Isolation of Human Skin Lymphatic Endothelial Cells and 3D Reconstruction of the Lymphatic Vasculature In Vitro
Studies of lymphangiogenesis and lymphatic endothelial biology in vitro require pure cultures of lymphatic endothelial cells and 3D vascular constructs, which closely resemble native human lymphatic vasculature. We describe a method for the isolation of human dermal microvascular lymphatic endothelial cells and generation of a 3D lymphatic capillary network. The lymphatic vascular construct is generated by coculturing primary lymphatic endothelial cells and fibroblasts in their native matrix, without the use of synthetic scaffolds or exogenous factors. The tissue is stable over many weeks and accurately recapitulates features of human dermal lymphatic microvasculature.
Key wordsHuman lymphatic endothelial cells Lymphatic endothelial cell isolation Lymphangiogenesis in vitro Lymphatic microvascular network in vitro 3D lymphatic capillary construct Self-assembly
This work was supported by the DOD grant BC044819, the Susan G. Komen grant IBC17512321, by the DRPPP Pilot Project Program, Dept. Oncological Sciences, Mount Sinai (M.S.), and the CIHR grant SAC92850 and the Fondation d’entreprise SILAB-Jean Paufique (F.A.).
(MOV 15607 kb)
- 3.Makinen T, Veikkola T, Mustjoki S, Karpanen T, Catimel B, Nice EC, Wise L, Mercer A, Kowalski H, Kerjaschki D, Stacker SA, Achen MG, Alitalo K (2001) Isolated lymphatic endothelial cells transduce growth, survival and migratory signals via the VEGF-C/D receptor VEGFR-3. EMBO J 20(17):4762–4773. https://doi.org/10.1093/emboj/20.17.4762 CrossRefPubMedPubMedCentralGoogle Scholar
- 7.Bruyere F, Melen-Lamalle L, Blacher S, Roland G, Thiry M, Moons L, Frankenne F, Carmeliet P, Alitalo K, Libert C, Sleeman JP, Foidart JM, Noel A (2008) Modeling lymphangiogenesis in a three-dimensional culture system. Nat Methods 5(5):431–437. https://doi.org/10.1038/nmeth.1205 CrossRefPubMedGoogle Scholar
- 9.Gibot L, Galbraith T, Kloos B, Das S, Lacroix DA, Auger FA, Skobe M (2016) Cell-based approach for 3D reconstruction of lymphatic capillaries in vitro reveals distinct functions of HGF and VEGF-C in lymphangiogenesis. Biomaterials 78:129–139. https://doi.org/10.1016/j.biomaterials.2015.11.027 CrossRefPubMedGoogle Scholar