Improvement of Hepatocyte Recovery from Liver Tissue Suffered from Warm Ischemia by Citrate Phosphate Dextrose (CPD) Added Euro-Collins Perfusion Solution

Abstract

Because hepatocyte cell lines lack sufficient native functions, they are not suitable for hepatocyte transplantation, pharmaceutical research and biological study. Thus, primary human hepatocyte is attracting researchers interest these days. However, when we use surgically resected liver tissues or livers from non-heart beating donors as cell sources, unavoidable warm ischemia time interferes the efficacy of the collagenase digestion, and reduces the live cell recovery. We speculated that the impairment of collagenase digestion is due to microembolism that is formed by warm ischemia. To improve recovery rate of hepatocytes, the different perfusion solution was evaluated in rat. Nine-week old male SD rats were anesthetized with enflurane inhalation and received midline abdominal incision. Then, portal vein and hepatic artery were separated gently from surrounding connective tissue and 22G cannula was inserted into portal vein. The warm ischemia was started by ligating both portal vein and hepatic artery with 3-0 suture at the lower part of the cannulated point. Two perfusion solutions, Euro-Collins and ET-Kyoto solution, and two anti-coagulative agents, heparin and citrate phosphate dextrose solution (CPD), were examined comparing with the Seglen's original Ca2+-Mg2+-free Hank's solution (Seglen, Methods Cell Biol 13:29 1976). In result, Seglen's solution showed 2.27 ± 0.53 × 108 live cell yield (n=11) in non-warm ischemia control, but only 0.38 ± 0.17 × 108 (n=3) cells in 10min warm ischemia group (P=0.00001). Addition of heparin in Seglen's solution did not improve cell recovery very much (0.71 ± 0.40 × 108 (n=4)). In 10min warm ischemia groups, CPD added Euro-Collins solution brought about the best recovery, 1.41 ± 0.50 × 108 (n=7) cells that is 3.7 times to Seglen's solution (P=0.041). In 15min warm ischemia groups, CPD added Euro-Collins showed 1.37 ± 0.28 × 108 (n=3) cells recovery that is 1.69 time greater than CPD added ET-Kyoto solution although there was no statistical significance. Addition of heparin to Euro-Collins or ET-Kyoto solution showed only 0.19 ± 0.07 × 108 (n=3) cells and 0.16 ± 0.03 × 108 (n=3) cells respectively. Therefore, heparin is not likely to be suitable for hepatocyte isolation from warm ischemia liver. Actually, macroscopic observation also demonstrated good blood removal by CPD added solutions, suggesting restoration of better hepatic microcirculation. The present results demonstrate that CPD would be more effective on ensuring the microcirculation in the liver tissue suffered from warm ischemia to enable collagenase digestion. Our present protocol will be benefitial to isolate hepatocytes not only from surgically resected liver tissue but also livers from non-heart beating donors. In conclusion, CPD, not heparin, added Euro-Collins solution would be suitable for the hepatocyte isolation from liver tissue suffered from warm ischemia.