Optimizing fluorescent labeling of endothelial cells for tracking during long-term studies of autologous transplantation

Background. The fluorescent marker PKH26 has been demonstrated to be useful for the tracking of endothelial cells in short-term studies; however, the optimal labeling conditions for long-term implants have not been determined . This study was designed to evaluate the effects of PKH26 on endothelial c ell proliferation and to identify labeling conditions that would yield the greatest fluorescence over time without adversely affecting cell viability. Materials and methods. Canine jugular vein endothelial cells (CJVECs) were labeled with 0.04 mu M PKH26. Proliferation of labeled and control cells wa s assessed for 8 consecutive days by [H-3]thymidine uptake. In a second exp eriment, CJVECs were labeled at concentrations of 0, 5, 8, 10, and 20 mu mo l/L. Cells were maintained in culture for 60 days. The fluorescence intensi ty of each cell population was measured using two techniques. At baseline a nd at 60 days, fluorescence was measured using a fluorescence-activated cel l sorter. On days 14, 28, 45, and 60 fluorescence was measured by construct ing gray-scale histograms from photomicrographs taken of each flask under r hodamine illumination. Mean viable cell number for each concentration was d etermined after 60 days. Results. In the first experiment, PKH26-labeled and unlabeled CJVECs demons trated nearly identical growth curves, suggesting that PRH26 had no adverse effect on proliferation. In the second experiment, after 60 days, the 10 a nd 20 mu M groups displayed greater fluorescence by histogram than the 0, 5 , or 8 mu M groups; however, they were not significantly different from eac h other (mean intensity 8.2 vs 9.1, P > 0.05, Student-Newman-Heuls test for multiple comparisons). Over 60 days, the cells labeled with 20 mu M PKH26 experienced the only significant decrease in viable cells compared to the u nlabeled group (5.5 x 10(5) vs 9.6 x 10(5) cells/flask, P < 0.05). Importan tly, we observed no significant differences in cell number between the 10 m u M group and the lower concentrations compared to the unlabeled cells (P > 0.05). Conclusions. We conclude that a concentration of 10 mu M PKH26 provides the optimal labeling condition for endothelial cells when long-term tracking i s desired. (C) 1999 Academic Press.