Basic Study
Copyright ©The Author(s) 2020. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Stem Cells. Oct 26, 2020; 12(10): 1184-1195
Published online Oct 26, 2020. doi: 10.4252/wjsc.v12.i10.1184
Creating rat hepatocyte organoid as an in vitro model for drug testing
Yu-Ting He, Xing-Long Zhu, Sheng-Fu Li, Bing-Qi Zhang, Yi Li, Qiong Wu, Yun-Lin Zhang, Yan-Yan Zhou, Li Li, Ya-Na Qi, Ji Bao, Hong Bu
Yu-Ting He, Xing-Long Zhu, Bing-Qi Zhang, Yi Li, Qiong Wu, Yun-Lin Zhang, Yan-Yan Zhou, Li Li, Ji Bao, Laboratory of Pathology, Key Laboratory of Transplant Engineering and Immunology, NHC, West China Hospital, Sichuan University, Chengdu 610041, Sichuan Province, China
Sheng-Fu Li, Key Laboratory of Transplant Engineering and Immunology, NHC, West China Hospital, Sichuan University, Chengdu 610041, Sichuan Province, China
Ya-Na Qi, Chinese Evidence-based Medicine Center, West China Hospital, Sichuan University, Chengdu 610041, Sichuan Province, China
Hong Bu, Department of Pathology, West China Hospital, Chengdu 610041, Sichuan Province, China
Author contributions: He YT, Zhu XL, Li Y, Wu Q, Bu H, and Bao J designed and coordinated the study; He YT, Zhu XL, Li SF, Zhang BQ, Zhang YL, Li L, and Zhou YY performed the experiments, and acquired and analyzed the data; He YT, Li Y, Wu Q, Li L, Qi YN, Bu H, and Bao J interpreted the data; He YT, Wu Q, Qi YN, and Bao J wrote the manuscript; all authors approved the final version of the article.
Supported by National Natural Science Foundation of China, No. 81770618; Key R&D (Major Science and Technology Project) Project of Sichuan Science and Technology Department, No. 2019YFS0138; Technological Innovation Project of Chengdu New Industrial Technology Research Institute, No. 2018-CY02-00046-GX; and the 1.3.5 Project for Disciplines of Excellence, West China Hospital, No. ZYGD18012.
Institutional animal care and use committee statement: All procedures involving animals were reviewed and approved by the Institutional Animal Care and Use Committee of Animal Experiment Center of Sichuan University (IACUC protocol number: [2020007A]).
Conflict-of-interest statement: All the authors have nothing to disclose.
Data sharing statement: No additional data are available.
ARRIVE guidelines statement: The authors have read the ARRIVE guidelines, and the manuscript was prepared and revised according to the ARRIVE guidelines.
Open-Access: This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/
Corresponding author: Ji Bao, MD, PhD, Professor, Laboratory of Pathology, Key Laboratory of Transplant Engineering and Immunology, NHC, West China Hospital, Sichuan University, No. 37 Guoxue Alley, Wuhou District, Chengdu 610041, Sichuan Province, China. baoji@scu.edu.cn
Received: February 29, 2020
Peer-review started: February 29, 2020
First decision: April 25, 2020
Revised: May 15, 2020
Accepted: August 1, 2020
Article in press: August 1, 2020
Published online: October 26, 2020
Processing time: 240 Days and 3 Hours
ARTICLE HIGHLIGHTS
Research background

In all cell therapies for liver diseases, primary hepatocyte is accepted as the best cell source with high liver-specific functions compared to immortalized human hepatoblastoma cell lines, nonparenchymal cell, and differentiated cell. But a major challenge that has limited the advancement of these therapies is the propensity of hepatocytes to lose liver-specific functions and the ability to replicate in vitro without microenvironment. Recently, organoid technology has been applied to the study of human development and generation of models for disease and drug screening in different systems such as the brain, stomach, intestine, and kidney. Liver organoids are able to simulate liver development and differentiation, show interaction between cells and cells and between cells and matrix, and can be used in liver transplant, extracorporeal devices, and the generation of models for liver disease and drug screening.

Research motivation

Although many studies reported different liver organoids generated from Lgr5+ stem cells or induced pluripotent stem cell derived hepatocytes liver organoids, few studies have described the generation of liver organoids assembled using mesenchymal stem cells (MSCs) and primary hepatocytes. Besides, almost all studies used Matrigel to create organoids, and we attempted to find a new and reproducible method to generate hepatocytes organoids by co-culture with MSCs on an extracellular matrix (ECM) hydrogel from decellularization porcine liver.

Research objectives

The present study aimed to create hepatocyte organoids by co-culture of hepatocytes with MSCs on a porcine liver extracellular matrix (PLECM) hydrogel.

Research methods

Porcine liver was infused with Triton and sodium dodecyl sulfate to obtain the porcine liver extracellular matrix (PLECM) scaffold material. After perfusion and enzymatic hydrolysis, PLECM scaffold material formed hydrogel, which was pre-coated in 48 well plate for organoid culture. Rat primary hepatocytes were obtained by two step perfusion method. To create primary hepatocytes organoids, MSCs and rat primary hepatocytes were co-cultured on a PLECM-gel pre-coated plate. HE, PAS, and immunohistological staining, albumin detection, urea production assay, and quantitative PCR for alb, CYP450 gene markers, and urea cycle gene markers were performed to evaluate organoids function.

Research results

Decellularized liver maintained the ultrastructure of the extracellular matrix and most important components. MSCs and rat primary hepatocytes generated primary hepatocyte organoids on the PLECM-gel pre-coated plate in 48 h after seeding. MSC condensation on the extracellular matrix hydrogel contributed to the hepatocyte organoids generation. Primary hepatocyte organoids maintained high quality of liver special function of primary hepatocytes and prolonged the survival time of hepatocytes in vitro.

Research conclusions

This is the first study to create primary hepatocytes organoids by co-culture of hepatocytes and MSCs on a liver-derived extracellular matrix hydrogel. Hepatocyte organoids show long-term survival and stable function in vitro.

Research perspectives

In this study, we used rat hepatocytes to create hepatocytes organoids on a liver-derived extracellular matrix hydrogel. If this method is applied to patients’ liver cancer cells, cryopreserved mature human hepatocytes, and human hepatocytes derived from Fah-/-/Rag2-/-/Il2rg-/- mice, or combined with liver-on-a-chip technologies, it can also be used as models for liver disease and drug screening for individualized therapy.