Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1977 Sep 1;74(3):858–877. doi: 10.1083/jcb.74.3.858

Adult rat hepatocytes in primary monolayer culture. Ultrastructural characteristics of intercellular contacts and cell membrane differentiations

PMCID: PMC2110088  PMID: 903373

Abstract

Primary monolayer cultures were obtained in 60-mm petri dishes by incubating 3 X 10(6) isolated hepatocytes at 37 degrees C in Dulbecco's medium supplemented with 17% fetal calf serum. The ultrastructure of monolayer cells was examined after various incubation periods. Within 4 h of plating, the isolated spherical cells adhere to the plastic surface, establish their first contacts by numerous intertwined microvilli, and form new hemidesmosomes. After 12 h of culture, wide branched trabeculae of flattened polyhedral cells extend in all directions. Finally, after 24 h of culture, bile canaliculi are reconstituted, and a biliary polarity is recovered: the Golgi elements, which are scattered throughout the cytoplasm in the isolated cells, are reassembled in front of the newly formed bile canalculi, symmetrically in the adjacent cells; lysosomes are concentrated in that region, and microtubules reappear. Concomitantly, plasma membrane differentiations, namely desmosomes and tight junctions, develop. Tight junctions sealing the bile ducts constitute a barrier to the passage of ruthenium red and horseradish peroxidase. De novo formation of these junctions was studied by the freeze-etching technique: 10-nm particles compose a network of anastomosed linear arrays in the vicinity of the bile canaliculi; in the next step of differentiation, the particles fuse, form short ridge segments and finally continuous branched smooth strands, characteristic of the mature tight junction.

Full Text

The Full Text of this article is available as a PDF (8.4 MB).

