Stock solutions preparation
1. Dissolve COL H in 1.5 ml sterile H2O and make 3 aliquots of 500 μl (each aliquot is the Solution A) and store at -20°C 2. Dissolve the Thermolysin in 300 μl sterile H2O and make 3 aliquots of 100 μl (each aliquot is the Solution B) and store at -20°C.
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Isolation of chondrocytes from 1 gr cartilage
DIGESTION SOLUTION FOR 1 gr OF CARTILAGE: Solubilize one aliquot of Solution A in 10 ml DMEM and put it on ice. Immediately before use add one aliquot Solution B.
1. Transfer 1 gr. of cartilage shavings into a 150-mm petri dish containing DMEM+ 1% Pen-Strep + 1% Fungizone solution.
2. Mince the cartilage shavings into small chips (0.5 to 1 mm) using two sterile stainless steel scalpels, and transfer them into petri dish.
3. Add to the chips the digestion solution + 1% Pen-Strep +1% Fungizone and digest in incubator for 12- 24 hrs at 37°C, 5% CO2.
4. Filter the collagenase/chips solution from step 4 through a 20-μm nylon filter membrane: dissociated chondrocytes will pass through the filter. Divide the cell into two 50 ml tube and wash with 40 ml DMEM plus 10% FCS.
5. Centrifuge for 10 min at 250g r.t. to obtain a pellet containing chondrocytes. Aspirate the supernatant and wash the cells twice in DMEM plus 10% FCS.
6. Add 2 ml medium and suspend cells by pipetting up and down. Count cells.
7. Plate the chondrocytes in cell culture plate for ~ 24 hrs to let primary chondrocytes completely attach. Then add an equivalent volume of medium, and refresh after 1 day.
