Lectins as Tools for the Purification of Liver Endothelial Cells

  • Daniel E. Gomez
  • Unnur P. Thorgeirsson
Part of the Methods in Molecular Medicine™ book series (MIMM, volume 9)

Abstract

Successful procedures for the isolation and culture of large-vessel endothelial cells (EC), were first reported in the early seventies (1,2). Since then, microvascular EC have been isolated from various organs, such as adrenal gland (3), brain (4), skin (5), retina (6), and myocardium (7). The initial steps of the conventional methods for EC isolation involve mechanical and/or enzymatic dissociation of the tissues, followed by filtration and pelleting of cells. A number of special techniques have been developed to eliminate contaminating cell types and enrich endothelial cells in mixed cell populations. These include: manual removal of nonendothelial cell types; use of selective media; plating cells on gelatin or fibronectin-coated dishes, and Percoll gradient centrifugation (8,9). The main problem with the conventional methods is that they are labor intensive and often do not produce pure EC populations. A more advanced approach is to use fluorescent-activated cell sorting (FACS) which allows sorting based on specific surface antigens or metabolic differences. Auerbach et al. used FACS for EC isolation, using an antibody against angiotensin converting enzyme (10). Later, Voyta et al. sorted EC, based on their uptake of acetylated low-density lipoprotein (11). Cell separation techniques using magnetic affinity are based on similar principles as the FACS, but do not involve expensive equipment. In this chapter, we describe liver endothelial cell isolation, using lectin-coated magnetic beads (12).

