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

Journal name:
Nature Protocols
Volume:
12,
Pages:
1077–1088
Year published:
DOI:
doi:10.1038/nprot.2017.025
Published online

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.

At a glance

Figures

  1. Workflow of the protocol for in vitro reconstruction of a human 3D lymphatic microvascular network.
    Figure 1: Workflow of the protocol for in vitro reconstruction of a human 3D lymphatic microvascular network.

    Stage 1: primary fibroblasts and LEC isolation. Stage 2: generation of dermal cell sheet rich in extracellular matrix. Stage 3: LEC seeding and organization. Stage 4: stacking and air–liquid maturation of the tissue. Stage 5: immunostaining, imaging and data analysis. BEC, blood endothelial cell; ECM, extracellular matrix; HMVEC, human microvascular endothelial cell; LEC, lymphatic endothelial cell; LYVE-1, lymphatic vessel endothelial hyaluronan receptor-1.

  2. Anchoring paper system and air-liquid supports.
    Figure 2: Anchoring paper system and air–liquid supports.

    (a) Anchoring paper. (b) Plastic ring weight made from a Falcon 50-ml tube cap. (c) Ring weight is placed on an anchoring paper to avoid floating in six-well plates. (d) Top view of the air–liquid support. (e) Lateral view of an air–liquid support. (f) Tissue culture at the air–liquid interface (stage 4). Scale bars, 1 cm.

  3. LEC organization after seeding on fibroblast cell sheet (Step 3).
    Figure 3: LEC organization after seeding on fibroblast cell sheet (Step 3).

    LECs were detected by immunofluorescence staining against LYVE-1 (green). Representative samples imaged by wide-field fluorescence microscopy. Scale bars, 200 μm. The primary cells used are exempt from IRB review, as defined by DHHS regulation 45 CFR 46.101(b)(4). Human Research Exemption Determination was obtained from Mount Sinai's Program for the Protection of Human Subjects Institutional Review Board. The protocol was approved by the institution's committee for the protection of human subjects (comité d'éthique de la recherche du Centre de recherche du CHU de Québec-Université Laval).

  4. Lymphatic network characteristics in whole-mount tissue.
    Figure 4: Lymphatic network characteristics in whole-mount tissue.

    (a,b) Lymphatic vascular network visualized by immunofluorescence staining for CD31 (red), imaged by confocal microscopy (a), or computationally reconstructed using Imaris software (b). (c) Quantification of lymphatic network volume using Imaris software. Each dot represents one tissue construct; n = 4, mean is indiscated by line (c). (d–f) Network details at higher magnification showing lumens (d, stars), blind ends (e, arrows) and filopodia (f). (g–i) Double-immunofluorescence staining for podoplanin (g) and CD31 (h), merged in i. Scale bars, 200 μm (a,b); 100 μm (d,e,gi); 10 μm (f). The primary cells used are exempt from IRB review, as defined by DHHS regulation 45 CFR 46.101(b)(4). Human Research Exemption Determination was obtained from Mount Sinai's Program for the Protection of Human Subjects Institutional Review Board. The -protocol was approved by the institution's committee for the protection of human subjects (comité d'éthique de la recherche du Centre de recherche du CHU de Québec-Université Laval).

  5. Extracellular matrix and lymphatic markers in tissue sections of lymphatic vascular constructs.
    Figure 5: Extracellular matrix and lymphatic markers in tissue sections of lymphatic vascular constructs.

    (a–d) Immunofluorescence staining for collagen I (a), fibronectin (b), nuclei stained with Hoechst (c) and merged image (d), all observed by confocal microscopy. (e–h) Immunostaining for podoplanin (e), CD31 (f), Prox1 (g) and Ki67 (h), imaged by wide-field microscopy. Arrows indicate positively stained nuclei. Scale bars, 50 μm. The primary cells used are exempt from IRB review, as defined by DHHS regulation 45 CFR 46.101(b)(4). Human Research Exemption Determination was obtained from Mount Sinai's Program for the Protection of Human Subjects Institutional Review Board. The protocol was approved by the institution's committee for the protection of human subjects (comité d'éthique de la recherche du Centre de recherche du CHU de Québec-Université Laval).

  6. In vitro-reconstructed microvascular lymphatic network is functionally responsive to inhibitors of lymphangiogenesis.
    Figure 6: In vitro-reconstructed microvascular lymphatic network is functionally responsive to inhibitors of lymphangiogenesis.

    (a–c) Lymphatic microvascular network in control (a), after 3 weeks of treatment with an anti-VEGFR3 blocking antibody (2.5 μg/ml) (b) or with c-Met inhibitor SU11274 (1 μM) (c). Inhibitors were added 4 h after the seeding of LECs on day 21. Lymphatic vasculature is visualized by immunostaining of the whole construct with a CD31 antibody and reconstructed in 3D with the Imaris software. Note reduction in vessel densities in b and c, and thinner vessels in b, but not in c. (d) Reduction in lymphatic network volume as compared with that in the control after treatment with an anti-VEGFR3 blocking antibody (2.5 μg/ml) or c-Met inhibitor (1 μM). Total network volume was quantified for each construct using Imaris software. Four constructs were analyzed for each condition, and the means are shown. Scale bars, 100 μm. The primary cells used are exempt from IRB review, as defined by DHHS regulation 45 CFR 46.101(b)(4). Human Research Exemption Determination was obtained from Mount Sinai's Program for the Protection of Human Subjects Institutional Review Board. The protocol was approved by the institution's committee for the protection of human subjects (comité d'éthique de la recherche du Centre de recherche du CHU de Québec-Université Laval).

Videos

  1. Stacking of cell layers. Cell layers are stacked to create a 3D construct (Steps 1215).
    Video 1: Stacking of cell layers. Cell layers are stacked to create a 3D construct (Steps 12–15).

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Author information

  1. These authors contributed equally to this work.

    • Mihaela Skobe &
    • François A Auger

Affiliations

  1. Institut de Pharmacologie et de Biologie Structurale (IPBS), Centre National de la Recherche Scientifique (CNRS), Université Paul Sabatier, Toulouse, France.

    • Laure Gibot
  2. Centre LOEX de l'Université Laval, Regenerative Medicine Section of the FRQS Research Center of the CHU de Québec, Quebec, Quebec, Canada.

    • Todd Galbraith,
    • Jennifer Bourland &
    • François A Auger
  3. Department of Oncological Sciences and Tisch Cancer Center, Icahn School of Medicine at Mount Sinai, New York, New York, USA.

    • Anita Rogic &
    • Mihaela Skobe

Contributions

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.

Competing financial interests

The authors declare no competing financial interests.

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