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 cell 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 microM PKH26. Proliferation of labeled and control cells was assessed for 8 consecutive days by [(3)H]thymidine uptake. In a second experiment, CJVECs were labeled at concentrations of 0, 5, 8, 10, and 20 micromol/L. Cells were maintained in culture for 60 days. The fluorescence intensity of each cell population was measured using two techniques. At baseline and at 60 days, fluorescence was measured using a fluorescence-activated cell sorter. On days 14, 28, 45, and 60 fluorescence was measured by constructing gray-scale histograms from photomicrographs taken of each flask under rhodamine illumination. Mean viable cell number for each concentration was determined after 60 days.

RESULTS

In the first experiment, PKH26-labeled and unlabeled CJVECs demonstrated nearly identical growth curves, suggesting that PKH26 had no adverse effect on proliferation. In the second experiment, after 60 days, the 10 and 20 microM groups displayed greater fluorescence by histogram than the 0, 5, or 8 microM groups; however, they were not significantly different from each other (mean intensity 8.2 vs 9.1, P > 0.05, Student-Newman-Keuls test for multiple comparisons). Over 60 days, the cells labeled with 20 microM PKH26 experienced the only significant decrease in viable cells compared to the unlabeled group (5.5 x 10(5) vs 9.6 x 10(5) cells/flask, P < 0.05). Importantly, we observed no significant differences in cell number between the 10 microM group and the lower concentrations compared to the unlabeled cells (P > 0.05).

CONCLUSIONS

We conclude that a concentration of 10 microM PKH26 provides the optimal labeling condition for endothelial cells when long-term tracking is desired.