Jacobs: Isolation of Primary Bone Cells

1. Kristen Lee and An Nguyen
2. 2/10/11
3. Primary Osteoblast/Osteocyte Isolation Protocol

Materials:

1. Sterile beaker
2. 4x 50ml conical tubes
3. Ice bucket and ice
4. Sterile forceps, scalpels, blue underpads
5. Filter .45 micron Steriflip
6. Alpha-MEM, FBS, CS, Pen-Strep
   • Collagenase type 2 (Worthington Biochemical Corp (4176, 40E11914)
7. DMEM
8. .05% Trypin/EDTA 4mM Solution
9. Shaking water bath at 37C
10. Sterile 50ml (small but wide) bottles
11. Day 8 neonatal mice

Medium Prep Before Isolation

1. For osteocyte culture: Use normal MLO-Y4 media, alpha-MEM, 1% PS, 5% CS, 5% FBS
2. For osteoblasts culture: Use normal MC3T3 media, alpha-MEM, 1% PS, 10% FBS
3. 20 ml DMEM with 1% PS in a sterile beaker/petri dish
4. Digestion Media
5. 1. Add collagenase type 2 at 2 mg/ml to 25 mL DMEM containing 1% PS
6. 2. Mix and filter and transfer to a 50 ml tube for cell digestion

Dissection and Isolation

1. 1) Spray head with 70% ethanol
2. 2) Dissect calvariae from 10-20 6 to 9-day old mice
3. 3) Clean periostium from bones and transfer bones to Beaker/Petri dish with DMEM with 1% PS
4. 4) Bring petri dishes to the cell culture hood
5. 5) Mince bones with scissors into small fragments (1 x 1 mm)
6. 6) Transfer to a sterile bottle with Digestion media (5-10 mL are needed depending on how many calvariae)
7. 7) Place bottle in shaking water bath for 20 minutes
8. 8) Collect media. Wash bone fragments with 5-10mL of Osteoblast media. This collection of media= fraction 1.
9. 9) Repeat again= fraction 2. Discard fraction 1 and 2.
10. 10) Repeat Step 7-8 but SAVE Digestion media. Collect and filter media and wash=fraction 3 and place in a 50 ml conical tube. Spin down cells at F3 for 5 min at 500rpm to form a pellet.
11. 11) Resuspend pellet in Osteoblast media and transfer to a TC dish.
12. 12) Repeat Steps 9-10 for F4-F6.
13. 13) Incubate bone fragments in 5-20 mL Trpsin/EDTA for 20 minutes. Repeat step 7-11 for F7.
14. a. Place dish in incubator for 30 minutes.
15. b. Remove from incubator. Gently pipette up media, as osteoblasts should have adhered while osteocytes remain unattached. (Not many osteoblasts will remain, but you can use them)
16. c. Spin down the osteocyte pellet and replate. Seed “osteocytes” on collagen-coated TC dishes with Osteocyte media.
17. 14) Repeat Steps 6-11 for F8-9.
18. a. Place dish in incubator for 30 minutes.
19. b. Remove from incubator. Gently pipette up media, as osteoblasts should have adhered while osteocytes remain unattached. (Not many osteoblasts will remain, but you can use them)
20. c. Spin down the osteocyte pellet and replate. Seed “osteocytes” on collagen-coated TC dishes with Osteocyte media.
21. 15) Cut up any remaining bone fragments as much as possible. Seed bone fragments on collagen-coated TC dishes with Osteocyte media = Bone crawl.

Notes:

1. The 30 minute lifting method was used on F3-F6 previously, but it did not seem to yield many osteocytes. Additionally, earlier fractions will have a high percentage of osteoblasts.

1. F3-F6 can be combined for a total osteoblast population.

1. The bone crawl method will result in very packed colonies of osteocytes exiting the tissue. We have added a small amount of Trypsin/EDTA to break up these cells and kept them in culture for a more homogenous layer of cells.

1. Do not let osteocytes in culture pass 60% confluency, do not let osteoblasts in culture pass 80% confluency.

1. Long bones and Calvariae can be used for maximum yield…but long bones will take longer to digest unless you cut them up very well. Make sure you flush out bone marrow first.

References

1. Zhou

1. Calvaria:
2. Tanaka et al. (1995)
3. Henley (1987)
4. Bakker
5. Klein-Nulend and Bonewald (2006)
6. Long bones:
7. Gu (2006)