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. 1988 Aug;82(2):450–459. doi: 10.1172/JCI113618

Collagen measured in primary cultures of normal rat hepatocytes derives from lipocytes within the monolayer.

J J Maher 1, D M Bissell 1, S L Friedman 1, F J Roll 1
PMCID: PMC303534  PMID: 3042806

Abstract

The cellular origin of hepatic collagen is under active investigation. Several recent studies using cells in primary culture suggest that hepatocytes are the source of much of the collagen in normal rat liver. In view of other data indicating that lipocytes produce substantial amounts of this protein, we have reexamined collagen biosynthesis in hepatocyte cultures that have been carefully characterized with respect to the presence of lipocytes. We find that routinely prepared hepatocyte isolates contain, by number, approximately 10% lipocytes. Lipocytes in early culture are difficult to visualize by phase-contrast microscopy but after 4 d proliferate and eventually replace the parenchymal cells. The size of the lipocyte subpopulation in these cultures correlates positively with collagen production. Similarly, removal of lipocytes by further processing of the initial hepatocyte isolate significantly reduces collagen production. Moreover, the only cells within hepatocyte cultures that display type I collagen by immunohistochemistry are lipocytes. We conclude that lipocytes are the principal source of collagen in primary hepatocyte cultures. The findings indicate also that these cells are the previously described "fibroblast" that appear in relatively long-term hepatocyte cultures.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bissell D. M., Arenson D. M., Maher J. J., Roll F. J. Support of cultured hepatocytes by a laminin-rich gel. Evidence for a functionally significant subendothelial matrix in normal rat liver. J Clin Invest. 1987 Mar;79(3):801–812. doi: 10.1172/JCI112887. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bissell D. M., Guzelian P. S. Phenotypic stability of adult rat hepatocytes in primary monolayer culture. Ann N Y Acad Sci. 1980;349:85–98. doi: 10.1111/j.1749-6632.1980.tb29518.x. [DOI] [PubMed] [Google Scholar]
  3. Bissell D. M., Hammaker L. E., Meyer U. A. Parenchymal cells from adult rat liver in nonproliferating monolayer culture. I. Functional studies. J Cell Biol. 1973 Dec;59(3):722–734. doi: 10.1083/jcb.59.3.722. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bissell D. M., Tilles J. G. Morphology and function of cells of human embryonic liver in monolayer culture. J Cell Biol. 1971 Jul;50(1):222–231. doi: 10.1083/jcb.50.1.222. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chojkier M., Bateman J., Phang J. M., Peterkofsky B. Formation of proline metabolites in chick embryo bone: interference with the measurement of free hydroxyproline by ion-exchange chromatography. Anal Biochem. 1982 Mar 1;120(2):330–338. doi: 10.1016/0003-2697(82)90354-2. [DOI] [PubMed] [Google Scholar]
  6. Clement B., Grimaud J. A., Campion J. P., Deugnier Y., Guillouzo A. Cell types involved in collagen and fibronectin production in normal and fibrotic human liver. Hepatology. 1986 Mar-Apr;6(2):225–234. doi: 10.1002/hep.1840060212. [DOI] [PubMed] [Google Scholar]
  7. Clement B., Guguen-Guillouzo C., Campion J. P., Glaise D., Bourel M., Guillouzo A. Long-term co-cultures of adult human hepatocytes with rat liver epithelial cells: modulation of albumin secretion and accumulation of extracellular material. Hepatology. 1984 May-Jun;4(3):373–380. doi: 10.1002/hep.1840040305. [DOI] [PubMed] [Google Scholar]
  8. Clement B., Rissel M., Peyrol S., Mazurier Y., Grimaud J. A., Guillouzo A. A procedure for light and electron microscopic intracellular immunolocalization of collagen and fibronectin in rat liver. J Histochem Cytochem. 1985 May;33(5):407–414. doi: 10.1177/33.5.3886779. [DOI] [PubMed] [Google Scholar]
  9. Clément B., Emonard H., Rissel M., Druguet M., Grimaud J. A., Herbage D., Bourel M., Guillouzo A. Cellular origin of collagen and fibronectin in the liver. Cell Mol Biol. 1984;30(5):489–496. [PubMed] [Google Scholar]
  10. Diegelmann R. F., Guzelian P. S., Gay R., Gay S. Collagen formation by the hepatocyte in primary monolayer culture and in vivo. Science. 1983 Mar 18;219(4590):1343–1345. doi: 10.1126/science.6828863. [DOI] [PubMed] [Google Scholar]
