I am trying to isolate chondrocytes from articular cartilage and studied the isolation protocols from different papers. Almost all papers have common method of isolation but I am unable to get cells in the filtrate. needs your suggestions.
Hi Muhammad,
I am glad to help you because I was also dealing with optimization of chondrocytes isolation from knee or hip joints. Here's my protocol:
After cartilage fragmentation into 1 mm3 pieces and wash with cold PBS without Ca2+ and Mg2+, the digenstion with collagenase I (1.5 mg/ml) and collagenase II (1.5 mg/ml) was performed for 16h at 37C and 5% CO2 (in cell incubator). Afterwards cell suspension was collected above the cartilage leftovers, the enzymes were inactivated with DMEM 10% FBS with pen/strep and spinned at 1800 rpm for 8 minutes. Cell pellet was resuspended in 3 ml PBS without Ca2+ and Mg2+ but with pen/strep and then filtered with 70 uM nylon mesh. After centrifugation in the same conditions as before cells were resuspended in cell culture medium, counted and seeded at denisty 10^4/ cm2.
In my opition crutial steps are:
a) cutting cartilage in as small pieces as it is possible
b) digestion on Petri dish in order to provide good penetration of digestion enzymes
c) after overnight digestion making sure that you have visible cell suspension above cartilage pieces, if not incubate it longer or exchange your digestion mix (without disposal of the previous one)
d) when you have cell suspension and want to filtrate it through the mesh the suspension density is very importatnt - maybe it is too high, try dilution
Wish you luck!
Thanks Katarzyna Dziedzic
I have also incubated for 16 hrs and got complete digest in the form of suspension but during filtration the debris also comes with filtrate which is not allowing cells to grow. how to get rid of this debris?
Hi,
please, may you upload your protocol? So we can see what might goes wrong...
Lukas Kucera here is my protocol.
Cut the cartilage into 2-3 mm pieces and washed with PBS pH 7.4.
the minced cartilage pieces were digested with 1 mg/ml pronase at 37 C for 1 hr .
After that the these pieces were digested with 1 mg/ml collagenase type I at 37 C for 5-15 hrs.
After complete digestion the suspension was filtered with 70 micron strainer and the filtrate was centrifuged at 1500 - 2000 rpm for 5 and 7 min respectively.
the pellets were washed with PBS and grown on DMEM.
Note. FBS and Pen/Strep were aded to DMEM at 10% and 1% conc respectively.
Hi Muhammad,
thanks for your protocol. I have few notes to this.
First, you can try collagenase type II as this type of collagen is prominent in cartilage. As it is enzyme, the activity varies from batch to batch, from vendor to vendor. Also the activity of collagenase is dependent on the concentration of calcium ions. Once I dissolved the collagenase in DMEM/F12 instead of F12 media (higher conc. of calcium) and after overnight incubation the cartilage was completely dissolved and chondrocytes were dead. So you need to check your process frequently. The first enzyme (pronase, dispase, etc..) is not so crucial and you can release chondrocytes even without it, however it increases the yield of cells generally. Use the shaker during the digestion and place tissue pieces into Petri dish or tissue culture flask, not in the tube. Please, see the protocol from my paper below.
Good luck!
Lukas
Cartilage pieces were digested in 4 mg/ml pronase E solution in F12 media (Gibco) for 1 hour at 37˚C and then in collagenase type II solution (1 mg/ml in F12 media, Gibco) for 14 hours at 37°C. Released cells were separated from undigested pieces of cartilage with a 70 µm nylon mesh cell strainer (BD Biosciences, USA) and the cell suspension was washed two times with phosphate buffered saline (PBS) before seeding. Cells were grown in DMEM/F12 media with Glutamax (Gibco, USA) supplemented with 10% fetal bovine serum, 1% Insulin-transferrin-sodium selenite supplement (Gibco, USA), and 1% Antibiotics-Antimycotics (Gibco, USA) in 75 cm2 tissue culture flasks (TPP, Switzerland)
Hi, Muhammad Samie Lukas Kucera
May I know
1. The concentration of collagenase II used. I am using 100 Units/ml and unable to get filtrate.
2. How much volume of collagenase II to be added for digestion with respect to the amount of tissue used.
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