Mass Preparation of Primary Porcine Hepatocytes and the Design of a Hybrid Artificial Liver Module using Spheroid Culture for a Clinical Trial
Mass Preparation of Primary Porcine Hepatocytes and the Design of a Hybrid Artificial Liver Module using Spheroid Culture for a Clinical Trial
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原代猪肝细胞的大量制备和使用球体培养的混合人工肝模块的设计用于临床试验
DOI：
10.1177/039139880102401104
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发表时间：
2001
期刊：
影响因子：
--
通讯作者：
Kazumori Funatsu
中科院分区：
文献类型：
--
作者：
J. Fukuda;R. Sakiyama;K. Nakazawa;H. Ijima;Y. Yamashita;M. Shimada;K. Shirabe;E. Tsujita;K. Sugimachi;Kazumori Funatsu
为了大量分离猪肝细胞，我们首次提出了一种大批量制备方法，将通常的全肝胶原酶灌注法和肝脏灌流后组织碎片胶原酶再消化法相结合。用10 kg的猪，胶原酶灌注法仅获得63±78×108个总细胞，存活率为69.2±25.3%，而我们的联合方法获得167±31×108个总细胞，存活率为87.9±4.4%(均值±SD)。同时将该方法应用于两头体重为10 kg的猪，11次分离得到387±×108肝细胞，存活率为87.1±6.9%，纯度为93.6±2.8%。我们设计了一种包含1×1010猪肝细胞的大型多毛细管聚氨酯泡沫填充床组件(MC-PUF)。组件中的猪肝细胞形成直径200-500μm的球形多细胞聚集体(球体)。荧光素二醋酸酯和溴化乙锭染色判断大多数形成球体的肝细胞是存活的。与含有2×108猪肝细胞的小型MC-PUF组件相比，大型MC-PUF组件的氨去除、白蛋白分泌和耗氧量的活性相同，并可保持培养至少9天。这些结果表明，一个大型MC-PUF组件成功地放大了50倍。综上所述，我们成功地开发了猪肝细胞的批量制备方法和临床试验规模的大型杂交人工肝模块。
To isolate a large number of porcine hepatocytes, we originally developed a mass preparation method that combined the usual collagenase perfusion method of a whole liver with a collagenase redigestion method of tissue fragments after liver perfusion. Using a pig of 10kg, collagenase perfusion only resulted in a yield of 63 ± 78 x 108 total cells with a viability of 69.2 ± 25.3 %, but our combined method had a yield of 167 ± 31 x 108 total cells with a viability of 87.9 ± 4.4 % (mean ± SD). Also, the combined method was applied to two pigs of 10kg body weight at the same time, and isolated 387 ± 89 x 108 hepatocytes with a viability of 87.1 ± 6.9 % and a purity of 93.6 ± 2.8 % in 11 experiments. We designed a large multi-capillary polyurethane foam (MC-PUF) packed-bed module containing 1 x 1010 porcine hepatocytes on a clinical trial scale. The porcine hepatocytes in the module formed spherical multicellular aggregates (spheroids) of 200 – 500 μm diameter. Most hepatocytes forming spheroids were viable judged by fluorescein diacetate and ethidium bromide staining. The activities of ammonia removal, albumin secretion and oxygen consumption of the large MC-PUF module were the same as for a small MC-PUF module containing 2 x 108 porcine hepatocytes, and were maintained for at least 9 days of culture. These results show that a large MC-PUF module is successfully scaled up 50 times. In conclusion, we succeeded in developing a mass preparation method of porcine hepatocytes and a large hybrid artificial liver module on a clinical trial scale.
生物人工肝治疗严重肝衰竭的临床经验。
影响因子：
9
作者：
Watanabe,FD;Mullon,CJ;Hewitt,WR;Arkadopoulos,N;Kahaku,E;Eguchi,S;Khalili,T;Arnaout,W;Shackleton,CR;Rozga,J;Solomon,B;Demetriou,AA
通讯作者：
Demetriou,AA