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 nuclearcytoplasmic 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

Keywords

• adipose tissue mesenchymal stem cells
• nucleus
• nuclear-cytoplasmic ratio
• coefficient of proliferation
• viability
• apoptosis
• early passages
• late passages