A Primary Canine Hepatocyte Culture Model for Analysis of Cellular Toxicity and Inflammation
The liver plays a critical role in metabolism and detoxification of noxious agents. Liver tissue is capable of limited regeneration in vivo following injury. Hepatocytes, the major cell type in the liver, are considered an appropriate cellular model as they reflect the metabolic profiles in vivo. However, hepatocytes are difficult to propagate in vitro. They do not readily attach nor proliferate outside the liver and are phenotypically unstable. To be useful for metabolic studies in vitro, there is a need to extend hepatocyte survival and stabilize their phenotype. We have established a culture system that facilitates cell attachment, proliferation, and maintenance of the liver phenotype using type I collagen as substrate. Type I collagen, a major component comprising liver extracellular matrix, has been reported to support hepatocyte survival. Here we evaluated whether primary canine hepatocytes seeded on collagen films will continue to proliferate, maintain their phenotype, and respond to interleukin-1 beta (IL-1β). We also determined whether hepatocyte response to IL-1β can be modulated by the hepatoprotective agent silybin phosphatidylcholine complex (SPC). SPC has been shown to promote liver health and manage liver disorders (Marin®).
Primary canine hepatocytes were seeded onto 0.1% rat-tail type I collagen films and propagated in media fortified with growth factors. Cells were pretreated with (a) 298 ng/ml of SPC, (b) 298 ng/ml of its constituent silybin (SB), or (c) control media alone for 24 hrs. Cultures were next activated with IL-1β (10 ng/ml) for up to 72 hrs. Cell proliferation was measured by BrdU labeling. Phenotype was evaluated by albumin and cytokeratin 8 (CK8) immunostaining. Production of prostaglandin E-2 (PGE-2), as well as the chemokines (i) macrophage chemotactic protein 1 (MCP-1) and (ii) interleukin-8 (IL-8) were measured by ELISA. Statistical significance was set at p<0.05 using one-way ANOVA, followed by Tukey post-hoc analysis.
Hepatocytes aggregated into colonies 4 hrs after plating and formed finger-like extensions that disappeared by 48 hrs. Cells proliferated for up to 21 days and continued synthesis of albumin and CK8. Exposure to IL-1β significantly decreased hepatocyte viability to 40%. IL-1β treatment also induced significant increases in pro-inflammatory PGE-2, MCP-1, and IL-8 production. Pre-treatment with SPC and its SB constituent significantly inhibited the cytotoxic effect of IL-1β and reduced production of PGE-2, MCP-1, and IL-8. The present study demonstrates for the first time that our culture model facilitates hepatocyte proliferation and stable phenotype expression. The cell model helps maintain the ability of the hepatocytes to respond to noxious stimuli. We also show that hepatocyte response to IL-1β can be down regulated by the anti-inflammatory antioxidant compound SPC and its SB constituent. Our hepatocyte culture model serves as a useful tool to identify hepatoprotective agents and their potential mechanism of action.