Quality Control for Some Commonly Used Cell Lines: Mycoplasma Contamination Detection, DNA Sequence Analysis of Cytochrome B and Cytochrome Oxidase Subunit I Genes

Öz

Aim: Mycoplasma contamination controls, identity verification and clonality determination of some cell lines such as cervix epithelioid carcinoma (HeLa), human peripheral blood promyelocytic leukemia (HL-60), mouse C3 / a connective tissue (L929), madin darby canine kidney (MDCK), mouse neuroblastoma (Neuro-2a) that are frequently used in laboratories.

Method: Five cell lines from three different species were used in this study. Mycoplasma contamination controls of all studied lines were performed by deoxyribonucleic acid (DNA) fluorescent labeling with Bisbenzimid (Hoechst 33258). DNA isolation was performed on cell lines and DNA samples were obtained. Regional amplification of obtained DNA samples was performed as follows: L14816 and H15173 primers were used for cytochrome B (CYTB) gene; LCO 1490 and HCO 2198 primers were used for cytochrome oxidase subunit I (COI) gene. Results of DNA sequence analysis for respective amplifications were compared with reference sequences using bioinformatics tools.

Results: In the study, no mycoplasma contamination was observed as a result of staining relevant cells with Bisbenzimid (Hoechst 33258) at the concentration and time determined by the manufacturer's protocols. In addition, comparing the results for CYTB gene region with the reference sequence in the database resulted in 97% similarity for HL-60, and 98% similarity for “HeLa, L929, MDCK, Neuro-2a” cell lines. Similarity rates for COI gene region were 95%, 99%, 96%, 96%, and 98% for “HeLa, HL-60, L929, MDCK and Neuro-2a” cell lines respectively.

Conclusion: In this context, Bisbenzimid (Hoechst 33258) labeling and DNA sequence analysis results obtained from the study provided an acceptable indicator and confidence for the quality of these cell lines used in many studies.

Anahtar Kelimeler

• Cell culture
• CYTB
• COI
• DNA sequence analysis
• mycoplasma
• Hoechst 33258

Sık Kullanılan Bazı Hücre Hatları için Kalite Kontrol: Mikoplazma Kontaminasyon Tespiti, Sitokrom B ve Sitokrom Oksidaz Alt Birim I Genlerinin DNA Dizi Analizlerinin Gerçekleştirilmesi

Öz

Amaç: Laboratuvarlarda sık kullanılan serviks epitelyal karsinom (HeLa), insan periferal kan promiyelösitik lösemi (HL-60), fare C3/bağ dokusu (L929), Madin Darby köpek böbrek (MDCK), fare nöroblastom (Neuro-2a) gibi bazı hücre hatlarının mikoplazma kontaminasyon kontrollerinin yapılması, kimlik doğrulamalarının gerçekleştirilmesi ve klonalitelerinin belirlenmesidir.

Yöntem: Bu çalışmada üç farklı türe ait beş hücre hattı kullanılmıştır. Çalışılan tüm hatların Bisbenzimid (Hoechst 33258) ile deoksiribonükleik asit (DNA) floresan işaretlemesi yapılarak mikoplazma kontaminasyonu kontrolleri gerçekleştirilmiştir. Hücre hatlarından DNA izolasyonları yapılmış, elde edilen DNA örneklerinden sitokrom B (CYTB) geninin bölgesel amplifikasyonu için L14816 ve H15173 primerleri; sitokrom oksidaz alt birim I (COI) geni için ise LCO 1490 and HCO 2198 primerleri kullanılmıştır. İlgili amplifikasyonların DNA dizi analiz sonuçları, biyoinformatik araçlar kullanılarak referans dizilerle karşılaştırmalı olarak değerlendirilmiştir.

Bulgular: Çalışmada ilgili hücrelerin, Bisbenzimid (Hoechst 33258) ile üretici firmanın protokollerine göre belirlenen konsantrasyon ve sürede yapılan boyama sonucunda mikoplazma kontaminasyonuna rastlanılmamıştır. Ayrıca CYTB gen bölgesi için veritabanında yer alan referans dizi ile yapılan karşılaştırma sonucu HL-60 için %97; "HeLa, L929, MDCK, Neuro-2a” hücre hatları için ise %98 oranında benzerlik bulunmuştur. COI gen bölgesi için ise bu benzerlik oranları “HeLa, HL-60, L929, MDCK ve Neuro-2a” hücre hatları için sırasıyla %95, %99, %96, %96 ve %98 olarak bulunmuştur.

Sonuç: Bu bağlamda, çalışmadan elde edilen Bisbenzimid (Hoechst 33258) işaretleme ve DNA dizi analiz sonuçları, pek çok araştırmada kullanılan bu hücre hatlarının kalitesi konusunda kabul edilebilir bir belirteç ve güven sağlamıştır.   

