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Isolation of Human Skin Lymphatic Endothelial Cells and 3D Reconstruction of the Lymphatic Vasculature In Vitro

Part of the Methods in Molecular Biology book series (MIMB,volume 1846)

Abstract

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 words

  • Human lymphatic endothelial cells
  • Lymphatic endothelial cell isolation
  • Lymphangiogenesis in vitro
  • Lymphatic microvascular network in vitro
  • 3D lymphatic capillary construct
  • Self-assembly

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  • DOI: 10.1007/978-1-4939-8712-2_18
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References

  1. Podgrabinska S, Braun P, Velasco P, Kloos B, Pepper MS, Skobe M (2002) Molecular characterization of lymphatic endothelial cells. Proc Natl Acad Sci U S A 99(25):16069–16074. https://doi.org/10.1073/pnas.242401399

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  2. Kriehuber E, Breiteneder-Geleff S, Groeger M, Soleiman A, Schoppmann SF, Stingl G, Kerjaschki D, Maurer D (2001) Isolation and characterization of dermal lymphatic and blood endothelial cells reveal stable and functionally specialized cell lineages. J Exp Med 194(6):797–808

    CAS  CrossRef  Google Scholar 

  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

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  4. Helm CL, Zisch A, Swartz MA (2007) Engineered blood and lymphatic capillaries in 3-D VEGF-fibrin-collagen matrices with interstitial flow. Biotechnol Bioeng 96(1):167–176. https://doi.org/10.1002/bit.21185

    CAS  CrossRef  PubMed  Google Scholar 

  5. Dixon JB, Raghunathan S, Swartz MA (2009) A tissue-engineered model of the intestinal lacteal for evaluating lipid transport by Lymphatics. Biotechnol Bioeng 103(6):1224–1235. https://doi.org/10.1002/Bit.22337

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  6. Bruyere F, Noel A (2010) Lymphangiogenesis: in vitro and in vivo models. FASEB J 24(1):8–21. https://doi.org/10.1096/fj.09-132852

    CAS  CrossRef  PubMed  Google 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

    CAS  CrossRef  PubMed  Google Scholar 

  8. Marino D, Luginbuhl J, Scola S, Meuli M, Reichmann E (2014) Bioengineering dermo-epidermal skin grafts with blood and lymphatic capillaries. Sci Transl Med 6(221):221–214. https://doi.org/10.1126/scitranslmed.3006894

    CAS  CrossRef  Google 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

    CAS  CrossRef  PubMed  Google Scholar 

  10. Gibot L, Galbraith T, Bourland J, Rogic A, Skobe M, Auger FA (2017) Tissue-engineered 3D human lymphatic microvascular network for in vitro studies of lymphangiogenesis. Nat Protoc 12(5):1077–1088. https://doi.org/10.1038/nprot.2017.025

    CrossRef  Google Scholar 

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Acknowledgements

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.).

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Correspondence to Mihaela Skobe .

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Rogic, A., Auger, F., Skobe, M. (2018). Isolation of Human Skin Lymphatic Endothelial Cells and 3D Reconstruction of the Lymphatic Vasculature In Vitro. In: Oliver, G., Kahn, M. (eds) Lymphangiogenesis. Methods in Molecular Biology, vol 1846. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8712-2_18

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  • DOI: https://doi.org/10.1007/978-1-4939-8712-2_18

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-8711-5

  • Online ISBN: 978-1-4939-8712-2

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