Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1969 Dec 1;43(3):506–520. doi: 10.1083/jcb.43.3.506

HIGH-YIELD PREPARATION OF ISOLATED RAT LIVER PARENCHYMAL CELLS

A Biochemical and Fine Structural Study

M N Berry 1, D S Friend 1
PMCID: PMC2107801  PMID: 4900611

Abstract

A new technique employing continuous recirculating perfusion of the rat liver in situ, shaking of the liver in buffer in vitro, and filtration of the tissue through nylon mesh, results in the conversion of about 50% of the liver into intact, isolated parenchymal cells. The perfusion media consist of: (a) calcium-free Hanks' solution containing 0.05% collagenase and 0.10% hyaluronidase, and (b) magnesium and calcium-free Hanks' solution containing 2 mM ethylenediaminetetraacetate. Biochemical and morphologic studies indicate that the isolated cells are viable. They respire in a medium containing calcium ions, synthesize glucose from lactate, are impermeable to inulin, do not stain with trypan blue, and retain their structural integrity. Electron microscopy of biopsies taken during and after perfusion reveals that desmosomes are quickly cleaved. Hemidesmosome-containing areas of the cell membrane invaginate and appear to pinch off and migrate centrally. Tight and gap junctions, however, persist on the intact, isolated cells, retaining small segments of cytoplasm from formerly apposing parenchymal cells. Cells which do not retain tight and gap junctions display swelling of Golgi vacuoles and vacuoles in the peripheral cytoplasm. Cytoplasmic vacuolization in a small percentage of cells and potassium loss are the only indications of cell injury detected. By other parameters measured, the isolated cells are comparable to normal hepatic parenchymal cells in situ in appearance and function.

Full Text

The Full Text of this article is available as a PDF (2.0 MB).

