Long-term viability of cryopreserved cultured epithelial grafts

Human cultured epithelial grafts are frozen for long-term preservation. To assess the viability of these stored grafts, their cell survival rate and colony-forming efficiency of grafts cryopreserved at -135 degrees C and at -80 degrees C were followed over time. Flow cytometry showed that the cell survival rate of the grafts cryopreserved at -135 degrees C for 1 month, 6 months and 1 year averaged 89.3%, 61.7% and 61.6%. Cryopreservation at -80 degrees C maintained cell survival rate as well for 1 month, but after 6 months of cryopreservation survival was reduced at -80 degrees C (35.2%) compared with that of -135 degrees C. In histological examination, the cell structure and basal layer were very well preserved after 6 months of storage at -135 degrees C, but not at -80 degrees C. Cell survival rate at -135 degrees C was also assessed by colony-forming efficiency. Colony-forming efficiency of the grafts cryopreserved for 1 month, 6 months and 1 year averaged 66.1%, 58.5% and 55.1% of control (noncryopreserved) grafts. These findings suggest that, even when cultured epithelial grafts are subjected to long-term cryopreservation, cell viability remains sufficient, reculturing is possible, and that graft banking could be used for clinical applications.