Isolation of Bone Marrow and Stromal Cells from Intact Bone

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Media:

DMEM + 10% IFS + 50ug/ml L-ascorbic acid

(Pen, Strp, Fung)

  1. Keep bone in serum free media or PBS at 4oC until use. (Can hold up to 24hrs after harvest).
  2. Transfer the bone pieces to a clean Petri dish containing 2-3ml PBS and cut the bone into 3-5mm pieces.
  3. Transfer the bone pieces to a 50ml conical (polypropylene tube) containing 15-20ml PBS.
  4. Vortex tube vigorously for 10 seconds and then leave to stand for 30 seconds to allow bone fragments to settle.
  5. Carefully decant the supernatant to a fresh tube. This contains the haematopietic tissue and dislodged cells.
  6. Repeat steps 4 & 5 2 more times for a minimum of 3 washes or until no remaining haematopoitietic marrow is visible and the bone fragments have assumed a white ivory-like appearance.
  7. Culture the washed bone fragments in media at a density of 200mg-600mg of tissue/ 80cm2 flask.
  8. Centrifuge the tubes containing the marrow for 5min and freeze in freeze media.