Abstract: BACKGROUND: Knee cartilage loss certainly occurs in aged mice, so it is difficult to extract the articular cartilage from the mice. In particular, it is difficult and rarely reported to extract chondrocytes from the small joint on the articular cartilage surface of the ankle joint.  
OBJECTIVE: To explore the effect of an improved two-step enzyme digestion method to extract chondrocytes from the ankle joint of aged mice, and to observe and evaluate their biological characteristics in vitro. 
METHODS: The ankle joints of 46-week-old mice were selected, separated and extracted sequentially by the improved two-step enzymatic digestion method in the laboratory with 0.25% pancreatin EDTA and 2g/L type II collagenase. Extracted cells were cultured in vitro and cell growth morphology was observed with an inverted microscope. After trypan blue staining, the number of primary adherent cells was calculated. Toluidine blue and type II collagen immunofluorescence staining was conducted. Chromogenic method using dimethyl methylene blue was used to determine the content of glycosaminoglycan sulfate in the primary adherent cell culture and digestive fluid. Proliferation of chondrocytes was detected using cell counting kit-8 to identify and evaluate the biological characteristics of chondrocytes.
RESULTS AND CONCLUSION: After the two-step enzymatic digestion of the articular cartilage, all cells were dissociated and released. An average of 9×105 primary adherent cells were harvested from each ankle joint of aged mice. Primary cells and the first- and second-generation cells displayed spindle and polygonal morphology. Toluidine blue staining showed blue-purple particles that were easily dyed. Type II collagen immunofluorescence staining revealed the positive expression of red fluorescence. The contents of glycosaminoglycan sulfate in the primary adherent cell culture fluid and digestive fluid were (42.41±15.00) and (65.63±10.00) mg/L, which were (0.35±3.78) and (0.21±2.56) mg/L in the negative control culture fluid and digestive fluid, respectively. The number of primary cells reached the maximum on the 6th day, and the number of the first- and second-generation cells reached the maximum on the 5th day. Meanwhile, the cell viability of the first-generation cells was greater than that of the second-generation cells and the primary cells. To conclude, the improved two-step enzymatic digestion method can successfully isolate chondrocytes from the ankle joint of aged mice. Large amounts of adherent cells with high purity and good biological characteristics can successfully synthesize and secrete type II collagen and proteoglycan. The first-generation chondrocytes have the highest viability, followed by the second-generation cells, which can be used for experimental research.

Key words: aged mice, ankle joint, chondrocyte, enzyme digestion method, cell culture