Bulletin "Veterinary biotechnology"

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Veterynarna biotehnologija – Veterinary biotechnology, 2018, 33, 45-54 [in Ukrainian]. https://doi.org/10.31073/vet_biotech33-06

KOVPAK V.V., e-mail: This email address is being protected from spambots. You need JavaScript enabled to view it., MAZURKEVICH A.I., e-mail: This email address is being protected from spambots. You need JavaScript enabled to view it., KOVPAK O.S., e-mail: This email address is being protected from spambots. You need JavaScript enabled to view it.

National University of Life and Environmental Sciences of Ukraine

TARASOV O.A., e-mail: This email address is being protected from spambots. You need JavaScript enabled to view it.

Institute of Veterinary Medicine of the NAAS

INFLUENCE OF CRYOPRESERVATION ON THE GENETIC STABILITY OF THE CAT STEM CELLS IN DEPENDENCE ON THEIR SOURCE

Introduction. An important issue of the cell therapy is the long-term preservation of the cell material. Currently, cryopreservation is used for this purpose. In the process of the cell material freezing, there are two main problems of its safe use: morphological and chromosomal abnormalities. This in turn leads to a change in the functional activity of the cells such as proliferative activity, differentiation and, as a consequence, ability to survive. In the process of freezing, and especially unfreezing, the important role is played by formation of ice crystals and gas bubbles both intra- and extracellularly, which in turn induces anomalous segregation of chromosomes by destroying the spindle microtubules. Slow freezing of the cell material prevents formation of intracellular ice (which can cause rupture of the cell membrane), but such cryopreservation method can lead to cell dehydration by formation of extracellular ice. However, the combined use of slow freezing and cryoprotectants reduces a significant part of these disorders and has a stabilizing effect on microtubules. Over the past 40 years, various types of cryoprotectants have been used, but dimethylsulfoxide (DMSO) yields the best results for today. However, in spite of the DMSO toxicity, additional components that contain cryoprotective properties are often introduced into the medium, most often these are protein compounds (for our experience we used fetal calf serum). In view of the foregoing, the purpose of our research was to study the effect of dimethylsulfoxide (DMSO) on the genetic stability of the cat stem cells, depending on their source.

Materials and methods. During the experiment, we used fat tissue, bone marrow and pancreas of cats to obtain cell cultures. The material was obtained in parallel, during planned surgical operations. All manipulations with animals were carried out with the prior consent of their owners and in compliance with the requirements of the Law of Ukraine "On the Protection of Animals from Cruel Treatment" (Article 230 dated on 2006). During cryopreservation, cells (concentration 1×106 cells/cm3) were diluted with a cryopreservation medium (10% DMSO + 90% FBS) and subjected to slow freezing. Before further studies, the samples were stored during at least 1 month at -196 °C. Cytogenetic analysis was performed on 50 metaphase plates of each group (n = 3) according to a standard procedure. The studies were carried out on cells of passage IV.

Results of research and discussion. In culture of the bone marrow stem cells the number of cells with altered karyotype increased to 36.0±1.3%, which is in 1.7 times (p<0.001) more than in the control group (without cryopreservation). Whereas in culture of the fat tissue stem cells this indicator increased in 1.5 times (p<0.05) in comparison with the control and was 22.7±1.3%. Culture of the pancreas proved to be the most resistant to the toxic effect of DMSO, in the experimental group of cups the number of cells with altered karyotype increased in 1.4 times (p <0.01) as compared to the control group and was 26.7±0.9%.

Conclusions and prospects for further research: 1. According to the results of cytogenetic analysis, it was established that in the culture of the bone marrow stem cells in the process of cryopreservation-defrosting, the percentage of cells with altered karyotype (in comparison with the control) increases in 1.7 times. 2. In the culture of the fat tissue stem cells, this indicator increased in 1.5 times (in comparison with the control). 3. The culture of the pancreas stem cells turned out to be the most resistant to the effects of cryopreservation, the number of cells with a changed number of chromosomes increased in 1.4 times in comparison with the control. The obtained data on the differences in the effects of cryopreservation on culture of stem cell bone marrow, fat tissue and pancreas will be used to establish biosafety of these cultures.

Keywords: cryopreservation, culture of the bone marrow stem cells, culture of the fat tissue stem cells, culture of the pancreas stem cells, genetic stability, cat.

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