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Tissue-engineered 3D human lymphatic microvascular network for in vitro studies of lymphangiogenesis

Abstract

This protocol describes a unique in vitro method for the generation of a 3D human lymphatic network within native connective tissue devoid of any exogenous material such as scaffolds or growth factors. In this five-stage protocol, human lymphatic endothelial cells (LECs) cocultured with dermal fibroblasts spontaneously organize into a stable 3D lymphatic capillary network. Stage 1 involves the isolation of primary fibroblasts and LECs from human skin. Fibroblasts are then cultured to produce connective tissue rich in extracellular matrix (stage 2), onto which LECs are seeded to form a network (stage 3). After stacking of tissue layers and tissue maturation at the air–liquid interface (stage 4), the 3D construct containing the lymphatic microvascular network can be analyzed by microscopy (stage 5). Lymphatic vasculature generated by this approach exhibits the major cellular and ultrastructural features of native in vivo human dermal lymphatic microvasculature and is stable over many weeks. The protocol for generating a 3D construct takes 6 weeks to complete, and it requires experience in cell culture techniques. The system described here offers a unique opportunity to study the mechanisms underlying lymphatic vessel formation, remodeling and function in a human cell context.

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Figure 1: Workflow of the protocol for in vitro reconstruction of a human 3D lymphatic microvascular network.
Figure 2: Anchoring paper system and air–liquid supports.
Figure 3: LEC organization after seeding on fibroblast cell sheet (Step 3).
Figure 4: Lymphatic network characteristics in whole-mount tissue.
Figure 5: Extracellular matrix and lymphatic markers in tissue sections of lymphatic vascular constructs.
Figure 6: In vitro-reconstructed microvascular lymphatic network is functionally responsive to inhibitors of lymphangiogenesis.

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Acknowledgements

This work was supported by US Department of Defense grant BC044819 and by the DRPPP Pilot Project Program, Department of Oncological Sciences, Mount Sinai (M.S., A.R.), Canadian Institute of Health Research (CIHR) grant SAC92850 (F.A.A.) and the Fondation d'entreprise SILAB-Jean Paufique (L.G.).

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L.G., F.A.A. and M.S. designed the study. L.G., T.G., J.B. and A.R. conducted the experiments. L.G., T.G., J.B., A.R., M.S. and F.A.A. analyzed the data. L.G., T.G. and M.S. wrote the manuscript.

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Correspondence to Mihaela Skobe or François A Auger.

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The authors declare no competing financial interests.

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

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