Osteoclast precursor culture and differentiation

Materials
Dissection

• Clean scissors
• Clean forceps
• 70% Ethanol
• Dissecting board (cork, Styrofoam, etc.)
• Push pins
• Absorptive disposable cloth (like bench liner)
• 10mL syringe (Fisher Scientific – 14-823-2A)
• 25-guage needle (Fisher Scientific – 14-821-13D)

Culture

• α-MEM
  • Stericup 0.22-micron PVDF filter with 1L bottle (Millipore – SCGVU11RE)
  • 1 jar Minimum Essential Medium powder (Sigma-Aldrich –  M0894-10X1L)
  • 880mL deionized water
  • 10mL 200mM L-glutamine (ThermoFisher – 25030081).
  • 10mL 10,000IU Penicillin/10,000μg Streptomycin (ThermoFisher – 15140122)
  • 1.98g Sodium Bicarbonate (Fisher Scientific – BP328-500)
  • Add 100mL heat inactivated fetal bovine serum
  • Mix and sterile filter into a new bottle
• Freezing medium
  • 90% α-MEM
  • 10% Dimethyl sulfoxide (Fisher Scientific – BP231-100)

• Phosphate Buffered Saline without Ca⁺⁺ and Mg⁺⁺ (ThermoFisher – 14190235)
• 100mm tissue culture-treated dishes (Denville Scientific – T1110)
• 60mm untreated suspension culture dishes (Fisher Scientific – 08-772-31)
• 24-well tissue culture plates (Denville Scientific – T1024)
• 50mL and 15mL tubes
• 5mL and 10mL serological pipettes
• 37^oC water bath
• ACK lysing buffer (ThermoFisher – A1049201)
• Recombinant mouse Monocyte/Macrophage Colony-Stimulating Factor (MCSF) (Biolegend – 576406)
• Recombinant mouse Receptor Activator of Nuclear Factor κB Ligand (RANKL) (Biolegend – 769404)
• Accutase cell dissociation reagent (ThermoFisher – A1110501)
• Trypan blue

Method
Day 1

1. Sacrifice a mouse (C57Bl/6 work well) via CO₂ inhalation followed by cervical dislocation
2. Using clean technique, dissect out and separate the tibiae and femora.
3. Cut the ends off femora and tibiae to facilitate flushing
4. Working in the hood, fill a 10mL syringe with 10mL α-MEM.
5. Pick up a bone with forceps and insert syringe needle into one end.
6. Flush ~2.5mL α-MEM through the bone into a 15mL tube. Note: you’ll know you’ve flushed out the marrow if the bone changes from a reddish color to a more white color.
7. Repeat the flushing procedure for remaining bones adding all marrow flushes from each mouse into a single tube.
8. Using a 10mL serological pipette, transfer medium with marrow cells into a clean 15mL tube. Take care not to transfer any debris from the bone that may have settled to the bottom of the tube.
9. Pellet cells at 300RCF for 5 minutes.
10. Aspirate medium and resuspend pellet in 500μL ACK lysing buffer and incubate at 37^oC for 2 minutes to lyse red blood cells.
11. Add 10mL PBS to cells and pellet at 300RCF for 5 minutes.
12. Resuspend pellet in 10mL α-MEM and transfer to a 100mm tissue culture-treated dish.
13. Incubate plate in a 37^oC humidified incubator at 5% CO₂ This will allow osteoblasts, stroma, fibroblasts, and all other adherent cells that may be in the flush to attach to the plate.

Day 2

1. Transfer non-adherent cells to a 15mL tube.
2. Add α-MEM to a volume of 10mL.
3. Add MCSF to a final concentration of 25ng/mL.
4. Deposit cells to 100mm petri dish (non-culture treated, non-pyrogenic).
5. Incubate dishes in a 37^oC humidified incubator at 5% CO₂ overnight.

Day 3

1. Refresh medium on cells.

Day 5

1. When plates are 75% confluent, wash with PBS, and add 1mL Accutase. It usually takes ~5min at room temperature for Accutase to weaken the attachment enough to lift the cells.
2. Add 4mL α-MEM to cells and use gentle pipette motion to lift. Move cells to a 15mL tube.
3. Make a 2-fold dilution of cells (15μL cells + 15μL trypan blue) and count cells using a hemocytometer.
4. To make osteoclasts in the following plates use these numbers of macrophages (~26000 cells per cm² of culture area):
   • 96-well culture treated plate – 10 x 10³ (seed in 200μL)
   • 24-well culture treated plate – 5 x 10⁴ (seed in 1mL)
   • 35mm culture treated dish – 2.5 x 10⁵ (seed in 2mL)
   • 60mm culture treated dish – 7.4 x 10⁵ (seed in 3mL)
   • 100mm culture treated dish – 2 x 10⁶ (seed in 10mL)
5. Prepare a sufficient volume of cells for seeding and add MCSF to a final concentration of 25ng/mL and RANKL to a concentration between 6.25 and 25ng/mL.
   • In our experience, 25ng/mL RANKL produces maximal osteoclast differentiation. If you expect your experimental group to demonstrate enhanced differentiation, use a lower concentration.
6. Alternatively, cells can be seeded on bone or dentine slices. Use the same cell density of the culture vessel the slice is kept in (usually a 24-well plate)
7. Incubate dishes in a 37^oC humidified incubator at 5% CO₂ for 2 days.

Day 6

1. Refresh the medium on the cells – α-MEM w/ 25ng/mL M-CSF + 6.25-25ng/mL RANKL
2. Late in the afternoon, you may see some multinuclear cells.

Day 7

1. Osteoclastogenesis should be complete in the AM
2. Osteoclasts don’t live long in culture, so watch your cells closely.
3. Osteoclasts on bone slices usually take 1 more day to mature than those on culture plastic.
4. Osteoclasts can be identified using a TRAP stain (sample staining protocol).

Mature osteoclasts stained for Tartrate-Resistant Acid Phosphatase (TRAP) activity.