Optimization of Primary Tupaia Hepatocytes’ Isolation and Culture Condition
Abstract: Objectives: To get a long-term and stable culture of primary Tupaia hepatocytes (PTH) in vitro, the isolation and culture conditions were optimized through the single factor variation experiments. Methods: The isolation conditions of PTH, such as the formula of liver perfusate and enzyme digestion, the centrifugal factors, and the culture conditions such as cell culture density and medium component have been changed, and the counts, viability and growth status of cultured PTH was evaluated with trypan blue staining and cell counting, MTT detection and EDU labeling method. Results: We found that we can obtain liver cells at the highest yield and dynamic generation by perfusing with D-Hank’s solution with glucose, digesting with D-Hank’s solution containing 0.005 mol/L CaCl2 and 12 × 104 units/L collagenase IV at 37˚C for 15 to 20 minutes, washing the obtained cells by gradient centrifugation three times. In addition, the primary tree shrew hepatocytes cultured in Williams’ ME basic medium which supplemented with growth factors (10 ng/ml), glucose (0.25%), ITS-X (1 × multiple), 1% of penicillin and 2% DMSO were able to maintain stable growth up to 42 days in vitro. Conclusion: Therefore, the optimized isolation and culture conditions for primary Tupaia hepatocytes cannot only improve the cell yield greatly and prolong the stable cell proliferation and growth time, but also give a hand to the researches which study the physiological and metabolic properties of hepatocytes and the infection mechanism of hepatotropic virus in tree shrew.
文章引用: 薛兰洁 , 冯悦 , 徐明 , 孙晓梅 , 张华堂 , 代解杰 , 夏雪山 (2013) 树鼩原代肝细胞分离培养条件优化。 生物过程， 3， 1-8. doi: 10.12677/BP.2013.31001
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