Rapid and simple isolation of precartilaginous stem cells: A novel approach without immunomagnetic bead sorting.

Objective: The present investigation was designed to devise a rapid and straightforward technique for the isolation of rat precartilaginous stem cells (PCSCs) that eschews the use of immunomagnetic bead sorting. Method: Rat neonates within 24 h of birth were selected for this study. Microsurgical techniques were used to harvest the femur, tibia, and the musculature of the knee joint. The ring of LaCroix between the metaphysis of the femur and the epiphysis was excised and divided into fragments of approximately 1 mm³. Tissue sections were cultured in Dulbecco's modified Eagle's medium(DMEM)/F12 medium supplemented with 20 % fetal bovine serum and 1 % penicillin-streptomycin. Cell digestion and passaging were performed using trypsin when cells reached 70%-80 % confluence. Third-generation cells underwent immunofluorescence staining and flow cytometry to evaluate fibroblast growth factor receptor-3(FGFR-3) and proliferating cell nuclear antigen(PCNA) expression, while β-galactosidase staining was used to determine cellular senescence. Results: Within two days of isolation, numerous short spindle-shaped cells exhibiting distinct refractive properties were observed around the tissue fragments. These cells began to proliferate within 2-3 days and displayed ample cytoplasm. Adherent cells adopted various morphologies, including angular, triangular, and elongated spindles. By the fifth day, more than 80 % of the culture dish surface was covered with elongated cells, with some arranged in patterns reminiscent of whirlpools. Significant FGFR-3 and PCNA expression was confirmed via immunofluorescence in the third-generation cells. Additionally, flow cytometry identified that the proportion of cells positive for FGFR-3 and PCNA exceeded 98 %. Notably, the cells preserved their proliferative capacity through nine passages in vitro, with a marginal proportion showing senescence as indicated by β-galactosidase staining alone. Conclusion: The developed tissue adherence protocol was used to successfully isolate PCSCs with positive FGFR-3 and PCNA expression, rendering the immunomagnetic bead sorting superfluous. The expression of FGFR-3 and PCNA in the isolated cells persisted through the ninth passage in vitro with minimal senescence.