Intravital fluorescence microscopy of endothelial cells on vascular grafts

The ability to evaluate the extent of initial endothelial cell coverage on a vascular graft subsequent to an endothelial cell seeding technique would be desirable to substantiate the durability of the endothelial cell lining. To this end, we have evaluated the use of intravital fluorescence microscopy to assess human endothelial cell interaction with vascular grafts. Five fluorescent stains, mithramycin, Hoechst 33342, sulfofluorescein diacetate, Nile red, and rhodamine 123 were evaluated for their ability to fluorescently label human endothelial cells. The staining capability of each dye was also evaluated with respect to accuracy in determining seeded cell number and cell spreading. Of the stains evaluated, rhodamine 123 produced the most desirable characteristics. We observed excellent cell visualization after a 30-min incubation. Unlike the other four stains, rhodamine 123 exhibited a bright orange fluorescence emission at a 510-nm excitation wavelength while the underlying dacron or expanded polytetrafluoroethylene demonstrated minimal autofluorescence. Rhodamine 123 also exhibited no inhibitory effect on cell attachment to plastic or subsequent cell growth in culture. Intravital fluorescence microscopy could be easily utilized in the operating room to visualize part or all of an endothelial cell-seeded graft prior to implantation and would permit a quantitative as well as qualitative evaluation of the seeding process. Since intravital fluorescence imaging does not require tissue fixation, the same surface as that evaluated for seeding efficiency can be directly implanted.