| Hepatocytes are the key material of bioartificial liver (BAL). The liver supporting effect of BAL in the treatment of the patients with liver failure counts on the biological function of the hepatocytes completely. Now, porcine hepatocytes are the most utilized cells in the research of BAL owing to the shortage of human hepatocytes, the ethical issue of the use of human fetal hepatocytes, and the oncoma origin of liver cell lines. Accompanied by the increasing of successful clinical research and applications, there are a lot of problems to be solved in the future, such as the source of hepatocytes in the BAL system, the mass exchange of bioreactor, and the preservation and transportation of hepatocytes.In this study, proliferation abilities of the suckling porcine hepatocytes during the primary culture were observed firstly. Both the synthesis and biotransformation function of the cultured hepatocytes were detected in order to evaluate the applicability of them to BAL. Then, the morphological and functional changes of the neonatal porcine hepatocytes were investigated under an extracorporeal circulation of the hepatocyte suspensions. The data could be available for the construction of bidirection circulation system for BAL which could solve the problem of mass exchange in the bioreactor. Finally, Suckling porcine hepatocytes were cultured on a mild hypothermic condition. The influence of mild hypothermy on the morphology and function of hepatocytes were observed before and after rewarming with the purpose of exploring a convenient and effective method of the hepatocytes' preservation and transportation for the clinical application of BAL.Our experiment methods and results are following.The isolated porcine hepatocytes were cultivated by RPMI-1640 medium at 37℃, 5%CO2. The changes of the cells' morphology and quantity were observed, the synthesis of albumin and urea were detected, and the clearance of ammonia, and the transformation 7-methoxylresorufine and p-nitrophenol were analyzed. In this experiment, seven foldsproliferation of hepatocytes were found. The growth curve shows an obvious exponential phase of cell growth and stationary phase. There isn't a marked stage of latency in the growth curve. Significant superiority of suckling porcine hepatocytes was found in the cultivation period when compared with fetal liver cells, such as synthesis ratio of urea, clearance rate of ammonia, activities of CYP and UGT. All above suggests that the suckling porcine hepatocytes have good proliferation abilities and biotransformation function.Hepatocyte circulation system was constructed by Cello artificial capillary culture equipment and freshly isolated hepatocyte suspensions were circulated by 50ml/min in vitro. After 4hr's circulation, the viability and adherence ratio of hepatocytes were 76.1±1.4% 62.8±1.8% respectively and the ratio of amino clearance was about 60% of the control group. These results implied that under a circulation condition hepatocytes still maintained cell activity and function after 4hr's experiment. But, along with time, many pieces of hepatocytes were observed in circulation suspensions as well as LDH and AST increased significantly. The cell viability and adherence ratio, synthesis of urea and albumin and the clearance rate of ammonia decreased also markedly. Certainly, hepatocyte suspensions circulation is harmful to the cells according to the experiment.Freshly isolated porcine hepatocytes were cultured in 37 for 4h at first. The following 24h, hepatocytes were spent in 25 28 , and 33 respectively and then hepatocytes returned to 37 and cultured 24h. Under the mild hypothermy, both the ability of cell proliferation and function of hepatocytes were decreased in different degree, yet fast proliferation and notable strengthen of the cellular viability and function were found when returned to 37 , especially in the 28 and 33 groups. Results of further experiments on different culture time such as 24h, 48h, and 72h under 28 revealed no difference according the observation of the cells' morphology and function, which implied that hepatocytes under 28 could be stored for 72h stably without obvious cellular damage, so that it is lasting long for the cells' transportation by this method.In summary, 1. Neonatal porcine hepatocytes possess a fairly good proliferation abilities and synthesis and biotransformation function, which accord with the requirements of BAL treatment for patients with liver failure. 2. Porcine hepatocyte suspensions could be applied to BAL system in a circulation condition in order to strengthen the mass exchange. But these cells need to be changed regularly for the maintenance of the cellular viabilityand the supporting effects of BAL. 3. Mild hypothermic culture could maintain cell morphology and function especially under 28 and 33 which could be utilized for the preservation and transportation of hepatocytes in a short period. |
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