INVESTIGATION OF ENZYMATIC ACTIVITY IN HUMAN DERMAL FIBROBLASTS DURING VARIOUS CULTIVATION PERIODS
Journal: Biotechnologia Acta (Vol.17, No. 2)Publication Date: 2024-04-30
Authors : A.A. TERESHCHENKO O.K. VORONINA Y.M. KHMELNYTSKA D.M. PYKHTIEIEV;
Page : 78-80
Keywords : human dermal fibroblasts; senescence; cell proliferation; metabolic activity.;
Abstract
Aim. Research into the change in enzymatic indicators of cell activity during the aging of human dermal fibroblasts in culture from 3 to 15 passages to determine the most optimal terms for cell transplantation to patients for further cell therapy. Methods: Dermal fibroblasts of donors A, B, and C aged from 40 to 60 years were cultivated in сulture medium containing FBS, antibiotics, bFGF. To determine the cell cycle, cells were fixed with formalin, stained with propidium iodide/RNase buffer and analyzed on a flow cytometer. To study the enzymatic activity of mitochondria, fibroblasts were seeded in microplate, MTT was added, followed by DMSO and glycine. To determine the activity of lysosomal enzymes, fibroblasts were fixed with formalin, stained with X-Gal and photographed on a microscope. A statistical analysis of the results was carried out. Results: Dermal fibroblasts retain their mitotic activity from early to late passages. The average percentage of mitotic cells was higher than the average value of the cells at the interphase. Optical density did not reveal significant changes with the change in the cultivation term. The increase in formazan level corresponds to a percentage of cells in mitosis. Studying microphotographs of early and late passages to detect cells with enhanced β-galactosidase secretion, no signs of aging of dermal fibroblasts of donors were noticed. Conclusions: Using various cytochemical methods, it has been proven that the culture of human dermal fibroblasts from donors of the age group from 40 to 60 years maintains stability during their cultivation from 3 to 15 passages.
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