PKH26 and125I-PKH95: Characterization and Efficacy as Labels forin Vitroandin VivoEndothelial Cell Localization and Tracking

PKH26, a fluorescent cell label, and PKH95, a125I-radioactive cell label, are both potentially valuable endothelial cell labels because they bind irreversibly within cell membranes. These labels would be particularly well suited to tract transplanted endothelial cellsin vivo.However, no previous studies documenting lack of transfer of the label to unlabeled endothelial cells, as well as the effect of the label on endothelial cell function, have been undertaken. The purpose of this study was to determine the optimal method of endothelial cell (EC) labeling with PKH26 and PKH95, whether significant EC-to-EC transfer of the label occurs, the effects of these labels on EC proliferation and membrane function, and the feasibility of using these labels for long-term quantitative EC trackingin vivo.Canine ECs in confluent monolayers or in cell suspension were labeled by exposure to 1.0 or 5.0 μMPKH26 for 1, 3, or 5 min. Cell viability was determined by trypan blue exclusion. The percentage of cells labeled and their fluorescence intensity were determined in a fluorescent-activated cell sorter (FACS). Effect of the label on cell function was assessed by measuring EC proliferation rates as well as intercellular adhesion molecule (ICAM) expression before and after induction with tumor necrosis factor (TNF). To determine if transfer of the label occurs, both labeled and nonlabeled ECs were grown in coculture and subjected to FACS analysis. Forin vivocell tracking, doubly labeled ECs were injected into the femoral artery of rat hindlimbs, and whole-body tissue analysis was undertaken to determine labeled-EC distribution at 60 days. Endothelial cells were labeled with equal efficiency as monolayers or in suspension. Labeling had no effect on EC proliferation rates nor on TNF-induced upregulation of ICAM expression. Coculture experiments revealed no significant label transfer to nonlabeled ECs.In vivocell tracking studies documented that 8% of label remained within the skeletal muscle capillaries at 60 days after injection. PKH26 and PKH95 labels incorporate stably into EC membranes, do not alter endothelial cell function, and provide a precise means for quantitative EC tracking and histologic localization bothin vitroandin vivo.These labels should prove to be very useful for studies of endothelial cell biology and transplantation.