Automated Liver Cell Processing Facilitates Large Scale Isolation and Purification of Porcine Hepatocytes
Automated Liver Cell Processing Facilitates Large Scale Isolation and Purification of Porcine Hepatocytes
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自动化肝细胞处理促进猪肝细胞的大规模分离和纯化
DOI：
10.1097/00002480-199506000-00006
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发表时间：
1995
期刊：
影响因子：
4.2
通讯作者：
A. Demetriou
中科院分区：
文献类型：
--
作者：
E. Morsiani;J. Rozga;Henri C. Scott;L. Lebow;A. Moscioni;L. Kong;M. McGrath;A. Demetriou
本文介绍了一种大规模分离纯化猪肝细胞的自动化方法。采用乙二胺四乙酸酯和胶原酶两步门静脉灌流的方法获取肝细胞。使用标准的人工处理方法(程序A)或使用滤室和可编程细胞洗涤器的自动处理方法(程序B)进行肝细胞纯化。两种方法均可获得较高的细胞产量(A组：1.30±0.55×1010个活肝细胞/肝；B组：1.38±0.32×1010个活肝细胞/肝)和存活率(A组：89±6.5%；B组：92±3.9%)。B组术后肝细胞纯度显著高于A组(93.1±3.1%比83.1±3%，p<；0.01)。用这两种方法分离的肝细胞形态完好，透射电子显微镜显示细胞质膜和细胞内细胞器完整。两种方法分离的肝细胞功能均无异常，但A法分离的肝细胞线粒体7-乙氧基香豆素-O-脱乙基酶活性(p<；0.05)和线粒体琥珀酸脱氢酶活性(p<；0.01)显著降低。综上所述，使用自动化肝细胞处理方法可以有效地大规模制备猪肝细胞，纯度更高，对分化的肝脏代谢功能的保留更大，耗时更少，劳动强度更小。《ASAIO期刊》1995；41：155-161。
&NA; An automated method for large scale isolation and purification of porcine hepatocytes is described. Liver cells were harvested by a two‐step portal vein perfusion with ethylene‐diaminetetraacetate and collagenase. Hepatocyte purification was carried out using either a standard manual processing method (Procedure A) or an automated processing method using a filtration chamber and a programmable cell washer (Procedure B). Both methods produced high cell yields (Procedure A: 1.30 ± 0.55 × 1010 viable hepatocytes/liver; Procedure B: 1.38 ± 0.32 × 1010 viable hepatocytes/liver) and viability (Procedure A:89 ± 6.5%; Procedure B: 92 ± 3.9%). Hepatocyte purity was significantly greater after Procedure B than after Procedure A (93.1 ± 3.1% versus 83.1 ± 3%, p < 0.01). Isolated hepatocytes by either method were morphologically intact, as demonstrated by transmission electron microscopy showing integrity of plasma membranes and intracellular organelles. Cultured hepatocytes isolated by either method were functionally intact, although those isolated by Procedure A showed significantly lower activity of microsomal 7‐ethoxycoumarin‐O‐deethylase activity (p < 0.05) and mitochondrial succinate dehydrogenase activity (p < 0.01). In conclusion, use of the automated hepatocyte processing method resulted in efficient large scale preparation of porcine hepatocytes, with higher purity and greater retention of differentiated liver metabolic functions, and was found to be less time consuming and less labor intensive. ASAIO Journal 1995; 41:155‐161.