A scheme outlining the culture technique is shown in Fig. 1.
1. Transfer tissue, removed at surgery or biopsy, into a sterile container with PBS or serum-free medium (SFM) for transport to the laboratory with minimal delay, preferably on the same day (see Note 6). An excellent source is the upper femur of patients undergoing total hip replacement surgery for osteoarthritis. Cancel-lous bone that would otherwise be discarded is removed from this site prior to the insertion of the femoral prosthesis. The tissue obtained is remote from the hip joint itself, and thus from the site of pathology, and is free of contaminating soft tissue (see Note 7).
2. Remove soft connective tissue from the outer surfaces of the bone by scraping with a sterile scalpel blade.
3 Rinse the tissue in sterile PBS and transfer to a sterile Petri dish containing a small volume of PBS (5-20 mL, depending on the size of the specimen). If the bone sample is a femoral head, remove cancellous bone directly from the open end using sterile bone rongeurs or a solid stainless steel blade with integral handle. Disposable scalpel blades may shatter during this process. With some bone samples (e.g., rib), it may be necessary to gain access to the cancellous bone by breaking through the cortex with the aid of the sterile surgical bone rongeurs.
4. Transfer the cancellous bone fragments to a clean Petri dish containing 2-3 mL of PBS and dice into pieces 3-5 mm in diameter. This can be achieved in two stages using a scalpel blade first, and then fine scissors.
5. Decant the PBS and transfer the bone chips to a sterile 30-mL "universal container" with 15-20 mL of PBS.
6. Vortex-mix the tube vigorously three times for 10 sec and then leave to stand for 30 sec to allow the bone fragments to settle. Carefully decant off the supernatant containing hematopoietic tissue and dislodged cells, add an additional 15-20 mL of PBS, and vortex-mix the bone fragments as before. Repeat this process a minimum of three times, or until no remaining hematopoietic marrow is visible and the bone fragments have assumed a white, ivory-like appearance.
7. Culture the washed bone fragments as explants at a density of 0.2-0.6 g of tissue/ 100-mm diameter Petri dish or 75-cm2 flask (see Note 4) in 10 mL of medium at 37° in a humidified atmosphere of 95% air, 5% CO2.
8. Leave the cultures undisturbed for 7 d, after which time replace the medium with an equal volume of fresh medium taking care not to dislodge the explants.
9. Check for outgrowth of cells at 7-10 d (see Note 8).
10. Replace the medium at 14 d and twice weekly thereafter until the desired cell density has been attained.
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