An efficient and simple co-culture method for isolating primary human hepatic cells: Potential application for tumor microenvironment research
An efficient and simple co-culture method for isolating primary human hepatic cells: Potential application for tumor microenvironment research
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DOI：
10.3892/or.2016.4979
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发表时间：
2016-10-01
期刊：
影响因子：
4.2
通讯作者：
Feng, Hao
中科院分区：
文献类型：
--
作者：
Dong, Wei;Lu, Aiguo;Feng, Hao
来自同一供体的非实质细胞 (NPC) 和肿瘤细胞的共培养对于转移性癌症研究非常重要。本研究旨在优化肝脏 NPC 分离方案。使用两种新颖的 3D 器官共培养模型来分离肝细胞、内皮细胞 (EC) 和库普弗细胞 (KC)。建立了长期细胞共培养、密度梯度离心和磁激活细胞分选（MACS）。 ECs从共培养系统中分离出来； EC 的纯度为 92 +/- 1.2%。在3D共培养系统中观察到肝细胞的岛状形状，并且在其余空间中发现梭形细胞。免疫荧光分析显示呈网状结构；结缔组织经VE-cadherin或CD68染色呈阳性，分别为ECs和KCs/巨噬细胞。 KC 在该系统中得到富集，并通过选择性粘附到塑料上进行分离。 Clec4f(+) KC 由这些细胞的 87 +/- 6.3% 组成。检测到异质内皮细胞群，包括血窦内皮细胞、微血管内皮细胞和肝淋巴管上皮细胞。此外，分离肝祖细胞并分化为成肝细胞。通过密度梯度离心和磁珠分选进一步分离树突状细胞（DC）、不变自然杀伤T（iNKT）细胞。在本研究中，在肝星状细胞（HSC）中观察到结蛋白和 GFAP 的高蛋白表达水平。大多数 HSC 是 α-SMA 阳性细胞，这强调了活化 HSC 的身份。肝内人胆管上皮细胞 (hBEC) 通过 Percoll 梯度离心进行半纯化，并进一步进行免疫纯化。总之，我们提供了一种有效的长期培养方法来获得足够数量和纯度的肝脏 NPC。
Co-cultivation of non-parenchymal cells (NPCs) and tumor cells from the same donor is important for metastatic cancer research. This study aimed to optimize a protocol for liver NPC isolation. Two novel 3D organotypic co-culture models for hepatocyte, endothelial cell (EC) and Kupffer cell (KC) isolation were used. Long-term cell co-culture, density gradient centrifugation and magnetic-activated cell sorting (MACS) were established. ECs were isolated from the co-culture system; the purity of the ECs was 92 +/- 1.2%. The island-like shape of hepatocytes was noted in the 3D co-culture system, and spindle cells were found in the rest space. Immunofluorescence analysis showed a net structure; the connective tissue was positively stained with VE-cadherin or CD68, which were ECs and KCs/macrophages. KCs were enriched in this system and separated by using selective adherence to plastic. Clec4f(+) KCs consisted of 87 +/- 6.3% of these cells. Heterogeneous endothelium populations were detected, including sinusoid ECs, microvascular ECs and hepatic lymphatic vessel epithelial cells. In addition, hepatic progenitor cells were isolated and differentiated into hepatoblasts. Dendritic cells (DCs), invariant natural killer T (iNKT) cells were further separated by density gradient centrifugation and magnetic bead sorting. In the present study, high protein expression levels of desmin and GFAP were observed in the hepatic stellate cells (HSCs). Most of the HSCs were alpha-SMA-positive cells, which underlined the identity of activated HSCs. Intrahepatic human biliary epithelial cells (hBECs) were semi-purified by centrifugation on a Percoll gradient and were further immunopurified. In conclusion, we provide an efficient long-term culture method to obtain liver NPCs in sufficient number and purity.