Abstract
A protocol describing the isolation of foreskin lymphatic endothelial cells (LECs) and lymphatic malformation lymphatic endothelial cells (LM LECs) is presented herein. To isolate LECs and LM LECs, tissues are mechanically disrupted to make a single-cell suspension, which is then enzymatically digested in dispase and collagenase type II. LECs and LM LECs, in the resulting single-cell suspension, are then sequentially labeled with antibodies recognizing fibroblast and endothelial cell surface antigens CD34 and CD31 and separated from the remaining components in the cell suspension by capture with magnetic beads. Viable LECs and LM LECs are then seeded and expanded on fibronectin-coated flasks. LEC and LM LEC purity is determined immunohistochemically using cell surface markers CD31, CD34, podoplanin, VEGFR-3 and nuclear marker PROX-1. Cells whose purity is >98 % are used for experiments between passage 4 and 6.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Baluk P, Fuxe J, Hashizume H, Romano T, Lashnits E, Butz S et al (2007) Functionally specialized junctions between endothelial cells of lymphatic vessels. J Exp Med 204(10):2349–2362
Alitalo K (2011) The lymphatic vasculature in disease. Nat Med 17(11):1371–1380
Ferrell RE, Kimak MA, Lawrence EC, Finegold DN (2008) Candidate gene analysis in primary lymphedema. Lymph Res Biol 6(2):69–76
Greene AK, Perlyn CA, Alomari AI (2011) Management of lymphatic malformations. Clin Plastic Surg 38(1):75–82
Lokmic Z, Mitchell GM (2011) Visualisation and stereological assessment of blood and lymphatic vessels. Histol Histopathol 26(6):781–796
Lokmic Z, Mitchell GM, Koh Wee Chong N, Bastiaanse J, Gerrand YW, Zeng Y et al (2014) Isolation of human lymphatic malformation endothelial cells, their in vitro characterization and in vivo survival in a mouse xenograft model. Angiogenesis 17(1):1–15
Hirakawa S, Hong YK, Harvey N, Schacht V, Matsuda K, Libermann T et al (2003) Identification of vascular lineage-specific genes by transcriptional profiling of isolated blood vascular and lymphatic endothelial cells. Am J Pathol 162(2):575–586
Baudin B, Bruneel A, Bosselut N, Vaubourdolle M (2007) A protocol for isolation and culture of human umbilical vein endothelial cells. Nat Protoc 2(3):481–485
Acknowledgment
This work is supported by Jigsaw Foundation Research Grant and “Women in Science” Fellowship Award.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2016 Springer Science+Business Media New York
About this protocol
Cite this protocol
Lokmic, Z. (2016). Isolation, Identification, and Culture of Human Lymphatic Endothelial Cells. In: Martin, S., Hewett, P. (eds) Angiogenesis Protocols. Methods in Molecular Biology, vol 1430. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3628-1_5
Download citation
DOI: https://doi.org/10.1007/978-1-4939-3628-1_5
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-3626-7
Online ISBN: 978-1-4939-3628-1
eBook Packages: Springer Protocols