Selected References

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

  1. Alwen J., Gallhai-Atchard J. J. A method of maintaining parenchymal cells from adult rat liver in vitro. J Cell Sci. 1972 Jul;11(1):249–260. doi: 10.1242/jcs.11.1.249. [DOI] [PubMed] [Google Scholar]
  2. Bernaert D., Wanson J. C., Drochmans P., Popowski A. Effect of insulin on ultrastructure and glycogenesis in primary cultures of adult rat hepatocytes. J Cell Biol. 1977 Sep;74(3):878–900. doi: 10.1083/jcb.74.3.878. [DOI] [PMC free article] [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. Bruni C., Porter K. R. The Fine Structure of the Parenchymal Cell of the Normal Rat Liver: I. General Observations. Am J Pathol. 1965 May;46(5):691–755. [PMC free article] [PubMed] [Google Scholar]
  5. Chapman G. S., Jones A. L., Meyer U. A., Bissell D. M. Parenchymal cells from adult rat liver in nonproliferating monolayer culture. II. Ultrastructural studies. J Cell Biol. 1973 Dec;59(3):735–747. doi: 10.1083/jcb.59.3.735. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Claude A. Growth and differentiation of cytoplasmic membranes in the course of lipoprotein granule synthesis in the hepatic cell. I. Elaboration of elements of the Golgi complex. J Cell Biol. 1970 Dec;47(3):745–766. doi: 10.1083/jcb.47.3.745. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Drochmans P., Wanson J. C., Mosselmans R. Isolation and subfractionation on ficoll gradients of adult rat hepatocytes. Size, morphology, and biochemical characteristics of cell fractions. J Cell Biol. 1975 Jul;66(1):1–22. doi: 10.1083/jcb.66.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Ehrenreich J. H., Bergeron J. J., Siekevitz P., Palade G. E. Golgi fractions prepared from rat liver homogenates. I. Isolation procedure and morphological characterization. J Cell Biol. 1973 Oct;59(1):45–72. doi: 10.1083/jcb.59.1.45. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Elias P. M., Friend D. S. Vitamin-A-induced mucous metaplasia. An in vitro system for modulating tight and gap junction differentiation. J Cell Biol. 1976 Feb;68(2):173–188. doi: 10.1083/jcb.68.2.173. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. FARQUHAR M. G., PALADE G. E. Junctional complexes in various epithelia. J Cell Biol. 1963 May;17:375–412. doi: 10.1083/jcb.17.2.375. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Geelen M. J., Gibson D. M. Lipogenesis in maintenance cultures of rat hepatocytes. FEBS Lett. 1975 Oct 15;58(1):334–339. doi: 10.1016/0014-5793(75)80292-4. [DOI] [PubMed] [Google Scholar]
  12. Glaumann H., Bergstrand A., Ericsson J. L. Studies on the synthesis and intracellular transport of lipoprotein particles in rat liver. J Cell Biol. 1975 Feb;64(2):356–377. doi: 10.1083/jcb.64.2.356. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Graham R. C., Jr, Karnovsky M. J. The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: ultrastructural cytochemistry by a new technique. J Histochem Cytochem. 1966 Apr;14(4):291–302. doi: 10.1177/14.4.291. [DOI] [PubMed] [Google Scholar]
  14. Guguen C., Guillouzo A., Boisnard M., Le Cam A., Bourel M. Etude ultrastructurale de monocouches d'hépatocytes de rat adulte cultivés en présence d'hémisuccinate d'hydrocortisone. Biol Gastroenterol (Paris) 1975 Jul-Aug;8(3):223–231. [PubMed] [Google Scholar]
  15. Hamilton R. L., Regen D. M., Gray M. E., LeQuire V. S. Lipid transport in liver. I. Electron microscopic identification of very low density lipoproteins in perfused rat liver. Lab Invest. 1967 Feb;16(2):305–319. [PubMed] [Google Scholar]
  16. Howard R. B., Lee J. C., Pesch L. A. The fine structure, potassium content, and respiratory activity of isolated rat liver parenchymal cells prepared by improved enzymatic techniques. J Cell Biol. 1973 Jun;57(3):642–658. doi: 10.1083/jcb.57.3.642. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Iype P. T. Cultures from adult rat liver cells. I. Establishment of monolayer cell-cultures from normal liver. J Cell Physiol. 1971 Oct;78(2):281–288. doi: 10.1002/jcp.1040780217. [DOI] [PubMed] [Google Scholar]
  18. Jeejeebhoy K. N., Ho J., Breckenridge C., Bruce-Robertson A., Steiner G., Jeejeebhoy J. Synthesis of VLDL by isolated rat hepatocytes in suspension. Biochem Biophys Res Commun. 1975 Oct 27;66(4):1147–1153. doi: 10.1016/0006-291x(75)90478-7. [DOI] [PubMed] [Google Scholar]
  19. Jones A. L., Ruderman N. B., Herrera M. G. Electron microscopic and biochemical study of lipoprotein synthesis in the isolated perfused rat liver. J Lipid Res. 1967 Sep;8(5):429–446. [PubMed] [Google Scholar]
  20. Kuroda Y., Nagatani T. Experimentally induced changes in fructose-1,6-diphosphatase activity in embryonic chick liver and heart cells in vitro. Dev Biol. 1965 Jun;11(3):335–351. doi: 10.1016/0012-1606(65)90043-6. [DOI] [PubMed] [Google Scholar]
  21. Luft J. H. Ruthenium red and violet. I. Chemistry, purification, methods of use for electron microscopy and mechanism of action. Anat Rec. 1971 Nov;171(3):347–368. doi: 10.1002/ar.1091710302. [DOI] [PubMed] [Google Scholar]
  22. Mahley R. W., Hamilton R. L., Lequire V. S. Characterization of lipoprotein particles isolated from the Golgi apparatus of rat liver. J Lipid Res. 1969 Jul;10(4):433–439. [PubMed] [Google Scholar]
  23. Montesano R., Friend D. S., Perrelet A., Orci L. In vivo assembly of tight junctions in fetal rat liver. J Cell Biol. 1975 Nov;67(2PT1):310–319. doi: 10.1083/jcb.67.2.310. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Moscona A. A. Cell aggregation: properties of specific cell-ligands and their role in the formation of multicellular systems. Dev Biol. 1968 Sep;18(3):250–277. doi: 10.1016/0012-1606(68)90035-3. [DOI] [PubMed] [Google Scholar]
  25. REYNOLDS E. S. The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J Cell Biol. 1963 Apr;17:208–212. doi: 10.1083/jcb.17.1.208. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Seglen P. O. Preparation of rat liver cells. 3. Enzymatic requirements for tissue dispersion. Exp Cell Res. 1973 Dec;82(2):391–398. doi: 10.1016/0014-4827(73)90357-1. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES