Protocol for Isolation of Primary Human Hepatocytes and Corresponding Major Populations of Non-parenchymal Liver Cells
Protocol for Isolation of Primary Human Hepatocytes and Corresponding Major Populations of Non-parenchymal Liver Cells
复制标题
DOI：
10.3791/53069
复制
发表时间：
2016-03-01
影响因子：
1.2
通讯作者：
Damm, Georg
中科院分区：
文献类型：
--
作者：
Kegel, Victoria;Deharde, Daniela;Damm, Georg
除了实质肝细胞外，肝脏还包括非实质细胞（NPC），即枯否细胞（KC）、肝内皮细胞（LEC）和肝星状细胞（HSC）。原代人肝细胞的二维培养仍然被认为是体外药物代谢和肝毒性检测的“金标准”。众所周知，PHH的2D单一培养遭受去分化和功能丧失。近年来研究表明，肝NPC在肝脏病理生理和PHH功能的维持中起着重要作用。目前的研究重点是通过3D和共培养模型重建体内组织结构，以克服2D单一培养的局限性。此前，我们在《实验生物学和医学》1中发表了一种分离人肝细胞的方法，并研究了这些细胞在细胞培养中的适用性。基于广泛的兴趣，在这项技术的目的是提供一个更详细的协议，肝细胞的分离过程中，包括一个视频，这将使一个容易再现的技术。人肝细胞分离的人肝组织样本的外科干预两步EGTA/胶原酶P灌注技术。通过在50 x g下初始离心将PHH与NPC分离。密度梯度离心步骤用于去除死细胞。使用特定的细胞特性和细胞分选程序从富集的NPC级分中分离单个肝细胞群。除PHH分离外，我们还能够分离KC、LEC和HSC用于进一步培养。总之，所提出的方案允许从一个供体组织样品中高质量和数量地分离PHH和NPC。获得纯化的肝细胞群体可以允许创建体内类似人类肝脏模型。
Beside parenchymal hepatocytes, the liver consists of non-parenchymal cells (NPC) namely Kupffer cells (KC), liver endothelial cells (LEC) and hepatic Stellate cells (HSC). Two-dimensional (2D) culture of primary human hepatocyte (PHH) is still considered as the "gold standard" for in vitro testing of drug metabolism and hepatotoxicity. It is well-known that the 2D monoculture of PHH suffers from dedifferentiation and loss of function. Recently it was shown that hepatic NPC play a central role in liver (patho-) physiology and the maintenance of PHH functions. Current research focuses on the reconstruction of in vivo tissue architecture by 3D- and co-culture models to overcome the limitations of 2D monocultures. Previously we published a method to isolate human liver cells and investigated the suitability of these cells for their use in cell cultures in Experimental Biology and Medicine1. Based on the broad interest in this technique the aim of this article was to provide a more detailed protocol for the liver cell isolation process including a video, which will allow an easy reproduction of this technique.Human liver cells were isolated from human liver tissue samples of surgical interventions by a two-step EGTA/collagenase P perfusion technique. PHH were separated from the NPC by an initial centrifugation at 50 x g. Density gradient centrifugation steps were used for removal of dead cells. Individual liver cell populations were isolated from the enriched NPC fraction using specific cell properties and cell sorting procedures. Beside the PHH isolation we were able to separate KC, LEC and HSC for further cultivation.Taken together, the presented protocol allows the isolation of PHH and NPC in high quality and quantity from one donor tissue sample. The access to purified liver cell populations could allow the creation of in vivo like human liver models.