Isolation, ex vivo expansion and characterization of rabbit bone marrow derived mesenchymal stem cells (rBM-MSCs)

The study was conducted on 32 clinically healthy New Zealand White (six to seven months old) rabbits of either sex, weighing 1.4–2.2 kg. Five ml bone marrow aspirate was collected from the iliac crests of each animal. Mono-nucleated cells were separated using Ficoll-Paque plus density gradient and cultured in Dulbecco's Modified Eagle's Medium - low glucose (DMEM-LG). A total of 62.5% BMMSCs culture showed excellent to good quality growth rate. Cells started to adhere to tissue culture flask surface on third day and begin to change morphology from round to spindle shape on day five to seven. After seven to ten days, the homogenous spindle shape cells formed 4–6 small colonies in the tissue culture flasks. These cells showed 80–90% confluency after 14 days. Second passage mesenchymal stem cells showed high alkaline phosphatase activity and express osteopontin gene. The cells were grown in osteogenic differentiation medium for 28 days. The osteoblastic differentiation was confirmed by the presence of mineralized extracellular matrix by staining with von Kossa technique. It was concluded that rabbit bone marrow derived mesenchymal stem cells express alkaline phosphatase activity and osteopontin gene and calcium deposition following osteoblastic differentiation.