Abstract:

BACKGROUND: In recent years, there are numerous studies on bone marrow mesenchymal stem cells (BMSCs) from small animals such as rats and rabbits, but reports addressing BMSCs from large animals, such as sheep, are rare.
OBJECTIVE: To observe the effect of two isolated methods on the biological activity of sheep BMSCs.
METHODS: Fresh bone marrow of 10 mL was extracted from the posterior superior iliac spine of a healthy Altay sheep aged two months by puncture following anesthesia, and BMSCs were obtained by the whole bone marrow culture method and density gradient centrifugation method. The shape and adherent growth of primary BMSCs was observed under inverted fluorescence microscope. Primary culture time in two groups was recorded, and the CD44 antigen expression was identified by flow cytometry. Cell growth curve was measured by MTT assay. The potential of osteogenic differentiation was examined by Von Kossa’s staining.
RESULTS AND CONCLUSION: Using both of these two separation methods, more uniform spindle, triangle and polygon BMSCs could be harvested, the nuclei were big and cytoplasm was rich, which were positive for CD44 antigen. The whole marrow culture was better than the density gradient centrifugation in cells quantity, slightly early in culture time. Von Kossa staining was positive in the two groups after BMSCs were cultured for 14-21 days. The adherent growth of passaged BMSCs obtained by using the whole bone marrow culture method was faster than that using the density gradient centrifugation culture method, and the cell vitality was also better in the former. It is indicated that the whole marrow culture is an effective method of extraction.