正常兔膝关节软骨细胞体外培养体系的建立和鉴定

Objective:Cultivate normal chondrocytes in vitro culture system and provide chondrocyte for the subsequent experience.Methods:1.Operation under sterile conditions,obtain the full-thickness cartilage from bilateral knee femoral condyle and tibial plateau of rabbits,gather the chondrocytes from digested cartilage by trypsin and type II collagenase and culture and passaged in DMEM low glucose medium containing 10% FBS. Identify the second generation chondrocytes by toluidine blue, safranin O, type II collagen staining and use MTT estimate cell proliferation.Results: After 12 hours, primary culture cells began adherent, all cells were adherent at 24 hours, the number of cells were increased after 48 hours and the cells appeared irregular polygonal shape. There were a large number of cell colonies after 3-4 days proliferated rapidly. The cells covered the bottom and fusion after 5 days. When the cells fusion was up to 80% after 6-7 days, primary culture cells were subcultured. After passage of three generation,the cells began hypertrophy and the growth slows down. The second generation chondrocytes were stained positive by toluidine blue, safranin O and type II collagen staining. In the first four days,MTT assay cells began to enter the logarithmic growth phase, the first eight days into the plateau phase.Conclusion:1. The method of Improved enzymatic digestion can be successfully cultured and passaged chondrocytes. Identification the cultured chondrocytes by staining were positive. In the first four days , chondrocytes come into the logarithmic growth phase. After passaged to second generation,the chondrocytes begin to differentiate.