Cryopreservation of an engineered skin equivalent: the apoptosis paradigm

The emergence of the tissue engineering sciences has led to an immediate need for cryopreservation protocols compatible with cell survival, cell-matrix binding and 'scaffold' integrity. While numerous problems exist related to directional restrictions in cryoprotectant transport in tissue models, recent findings have demonstrated that the induction of specific molecular events, especially gene regulated cell death (apoptosis) is of significance. Accordingly, we report on a new strategy for the successful cryopreservation of a human skin equivalent. The integration of an intracellular-type cryopreservation solution containing dimethyl sulfoxide with and without apoptotic inhibitors provides a successful model for tissue preservation. With optimized formulations, post-thaw improvement of viability upwards of three-fold has been demonstrated when compared with traditional preservation methodologies. We conclude that 1) the use of an intracellular-type cryopreservation medium, Hypothermosol, can improve post-thaw tissue construct integrity and cell viability, and 2) the inclusion of apoptotic inhibitors significantly improves cryopreservation outcome.