Organoid Establishment of Long-Term Culture Using Primary Mouse Hepatocytes and Evaluation of Liver Function

Hye Mi Kim; Yerin Kim; Yuri Kim; Young Jun Kim; Kwang Suk Ko

doi:10.3746/pnf.2023.28.3.360

Organoid Establishment of Long-Term Culture Using Primary Mouse Hepatocytes and Evaluation of Liver Function

Prev Nutr Food Sci. 2023 Sep 30;28(3):360-369. doi: 10.3746/pnf.2023.28.3.360.

Authors

Hye Mi Kim^{1

2}, Yerin Kim^{1

3}, Yuri Kim^{1

2}, Young Jun Kim⁴, Kwang Suk Ko^{1

2}

Affiliations

¹ Department of Nutritional Science and Food Management, Ewha Womans University, Seoul 03760, Korea.
² Graduate Program in System Health Science and Engineering, Ewha Womans University, Seoul 03760, Korea.
³ Department of Medicine, Catholic Kwandong University College of Medicine, Gangwon 25601, Korea.
⁴ Department of Food and Biotechnology and Food and Regulatory Science, Korea University, Sejong 30019, Korea.

Abstract

Primary hepatocytes and various animal models have traditionally been used in liver function tests to assess the effects of nutrients. However, these approaches present several limitations such as time consumption, high cost, the need for facilities, and ethical issues in primary mouse hepatocytes and animal models. In this study, we constructed liver organoids from primary mouse hepatocytes (OrgPH) to replace primary hepatocytes and animal models. We isolated primary mouse hepatocytes from 6- to 10-week-old male C57BL/6J mice using the two-step collagenase method, and generated liver organoids by clustering the cells in Matrigel. To assess the hepatic function of OrgPH, we examined specific liver markers and gene expressions related to hepatic glucose, ethanol, and cholesterol metabolism. Over a 28-day culture period, liver-specific markers, including Alb, Arg1, G6pc, and Cyp1a1, increased or remained stable in the OrgPH. However, they eventually decreased in primary hepatocytes. Glucose and ethanol metabolism-related gene expression levels exhibited a similar tendency in AML12 cells and OrgPH. However, the expression levels of cholesterol metabolism-related genes displayed an opposite trend in OrgPH compared with those in AML12 cells. These results agree with those of previous studies involving in vivo models. In conclusion, our study indicates that OrgPH can retain liver function and mimic the hepatocytic physiology of mouse in vivo models. Therefore, organoids originating from primary mouse hepatocytes are potentially useful as an animal-free method for evaluating the safety and toxicity of health functional foods and a replacement for animal models.

Keywords: liver function; long-term culture; organoid; primary hepatocyte.