BIOLOGICAL PECULIARITIES OF ADIPOSE TISSUE-DERIVED MESENCHYMAL STEM CELLS AT DIFFERENT PASSAGES OF CULTIVATION

Abstract

The studies were conducted on 2–3-months-old males of C57BL/6 mice weighing 20–24 g. Obtaining and operating with adipose tissue-derived mesenchymal stem cell (MSC) culture was performed in a sterile laminar box under conditions of asepsis and antiseptics. The adipose tissue-derived MSC 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 were calculated using the Axiovision light microscope (Carl Zeiss, Germany) and Image J 1.45 software. Trypan blue dye used for investigation of the viability of MSC. The morphological characteristics of adipose tissue-derived MSC during the process of cultivation changes: at the first passages of cultivation, the cells are spindle-shaped with two, at least three, long cytoplasmic processes, which are 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 the 7th passage to 0.2189 ± 0.0122 (P < 0.01), and at the 12th passage to 0.1111 ± 0.0086 (P < 0.001) compared to the 2nd passage. The coefficient of proliferation of adipose tissue-derived MSC is significantly reduced at 12th passage. The viability of MSC 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 cultivation significantly increased at the 7th and 12th passages and was 21.33 ± 1.36 (P < 0.05) and 23.67 ± 0.97% (P < 0.05), respectively.