Cell and Organ Transplantology. 2016; 4(1):66-69.
Cytogenetic analysis of rat pancreatic cell cultures at early passages
Kovpak V. V., Kovpak O. S.
National University of Life and Environmental Sciences of Ukraine, Kyiv, Ukraine
Cell culture obtained from the pancreas can serve as a source of physiologically competent substitute for primary islets of Langerhans in the treatment of diabetes. However, it is possible to obtain the required number of cells only at long-term cultivation in vitro. Therefore, it is necessary to investigate the risks of neoplastic transformation of cells in vitro before transplantation.
Materials and methods. Cell culture was obtained by explant method from pancreas of 12-day-old rats. Cell cultures of the first to sixth passages were used for the cytogenetic analysis. In this study the number of cells with altered karyotype, cells with micronuclei, binucleated cells and the cells in a state of apoptosis were considered, mitotic index was calculated.
Results. Aneuploid cells were noted at all passages in an amount of 2.2 % (1st) to 16.6 % (4th). Polyploidy manifested in a population of cells from the second (1.1 %) to the sixth passage (4.4 %) with a maximum at passage four (7.8 %). A significant increase in their number was observed since the second passage (0.3 %). We have seen a significant increase in the number of binucleated cells from the first (0.1 %) to the sixth passage (0.8 %). During the study there was a decrease in mitotic index from the first (2.7 %), to the third passage (1.5 %) and its gradual increase in fourth (1.7 %) and sixth (2.0 %) passages. In addition, there was discovered a small percentage of cells in apoptosis, their number gradually increased to the 4th passage (0.5 %). The 5th-6th passages showed decrease in the number of apoptotic cells to 0.1 %.
Conclusion. There have been revealed changes in the rat pancreatic cells culture as aneuploidies, polyploidies and micronuclei, the intensity of which varied depending on the passage. However, karyotype variability of mentioned cell did not exceed the level of spontaneous mutations characteristic of mammalians.
Keywords: pancreatic cells culture, cytogenetic analysis, micronucleus testFull Text PDF
|1. Ametov AS. Perspektivy razvitiya diabetologii [Prospects of Development of diabetology]. Terapevticheskiy arkhiv – Therapeutic archive. 2005; 10: 5-9 [in Russian].|
|2. Bersenev AV. Izuchenie spontannoy onkogeneticheskoy transformatsii mezenkhimal’nykh stvolovykh kletok cheloveka v kul’ture [The study of spontaneous oncogenetic transformation of human mesenchymal stem cells in culture]. Kletochnaya transplantologiya i tkanevaya inzheneriya – Cellular Transplantation and Tissue Engineering. 2005; 1: 14–16 [in Russian].|
|3. Kolmakova TS, Belik SN, Morgul EV, et al. Ispol’zovanie mikroyadernogo testa dlya otsenki effektivnosti lecheniya allergii u detey: metodicheskie rekomendatsii [Using the micronucleus test to assess the effectiveness of the treatment of allergies in children: guidelines]. Rostov na Donu: Izd-vo RostGMU. – Rostov-on-Don: Publisher RostGMU, 2013. 31 p. [in Russian].|
|4. Kovaleva OA. Tsitogeneticheskie anomalii v somaticheskikh kletkakh mlekopitayushchikh [Cytogenetic abnormalities in somatic mammalian cells]. Tsitologiya i genetika – Cytology and Genetics. 2008; 1: 58-72 [in Russian].|