Selected References

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

  1. ANDERSON N. G. The mass isolation of whole cells from rat liver. Science. 1953 Jun 5;117(3049):627–628. doi: 10.1126/science.117.3049.627. [DOI] [PubMed] [Google Scholar]
  2. BRANSTER M. V., MORTON R. K. Isolation of intact liver cells. Nature. 1957 Dec 7;180(4597):1283–1284. doi: 10.1038/1801283a0. [DOI] [PubMed] [Google Scholar]
  3. CHANEY A. L., MARBACH E. P. Modified reagents for determination of urea and ammonia. Clin Chem. 1962 Apr;8:130–132. [PubMed] [Google Scholar]
  4. CONWAY E. J., DOWNEY M. An outer metabolic region of the yeast cell. Biochem J. 1950 Sep;47(3):347–355. doi: 10.1042/bj0470347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. 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]
  6. FAWCETT D. W. Observations on the cytology and electron microscopy of hepatic cells. J Natl Cancer Inst. 1955 Apr;15(5 Suppl):1475–1503. [PubMed] [Google Scholar]
  7. HANKS J. H., WALLACE R. E. Relation of oxygen and temperature in the preservation of tissues by refrigeration. Proc Soc Exp Biol Med. 1949 Jun;71(2):196–200. doi: 10.3181/00379727-71-17131. [DOI] [PubMed] [Google Scholar]
  8. HENLEY K. S., POLLARD H. M. A new method for the determination of glutamic oxalacetic and glutamic pyruvic transaminase in plasma. J Lab Clin Med. 1955 Nov;46(5):785–789. [PubMed] [Google Scholar]
  9. HENLEY K. S., WIGGINS H. S., POLLARD H. M., DULLAERT E. The transaminase content of parenchymatous liver cells. Gastroenterology. 1959 Jan;36(1):1–6. [PubMed] [Google Scholar]
  10. 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]
  11. Heath T., Wissig S. L. Fine structure of the surface of mouse hepatic cells. Am J Anat. 1966 Jul;119(1):97–127. doi: 10.1002/aja.1001190107. [DOI] [PubMed] [Google Scholar]
  12. Hems R., Ross B. D., Berry M. N., Krebs H. A. Gluconeogenesis in the perfused rat liver. Biochem J. 1966 Nov;101(2):284–292. doi: 10.1042/bj1010284. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Howard R. B., Christensen A. K., Gibbs F. A., Pesch L. A. The enzymatic preparation of isolated intact parenchymal cells from rat liver. J Cell Biol. 1967 Dec;35(3):675–684. doi: 10.1083/jcb.35.3.675. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Howard R. B., Pesch L. A. Respiratory activity of intact, isolated parenchymal cells from rat liver. J Biol Chem. 1968 Jun 10;243(11):3105–3109. [PubMed] [Google Scholar]
  15. JACOB S. T., BHARGAVA P. M. A new method for the preparation of liver cell suspensions. Exp Cell Res. 1962 Sep;27:453–467. doi: 10.1016/0014-4827(62)90011-3. [DOI] [PubMed] [Google Scholar]
  16. Jacob S. T., Bhargava P. M. Effect of chloramphenicol on ribonucleic acid synthesis in liver cells in suspension. Biochem J. 1965 Oct;97(1):67–73. doi: 10.1042/bj0970067. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Jones A. L., Ruderman N. B., Herrera M. G. An electron microscopic study of lipoprotein production and release by the isolated perfused rat liver. Proc Soc Exp Biol Med. 1966 Oct;123(1):4–9. doi: 10.3181/00379727-123-31388. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. KALTENBACH J. P. The preparation and utilization of whole cell suspensions obtained from solid mammalian tissues. Exp Cell Res. 1954 Nov;7(2):568–571. doi: 10.1016/s0014-4827(54)80101-7. [DOI] [PubMed] [Google Scholar]
  20. KULKA R. G. Colorimetric estimation of ketopentoses and ketohexoses. Biochem J. 1956 Aug;63(4):542–548. doi: 10.1042/bj0630542. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. LAWS J. O., STRICKLAND L. H. Metabolism of isolated liver cells. Nature. 1956 Aug 11;178(4528):309–310. doi: 10.1038/178309a0. [DOI] [PubMed] [Google Scholar]
  22. LONGMUIR I. S., REES W. A. P. Preparation of cell suspensions from rat livers. Nature. 1956 May 26;177(4517):997–997. doi: 10.1038/177997a0. [DOI] [PubMed] [Google Scholar]
  23. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  24. LUFT J. H. Improvements in epoxy resin embedding methods. J Biophys Biochem Cytol. 1961 Feb;9:409–414. doi: 10.1083/jcb.9.2.409. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. MILLER L. L., BLY C. G., WATSON M. L., BALE W. F. The dominant role of the liver in plasma protein synthesis; a direct study of the isolated perfused rat liver with the aid of lysine-epsilon-C14. J Exp Med. 1951 Nov;94(5):431–453. doi: 10.1084/jem.94.5.431. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. MORTIMORE G. E. Effect of insulin on potassium transfer in isolated rat liver. Am J Physiol. 1961 Jun;200:1315–1319. doi: 10.1152/ajplegacy.1961.200.6.1315. [DOI] [PubMed] [Google Scholar]
  27. NOVIKOFF A. B., GOLDFISCHER S. Nucleosidediphosphatase activity in the Golgi apparatus and its usefulness for cytological studies. Proc Natl Acad Sci U S A. 1961 Jun 15;47:802–810. doi: 10.1073/pnas.47.6.802. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Ontko J. A. Chylomicron, free fatty acid and ketone body metabolism of isolated liver cells and liver homogenates. Biochim Biophys Acta. 1967 Feb 14;137(1):13–22. doi: 10.1016/0005-2760(67)90003-3. [DOI] [PubMed] [Google Scholar]
  29. Overton J. The fat of desmosomes in trypsinized tissue. J Exp Zool. 1968 Jun;168(2):203–214. doi: 10.1002/jez.1401680208. [DOI] [PubMed] [Google Scholar]
  30. Revel J. P., Karnovsky M. J. Hexagonal array of subunits in intercellular junctions of the mouse heart and liver. J Cell Biol. 1967 Jun;33(3):C7–C12. doi: 10.1083/jcb.33.3.c7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. SCHMIDT F. H. [Enzymatic determination of glucose and fructose simultaneously]. Klin Wochenschr. 1961 Dec 1;39:1244–1247. doi: 10.1007/BF01506150. [DOI] [PubMed] [Google Scholar]
  32. TAKEDA Y., ICHIHARA A., TANIOKA H., INOUE H. THE BIOCHEMISTRY OF ANIMAL CELLS. I. THE EFFECT OF CORTICOTEROIDS ON LEAKAGE OF ENZYMES FROM DISPERSED RAT LIVER CELLS. J Biol Chem. 1964 Oct;239:3590–3596. [PubMed] [Google Scholar]
  33. WERKHEISER W. C., BARTLEY W. The study of steady-state concentrations of internal solutes of mitochondria by rapid centrifugal transfer to a fixation medium. Biochem J. 1957 May;66(1):79–91. doi: 10.1042/bj0660079. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. WROBLEWSKI F., LADUE J. S. Lactic dehydrogenase activity in blood. Proc Soc Exp Biol Med. 1955 Oct;90(1):210–213. doi: 10.3181/00379727-90-21985. [DOI] [PubMed] [Google Scholar]
  35. ZIMMERMAN M., DEVLIN T. M., PRUSS M. P. Anaerobic glycolysis of dispersed cell suspensions from normal and malignant tissues. Nature. 1960 Jan 30;185:315–316. doi: 10.1038/185315a0. [DOI] [PubMed] [Google Scholar]

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

RESOURCES