Biological peculiarities of adipose tissue-derived mesenchymal stem cells at different passages of cultivation

Authors

  • L. V. Kladnytska National University of Life and Environmental Sciences of Ukraine, Kyiv, Ukraine, 03041, Heroev oborony, 15
  • A. Y. Mazurkevych National University of Life and Environmental Sciences of Ukraine, Kyiv, Ukraine, 03041, Heroev oborony, 15
  • S. V. Velychko Hospital of Veterinary Medicine, Нolosiivskyi Avenue, 105 b, Kyiv 03127, Ukraine
  • L. V. Garmanchuk National Taras Shevchenko University, Educational and Scientific Center "Institute of Biology and Medicine, Kyiv 01033, Ukraine
  • М. О. Maluk National University of Life and Environmental Sciences of Ukraine, Kyiv, Ukraine, 03041, Heroev oborony, 15
  • Т. A. Mazurkevych National University of Life and Environmental Sciences of Ukraine, Kyiv, Ukraine, 03041, Heroev oborony, 15
  • V. V. Kovpak National University of Life and Environmental Sciences of Ukraine, Kyiv, Ukraine, 03041, Heroev oborony, 15
  • T. V. Kozytska Bogomolets National Medical University, Medical Faculty No 3, Department Of Histology And Embryology, Shevchenko blvr., 13, 01601, Kyiv, Ukraine, Kyiv, Ukraine.
  • Yu. O. Kharkevych National University of Life and Environmental Sciences of Ukraine, Kyiv, Ukraine, 03041, Heroev oborony, 15
  • R. R. Bokotko National University of Life and Environmental Sciences of Ukraine, Kyiv, Ukraine, 03041, Heroev oborony, 15
  • T. L. Savchuk National University of Life and Environmental Sciences of Ukraine, Kyiv, Ukraine, 03041, Heroev oborony, 15

Abstract

The studies were conducted on 2-3-months-old males of C57BL/6 mice weighing 20-24 g. Obtaining and working with mesenchymal stem cells of adipose tissue culture (AD MSCs) was performed in a sterile laminar box under 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 Image J 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).

Key words: adipose tissue mesenchymal stem cells, nucleus, nuclear-cytoplasmic ratio, coefficient of proliferation, viability, apoptosis, early passages, late passages

 

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Published

2021-12-04

Issue

Vol. 12 No. 4 (2021)

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Article

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