· EXTRACTION OF CHONDROCYTES
1. The extracted rabbit knee is further accessed by removing the connective tissues adhesions with 11-blade till transparent white flexible material joining hind limb bones are visible (Figure 1).
2. Excise thin slices of articular cartilage using the sample blade (Figure 2).
² Gently cut the superficial part of articular cartilage and avoid excavating it deeper or otherwise your sample will be contaminated with other cells along with chondrocytes.
² The excised slices should be not too small nor too big as it affects the chondrocytes survival. Small slices are readily degraded upon collagenase treatment causing harm to chondrocytes integrity whereas too big slices take considerable amount of time to melt during collagenase treatment.
3. Excised slices of articular cartilages are then placed on the dish containing phosphate-buffered saline (PBS) containing 2% penicillin and streptomycin.
4. Dish containing cartilages slices are then transferred to the falcon tube containing 20 ml of PBS for washing. The process is repeated thrice to ensure complete cleaning and decontamination of articular cartilages.
5. With the help of pipette, remove the supernatant and add 1 % of collagenase diluted in TESCA buffer of about 800 µl in the same tube along with 1.6 ml of Dulbecco’s Modified Eagle Medium (DMEM) containing penicillin (P) and streptomycin (S) (Figure 3).
6. Then the samples are then incubated at 37 oC-5% CO2 in the incubation chamber for 6-8 hours.
² Gently mix your samples every hour to avoid cell clumping to extract as many chondrocytes as possible. Do not tightly close the lid of the tube to allow the entrance of air inside the tube.
7. After incubation, add 3 ml of DMEM containing penicillin and streptomycin in the samples and centrifuge it at 1000 RPM for 10 seconds.
8. The supernatant is transferred to another falcon tube and the pellet is discarded and fresh media of 3 ml (DMEM+P.S) is added on to the pellet.
² The process is repeated thrice to remove any undigested cartilaginous debris and other chemicals from the samples to obtain fresh and viable cells from the media.
9. The supernatant obtained from previous steps are resubjected to centrifugation at 1000 RPM for 10 mins.
10. This time, chondrocytes will accumulate in the pellet and the supernatant will contain remaining debris which should be discarded.
· CALCULATION OF LIVE CHONDROCYTES AND CELL CULTURE
1. Fresh media containing (DMEM+P.S+Calf serum) is then added on to the pellet to stimulate the growth and prevent death of chondrocytes.
² The suspended pellet immersed in DMEM+P.S+Calf serum is then gently tapped at the bottom to disintegrate chondrocytes adhesions with one another and to obtain individual cells.
2. Afterwards, 10 µl of sample is taken from the sample (DMEM+P.S+Calf serum) with the aid of pipette containing chondrocytes and then added to the diluted 0.25 % trypan solution (90 µl) prepared in microfuge tube.
3. Addition of samples in trypan blue solution is gently mixed with the help of pipette and then flow cytometry should be performed to estimate the concentration of live cells.
² The calculation of live cells provides scientists to prepare the media accordingly for cell propagation and prevent cell death and loss of cells phenotype from happening.
4. Cell seeding is then performed by preparing fresh DMEM+P.S+Calf serum media and transferring 1 ml from the previous media containing extracted chondrocytes on to the fresh media.
5. The propagated cells are then incubated at 37 oC with 5% CO2 in incubation chamber for 12 hours. Afterwards, the spent media is then discarded via a sucker and fresh media is added through pipette again to allow an exponential growth of chondrocytes for 2 days to reach the confluence level up to 70-80 %.
· TREATMENT OF CELLS
1. Following the third day, the chondrocytes are exposed with dedifferentiating agent (2-deoxy-D-glucose) in dose and time dependent manner.
· CONFIRMATION OF DEDIFFERENTIATION
1. The status of dedifferentiation can be evaluated by Type II collagen through western blotting.
2. Alcian Blue staining can also be performed to check the proteoglycan levels. Reduction in the expression of these proteins indicate that chondrocytes are dedifferentiating and the vice versa.