Rapid isolation of muscle-derived stem cells by discontinuous Percoll density gradient centrifugation.

We investigated relationship between the maturity and density of muscle cells and developed a rapid isolation method to acquire stem cells from skeletal muscle. Mononuclear cells were isolated from the lower hind-limb muscles of 7-d-old male Sprague-Dawley rats and separated by Percoll density gradient centrifugation. After centrifugation, the cells were layered in the interfaces between each Percoll density layer. Flow cytometry was used to investigate the Sca-1, Pax7, CD34, CD45, M-cadherin, and myosin expression of the cells in each density layer. We found that CD45-positive cells were not present in freshly isolated muscle cells. CD34-, Pax7-positive cells were mainly observed at the interface between the 15% and 25% Percoll layers and had a density of 1.0235-1.0355 g/ml. Cells positive for M-cadherin were at the 25-35% Percoll density interface and had a density of 1.0355-1.0492 g/ml. We conclude that because there appears to be a correlation between maturity and density, muscle-derived stem cells may be isolated successfully from the 15-25% Percoll interface.