Cytogenetic research of mesenchymal stem cells from rat umbilical cord
in cultivated in vitro
Authors: Zahrychuk Oksana , Palii Ilona , Dovgalyuk Alina , Kramar S.B. , Lavrenchuk Halina 
Published: March 2024

Summary. The results of preclinical researches and clinical trials
indicate the high efficiency of the use of stem cells to restore
pathologically affected body structures. To obtain high-quality cellular
material for regenerative medicine, the important condition is to maintain
the cytogenetic stability of stem cell culture in vitro. The aim of the
study – cytogenetic analysis of the culture of umbilical cord cells of rat
embryos at different passages of cultivation and assessment of the karyotype
stability of cultivated stem cells. Materials and Methods. To obtain
mesenchymal stem cells (MSCs), the umbilical cords of WISTAR Rattus
norvegicus Berkenhout rats were used. The analysis was performed during 8
passages. Metaphase plates were obtained by a modified standard karyotyping
technique. After three hours of incubation in 10-7 M colchicine solution,
trypsinization of the material was performed. Next, the enzyme was
neutralized with conditioned medium and warm hypotonic KCl solution (0.075
M) was added to the cells. The material was fixed with acetic methanol (1:3)
on melting ice. The fixing solution was replaced three times, then the cell
suspension was dripped onto cold wet slides. The samples were stained with
Romanowski-Gimza solution. Cytogenetic analysis of MSC was performed on 30
metaphase plates at each passage. Quantitative abnormalities of the
chromosome set (aneuploidy (AP), polyploidy (PP)) were detected in the
obtained samples and the number of cells with micronuclei (MN) and mitotic
index (MI) were counted. The frequency of AP, PP and MN was calculated per
500 cells (in %). Results. In the study of MSC of the rat umbilical cord in
the first passages single disorders in the chromosomal set, such as AP and
PP were revealed. The number of cells with cytogenetic abnormalities
gradually increased with increasing duration of cultivation, but the
percentage of cells with normal karyotype did not decrease until the eighth
passage by more than 10 %. The percentage of AP in the first two passages
was increasing from 1.5 % to 1.9 %. With further cultivation to 6 passages,
the level of AP increased almost 3 times. Over the next two passages, the
number of APs has hardly changed. In the first two passages, the percentage
of PP was minimal – 1.1–1.3 %. Till the sixth passage, there was an increase
in the number of such cells by 2.8 %. When cultivated to the eighth passage,
the doubling of the number of chromosomes increased 1.1 times. At the first
passage in the culture of MSC a very small percentage of MN (≈0.2 %) was
registrated. As the passages continued, the MN number increased by 2.9 %.
The MSC's mitotic index was rising from 3.9 to 4.8 % from the first to the
fifth passage. With each subsequent passage, the mitotic potential of the
cells decreased and by the end of the analyzed cultivation period it was 3.1
%. Conclusions. Karyotyping the rat umbilical cord cells under the selected
cultivation conditions showed a slight increase in the percentage of AP and
PP. A low percentage of micronuclear cells was also observed. Therefore, in
early passages this cell line can be safely used for therapeutic purposes
for the treatment of simulated pathologies in rats