Morphological peculiarites and functional activity of adipose-derived mesenchimal stem cells during in vitro cultivation conditions
The studies were conducted on 2-3-months-old males of C57BL/6 mice weighing 20–24 g. Obtaining and cultivating of adipose-derived mesenchimal stem cells (AD MSCs) were carried out in a sterile laminar box with compliance of conditions of asepsis and antiseptics. AD MSCs of the 2, 4, 7 and 12 passages were analyzed. Morphometric analysis was performed using a light microscopy. Morphometric parameters such as cell and nucleus area or nuclear-cytoplasmic ratio (NCR) were calculated using the Axiovision light microscope (Carl Zeiss, Germany) and ImageJ 1.45 software. Trypan blue dye used for investigation of the viability of MSC. The morphological characteristics of mesenchymal stem cells from adipose tissue during the process of cultivation changes: at the first passages of cultivation, the cells are spindle-shaped with two, at least three, long long cytoplasmic processes, located bipolar. Near the nucleus the Golgi complex is clearly visible – a sign of active cells. At later passages cells have a small cytoplasmic processes and the bipolar arrangement of processes changes by stellar arrangement. Golgi complex is also clearly visualized. The indicator of the nuclear-cytoplasmic ratio in MSC from adipose tissue is significantly reduced at 7 passage to 0.2189 ± 0.0122 (P < 0.01), and at 12 passage to 0.1111 ± 0.0086 (P < 0.001) compared to the 2 passage. The coefficient of proliferation of MSC from adipose tissue is significantly reduced at 12th passage. The viability of mesenchymal stem cells from adipose tissue with an increasing of a number of passages significantly reduces and at the 12th passage of cultivation reaches 84,67 ± 1,36* (P < 0.05). The content of apoptotic cells that exhibited sensitivity to serum-free significantly increased at 7 and 12 passages and was respectively 21.33 ± 1.36 (P < 0.05) and 23.67 ± 0.97% (P < 0.05).
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