Long-term coculture strategies for primary hepatocytes and liver sinusoidal endothelial cells

Tissue Eng Part C Methods. 2015 Apr;21(4):413-22. doi: 10.1089/ten.TEC.2014.0152. Epub 2014 Nov 6.

Abstract

Hepatocytes and their in vitro models are essential tools for preclinical screening studies for drugs that affect the liver. Most of the current models primarily focus on hepatocytes alone and lack the contribution of non-parenchymal cells (NPCs), which are significant through both molecular and the response of the NPCs themselves. Models that incorporate NPCs alongside hepatocytes hold the power to enable more realistic recapitulation and elucidation of cell interactions and cumulative drug response. Hepatocytes and liver sinusoidal endothelial cells (LSECs) account for ∼ 80% of the liver mass where the LSECs line the walls of blood vessels, and act as a barrier between hepatocytes and blood. Culturing LSECs with hepatocytes to generate multicellular physiologically relevant in vitro liver models has been a major hurdle since LSECs lose their phenotype rapidly after isolation. To this end, we describe the application of collagen gel (1) in a sandwich and (2) as an intervening extracellular matrix layer to coculture hepatocytes with LSECs for extended periods. These coculture configurations provide environments wherein hepatocyte and LSECs, through cell-cell contacts and/or secretion factors, lead to enhanced function and stability of the cocultures. Our results show that in these configurations, hepatocytes and LSECs maintained their phenotypes when cultured together as a mixture, and showed stable secretion and metabolic activity for up to 4 weeks. Immunostaining for sinusoidal endothelial 1 (SE-1) antibody demonstrated retention of LSEC phenotype during the culture period. In addition, LSECs cultured alone maintained high viability and SE-1 expression when cultured within a collagen sandwich configuration up to 4 weeks. Albumin production of the cocultures was 10-15 times higher when LSECs were cultured as a bottom layer (with an intervening collagen layer) and as a mixture in a sandwich configuration, and native CYP 1A1/2 activity was at least 20 times higher than monoculture controls. Together, these data suggest that collagen gel-based hepatocyte-LSEC cocultures are highly suitable models for stabilization and long-term culture of both cell types. In summary, these results indicate that collagen gel-based hepatocyte-LSEC coculture models are promising for in vitro toxicity testing, and liver model development studies.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Communication*
  • Cells, Cultured
  • Coculture Techniques / methods*
  • Drug Evaluation, Preclinical / methods
  • Endothelial Cells* / cytology
  • Endothelial Cells* / metabolism
  • Female
  • Hepatocytes* / cytology
  • Hepatocytes* / metabolism
  • Liver* / cytology
  • Liver* / metabolism
  • Models, Biological*
  • Rats
  • Rats, Inbred Lew
  • Time Factors