Equine bone marrow-derived mesenchymal stem cells: optimization of cell density in primary culture.

Background: The primary cell seeding density of bone marrow-derived mononuclear cells (BM-MNCs) affects several cellular behaviors, including attachment to the culture dish, proliferation, and differentiation.

Methods: The aim of this study was to determine the best density of equine BM-MNCs in primary culture (P0) for obtaining the maximum bone marrow-derived mesenchymal stem cell (BM-MSC) yields at the end of P0. Bone marrow samples of two healthy mares were aspirated. The MNCs were isolated and cultured at different densities (1×105 , 2×105 , 4×105 , 8×105 , and 1×106 cells/cm2 ). Within the 7th and 14th days after seeding, the colonies containing more than 15 cells were counted and the percentage of confluency and the number of cells were calculated on day 21.

Results: The lowest density of MNCs was associated with the least number of colonies, number of adherent cells, and confluency percentage, whereas the highest density was associated with the maximum number of colonies and confluency percentage (P<0.05). However, the maximum number of cells at the end of P0 was associated with the intermediate (4×105 cells/cm2 ) and the highest concentration (P<0.05).

Conclusions: The maximum number of MSCs at the end of P0 was obtained at the densities of 1×106 and, especially, at 4×105 cells/cm2 .