PMID- 27498714
OWN - NLM
STAT- MEDLINE
DCOM- 20170303
LR  - 20170303
IS  - 1791-2431 (Electronic)
IS  - 1021-335X (Linking)
VI  - 36
IP  - 4
DP  - 2016 Oct
TI  - An efficient and simple co-culture method for isolating primary human hepatic 
      cells: Potential application for tumor microenvironment research.
PG  - 2126-34
LID - 10.3892/or.2016.4979 [doi]
AB  - 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.
FAU - Dong, Wei
AU  - Dong W
AD  - Heart Center, Shanghai Children's Medical Center, Shanghai Jiao Tong University 
      School of Medicine, Shanghai 200127, P.R. China.
FAU - Lu, Aiguo
AU  - Lu A
AD  - Department of General Surgery, Ruijin Hospital, Shanghai Jiao Tong University 
      School of Medicine, Shanghai 200025, P.R. China.
FAU - Zhao, Jingkun
AU  - Zhao J
AD  - Department of General, Visceral, Transplantation and Vascular Surgery, University 
      Hospital of Munich (Ludwig Maximilian University of Munich), D‑81377 Munich, 
      Germany.
FAU - Yin, Shuai
AU  - Yin S
AD  - Department of General, Visceral, Transplantation and Vascular Surgery, University 
      Hospital of Munich (Ludwig Maximilian University of Munich), D‑81377 Munich, 
      Germany.
FAU - Ou, Baochi
AU  - Ou B
AD  - Heart Center, Shanghai Children's Medical Center, Shanghai Jiao Tong University 
      School of Medicine, Shanghai 200127, P.R. China.
FAU - Feng, Hao
AU  - Feng H
AD  - Heart Center, Shanghai Children's Medical Center, Shanghai Jiao Tong University 
      School of Medicine, Shanghai 200127, P.R. China.
LA  - eng
PT  - Journal Article
DEP - 20160728
PL  - Greece
TA  - Oncol Rep
JT  - Oncology reports
JID - 9422756
SB  - IM
MH  - Cell Separation/methods
MH  - Coculture Techniques/*methods
MH  - *Endothelial Cells
MH  - Flow Cytometry
MH  - Fluorescent Antibody Technique
MH  - *Hepatocytes
MH  - Humans
MH  - *Kupffer Cells
MH  - Liver/cytology
MH  - Liver Neoplasms/pathology
MH  - *Tumor Microenvironment
EDAT- 2016/08/09 06:00
MHDA- 2017/03/04 06:00
CRDT- 2016/08/09 06:00
PHST- 2016/02/22 00:00 [received]
PHST- 2016/04/07 00:00 [accepted]
PHST- 2016/08/09 06:00 [entrez]
PHST- 2016/08/09 06:00 [pubmed]
PHST- 2017/03/04 06:00 [medline]
AID - 10.3892/or.2016.4979 [doi]
PST - ppublish
SO  - Oncol Rep. 2016 Oct;36(4):2126-34. doi: 10.3892/or.2016.4979. Epub 2016 Jul 28.