Cultivation and identification of biologic activity of minitype porcine hepatocytes on microcarriers

Objective To explore a method for culture of minitype porcine hepatocytes on microcarriers and to investigate the functional features of the cultured hepatocytes. Methods Liver cells from minitype porcine liver were harvested by using a two - step extracorporeal perfusion apparatus with ethylene diamineteraacetic acid and collagenase soiutions. The isolated hepatocytes were inoculated in the fresh RPMI 1640 medium supplemented with 2 g/L cytodexTM 3, 100mL/ 1 calf serum and so on. The morphologic features of cultured hepatocytes were recorded. The biological function of biosynthesis and biotransformation of hepatocytes during different cultural intervals was evaluated. Results The harvested primary hepatocytes consisted of 99 % of the total isolated mass liver cells with the cellular viability as high as 96.25 % and gave an average yield of 7.58×109 cells each porcine liver, Primary hepatocytes in vitro cultured possessed the features of morphology of normal porcine primary hepatocytes; The primary hepatocytes attached to cytodexTM 3 after 3h of incubation, and began to actively proliferate after 20h of incubation in a mode of similar monolayer growth; The culture mass of primary hepatocytes has been identified to have the active synthetic and metabolic functions, e.g. synthesized urea and albumin being 1.22mmol/L and 0.075 g/L in 3.2×107 hepatocytes within 24 hours, respectively, and the transform rate of lidocanine into 3 - hydroxy - lidocanine being 100% in 3 .2×107 hepatocytes within 24 hours. Conclusions The primary hepatocytes from minitype porcine on microcarriers posses the features of active proliferation activity, morphology of normal porcine primary hepatocytes, active biosynthesis and biotransformation, which may be served as the better support model of extracorporeal bioartifical liver support system for patients with hepatic failure.