Long-Term Culture of Functional Liver Tissue: Three-Dimensional Coculture of Primary Hepatocytes and Stellate Cells

Abstract

One of the greatest challenges in the attempt to create functional liver tissue in vitro is the maintenance of hepatocyte-specific functions. The pharmaceutical industry has long awaited the development of engineered liver tissue, which could represent a long-term, inducible, high-fidelity model for high-throughput screening of new drug compounds. It is also anticipated that such engineered models could one day be used in liver transplants, where replacement is limited by chronic donor shortages. As isolated hepatocytes dedifferentiate rapidly in culture the use of hepatocytes in long-term studies has proved to be a difficult challenge. Here we report a system of rat hepatocytes cocultured with primary rat hepatic stellate cells on a biodegradable poly(DL-lactic acid) substratum. These coculture conditions were found to encourage the rapid self-organization of three-dimensional spheroids. The spheroids formed exhibit hepatocyte-specific functionality (CYP-450 activity and albumin secretion) after almost 2 months in static culture.

A Mathematical Model of Liver Cell Aggregation In Vitro

“…This is in good qualitative agreement with images of aggregates cultured in vitro (Riccalton-Banks et al, 2003;Thomas et al, 2005).…”

“…Recent experimental research has looked at the effect of co-culturing hepatocytes with other cell types, such as hepatic stellate cells (Riccalton-Banks et al, 2003), fibroblasts (Bhandari et al, 1997) and pancreatic islet cells (Lee et al, 2004). Under such conditions, spheroids appear to form more quickly, and may also be larger than those which arise in hepatocyte-only cultures.…”

“…These epithelial cells perform most of the liver's important functions (Selden et al, 1999), and hence have received the greatest attention from tissue engineers. Hepatocytes cultured in vitro often form a monolayer, have a tendency to dedifferentiate (lose their ability to function normally) within hours, and die after a few days (Riccalton-Banks et al, 2003;Bhandari et al, 1997). Techniques that have been developed to overcome this problem include co-culture with other cell types (such as stellate cells, also found in the liver), culture on polymer scaffolds and the use of growth factors and cytokines (Bhandari et al, 1997).…”

Characterisation of the NES2Y cell line and its use in the production of human glucose-responsive insulin producing (hGRIP) cell lines by cell-cell fusion

“…These morphological characteristics emphasise their capacity to organize into threedimensional structures, which could be important in the development and bio-engineering of NES2Y cells and hybrids derived from them. 33,34 In demonstrating that the NES2Y cells had the important characteristics of human endocrine cells and were able to proliferate in culture, in order to use them as fusion partners to produce insulin-producing hybrids, it was necessary to define their susceptibility to selection in HAT and HA media as shown in Figure 3.…”

Rat Carboxylesterase ES-4 Enzyme Functions as a Major Hepatic Neutral Cholesteryl Ester Hydrolase

“…To isolate rat primary stellate cells, collagenasetreated liver was filtered through a 70-mesh and gently centrifuged (50 ϫ g) to remove hepatocytes. The remaining cells in the supernatant were subject to further centrifugation steps and pelleted at (200 ϫ g) (23). Cells were then plated on collagen-coated plates.…”