Endothelial cell viability and histology as parameters for ex-vivo mouse heart perfusion system optimization.

Abstract

Heart failure is a main cause of morbidity and mortality worldwide. To reduce the disparity between the waiting list and available donor hearts, extended criteria donor hearts are an alternative, because the development of ex-vivo heart perfusion in many countries has led to utilisation of extended criteria donor organs recently. Compared to SCS and hypothermic machine perfusion, normothermic machine perfusion provides a higher protective capacity, a superior prediction of the early graft function, and more accurate assessment of donor heart functionality. Importantly, the endothelium should also be considered specifically when optimising preservation solutions. In this work, mouse hearts were perfused in KHB solution at normothermic temperature. Histology and microscopy analysis revealed no statistically significant differences in SWT, AWT, and LVWA when comparing different perfusion times and switching from Langendorff to working perfusion mode. In addition, the degree of ischemia was lowest after 15-20 minutes of Langendorff perfusion and increased with 2-hour Langendorff perfusion, but fluctuated afterwards, suggesting that ischemia is a dynamic process. Furthermore, no statistically significant differences were found in AWT and LVWA for mouse hearts exposed to low oxygen levels in the KHB solution compared to high oxygen levels. However, a statistically significant higher SWT was observed in mouse hearts exposed to a low oxygen level compared to the hearts exposed to a high oxygen level. A higher oxygen level seems to preserve mouse hearts better. Moreover, the average cardiomyocyte size showed no statistically significant difference between mouse hearts exposed to either high or low oxygen levels. After performing flow cytometry and dataanalysis, no statistically significant difference in HMEC-1 cell viability was found when exposed to cold preservation solution St Thomas cardioplegia, although an indication might be found for the cells to be not affected during the different incubation periods. Using the RTCA xCELLigence system (Roche) to determine the viability of HMEC-1 cells over time when exposed to different colloids and different perfusion fluids, showed that albumin might be the preferred colloid and KHB seem to preserve the viability better than Steen solution based on the experimental data. To conclude, the results suggest that better preservation could be achieved with normothermic perfusion of mouse hearts in an ex-vivo heart perfusion system at high oxygen level compared to a low oxygen level. Moreover, ischemia for perfusion system optimisation may be important to consider. Furthermore, from the in-vitro experiments can be concluded that the viability of endothelial cells might not be affected over different incubation periods with St Thomas cardioplegia. Also, albumin may be preferred over the artificial colloids when only considering the experimental results, although for determining whether KHB or Steen solution is more optimal as perfusate on the system, additional experiments are necessary.