PRESERVATION OF EMBRYONIC KIDNEYS FOR TRANSPLANTATION

Abstract

P30 Aims: Long-term storage of embryonic kidneys is crucial for the organisation of transplantation and organ banking. To date, ex vivo preservation of rat embryonic kidneys has proved feasible for three days in ice cold University of Wisconsin solution with added growth factors. In this present study, the effects of controlled rate freezing and ice-free vitrification on metanephroi (mn) viability were investigated. Vitrification is amorphous solidification of a supercooled liquid and it is achieved by adjusting the solute composition and the cooling rate such that nucleation and growth of ice crystals is prevented. Vitrification typically employs high concentrations of cryoprotectants to prevent ice formation with the risk of cryoprotectant-induced cytotoxicity. Methods: Metanephroi were isolated from 15-day (E15) timed pregnant Lewis rats, these were then randomly divided between three groups: control (C), controlled rate freezing (CRF) and vitrification (V). CRF metanephroi were cryopreserved using a DMSO/FCS/RPMI solution and controlled rate freezing at –0.3°C/min to -40°C prior to storage at -135°C for 48 hours. Mn were re-warmed by agitation in a 37°C waterbath until thawed, cryoprotectant was them removed and mn washed prior to re-suspension in media and subsequent viability analysis. Vitrified samples were cooled to -100°C in cryoprotectant (VS55, consisting of an 8.4M mixture of DMSO, formamide, and propanediol in EuroCollins solution) using an isopentane bath, then vitrified to -120°C and stored for 48 hours at -135°C. After storage mn were thawed to -35°C in a 30% DMSO bath then the cryoprotectant was diluted out by addition of mannitol in Unisol. The supernatant was removed and mn were resuspended in media prior to viability analysis. Mn viability was analysed for cell viability via an AlamarBlue assay, histology (light microscopy, T.E.M. and cryosubstitution) and transplantation to adult Lewis recipients. Results: Following normalisation, there was statistically no difference in embryonic kidney metabolic activity of either of the cryopreserved mn group relative to the control untreated group. However, cryosubstitution demonstrated the presence of significant ice formation during controlled-rate freezing resulting in ‘scattered’ tissue and water inside the cells post-thawing. In contrast, the amount of ice was significantly reduced by vitrification. This was supported by light microscopy demonstrating significant vacuolation of the cytoplasm of control-rate frozen metanephroi following re-warming. By comparison, the re-warmed vitrified metanephroi had little cytoplasmic disruption. However, T.E.M demonstrated mitochondrial and nuclear injury at the cellular level. Transplantation of the mn is the ultimate test of viability, statistically less CRF metanephroi grew compared with control and vitrified metanephroi (p<0.05). Conclusions: There is a need for long-term storage of organs to make mn transplantation a realistic possibility. This study demonstrates that standard control-rate freezing methods are unsuitable for this purpose. Vitrification of mn using a protocol developed for blood vessels yielded more promising results but further development of the vitrification solution, exposure time and loading steps are required to reduce the observed toxicity and subsequent reduced viability.