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.
Keywords: mesenchimal stem cells, morphometric analysis,
viability, apoptosis
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