Abstract
Hepatic stellate cells (HSCs) are routinely prepared by collagenase/pronase digestion of liver using a perfusion system and subsequent fractionation of the heterogenous cell suspension on continuous density gradients made out of Nycodenz, metrizamide, stractan, or percoll. Because of their lipid content, stellate cells are the least dense fraction of the nonparenchymal cells, and during centrifugation they float effectively away from other hepatic cells resulting in preparations containing almost 80% stellate cells. The degree of purity can be increased by further enrichment of cells by methods like centrifugal elutriation or Scatter-activated cell sorting. We present a detailed protocol from our laboratory to obtain a high number of pure, viable, freshly isolated hepatic stellate cells from rat liver. This two-step protocol (collagenase/pronase digestion and Nycodenz gradient) yields a preparation of approx 4–5 ×107 cells enriched in 74% HSC having a viability of at least 76% as estimated by Trypan blue exclusion test. Further purification by centrifugal elutriation results in virtually pure HSC preparations (>98%).
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References
Hendriks, H. F., Verhoofstad, W. A., Brouwer, A., de Leeuw, A. M., and Knook, D. L. (1985) Perisinusoidal fat-storing cells are the main vitamin A storage sites in rat liver. Exp. Cell Res. 160, 138–149.
Friedman, S. L. (2000) Molecular regulation of hepatic fibrosis, an integrated cellular response to tissue injury. J. Biol. Chem. 275, 2247–2250.
Geerts, A. (2001) History, heterogeneity, developmental biology, and functions of quiescent hepatic stellate cells. Sem. Liv. Dis. 21, 311–335.
Hepatic stellate cell nomenclature (1996) [Letter] Hepatology 23, 193.
Ballardini, G., Groff, P., Badiali de Giorgi, L., Schuppan, D., and Bianchi, F. B. (1994) Ito cell heterogeneity: desmin-negative Ito cells in normal rat liver. Hepatology 19, 440–446.
Zhou, Z., Ekataksin, W., and Wake, K. (1998) Zonal and regional differences identified from precision mapping of vitamin A-storing lipid droplets of the hepatic stellate cells in pig liver: a novel concept of addressing the intralobular area of heterogeneity. Hepatology 27, 1098–1108.
Giampieri, M. P., Jezequel, A. M., and Orlandi, F. (1981) The lipocytes in normal human liver. A quantitative study. Digestion 22, 165–169.
Schafer, S., Zerbe, O., and Gressner, A. M. (1987) The synthesis of proteoglycans in fat-storing cells of rat liver. Hepatology 7, 680–687.
Grabske, R. J., Lake, S., Gledhill, B.L., and Meistrich, M. L. (1975) Centrifugal elutriation: separation of spermatogenic cells on the basis of sedimentation velocity. J. Cell Physiol. 86, 177–189.
Galambos, J. T., Hollingsworth, M. A. Jr, Falek, A., Warren, W. D., and McCain, J. R. (1977) The rate of synthesis of glycosaminoglycans and collagen by fibroblasts cultured from adult human liver biopsies. J. Clin. Invest. 60, 107–114.
Beckman Instruments: The JE-5.0 elutriation system for use with J6 series centrifuges. Instruction manual JE5-IM-7 (Beckman Coulter, Palo Alto, CA).
Geerts, A., Niki, T., Hellemans, K., et al. (1998) Purification of rat hepatic stellate cells by side scatter-activated cell sorting. Hepatology 27, 590–598.
Wake, K. (1980) Perisinusoidal stellate cells (fat-storing cells, interstitial cells, lipocytes), their related structure in and around the liver sinusoids, and vitamin A-storing cells in extrahepatic organs. Int. Rev. Cytol. 66, 303–353.
de Leeuw, A. M., McCarthy, S. P., Geerts, A., and Knook D. L. (1984) Purified rat liver fat-storing cells in culture divide and contain collagen. Hepatology 4, 392–403.
Borojevic, R., Monteiro, A. N., Vinhas, S. A., et al. (1985) Establishment of a continuous cell line from fibrotic schistosomal granulomas in mice livers. In Vitro Cell. Dev. Biol. 21, 382–390.
