Skip to main content
SpringerLink
Log in

An improved method for isolation of microvascular endothelial cells from normal and inflamed human lung

  • Cellular Models
  • Published:
In Vitro Cellular & Developmental Biology - Animal Aims and scope Submit manuscript

Summary

Microvascular endothelial cells (MVEC), which differ from large vessel endothelial cells, have been isolated successfully from lungs of various species, including man. However, contamination by nonendothelial cells remains a major problem in spite of several technical improvements. In view of the organ specificity of MVEC, endothelial cells should be derived from the tissue involved in the diseases one wishes to study. Therefore, to investigate some of the immunopathological mechanisms leading to acute respiratory distress syndrome (ARDS), we have attempted to isolate lung MVEC from patients undergoing thoracic surgery for lung carcinoma and patients dying of ARDS. The method described here includes four main steps: (1) full digestion of pulmonary tissue with trypsin and collagenase, (2) aggregation of MVEC induced by human plasma, (3) Percoll density centrifugation, and (4) selection and transfer of MVEC after local digestion with trypsin/EDTA under light microscopy. Normal and ARDS-derived lung MVEC purified by this technique presented contact inhibition (i.e., grew in monolayer), and expressed classical endothelial markers, including von Willebrand factor (vWF), platelet endothelial cell adhesion molecule 1(PECAM-1, CD31), and transcripts for the angiogensin converting enzyme (ACE). The cells also formed capillarylike structures, took up high levels of acetylated low-density lipoprotein (Ac-LDL), and exhibited ELAM-1 inducibility in response to TNF. Contaminant cells, such as fibroblasts, smooth muscle cells, or pericytes, were easily recognized on the basis of morphology and were eliminated by selection of plasma-aggregated cells under light microscopy. The technique presented here allows one to study the specific involvement and contribution of pulmonary endothelium in various lung diseases.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Beekhuizen, H.; Vanfurth, R. Growth characteristics of cultured human microvascular venous and arterial and microvascular endothelial cells. J. Vasc. Res. 31:230–239; 1994.

    Article  PubMed  CAS  Google Scholar 

  2. Belloni, P. N.; Carney, D. H.; Nicolson, G. L. Organ-derived microvessel endothelial cells exhibit differential responsiveness to thrombin and other growth factors. Microvas. Res. 43:20–45; 1992.

    Article  CAS  Google Scholar 

  3. Carlay, W. C. M.; Niedbala, J.; Gerritsen, M. E. Isolation, cultivation and partial characterization of microvascular endothelium derived from human lung. Am. J. Respir. Cell Mol. Biol. 7:620–630; 1992.

    Google Scholar 

  4. Charo, I.; Karasek, S.; Davision, M. A., et al. Prostaglandins I2 is not a major metabolite of arachidonic acid in cultured endothelial cells from human foreskin microvessels. J. Clin. Invest. 74:914–919; 1984.

    PubMed  CAS  Google Scholar 

  5. Chomczynski, P.; Sacchi, N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162:156–159; 1987.

    Article  PubMed  CAS  Google Scholar 

  6. Chung-Welch, N.; Patton, W. F.; Ameia Yen-Patton, G. P., et al. Phenotypic comparison between mesothelial and microvascular endothelial cell lineage using conventional endothelial cell markers, cytoskeletal protein markers, and in vitro assays of angiogenic potential. Differentiation 42:44–53; 1989.

    Article  PubMed  CAS  Google Scholar 

  7. Chung-Welch, N.; Shepro, D.; Dunham, B., et al. Prostacyclin and prostaglandin E2 secretion by bovine pulmonary microvascular endotheliial cells are altered by changes in culture conditions. J. Cell. Physiol. 135:224–234; 1988.

    Article  PubMed  CAS  Google Scholar 

  8. Danielsen, H.; Funderud, S.; Nustad, K., et al. The interaction between cell-surface antigens and antibodies bound to monodisperse polymer particles in normal and malignant cells. Scand. J. Immunol. 24:179–187; 1986.

    Article  PubMed  CAS  Google Scholar 

  9. Davison, P. M.; Bensch, K.; Karasek, M. A. Isolation and growth of endothelial cells from the microvessels of newborn human foreskin in cell culture. J. Invest. Dermatol. 75:316–321; 1980.

