To develop an experimental method for routine isolation and short‐term culture of primary lymphatic endothelial cells from specific collecting vessels.

Lymphatic endothelial cell tubes (LECTs) were isolated from micro‐dissected collecting vessels. LECTs were allowed to attach and grow for ~3 weeks before being passaged. Non‐purified cultures were partially characterized by immunofluorescence and RT‐PCR at passages 1–2.

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.

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.