Chondrocyte Isolation

Dominik Haudenschild, August 3 2010 at UC Davis

NOTE: Person isolating chondrocytes is responsible for expanding cells to P2 as described here, then either using cells for experiments or freezing them as Lab Stock. Please do not use P0 or P1 cells for experiments; we do not have enough cartilage donors for this.

1. Wash Cartilage or Osteochondral piece 3-8 times in HBSS with Pen/Strep/Fungizone

2. Cut Cartilage from bone using sterile scalpel

3. Pin Cartilage pieces onto petri dish using syringe-needle, and mince cartilage into cubes about 1mm

4. Trypsinize with 10% Trypsin for 10-15 minutes at 37 degrees

a. This is to remove any fibroblasts or non-chondrocytes

b. Use 1 part Trypsin and 9 parts DMEM (Sigma T-4549, which is 25g/L porcine Trypsin)

5. Remove Trypsin and Fibroblasts, digest with Collagenase

a. Make 2mg/ml Type IV Collagenase in 5-10% calf serum in DMEM + antibiotics

b. Use 2 volumes collagenase for 1 volume minced cartilage

c. Digest on shaker at 180rpm 37 degrees, in 50ml conical tube, nearly horizontal

d. Digest until no more cartilage clumps, (Max - o/n for older donors, 6-8hrs for younger)

e. Cartilage Pieces should be mostly digested

6. Pellet cells, wash once with DMEM, resuspend cells in DMEM and count

7. Plate cells at 3,000 (max 6,000) cells/cm², corresponding to ~500,000 cells (1x10⁶ max) per large flask.

a. Use T-150 screw-cap flasks with vented cap for P0 human cells, as some may be infected

b. Plate cells 30 minutes to 3 hours in 0% serum, to promote rapid cell attachment

8. Add DMEM/F12 complete with 10% calf serum and 25µg/ml ascorbic acid, 1ng/ml FGF2 optional

9. Change media every 2-4 days until nearly confluent

10. Passage cells to P1 at 1:4 to 1:6 using 2ml Trypsin (Sigma T4549)

a. Seed cells at 3,000cells/cm² (max 6000)

11. Freeze cells at P2 for lab stock

a. Freeze cells in DMEM/F12 + 20% Serum + 10% DMSO

Sources:

· Type  IV collagenase                                      Sigma C-5138

· Iron-Supplemented Calf Serum                Atlanta Biologicals S11950

· DMEM/F12                                                    HyClone SH30023.01

· Ascorbic Acid                                                 Sigma A-8960

· 10x Trypsin                                                    Sigma T-4549