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Biological, cytomorphological and karyological heterogeneity of transformed cell lines derived from domestic pig (Sus scrofa L.) organs

https://doi.org/10.29326/2304-196X-2023-12-1-13-22

Abstract

The main advantage of transformed cell lines as compared to primary ones is that they allow generation of the stable material suitable for long-term research and practical use. Therefore, development of new continuous cell cultures from various animal tissues is of great practical importance. Results of examination of transformed cell lines derived from organs of domestic pigs (Sus scrofa L.) for their biological, cytomorphological and karyological features are described in the paper. The said cell cultures are confirmed to be susceptible to various animal viruses. Also, a procedure for preparation of new diploid cell culture from porcine spleen (SSs – Splеen Sus scrofa) is described. Based on the obtained data analysis it was concluded that the epithelial cells derived from trypsinized porcine spleens could be successfully immortalized. All transformed cell lines of porcine origin have similar morphology with predominated epithelium-like forms. Some of them – SPEV, А4 С2 , RSK – tend to adopt a spherical shape in suspension. Such cell lines as PSGK-30 and PPES cell lines form partial multilayer or they are characterized by significant monolayer compaction with pseudosyncytium formation. Only pseudodiploid cell culture (SPEV cell culture) tends to grow in suspension, it also grows in rotating culture flasks. Karyological transformations in different cell cultures stabilized at certain level. Spontaneous increase in chromosome numbers in the main population of transformed cell lines towards triploidy resulted in stabilization of culture properties and increase in proliferation. PSGK-30 cell culture has the highest modal class – 64 chromosomes. Near-diploid cultures (А4 С2 , RSK) demonstrate stable growth properties and are similar to SPEV cell culture in adopting spherical cell forms in medium, monolayer character and cell morphology. PK-15 cell culture having a distinct karyotype under different cultivation conditions while retaining other culture properties is found to be the most adaptive. A new transformed diploid SSs cell culture is developed by long-term incubation, subcultivation (more than 80 passages) and selection at the FGBI “ARRIAH” laboratory; it can remain diploid or may spontaneously become heteroploid-immortalized during further passaging. The cell hyperploidy is very likely to enhance telomerase activity, which in turn stabilizes immortalization and results in proliferative activity increase The cell viability has been maintained so far by regular reseedings (split ratio – 1:2–1:3) performed 1–2 times a week.

About the Authors

B. L. Manin
FGBI “Federal Centre for Animal Health” (FGBI “ARRIAH”)
Russian Federation

Boris L. Manin, Candidate of Science (Biology)

Vladimir



E. A. Trofimova
FGBI “Federal Centre for Animal Health” (FGBI “ARRIAH”)
Russian Federation

Elena A.  Trofimova, Head of  Sector, Cell Cultivation Unit

Vladimir



V. L. Gavrilova
FGBI “Federal Centre for Animal Health” (FGBI “ARRIAH”)
Russian Federation

Vera L. Gavrilova, Candidate of Science (Biology), Reference Laboratory for African Swine Fever

Vladimir



O. S. Puzankova
FGBI “Federal Centre for Animal Health” (FGBI “ARRIAH”)
Russian Federation

Olga S. Puzankova, Candidate of Science (Veterinary Medicine), Senior Researcher, Reference Laboratory for African Swine Fever

Vladimir



References

1. Animal cell in culture (methods and implementation in biotechnology). Ed. by L. P. Dyakonov; Russian Academy of Agricultural Sciences. 2nd ed., enlarged. Moscow: Sputnik+; 2009. 652 p. (in Russ.)

2. Belun О. V., SokovaV. А., Kurnosov А. N. Klonirovanie perevivaemoi linii kletok pochki porosenka = Cloning of continuous porcine cell line. Voprosy veterinarnoi virusologii, mikrobiologii i epizootologii: tezisy dokladov nauchnoi konferentsii = Veterinary virology, microbiology and epidemiology aspects: Abstractsforscientific conference. Pokrov: VNIIVViM; 1978; 16–17. (in Russ.)

3. Kolbasova O. L. Karyological and cytochemical characterization of continuous cell lines permissive and non-permissive (resistant) to classical swine fever virus: Author’s abstract, Thesis for degree of Candidate of Science (Biology). Pokrov; 2000. 21 p. (in Russ.)

4. Filina A. Yu., Gerasimov V. N., Baibikov T. Z., Yegorova A. I. Cultivation of different classical swine fever virus strains in continuous cell cultures. Proceedings of the Federal Centre for Animal Health. 2007; 5: 278–284. eLIBRARY ID: 14454067. (in Russ.)

5. Stroganova I. Ya., Trukhonenko А. А. Use of cell culture in virology: methodical guidelines. Krasnoyarsk: KrasSAU; 2013. 48 p. Available at: http://www.kgau.ru/sveden/2017/ipbivm/mu_360501_9.pdf. (in Russ.)

