ISOLATION, PURIFICATION, AND HETEROGENEITY OF HUMAN LYMPHATIC ENDOTHELIAL CELLS FROM DIFFERENT TISSUES

E Garrafa, L Trainini, A Benetti, E Saba, L Fezzardi, B Lorusso, P Borghetti, T Bottio, E Cervi, N Portolani, S Bonardelli, SM Giulini, G Annibale, A Corradi, L Imberti, A Caruso

Abstract


Relatively few attempts have been madein the past to isolate and expand lymphaticendothelial cells (LECs). Recently this taskhas become feasible thanks to the identificationof new lymphatic markers such asPodoplanin, Lyve-1, Prox-1 and D2-40. Usinga two-step purification method based on thesorting of endothelial cells with UlexEuropaeus Agglutinin 1-coated beads followedby purification with monoclonal antibodyD2-40, we were able to purify and in vitroexpand human derived LECs from tissuessuch as lymph node, spleen, thymus, palatinetonsil and iliac lymphatic vessels. The isolatedLECs were expanded on collagen type 1 andfibronectin coated flasks for up to 8-10passages and then analyzed for phenotypicand functional properties. LECs were able toform a capillary like network, when seeded onCultrex BME, indicating their capability toform lymphatic vessels in vitro. Comparative studies were performed, and we found thatspecific lymphatic and vascular markers weredifferentially expressed by LECs preparedfrom different sources, clearly demonstratingthe phenotypic heterogeneity of LECs fromdifferent organs and different segments of thelymphatic vasculature. We here propose a newtechnique to make available ready sources ofabundant well-characterized human LECs toexamine normal profiles and behavior tocompare with abnormal conditions.

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