The development of cryopreservation (CP) strategies has traditionally focused on the cellular chemo-osmometric characteristics attendant to the freeze-thaw process. This approach coupled with a limited understanding of cellular physiological and biochemical responses to the CP process often yields sub-optimal cell survival. Recently, we have reported on the benefits of the utilization of an intracellular-like preservation solution, HypoThermosol (HTS), as well as incorporating a molecular approach to improving CP outcome [In Vitro Cell. Dev. Biol. Anim. 36(4) (2000) 262]. We now report on the elucidation of a cryoprotective agent (CPA)-dependent survival limit (cap) associated with standard CP methodologies. We further demonstrate an elevation and shift in the CP cap through the utilization of HTS coupled with a reduction in CPA levels necessary to achieve "successful" cell preservation.
Methods: Human fibroblasts, keratinocytes, hepatic, and renal cells were cryopreserved in a standard fashion (approximately 1 degrees C min-1 cooling and storage in LN2) in culture media (serum-free) or HTS with varying levels of dimethyl sulfoxide (Me2SO). Samples were allowed to recover for 24-h prior to survival assessment. Survival was assessed using alamarBlue (metabolic activity indicator) and calcien-AM (membrane integrity stain) in comparison with non-frozen controls.
Results: (1) A limit in cell survival was identified following CP in media-based CP solutions yielding a cell-type specific CPA-dependent survival limit, (2) peak cell survival resulted in the identification of "optimal" Me2SO concentrations for CP of each cell type, (3) incorporation of HTS as the carrier medium at typical Me2SO concentrations substantially elevated survival, and (4) utilization of HTS allowed for the successful preservation of all systems examined at significantly reduced Me2SO levels.
Conclusion: The data presented in this study illustrate that the utilization of HTS as the carrier medium during CP facilitated a significant improvement in efficacy at reduced Me2SO levels. Further, the utilization of HTS offers the potential for successful Me2SO-free CP. These findings may prove significant to the advancement in the development of cell-based clinical therapies by providing an improved biocompatible CP methodology.