Minimum Essential Medium Eagle

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	the trabecular bone was cut into small pieces and thoroughly washed with commercial standardized Joklik’s modified MEM serum-free medium, to remove no adherent marrow cells. The pieces were incubated with rotation at 37 °C for 30 min with the same medium containing 0.5 mg/ml type IV collagenase, and collagenase digestion was stopped by the addition of Iscove’s modified medium containing 10% fetal bovine serum (FBS). Between eight and ten pieces from each patient were then placed in 25 cm2 flasks and cultured in IMDM containing 10% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, 50 U/ml mycostatin, and 0.25 µg/ml amphotericin B until confluence; the culture medium was changed every 2–3 days.

Protocol tips

the trabecular bone was cut into small pieces and thoroughly washed with commercial standardized Joklik’s modified MEM serum-free medium, to remove no adherent marrow cells. The pieces were incubated with rotation at 37 °C for 30 min with the same medium containing 0.5 mg/ml type IV collagenase, and collagenase digestion was stopped by the addition of Iscove’s modified medium containing 10% fetal bovine serum (FBS). Between eight and ten pieces from each patient were then placed in 25 cm2 flasks and cultured in IMDM containing 10% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, 50 U/ml mycostatin, and 0.25 µg/ml amphotericin B until confluence; the culture medium was changed every 2–3 days.

Publication protocol

Human bone cell cultures were established by means of a modified version of the Gehron-Robey and Termine procedure [27] using trabecular bone samples obtained from waste material of female patients during orthopedic surgery for degenerative diseases or traumatic fractures of the femoral neck requiring osteotomy. None of the patients (aged 71–82 year) had any malignant bone diseases and all of them gave their written consent for the use of the waste material. The protocol was approved by the Institutional Ethical Committee (Protocol BMU-WNT, 25.03.2008; amendment 1, 29.9.2012). Briefly, the trabecular bone was cut into small pieces and thoroughly washed with commercial standardized Joklik’s modified MEM serum-free medium, to remove no adherent marrow cells. The pieces were incubated with rotation at 37 °C for 30 min with the same medium containing 0.5 mg/ml type IV collagenase, and collagenase digestion was stopped by the addition of Iscove’s modified medium containing 10% fetal bovine serum (FBS). Between eight and ten pieces from each patient were then placed in 25 cm2 flasks and cultured in IMDM containing 10% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, 50 U/ml mycostatin, and 0.25 µg/ml amphotericin B until confluence; the culture medium was changed every 2–3 days. The cell population was tested for alkaline phosphatase (ALP) and osteocalcin (BGP) production after 1,25(OH)2D3 10−8 M to ensure that the cells were endowed with osteoblast characteristics. ALP and BGP were measured by means of a multianalyzer COBAS (Roche Diagnostics SpA, Monza, Italy). Cells were used at first passage to reduce the possibility of phenotype changes.

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