title: Isolation, Identification, and Culture of Human Lymphatic Endothelial Cells.
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.
authors.affiliation: Murdoch Childrens Research Institute, The Royal Children's Hospital Melbourne, 50 Flemington Rd, Parkville, 3052, VIC, Australia. zerina.lokmic@mcri.edu.au.|Department of Paediatrics, Faculty of Medicine, Dentistry and Health Sciences, The University of Melbourne, Parkville, 3052, VIC, Australia. zerina.lokmic@mcri.edu.au.
authors.forename: Zerina
authors.initials: Z
authors.lastname: Lokmic
mesh_terms: D018952:Antigens, CD34 / Q000378:metabolism; D015415:Biomarkers / Q000378:metabolism*; D018929:Cell Culture Techniques; D049109:Cell Proliferation; D002469:Cell Separation; D002470:Cell Survival; D042783:Endothelial Cells / Q000166:cytology* / Q000378:metabolism; D005434:Flow Cytometry / Q000379:methods; D052816:Foreskin / Q000166:cytology; D006801:Humans; D042601:Lymphatic Vessels / Q000002:abnormalities / Q000166:cytology* / Q000378:metabolism; D008297:Male; D019408:Platelet Endothelial Cell Adhesion Molecule-1 / Q000378:metabolism
chemical_list: D018952:Antigens, CD34; D015415:Biomarkers; D019408:Platelet Endothelial Cell Adhesion Molecule-1
keywords: Cell culture; Foreskin; Human lymphatic endothelial cells; Isolation; Lymphatic malformation; Magnetic beads