Isolation, Culture and Characterization of Endothelial Cells from Capillary Lymphantic Vessels of Human Skin / 实用妇产科杂志

ABSTRACT

Objective:Human capillary lymphatic endothelial cells (LECs) were isolated and cultured to assist further investigation of the function of lymphatic vessels generation during cancer metastasis. Methods:Human skin was digested by type I collagenase. Cells were isolated using magnetic beads which were marked by monoclonal antibodies against the extracellular domain of VEGFR-3, and purified by cloning col-umn. The morphology and structure of cells were observed by microscopy. Immunophenotype was identified by immunofluorescence. Cell growth curve was recorded to measure the effect of VEGF-C protein. Results:LECs exhibited the typical cobblestone morphology as monolayer growth pattem under microscopy. Enlarged nucleus and rich cytoplasm with bubbles were found under electromicroscopy. LECs specific markers inclu-ding VEGFR-3, LYVE-1, Podoplaninand D2-40 all were positive. VIII factor as specific marker of blood ves-sel endothelium cells (BVECs) was negative. VEGF-C induced a marked increase of cell proliferation. Con-clusions:Human dermal LECs could be harvested successfully using collagenase digestion, immunomagnet-ic beads sorting and clonic column purification.