Skip to main content
Log in

Isolation and culture of rat microvascular endothelial cells

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

Summary

The purpose of this study is to identify the separation techniques that result in pure cultures of rat microvascular endothelial cells (MECs). A multistep process is used to optimize the separation of the cells from rat epididymal fat pads, obtaining as pure a culture as possible within a relatively short processing time. The process initially employs the digestion, filtration, and density gradient separation steps. We further describe the use of an attachment phase that allows the differential adherence of contaminating cell types. Immunomagnetic purification is the final step in the process and is performed using anti-PECAM-1 (CD31) monoclonal antibody-labeled DynaBeads.

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

Access this article

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

Instant access to the full article PDF.

Similar content being viewed by others

References

  • Arts, C. H. P.; Heijnen-Snyder, G.; Joosten, P. Ph. A. H.; Verhagen, H. J. M.: Eikelboom, B. C.; Sixma, J. J.; de Groot, P. G. A novel method for isolating pure microvascular endothelial cells from subcutaneous fat tissue ideal for direct cell seeding. Lab. Invest. 81(10):1461–1465; 2001.

    PubMed  CAS  Google Scholar 

  • Brey, E. M.; King, T. W.; Johnston, C.; McIntire, L. V.; Reece, G. P.; Patrick, C. W. Jr.. A technique for quantitative 3D analysis of microvascular structure. Microvasc. Res., 63:279–244, 2002.

    Article  PubMed  Google Scholar 

  • Dejana, E.; Corada, M.; Grazia Lampugnani, M. Endothelial cell-to-cell junctions. FASEB J. 9:910–918; 1995.

    PubMed  CAS  Google Scholar 

  • Gargett, C. E.; Bucak, K.; Rogers, P. A. W. Isolation, characterization and long-term culture of human myometrial microvascular endothelial cells. Hum. Reprod. 15(2):293–301; 2000.

    Article  PubMed  CAS  Google Scholar 

  • Gerritsen, M. E.; Shen, C.-P.; Atkinson, W. J.; Padgett, R. C.; Gimbrone, M. A. S., Jr., Microvascular endothelial cells from E-selectin-deficient mice form tubes in vitro. Lab. Invest. 75:175–184; 1996.

    PubMed  CAS  Google Scholar 

  • Hannum, R. S.; Ojeifo, J. O.; Zwiebel, J. A.; McLeskey, S. W. Isolation of tumor-derived endothelial cells. Microvasc. Res. 61:287–290; 2001.

    Article  PubMed  CAS  Google Scholar 

  • Hewett, P. W.; Murray, J. C. Immunomagnetic purification of human microvessel endothelial cells using Dynabeals coated with monoclonal antibodies to PECAM-1. Eur. J. Cell Biol. 62:451–454; 1993.

    PubMed  CAS  Google Scholar 

  • Jones, M. K.; Wang, H.; Tomikawa, M.; Szabo, I. L.; Kawanaka, H.; Sarfeh, I. J.; Tarnawski, A. S. Isolation and characterization of rat gastric microvascular endothelial cells as a model for studying gastric angiogenesis in vitro. J. Physiol. Pharmacol. 51:813–820; 2000

    PubMed  CAS  Google Scholar 

  • Kader, K. N.; Moore, L. R.; Saul, J. M.; Zborowski, M.; Ziats, N. P.; Bellamkonda, R. V. Isolation and purification of canine adipose microvascular endothelial cells. Microvasc. Res. 61:220–226; 2001.

    Article  PubMed  CAS  Google Scholar 

  • Karasek, M. A. Microvascular endothelial cell culture. J. Invest. Dermatol. 93:33S-38S; 1989.

    Article  PubMed  CAS  Google Scholar 

  • King, T. W.; Brey, E. M.; Youssef, A.; Johnston, C.; Patrick, C. W., Jr. Quantification of vascular density using a semiautomated technique for immunostained specimens. Anal. Quant. Cytol Histol., 24:39–48, 2002.

    PubMed  Google Scholar 

  • Kriehuber, E.; Breiteneder-Geleff, S.; Groeger, M.; Soleiman, A.; Schoppmann, S. F.; Stingl, G.; Kerjaschki, D.; Maurer, D. Isolation and characterization of dermal lymphatic and blood endothelial cells reveal stable and functionally specialized cell lineages. J. Exp. Med. 194(6):797–808; 2001.

    Article  PubMed  CAS  Google Scholar 

  • Krump-Konvalinkova, V.; Bittinger, F.; Unger, R. E.; Peters, K.; Lehr, H.-A.; Kirpatrick, C. J. Generation of human pulmonary microvascular endothelial cell lines. Lab. Invest. 81:1717–1727; 2001.

    PubMed  CAS  Google Scholar 

  • Lamszus, K.; Schmidt, N. O.; Ergun, S.; Westphal, M. Isolation and culture of human neuromicrovascular endothelial cells for the study of angiogenesis in vitro. J. Neurosci. Res. 55:370–381; 1999.

