An Experimental Study on Bovine Nucleus Pulposus Cells Labelled with PKH26 in Vitro.

Objective To investigate the application of PKH26 fluorescent labeling on nucleus pulposus cells isolated from bovine coccyx disc, and to provide nucleus pulposus tissue engineering with traceable nucleus pulposus cells by PKH26 fluorescence labelling. 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 methods used to stain for passage one generation cells. Nucleus pulposus cells were labeled with PKH26 fluorescence in accordance with the instructions. The cell activity, fluorescence intensity at d0, d14 and d28 of culture, characteristics of proliferation and the expression of gene in labeled cells were assessed. Results Isolated nucleus pulposus cells amounted to (1.56 ± 0.35) x 106/g. Under the inverted microscope, primary cells adhered at the 4 th day of culture, grew in groups, and covered the bottom of culture flask at the 13 th 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 results. The cell activity before and after PKH26 labeling showed more than 95%, and the fluorescence intensity at d0, d14 and d28 performed a decreasing trend, but still showed detect strong fluorescence at d28. There were no significant differences in proliferation and the expression of gene (collagen type I and II, aggrecan) before and after cell labeling (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. [ABSTRACT FROM AUTHOR]