Isolation, characterization, and cultivation of human hepatocytes and non-parenchymal liver cells
Isolation, characterization, and cultivation of human hepatocytes and non-parenchymal liver cells
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DOI：
10.1177/1535370214558025
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发表时间：
2015-05-01
影响因子：
3.2
通讯作者：
Damm, Georg
中科院分区：
文献类型：
--
作者：
Pfeiffer, Elisa;Kegel, Victoria;Damm, Georg
原代人肝细胞（PHH）被认为是体外检测外源性代谢和肝毒性的金标准。然而，在2D单培养物中培养PHH导致去分化和功能丧失。肝非实质细胞（NPC），如枯否细胞（KC）、肝内皮细胞（LEC）和肝星状细胞（HSC），在维持PHH功能中起核心作用。本研究的目的是建立一个协议，同时分离人类PHH和NPC从同一组织标本，并测试其适用于在体外共培养。通过两步EDTA/胶原酶灌注技术从部分肝脏切除获得的组织中分离出人PHH和NPC。通过Percoll密度梯度离心纯化获得的细胞级分。使用特异性粘附特性和磁激活细胞分选（MACS（R））分离NPC级分中所含的KC、LEC和HSC。经鉴定的NPC显示每克肝组织产率为1.9 × 10（6）KC、2.7 × 10（5）LEC和4.7 × 10（5）HSC，显示活力> 90%。这些NPC的表征表明，所有群体都经历了一个激活过程，这影响了细胞的命运。KC的激活强烈依赖于组织质量和供体记忆。KC在培养物中被激活，并在4-5天内丧失活力。LEC在培养过程中失去了特定的功能，而HSC经历了转化成肌成纤维细胞的过程。不同培养条件对HSC的检测表明，它们可以减弱HSC，但不能阻止体外去分化。总之，所描述的方法允许从同一供体中高质量和高数量地分离和分离PHH和NPC。
Primary human hepatocytes (PHH) are considered to be the gold standard for in vitro testing of xenobiotic metabolism and hepatotoxicity. However, PHH cultivation in 2D mono-cultures leads to dedifferentiation and a loss of function. It is well known that hepatic non-parenchymal cells (NPC), such as Kupffer cells (KC), liver endothelial cells (LEC), and hepatic stellate cells (HSC), play a central role in the maintenance of PHH functions. The aims of the present study were to establish a protocol for the simultaneous isolation of human PHH and NPC from the same tissue specimen and to test their suitability for in vitro co-culture. Human PHH and NPC were isolated from tissue obtained by partial liver resection by a two-step EDTA/collagenase perfusion technique. The obtained cell fractions were purified by Percoll density gradient centrifugation. KC, LEC, and HSC contained in the NPC fraction were separated using specific adherence properties and magnetic activated cell sorting (MACS (R)). Identified NPC revealed a yield of 1.9 x 10(6) KC, 2.7 x 10(5) LEC and 4.7 x 10(5) HSC per gram liver tissue, showing viabilities >90%. Characterization of these NPC showed that all populations went through an activation process, which influenced the cell fate. The activation of KC strongly depended on the tissue quality and donor anamnesis. KC became activated in culture in association with a loss of viability within 4-5 days. LEC lost specific features during culture, while HSC went through a transformation process into myofibroblasts. The testing of different culture conditions for HSC demonstrated that they can attenuate, but not prevent dedifferentiation in vitro. In conclusion, the method described allows the isolation and separation of PHH and NPC in high quality and quantity from the same donor.