Advertisement

Fibrosis pp 165-191 | Cite as

Isolation and Culture of Primary Murine Hepatic Stellate Cells

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1627)

Abstract

Hepatic stellate cells (HSCs) are found in the perisinusoidal space of the liver (i.e., the space of Dissé). They represent 5–8% of the total number of liver cells. In normal liver, these cells have a quiescent phenotype and are characterized by numerous fat vacuoles that store vitamin A in a form of retinyl ester. In injured liver, these cells transdifferentiate into a myofibroblast phenotype, become highly proliferative and are responsible for excess collagen synthesis and deposition during fibrosis. Due to their exceptional pathophysiological relevance, several isolation and purification protocols of primary HSCs have been established that provide the basis for studying HSC biology in vitro. We here describe a method for high-purity isolation of HSCs from mice. This protocol includes the enzymatic digestion of the liver tissue by pronase and collagenase, cellular enrichment by centrifugation of the crude cell suspension through a Nycodenz density gradient, and a final (optional) flow cytometric enrichment that allows generating ultrapure HSC fractions.

Key words

Hepatic stellate cells Myofibroblasts Nycodenz FACS Density gradient Desmin Phalloidin 

Notes

Acknowledgments

This work was supported by grants from the German Research Foundation (SFB/TRR 57 projects P09, P13, and Q3).

