Characterization of In Vitro 3D Cell Model Developed from Human Hepatocellular Carcinoma (HepG2) Cell Line.

Martina Štampar,Bojana Žegura,Barbara Breznik,Metka Filipič

doi:10.3390/cells9122557

Martina Štampar, Bojana Žegura + Show 2 more

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https://doi.org/10.3390/cells9122557

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Abstract

In genetic toxicology, there is a trend against the increased use of in vivo models as highlighted by the 3R strategy, thus encouraging the development and implementation of alternative models. Two-dimensional (2D) hepatic cell models, which are generally used for studying the adverse effects of chemicals and consumer products, are prone to giving misleading results. On the other hand, newly developed hepatic three-dimensional (3D) cell models provide an attractive alternative, which, due to improved cell interactions and a higher level of liver-specific functions, including metabolic enzymes, reflect in vivo conditions more accurately. We developed an in vitro 3D cell model from the human hepatocellular carcinoma (HepG2) cell line. The spheroids were cultured under static conditions and characterised by monitoring their growth, morphology, and cell viability during the time of cultivation. A time-dependent suppression of cell division was observed. Cell cycle analysis showed time-dependent accumulation of cells in the G0/G1 phase. Moreover, time-dependent downregulation of proliferation markers was shown at the mRNA level. Genes encoding hepatic markers, metabolic phase I/II enzymes, were time-dependently deregulated compared to monolayers. New knowledge on the characteristics of the 3D cell model is of great importance for its further development and application in the safety assessment of chemicals, food products, and complex mixtures.

Highlights

In recent years, considerable efforts have been made to develop hepatic in vitro 3D cell models with higher predictability for detecting the genotoxic effects of chemicals and environmental particular liver, which is the main target organ of chemical activation and detoxification processes [1,2]
It is widely recognised that human primary hepatocytes (PHH) are the golden standard for studying metabolism and toxicity, as they are of human origin and express metabolic enzymes that are relevant for human metabolism of xenobiotics [5,6,7,8,9]
Before 3D cell models can be integrated for genotoxicity testing research, there is a need for the development and subsequent standardisation of robust models that accurately predict the possible effects of studied compounds [45]

Summary

Introduction

Considerable efforts have been made to develop hepatic in vitro 3D cell models with higher predictability for detecting the genotoxic effects of chemicals and environmental particular liver, which is the main target organ of chemical activation and detoxification processes [1,2]. Hepatic carcinoma-derived cell lines, such as HepG2, C3A, HepaRG, HuH6, and many others, are frequently used in genotoxicity studies, due to their unlimited growth, availability, and high reproducibility of results. These cell lines [13,14,15,16] have several phenotypic characteristics and some functional properties of liver cells [17,18] and represent an effective compromise between the ease of culturing and the expression of several key enzymes involved in xenobiotic metabolism [19,20,21]. It is highly plausible that such cell models give inaccurate and false-positive results [23]

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