Cryopreservation of porcine aortic heart valve leaflet-derived myofibroblasts.

Cryopreservation by freezing is usually employed for storage of allogeneic valves destined for clinical use; however, disruption of leaflet extracellular matrices by ice may occur. This study was performed to determine the effects of cryoprotectants employed in VS55 for ice-free, vitreous cryopreservation upon adherent porcine heart valve leaflet-derived myofibroblasts in culture. Low-passage myofibroblasts exhibiting strong actin and myosin staining were employed for these experiments. Three cryoprotectants, dimethylsulfoxide, formamide, and propanediol, were tested individually and in combination. Exposure experiments demonstrated that the individual cryoprotectants (0-5 M) were generally cytotoxic in a dose-dependent manner with little if any loss of cells as determined by measuring metabolic activity and DNA content. Exposure to formamide resulted in the greatest loss of cells and reduction in viability. Combination of the three cryoprotectants demonstrated that the cytotoxic effects of each cryoprotectant were not cumulative. Cell viability and DNA content were equivalent to dimethylsulfoxide and propanediol and higher than formamide alone over most of the 0-5 M dose-response curve. After cryopreservation by slow-rate freezing, the benefits of the combination of cryoprotectants over individual cryoprotectants were demonstrated at the 4 M concentration range for both cell viability and cell retention. In conclusion, these studies demonstrated that, with the exception of the lower concentrations of propanediol, the combination of cryoprotectants employed in these studies result in equivalent or better cell viability and attachment than individual cryoprotectants.

Preservation of embryonic kidneys for transplantation.

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 degrees C/min, from -10 degrees to -40 degrees C; or (ii) cryopreserved in an ice-free state by rapid cooling to -100 degrees C in cryoprotectant (VS55), followed by vitrification to -120 degrees C. After cryopreservation, the metanephroi were stored at -135 degrees 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.

Hepatic function in hypothermically stored porcine livers: comparison of hypothermic machine perfusion vs cold storage.

Hypothermic machine perfusion (HMP) has a potential to relieve the current donor liver crisis by providing an improved and extended preservation method. This study examined the effect of HMP on hepatocellular functions, using a prototype liver transporter capable of preserving livers for 24 hours. Livers obtained from adult farm pigs (28 to 32 kg body weight) were divided into three groups: fresh control, HMP, and simple cold storage (n = 4 each). A 4-hour normothermic reperfusion of livers was conducted to assess hepato-metabolic and cellular functions. The hepatic transport function, as indicated by canalicular excretion of indocyanine green, was improved in the HMP group than in the SCS group. The overall tissue viability, as indicated by oxygen consumption levels, was notably improved in HMP and control livers as compared to the SCS group. Higher bile production in both the preserved groups as compared to the fresh control livers could be a result of biliary edema and leakage of plasma into the canaliculus. The hepato-cellular injury, measured by ALT, release was significantly greater in the SCS group as compared to the HMP and control groups. These findings suggest that HMP could be a better method to preserve hepatic function and overall tissue viability as compared to SCS. Improved hepatic functions are indirect indicators of superior microcirculation and sinusoidal endothelial cell functions. Further studies in progress will evaluate these functions to confirm the significance of these observations.