An efficient and simple co-culture method for isolating primary human hepatic cells: Potential application for tumor microenvironment research

Dong,Wei; Lu,Aiguo; Zhao,Jingkun; Yin,Shuai; Ou,Baochi; Feng,Hao

doi:10.3892/or.2016.4979

An efficient and simple co-culture method for isolating primary human hepatic cells: Potential application for tumor microenvironment research

Authors:
- Wei Dong
- Aiguo Lu
- Jingkun Zhao
- Shuai Yin
- Baochi Ou
- Hao Feng
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Affiliations: Heart Center, Shanghai Children's Medical Center, Shanghai Jiao Tong University School of Medicine, Shanghai 200127, P.R. China, Department of General Surgery, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, P.R. China, Department of General, Visceral, Transplantation and Vascular Surgery, University Hospital of Munich (Ludwig Maximilian University of Munich), D‑81377 Munich, Germany
Published online on: July 28, 2016 https://doi.org/10.3892/or.2016.4979
Pages: 2126-2134

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Abstract

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 α‑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.

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