Characterizing the BHK-21 C5 cell line and determining cellular sensitivity to rubella virus compared with the routine cell (RK13)

Document Type : Original Articles


1 Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran

2 Department of Bio bank, Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Extension Organization (AREEO), P.O. Box 31975-148, Karaj, Iran


The World Health Organization has strict rules and recommendations on the selection and use of cell substrates in laboratories. Given the widespread use of safe and secure cell substrates in the production and quality control of viral vaccines and also the high demand for vaccines against viral diseases, obligating the selection of a suitable cell substrate for cultivation and production of biological products. Animal cell lines play a valuable role in the preparation and propagation of viral seeds; thus, the current study used the BHK-21 cell line among others for viral checking with the aim of replacing the BHK-21 C5 cell line with the RK13 cell line to investigate the cytopathic effects of the rubella virus. To this end, attempts were made to determine the characteristics of the BHK-21 C5 cell line including cell growth characteristics and sterility tests to validate its safety and security. Then, by culturing the cells in a 96-well microplate, titration of the rubella virus was subsequently performed by preparing serial dilutions of the virus from 10-1 to 10-5 and inoculated to cell lines in order to compare the sensitivity of BHK-21 C5 and RK13 cell lines to rubella virus. Data analysis according to the results of the tests by ahead default, p-value < 0/05 was equal to p-value = 0.01 based on SPSS analysis with the paired-sample t-test. In addition, the box-plot diagram indicated a significant difference between these cell lines. Based on the results, the BHK-21 C5 cell line seems to be more sensitive to the rubella virus than others. Therefore, it can be used for production and quality control of the vaccine and in research and diagnosis of rubella.


Main Subjects

Article Title [French]

Caractériser la Lignée Cellulaire BHK-21 C5 et Déterminer la Sensibilité Cellulaire au Virus de la Rubéole par Rapport à la Cellule de Routine (RK13)

Abstract [French]

L'Organisation mondiale de la santé a des règles et des recommandations strictes sur la sélection et l'utilisation de substrats cellulaires dans les laboratoires. L'utilisation généralisée de substrats cellulaires sûrs et sécurisés dans la production et le contrôle de la qualité des vaccins viraux ainsi que la forte demande de vaccins contre les maladies virales nous obligent à sélectionner un substrat cellulaire approprié pour la culture et la production de produits biologiques. Les lignées cellulaires animales jouent un rôle précieux dans la préparation et la propagation des graines virales; ainsi, la présente étude a utilisé la lignée cellulaire BHK-21 entre autres pour le contrôle viral dans le but de remplacer la lignée cellulaire BHK-21 C5 par la cellule RK13 pour étudier les effets cytopathiques du virus de la rubéole. À cette fin, des tentatives ont été faites pour déterminer les caractéristiques de la lignée cellulaire BHK-21 C5, y compris les caractéristiques de croissance cellulaire et les tests de stérilité pour valider sa sûreté et sa sécurité. Ensuite, en cultivant les cellules dans une microplaque à 96 puits, le titrage du virus de la rubéole a ensuite été effectué en préparant des dilutions en série du virus de 10-1 à 10-5 et inoculé à des lignées cellulaires afin de comparer la sensibilité de BHK-21 lignées cellulaires C5 et RK13 au virus de la rubéole. L'analyse des données selon les résultats des tests par défaut anticipé, la valeur p<0/05 était égale à la valeur p = 0.01 sur la base de l'analyse SPSS avec le test t pour échantillons appariés. De plus, le diagramme en boîte a indiqué une différence significative entre ces lignées cellulaires. D'après les résultats, la lignée cellulaire BHK-21 C5 semble être plus sensible au virus de la rubéole que les autres. Par conséquent, elle peut être utilisée pour la production et le contrôle qualité du vaccin et dans la recherche et le diagnostic de la rubéole.

Keywords [French]

