Characterization and reproducibility of HepG2 hanging drop spheroids toxicology in vitro

Abstract

Hepatotoxicity remains a major challenge in drug development despite preclinical toxicity screening using hepatocytes of human origin. To overcome some limitations of reproducing the hepatic phenotype, more structurally and functionally authentic cultures in vitro can be introduced by growing cells in 3D spheroid cultures. Characterisation and reproducibility of HepG2 spheroid cultures using a high-throughput hanging drop technique was performed and features contributing to potential phenotypic variation highlighted. Cultured HepG2 cells were seeded into Perfecta 3D® 96-well hanging drop plates and assessed over time for morphology, viability, cell cycle distribution, protein content and protein-mass profiles. Divergent aspects which were assessed included cell stocks, seeding density, volume of culture medium and use of extracellular matrix additives. Hanging drops are advantageous due to no complex culture matrix being present, enabling background free extractions for downstream experimentation. Varying characteristics were observed across cell stocks and batches, seeding density, culture medium volume and extracellular matrix when using immortalized HepG2 cells. These factors contribute to wide-ranging cellular responses and highlights concerns with respect to generating a reproducible phenotype in HepG2 hanging drop spheroids.

Hepato(Geno)Toxicity Assessment of Nanoparticles in a HepG2 Liver Spheroid Model

“…The presence of non-parenchymal cells can possibly explain the higher response to the NPs in the InSphero model compared to the HepG2 spheroids. The HepG2 spheroids show relatively high metabolic capacity and appear to be a good advanced in vitro model for the liver [21][22][23][24]. The commercial primary cell InSphero co-culture model is more complex than the HepG2 spheroids.…”

“…However, when comparing in vitro cell culture models in standard two-dimensional (2D) monolayers with complex organs, the cell lines in 2D culture display a limited hepatocytic functionality [21].The liver-like functionality of the human hepatocellular carcinoma cell line HepG2 is enhanced when the cells are cultured in a three-dimensional (3D) arrangement. This increases the cell-to-cell contacts and intercellular communication [22] and changes the protein expression and metabolic status of the cells [21][22][23]. HepG2 cells in 3D cultures show upregulation of genes involved in liver-specific xenobiotic and lipid metabolism, whereas genes related to the extracellular matrix, cytoskeleton and cell adhesion have higher expression in 2D cultures [22,24].The use of spheroids as 3D cultures in hepatotoxicity assessment is an increasing field of interest, and HepG2 spheroids, prepared with and without using scaffolds, have been applied for toxicity experiments with both NPs [6,25,26] and chemicals [27,28].…”

“…The liver-like functionality of the human hepatocellular carcinoma cell line HepG2 is enhanced when the cells are cultured in a three-dimensional (3D) arrangement. This increases the cell-to-cell contacts and intercellular communication [22] and changes the protein expression and metabolic status of the cells [21][22][23]. HepG2 cells in 3D cultures show upregulation of genes involved in liver-specific xenobiotic and lipid metabolism, whereas genes related to the extracellular matrix, cytoskeleton and cell adhesion have higher expression in 2D cultures [22,24].…”

The study of establishment of an in vivo tumor model by three-dimensional cells culture systems methods and evaluation of antitumor effect of biotin-conjugated pullulan acetate nanoparticles

“…Since the survival advantages of HepG2 cells studies have been reported, it has been shown that more cells in the 3D accumulated in the G1 phase of the cell cycle as compared to the 2D cells. 26,27 The adherent HepG2 cells in 2D culture environment were polygonal-shaped on planar rigid surface (Figure 4(a)), and in 3D growing into the sphericals and gathered to form homogenous multicellular spheroids (Figure 4(b,c)). We found that cells in 3D culture environment showed higher formation rate of spheroids.…”

Development of a tunable method to generate various three-dimensional microstructures by replenishing macromolecules such as extracellular matrix components and polysaccharides

“…Laschke et al 5 reviewed various methods to assemble spheroids and discussed their advantages. For example, spheroids of the hepatoma cell lines Hep G2 and HuH-7 were successfully generated in hanging drops or on low adhesion surfaces [6][7][8][9][10] . However, these culture systems do not fully consider the influence of the ECM.…”