Primary source gender and cell line confirmations

Murine Femur/Tibia Bone Marrow Isolation for Osteoblast, Osteoclast, and Adipocyte Culture

Rosen Lab, MaineHealth Institute for Research – Edited by EGE 12/01/21

Bone Marrow Isolation 

Materials

-Mice (n = 3 littermates) around 8 weeks of age   -70% EtOH 

-Sterile scissors & tweezers-Sterile chilled PBS

-1mL syringes & 25G needles        -Sterile petri dishes

-1.5mL microcentrifuge tubes      -15 & 50mL conical tubes             

-Kimwipes                      -70µm cell strainers            

-Trypan Blue & Hemocytometer      -Culture media: αMEM+10%FBS/1% Pen-Strep

Limb Collection (chemistry hood)

1. Sac animals via CO₂ followed by cervical dislocation – spray thoroughly with 70% EtOH
2. Pinching skin of lower back, make cut with scissors
3. Peel back skin to widen incision completely around body, then pull down off limbs to feet
4. Cut just below ankle joint to remove feet and attached skin
5. Place index finger at back of ileac crest and apply pressure to front with thumb to dislocate hip
6. Cut through muscle around exposed femoral head to free limb, place in sterile cold PBS
    

Marrow Collection (tissue culture hood)

1. Spray down hood surfaces and all equipment with 70% EtOH
2. Manually remove muscle from limbs with kimwipe, place bones in petri dish with sterile PBS
3. Prepare microcentrifuge tubes with 200µL culture media and trimmed 200µL pipette tip
4. Isolate femur and tibia by cutting below femoral head, above/below knee, and below ankle
5. Place 2 femur and 2 tibia per microcentrifuge tube tip insert, packed in securely
6. Perform quick spin (15s @ 13Krpm) to remove marrow from the femur and tibia
7. Remove bones from pipette tip with tweezers, flush remaining marrow from tip with pipette
8. Combine and transfer all marrow samples in group into 15mL conical tube
9. Add 10mL culture media, pipet to homogenize, filter via 70µm cell strainer into 50mL conical
10. Add 20mL culture media and plate into one T150 or two T75 flasks
     

Bone Cell Differentiation 

After 48hrs of culture, flasks now contain adherent MSCs (osteoblast & adipocyte progenitors) on surface and non-adherent HSCs (osteoclast progenitors) in culture media
 

Osteoclast Culture

RANKL StockM-CSF Stock

10µg RANKL (PeproTech 310-01)             10µg m-CSF (PeproTech 315-02)

+400µl sterile water                                  +400uL sterile water

 Orbital shake on ice for 30 min              Orbital shake on ice for 30 min

 Aliquot 50µl, store at -80°C                    Aliquot 25µl, store at -80°C

Osteoclast Differentiation Media (OCL-DM)

Per 1mL culture media (pre-filtered):

+4µl RANKL

+1.2µl M-CSF

Make desired volume & use fresh, or store @ 4°C for up to a week

1. Collect culture media with non-adherent cells from bone marrow flasks, spin down (1.5Krpm for 10min), resuspend in 1mL OCL-DM, count, and plate in osteoclast media at 1.56x10⁵ cells/cm²
   • 96-well plate: 50K cell/well
   • 12-well plate: 600K cell/well
2. Culture in OCL-DM, observing for first appearance of fused cells within 3-5 days
3. Widespread fusion of large osteoclasts should occur by 7-10 days
4. Wash wells with PBS and fix in 10% formalin, then stain for mature osteoclasts with TRAP+ kit
5. Image wells, counting blinded for stain+ cells with >3 nuclei to quantify mature osteoclasts/well
    

Osteoblast Culture

Ascorbic Acid Stock       β-glycerol Phosphate Stock    Dexamethasone Stock (1mM)

50mg asc. acid (Sigma A4544)    2.16g β-gp (Sigma G9422)  3.9mg dexamethasone

+2mL sterile dH₂O                       +10mL PBS                         +10mL 100% EtOH

Make fresh, protect from light      Sterile filter, store @ 4°C     Store @ -20°C

Osteoblast Differentiation Media (OBL DM)

88mL αMEM

+10mL FBS

+1mL Pen-Strep

+800µL β-glycerol phosphate

+200µL Ascorbic acid

+100µL 1mM Dexamethasone stock (for 1µM final)

Sterile filter, store @ 4°C up to one week

1. After removing cell culture media from bone marrow flask for osteoclast culture or discarding feed flask and culture additional 48hr for higher confluency if large number of cells needed
2. When flasks achieve desired confluency, wash with PBS gently to not disturb attached cells
3. Remove PBS and apply 0.25% Trypsin EDTA (see chart on fridge for volume/flask size) for 5 min at 37°C
4. Quench trypsin with equal volume culture media, spin @ 1.5Krpm for 10min, resuspend in 1mL culture media
5. Plate 1x10⁶ cells/well in 6-well plate in culture media
6. Culture until confluent (~48-72hr), then switch to OBL-DM
7. Culture in OBL-DM, observing for dense cuboidal osteoblasts (~2 weeks) and formation of dark mineralized nodules (~3 weeks)
8. Terminate culture ~ Day 21 upon observation of widespread mineralization, or earlier if cell layer peeling necessitates (Dexamethasone should help prevent this)
9. Stain cells for qualitative assessment of mature osteoblasts (ALP) and mineralization (Von Kossa)
    

Adipocyte Culture

IBMX Stock (62.5mM)                       Dexamethasone Stock (1mM)

9.5mg 3-Isobytyl-1-methylxathine (IBMX, Sigma I7018)   3.9mg dexamethasone (Sigma D-4902)

+684µL 70% EtOH                                                              +10mL 100% EtOH

Insulin Stock (10mg/mL)       Rosiglitazone Primary Stock (20mM)

10mg insulin (Sigma I6634)      5mg rosiglitazone (Cayman Chemical 717410) +700µL DMSO

+1mL 0.01N HCl                        Secondary (1mM): 5µL Primary + 95µL DMSO

Aliquot 25µL, store @ -20°C up to a year  Store @ -20°C up to 2 years

Note: Rosiglitazone for BMSC cultures only, not 3T3L1

Adipocyte Base Media

High Glucose (4.5 g/L) DMEM +10%FBS/1% Pen-Strep

1. Following adherent bone marrow cell culture and trypsinization in osteoblast protocol, plate 1x10⁶ cells/well in 6-well plate in culture media and culture until confluence (~48-72hr)

2. When confluent, switch media and culture in Adipocyte Differentiation Media (ADP-DM) as follows:

Days 0, 2

25mL Base Media

+200µL IBMX (0.5mM final)

+25µL Dexamethasone (1µM final)                                   

+25µL Insulin (10µg/mL final)

+25µL Rosiglitazone (1µM final)

Day 4

25mL Base Media

+25µL Insulin (10µg/mL final)

+25µL Rosiglitazone (1µM final)

Day 7

25mL Base Media

+25µL Insulin (10µg/mL final)

3. Observe for adipocytes with lipid droplets after ~1 week, remove media (do not wash) and fix cells with 10% formalin, then stain for qualitative assessment of adipocyte/lipid formation (Oil Red O)