  • caractérisation
  • lignée cellulaire BHK-21 C5
  • virus de la rubéole
  • test de titrage
  • lignée cellulaire RK13
  1. Aubrit F, Perugi F, Léon A, Guéhenneux F, Champion-Arnaud P, Lahmar M, et al. Cell substrates for the production of viral vaccines. Vaccine. 2015;33(44):5905-12.
  2. Petricciani J. Cell substrates: Where do we stand after 50 years of discussion? Dev Biol. 2006;123:11.
  3. Montagnon B, Fanget B, Nicolas A. The large-scale cultivation of VERO cells in micro-carrier culture for virus vaccine production. Preliminary results for killed poliovirus vaccine. Dev Biol Stand. 1981;47:55-64.
  4. Zahoor MA, Khurshid M, Qureshi R, Naz A, Shahid M. Cell culture-based viral vaccines: current status and future prospects. Future Virol. 2016;11(7):549-62.
  5. Plotkin SA. Vaccines: past, present and future. Nat Med. 2005;11(4s):S5.
  6. Whitford W. Using Disposables in Cell-Culture–Based Vaccine Production. BioProcess Int. 2010;8(4).
  7. Merten O-W. Advances in cell culture: anchorage dependence. Philosophical Transactions of the Royal Society B: Biological Sciences. 2015;370(1661):20140040.
  8. Hudu SA, Alshrari AS, Syahida A, Sekawi Z. Cell culture, technology: enhancing the culture of diagnosing human diseases. J Clin Diagn Res. 2016;10(3):DE01.
  9. Hernandez R, Brown DT. Growth and maintenance of baby hamster kidney (BHK) cells. Curr Protoc Microbiol. 2010;17(1):A. 4H. 1-A. 4H. 7.
  10. Lennette EH, Schmidt NJ, Lennette DA, Emmons RW, Lennette ET. Diagnostic Procedures for Viral, Rickettsial, and Chlamydial Infections: American Public Health Association; 1995.
  11. Cunningham AL, Fraser J. Persistent rubella virus infection of human synovial cells cultured in vitro. J Infect Dis. 1985;151(4):638-45.
  12. Wang D-Y, Yeh S-Y, Chou C-P, Cheng H-F, Hsieh J-T, Lin C-P. Evaluation and validation of potency testing method for live rubella virus vaccine. J Food Drug Anal. 2001;9(4):183-190.
  13. Kallel H, Jouini A, Majoul S, Rourou S. Evaluation of various serum and animal protein free media for the production of a veterinary rabies vaccine in BHK-21 cells. J Biotechnol. 2002;95(3):195-204.
  14. Freshney RI. Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications: John Wiley & Sons; 2011.
  15. Abercrombie M. Fibroblasts. J Clin Pathol Suppl (Royal College of Pathologists). 1978;12:1.
  16. DeliveReD G. Animal Cell Culture Guide. 2012.
  17. Coecke S, Balls M, Bowe G, Davis J, Gstraunthaler G, Hartung T, et al. Guidance on good cell culture practice: a report of the second ECVAM task force on good cell culture practice. Altern Lab Anim. 2005;33(3):261-87.
  18. Nema R, Khare S. An animal cell culture: Advance technology for modern research.Sci Res. 2012;3(2)219-226.
  19. Phelan M, Lawler G. Cell counting. Current protocols in cytometry. 2001:Appendix 3A.
  20. Strober W. Trypan blue exclusion test of cell viability. Curr Protoc Immunol. 2015;111(1):3B.
  21. Nema R, Khare S. An animal cell culture: Advance technology for modern research. Adv Biosci Biotechnol. 2012;3(3):219.
  22. Kaplitt MG, Loewy AD. Viral vectors: gene therapy and neuroscience applications: Academic Press; 1995.
  23. Murphy FA, Halonen PE, Harrison AK. Electron microscopy of the development of rubella virus in BHK-21 cells. J Virol. 1968;2(10):1223.
  24. Charretier C, Saulnier A, Benair L, Armanet C, Bassard I, Daulon S, et al. Robust real-time cell analysis method for determining viral infectious titers during development of a viral vaccine production process. J Virol Methods. 2018;252:57-64.
  25. Zimmerman JJ, Hill HT, Beran GW, Meetz MC. Serologic diagnosis of encephalomyocarditis virus infection in swine by the microtiter serum neutralization test. J Vet Diagn Invest. 1990;2(4):347-50.
  26. Fogel A, Plotkin SA. Markers of rubella virus strains in RK13 cell culture. J Virol. 1969;3(2):157-63.
  27. Salas-Benito JS, Nova-Ocampo D. Viral interference and persistence in mosquito-borne flaviviruses. J Immunol Res. 2015;2015:873404.
  28. Petricciani J, Sheets R. An overview of animal cell substrates for biological products. Biologicals. 2008;36(6):359-62.
  29. Oyeleye O, Ola S, Omitogun O. Basics of animal cell culture: Foundation for modern science. Biotechnol Mol Biol Rev. 2016;11(2):6-16.
  30. Betakova T, Svetlikova D, Gocnik M. Overview of measles and mumps vaccine: origin, present, and future of vaccine production. Acta Virol. 2013;57(2):91-6.
  31. Hess RD, Weber F, Watson K, Schmitt S. Regulatory, biosafety and safety challenges for novel cells as substrates for human vaccines. Vaccine. 2012;30(17):2715-27.
  32. Schiff LJ. production, characterization, and testing of banked mammalian cell substrates used to produce biological products. In Vitro Cell Dev Biol Anim. 2005;41(3-4):65-70.
  1. Srček VG, Čajavec S, Sladić D, Kniewald Z. BHK 21 C13 cells for Aujeszky’s disease virus production using the multiple harvest process. Cytotechnology. 2004;45(3):101-6.
  2. Singh S, Rajaram S. Devlopment and Commercialization of Cell Based Viral Vaccines for Animal Health in National Immunization in India. Int J Vaccin. 2016;3(3):00066.
  3. Bushar G, Sagripanti J-L. Method for improving accuracy of virus titration: standardization of plaque assay for Junin virus. J Virol Methods. 1990;30(1):99-107.