Abstract
Hepatocytes are an important research tool used for numerous applications. However, a short life span and a limited capacity to replicate in vitro limit the usefulness of primary hepatocyte cultures. We have hypothesized that in vivo priming of hepatocyte could make them more susceptible to growth factors in the medium for continuous proliferation in vitro. Here, a novel approach used to establish hepatocyte cell lines that included hepatocyte priming in vivo prior to culture with a 3,5-diethoxycarbonyl-1,4-dihydrocollidine diet was attempted. The cell line grew in a monolayer while maintaining a granular cytoplasm and a round nucleus. Electron microscopy displayed hepatocyte-like features including mitochondria, glycogen granules, and the presence of bile canaliculi. This cell line expressed many mature hepatocyte-specific genes including albumin, alpha1-antitrypsin, glucose 6-phosphatase, and tyrosine aminotransferase. Functional characteristic of hepatocytes like the ability to store glycogen, lipid, and synthesis of urea is well demonstrated by this cell line. These cells demonstrated anchorage dependent growth properties in soft agar and did not form tumors after transplantation into nude mice. This cell line can be sustained in culture for more than 100 passages (>1.5 years) without undergoing noticeable morphological changes or transformation. This novel method resulted in the establishment of an immortal, non-transformed hepatocyte cell line with functional characteristics that may aid research of cell metabolism, toxicology, and hepatocyte transplantation.
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Acknowledgements
This work was supported by grants for research from the Research Institute of Clinical Medicine, Chonbuk National University Hospital. The authors are grateful to Professor P.H. Hwang for helpful discussion, Dr. S.Y. Kim, Dr. L.M. Jung, Mrs. J. H. Koo, and Ms. S. J. Jung for the technical assistance.
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Editor: T. Okamoto
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Supplementary Figure 1
Immunofluoresence analysis showed no expression of K-19 and AFP in PMH08 cells. MCT3T-E1 was used as negative control and HepG2 (human liver cancer cell line) was used as positive control. (JPEG 22 kb)
Supplementary Figure 2
A Mice injected with the Hep3B cell line, photograph at 10 wk after cell line injection. B Mice injected with PMH08 cell line, photograph 28 wk after injection. C Hep3B illustrated by the black color colonies, D PMH08 cell line formed no colonies, E control without cells in soft agar culture. (JPEG 31 kb)
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Risal, P., Cho, B.H., Sylvester, K.G. et al. The establishment and characterization of immortal hepatocyte cell lines from a mouse liver injury model. In Vitro Cell.Dev.Biol.-Animal 47, 526–534 (2011). https://doi.org/10.1007/s11626-011-9445-0
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DOI: https://doi.org/10.1007/s11626-011-9445-0