  11. Diegelmann R. F., Peterkofsky B. Inhibition of collagen secretion from bone and cultured fibroblasts by microtubular disruptive drugs. Proc Natl Acad Sci U S A. 1972 Apr;69(4):892–896. doi: 10.1073/pnas.69.4.892. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Friedman S. L., Roll F. J., Boyles J., Bissell D. M. Hepatic lipocytes: the principal collagen-producing cells of normal rat liver. Proc Natl Acad Sci U S A. 1985 Dec;82(24):8681–8685. doi: 10.1073/pnas.82.24.8681. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Friedman S. L., Roll F. J. Isolation and culture of hepatic lipocytes, Kupffer cells, and sinusoidal endothelial cells by density gradient centrifugation with Stractan. Anal Biochem. 1987 Feb 15;161(1):207–218. doi: 10.1016/0003-2697(87)90673-7. [DOI] [PubMed] [Google Scholar]
  14. Green H., Todaro G. J., Goldberg B. Collagen synthesis in fibroblasts transformed by oncogenic viruses. Nature. 1966 Feb 26;209(5026):916–917. doi: 10.1038/209916a0. [DOI] [PubMed] [Google Scholar]
  15. Guguen-Guillouzo C., Guillouzo A. Modulation of functional activities in cultured rat hepatocytes. Mol Cell Biochem. 1983;53-54(1-2):35–56. doi: 10.1007/BF00225245. [DOI] [PubMed] [Google Scholar]
  16. Guzelian P. S., Lindblad W. J., Diegelmann R. F. Glucocorticoids suppress formation of collagen by the hepatocyte. Studies in primary monolayer cultures of parenchymal cells prepared from adult rat liver. Gastroenterology. 1984 May;86(5 Pt 1):897–904. [PubMed] [Google Scholar]
  17. Guzelian P. S., Qureshi G. D., Diegelmann R. F. Collagen synthesis by the hepatocyte: studies in primary cultures of parenchymal cells from adult rat liver. Coll Relat Res. 1981;1(1):83–93. doi: 10.1016/s0174-173x(80)80010-0. [DOI] [PubMed] [Google Scholar]
  18. Hata R., Ninomiya Y., Sano J., Konomi H., Hori H., Sunada H., Tanaka S., Kabuki K., Nagai Y., Tsukada Y. Activation of collagen synthesis in primary culture of rat liver parenchymal cells (hepatocytes). J Cell Physiol. 1985 Mar;122(3):333–342. doi: 10.1002/jcp.1041220302. [DOI] [PubMed] [Google Scholar]
  19. Hensgens H. E., Meijer A. J., Williamson J. R., Gimpel J. A., Tager J. M. Prolone metabolism in isolated rat liver cells. Biochem J. 1978 Mar 15;170(3):699–707. doi: 10.1042/bj1700699. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Irving M. G., Roll F. J., Huang S., Bissell D. M. Characterization and culture of sinusoidal endothelium from normal rat liver: lipoprotein uptake and collagen phenotype. Gastroenterology. 1984 Dec;87(6):1233–1247. [PubMed] [Google Scholar]
  21. Jefferson D. M., Reid L. M., Giambrone M. A., Shafritz D. A., Zern M. A. Effects of dexamethasone on albumin and collagen gene expression in primary cultures of adult rat hepatocytes. Hepatology. 1985 Jan-Feb;5(1):14–20. doi: 10.1002/hep.1840050105. [DOI] [PubMed] [Google Scholar]
  22. Kawase T., Shiratori Y., Sugimoto T. Collagen production by rat liver fat-storing cells in primary culture. Exp Cell Biol. 1986;54(4):183–192. doi: 10.1159/000163355. [DOI] [PubMed] [Google Scholar]
  23. Kent G., Gay S., Inouye T., Bahu R., Minick O. T., Popper H. Vitamin A-containing lipocytes and formation of type III collagen in liver injury. Proc Natl Acad Sci U S A. 1976 Oct;73(10):3719–3722. doi: 10.1073/pnas.73.10.3719. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Labarca C., Paigen K. A simple, rapid, and sensitive DNA assay procedure. Anal Biochem. 1980 Mar 1;102(2):344–352. doi: 10.1016/0003-2697(80)90165-7. [DOI] [PubMed] [Google Scholar]
  25. Leffert H. L., Paul D. Studies on primary cultures of differentiated fetal liver cells. J Cell Biol. 1972 Mar;52(3):559–568. doi: 10.1083/jcb.52.3.559. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Madri J. A., Williams S. K. Capillary endothelial cell cultures: phenotypic modulation by matrix components. J Cell Biol. 1983 Jul;97(1):153–165. doi: 10.1083/jcb.97.1.153. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Maher J. J., Friedman S. L., Roll F. J., Bissell D. M. Immunolocalization of laminin in normal rat liver and biosynthesis of laminin by hepatic lipocytes in primary culture. Gastroenterology. 1988 Apr;94(4):1053–1062. doi: 10.1016/0016-5085(88)90566-5. [DOI] [PubMed] [Google Scholar]
