In Vitro

, Volume 17, Issue 4, pp 353–362 | Cite as

Primary culture of capillary endothelium from rat brain

  • Phillip D. Bowman
  • A. Lorris Betz
  • Diane aR
  • Jerry S. Wolinsky
  • Jack B. Penney
  • Richard R. Shivers
  • Gary W. Goldstein


To provide an in vitro system for studies of brain capillary function we developed a method for culture of brain capillary endothelial cells. Capillaries were isolated from rat brain and enzymatically treated to remove the basement membrane and contaminating pericytes. Subsequent Percoll gradient centrifugation resulted in a homogeneous population of capillary endothelial cells that attached to a collagen substrate and incorporated [3H]thymidine. Evidence for the endothelial nature of these cells was provided by the presence of Factor VIII antigen and angiotensin converting enzyme activity and by the failure of platelets to adhere to the cell surface. In addition, the cells were joined together by tight junctions. Thus, primary cultures of these cells retained both endothelial and blood-brain barrier features.

Key words

rat brain endothelial cell culture primary culture Factor VIII angiotensin converting enzyme tight junctions 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Rapoport, S. I. Blood-brain barrier in physiology and medicine. New York: Raven; 1976.Google Scholar
  2. 2.
    Goldstein, G. W.; Wolinsky, J. S.; Csejtey, J.; Diamond, I. Isolation of metabolically active capillaries from rat brain. J. Neurochem. 25: 715–717; 1975.PubMedCrossRefGoogle Scholar
  3. 3.
    Betz, A. L.; Goldstein, G. W. Polarity of the blood-brain barrier: Neutral amino acid transport into isolated brain capillaries. Science 202: 225–227; 1978.PubMedCrossRefGoogle Scholar
  4. 4.
    Betz, A. L.; Csejtey, J.; Goldstein, G. W. Hexose transport and phosphorylation by capillaries isolated from rat brain. Am. J. of Physiol. 236: C96-C102; 1979.Google Scholar
  5. 5.
    Goldstein, G. W. Relation of potassium transport to oxidative metabolism in isolated brain capillaries. J. Physiol. 286: 185–195; 1979.PubMedGoogle Scholar
  6. 6.
    Hjelle, J. T.; Baird-Lambert, J.; Cardinale, G.; Spector, S.; Udenfriend, S. Isolated microvessels: The blood-brain barrier in vitro. Proc. Natl. Acad. Sci. USA 75: 4544–4548; 1978.PubMedCrossRefGoogle Scholar
  7. 7.
    Herbst, T. J.; Raichle, M. E.; Ferrendelli, J. A. Beta-adrenergic regulation of adenosine 3′,5′-monophosphate concentration in brain microvessels. Science 204: 330–332; 1979.PubMedCrossRefGoogle Scholar
  8. 8.
    Brendel, K.; Meezan, E.; Carlson, E. C. Isolated brain microvessels: A purified, metabolically active preparation from bovine cerebral cortex. Science 185: 953–955; 1974.PubMedCrossRefGoogle Scholar
  9. 9.
    Eisenberg, H. M.; Suddith, R. L. Cerebral vessels have the capacity to transport sodium and potassium. Science 206: 1083–1085; 1979.PubMedCrossRefGoogle Scholar
  10. 10.
    Debault, L. E.; Kahn, L. E.; Frommes, S. P.; Cancilla, P. A. Cerebral microvessels and derived cells in tissue culture: Isolation and preliminary characterization. In Vitro 15: 473–487; 1979.PubMedGoogle Scholar
  11. 11.
    Panula, P.; Joo, F.; Rechardt, L. Evidence for the presence of viable endothelial cells in cultures derived from dissociated rat brain. Experimentia 34: 95–97; 1978.CrossRefGoogle Scholar
  12. 12.
    Phillips, P.; Kumar, P.; Kumar, S.; Waghe, M. Isolation and characterization of endothelial cells from rat and cow brain white matter. J. Anat. 129: 261–272; 1979.PubMedGoogle Scholar
  13. 13.
    Bowman, P. D.; Betz, A. L.; Goldstein, G. W. Characteristics of cultured brain capillaries. J. Cell Biol. 83: 95a; 1979.Google Scholar
  14. 14.
    Penney, J. B.; Bowman, P. D.; Betz, A. L.; Wolinsky, J. S.; Goldstein, G. W. Ultrastructural features of endothelial cells cultured from rat brain capillaries. Neurology 30: 407; 1980.Google Scholar
  15. 15.
    Pertoff, H.; Laurent, T. C. Isopynic separation of cells and cell organelles by centrifugation in modified colloidal silca gradients. Catsimpoolas, N. ed. Methods of cell separation. New York: Plenum Press; 1977: 25.Google Scholar
  16. 16.
    Absher, M. Hemocytometer counting. Kruse, P. F., Jr.; Patterson, M. K., Jr. eds. Tissue culture methods and applications. New York: Academic Press; 1973: 395–397.Google Scholar
  17. 17.
    Michelopoulis, G.; Pitot, H. Primary culture of parenchymal liver cells on collagen membranes. Exp. Cell. Res. 94: 70–78; 1975.CrossRefGoogle Scholar
  18. 18.
    Karnovsky, M. J. A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy. J. Cell Biol. 27: 137; 1965.Google Scholar
