Method of chondrocytes isolation from hyaline cartilage

Abstract

The aim of this study is isolation of chondrocytes from articular hyaline cartilage and their expansion in cell cultures for further transplantation in a cartilage defect. Materials and methods: The study was performed on 9 New Zealand White rabbit 6 months old. Under sterile conditions, slices of hyaline cartilage were harvested from unbearing area of knee joint, followed by 0,25% tripsină-EDTA treatment for 30 min and 0,6% collagenase for 6 hours. The cells were cultivated in cell culture flasks by 10000 ±500 cell/cm2 and incubated at 37 ° C with 5% CO2 in DMEM with 10% FBS. The cells were expanded in culture up to 21 days to a confluence of 80%. The cells was counted by a hemocytometer. The chondrocytes were stained with Safranin O and toluidine blue/fast green. Results: From approximately 50±10 mg of cartilage were isolated 4x105 ±5x104 cells. At staining chondrocytes with Safranin O, the nuclei were black, the cytoplasm gray-green and and cartilage, mucin were orange to red. At staining chondrocytes with toluidine blue/fast green, the nuclei appeared dark blue, the cartilage blue, deep purple and background green. Conclusion The method of chondrocytes isolation from hyaline cartilage is efficient and it was confirmed by in vitro cell staining with Safranin O and toluidine blue/fast green. Our further purpose is implantation in vitro of expanded chondrocytes on tridimensional scaffold and their transplantation in an osteochondral defect.