Preservation of embryonic kidneys for transplantation

Abstract

Background Long-term storage of embryonic kidneys is crucial for the organization of transplantation and organ banking. In this study, we investigated the effects of controlled-rate freezing and ice-free vitrification on metanephroi (MN) viability. Methods Metanephroi isolated from 15-day (E15) timed pregnant Lewis rats were either: (i) frozen, using a DMSO/FCS/RPMI solution and a controlled freezing rate of −0.3°C/min, from −10° to −40°C; or (ii) cryopreserved in an ice-free state by rapid cooling to −100°C in cryoprotectant (VS55), followed by vitrification to −120°C. After cryopreservation, the metanephroi were stored at −135°C for 48 hours. After storage the MN were rewarmed, resuspended in culture media, and their viability was assessed using the AlamarBlue assay and histology (light microscopy, TEM, and cryosubstitution). Results There was statistically no difference in embryonic kidney metabolic activity of either of the cryopreserved MN groups relative to the control untreated group. However, cryosubstitution demonstrated the presence of significant ice formation during controlled-rate freezing, yet in contrast the amount of ice was significantly reduced by vitrification. This was confirmed by TEM, where vacuolation of the cytoplasm of controlled-rate frozen metanephroi was observed, whereas vitrified metanephroi had little cytoplasmic disruption. However, vitrified metanephroi showed mitochondrial and nuclear injury at the cellular level. Conclusions There is a need for long-term storage of organs to make MN transplantation a reality. This study demonstrates that standard freezing methods are unsuitable for this purpose. Vitrification yielded more promising results, but further development is required.