3D hepg2 cell-based cell transformation assay.

Abstract

Monolayer in vitro cultures of hepatic cells, so-called two-dimensional (2D) liver models do not adequately mimic the natural cell microenvironment because of the lack of different biological features and functions such as cell-to-cell and cell-to-matrix interactions. That is why there is a need to develop and improve three-dimensional (3D) hepatic cell (liver) models, characterized by higher expression of liver cell markers and cell interactions, although they still present some limitations and special requirements. This study aims to optimize and adapt 3D HepG2 cell-based transformation assay applicable for biomedicine, carcinogenicity research and toxicology. Within the proposed protocol, HepG2 cells spheroids were first exposed to an initiator (3-methylcholanthrene, 3-MC) followed by the exposure to promotor (12-O-tetradecanoylphorbol-13- acetate, TPA) to create a tumor environment and characterize a new hepatocellular carcinoma in vitro model. The spheroids were monitored within a culture with a non-destructive and non-invasive bright field microscopy-based assay. The analysis is suitable with manual high-speed automated microscopic image acquisition and automated analysis using an in-house built macro 'Spheroid_Finder' in Fiji/ImageJ software. This protocol was essential to characterize and quantify 3D spheroid formation, size and shape. The results show that spheroids treated with solvent controls did not differ from non-treated spheroids in size or shape. Interestingly, the spheroids treated with 3-MC alone and with the combination with TPA were smaller than the control spheroids, and their shape and compactness were also affected. On the other hand, spheroids treated with TPA alone were mildly bigger than the control ones, and their shape and compactness were changed. It should be emphasized that to confirm cell transformation, the gene expression related to neoplastic and hepatocellular carcinoma-derived cell lines phenotypes will be further evaluated with RT-qPCR, as well as spheroids functionality by measuring albumin, urea or lactate in the collected medium. Despite this assay needing further optimization and validation to confirm its potential, we conclude that CTA assay utilizing HepG2 3D spheroids can be a promising tool for screening carcinogenicity potential of chemicals or new anti-cancer in biomedical, pharmacological, and toxicological research.