Lymphatic endothelial cells: establishment of primaries and characterization of established lines

This chapter describes detailed methods for the isolation of primary human lymphatic endothelial cells from neonatal foreskin. We also provide protocols and information for their characterization and propagation. Isolation of primary human lymphatic endothelial cells requires a two-step process: mechanical and enzymatic digestion of human foreskins and cell sorting by fluorescence-activated cell sorting of CD31+/podoplanin+/CD45- cells. Characterization of these cells requires an assessment of the expression of several markers specific for lymphatic endothelium. This is determined by fluorescence-activated cell sorting, immunocytochemistry, and polymerase chain reaction. All procedures are based on simple laboratory techniques and, with the exception of a cell sorter and the skills to use it, do not require specialized equipment.