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Optimization of bone marrow stromal cell culture |
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Bone marrow stromal cells (MSC) are multipotent stem cells that can be readily isolated from adult bone marrow. In recent years, MSC have emerged as promising candidates for cell therapy for CNS disorders including spinal cord injury. While the isolation methods used by different laboratories are relatively standard, protocols for culturing of MSC vary widely. It has been suggested that very low initial plating density results in increased rates of cell growth and that maintaining low-density conditions increases the proportion of multipotent cells, thus lowering the heterogeneity of MSC cultures. In this study, we used rat MSC to test the effects of different initial plating densities (20 to 2000 cells/cm2) on doubling time and cell morphology over multiple passages. We found that an initial plating density of 200 cells/cm2 resulted in the fastest doubling time over 45 population doublings. Cultures derived from all initial plating densities showed an increased proportion of large, flat cells over time. MSC sustained the potential for all three mesenchymal phenotypes through at least passage 5. Our findings suggest that the initial plating density does not critically affect MSC growth kinetics, and that careful expansion of these cells, even at higher initial plating densities, results in rapidly growing cultures that retain the ability to differentiate in vitro to cartilage, bone, and fat.
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Micrographs show examples of typical immature and mature MSC morphology. Immature MSC are generally small, spindle-shaped cells, whereas mature MSC are large cells with a flat, polygonal morphology.
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Growth kinetics are graphed as cumulative population doublings over multiple passages (MSC were passaged at 50% confluency, each point on the graph represents a passage). MSC plated at 200 cells/cm2 have the fastest doubling time over 45 population doublings. |
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The number of mature cells (red) increased over time in culture (passage 10) compared to the number of immature cells (blue). This increase was independent of initial plating density. |
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MSC were assayed in vitro for adipogenesis, osteogenesis and chondrogenesis. MSC formed lipid vesicles (red, A) when treated with adipogenic supplements. MSC deposited calcium phosphate in an osteo assay (black, B). The micromass pellet obtained after chondro assay was positive for Type II collagen (C) and anionic proteoglycans (D). Histogram shows the amounts of calcium deposition over multiple passages. |
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