Comparative analysis of 3D culture methods on human HepG2 cells

Arch Toxicol. 2017 Jan;91(1):393-406. doi: 10.1007/s00204-016-1677-z. Epub 2016 Feb 12.


Human primary hepatocytes represent a gold standard in in vitro liver research. Due to their low availability and high costs alternative liver cell models with comparable morphological and biochemical characteristics have come into focus. The human hepatocarcinoma cell line HepG2 is often used as a liver model for toxicity studies. However, under two-dimensional (2D) cultivation conditions the expression of xenobiotic-metabolizing enzymes and typical liver markers such as albumin is very low. Cultivation for 21 days in a three-dimensional (3D) Matrigel culture system has been reported to strongly increase the metabolic competence of HepG2 cells. In our present study we further compared HepG2 cell cultivation in three different 3D systems: collagen, Matrigel and Alvetex culture. Cell morphology, albumin secretion, cytochrome P450 monooxygenase enzyme activities, as well as gene expression of xenobiotic-metabolizing and liver-specific enzymes were analyzed after 3, 7, 14, and 21 days of cultivation. Our results show that the previously reported increase of metabolic competence of HepG2 cells is not primarily the result of 3D culture but a consequence of the duration of cultivation. HepG2 cells grown for 21 days in 2D monolayer exhibit comparable biochemical characteristics, CYP activities and gene expression patterns as all 3D culture systems used in our study. However, CYP activities did not reach the level of HepaRG cells. In conclusion, the increase of metabolic competence of the hepatocarcinoma cell line HepG2 is not due to 3D cultivation but rather a result of prolonged cultivation time.

Keywords: 3D cultivation; Cytochrome P450; HepG2 cells; Liver cell model; Xenobiotic metabolism.

Publication types

  • Comparative Study

MeSH terms

  • Animals
  • Biomarkers / metabolism
  • Cell Culture Techniques*
  • Cell Shape
  • Cell Survival / drug effects
  • Cells, Cultured
  • Collagen / chemistry
  • Drug Combinations
  • Gene Expression Profiling
  • Gene Expression Regulation, Enzymologic* / drug effects
  • Hep G2 Cells
  • Hepatocytes / cytology
  • Hepatocytes / drug effects
  • Hepatocytes / metabolism*
  • Humans
  • Imaging, Three-Dimensional
  • Laminin / chemistry
  • Microscopy, Confocal
  • Proteoglycans / chemistry
  • RNA, Messenger / metabolism
  • Time Factors
  • Tissue Scaffolds / chemistry*
  • Toxicity Tests*
  • Xenobiotics / toxicity


  • Biomarkers
  • Drug Combinations
  • Laminin
  • Proteoglycans
  • RNA, Messenger
  • Xenobiotics
  • matrigel
  • Collagen