|5. Mazurkevich AY, Malyuk MO, Starodub LF. Karіotipoviy analіz mezenkhіmal’nikh stovburovikh klіtin kіstkovogo mozku krolіv za rіznikh metodіv disotsіatsії klіtinnogo monosharu na rannіkh pasazhakh kul’tivuvannya in vitro [Karyotype analysis of mesenchymal bone marrow stem cells of rabbits in various methods of dissociation of cell monolayer at early passages of cultivation in vitro]. Naukoviy vіsnik NUBіP Ukraїni Serіya: Veterinarna meditsina, yakіst’ і bezpeka produktsії tvarinnitstva – Scientific Journal NULES of Ukraine. Series: Veterinary Medicine, quality and safety of animal products, 2015. Issue 227 [in Ukranian].|
|6. Mazurkevich AY, Kovpak VV, Danilov VB. Klіtinnі tekhnologії u veterinarnіy meditsinі [Cellular technology in veterinary medicine]. Kiїv: Komprint – Кyiv: Komprint, 2014. 132 p. [in Ukranian].|
|7. Kovaljova O, Kobozeva N, Burdo E, et al. Mikrojadernyj test kak metod opredelenija sezonnoj izmenchivosti citogeneticheskih pokazatelej u mlekopetajushhih [Micronucleus test as a method of determination of seasonal variability of cytogenetic indices in mammals]. Rarіtetna terіofauna ta її ohorona. Pracі Terіologіchnoї shkoli, Lugans’k. – Rare Theriofauna and its protection. Proceedings of Theriological school, Lugansk. 2008; 9: 266-269 [in Russian].|
|8. Mushkambarov NN, Kuznetsov SL. Molekulyarnaya biologiya [Molecular biology]. Moskva, «Meditsinskoe informatsionnoe agentstvo» – Moscow, «Medical News Agency», 2013. 544 p. [in Russian].|
|9. Tron’ko ND, Paster IP. Dostizheniya regenerativnoy meditsiny v terapii sakharnogo diabeta 1 tipa. I. Istochniki polucheniya b-kletok (1 chastina) [Advances of regenerative medicine in the treatment of type 1 diabetes. I. Sources of b-cells (1 Chapter)]. Endokrinologіya – Endocrinology. 2012; 17(2): 66-73 [in Russian].|
|10. Tron’ko ND, Paster IP. Dostizheniya regenerativnoy meditsiny v terapii sakharnogo diabeta 1 tipa. II. Istochniki polucheniya b-kletok (2 chastina) [Advances of regenerative medicine in the treatment of type 1 diabetes. II. Sources of b-cells (2 Chapter)]. Endokrinologіya – Endocrinology. 2012; 17(3): 74-84 [in Russian].|
|11. Ernst LK, Zhigachev AI. Monitoring geneticheskikh bolezney zhivotnykh v sisteme krupnomasshtabnoy selektsii [Monitoring of animal genetic diseases in the system of large-scale selection]. Moskva, Rossel’khozakademiya – Moskow, Rosselkhozakademia, 2006. 382 p. [In Russian].|
|12. Burns CJ, Persaud SJ, Jones PM. Stem cell therapy for diabetes: do we need to make beta cells? J. Endocrinol. 2004; 183: 437-43.
|13. Foudah D, Redaelli S, Donzelli E, et al. Monitoring the genomic stability of in vitro cultured rat bone-marrow-derived mesenchymal stem cells. Chromosome Res. 2009; 17(8): 1025–39.
|14. Izadpanah R, Kaushal D, Kriedt C, et al. Long-term In vitro Expansion Alters the Biology of Adult Mesenchymal Stem Cells. Cancer Res. 2008; 68(11): 4229-238.
|15. Mareschi K, Ferrero I, Rustichelli D, et al. Expansion of mesenchymal stem cells isolated from pediatric and adult donor bone marrow. J. Cell. Biochem. 2006; 97(4): 744–54.|
|16. Miura M, Miura Y, Padilla NM, et al. Accumulated chromosomal instability in murine bone marrow mesenhymal stem cells leads to malignant transformation. Stem. Cells. 2005; 24: 1095–103.|
|17. Nathan DM. Long-term complications of diabetes mellitus. N. Engl. J. Med. 1993; 328: 1676-85.|
|18. Rubio D, GarciaCastro J, Martin MC, et al. Spontaneous human stem cell trans formation. Cancer Res. 2005; 65: 3035–39.|
|19. The DCCT Research Group. The effect of intensive treatment of diabetes on the development of long-term complications in insulin-dependent diabetes mellitus. N. Engl. J. Med. 1993; 29: 977-86.|
|20. Xikum X, Liming S. Observations on micronuclei germ cells. Zool. Res. 1990; 11(4): 343.|
Kovpak VV, Kovpak OS. Cytogenetic analysis of rat pancreatic cell cultures at early passages. Cell and Organ Transplantology. 2016; 4(1):66-69. doi: 10.22494/COT.V4I1.8