Nature Methods
- 5, 431 - 437 (2008)
Published online: 20 April 2008; | doi:10.1038/nmeth.1205
Modeling lymphangiogenesis in a three-dimensional culture systemFrançoise Bruyère1, Laurence Melen-Lamalle1, Silvia Blacher1, Guy Roland1, Marc Thiry2, Lieve Moons3, Francis Frankenne1, Peter Carmeliet4, 5, Kari Alitalo6, Claude Libert7, 8, Jonathan P Sleeman9, 10, Jean-Michel Foidart1, 11 & Agnès Noël11
Laboratory of Tumor and Developmental Biology, Groupe Interdisciplinaire de Génoprotéomique Appliqué–Cancer, University of Liège, Avenue de l'Hôpital 3, B-4000 Liège, Belgium. 2
Laboratory of Cell and Tissue Biology, University of Liege, rue de Pitteurs 20, B-4020, Liège, Belgium. 3
Laboratory of Neural Circuit Development and Regeneration, Zoological Institute, Katholieke Universiteit Leuven, Naamsestraat 61, B-3000 Leuven, Belgium. 4
Department for Transgene Technology and Gene Therapy, Vlaams Instituut voor Biotechnologie, Onderwijs en Navorsing building I, Herestraat 49, B-3000 Leuven, Belgium. 5
Center for Transgene Technology and Gene Therapy, Katholieke Universiteit Leuven, Naamsestraat 61, B-3000 Leuven, Belgium. 6
Molecular Cancer Biology Program, Ludwig Institute for Cancer Research, Biomedicum Helsinki, Haartman Institute, P.O. Box 63 (Haartmaninkatu 8), FI-00014 University of Helsinki, Finland. 7
Department for Molecular Biomedical Research, Universiteit Gent Vlaams Instituut voor Biotechnologie, Technologiepark 927, B-9052 Gent, Belgium. 8
Department of Molecular Biology, Ghent University, Technologiepark Zwijnaarde 927, B-9052 Ghent, Belgium. 9
Forschungszentrum Karlsruhe, Institut fur Toxikologie und Genetik, Postfach 3640, D-76021 Karlsruhe, Germany. 10
Medical Faculty Mannheim, University of Heidelberg, Theodor-Kutzer-Ufer 1-3, D-68135 Mannheim, Germany. 11
Department of Gynecology, Centre Hospitalier Universitaire, Blvd. du 12ème de Ligne, B-4000 Liège, Belgium.
Correspondence should be addressed to Agnès Noël agnes.noel@ulg.ac.be A lack of appropriate in vitro models of three-dimensional lymph vessel growth hampers the study of lymphangiogenesis. We developed a lymphatic ring assay—a potent, reproducible and quantifiable three-dimensional culture system for lymphatic endothelial cells that reproduces spreading of endothelial cells from a pre-existing vessel, cell proliferation, migration and differentiation into capillaries. In the assay, mouse thoracic duct fragments are embedded in a collagen gel, leading to the formation of lumen-containing lymphatic capillaries, which we assessed by electron microscopy and immunostaining. We developed a computerized method to quantify the lymphatic network. By applying this model to gene-deficient mice, we found evidence for involvement of the matrix metalloproteinase, MMP-2, in lymphangiogenesis. The lymphatic ring assay bridges the gap between two-dimensional in vitro models and in vivo models of lymphangiogenesis, can be used to exploit the potential of existing transgenic mouse models, and rapidly identify regulators of lymphangiogenesis.
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