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目的:获得一个具有丰富来源的肝细胞生产技术。方法:取本地杂种小猪作为肝细胞供体,用胶原酶原位门腔循环灌注法分离获取肝细胞。置于 DMEM-猪门静脉血清体系中进行微载体粘附悬浮培养。观察细胞生长情况并动态检测培养上清中白蛋白及尿素浓度。结果:每只猪肝获取肝细胞数为(4.26±0.75)×10~(10),活率为(91.22±0.83)%。培养7天后粘附肝细胞数为(5.36±0.69)×10~7/mL,15天后达(3.18±0.71)×10~8/mL,23天后为(2.62±0.52)×10~7/mL。于培养第15天肝细胞的白蛋白及尿素合成功能达峰值。结论:采用 IV 型胶原酶原位门腔循环灌注可分离获取大量、高活性的原代正常猪肝细胞,经微载体粘附悬浮培养后可达到高密度、高活性、长期离体培养的目的,可满足生物人工肝对生物材料的要求。
Objective: To obtain a rich source of hepatocyte production technology. Methods: Local hybrid piglets were used as hepatocyte donors, and hepatocytes were isolated by collagenase in situ portal perfusion. Placed in DMEM-porcine portal vein serum system for microcarrier adhesion suspension culture. Observation of cell growth and dynamic detection of albumin and urea concentration in culture supernatant. Results: The number of liver cells per pig was (4.26 ± 0.75) × 10 ~ (10) and the survival rate was (91.22 ± 0.83)%. The number of adherent hepatocytes reached (5.36 ± 0.69) × 10 ~ 7 / mL after 7 days of culture, reaching (3.18 ± 0.71) × 10 ~ 8 / mL after 15 days and (2.62 ± 0.52) × 10 ~ 7 / mL after 23 days . Hepatic albumin and urea synthesis peaked on day 15 of culture. CONCLUSION: A large number of high-activity primary normal porcine hepatocytes can be isolated and isolated by in-situ portal venous perfusion of type IV collagenase, which can achieve high density and high activity after long-term in vitro culture by microcarrier adhesion suspension culture , To meet the bio-artificial liver biological material requirements.