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L. Chen; Isolation, Expansion, and Characterization of Lymphatic Endothelial Cells Using a Newly Established System. Invest. Ophthalmol. Vis. Sci. 2007;48(13):195. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Lymphatic research has progressed exponentially in the past few years owning to the discovery of several lymphatic specific markers. However, there are relatively few studies on the isolation, characterization, and cultivation of lymphatic endothelial cells (LEC), hindering the development of an in vitro tool to investigate the fundamental processes involved in lymphatic vessel growth (lymphangiogenesis, LG). The purpose of this study was to establish a valid system for LEC isolation, characterization, and expansion.
LECs from human dermal microvascular endothelial cells (HMVEC) were isolated with a monoclonal antibody (D2-40) using the fluro MoFlo High Performance Cell Sorter. The cells were cultured and expanded in EBM-2 medium. For phenotyping purpose, HMVECs before sorting were stained with anti-CD31 (panendothelial marker) and D2-40 antibodies for flow cytometric analysis. After sorting, LECs were tested by epifluorescent microscopic studies using a panel of lymphatic specific markers including vascular endothelial growth factor receptor-3 (VEGFR-3), lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1) and D2-40.
LECs were successfully isolated from HMVECs, and 99% of HMVECs were CD-31+, confirming their endothelial cell phenotype. 22% of these cells were D2-40+, indicating a lymphatic endothelial population. The sorted LECs survived and expanded in the culture for several passages studied, and these cells expressed the panel of lymphatic markers tested, confirming their LEC phenotype.
This new system of isolating and maintaining LECs in culture provides a potentially powerful tool for functional studies of LG in vitro. Results from the studies using this system can be applied to the development of new therapeutic protocols for lymphatic related disorders, including corneal transplant rejection (especially where grafting is performed on the inflamed and lymphatic-rich host beds) as well as a diverse array of non-ocular diseases, such as cancer metastasis, lymphedema, diabetics, delayed wound healing, and AIDS, among many others.
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