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
Abstract
BACKGROUND

Liver organoids have recently been applied as models for liver disease and drug screening, especially when combined with liver-on-a-chip technologies. Compared to hepatocyte-like cells, primary hepatocytes have high functionality but cannot maintain their function when cultured in vitro. Mesenchymal stem cells (MSCs) enhance hepatocyte function and maintain hepatocyte metabolism when co-cultured with hepatocytes. MSCs can help induced pluripotent stem cells to generate an organoid structure via the MSC-based traction force triggered by extracellular matrix (ECM) proteins. In this study, primary hepatocytes were co-cultured with MSCs on a liver-derived ECM to generate liver organoids within a short duration.

AIM

To create hepatocyte organoids by co-culturing primary hepatocytes with MSCs on a porcine liver extracellular matrix (PLECM) gel.

METHODS

Perfusion and enzymatic hydrolysis were used to form the PLECM gel. Rat hepatocytes and human MSCs were mixed and plated on pre-solidified PLECM gel in a 48-well plate for 48 h to generate organoids. Generated organoids were evaluated through hematoxylin and eosin, periodic acid-Schiff, immuno-histological, and immunofluorescence staining, and quantitative PCR for alb, CYP450 gene markers, and urea cycle genes. Culture medium was collected to detect albumin (ALB) and urea production on days 2, 4, 6, 8, 14, and 20.

RESULTS

The whole porcine liver was perfused and enzymatically hydrolyzed to form a PLECM gel. The structural components and basement membrane composition of the ECM, such as collagen type I, collagen type IV, fibronectin, and laminin, were demonstrated to be retained. Through interaction of human MSCs with the liver-derived ECM, primary hepatocytes and human MSCs assembled together into a 3D construction and generated primary hepatocyte organoids for 48 h. The mRNAs of the gene alb, the CYP450 gene markers cyp1a1, cyp1a2, and cyp3a2 as well as urea cycle genes arg-1, asl, ass-1, cps-1, nags were highly expressed in hepatocyte organoids. Long-term survival of the primary hepatocyte organoids, as well as stable functionality, was demonstrated via ALB and urea production in vitro.

CONCLUSION

Our new method of creating primary hepatocyte organoids by co-culturing hepatocytes with MSCs on liver-derived ECM hydrogels could be used to develop models for liver disease and for drug screening.

Keywords: Organoid; Primary hepatocytes; Stem cells; Liver therapies; Extracellular matrix; Drug screening

Core Tip: Mesenchymal stem cells (MSCs) can help primary hepatocytes to create hepatocyte organoids by interacting with a liver-derived extracellular matrix. MSCs and hepatocytes self-assembled together into hepatocyte organoids via MSC-derived condensation related to myosin-II regulatory light chain. The hepatocyte organoids can survive for a long time and maintain the functionality of the hepatocytes while avoiding the limitation of rapid function loss of primary hepatocytes in vitro. This hepatocyte organoid technology can also be used to develop models for liver disease and drug screening.