The development and identification of animal osteoblasts from bone marrow culture

This study was aimed to evaluate a modified method for animal osteoblast marrow culture on the basis of previously published method. The marrow mononuclear cells(MMNC) in femoral bone-marrow of SD rat were gained with Ficoll-Hapaque centrifuge, which were then cultured in 5 x 10(5) cells/cm3 on the 96-well culture plates with RPMI 1640 medium containing 30% horse serum in a humidified atmosphere of 5% carbon dioxide at 37 degree Celsius. The medium was changed every three days. Cultured cells were identified with inverted microscopy for observing the shape of living cells, and with Wright-Geimsa mixture stain and Alkaline phosphatase (ALP) stain. In order to avoid false positive reaction, we caused damage to the ALP in cultured cells and stained the cells with ALP again for comparison. The results of inverted microscopy showed that the small spherical mononuclear cells became polygonal, spindle-shaped or squamous successively from the fifth day onward. Wright-Geimsa staining showed the specific property of osteoblast. ALP stain positive reaction was between "2+" and "3+", then ALP stain negative reaction was noticed after the damage to ALP. These data demonstrated that MMNC in SD rat could become osteoblast in RPMI 1640 medium containing 30% horse serum. The modified osteoblast marrow culture method has the advantages of short time, less risk of contamination and higher efficiency.