Anahtar Kelimeler

• Hücre kültürü
• CYTB
• COI
• DNA dizi analizi
• mikoplazma
• Hoechst 33258

Kaynakça

1. Losi CG, Ferrari S, Sossi E, Villa R, Ferrari M. An alternative method to isoenzyme profile for cell line identification and interspecies cross-contaminations: Cytochrome b PCR-RLFP analysis. In Vitro CellDevBiol-Animal. 2008;44(8-9):321-329. doi:10.1007/s11626-008-9125-x.
2. Huang Y, Liu Y, Zheng C, Shen C. Investigation of cross-contamination and misidentification of 278 widely used tumor cell lines. Talamas-Rohana P, ed. PLoS ONE. 2017;12(1):e0170384. doi:10.1371/journal.pone.0170384.
3. Yin C, Liu Y, Guo X, et al. An Effective strategy to eliminate inherent cross-contamination in mtDNA next-generation sequencing of multiple samples. The Journal of Molecular Diagnostics. 2019;21(4):593-601. doi:10.1016/j.jmoldx.2019.02.006.
4. Weissensteiner H, Forer L, Fendt L, et al. Contamination detection in sequencing studies using the mitochondrial phylogeny. Genome Res. 2021;31(2):309-316. doi:10.1101/gr.256545.119.
5. Souren NY, Fusenig NE, Heck S, et al. Cell line authentication: A necessity for reproducible biomedical research. The EMBO Journal. 2022;41(14):e111307. doi:10.15252/embj.2022111307.
6. Editorial. Reading between the lines. Nature. 2018;560(7718):281-281. doi:10.1038/d41586-018-05935-y. Published: 15 August 2018. Accessed: 20 November 2022.
7. Johnen G, Rozynek P, von der Gathen Y, et al. Cross-contamination of a UROtsa stock with T24 cells – molecular comparison of different cell lines and stocks. Cotterill S, ed. PLoS ONE. 2013;8(5):e64139. doi:10.1371/journal.pone.0064139.
8. Baghbaderani BA, Syama A, Sivapatham R, et al. Detailed characterization of human induced pluripotent stem cells manufactured for therapeutic applications. Stem Cell Rev and Rep. 2016;12(4):394-420. doi:10.1007/s12015-016-9662-8.