6. Pankova G. E. Cell population selection and morphologic characteristics of clonesfrom the PP(RS) and PK-15 transplantable swine kidney cell lines. Tsitologiya. 1976; 18 (8): 1036–1039. PMID: 988662. (in Russ.)

7. Polyanskya G. G. Cell line generation, main characteristics and variability. In: Cell cultivation methods. Saint Petersburg: Polytechnic University; 2008; 22–40. (in Russ.)

8. Ruggli N., Summerfield A., Häni R. E. From pigs to cells: Virulence of classical swine fever virus is predicable in cell cultures. 3R-Info-Bulletin. 2010; 44. Available at: https://www.forschung3r.ch/data/publications/Ruggli-Bul44.pdf.

9. Kasza L., Shadduck J. A., Christofinis G. J. Establishment, viral susceptibility and biological characteristics of a swine kidney cell line SK-6. Res. Vet. Sci. 1972; 13 (1): 46–51. PMID: 4336054.

10. Galnbek T. V. Porcine thyroid and intestinal cell cultures and their use in virology and biotechnology: Author’s abstract, Thesis for degree of Candidate of Science (Biology). Moscow; 1991. 25 p. (in Russ.)

11. Dyakonov L. P., Subaev G. H., Galnbek T. V., Taktashev Sh. S., Nepoklonov E. A., Fedorova E. S., Rasulev О. Sh. Methodical recommendations for preparation of cells from porcine thyroid and other organs and their cultivation. Moscow; 1985. 19 p. Available at: https://meganorm.ru/ Data2/1/4293737/4293737310.pdf. (in Russ.)

12. Zhdanova N. A. Preparation of suspension continuous porcine testicular cell line and optimization of its cultivation conditions: Author’s abstract, Thesis for degree of Candidate of Science (Biology). Pokrov; 2009. 25 p. (in Russ.)

13. Makaryan E. A., Balayan O. R., Dichenskij A. V., Abylkasimov D. A., Degtyarev V. P., Fedotov S. V. Method for obtaining of preparation based on stem cells selected from pigs spleen tissue, for prevention and treatment of infectious and non-infectious diseases of domestic and farm animals. Patent No. 2611205 Russian Federation, Int. Cl. C12N 5/077 (2010.01), C12N 5/0797 (2010.01), A61P 19/02 (2006.01), A61P 31/00 (2006.01). FGBOU VO “Tverskaya gosudarstvennaya selskokhozyajstvennaya akademiya”. No. 2015148589. Date of filing: 12.11.2015. Date of publication: 21.02.2017. Bull. No 6. Available at: https://patents.s3.yandex.net/ RU2611205C1_20170221.pdf. (in Russ.)

14. Kolbasova O. L., Jurkov S. G., Neverovskaja N. S., Dmitrenko V. V., Lyska V. M. Strain of diploid cells of synovial membrane of young pig Sus scrofa, used for virology. Patent No. 2506310 Russian Federation, Int. Cl. C12N 5/077 (2010.01), G01N 33/569 (2006.01). GNU Vserossijskij nauchno-issledovatel’skij institute veterinarnoj virusologii i mikrobiologii Rossel’khozakademii. No. 012131176/10. Date of filing: 23.07.2012. Date of publication: 10.02.2014. Bull. No 4. Available at: https://patents.s3.yandex. net/RU2506310C1_20140210.pdf. (in Russ.)

15. Portugal R., Goatley L. C., Husmann R., Zuckermann F. A., Dixon L. K. A porcine macrophage cell line that supports high levels of replication of OURT88/3, an attenuated strain ofAfrican swine fever virus. Emerg. Microbes Infect. 2020; 9 (1): 1245–1253. DOI: 10.1080/22221751.2020.1772675.

16. Weingartl H. M., Sabara M., Pasick J., van Moorlehem E., Babiuk L. Continuous porcine cell lines developed from alveolar macrophages: partial characterization and virus susceptibility. J. Virol. Methods. 2002; 104 (2): 203–216. DOI: 10.1016/s0166-0934(02)00085-x.

17. Lee Y. J., Park C. K., Nam E., Kim S. H., Lee O. S., Lee du S., Lee C. Generation of a porcine alveolar macrophage cell line for the growth of porcine reproductive and respiratory syndrome virus. J. Virol. Methods. 2010; 163 (2): 410–415. DOI: 10.1016/j.jviromet.2009.11.003.

18. Chitko-McKown C. G., Chapes S. K., Miller L. C., Riggs P. K., Ortega M. T., Green B. T., McKown R. D. Development and characterization oftwo porcine monocyte-derived macrophage cell lines. Results Immunol. 2013; 3: 26–32. DOI: 10.1016/j.rinim.2013.03.001.

19. Kadoi K., Tsukise A., Shiba H., Ikeda K., Seki T., Ariga T. Establishment of a swine monocyte cell line. New Microbiol. 2001; 24 (3): 243–247. PMID: 11497081.

