Cytotoxicity effects of cryoprotectants as single-component and cocktail vitrification solutions

Cryoprotectant (CPA) cytotoxicity constitutes a challenge in developing cryopreservation protocols, specifically in vitrification where high CPA concentrations are necessary to achieve the ice-free, vitreous state. Few cytotoxicity studies have investigated vitrification-relevant concentrations of CPAs, and the benefits and disadvantages of cocktail solutions and of incorporating non-permeating solutes have not been fully evaluated. In this study, we address these issues by determining the cytotoxicity kinetics for dimethylsulfoxide (Me(2)SO) and 1,2-propanediol (PD) on alginate-encapsulated βTC-tet mouse insulinomas for a range of concentrations and temperatures. Cytotoxicity kinetics were also determined for two cocktails, DPS (3M Me(2)SO+3M PD+0.5M sucrose) and PEG400 (1M Me(2)SO+5M PD+0.34M poly(ethylene)glycol with M.W. of 400). PD was found to be more cytotoxic than Me(2)SO at higher concentrations and temperatures. This was reflected in PEG400 being more cytotoxic at room temperature than PEG400 at 4°C or DPS at either temperature. Addition of non-permeating solutes increased the cytotoxicity of cocktails. Furthermore, results indicate that CPA cytotoxicity may not be additive and that combining CPAs may increase cytotoxicity synergistically. Finally, when comparing cytotoxic effects towards encapsulated HepG2 and βTC-tet cells, and towards βTC-tet cells in capsules and in monolayers, CPAs appear more cytotoxic towards cells with higher metabolic activity. The incorporation of these results in the rational design of CPA addition/removal processes in vitrification is discussed.