Abstract: 【Abstract】 Objiective To investigate the application of PKH26 fluorescent labeling on nucleus pulposus cells isolated from bovine Coccyx disc. Methods Nucleus pulposus primary cells were isolated from the nucleus pulposus tissue detached from bovine Coccyx disc by enzymatic digestion, and observed under the inverted microscope. Safranin O, toluidine blue and type II collagen immunocytochemistry were stained for passage one generation cells. Nucleus pulposus cells were labeled with PKH26 fluorescence in accordance with the instructions, and labeled cells’ activity, fluorescence intensity at days 0, 14, 28 of culture and characteristics of proliferation were assessed. Results Isolated nucleus pulposus cells amounted to (1.56 ± 0.35) × 106/g. Under the inverted microscope, primary cells adhered at 4th day of culture, grew in groups, and cells covered the bottom of culture flask at 13th day, both primary cells and the P1 generation cells were chondrocyte-like morphology. The staining of safranin O, toluidine blue and type II collagen immunocytochemistry for P1 generation of nucleus pulposus cells showed positive result. Cell activity before and after PKH26 label showed more than 95%, the fluorescence intensity at 0d, 14d, 28d performed a decreasing trend, but 28d can still detect strong fluorescence, there were no significant differences in growth curve between before and after labeled cells (P > 0.05). Conclusion As the seed cells of tissue engineering, nucleus pulposus cells isolated from bovine Coccyx can reach a satisfactory number and maintain cartilage-like phenotype, and no changes shown in the biological characteristics after labeling. PKH26 labeled nucleus pulposus cells are suitable for the traceable cells in vivo study.

Key words: Bovine Coccyx, Nucleus pulposus cells, Tissue engineering, intervertebral disc, PKH26