树鼩原代肝细胞分离培养条件优化

Optimization of Primary Tupaia Hepatocytes’ Isolation and Culture Condition

摘要: 目的：通过优化树鼩原代肝细胞的分离及体外培养条件，获得能够在体外进行长期稳定培养的树鼩原代肝细胞。方法：优化肝脏灌流液、酶消化液及细胞离心速度等原代肝细胞分离条件，培养细胞密度和培养基配方等原代肝细胞培养条件，用台盼蓝染色细胞计数法、MTT检测法及EDU标记法评价所分离、培养树鼩原代肝细胞的数量、活力及生长状态。结果：添加葡萄糖的D-Hank’s液作为灌流液，含有0.005 mol/L CaCl2及12 × 104 units/L胶原酶IV的D-Hank’s液作为消化液，37℃消化15~20分钟，所获得细胞梯度离心洗涤三次，可获得最高得率及活力的原代肝细胞。在Williams’ ME基础培养基中添加生长因子(10 ng/ml)，葡萄糖(0.25%)、ITS-X(1 × multiple)、1%青链霉素及2% DMSO时可以维持树鼩原代肝细胞体外稳定增殖生长达42天。结论：树鼩原代肝细胞分离与体外培养条件的优化，可大大提高细胞得率，延长细胞稳定增殖生长时间，有助于以树鼩为模型研究肝细胞生理代谢特性及嗜肝病毒感染机制。

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.