The diaphragmatic lymphatic network plays a pivotal role in setting pleural fluid homeostasis, contributing to the maintenance of a proper lung-chest wall mechanical coupling. The wall of diaphragmatic lymphatics is essentially delimited by a discontinuous layer of lymphatic endothelial cells (LECs), a basement membrane and only few smooth muscle cells. Lymph formation, propulsion and flow modulation may depend upon: a) external tissue displacement and local tissue stress, and/or b) intrinsic contractility of smooth muscle cells. No data are available at present on the potential role of LECs in modulating lymph flow in response to intraluminar and/or interstitial mechanical and/or chemical factors. No diaphragmatic LEC cell line is available at present. Therefore, to study their functions,we developed a new approach to isolate, characterise and maintain in long term primary cultures LECs deriving from in vivo FITC-dextran or anti-podoplanin stained rats and mice diaphragms. In vitro, LECs start to spread out from tissue samples in 5 to 10 days, either as a monolayer (2D) or as organized vessels (3D matrigel cultures). Their lymphatic nature was confirmed by immunostaining with specific markers (anti-podoplanin, anti-LYVE-1 and anti-PROX-1 antibodies). After 3–4 weeks, sprouting vessels begin to display a self-sustained contractile activity typical, "in situ" of vessels equipped with smooth muscle support.