Assessment of cellular proliferation, death and senescence in a model of steatosis in vitro

Abstract

Introduction and objectives: Hepatocyte cell culture in steatogenic medium is a useful, reproducible tool. During NAFLD, cells undergo processes in response to the steatogenic input. Here we aimed to study cellular proliferation, death, and senescence in an in vitro model of steatosis. Materials and Methods: HepG2 hepatocytes were cultured in standard RPMI1640. Steatogenic medium was prepared by supplementing RPMI1640 with lipids in two levels: Mild steatosis (MS: 50 µM sodium oleate/sodium palmitate (OA/PA) at 2:1 ratio) and Severe steatosis (SS: 500 µM OA/PA 2:1). A control (C) group (RPMI1640) was included. 105 cells per well were allowed to attach for 24 h in RPMI1640 at 37°C and 5% CO2, then incubated in MS or SS medium for up to 96 h. Steatogenic medium was refreshed daily. Viability and mortality rates were assessed, and proliferation and senescence were analyzed by commercial kits, followed by a morphometric analysis. All assays were performed in triplicates. Data: Mean ± SD. 2-way ANOVA followed by Tukey. P<0.05. Results: MS and SS showed significantly lower cell viability versus C. Mortality rates were increased in MS and SS. Proliferation was significantly decreased in MS and SS compared with C. MS showed a significantly increased senescence from 48 h versus C, whereas in SS decreased compared with C and MS. Conclusion: MS showed an increment in senescence compared with C and might be considered a mechanism aimed at avoiding damaged-cell proliferation. In contrast, SS showed an increased mortality rate and decreased senescence, suggesting activation of death pathways as a response to lipid overexposure. Funding: This work was funded by Conacyt (CB-221137) Declaration of interest: The authors declare no potential conflicts of interest.