Isolation of Cartilage Progenitor Cells from Human Articular Cartilage

Isolation of Cartilage Progenitor Cells from Human Articular Cartilage

Reagents and materials:
1. The uninvolved part of the knee joint hemisection in patients with osteoarthritis;
2. Pronase digestion medium: DMEM/F12 (1:1), 5% FBS, ascorbic acid 50μg/ml, glucose 1mg/ml, gentamicin (50mg/ml), 0.2% (final concentration 100μg/ ml), Pronase 70U/ml, HEPES 10mmol/L;
3. Collagenase digestion medium: DMEM/F12 (1:1), 5% FBS, ascorbic acid 50μg/ml, glucose 1mg/ml, gentamicin (50mg/ml), 0.2% (final concentration 100μg/ml ), Type I collagenase 300U/ml, HEPES 10mmol/L;
4. Trypsin/EDTA: Trypsin (0.05%) and EDTA (0.53mmol/L) PBSA preparation;
5. PBSC: Dulbecco’s phosphate buffer containing 1mmol/L CaCl2, 1mmol/L MgCl2;
6. PBSA: Dulbecco’s phosphate buffer without Ca2+, Mg2+;
7. Fetal bovine serum;
8. Bovine serum albumin (BSA), 10μg/ml and 1mg/ml, prepared with PBSC;
9. Serum fibronectin, 10μg/ml, prepared with PBSC;
10. Nylon membrane, 40μm mesh;
11. Ordinary utensils, 30ml, or centrifuge tube 50ml, with a conical bottom to facilitate cell centrifugation;
12. Scalpel blade, size 10;
13. Scalpel handle, size 3;
14. Tweezers, 2 pairs (1 large and 1 small);
15. Mineral oil gel (Vaseline);
16. Cloning cup, 6mm;
17. Chain mail gloves for separating cartilage;
experimental method:
1. Tissue collection and cell separation;
(1) Under aseptic conditions, use a No. 10 scalpel blade to carefully dissect the cartilage from under the subchondral bone. Place the cartilage sheet in a 9cm Petri dish containing PBSA;
(2) Use tweezers and a blade to carefully cut the cartilage into small pieces of 1mm3;
(3) Transfer the small pieces of cartilage to ordinary utensils or centrifuge tubes containing 18ml streptin digestion medium;
(4) Place it in a tumbling machine at 37°C for 1h;
(5) Aspirate and discard the digestive juice;
(6) Add 18ml of collagenase digestion medium and place it on the tumbling machine again at 37°C for 1h;
(7) 40μm cell strainer filter;
(8) Use a blood cell counter to count cells;
(9) Add adhesion assay medium, centrifuge at 620g for 10 min to wash, and resuspend cells (4000 cells) with adhesion medium at a density of 2000 cells/ml;
2. Differential adhesion measurement;
(1) Coat a 3.5cm Petri dish with 10μg/ml fibronectin at 4°C overnight. The negative control uses 10μg/ml BSA;
(2) Aspirate the liquid, and seal the petri dish with PBSC containing 1mg/ml BSA for 30 minutes;
(3) Add 2ml of culture medium to resuspend the cell suspension and let it stand for 20 minutes;
(4) Aspirate the medium and non-adherent cells and place them in the second petri dish for 20 minutes;
(5) Add 2ml of growth medium to the first petri dish and place it in the incubator;
(6) Aspirate the medium and non-adherent cells from the second petri dish to the third petri dish;
(7) Add 2ml of the growth technique to the second petri dish and place it in the incubator;
(8) Add 200μl FBS to the last petri dish and place it in the incubator;
(9) After 3 hours, use a phase contrast microscope to count the initially adherent cells;
(10) It usually takes 10 days to culture the cells until a colony of more than 32 cells is visible;
3. Colony expansion;
(1) Use a phase-contrast microscope to identify cell colonies with more than 32 cells, and mark them in a circle with a marker under the petri dish;
(2) Count and record the number of cells in each colony;
(3) Aspirate the medium from the Petri dish and rinse with PBSA;
(4) Apply petroleum jelly on the cloning ring aseptically, or use sterile tweezers to directly immerse it in petroleum jelly;
(5) Place the cloning ring on the colony, add trypsin/EDTA solution to the ring, and let it stand for 5-10 minutes-observe that the cells have fallen off with a phase contrast microscope;
(6) Resuspend the cells, place them in an Eppendorf tube, and centrifuge at 300g for 5 minutes;
(7) Wash the growth medium and repeat the above centrifugation;
(8) Dilute the growth medium, and inoculate 1 colony per well in a 12-well plate;
(9) When the cells are confluent, they are inoculated into a 3.5cm petri dish (or 6-well plate), and then transferred to a larger culture flask.