Horie, S., Kitamura, Y., Kawasaki, H., and Terada, T. (2000) Inhibitory effects of antisense oligonucleotides on the expression of procollagen type III gene in mouse hepatic stellate cells transformed by simian virus 40. Pathol. Int. 50, 937–944.
Greenwel, P., Rubin, J., Schwartz, M., Hertzberg, E. L., and Rojkind, M. (1993) Liver fat-storing cell clones obtained from a CCl4-cirrhotic rat are heterogeneous with regard to proliferation, expression of extracellular matrix components, interleukin-6, and connexin 43. Lab. Invest. 69, 210–216.
Greenwel, P., Schwartz, M., Rosas, M., Peyrol, S., Grimaud, J. A., and Rojkind, M. (1991) Characterization of fat-storing cell lines derived from normal and CCl4-cirrhotic livers. Differences in the production of interleukin-6. Lab. Invest. 65, 644–653.
Kim, Y., Ratziu, V., Choi, S. G., et al. (1998) Transcriptional activation of transforming growth factor beta1 and its receptors by the Kruppel-like factor Zf9/core promoter-binding protein and Sp1. Potential mechanisms for autocrine fibrogenesis in response to injury. J. Biol. Chem. 273, 33,750–33,758.
Sauvant, P., Sapin, V., Abergel, A., et al. (2002) PAV-1, a new rat hepatic stellate cell line converts retinol into retinoic acid, a process altered by ethanol. Int. J. Biochem. Cell. Biol. 34, 1017–1029.
Murakami, K., Abe, T., Miyazawa, M., et al. (1995) Establishment of a new human cell line, LI90, exhibiting characteristics of hepatic Ito (fat-storing) cells. Lab. Invest. 72, 731–739.
Weill, F. X., Blazejewski, S., Blanc, J. F., et al. (1997) Characterization of a new human liver myofibroblast cell line: transcriptional regulation of plasminogen activator inhibitor type I by transforming growth factor beta 1. Lab. Invest. 77, 63–70.
Xu, L., Hui, A. Y., Albanis, E., et al. (2005) Human hepatic stellate cell lines, LX-1 and LX-2: new tools for analysis of hepatic fibrosis. Gut 54, 142–151.
Friedman, S. L., Rockey, D. C., McGuire, R. F., Maher, J. J., Boyles, J. K., and Yamasaki, G. (1992) Isolated hepatic lipocytes and Kupffer cells from normal human liver: morphological and functional characteristics in primary culture. Hepatology 15, 234–243.
Blazejewski, S., Preaux, A. M., Mallat, A., et al. (1995) Human myofibroblastlike cells obtained by outgrowth are representative of the fibrogenic cells in the liver. Hepatology 22, 788–797.
Friedman, S. L. and Roll, F. J. (1987) Isolation and culture of hepatic lipocytes, Kupffer cells, and sinusoidal endothelial cells by density gradient centrifugation with Stractan. Anal. Biochem. 161, 207–218.
Pinzani, M., Gesualdo, L., Sabbah, G. M., and Abboud, H. E. (1989) Effects of platelet-derived growth factor and other polypeptide mitogens on DNA synthesis and growth of cultured rat liver fat-storing cells. J. Clin. Invest. 84, 1786–1793.
Blomhoff, R. and Berg, T. (1990) Isolation and cultivation of rat liver stellate cells. Meth. Enzymol. 190, 58–71.
Neyzen, S., Van de Leur, E., Hollweg, G., Gressnerm, A. M., and Weiskirchen, R. (2005) Cryopreservation of hepatic stellate cells: methodology to conserve fibrogenic progenitor cells. Submitted.
Acknowledgments
The author’s work is supported by grants from the Deutsche Forschungs-gemeinschaft (DFG) and from the Federal Ministry of Education and Research of Germany (BMBF), e.g., IZKF Biomat and Hep-Net.
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Weiskirchen, R., Gressner, A.M. (2005). Isolation and Culture of Hepatic Stellate Cells. In: Varga, J., Brenner, D.A., Phan, S.H. (eds) Fibrosis Research. Methods in Molecular Medicine, vol 117. Humana Press. https://doi.org/10.1385/1-59259-940-0:099
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DOI: https://doi.org/10.1385/1-59259-940-0:099
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Print ISBN: 978-1-58829-479-1
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