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Isolation and cultivation of porcine hepatocytes for extracorporeal artificial liver support system
作者姓名:周晓东  刘丽
作者单位:日本通商产业省工业技术院产业技术融合领域研究所仿生组日本
基金项目:ThisworkwassupportedbytheNationalInstituteforAdvancedInterdisciplinaryResearch,China (No .985 15 45 6 )
摘    要:目的 开发从屠宰器官进行高活性猪肝细胞规模分离的优化程序。方法 采用部分肝叶逆行灌注、机械及组酶肝消化并经密度梯度离心获取高产量、高纯度肝实质性细胞。结果 部分肝叶经消化可获取 1 39× 10 9肝细胞的平均产量 (9 9× 10 6肝细胞 /克组织 )及 92 5 %的平均细胞存活率。经组合性dispase/胶原酶的消化处理肝细胞的收获量及存活率明显增加。对细胞灌流及分离液进行氧化处理能阻止泡状细胞的发生。分离的猪肝细胞经原代培养其活性及机能保持着与鼠肝细胞一致的水平 ,且随时间的推移而减退。结论 通过我们改良的方法可收获高产量成年猪肝细胞 ,并保持良好的活性与分化机能。因此 ,成年猪肝细胞被证实是人工肝脏辅助支持系统中生物组的一个很有价值的肝细胞来源。

关 键 词:肝细胞分离  人工肝脏原代培养  成年猪

Isolation and cultivation of porcine hepatocytes for extracorporeal artificial liver support system
ZHOU Xiaodong,LIU Li,Juno Kano,Toshiyuki Mukaiyama,Takayoshi Tokiwa.Isolation and cultivation of porcine hepatocytes for extracorporeal artificial liver support system[J].Chinese Medical Journal,2001,114(9):946-949.
Authors:ZHOU Xiaodong  LIU Li  Juno Kano  Toshiyuki Mukaiyama  Takayoshi Tokiwa
Institution:Bionic Design Group, National Institute for Advanced Interdisciplinary Research, 1-1-4 Higashi, Tsukuba, Ibaraki 305-8562, Japan;Bionic Design Group, National Institute for Advanced Interdisciplinary Research, 1-1-4 Higashi, Tsukuba, Ibaraki 305-8562, Japan;Bionic Design Group, National Institute for Advanced Interdisciplinary Research, 1-1-4 Higashi, Tsukuba, Ibaraki 305-8562, Japan;Bionic Design Group, National Institute for Advanced Interdisciplinary Research, 1-1-4 Higashi, Tsukuba, Ibaraki 305-8562, Japan;Bionic Design Group, National Institute for Advanced Interdisciplinary Research, 1-1-4 Higashi, Tsukuba, Ibaraki 305-8562, Japan
Abstract:Objective To develop procedures for the successful harvesting of large quantities of viable and functional pig liver cells from abattoir organs. Methods The procedure included partial liver lobe retrograde perfusion and mechanical/enzymatic digestion of the liver tissue, followed by separation of the hepatocytes, based on size and density, from contaminating cell types. Results Digestion of the partial liver lobe resulted in an average yield of 1.39×10(9) cells (9.9×10(6)cells/g liver) with an average viability of 92.5%. The yield and viability of cells were improved by dispase/collagenase resultant digestion. The emergence of blebby cells was blocked by supplying oxygen to the cell isolation buffers. Isolated hepatocytes seeded onto polystyrene surfaces remained viable and functional at a level comparable to that of rat hepatocytes, although their function decreased over time.Conclusions Adult pig hepatocytes can be harvested with high yields and retain viability and differentiated function using this method. Abattoir pig livers can be an excellent source of hepatocytes for use as the biological component of artificial liver assist devices.
Keywords:hepatocyte isolation  liver assist devices  primary culture  adult pig
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