    Article  PubMed  CAS  Google Scholar 

  10. de Kossodo, S.; Grau, G. E. Profiles of cytokine production in relation with susceptibility to experimental cerebral malaria. J. Immunol. 151:4811–4820; 1993.

    PubMed  Google Scholar 

  11. Delvecchio, P. J.; Siffingerbirnboim, A.; Belloni, P. N., et al. Culture and characterization of pulmonary microvascular endothelial cells. In Vitro Cell. Dev. Biol. 28A:711–715; 1992.

    CAS  Google Scholar 

  12. Gerritsen, M. E.; Printz, M. P. Site of prostaglandin synthesis in bovine heart and isolated bovine coronary microvessels. Circ. Res. 5:1152–1163; 1981.

    Google Scholar 

  13. Gitlin, J. D.; D’Amore, P. A. Culture of retinal capillary cells using selective growth media. Microvas. Res. 26:74–80; 1983.

    Article  CAS  Google Scholar 

  14. Grau, G. E.; Lou, J. TNF in vascular pathology: importance of plateletendothelium interactions. Res. Immunol. 144:355–363; 1993.

    Article  PubMed  CAS  Google Scholar 

  15. Grau, G. E.; Mili, N.; Lou, J. N., et al. Phenotypic and functional analysis of pulmonary microvascular endothelial cells from patients with acute respiratory distress syndrome. Lab. Invest. 74:761–770; 1996.

    PubMed  CAS  Google Scholar 

  16. Hewett, P. W.; Murray, J. C. Human lung microvessel endothelial cells—isolation, culture, and characterization. Microvas. Res. 46:89–102; 1993.

    Article  CAS  Google Scholar 

  17. Hewett, P. W.; Murray, J. C.; Price, E. A., et al. Isolation and characterization of microvessel endothelial cells from human mammary adipose tissue. In Vitro Cell. Dev. Biol. 29A:325–331; 1993.

    Article  CAS  Google Scholar 

  18. Hviid, L.; Elhassan, I. M.; Dodoo, D., et al. Differential t-cell expression of LFA-1 in residents from Africa and Denmark—description of the phenomenon and its possible basis. Immunol. Lett. 39:147–151; 1994.

    Article  PubMed  CAS  Google Scholar 

  19. Jaffe, E. A.; Nachman, R. L.; Becker, C. G. Culture of human endothelial cells derived from umbilical veins: identification by morphologic criteria. J. Clin. Invest. 52:2745–2758; 1973.

    Article  PubMed  CAS  Google Scholar 

  20. Jonjic, N.; Peri, G.; Bernasconi, S., et al. Expression of adhesion molecules and chemotactic cytokines in cultured human mesothelial cells. J. Exp. Med. 176:1165–1174; 1989.

    Article  Google Scholar 

  21. Kern, P. A.; Knedler, A.; Eckel, R. H. Isolation and the culture of the microvascular endothelium from adipose tissue. J. Clin. Invest. 71:1822–1829; 1983.

    PubMed  CAS  Google Scholar 

  22. Latron, Y.; Alessi, M. C.; George, F., et al. Characterization of epithelial cells from human omentum: comparison with endothelial cells from umbilical veins. Thromb. Haemostasis 66:361–367; 1991.

    CAS  Google Scholar 

  23. Ley, K.; Gaehtgens, P.; Spanelborowski, K. Differential adhesion of granulocytes to five distinct phenotypes of cultured microvascular endothelial cells. Microvas. Res. 43:119–133; 1992.

    Article  CAS  Google Scholar 

  24. Marks, R. M.; Czerniecki, M.; Penny, R. Human dermal microvascular endothelial cells: an improved method for tissue culture and a description of some singular properties in culture. In Vitro Cell. Dev. Biol. 21:627–635; 1985.

    Article  PubMed  CAS  Google Scholar 

  25. Meyrick, B.; Hoover, R.; Jones, M. R., et al. In vitro effects of endotoxin on bovine and sheep lung microvascular and pulmonary artery endothelial cells. J. Cell. Physiol. 138:165–174; 1989.

