Extracting MSCs from Bone Marrow

(Dominik at UCD, 2010)

NOTE: Person isolating BMSC is responsible for expanding cells to P3 as described here, then either using cells for experiments or freezing them as Lab Stock. Please do not use P0, P1 or P2 cells for experiments; we do not have enough marrow donors for this.

Solutions:

• HBSS.
• 1.5-M NaCl.
• Percoll.
• 90% Percoll Stock.
• 45-mL of 100% Percoll + 5-mL 1.5-M NaCl.
• 58.1% Percoll Solution.
• 29.05-mL of 90% Percoll, 2.1-mL 1.5 NaCl + 18.85-mL water.
• The MSCGM™ BulletKit^® (PT-3001), which contains one bottle of MSCBM™ (PT-3238) and one MSCGM™ SingleQuots^® Kit (PT-4105) for about $112 for 500ml

Materials:

• Sterile 50ml tubes
• Cell strainer for 50ml tubes. (such as the one at VMCS)
• Centrifuge for 50ml tubes

Procedure:

1. Obtain sample as fresh as possible, same day is best, overnight is absolute longest.
2. Estimate volume of marrow.
3. Add 1 volume of HBSS.
4. Pipette repeatedly to break up clumps.
5. Pipette onto cell strainer placed on 50-mL tube.
6. Estimate volume of recovered cell solution and add 1 new volume of HBSS.
7. Spin at 1000-g for 5 minutes (2100-rpm on Sorvall RT-7).
8. Aspirate most of the supernatant and all of fat.
9. Loosen pellet by gentle flicking.
10. Resuspend in 2 new volumes of HBSS.
11. Mix by pipetting.
12. In fresh tube, put 10-mL of 58.1% Percoll solution (as described above).
13. VERY SLOWLY layer cell suspension onto Percoll.
1. Hold tube at 45 degree angle.
2. Do NOT allow mixing of Percoll and cell suspension.
14. GENTLY put tube in centrifuge and spin 30-minutes at 1100-g (2300-rpm on Sorvall RT-7).
1. Turn brake OFF.
2. Room temperature is best.
15. Aspirate the top of the liquid to about 10-mL above top of cell layer.
1. Cells of interest are topmost, right above Percoll.
16. With disposable transfer pipette, take off topmost cell layer, discard pellet and Percoll.
17. Add HBSS to 35-mL and centrifuge at 1000-g for 5 minutes.
18. Wash 2 to 3 times in HBSS.
19. Plate cells onto one T-150 flasks per 5ml of bone marrow, using 20-mL MSCGM media (Lonza) supplemented with 1ng/ml FGF2
1. Recommend using Flasks with Vent-Cap, since P0 cells may not be sterile.
20. After ~2 days, wash away non-adherent cells.
21. Passage cells using Accutase to release.
1. Replenish media every 2-3 days until ready to passage
2. Passage at ~80% confluent - DO NOT GROW TO CONFLUENCY!!
3. When passaging, seed 500,000 per T150 flask.
4. Freeze at P3 for general lab use, document location.

NOTE: Person isolating BMSC is responsible for expanding cells to P3 as described here, then either using cells for experiments or freezing them as Lab Stock. Please do not use P0, P1 or P2 cells for experiments; we do not have enough marrow donors for this.