Everything you need to know about Hepatocyte

The liver is an organ that is part of the digestive system in vertebrates. It is composed of 4 different types of cells, hepatocytes (HCs), hepatic stellate cells (HSCs), Kupffer cells (KC) and liver sinusoidal endothelial cells (LSECs). Hepatocytes are the predominant cells in the liver and are responsible for regulation of metabolism, detoxification, production of bile and manufacture of essential proteins (1). Thus it serves as the main cell in studies involving liver disease, toxicology screens quantifying drug-induced liver injury during drug discovery and development.

2D vs. 3D Primary Hepatocyte cell culture

Primary hepatocyte culturing often faces difficulties in prolonging cell viability and function in traditional 2D cultures (2-4). The use of collagen substrate and matrigel overlay has helped in improving cell viability, albeit with some hepatic gene expression changes, thus limiting its use in liver toxicology studies (5). 3D culturing of primary heptaocytes has provided an alternative approach in improving cell viability and function with high throughput capacity.

Scaffold free Liver Spheroids

In addition to maintaining cell viability for more than 4 weeks, liver spheroids were found to preserve hepatic cellular phenotype comprised of in-vivo like 3D organization and intracellular interactions (6-8). Protein and gene expression studies were similar to the native state (9,10). Further technological advances in novel 96 well-plates have improved the quality of dog 3D spheroids that were once difficult to culture in traditional low attachment 96- well plates, thus improving its potential for use in drug induced liver toxicity studies (12).

Cryopreserved cynomolgus monkey hepatocytes and primary human hepatocytes can be successfully cultured in spheroid form on highly defined u-bottom plates coated with a specialized polymer solution to render a physiologically relevant environment (13). In this manner liver spheroids serves as an excellent in-vitro system that can be used in toxicology studies and as well as to model liver diseases such as fatty liver disease (11,13).


  1. Chen Ding, Yanyan Li, Feifei Guo, Ying Jiang, Wantao Ying, Dong Li, Dong Yang, Xia Xia, Wanlin Liu, Yan Zhao, Yangzhige He, Xianyu Li, Wei Sun, Qiongming Liu, Lei Song, Bei Zhen, Pumin Zhang, Xiaohong Qian, Jun Qin, Fuchu He. A Cell-type-resolved Liver Proteome. Mol Cell Proteomics. 2016 Oct; 15(10): 3190–3202.
  2. Y. Kono, S. Yang, E.A. Roberts. Extended primary culture of human hepatocytes in a collagen gel sandwich system. In Vitro Cell Dev Biol Anim, 33 (1997), pp. 467-472
  3. G. Elaut, T. Henkens, P. Papeleu, et al. Molecular mechanisms underlying the dedifferentiation process of isolated hepatocytes and their cultures. Curr Drug Metab, 7 (2006), pp. 629-660
  4. M.J. Gomez-Lechon, L. Tolosa, I. Conde, M.T. Donato. Competency of different cell models to predict human hepatotoxic drugs. Expert Opin Drug Metab Toxicol, 10 (2014), pp. 1553-1568
  5. C.C. Bell, A.C.A. Dankers, V.M. Lauschke, et al. Comparison of hepatic 2D sandwich cultures and 3D spheroids for long-term toxicity applications: a multicenter study. Toxicol Sci, 162 (2018), pp. 655-666
  6. S. Messner, I. Agarkova, W. Moritz, J.M. Kelm Multi-cell type human liver microtissues for hepatotoxicity testing. Arch Toxicol, 87 (2013), pp. 209-213
  7. C.C. Bell, D.F. Hendriks, S.M. Moro, et al. Characterization of primary human hepatocyte spheroids as a model system for drug-induced liver injury, liver function and disease. Sci Rep, 6 (2016), p. 25187
  8. D.F. Hendriks, L. Fredriksson Puigvert, S. Messner, W. Mortiz, M. Ingelman-Sundberg. Hepatic 3D spheroid models for the detection and study of compounds with cholestatic liability. Sci Rep, 6 (2016), p. 35434
  9. C.C. Bell, V.M. Lauschke, S.U. Vorrink, et al. Transcriptional, functional, and mechanistic comparisons of stem cell-derived hepatocytes, HepaRG cells, and three-dimensional human hepatocyte spheroids as predictive in vitro systems for drug-induced liver injury. Drug Metab Dispos, 45 (2017), pp. 419-429
  10. S. Messner, I. Agarkova, W. Moritz, J.M. Kelm. Transcriptomic, proteomic, and functional long-term characterization of multicellular three-dimensional human liver microtissues. Appl In Vitro Toxicol, 4 (2018), pp. 1-12
  11. M. Kozyra, I. Johansson, A. Nordling, S. Ullah, V.M. Lauschke, M. Ingelman-Sundberg. Human hepatic 3D spheroids as a model for steatosis and insulin resistance. Sci Rep, 8 (2018), p. 14297
  12. Raic, A., Oberfrank, C., Birk, B. et al. 3D-Hunde-Lebersphäroide für die präklinische in vitro-Testung. Biospektrum 27, 736–738 (2021). https://doi.org/10.1007/s12268-021-1661-x
  13. Ullrich A., Dieckhoff J., Schwartz V., Runge D., Hewitt P., Mentzel T. A novel polymer solution to generate ultra-low cell attachment surfaces and highly uniform spheroids in 3D primary cell cultures. (2021) https://www.swiftanalytical.com/wp-content/uploads/2021/02/Application-Note-Primary-Cells.pdf