Abstract: Objective Construction of mouse liver senescence model induced rapidly in vitro by 3D organoid culture. Methods Mouse hepatocytes were collected by two-step perfusion method and cultured in 3D. Organoids were preliminarily identified by morphology, qPCR and immunofluorescence. The function of hepatocyte organoid was evaluated by their uptake of LDL. The hepatocyte organoids were treated with oleic acid, and their clonal growth, senescence related secretory phenotype, LDL uptake and ROS level of liver cells were detected. The hepatocytes were further treated with N-acetylcysteine (NAC) to detect the above phenotypic and functional changes. Results A large number of active primary mouse hepatocytes were obtained by two-step perfusion method. The hepatocyte organoid with self-reproduction ability was collected by 3D culture in the matrix gel. The expression of liver marker genes in hepatocyte organoid and primary hepatocytes was shown by qPCR detection. The immunofluorescence results showed that it highly expressed ALB protein. Moreover, hepatocyte organoid had normal uptake function of LDL. After treatment with oleic acid, the growth of hepatocyte organoid was slowed down, the expression of senescence related secretory phenotype genes was up-regulated(P＜0.001), the uptake of LDL was reduced(P＜0.001), and ROS was significantly increased(P＜0.001). The above phenotypes were alleviated by NAC treatment(P＜0.01 or P＜0.001). Conclusions Oleic acid treatment can successfully construct the aging model of mouse liver in vitro. Adding NAC can clear ROS and alleviate the occurrence of aging.

Key words: primary hepatocyte, organoid, oleic acid, reactive oxygen species, aging