Keywords

Vortex Filtration Carbohydrate Hydrocortisone Sedimentation 

References

  1. 1.
    Jaffe, E. A., Nachman, R L, Becker, C G, and Minick, C R (1973) Culture of human endothelial cells derived from umbilical veins Identification by morphologic and immunologic criteria. J Clin Invest 52, 2745–2756.PubMedCrossRefGoogle Scholar
  2. 2.
    Gimbrone, M. A., Jr. (1975) Culture of vascular endothelium, in Progress in Hemostasis and Thrombosis, vol 3 (Spaet, T., ed) Grune Stration, Orlando, FL, pp 1–28.Google Scholar
  3. 3.
    Del Vecchio P. J., Ryan, U. S, and Ryan, J. W. (1977) Isolation of capillary segments from rat adrenal gland J Cell Biol 75, 73a, abstract no CU041.Google Scholar
  4. 4.
    Brendel, K, Meezan, E, and Carlson, E. C (1974) Isolated brain microvessels a purified, metabolically active preparation from bovine cerebral cortex. Science 185, 953–955.PubMedCrossRefGoogle Scholar
  5. 5.
    Folkman, J, Haudenschild, C C., and Zetter, B R (1979) Long-term culture of capillary endothelial cells Proc Natl Acad Sci USA 76, 5217–5221.PubMedCrossRefGoogle Scholar
  6. 6.
    Buzney, S. M. and Massicotte, S J. (1979) Retinal vessels proliferation of endothelium in vitro Invest Opthalmol Vis Sci 18, 1191–1195.Google Scholar
  7. 7.
    Gerritsen, M. E. and Cheli, C. D. (1983) Arachidonic acid and prostaglandin endoperoxide metabolism in isolated rabbit and coronary microvessels and isolated and cultivated coronary microvessel endothelial cells J Clin Invest 72, 1658–1671.PubMedCrossRefGoogle Scholar
  8. 8.
    Balconi, G and Dejana, E (1986) Cultivation of endothelial cells: limitations and perspectives (review) Medical Biol 64, 231–245.Google Scholar
  9. 9.
    Gerritsen, M. E (I988) Microvascular endothelial cells isolation, identification, and cultivation Adv Cell Cult 6, 35–67.Google Scholar
  10. 10.
    Auerbach, R L, Alby, J, Grieves, J, Joseph, C., Lindgren, L W., Morrissey, Y. A, Sidky, M, Tu. Z., and Watt, S L. (1982) Monoclonal antibody against angiotensin-converting enzyme its use as a marker for murine, bovine and human endothelial cells. Proc Natl Acad Sci USA 79, 7891–7895.PubMedCrossRefGoogle Scholar
  11. 11.
    Voyta, J. C., Via, D P., Butterfield, C E.. and Zetter, B R (1984) Identification and isolation of endothelial cells based on their increased uptake of acetylated low-density lipoprotein J Cell Biol 99, 2034–2040.PubMedCrossRefGoogle Scholar
  12. 12.
    Gomez, D E, Hartzler, J L, Corbitt, R. H, Nason, A. M, and Thorgeirsson, U P (1993) Immunomagnetic separation as a final purification step of liver endothelial cells In Vitro Cell Dev Biol 29A, 451–455.CrossRefGoogle Scholar
  13. 13.
    Holthofer, H, Virtanen. I, Kariniemi, A., Hormia, M. Linder, E, and Miettinen, A (1982) Ulex Europaeus I lectin as a marker for vascular endothelium in human tissues. Lab Invest 47, 60–66.PubMedGoogle Scholar
  14. 14.
    Roussel, F and Dalion, J (1988) Lectins as markers of endothelial cells, comparative study between human and animal cells Lab Anim 22, 135–140.PubMedCrossRefGoogle Scholar
  15. 15.
    Jackson, C J, Garbett, P K, Nissen, B, and Schrieber, L (1990) Binding of human endothelium to Ulex europaeus I coated Dynabeads: application to the isolation of microvascular endothelium. J Cell Sci 96, 257–262.PubMedGoogle Scholar
  16. 16.
    Laitinen, L (1987) Griffonia simplicifolia lectins bind specifically to endothelial cells and some epithelial cells in mouse tissues Histochem J 19, 225–234.PubMedCrossRefGoogle Scholar
  17. 17.
    Porter, G A, Palade, G E, and Milici, A J (1990) Differential binding of the lectins Griffonia simplicifolia I and Lycopersicon esculentum to microvascular endothelium organ-specific localization and partial glycoprotein characterization. Eur J Cell Biol 51, 85–95.PubMedGoogle Scholar
  18. 18.
    Kataoka, M. and Tavassoli, M (1985) Identification of lectin-like substances recognizing galactosyl residues of glycoconjugates on plasma membrane of marrow sinus endothelium Blood 65, 1163–1171.PubMedGoogle Scholar
  19. 19.
    Bevilacqua, M P, Stengelin, S, Gimbrone, M Y, and Seed, B (1989) Endothelial leukocyte adhesion molecule 1 an inducible receptor for neutrophils related to complement regulatory proteins and lectins Science 243, 1160–1164.PubMedCrossRefGoogle Scholar
  20. 20.
    Johnston, G, Cook, R, and MacEver, R P. (1989) Cloning of GMP-140, a granule membrane protein of platelets and endothelium sequence similarity to proteins involved in cell adhesion and inflammation Cell 56, 1033–1044.PubMedCrossRefGoogle Scholar
  21. 21.
    Padmanabhan, R, Corsico, C, Holter, W, Howard, T, and Howard, B (1989) Purification of transiently transfected cells by magnetic-affinity cell sorting J Immunogenet 16, 91–102.PubMedCrossRefGoogle Scholar
  22. 22.
    Kemshead, J T and Ugelstad, J. (1975) Magnetic separation techniques their application to medicine. Mol Cell Biochem 67, 11–16.Google Scholar
  23. 23.
    Kemshead, J T., Heath, L, Gibson, F. M, Katz, F., Richmond, F., Treleaven, J, and Ugelstad, J (1986) Magnetic microspheres and monoclonal antibodies for the depletion of neuroblastoma cells from bone marrow experiences, improvements, and observations Br J Cancer 54, 771–778.PubMedCrossRefGoogle Scholar
  24. 24.
    Knook, D L, Blanjaar, N, and Sleyter, C H (1977) Isolation and characterization of Kupffer and endothelial cells from the rat liver Exp Cell Res 109, 317–329.PubMedCrossRefGoogle Scholar
  25. 25.
    Morin, O., Patry, P. and Lafleur, L (1984) Heterogeneity of endothelial cells of adult rat liver as resolved by sedimentation velocity and flow cytometry J Cell Physiol 119, 327–334.PubMedCrossRefGoogle Scholar
  26. 26.
    Carpena, T J and Garvey, J S (1982) Antigen handling in aging II. The role of Kupffer and endothelial cells in antigen processing in Fischer 344 rats Mech Ageing Dev 20, 205–221.CrossRefGoogle Scholar
  27. 27.
    Blomhoff, R, Smedsrod. B, and Eskild, W (1984) Preparation of isolated liver endothelial cells and Kupffer cells in high yield by means of an enterotoxin Exp Cell Res 150, 194–204.PubMedCrossRefGoogle Scholar
  28. 28.
    Nagelkerke, J F, Barto, K P, and VanBerkel, T J C (1983) In vivo and in vitro uptake and degradation of acetylated low-density lipoprotein by rat liver endothelial, Kupffer, and parenchymal cells J Biol Chem 258, 12,221–12,227.PubMedGoogle Scholar
  29. 29.
    Nnalue, N A., Shnyra, A, Hultenby, K., and Lindberg, A A (1992) Salmonella choleraesuis and salmonella typhimurium associated with liver cells after intravenous inoculation of rats are localized mainly in Kupffer cells and multiply intracellularly Infect immun 60, 2758–2768.PubMedGoogle Scholar
  30. 30.
    Smedsrod, B and Pertoft, H (1985) Preparation of pure hepatocytes and reticuloendothelial cells in high yield from a single rat liver by means of Percoll centrifugation and selective adherence J Leukocyte Biol 38, 213–230.PubMedGoogle Scholar

Copyright information

© Humana Press Inc. 1998

Authors and Affiliations

  • Daniel E. Gomez
    • 1
  • Unnur P. Thorgeirsson
    • 2
  1. 1.Department of Science and TechnologyUniversidad Nacional de QuilmesBuenos AiresArgentina
  2. 2.Division of Cancer EtiologyNational Cancer InstituteBethesda

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