043 Antioxidants as additional additives to CPA may help to minimize epigenetic modifications caused by Me2SO

Cryopreservation plays an important role in the long-term storage of cells and tissues. A high survival rate is a function of optimal cooling rate, appropriate cryoprotective agent (CPA) and its adjusted concentration. The most widely used CPA, dimethyl sulfoxide (Me2SO), however is toxic at high concentrations and has detrimental effects on the biological functioning of a cell. Therefore, it is of great interest to develop new cryoprotective strategies to replace currently used CPAs or to reduce their concentration. Since one of the major damaging factors is the occurence of ROS during and after thawing we have investigated the addition of ascorbic acid and α-tocopherol as potential antioxidants. Mesenchymal stem cells (MSC) of the common marmoset monkey were frozen in 200 μl PCR-tubes (5x105 cells/ml) with optimal cooling rate (Pogozhykh et al., Reg. Med. 6 (6s2) (2011) 229) in a μ-freezer device. Different concentrations of ascorbic acid (50, 100 and 250 mM) and α-tocopherol (100, 200 and 500 μM) were studied exclusive or in combination and with Me2SO (2,5 and 5% v/v). Cells attached to the culture flask surface after 24 h recultivation were considered as viable, the survival rate was compared to that of cells frozen with Me2SO as positive control. Surviving cells were further tested for differentiation capability. The addition of α-tocopherol improved the survival of primate MSCs after cryopreservation even with 2,5% Me2SO. With 100 μM α-tocopherol and 5% Me2SO survival was more than two fold higher than with 5% Me2SO alone. Ascorbic acid had no considerable effect on the survival rate of MSCs but in combination with tocopherol its addition stabilized the positive effects. Intracellular lipid vesicles were clearly stained by Oil Red O and proved the adipogenic differentiation capability of marmoset MSCs after cryopreservation with antioxidant addition. Conclusions: Accessory application of α-tocopherol and ascorbic acid improved survival and proliferation of MSCs from the marmoset monkey after cryopreservation. Me2SO could be reduced to moderate concentrations. These findings will help to avoid epigenetic modifications caused by Me2SO which preliminary results showed. We thank L. Smits, S. Hüper and M. Abbetmeier-Basse for their outstanding technical assistance. Source of funding: This work is supported by funding from the Deutsche Forschungsgemeinschaft for the Cluster of Excellence REBIRTH (EXC 62/1). Conflict of interest: None declared. [email protected]