9. Araújo SBD, Patricio GF, Simoni IC, Rivas EB, Fernandes MJB. Isoenzyme and molecular approach for authenticating and monitoring of animal cell lines. An Acad Bras Ciênc. 2019;91(3):e20180487. doi:10.1590/0001-3765201920180487.
10. Ali S. Microbial and viral contamination of animal and stem cell cultures: Common contaminants, detection and elimination. JSRT. 2017;2(5):149-155. doi:10.15406/jsrt.2017.02.00078.
11. Shrader HR, Miller AM, Tomanek-Chalkley A, et al. Effect of bacterial contamination in bile on pancreatic cancer cell survival. Surgery. 2021;169(3):617-622. doi:10.1016/j.surg.2020.09.029.
12. Hoff FW, Hu CW, Qutub AA, et al. Mycoplasma contamination of leukemic cell lines alters protein expression determined by reverse phase protein arrays. Cytotechnology. 2018;70(6):1529-1535. doi:10.1007/s10616-018-0244-2.
13. Feng N, Huang X, Jia Y. Mycoplasma contamination affects cell characteristics and decreases the sensitivity of BV2 microglia to LPS stimulation. Cytotechnology. 2019;71(2):623-634. doi:10.1007/s10616-019-00311-8.
14. Dennert K, Kumar R. Traceability methods for cell line authentication and mycoplasma detection. SLAS Technology. 2021;26(6):630-636. doi:10.1177/24726303211030290.
15. Nikfarjam L, Farzaneh P. Prevention and detection of mycoplasma contamination in cell culture. Cell J. 2012;13(4):203-212.
16. Doyle C, Nakamura R, Bing R, Rousseau B, Branski RC. Mycoplasma affects baseline gene expression and the response to glucocorticoids in vocal fold fibroblasts. Journal of Medical Microbiology. 2021;70(5):001362-8. doi:10.1099/jmm.0.001362.
17. Fratz‐Berilla EJ, Faison T, Kohnhorst CL, et al. Impacts of intentional mycoplasma contamination on CHO cell bioreactor cultures. Biotechnology and Bioengineering. 2019;116(12):3242-3252. doi:10.1002/bit.27161.
18. Fratz‐Berilla EJ, Angart P, Graham RJ, et al. Impacts on product quality attributes of monoclonal antibodies produced in CHO cell bioreactor cultures during intentional mycoplasma contamination events. Biotechnology and Bioengineering. 2020;117(9):2802-2815. doi:10.1002/bit.27436.
19. Soheily Z, Soleimani M, Keivan MA. Detection of mycoplasma contamination of cell culture by A loop-mediated isothermal amplification method. Cell J. 2019;21(1):43-8. doi:10.22074/cellj.2019.5624.
20. Baaity Z, Breunig S, Önder K, Somogyvári F. Direct qPCR is a sensitive approach to detect mycoplasma contamination in U937 cell cultures. BMC Res Notes. 2019;12(1):720-5. doi:10.1186/s13104-019-4763-5.
21. Russell BJ, Horiuchi K, Velez JO, Goodman CH, Johnson BW. Mycoplasma detection in a historical arbovirus repository: Commercial kit comparison and implications for improved repository management. Journal of Virological Methods. 2020;276:113769-76. doi:10.1016/j.jviromet.2019.113769.
22. Young L, Sung J, Stacey G, Masters JR. Detection of mycoplasma in cell cultures. Nat Protoc. 2010;5(5):929-934. doi:10.1038/nprot.2010.43.
23. Sugita S, Hono A, Fujino S, et al. Detection of mycoplasma contamination in transplanted retinal cells by rapid and sensitive polymerase chain reaction Test. IJMS. 2021;22(22):12555-67. doi:10.3390/ijms222212555.
24. Greenfield EA. Testing hybridoma cells for Mycoplasma contamination. Cold Spring Harb Protoc. 2021;2021(7):pdb.prot103283. doi:10.1101/pdb.prot103283.
25. Li P, Li D, Hong Y, et al. Combining DNA mini-barcoding and species-specific primers PCR technology for identification of heosemys grandis. Front Ecol Evol. 2022;10:822871. doi:10.3389/fevo.2022.822871.
26. Tsoupas A, Papavasileiou S, Minoudi S, et al. DNA barcoding identification of Greek freshwater fishes. Peng Z, ed. PLoS ONE. 2022;17(1):e0263118. doi:10.1371/journal.pone.0263118.
27. Dave AR, Chaudhary DF, Mankad PM, Koringa PG, Rank DN. Genetic diversity among two native Indian chicken populations using cytochrome c oxidase subunit I and cytochrome b DNA barcodes. Vet World. 2021:1389-1397. doi:10.14202/vetworld.2021.1389-1397.
28. Savolainen V, Cowan RS, Vogler AP, Roderick GK, Lane R. Towards writing the encyclopaedia of life: An introduction to DNA barcoding. Philos Trans R Soc Lond B Biol Sci. 2005;360(1462):1805-1811. doi:10.1098/rstb.2005.1730.
29. Ahmed S, Ibrahim M, Nantasenamat C, et al. Pragmatic applications and Universality of DNA barcoding for substantial organisms at species level: A review to explore a way forward. Khan S, ed. BioMed Research International. 2022;2022:1-19. doi:10.1155/2022/1846485.
30. Segeritz CP, Vallier L. Cell culture. In: Basic Science Methods for Clinical Researchers. Elsevier. 2017:151-172. doi:10.1016/B978-0-12-803077-6.00009-6.
31. Huang YC, Kuo CL, Lu KW, et al. 18α-Glycyrrhetinic acid induces apoptosis of HL-60 human leukemia cells through caspases- and mitochondria-dependent signaling pathways. Molecules. 2016;21(7):872-85. doi:10.3390/molecules21070872.
32. Yahia D, Kamata Y. PCR-RFLP and Sequence analysis of hair cytochrome b gene for identification of animal species. Asian J of Animal and Veterinary Advances. 2018;13(2):155-165. doi:10.3923/ajava.2018.155.165.
33. Abd-El-Samie EM, Elkafrawy I, Osama M, Ageez A. Molecular phylogeny and identification of the Egyptian wasps (Hymenoptera: Vespidae) based on COI mitochondrial gene sequences. Egypt J Biol Pest Control. 2018;28(1):36-42. doi:10.1186/s41938-018-0038-z.
34. Halbedel S, Stülke J. Tools for the genetic analysis of mycoplasma. International Journal of Medical Microbiology. 2007;297(1):37-44. doi:10.1016/j.ijmm.2006.11.001.
35. Ligasová A, Vydržalová M, Buriánová R, et al. A New sensitive method for the detection of mycoplasmas using fluorescence microscopy. Cells. 2019;8(12):1510-26. doi:10.3390/cells8121510.
36. Tobe SS, Kitchener AC, Linacre AMT. Reconstructing mammalian phylogenies: A detailed comparison of the cytochrome b and cytochrome oxidase subunit i mitochondrial genes. DeSalle R, ed. PLoS ONE. 2010;5(11):e14156. doi:10.1371/journal.pone.0014156.
37. Weir JT, Schluter D. The latitudinal gradient in recent speciation and extinction rates of birds and mammals. Science. 2007;315(5818):1574-1576. doi:10.1126/science.1135590
38. Cooper JK, Sykes G, King S, et al. Species identification in cell culture: A two-pronged molecular approach. In Vitro CellDevBiol-Animal. 2007;43(10):344-351. doi:10.1007/s11626-007-9060-2.
39. Ramya R, Nagarajan T, Sivakumar V, et al. Identification of cross-contaminated animal cells by PCR and isoenzyme analysis. Cytotechnology. 2009;61(3):81-92. doi:10.1007/s10616-009-9245-5.
40. Parodi B, Aresu O, Bini D, et al. Species identification and confirmation of human and animal cell lines: A PCR-based method. BioTechniques. 2002;32(2):432-440. doi:10.2144/02322rr05.
41. Soleimani S, Ziyaeifar F, Lotfi M. Study on identification of Iranian BHK-21-C5 cell line by two steps PCR. Archives of Razi Institute. 2021;76(2):193-201. doi:10.22092/ari.2020.128637.1419