Functional activity and morphological peculiarities of mesenchimal stem cells during in vitro cultivation conditions

Abstract

The studies were conducted on 2-3-months-old males of C57BL/6 mice weighing 20-24 g. Obtaining and cultivating of mesenchimal stem cells (MSCs) were carried out in a sterile laminar box with compliance of conditions of asepsis and antiseptics. 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 features of cells during cultivation changes: at first cells have a spindle-like shape with two long cytoplasmic processes, located bipolar. In later passages, cells have a significant number of cytoplasm processes, bipolar arrangement of processes changes to stellar. The NCR index of MSC significant decreases at the 4 passage by 12,9 % (p ≤ 0,05), at the 7 passage - by 35,3 % (p <0,001), at the 12 passage - by 76,6 % (p <0,001) compared to the initial state. The proliferative activity of the MSC of the bone marrow during cultivation significantly dereases at the later passages. Cell resistance to apoptosis induced by cultivation in the serum-free medium is fairly high. The number of cells in the state of apoptosis was 14,0±1,74 at the 4 passage and was reliably increased at the 12 passage to 22,67±1,55 % (p ≤ 0,05) during cultivation.