Abbreviations: rBM-MSC, rat bone marrow derived mesenchymal stem cells; CFU-F, colony forming unit-fibroblast; DPBS, dulbecco's phosphate buffer saline; DMEM, dulbecco's modified eagles medium; EDTA, ethylene diamine tetra acetic acid IntroductionIn the past decade, the field of stem cells and cell-based therapies has undergone a remarkable evolution. The potential of stem cell to differentiate into various types of cells has revolutionized their use in clinics for the treatment of a variety of clinical conditions. Mesenchymal stem cells can be cultured and grown for many generations under appropriate conditions in the laboratory and still retain a stable morphology and chromosome complement.1 Bone marrow obtained mesenchymal stem cells are the most studied stem cell type that is capable of differentiating into variety of cell lineages. Differences in differentiation ability to osteogenic chondrogenic and adipogenic lineages of MSCs harvested from Murine species of various age groups and the number of passage of these cultured cells has been reported. Osteogenic and chondrogenic potential reduced with each and every age group and adipogenic differentiation ability reduced only in cells obtained from oldest donors. 2 The technique of bone marrow collection and stem cell culture vary for different species.3 Culturing of rodent bone marrow derived stem cell is a little bit difficult when compared to its human counterpart. 4 Here we describe a simple and easy technique of stem cells isolation and differentiation from adult Wistar rats. Materials and methods Bone marrow collectionTwo male Wistar rats of 14 weeks age were used for bone marrow collection. Prior permission was obtained from Institutional Animal Ethics Committee (IAEC) for conducting animal trials. Shortly after euthanizing the animal, back side extending from lumbar region to toes were shaved and aseptically prepared for dissection ( Figure 1A). Skin incision was given on the lateral aspect of the thigh and both tibia and femur were exteriorized after removing the muscular and tendinous attachment ( Figure 1B). Femur and tibia of both the legs were placed separately in a 50ml centrifuge tube containing DPBS (Dulbecco's Phosphate Buffered Saline) and antibiotics. It was then placed under laminar hood in petridish containing DMEM (Dulbecco's Modified Eagles Media) with antibiotics. With a scissor the metaphyseal region of both bones were cut. After inserting a needle into the medullary cavity the bone marrow was flushed out using DMEM into a 15ml centrifuge tube. Flushed out bone marrow was centrifuged for 5minutes at 1000rpm in order to concentrate the cells. The supernatant was decanted and 5ml of complete media was added to the tube and mixed well by pipetting. Cell suspension was layered over equal quantity of histopaque and again subjected for centrifugation at 2500rpm for half an hour. After this density gradient centrifugation mononuclear cells were collected from the interface and washed with Ca ++ and Mg ++ free DPBS ( Figure 1C). Obtained cell ...

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