Continual proteomic divergence of HepG2 cells as a consequence of long-term spheroid culture

Abstract

Three-dimensional models are considered a powerful tool for improving the concordance between in vitro and in vivo phenotypes. However, the duration of spheroid culture may influence the degree of correlation between these counterparts. When using immortalised cell lines as model systems, the assumption for consistency and reproducibility is often made without adequate characterization or validation. It is therefore essential to define the biology of each spheroid model by investigating proteomic dynamics, which may be altered relative to culture duration. As an example, we assessed the influence of culture duration on the relative proteome abundance of HepG2 cells cultured as spheroids, which are routinely used to model aspects of the liver. Quantitative proteomic profiling of whole cell lysates labelled with tandem-mass tags was conducted using liquid chromatography-tandem mass spectrometry (LC–MS/MS). In excess of 4800 proteins were confidently identified, which were shared across three consecutive time points over 28 days. The HepG2 spheroid proteome was divergent from the monolayer proteome after 14 days in culture and continued to change over the successive culture time points. Proteins representing the recognised core hepatic proteome, cell junction, extracellular matrix, and cell adhesion proteins were found to be continually modulated.

The Performance of HepG2 and HepaRG Systems through the Glass of Acetaminophen-Induced Toxicity

“…In in vitro models of primary human hepatocytes, immortalized human hepatic cell lines have been used, but they are limited regarding their viability, hepatic gene expression, and function [42]. Of the many options, three-dimensional (3D) models [19][20][21][22][23][24][25][26][27] and stem cell-derived models [43] have also become areas of significant interest. Developing appropriate toxicological model systems is not an easy task, but it will help the effectiveness of toxicological studies.…”

“…Developments in cell culture techniques aim at narrowing the gap between in vitro and in vivo models. Regarding hepatic in vitro models, 3D culture methods are extensively used to increase hepatic function [19][20][21][22][23][24][25][26][27].…”

“…As a result, there is a high pressure to replace PHHs in liver-related studies. Strategies to achieve better hepatocyte functions include genetic modification of long-established hepatoma cell lines or improvement of culture conditions, such as the development of cocultures and/or three-dimensional (3D) cultures [19][20][21][22][23][24][25][26][27].…”

Effect of HepG2 cell 3D cultivation on the metabolism of the anabolic androgenic steroid metandienone

“…The established methods for 3D cultivation can be allocated either to extracellular matrix‐based techniques or to scaffold‐free techniques based on self‐aggregation. For the latter group, cell aggregation can be achieved by using cell culture plastic ware with low‐attachment surface, external forces (magnetic forces, centrifugation) or by culturing as hanging drops 13,14 . As scaffold, hydrogels simulating the extracellular matrix, which might be of natural (e.g., Matrigel®) or synthetic origin, have been used for spheroid generation.…”

“…With regard to human hepatocellular HepG2 line, several studies investigated the usability of HepG2 spheroids as models for cytotoxicity and metabolism‐dependent drug‐induced liver injury. For the formation of the 3D HepG2 spheroids, the usage of low‐attachment culture plates, the hanging drop method and the cultivation on an extracellular matrix‐based hydrogen has been described in literature 11,14–17 …”