In Vitro Generation of Functional Liver Organoid-Like Structures Using Adult Human Cells

PLoS One. 2015 Oct 21;10(10):e0139345. doi: 10.1371/journal.pone.0139345. eCollection 2015.

Abstract

In this study we used differentiated adult human upcyte® cells for the in vitro generation of liver organoids. Upcyte® cells are genetically engineered cell strains derived from primary human cells by lenti-viral transduction of genes or gene combinations inducing transient proliferation capacity (upcyte® process). Proliferating upcyte® cells undergo a finite number of cell divisions, i.e., 20 to 40 population doublings, but upon withdrawal of proliferation stimulating factors, they regain most of the cell specific characteristics of primary cells. When a defined mixture of differentiated human upcyte® cells (hepatocytes, liver sinusoidal endothelial cells (LSECs) and mesenchymal stem cells (MSCs)) was cultured in vitro on a thick layer of Matrigel™, they self-organized to form liver organoid-like structures within 24 hours. When further cultured for 10 days in a bioreactor, these liver organoids show typical functional characteristics of liver parenchyma including activity of cytochromes P450, CYP3A4, CYP2B6 and CYP2C9 as well as mRNA expression of several marker genes and other enzymes. In summary, we hereby describe that 3D functional hepatic structures composed of primary human cell strains can be generated in vitro. They can be cultured for a prolonged period of time and are potentially useful ex vivo models to study liver functions.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adult
  • Albumins / genetics
  • Albumins / metabolism
  • Bioreactors
  • Cadherins / genetics
  • Cadherins / metabolism
  • Cell Differentiation
  • Cells, Cultured
  • Collagen
  • Cytochrome P-450 Enzyme System / genetics
  • Cytochrome P-450 Enzyme System / metabolism
  • Drug Combinations
  • Endothelial Cells / cytology
  • Endothelial Cells / metabolism
  • Endothelial Cells / physiology*
  • Hepatocytes / cytology
  • Hepatocytes / metabolism
  • Hepatocytes / physiology*
  • Humans
  • Immunohistochemistry
  • Laminin
  • Liver / cytology
  • Liver / metabolism
  • Liver / physiology*
  • Mesenchymal Stem Cells / cytology
  • Mesenchymal Stem Cells / metabolism
  • Mesenchymal Stem Cells / physiology*
  • Microscopy, Confocal
  • Organoids / cytology
  • Organoids / metabolism
  • Organoids / physiology*
  • Proteoglycans
  • Reverse Transcriptase Polymerase Chain Reaction
  • Time Factors
  • Tissue Engineering / methods
  • Zonula Occludens-1 Protein / genetics
  • Zonula Occludens-1 Protein / metabolism

Substances

  • Albumins
  • Cadherins
  • Drug Combinations
  • Laminin
  • Proteoglycans
  • TJP1 protein, human
  • Zonula Occludens-1 Protein
  • matrigel
  • Collagen
  • Cytochrome P-450 Enzyme System

Grant support

The work leading to these results has received funding from the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement n° 304961, the HeMiBio consortium (grant agreement no. HEALTH-F5-2010-266777) and from the “Ministerium für Wissenschaft, Forschung und Kunst Baden-Württemberg”, funding program “Entwicklung von Alternativmethoden zur Vermeidung von Tierversuchen" (KB-H). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Medicyte GmbH and upcyte technologies GmbH provided support in the form of salaries for authors [SDR, BM, SH, JB] and [SH], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the "author contributions" section.