Hi Maruf,

I've had success isolating costal chondrocytes from neonatal mice. If you're using older animals, the whole thing is more difficult, but the following protocol should give you a reference point at least.

1) Dissect out your cartilage, and clear away as much excess tissue as you can.

2) Wash in PBS w/o Mg/Ca

3) Digest with standard tissue culture trypsin with moderate shaking for 20-30 minutes @ 37°C. (This is to get rid of all connective tissue and muscle)

4) Replace with fresh trypsin, and shake again for 20-30 minutes (the sample might be a bit goopy, so take care with this replacement)

5) Manually dissect away any remaining tissue and transfer to a fresh tube

6) Wash in fresh α-MEM

7) Digest tissue in 0.5% hyaluronidase for 5-10 minutes @ RT X2 (this opens up the collagen for efficient digestion)

8) Wash in fresh α-MEM

9) Digest in 0.2% collagenase I diluted in α-MEM for 30 minutes with shaking @ 37°C (Mg/Ca are really important here)

10) Collect the released cells and add fresh collagenase I for another 30 minute digestion. (you can do this a third time if you think it's necessary, and for longer if the cartilage is quite thick. 12 hours seems like way to long to me though.)

11) Pass all of the collected cells through a 70 μm sieve into a fresh 50ml conical tube.

12) Count your cells, spin @ 200G for 10 min, and resuspend @ ~150K/ml in maintenance media (DMEM:F12 @ 1:1, 0.2 μg/ml Na-Selenite, 10 μg/ml Transferrin, 20 mM L-glutamate, 10% FBS, 50 μg/ml ascorbic acid, antibiotics)

13) Plate the cells at this concentration

14) Allow the cells to become heavily confluent over the next 4 days or so, changing media every other day

15) Begin differentiation by reducing serum to 1% and including 10 μg/ml insulin and 10 mM β-glycerol phosphate.

Personally, I think you're better off seeding the cells into 96 well round bottom plates with an ultra low attachment coating, and getting nice spheroids going, but it'll depend on your downstream application.

I hope this helps!

-Steve

Thanx to Steve for your reply and suggestions full of important informations

Maruf, twelve hours is a lot of time.i do cell culture on pork knees (articular cartilage to isolate chondrocytes) which takes 1:30 hour digestion in collagenase,i used to work on cow knees which took 3 hours digestion.so it depends on the animal and morbility for knee samples.

digesting with trypsin might not be the best thing to do, since it will degrade many receptors and surface proteins from the chondrocytes. thus, it will cause changes in gene transcription via stress pathways. Use collagenase type II, since collagen type II is the main structural component of at least the joint cartilage. we see a minimum of 8 hours of digestion is necessary to release more than 75% of cells. do it overnight to get 95% of cells. 1.5mg/mL of the enzyme should do it. sterile filter it in the medium you are using, next day, strain the digested material through a 100 um screen (from BD faclcon), then dilute with fresh buffer, spin down cells, resuspend and count.

Oh, we do overnight digest on the bovine articular cartilage, and we start with 5-10 grams of tissue, so you should scale it down, and possibly it will take less time as well.

Thanks to Derek Rosenzweig, i like your though and i think it is the best way to isolate Chondrocytes.

I too, i'm trying to isolate it from 1 month old rats