Isolation and Short‐Term Culturing of Primary Lymphatic Endothelial Cells from Collecting Lymphatics: A Techniques Study

Objective: To develop an experimental method for routine isolation and short-term culture of primary lymphatic endothelial cells from specific collecting vessels. Methods: Lymphatic endothelial cell tubes (LECTs) were isolated from micro-dissected collecting vessels. LECTs were allowed to attach and grow for similar to 3 weeks before being passaged. Non-purified cultures were partially characterized by immunofluorescence and RT-PCR at passages 1-2. Results: The method was validated in cultures of primary lymphatic endothelial cells (LECs) from male and female mice. After 1 or 2 passages, >60% of the LECs maintained expression of Prox1. Expression of 22 different genes was assessed using RT-PCR. Prox1, Vegfr3, eNos, Cdh5, Pecam1, Cx43, Cx37, and Cx47, among others, were expressed in these short-term cultured LECs, while Myh11, Cnn1, Desmin, and Cd11b were not detected. Prox1 expression, as determined by western blotting, was similar in cultured LECs from age-matched male and female mice. Confocal imaging of intracellular calcium in cultures of primary LECs from Cdh5-GCaMP8 mice demonstrated that a functional phenotype was maintained, similar to lymphatic endothelial cells in freshly isolated vessels. Conclusions: This method provides an innovative tool for routine isolation and study of primary LECs from specific collecting lymphatic vessels from any mouse, and in fact, from other species.