Establishing Methotrexate-Induced Liver Fibrosis In Vitro Model with Regulatory Guidelines

Abstract Methotrexate (MTX)-induced liver fibrosis poses significant challenges in hepatotoxicity research, requiring advanced experimental models to evaluate its effects on hepatocyte functionality and fibrosis progression. This study investigates the impact of MTX on 2D HepG2 cell cultures by integrating morphological, biochemical, and molecular analyses. The results reveal a progressive decline in hepatocyte-specific functionality and a concomitant increase in fibrotic remodelling. Albumin secretion decreased from 1.2 g/dL on day 1 to below 0.6 g/dL by day 3 in fibrotic conditions, compared to a steady increase to 1.8 g/dL in healthy controls. Urea synthesis also declined significantly, dropping from 1.2 mmol/L to below 0.6 mmol/L over the same period, reflecting impaired metabolic activity. LDH release, indicative of membrane integrity loss, peaked at 2.0 µmol/L by day 3 in fibrotic cultures, contrasting with a gradual rise to 2.3 µmol/L in healthy conditions. Gene expression analysis demonstrated a threefold upregulation of collagen I by day 3 in fibrotic conditions, signifying ECM remodelling and activation of pro-fibrotic pathways such as TGF-β signalling. Morphological assessments via H&E staining showed nuclear condensation, ECM deposition, and cell disintegration at higher MTX concentrations. The results underscore the dual impact of MTX, impairing hepatocyte functionality while promoting fibrotic remodelling. These findings emphasize the need for advanced models, including co-culture systems and 3D bioprinting technologies, to capture the dynamic interplay between hepatocyte-specific activities and fibrosis progression. Lay Summary Advancement in liver fibrosis is a major cause for serious liver disease progression such as cirrhosis, carcinoma, eventually leading to liver failure. Currently, there is a lack of effective in vitro models to understand liver disease progression and treatment parameters. This study focuses on the development of a 2D in vitro model for anti-fibrotic drug screening, where the well-known cell line HepG2 was used to mimic a fibrotic platform. MTX induced hepatotoxicity on the 2D model, successfully recapitulating key cellular and morphological features of early-stage fibrosis. This innovative platform was confirmed by various morphologic, molecular, and functional assays demonstrating its potential as a drug testing platform. The results in this study highlight the requirement of developing sophisticated 3D in vitro platforms, such as 3D bioprinting approaches, to replicate accurately the complex interactions between hepatocyte function during liver fibrosis. This study offers the significant progress in the interpretation and modelling fibrosis using in vitro cultures, to overcome the limitations of other present preclinical approaches. The main aim of this study is to optimize the concentration of the fibrotic drug for the futuristic application in the 3D model.