Mesenchymal stem cells (MSCs) are multipotent cells used in clinical trials for tissue regeneration, especially for treatment of locomotor disorders. Bone marrow (BM) is a typical source for isolation of MSCs. However, the available volume of bone marrow and the number of its cells are limited. In this study, we aimed to determine the effects of the cell density of the primary culture on the success of isolation of MSCs from bone marrow aspirates. For this purpose, the bone marrow samples aspirated from two horses underwent density centrifugation. Then, the buffy coat of mononuclear cells was collected, washed and cultured with different densities for 21 days as passage 0 (P0). At day 14, the number of colonies that had more than 15 cells was determined. Also, at the end of P0, the confluency was estimated and the total cell number was determined. The results showed that minimum percent of confluency, colony numbers and total cell number were obtained from samples with minimal density (1×105) and maximum numbers of colonies, total cell number and confluency belonged to the samples with the maximal cell density in primary culture (1×106/cm2). In conclusion, the number of the isolated mesenchymal stem cells is directly related to the density of the cells in primary culture i.e. a higher density yields a higher number of cells with a higher percentage of confluency. Nonetheless, even minimal cell number in primary culture (1×105/cm2) gave enough number of MSCs which can be used for either clinical applications or basic studies.

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