References

  1. 1.
    Tacke F, Weiskirchen R (2012) Update on hepatic stellate cells: pathogenic role in liver fibrosis and novel isolation techniques. Expert Rev Gastroenterol Hepatol 6(1):67–80. doi: 10.1586/egh.11.92 CrossRefPubMedGoogle Scholar
  2. 2.
    Knook DL, Seffelaar AM, de Leeuw AM (1982) Fat-storing cells of the rat liver. Their isolation and purification. Exp Cell Res 139(2):468–471. doi: 10.1016/0014-4827(82)90283-X CrossRefPubMedGoogle Scholar
  3. 3.
    Friedman SL, Roll FJ (1987) Isolation and culture of hepatic lipocytes, Kupffer cells, and sinusoidal endothelial cells by density gradient centrifugation with Stractan. Anal Biochem 161(1):207–218. doi: 10.1016/0003-2697(87)90673-7 CrossRefPubMedGoogle Scholar
  4. 4.
    Pinzani M, Gesualdo L, Sabbah GM et al (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(6):1786–1793. doi: 10.1172/JCI114363 CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    de Leeuw AM, McCarthy SP, Geerts A et al (1984) Purified rat liver fat-storing cells in culture divide and contain collagen. Hepatology 4(3):392–403. doi: 10.1002/hep.1840040307 CrossRefPubMedGoogle Scholar
  6. 6.
    Schafer S, Zerbe O, Gressner AM (1987) The synthesis of proteoglycans in fat-storing cells of rat liver. Hepatology 7(4):680–687. doi: 10.1002/hep.1840070411 CrossRefPubMedGoogle Scholar
  7. 7.
    Weiskirchen R, Gressner AM (2005) Isolation and culture of hepatic stellate cells. Methods Mol Med 117:99–113. doi: 10.1385/1-59259-940-0:099 PubMedGoogle Scholar
  8. 8.
    Maschmeyer P, Flach M, Winau F (2011) Seven steps to stellate cells. J Vis Exp (51). doi:10.3791/2710Google Scholar
  9. 9.
    Elsharkawy AM, Wright MC, Hay RT et al (1999) Persistent activation of nuclear factor-kappaB in cultured rat hepatic stellate cells involves the induction of potentially novel Rel-like factors and prolonged changes in the expression of IkappaB family proteins. Hepatology 30(3):761–769. doi: 10.1002/hep.510300327 CrossRefPubMedGoogle Scholar
  10. 10.
    Graham JM (2002) Fractionation of hepatic nonparenchymal cells. ScientificWorldJournal 2:1347–1350. doi: 10.1100/tsw.2002.283 CrossRefPubMedGoogle Scholar
  11. 11.
    Blomhoff R, Berg T (1990) Isolation and cultivation of rat liver stellate cells. Methods Enzymol 190:58–71. doi: 10.1016/0076-6879(90)90009-P CrossRefPubMedGoogle Scholar
  12. 12.
    D'Ambrosio DN, Walewski JL, Clugston RD et al (2011) Distinct populations of hepatic stellate cells in the mouse liver have different capacities for retinoid and lipid storage. PLoS One 6(9):e24993. doi: 10.1371/journal.pone.0024993 CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Geerts A, Niki T, Hellemans K et al (1998) Purification of rat hepatic stellate cells by side scatter-activated cell sorting. Hepatology 27(2):590–598. doi: 10.1002/hep.510270238 CrossRefPubMedGoogle Scholar
  14. 14.
    Paik YH, Iwaisako K, Seki E et al (2011) The nicotinamide adenine dinucleotide phosphate oxidase (NOX) homologues NOX1 and NOX2/gp91(phox) mediate hepatic fibrosis in mice. Hepatology 53(5):1730–1741. doi: 10.1002/hep.24281 CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Mederacke I, Dapito DH, Affo S et al (2015) High-yield and high-purity isolation of hepatic stellate cells from normal and fibrotic mouse livers. Nat Protoc 10(2):305–315. doi: 10.1038/nprot.2015.017 CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Bartneck M, Warzecha KT, Tag CG et al (2015) Isolation and time lapse microscopy of highly pure hepatic stellate cells. Anal Cell Pathol (Amst) 2015:417023. doi: 10.1155/2015/417023 Google Scholar
  17. 17.
    Wang YQ, Ikeda K, Ikebe T et al (2000) Inhibition of hepatic stellate cell proliferation and activation by the semisynthetic analogue of fumagillin TNP-470 in rats. Hepatology 32(5):980–989. doi: 10.1053/jhep.2000.18658
  18. 18.
    Davies D (2012) Cell separations by flow cytometry. Methods Mol Biol 878:185–199. doi: 10.1007/978-1-61779-854-2_12 CrossRefPubMedGoogle Scholar
  19. 19.
    Donnenberg AD, Donnenberg VS (2007) Rare-event analysis in flow cytometry. Clin Lab Med 27(3):627–652, viii. doi: 10.1016/j.cll.2007.05.013 CrossRefPubMedGoogle Scholar
  20. 20.
    Neyzen S, Van de Leur E, Borkham-Kamphorst E et al (2006) Cryopreservation of hepatic stellate cells. J Hepatol 44(5):910–917. doi: 10.1016/j.jhep.2005.07.008 CrossRefPubMedGoogle Scholar
  21. 21.
    Riccalton-Banks L, Bhandari R, Fry J et al (2003) A simple method for the simultaneous isolation of stellate cells and hepatocytes from rat liver tissue. Mol Cell Biochem 248(1-2):97–102. doi: 10.1023/A:1024184826728 CrossRefPubMedGoogle Scholar
  22. 22.
    Werner M, Driftmann S, Kleinehr K et al (2015) All-in-one: advanced preparation of human parenchymal and non-parenchymal liver cells. PLoS One 10(9):e0138655. doi: 10.1371/journal.pone.0138655 CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Mohar I, Brempelis KJ, Murray SA et al (2015) Isolation of non-parenchymal cells from the mouse liver. Methods Mol Biol 1325:3–17. doi: 10.1007/978-1-4939-2815-6_1 CrossRefPubMedGoogle Scholar
  24. 24.
    Nakamura A, Ueno T, Yagi Y et al (2010) Human primary cultured hepatic stellate cells can be cryopreserved. Med Mol Morphol 43(2):107–115. doi: 10.1007/s00795-009-0484-5 CrossRefPubMedGoogle Scholar
  25. 25.
    Herrmann J, Gressner AM, Weiskirchen R (2007) Immortal hepatic stellate cell lines: useful tools to study hepatic stellate cell biology and function? J Cell Mol Med 11(4):704–722. doi: 10.1111/j.1582-4934.2007.00060.x CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Jmol: an open-source Java viewer for chemical structures in 3D. http://jmol.sourceforge.net/. Accessed 26 Aug 2016
  27. 27.
    Lindgren E (1980) Iohexol, a non-ionic contrast medium: pharmacology and toxicology. Acta Radiol Suppl (Stockholm) 362:1–134Google Scholar
  28. 28.
    Rickwood D, Birnie GD (1975) Metrizamide, a new density-gradient medium. FEBS Lett 50(2):102–110. doi: 10.1016/0014-5793(75)80467-4 CrossRefPubMedGoogle Scholar
  29. 29.
    Corash LM, Piomelli S, Chen HC et al (1974) Separation of erythrocytes according to age on a simplified density gradient. J Lab Clin Med 84 (1):147-151. doi:0022-2143(74)90232-7 [pii]Google Scholar
  30. 30.
    Van Veldhoven PP, Baumgart E, Mannaerts GP (1996) Iodixanol (Optiprep), an improved density gradient medium for the iso-osmotic isolation of rat liver peroxisomes. Anal Biochem 237(1):17–23. doi: 10.1006/abio.1996.0194 CrossRefPubMedGoogle Scholar
  31. 31.
    Graham JM (2002) OptiPrep density gradient solutions for mammalian organelles. ScientificWorldJournal 2:1440–1443. doi: 10.1100/tsw.2002.840 CrossRefPubMedGoogle Scholar
  32. 32.
    Pertoft H, Laurent TC, Laas T et al (1978) Density gradients prepared from colloidal silica particles coated by polyvinylpyrrolidone (Percoll). Anal Biochem 88(1):271–282. doi: 10.1016/0003-2697(78)90419-0 CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  1. 1.Institute of Molecular Pathobiochemistry, Experimental Gene Therapy and Clinical ChemistryRWTH University Hospital AachenAachenGermany
  2. 2.Department of Internal Medicine IIIRWTH University Hospital AachenAachenGermany

Personalised recommendations