    Article  PubMed  CAS  Google Scholar 

  26. Newman, P. J.; Kawai, Y.; Montgomery, R. R., et al. Synthesis by cultured human umbilical vein endothelial cells of two proteins structurally and immunologically related to platelet membrane glycoproteins IIb and IIIa. J. Cell Biol. 103:81–86; 1986.

    Article  PubMed  CAS  Google Scholar 

  27. Rupnick, M. A.; Carey, A.; Williams, S. K. Phenotypic diversity in cultured cerebral microvascular endothelial cells. In Vitro Cell. Dev. Biol. 24:435–444; 1988.

    Article  PubMed  CAS  Google Scholar 

  28. Sato, N.; Goto, T.; Haranaka, K., et al. Actions of tumor necrosis factor on cultured vascular endothelial cells: morphologic modulation, growth inhibition, and cytotoxicity. J. Natl. Cancer Inst. 76:1113–1121; 1986.

    PubMed  CAS  Google Scholar 

  29. Scott, P. A. E.; Bicknell, R. The isolation and culture of microvascular endothelium. J. Cell Sci. 105:269–273; 1993.

    PubMed  Google Scholar 

  30. Spanel-Borowski, K.; Van der Bosch, J. Different phenotypes of cultured microvessel endothelial cells obtained from the bovine corpus luteum. Cell Tissue Res. 261:35–47; 1990.

    Article  PubMed  CAS  Google Scholar 

  31. Swerlick, R. A.; Lawley, T. J. Role of microvascular endothelial cells in inflammation. J. Invest. Dermatol. 100:S111-S115; 1993.

    Article  Google Scholar 

  32. Van Hinsbergh, V. W. M.; Kooistra, T.; Scheffer, M. A., et al. Characterization and fibrinolytic properties of human omental tissue mesothelial cells: comparison with endothelial cells. Blood 75:1490–1497; 1990.

    PubMed  Google Scholar 

  33. Visner, G. A.; Chesrown, S. E.; Monnier, J., et al. Regulation of manganese superoxide dismutase—il-1 and tnf induction in pulmonary artery and microvascular endothelial cells. Biochem. Biophys. Res. Commun. 188:453–462; 1992.

    Article  PubMed  CAS  Google Scholar 

  34. Voyta, J. C.; Via, D. P.; Butterfield, C. E., et al. Identification and isolation of endothelial cells based on their increased uptake of acetylated-low density lipoprotein. J. Cell Biol. 99:2034–2040; 1984.

    Article  PubMed  CAS  Google Scholar 

  35. Wautier, J. L.; Setiadi, H.; Vilette, D., et al. Leukocyte adhesion to endothelial cells. Biorheology 27:425–432; 1990.

    PubMed  CAS  Google Scholar 

  36. Woodley, N.; Barclay, J. K. Cultured endothelial cells from distinct vascular areas show differential responses to agonists. Can. J. Physiol. Pharmacol. 72:1007–1012; 1994.

    PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Division of Investigative Surgery, Department of Surgery, University Hospital, University of Geneva, 24, rue Micheli-du-crest 1211, Geneva 14, Switzerland

    Jin Ning Lou, Nabil Mili, Christine Decrind, Yves Donati, Sylvie Kossodo, Bara Ricou, Peter M. Suter & Denis R. Morel

  2. Division of Surgical Intensive Care, Clinics of Cardiovascular and Thoracic Surgery, University Hospital and University Medical Center, CH-1211, Geneva 4, Switzerland

    Anastase Spiliopoulos & Georges E. Grau

  3. Division of Investigative Surgery, Department of Surgery, University Hospital and University Medical Center, CH-1211, Geneva 4, Switzerland

    Jin Ning Lou & Philippe Morel

Authors
  1. Jin Ning Lou
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Nabil Mili
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Christine Decrind
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Yves Donati
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Sylvie Kossodo
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Anastase Spiliopoulos
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Bara Ricou
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Peter M. Suter
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Denis R. Morel
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Philippe Morel
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Georges E. Grau
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Lou, J.N., Mili, N., Decrind, C. et al. An improved method for isolation of microvascular endothelial cells from normal and inflamed human lung. In Vitro Cell.Dev.Biol.-Animal 34, 529–536 (1998). https://doi.org/10.1007/s11626-998-0112-z

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11626-998-0112-z

Key words

Search

Navigation