Cryobiological parameters of multipotent stromal cells obtained from different sources

Abstract

Stem cells are important for regenerative medicine mainly due to their multilineage differentiation capacity. However, the cells rapidly loose this capability during culturing. Cryopreservation preserves the differentiation potential of the cells, until they are needed. In this study, specific cell properties of multipotent stromal cells (MSCs), from the common marmoset monkey Callithrix jacchus MSCs derived from amnion (Am) and bone marrow (Bm) were studied in order to predict optimal cooling rates for cryopreservation. Cell volume behaviour in anisotonic media, hydraulic membrane permeability at supra as well as subzero temperatures, and time point of intracellular ice formation (IIF) were investigated by Coulter Counter and cryomicroscopy. Cryopreservation outcome was studied using the predicted and experimentally determined cooling rate followed by 24 h re-cultivation. Little differences in osmotically inactive volume were found between amnion (0.27 × V_o) and bone marrow (0.28 × V_o) derived MSCs. The activation energy for water transport at suprazero temperature was found to be similar for both cell types; 4.4 ± 0.2 and 5.0 ± 0.15 kcal mol^-1 for amnion and bone marrow derived MSCs, respectively. At subzero temperatures in the absence of dimethyl sulfoxide (Me₂SO), the activation energy for water transport increased to 24.8 ± 3 kcal mol^-1 and 27.4 ± 0.9 kcal mol^-1 for Am and BmMSCs respectively. In the presence of Me₂SO, activation energies were found to be 11.6 ± 0.3 kcal mol^-1 and 19.5 ± 0.5 kcal mol^-1 respectively. Furthermore, Me₂SO was found to decrease the incidence of intracellular ice formation. The predicted optimal cooling rates of 11.6 ± 0.9 °C/min (AmMSCs) and 16.3 ± 0.5 °C/min (BmMSCs) resulted in similar post-thaw viability values compared to the experimentally determined optimal cooling profiles of 7.5 °C/min to -30 °C, followed by 3 °C/min to -80 °C.

Keywords: Cellular dehydration; Cryomicroscopy; Induced nucleation; Intracellular ice formation; Multipotent stromal cells; Suprazero and subzero membrane hydraulic permeability.