Cryopreservation is one of the most practical and effective methods to preserve cells and tissues in the frozen state for extended periods of time to ensure off-shelf availability in tissue engineering, stem cell therapy and drug testing. However, stresses resulted from cryopreservation, such as osmotic and cold shock, can cause irreversible damage to cells.

Although human mesenchymal stem cells (hMSCs) isa good candidate in regenerative medicine, few reports are published on the cryopreservation of adherent hMSCs. Researchers with Institute of Process Engineering (IPE) investigated the responses of adherent hMSCs to the osmotic and cold shock during cryopreservation, including the changes in cell viability, intracellular properties and the capacity of recovery after cryopreservation.

The experiments maily included the following steps: maintenance of hMSC, hMSC culture for evaluation of the effect of cryopreservation procedures,CPA addition and CPA addition/removal without freezing,cryopreservation of hMSCs, assessment of cell morphology and cell viability, assessment of cell metabolic activity and proliferation, F-actin morphology, intracellular pH and mitochondria distribution and statistics analysis.

The results showed a significant decrease in cell viability around 30% after cryopreservation at the cooling rates of 1, 5 and 10 °C/min in comparison to the adherent cells and the cells in suspension, implicating that the adherent cells are more vulnerable than the suspension cells.

The osmotic shock and cold shock induced by freezing leaded to dramatic changes in the intracellular properties. The cooling rate of 10 °C/min resulted in acidification of intracellular pH, distortion and accumulation of filamentous actin, and aggregation of mitochondria. The findings also suggested that the cooling rate of 1 °C/min help to maintain cell morphology and attachment, integrity and uniformity of filamentous actin, and leaded to better cell recovery after cryopreservation.

The experiment results may help to increase the understanding of the cryopreservation of adherent cells. More information about this study please consult Journal of Biotechnology.