  28. Marinkovich M. P., Rocha V. Collagen synthesis and deposition during mammary epithelial cell spreading on collagen gels. J Cell Physiol. 1986 Jul;128(1):61–70. doi: 10.1002/jcp.1041280111. [DOI] [PubMed] [Google Scholar]
  29. Martinez-Hernandez A. The hepatic extracellular matrix. II. Electron immunohistochemical studies in rats with CCl4-induced cirrhosis. Lab Invest. 1985 Aug;53(2):166–186. [PubMed] [Google Scholar]
  30. McGowan J. A., Strain A. J., Bucher N. L. DNA synthesis in primary cultures of adult rat hepatocytes in a defined medium: effects of epidermal growth factor, insulin, glucagon, and cyclic-AMP. J Cell Physiol. 1981 Sep;108(3):353–363. doi: 10.1002/jcp.1041080309. [DOI] [PubMed] [Google Scholar]
  31. Rojkind M., Giambrone M. A., Biempica L. Collagen types in normal and cirrhotic liver. Gastroenterology. 1979 Apr;76(4):710–719. [PubMed] [Google Scholar]
  32. Roll F. J., Madri J. A., Furthmayr H. A new method of iodinating collagens for use in radioimmunoassay. Anal Biochem. 1979 Jul 15;96(2):489–499. doi: 10.1016/0003-2697(79)90611-0. [DOI] [PubMed] [Google Scholar]
  33. Saber M. A., Shafritz D. A., Zern M. A. Changes in collagen and albumin mRNA in liver tissue of mice infected with Schistosoma mansoni as determined by in situ hybridization. J Cell Biol. 1983 Oct;97(4):986–992. doi: 10.1083/jcb.97.4.986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Saber M. A., Zern M. A., Shafritz D. A. Use of in situ hybridization to identify collagen and albumin mRNAs in isolated mouse hepatocytes. Proc Natl Acad Sci U S A. 1983 Jul;80(13):4017–4020. doi: 10.1073/pnas.80.13.4017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Sanderson R. D., Fitch J. M., Linsenmayer T. R., Mayne R. Fibroblasts promote the formation of a continuous basal lamina during myogenesis in vitro. J Cell Biol. 1986 Mar;102(3):740–747. doi: 10.1083/jcb.102.3.740. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Schäfer S., Zerbe O., Gressner A. M. The synthesis of proteoglycans in fat-storing cells of rat liver. Hepatology. 1987 Jul-Aug;7(4):680–687. doi: 10.1002/hep.1840070411. [DOI] [PubMed] [Google Scholar]
  37. Seyer J. M., Hutcheson E. T., Kang A. H. Collagen polymorphism in normal and cirrhotic human liver. J Clin Invest. 1977 Feb;59(2):241–248. doi: 10.1172/JCI108634. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Sims D. E. The pericyte--a review. Tissue Cell. 1986;18(2):153–174. doi: 10.1016/0040-8166(86)90026-1. [DOI] [PubMed] [Google Scholar]
  39. Todaro G., Matsuya Y., Bloom S., Robbins A., Green H. Stimulation of RNA synthesis and cell division in resting cells by a factor present in serum. Wistar Inst Symp Monogr. 1967;7:87–101. [PubMed] [Google Scholar]
  40. Tseng S. C., Lee P. C., Ells P. F., Bissell D. M., Smuckler E. A., Stern R. Collagen production by rat hepatocytes and sinusoidal cells in primary monolayer culture. Hepatology. 1982 Jan-Feb;2(1):13–18. doi: 10.1002/hep.1840020104. [DOI] [PubMed] [Google Scholar]
  41. Tseng S. C., Savion N., Gospodarowicz D., Stern R. Modulation of collagen synthesis by a growth factor and by the extracellular matrix: comparison of cellular response to two different stimuli. J Cell Biol. 1983 Sep;97(3):803–809. doi: 10.1083/jcb.97.3.803. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Tseng S. C., Smuckler E. A., Stern R. Types of collagen synthesized by normal rat liver hepatocytes in primary culture. Hepatology. 1983 Nov-Dec;3(6):955–963. doi: 10.1002/hep.1840030613. [DOI] [PubMed] [Google Scholar]
  43. Wake K. 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. 1980;66:303–353. doi: 10.1016/s0074-7696(08)61977-4. [DOI] [PubMed] [Google Scholar]
  44. Yokoi Y., Namihisa T., Kuroda H., Komatsu I., Miyazaki A., Watanabe S., Usui K. Immunocytochemical detection of desmin in fat-storing cells (Ito cells). Hepatology. 1984 Jul-Aug;4(4):709–714. doi: 10.1002/hep.1840040425. [DOI] [PubMed] [Google Scholar]
  45. de Leeuw A. M., McCarthy S. P., Geerts A., Knook D. L. Purified rat liver fat-storing cells in culture divide and contain collagen. Hepatology. 1984 May-Jun;4(3):392–403. doi: 10.1002/hep.1840040307. [DOI] [PubMed] [Google Scholar]

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