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Enhancement of proliferation in a rat hepatocyte co-culture model after mitogenic stimulation.
Citation:
Hester, S., M. A. McVay, A. H. TENNANT, AND S. Khetani. Enhancement of proliferation in a rat hepatocyte co-culture model after mitogenic stimulation. Presented at Society of Toxicology (SOT) Annual Meeting, San Francisco, CA, March 11 - 15, 2012.
Impact/Purpose:
Primary mouse and rat hepatocyte cultures have long been the gold standard for assessment of cellular changes following chemical exposure. While helpful for assessing proliferative and responses in vitro, these cultures are limited to 1 or 2 days of incubation. Our motivation was to test whether extending the culture time beyond 3 days using micropatterned primary rat cultures coupled with quantitative high content imaging (HCI) technology could provide a more accurate assessment ofthe cell proliferative response after mitogenic stimulation
Description:
Primary mouse and rat hepatocyte cultures have long been the gold standard for assessment of cellular changes following chemical exposure. While helpful for assessing proliferative and responses in vitro, these cultures are limited to 1 or 2 days of incubation. Our motivation was to test whether extending the culture time beyond 3 days using micropatterned primary rat cultures coupled with quantitative high content imaging (HCI) technology could provide a more accurate assessment of the cell proliferative response after mitogenic stimulation. The HepatoPac® 96well co-culture system, utilizes primary Sprague-Dawley hepatocytes seeded on micropattemed islands of collagen, surrounded by non-parenchymal murine fibroblasts. In this model, hepatocytes have been shown to maintain extended metabolic activity for up to weeks. Following 8 days of stabilization, HepatoPac® cultures were incubated for 3, 5, and 7 days in serum-free media containing three mitogenic micromolar concentrations of Wyeth 14, 643 (WY) and Phenobarbital (PB) followed by parallel imaging analysis at 2 laboratories for Ki67 signal (a marker of proliferation present in GI, S, G2,and mitosis, but not in Go). PB results showed an enhancement of Ki67 signal reported as % ofcontrol: 153%, 146%, and 83% at 3, 5, and 7 d respectively. WY results showed a similar trend: 140%, 108%, and 111% at 3, 5, and 7 d respectively. These results show an improvement of proliferative response with this hepatocyte model when compared to previous experiments using mono-layered primary rat cultures that produced a 5-10% hepatocyte proliferative response when held for 48hrs. These experiments suggest that the cellular environment & extended incubation capacity provided in the micropatterned cultures is a useful feature when testing compounds for mitogenic activity. (This abstract does not reflect EPA policy and mention of trade names is not an endorsement for any product).