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

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.

Take a deep breath and digest the material: organoids and biomaterials of the respiratory and digestive systems

“…Liver organoids derived from primary ductal tissue were able to maintain and propagate hepatocytes in vitro, [76,79,80] which is in contrast to two-dimensional primary liver cultures where hepatocytes cannot be propagated. [91] Using donor tissue, isolated bile ducts were cultured as a bi-potent progenitor that can become bile duct cells called cholangiocytes or hepatocytes.…”

“…[76,[79][80][81][82][83][84][85] Hepatocyte cultures from primary tissue or from hPSCs provide models to study hepatocyte function, including production of serum proteins, detoxification of metabolites, and metabolism. [76,[79][80][81][82][83][84][85][86][87] Specifically hepatocytes derived from hPSCs have been used for drug testing and discovery. [82][83][84]88] When hepatocytes fail to function, the onset of liver disease occurs and in extreme cases requires organ transplantation.…”

“…[80,92] Similar approaches were applied to the adult liver cell line, UPcyte. [79] Overall, transplanted liver organoids derived from patient hepatocytes do not need a biomaterial in order to engraft. However, in vitro grown organoids are expanded in Matrigel.…”

International regulatory needs for development ofan IATA for non-genotoxic carcinogenic chemical substances

“…This level includes in vitro tests such as the CTAs, which may also be at Level 2: The SHE assay can be utilized for the first steps of the multistep carcinogenic process, whilst the Balb 3T3 and C3H10T1/2 CTA assays are designed to address the later steps of a carcinogenic process. At this level, also 3D liver cell models (Hengstler et al, 2015;Prestigiacomo and Suter-Dick, 2015;Ramachandran et al, 2015;van Grunsven, 2015) and isolated organ studies, such as tissue perfusion and histopathology from in vivo repeated dose studies would be relevant.…”

New Platforms For Drug Screening And Toxicology: Necessity Or Need?

“…Nowadays, using 3D cultures including organoids and spheroids can be cited due to similarity with in vivo structure and polarity preservation. Therefore, 3D culture systems have been introduced in order to long-term maintenance of liver-specific functions, increases in sensitivity for drug metabolism and prolonged expression of phase I and II enzymes (10,11).…”