  19. 19.
    Jaffe, E. A.; Hoyer, L. W.; Nachman, R. L. Synthesis of antihemophilic factor antigen by cultured human endothelial cells. J. Clin. Invest. 52: 2757–2764; 1973.PubMedCrossRefGoogle Scholar
  20. 20.
    Laskey, R. A.; Mills, A. D. Quantitative film detection of3H and14C in polyacrylamide gels by. fluorography. Eur. J. Biochem. 56: 335–341; 1975.PubMedCrossRefGoogle Scholar
  21. 21.
    Wechezak, H. R.; Holbrook, K. A.; Way, S. A.; Mansfield, P. B. Platelet adherence in endothelial cell cultures. Blood Vessels 16: 35–42; 1979.PubMedGoogle Scholar
  22. 22.
    Shipman, C. Evaluation of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) as a tissue culture buffer. Proc. Soc. Exp. Biol. Med. 130: 305–309; 1969.PubMedGoogle Scholar
  23. 23.
    Karnovsky, M. J. Use of ferrocyanide-reduced osmium tetroxide in electron microscopy. J. Cell Biol. 146a; 1971.Google Scholar
  24. 24.
    Boyde, A.; Maconnachie, E. Freon 113 freeze drying for scanning electron microscopy. Scanning 2: 164–168; 1979.Google Scholar
  25. 25.
    Gilden, D. H.; Wroblewska, Z.; Kindt, B.; Warren, K. G.; Wolinsky, J. S. Vericella-zoster virus infection of human brain cells and ganglion cells in tissue culture. Arch. Virol. 56: 105–112; 1978.PubMedCrossRefGoogle Scholar
  26. 26.
    Shivers, R. R.; Brightman, M. W. Trans-glial channels in ventral nerve roots of crayfish. J. Comp. Neurol. 167: 1–26; 1976.PubMedCrossRefGoogle Scholar
  27. 27.
    Friedland, J.; Silverstein, E. A sensitive fluorimetric assay for serum angiotensin-converting enzyme. Am. J. Clin. Pathol. 66: 416–424; 1976.PubMedGoogle Scholar
  28. 28.
    Caldwell, P. R. B.; Segal, B. C.; Hsu, K. C.; Das, M.; Soffer, R. L. Angiotensin-converting enzyme: Vascular endothelial localization. Science 191: 1050–1051; 1976.PubMedCrossRefGoogle Scholar
  29. 29.
    Hayes, L. W.; Goguen, C. A.; Ching, S-F.; Slakey, L. L. Angiotensin-converting enzyme: Accumulation in medium from cultured endothelial cells. Biochem. Biophys. Res. Commun. 82: 1147–1153; 1978.PubMedCrossRefGoogle Scholar
  30. 30.
    Shivers, R. R.; Goldstein, G. W. Isolated brain capillaries: Freeze-fracture confirmation of interendothelial integrity. Trans. Soc. Neurosci. Abs. 5: 433; 1979.Google Scholar
  31. 31.
    Weibel, E. R.; Palade, G. E. New cytoplasmic components in arterial endothelia. J. Cell Biol. 23: 101–112; 1964.PubMedCrossRefGoogle Scholar
  32. 32.
    Wagner, R. C.; Matthews, M. A. The isolation and culture of capillary endothelium from epididymal fat. Microvascular Res. 10: 286–297; 1975.CrossRefGoogle Scholar
  33. 33.
    Folkman, J.; Hauderschild, C. C.; Zetter, B. R. Long-term culture of capillary endothelial cells. Proc. Natl. Acad. Sci. USA 76: 5217–5221; 1979.PubMedCrossRefGoogle Scholar
  34. 34.
    Sherer, G. K.; Fitzharris, T. P.; LeRoy, E. C. Cultivation of microvascular endothelial cells from human preputial skin. In Vitro 15: 158; 1979.Google Scholar
  35. 35.
    Frank, R. N.; Kinsey, V. E.; Frank, K. W.; Mikus, K. P.; Randolph, A. In vitro proliferation of endothelial cells from kitten retinal capilaries. Invest. Ophthalmol. Visual Sci. 18: 1195–1200; 1979.Google Scholar
  36. 36.
    Buzney, S. M.; Massicotte, S. J. Retinal vessels: Proliferation of endothelium in vitro. Invest. Ophthalmol Visual Sci. 18: 1191–1195; 1979.Google Scholar
  37. 37.
    Booyse, F. M.; Sedlak, B. J.; Rafelson, M. E. Culture of arterial endothelial cells: Characterization and growth of bovine aortic cells. Diath. Haemmorrh. 74: 825–839; 1975.Google Scholar
  38. 38.
    Gimbrone, M. A.; Cotran, R. S.; Folkman, J. Human vascular endothelial cells in culture: Growth and DNA synthesis. J. Cell Biol. 60: 673–684; 1974.PubMedCrossRefGoogle Scholar
  39. 39.
    Ryan, U. S.; Clements, E.; Habliston, D.; Ryan, J. W. Isolation and culture of pulmonary artery endothelial cells. Tissue Cell 10: 535–554; 1978.PubMedGoogle Scholar
  40. 40.
    Wolff, J. R. Ultrastructure of the terminal vascular bed as related to function. Kaley, G.; Altura, B. M. eds. Microcirculation. vol. 1. Baltimore: University Park Press; 1977: 95–130.Google Scholar

Copyright information

© Tissue Culture Association, Inc 1981

Authors and Affiliations

  • Phillip D. Bowman
    • 1
  • A. Lorris Betz
    • 1
  • Diane aR
    • 1
  • Jerry S. Wolinsky
    • 2
  • Jack B. Penney
    • 1
  • Richard R. Shivers
    • 3
  • Gary W. Goldstein
    • 1
  1. 1.University of MichiganAnn Arbor
  2. 2.Johns Hopkins UniversityBaltimore
  3. 3.The University of WesternOntarioCanada
  4. 4.Department of PediatricsUniversity HospitalAnn Arbor

Personalised recommendations