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.
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.
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.
Dejana, E.; Corada, M.; Grazia Lampugnani, M. Endothelial cell-to-cell junctions. FASEB J. 9:910–918; 1995.
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.
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.
Hannum, R. S.; Ojeifo, J. O.; Zwiebel, J. A.; McLeskey, S. W. Isolation of tumor-derived endothelial cells. Microvasc. Res. 61:287–290; 2001.
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.
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
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.
Karasek, M. A. Microvascular endothelial cell culture. J. Invest. Dermatol. 93:33S-38S; 1989.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Polinger, I. S. Separation of cell types in embryonic heart cell cultures. Exp. Cell Res. 63:78–82; 1970.
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.
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.
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.
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.
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.
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.
Wagner, R. C.; Matthews, M. A. The isolation and culture of capillary endothelium from epididymal fat. Microvasc. Res. 10(3):286–297; 1975.
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.
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.
Author information
Authors and Affiliations
Corresponding author
Rights 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
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1290/1071-2690(2002)038<0208:IACORM>2.0.CO;2