C-2027: Enhancement of cell survival rate with active control of nucleation temperature

Abstract

For the field of regenerative medicine the preservation of cells and tissue in liquid nitrogen is the state of the art method for their long term storage. There are problems in conservation of complex tissues and organs, because mechanisms behind tissue damage are still not fully understood. At the moment it is only possible to preserve single cell suspensions and small tissue pieces with high viability. The viability of cryopreserved cells can be enhanced by optimizing the freezing parameters. These parameters include cooling/thawing rate, cryoprotective agent (CPA) concentration and osmotic response of the cell membrane (Mazur, 1984). In the last years a new parameter, nucleation, came into focus as a contrast to cell survival. Nucleation is the process of accumulation of water molecules to form a nucleation embryo. If the critical size is passed a phase change from liquid to solid occurs. There are many ways to induce nucleation such as seeding, ultra sound, bacteria, pressure shift and electrofreezing. In this work we used electrofreezing and seeding to induce nucleation. In the literature there are few studies to optimize the nucleation temperature. In this study we investigated the influence of different nucleation temperatures on mesenchymal stem cells derived from the common marmoset, Callithrix jacchus (cjMSC) concerning membrane activity, recultivation efficiency and metabolic activity. Cells were frozen under optimal conditions using a controlled rate freezer and a nucleation device (electro freezing) using different concentrations of dimethylsulfoxide (Me2SO). The effect of the nucleation temperature on cell viability and proliferation of cjMSC were studied by analyzing the membrane integrity with trypan blue staining, efficiency of recultivation and metabolic activity with Alamar blue assay. Our results showed that electro freezing with active controlled induced nucleation at the desired temperature has a positive effect on the survival rate of cryopreserved cells. Discloure: This work was funded by the excellence cluster REBIRTH (DFG EXC 62/1).