The cell cycle features of canine adipose-derived mesenchymal stem cells on different passages of cultivation


  • L.V. Kladnytska National University of Life and Environmental Sciences of Ukraine
Keywords: mesenchymal stem cells, adipose tissue, dog, cultivation, cell cycle, phases, aneuploids, diploid, proliferative pool

Abstract

The features of the cell cycle of culture of adipose-derived mesenchymal stem cells from the for different cultivating passages were studied. Mesenchymal stem cells were obtained from the adipose tissue of the dog under a laminar flow hood by an explant method in our modification. Cell cultivation was carried out at 37 °C, 100% moisture and 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% antibiotic-antimycotic. The culture medium was changed 2–3 times per week and the cells were selected by their capacity to attach to the flask surface. When culture flasks became 80% confluence, cells were detached with 0.25% trypsin containing 1 mmol/L EDTA and subsequently replayed at a concentration of 104 cells/cm2 for next passaging. A cells culture of adipose derived mesenchymal stem cells was obtained on the 2nd, 7th and 12th passages. The method of flow cytometry determined the level of aneuploid cells and the distribution in the cell cycle phases. The morphology of cells of different passages was studied using an inverted microscope Axiovert 40. It was investigated that the culture of mesenchymal stem cells from adipose tissue in the 2nd passage contains a significant number of the proliferative pool (S + G2/M) cells and it was 29.51 ± 3.56% of the total number of diploid cells. The number of aneuploid cells was 1.55 ± 0.43%. All cells had fibroblast-like morphology. It was established that in the middle passages (7th) in the culture of mesenchymal stem cells from the adipose tissue of the dog no significant changes were found in the distribution of cells in the phases of the cell cycle. The number of diploid cells of the proliferative pool S + G2/M and the G0/G1 pre-synthetic period remains unchanged. The level of aneuploidy increases only within the tendency. Morphologically, cells had fibroblast-like form. It was determined on 12th passage of cultivation, a significant decrease in the number of cells of the proliferative pool (S+G2/M), which was 18.93 ± 0.66% of the total number of diploid cells compared to the 2nd passage. The number of aneuploid cells increased and it was 3.49 ± 0.38%. Morphologically, separate cells had processes. The indicator of the effect of cells cultivation on the content of diploid cells of the proliferative pool (S+G2/M) in culture is ɳ2x = 70% (P < 0.05). So, first characteristic properties of the aging of the culture of canine adipose-derived mesenchymal stem cells appear on the 12th passage of cultivation.

Downloads

Download data is not yet available.

References

Marx, C., Silveira, M.D., Selbach, I., da Silva, A.S., Braga, L.M., Camassola, M., Nardi, N.B. (2014). Acupoint injection of autologous stromal vascular fraction and allogeneic adipose-derived stem cells to treat hip dysplasia in dogs. Stem Cells. 2014:391274.

Haghighat, A., Akhavan, A., Hashemi-Beni, B., Deihimi, P., Yadegari, A. (2011). Adipose derived stem cells for treatment of mandibular bone defects: An autologous study in dogs. Dent. Res. J. 8(1), 1–7.

Volk, S., Theoret, C. (2013). Translating stem cell therapies: the role of companion animals in regenerative medicine. Wound Repair Regen. 3, 382–394.

Kathrine, K.J., Christian, G., Jensen, D.H., Fischer-Nielsen, A., Hjuler, T., von Buchwald, C. (2017). Mesenchymal stem cell therapy for laryngotracheal stenosis: A systematic review of preclinical studies. PLOS ONE 21 Sep 2017 https://doi.org/10.1371/journal.pone.0185283

Arnhold, S., Wenisch, S. (2015). Adipose tissue derived mesenchymal stem cells for musculoskeletal repair in veterinary medicine. Am. J. Stem Cells. 4(1), 1–12.

Reich, C., Raabe, O., Wenisch, S., Bridger, P., Kramer, M., Arnhold, S. (2012). Isolation, culture and chondrogenic differentiation of canine adipose tissue- and bone marrow-derived mesenchymal stem cells a comparative study. Vet. Res. Commun. 2, 139–148.

Dmitrieva, R.I., Minullina, I.R., Bilibina, A.A., Tarasova, O.V., Anisimov, S.V., Zaritskey, A.Y. (2011). Bone marrow- and subcutaneous adipose tissue-derived mesenchymal stem cells: Differences and similarities. Cell Cycle. 11(2), 377–383.

Zhang, Z.X., Guan, L.X., Zhang, K., Wang, S., Cao, P.C., Wang, Y.H., Wang, Z., Dai, L.J. (2007). Cytogenetic analysis of human bone marrow-derived mesenchymal stem cells passaged in vitro. Cell Biol. Int. 31(6), 64, 5–8.

Kladnytska, L.V., Mazurkevych, A.Y., Velychko, S.V., Zhyhunova, O.V. (2016). Otrymannia kultury stovburovykh klityn iz zhyrovoi tkanyny sobaky. Visnyk Sumskoho natsionalnoho ahrarnoho universytetu. Seriia «Veterynarna medytsyna». 6(38), 19–24 (in Ukrainian).

Kladnytska, L.V., Mazurkevych, A.I., Velychko, S.V. (2016). Patent Ukrainy na korysnu model №109148.Sposib otrymannia mezenkhimalnykh stovburovykh klityn iz zhyrovoi tkanyny sobaky; zaiavnyk i vlasnyk Natsionalnyi universytet bioresursiv i pryrodokorystuvannia Ukrainy. № u201602329; zaiavl. 11.03.2016; opubl. 10.08.2016, Biul. № 15 (in Ukrainian).

Nicoletti, I., Migliorati, G., Pagliacci, M.C. (1991). A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. J. Immunol. Methods. 139(2), 271–280.

Abstract views: 102
PDF Downloads: 135
Published
2017-04-03
How to Cite
Kladnytska, L. (2017). The cell cycle features of canine adipose-derived mesenchymal stem cells on different passages of cultivation. Scientific Messenger of LNU of Veterinary Medicine and Biotechnologies. Series: Veterinary Sciences, 19(78), 36-40. https://doi.org/10.15421/nvlvet7808