To determine the best and simplest method for cryopreservation of pig hepatocytes, we compared immediate cryopreservation with cryopreservation after short-term culture. Suckling pig hepatocytes were isolated by a modified 2-step in situ collagenase perfusion method, suspended in serum-free medium, and preserved for 10 da by two cryopreservation methods. Serial measurements were made of cell viability, LDH release, synthesis of protein, urea and glucose, glucose-6-phosphatase (G-6-Pase) activity, and diazepam transformation after thawing. These measurements were performed on both groups of cultured hepatocytes, and on freshly isolated hepatocytes, which served as a control. High viability (>95%)of thawed hepatocytes was obtained and maintained in both cryopreservation groups. There were no significant differences in cell viability, protein synthesis, glucose synthesis, G-6-Pase activity, or diazepam transformation between the two cryopreservation groups. In the immediate cryopreservation group, urea synthesis was less than in the group with cryopreservation after short-term culture. Protein synthesis, glucose synthesis, and diazepam transformation were lower in both cryopreserved groups than in the controls. The results showed that a protocol of immediate cryopreservation of hepatocytes in RPMI-1640 medium containing 10% DMSO, hormones, growth factors, and 10% newborn bovine serum, together with rate-controlled freezing and rapid thawing, provides indices of cell viability and function during subsequent serum-free culture that are comparable to hepatocytes cryopreserved after short-term culture, except for lower urea production. This simple procedure can be used in studies of bioartificial liver and hepatocyte transplantation.

译文

为了确定猪肝细胞冷冻保存的最佳,最简单的方法,我们将短期培养后的立即冷冻保存与冷冻保存进行了比较。采用改良的2步原位胶原酶灌注法分离乳猪肝细胞,悬浮于无血清培养基中,通过两种冷冻保存方法保存10 da。对细胞活力,LDH释放,蛋白质,尿素和葡萄糖的合成,葡萄糖-6-磷酸酶 (G-6-Pase) 活性以及解冻后的地西epa转化进行了连续测量。这些测量是在两组培养的肝细胞以及作为对照的新鲜分离的肝细胞上进行的。在两个冷冻保存组中获得并维持解冻的肝细胞的高活力 (>95%)。两个冷冻保存组之间的细胞活力,蛋白质合成,葡萄糖合成,G-6-Pase活性或地西epa转化均无显着差异。在短期培养后,立即冷冻保存组的尿素合成少于冷冻保存组。两个冷冻保存组的蛋白质合成,葡萄糖合成和地西epa转化均低于对照组。结果表明,在含有10% DMSO,激素,生长因子和10% 新生牛血清的RPMI-1640培养基中立即冷冻保存肝细胞的方案,以及速率控制的冷冻和快速解冻,在随后的无血清培养过程中提供细胞活力和功能的指标,这些指标与短期培养后冷冻保存的肝细胞相当,但尿素产量较低。这种简单的方法可用于生物人工肝和肝细胞移植的研究。

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