    Article  PubMed  CAS  Google Scholar 

  • Lang, I.; Hoffman, C.; Olip, H.; Pabst, M.-A.; Hahn, T.; Dohr, G.; Desoye, G. Differential mitogenic responses of human macrovascular and microvascular endothelial cells to cytokines underline their phenotypic heterogeneity. Cell Prolif. 34:143–155; 2001.

    Article  PubMed  CAS  Google Scholar 

  • Launder, A. M.; Gegen, N. W.; Knedler, A.; Harbeck, R. J. The isolation and characterization of enriched microvascular endothelial cells from mouse adipose tissue. J. Immunol. Methods 102:45–52; 1987.

    Article  PubMed  CAS  Google Scholar 

  • 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. 34:529–536; 1998.

    CAS  Google Scholar 

  • Marelli-Berg, F. M.; Peek, L.; Lidington, E. A.; Stauss, H. J.; Lechler, R. I. Isolation of endothelial cells from murine tissue. J. Immunol. Methods 244:205–215; 2000.

    Article  PubMed  CAS  Google Scholar 

  • Ment, L.; Stewart, W. B.; Scaramuzzino, D.; Madri, J. A. A in vitro three-dimensional coculture model of cerebral microvascular angiogenesis and differentiation. In Vitro Cell. Dev. Biol. 33A:684–691; 1997.

    Google Scholar 

  • Patrick, C. W., Jr.; Chauvin, P. B.; Hobley, J.; Reece, G. P. Preadipocyte seeded PLGA scaffolds for adiposc tissue engineering. Tissue Eng. 5(2):139–151; 1999.

    Article  PubMed  CAS  Google Scholar 

  • Patrick, C. W., Jr.; Chauvin, P. B.; Robb, G. L. Tissue engineered adipose. In: Patrick, C. W., Jr.; Mikos A. G.; McIntire, L. V., ed. Frontiers in tissue engineering. Oxford: Elsevier Science; 1998;369–382.

    Google Scholar 

  • Patrick, C. W., Jr.; Wu, X.; Johnston, C.; Reece, G. P. Epithelial cell culture: breast. In: Atala, A.; Lanza, R., ed. Methods of tissue engineering. New York: Academic Press; 2001:143–154.

    Google Scholar 

  • Polinger, I. S. Separation of cell types in embryonic heart cell cultures. Exp. Cell Res. 63:78–82; 1970.

    Article  PubMed  CAS  Google Scholar 

  • Richard, L.; Velasco, P.; Detmar, M. A simple immunomagnetic protocol for the selective isolation and long-term culture of human dermal microvascular endothelial cells. Exp. Cell Res. 240:1–6; 1998.

    Article  PubMed  CAS  Google Scholar 

  • Sato, N.; Sawasaki, Y.; Senoo, A.; Fuse, Y.; Hirano, Y.; Goto, T. Development of capillary networks from rat microvascular fragments in vitro: the role of myofibroblastic cells. Microvasc. Res. 33:194–210; 1987.

    Article  PubMed  CAS  Google Scholar 

  • Schor, A. M.; Schor, S. L. The isolation and culture of endothelial cells and pericytes from the bovine retinal microvasculature: a comparative study with large vessel vascular cells. Microvasc. Res. 32:21–38; 1986.

    Article  PubMed  CAS  Google Scholar 

  • Scott, N. A.; Candal, F. J.; Robinson, K. A.; Ades, E. W. Seeding of intracoronary stents with immortalized human microvascular endothelial cells. Am. Heart J. 129:860–866; 1995.

    Article  PubMed  CAS  Google Scholar 

  • Stachon, A.; Schluter, T.; Koller, M.; Weisser, H.; Krieg, M. Primary culture of microvascular endothelial cells from human benign prostatic hyperplasia. Prostate 48:156–164; 2001.

    Article  PubMed  CAS  Google Scholar 

  • Trochta, O. A.; Jacob, R. M.; Jefferson, B. J. Isolation and short term culture of canine adrenal microvascular endothelial cells. Can. J. Vet. Res. 63(1):1–4; 1999.

    PubMed  CAS  Google Scholar 

  • Wagner, R. C.; Matthews, M. A. The isolation and culture of capillary endothelium from epididymal fat. Microvasc. Res. 10(3):286–297; 1975.

    Article  PubMed  CAS  Google Scholar 

  • Williams, S.; Rose, D.; Jarrell, B. Microvascular endothelial cell sodding of ePTFE vascular grafts: improved patency and stability of the cellular lining. J. Biomed. Mater. Res. 28:203–212; 1994.

    Article  PubMed  CAS  Google Scholar 

  • Young, C.; Jarrell, B. E.; Hoying, J. B.; Williams, S. K. A porcine model for adipose tissue-derived endothelial cell transplantation. Cell Transplant. 1:293–298; 1992.

    PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Charles W. Patrick Jr..

Rights and permissions

Reprints and permissions

About this article

Cite this article

Frye, C.A., Patrick, C.W. Isolation and culture of rat microvascular endothelial cells. In Vitro Cell.Dev.Biol.-Animal 38, 208–212 (2002). https://doi.org/10.1290/1071-2690(2002)038<0208:IACORM>2.0.CO;2

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1290/1071-2690(2002)038<0208:IACORM>2.0.CO;2

Key words

Navigation