Abstract
Chemically defined serum-free media for rat hepatocytes have been useful in identifying EGFR ligands and HGF/MET signaling as direct mitogenic factors for rat hepatocytes. The absence of such media for mouse hepatocytes has prevented screening for discovery of such mitogens for mouse hepatocytes. We present results obtained by designing such a chemically defined medium for mouse hepatocytes and demonstrate that in addition to EGFR ligands and HGF, the growth factors FGF1 and FGF2 are also important mitogenic factors for mouse hepatocytes. Smaller mitogenic response was also noticed for PDGF AB. Mouse hepatocytes are more likely to enter into spontaneous proliferation in primary culture due to activation of cell cycle pathways resulting from collagenase perfusion. These results demonstrate unanticipated fundamental differences in growth biology of hepatocytes between the two rodent species.
Highlights
Primary cultures of rodent and human hepatocytes have been used as tools for studies of metabolism, drug toxicity, bile acid synthesis, and many other aspects of hepatic physiology
Mouse hepatocytes on the other hand have highly variable rates in that regard, with BRDU labeling indices as high as 30%–40% in the first and second day in primary culture with substantial variation seen from one hepatocyte isolate to another
Our findings demonstrate that there are fundamental differences in response to growth factors between mouse and rat hepatocytes in culture, probably as a result of different responses to degradation of extracellular matrix and release of bound growth factors and/or integrin signaling via collagenase perfusion
Summary
Primary cultures of rodent and human hepatocytes have been used as tools for studies of metabolism, drug toxicity, bile acid synthesis, and many other aspects of hepatic physiology. Growth of rat hepatocytes in primary culture and in chemically defined (serum-free) media has been a very useful tool for determining growth factors (Hepatocyte Growth Factor (HGF), and Epidermal Growth Factor Receptor (EGFR) ligands) that are direct mitogens for rat hepatocytes [1]. These studies paved the progress made for determining the role of EGFR and MET (the HGF receptor) receptors and their ligands in liver regeneration and hepatocyte growth [2,3,4,5]. The work presented in this manuscript describes a chemically defined serum-free medium that allows growth of mouse hepatocytes in primary culture and high response to growth factors comparable to those seen with the rat. Our work describes technical difficulties in applying common methodologies used to assess cell proliferation in culture
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