great interest in the use of multipotent stromal cells (MSCs) for therapeutic, clinical and commercial purposes, suitable cryopreservation protocols for these cells exhibiting maximum cell viability without genetic and epigenetic alterations are necessary. Dimethyl sulfoxide (Me2SO) is the most commonly used cryoprotective agent (CPA) used for freezing MSCs. However, due to its time, temperature and concentration-dependent toxicity, alternative non-toxic CPAs are increasingly being studied for their potential to maintain cellular viability. In the present study, the cryoprotective ability of additional CPAs, including methylcellulose, poloxamer 188 and α-tocopherol has been investigated. In the absence of serum and at concentrations of 2.5% (v/v) Me2SO, the cell viability and differentiation potential of bone marrow derived MSCs from Callithrix jacchus could be maintained and was comparable to cells frozen with 5% (v/v) Me2SO and FBS using a two-step freezing protocol. The effects of cryopreservation on the expression of stress related genes and epigenetic changes were also investigated. Two time-points (12 hours and 2 weeks post-thaw) were chosen to study these effects. Stress related genes described to be up/downregulated after cryopreservation did show deviations from the non-frozen cells after 12 hours post-thaw, but expression was comparable to non-frozen control cells after two weeks of in vitro culture. Global DNA methylation and epigenetic related gene expression were completely restored to pre-freeze levels after long term culture. The present studies indicate that long term recultivation of MSCs (2 weeks) was enough for cells to have genetic and epigenetic expression comparable to fresh non-frozen cells. Taken together, these results provide important information for developing safe and efficacious cryopreservation procedures for MSCs.