Approaches of Mesenchymal Stem Cell Isolation from ancient Horse Muscle for Preventing Fungi Contamination

From ancient times to modern times, various species of creatures have lived and become extinct. The ancient animals have invaluable information about evolution and extinction of species. Furthermore, Yakutsk is one of the coldest area in the world and Yakutia horses have survived in the cold climate of Siberia and they have evolved to adapt this environment. So there are many of studies about these horses in relation to their external features, metabolic mechanisms and genetic changes. For this reason, we perform stem cells isolation to revive ancient horse, which lived in Yakutia. Mesenchymal stem cells (MSCs) are adult somatic stem cells which exhibit plastic attachment. These cells are potent donor cells for somatic cell nuclear transfer (SCNT) and also have been widely studied for genomic/proteomic analysis. However, long-term preservation of ancient animal carcass in nature leads to the fungi contamination of tissues, resulting in failure of MSCs primary culture. We established a strategy to prevent fungi contamination of MSCs isolation. The ancient horse muscle tissues were cultured with different concentration of amphotericin b and evaluated for fungi contamination. The treatment of different concentration of amphotericin b showed that 2.5 ug/ml amphotericin b was the most efficient to prevent fungi contamination. Furthermore, we analyze contamination between tissue piece culture condition and single cell culture condition. This evaluation certify that the single cell culture system largely preserves the anti-fungi contamination of MSCs isolation. These results suggest a practical strategy for establishment in vitro ancient animal primary culture system along with prevention of fungi contamination.

ancient horse, mesenchymal stem cell, somatic cell nuclear transfer, long-term preservation, muscle tissue, fungi contamination, amphotericin b, single cell culture, tissue attachment culture, primary culture system

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