Hepatocytes isolation from UW preserved rat liver / 한국간담췌외과학회지

ABSTRACT

BACKGROUND/AIMS: In the immunological and functional aspects, freshly isolated human hepatocytes are better than animal hepatocytes from other species such as the pig and hepatocytes from human tumor cell lines. Human liver tissue for hepatocyte isolation can be obtained either from surgical specimens of patients with liver disease, such as hepatocellular carcinoma or from donor livers that are not suitable for transplantation. In the latter case, the liver may be preserved in 4 degrees C UW solution. Therefore, studies of the limit on the preservation time to get clinically applicable hepatocytes, and proper isolation techniques improving cell viability are needed. In this study, the authors isolated hepatocytes from a few kinds of the liver with different UW preservation times and different isolation techniques. METHODS: Male Sprague-Dawley rats weighing 200g-250g were used. The hepatocytes were isolated from unpreserved livers(control), livers preserved for 24 hours in UW solution, and livers preserved for 48 hour in UW solution, with a modified Seglen's two step method. To improve cell viability, 5mM glycin was added to the EDTA solution in one group and in another, the pH of the EDTA solution was slowly changed from 6.5 to 7.25. RESULTS: The control group showed 80.0% of cell viability. 53.8% of isolated hepatocytes were viable even after 24 hours of preservation. The cell viability decreased to 36.1% and severe cellular damage was observed after 48 hours preservation. The glycin and pH change showed no protective effect on the isolation of hepatocytes from UW preserved livers. CONCLUSION: After 24 hours preservation in UW solution, many clinically useful hepatocytes were isolated with a modified Seglen's two step method. Further studies concerning the preservation of functions of hepatocytes isolated from UW preserved livers and on the technique of cell culture and cryopreservation are needed.