Importance of controlled ice formation for efficient cell biobanking.

Effective long-term storage of rare and clinically relevant cells depends on the cell type and thus requires optimization of the main process parameters involved in cryopreservation. Among these parameters, the cooling and thawing rates, as well as the temperature of nucleation can be adjusted by a specific cryopreservation method. In this work, we reveal the optimal conditions for cryopreservation of human fibroblasts (HF), human pulmonary microvascular endothelial cells (HPMECs), amnion (aMSCs), and bone marrow stem cells (bMSCs) using an electro-freezing method and applying 2.5 %, 5 % or 10 % (v/v) dimethyl sulfoxide (Me2SO) as a cryoprotective agent. The optimal nucleation temperature for freezing of HF was −10 °C for a cooling rate of 1 K·min-1 and −7.5 °C for 5 K·min-1 using 5 % Me2SO. Application of a cooling rate of 5 K·min-1 and induction of the ice formation at −12 °C resulted in 90 % of viable HPMECs. The aMSCs and bMSCs reflected the highest viability of 75 % after freezing using a two-step freezing protocol utilizing a cooling rate of 7.5 K·min-1 down to −30 °C and 3 K·min-1 down to −80 °C. The highest cell viability was observed while inducing the ice formation at −10 °C for both aMSCs and bMSCs.