20. TakenouchiT., Kitani H., Suzuki S., Nakai M., FuchimotoD. I., Tsukimoto M., et al. Immortalization and characterization of porcine macrophages that had been transduced with lentiviral vectors encoding the SV40 large T antigen and porcine telomerase reverse transcriptase. Front. Vet. Sci. 2017; 4:132. DOI: 10.3389/fvets.2017.00132.

21. Takenouchi T., Masujin K., Miyazaki A., Suzuki S., Takagi M., Kokuho T., Uenishi H. Isolation and immortalization of macrophages derived from fetal porcine small intestine and theirsusceptibility to porcine viral pathogen infections. Front. Vet. Sci. 2022; 9:919077. DOI: 10.3389/ fvets.2022.919077.

22. TalbotN. C., Paape M., Worku M. Selective expansion and continuous culture of macrophagesfrom adult pig blood. Vet. Immunol. Immunopathol. 1998; 64 (2): 173–190. DOI: 10.1016/s0165-2427(98)00128-7.

23. Wardley R. C., Lawman M. J., Hamilton F. The establishment of continuous macrophage cell linesfrom peripheal blood monocytes. Immunology. 1980; 39 (1): 67–73. PMID: 6769783.

24. Graphodatsky A. S., Radzhabli S. I. Farmed and laboratory mammal chromosomes: atlas. Novosibirsk: Nauka; 1988. 127 p. (in Russ.)

25. Dyakonov L. P., Galnbek Т. V., Akinshina G. Т., Abdrakhmanov I. К., Samuilenko А. Ya., Dagadanova А. V., et al. Specialized collection of continuoussomatic cell lines of livestock and game animals, RCCC(V) (Livestock animals Russian Academy of Agricultural Sciences): catalogue. 2nd ed., enlarged. Мoscow: VIEV; 2006. 115 p. (in Russ.)

26. Kuleshov K. V. Species identification of cell cultures with moleculargenetic methods: Author’s abstract, Thesis for degree of Candidate of Science (Biology). Shchyolkovo; 2009. 24 p. (in Russ.)

27. Freshney R. Ian. Culture of Animal Cells: A Manual of BasicTechnique. Hoboken: John Wiley and Sons, Inc.; 2005. 672 p.

28. Moorhead P. S., Nowell P. C., Mellman W. J., Battips D. M., Hungerford D. A. Chromosome preparations of leukocytes cultured from human peripheral blood. Exp. Cell. Res. 1960; 20: 613–616. DOI: 10.1016/0014- 4827(60)90138-5.

29. Mamaeva S. E. Chromosomal analysis of cultured cells. In: Methods of Cell Culture: Collection of Scientific Papers. Ed. by G. P. Pinaev. Leningrad: Nauka; 1988; 78–79. (in Russ.)

30. Prudnikova E. Ju., Balysheva V. I., Gal’nbek T. V., Balyshev V. M. Finite hybrid subline of cells A4 C2 /9k Sus scrofa, used for virological studies of African swine fever virus. Patent No. 2545720 Russian Federation, Int. Cl. C12N 5/073 (2010.01), C12N 7/00 (2006.01). GNU Vserossijskij nauchno-issledovatel’skij institute veterinarnoj virusologii i mikrobiologii Rossel’khozakademii. No. 2013153452/10. Date of filing: 03.12.2013. Date of publication: 04.10.2015. Bull. No. 10. Available at: https://patents.s3.yandex.net/RU2545720C1_20150410.pdf. (in Russ.)

31. D’jakonov L. P., Majdzhi E. V., Gerasimov V. N., Gal’nbek T. V., Dudar L. N., Soldatova N. V., Fedorova E. E., Egorova A. I. Strain of intraspecies hybrid cells of Suis domestica used forisolation and cultivation ofswine classic plague virus. Patent No. 2082758 Russian Federation, Int. Cl. C12N 5/06. No. 94026140/13. Date of filing: 14.07.1994. Date of publication: 27.06.1996. Available at: https://patents.s3.yandex.net/RU94026140A1_19960710.pdf. (in Russ.)

32. The European Collection of Authenticated Cell Cultures. Cell Lines and Hybridomas. Available at: https://www.culturecollections.org.uk/products/celllines/index.aspx (date of access: 12.08.2022).

33. Pan I. C., Shimizu M., Hess W. R. Replication of African swine fever virusin cell cultures. Am. J. Vet. Res. 1980; 41 (9): 1357–1367. PMID: 7004279.


Review

For citations:


Manin B.L., Trofimova E.A., Gavrilova V.L., Puzankova O.S. Biological, cytomorphological and karyological heterogeneity of transformed cell lines derived from domestic pig (Sus scrofa L.) organs. Veterinary Science Today. 2023;12(1):13-22. https://doi.org/10.29326/2304-196X-2023-12-1-13-22

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