Application of Epstein–Barr Virus for Optimization of Immortalized B-lymphocyte Production as a Positive Control in Genetic Studies

Document Type : Original Article

Authors
Behnaz Tousizadeh 1
Sharareh Moghim 2
Ahmad Reza Salehi Chaleshtori 3
Maryam Ghanbarian 4
Mina Mirian 5
Mansoor Salehi 6
Sepideh Tousizadeh 7
Fatemeh Zaboli 8
1 Department of Microbiology, Faculty of Basic Sciences, Ayatollah Amoli Azad University, Amol; Pediatric Inherited Disease Research Center, Isfahan, Iran
2 Department of Microbiology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran
3 Medical Genetics Center of Genome, Isfahan, Iran
4 Department of Microbiology, Faculty of Basic Science, Islamic Azad University, Falavarjan Branch, Isfahan, Iran
5 Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran
6 Department of Microbiology, Faculty of Basic Sciences, Ayatollah Amoli Azad University, Amol; Medical Genetics Center of Genome, Isfahan; Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran
7 Department of Health, School of Health, Shahrekord University of Medical Sciences, Shahrekord, Iran
8 Department of Microbiology, Faculty of Basic Sciences, Ayatollah Amoli Azad University, Amol, Iran
Abstract
Background: Infection of B-cells with Epstein–Barr virus (EBV) leads to more and subsequent immortalization. This is considered as the method of choice for generating lymphoblastoid cell lines (LCLs). Producing LCLs, although very useful but is very time consuming and troublesome, drives the requirement for quicker and more reliable methods for EBV-driven B-cell transformation.Materials and Methods: After successfully production of LCLs, different parameters including temperature, serum concentration, type of culture medium, and CO2concentration were evaluated on EBV-transformed B-cells. In this study, we were able to produce LCLs and optimize condition. Results: The best condition for generating LCLs was 37°C, 5% CO2, 20% fasting blood sugar, and RPMI 1640. The study results were to establish a reliable method for producing LCLs that can be used to produce immortalized B-cells from almost any sources. Conclusion: This can help with tumorgenecity studies, as well as producing control material for rare genetic disorders and so on. The aim of this study was to determine optimized condition for reliable and reproducible LCLs from different sources.

Keywords

1.
Megyola C, Ye J, Bhaduri-McIntosh S. Identification of a sub-population of B cells that proliferates after infection with Epstein-Barr virus. Virol J 2011;8:84.  Back to cited text no. 1
[PUBMED]    
2.
Subramanian A, Tamayo P, Mootha VK, Mukherjee S, Ebert BL, Gillette MA, et al. Gene set enrichment analysis: A knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci U S A 2005;102:15545-50.  Back to cited text no. 2
[PUBMED]    
3.
Sixbey JW, Nedrud JG, Raab-Traub N, Hanes RA, Pagano JS. Epstein-Barr virus replication in oropharyngeal epithelial cells. N Engl J Med 1984;310:1225-30.  Back to cited text no. 3
[PUBMED]    
4.
Hui-Yuen J, McAllister S, Koganti S, Hill E, Bhaduri-McIntosh S. Establishment of Epstein-Barr virus growth-transformed lymphoblastoid cell lines. J Vis Exp 2011. pii: 3321.  Back to cited text no. 4
    
5.
Callard RE, Lau YL, Shields JG, Smith SH, Cairns J, Flores-Romo L, et al. The marmoset B-lymphoblastoid cell line (B95-8) produces and responds to B-cell growth and differentiation factors: Role of shed CD23 (sCD23). Immunology 1988;65:379-84.  Back to cited text no. 5
[PUBMED]    
6.
Amoli MM, Carthy D, Platt H, Ollier WE. EBV Immortalization of human B lymphocytes separated from small volumes of cryo-preserved whole blood. Int J Epidemiol 2008;37 Suppl 1:i41-5.  Back to cited text no. 6
[PUBMED]    
7.
Mosier DE, Subbarao B. Thymus-independent antigens: Complexity of B-lymphocyte activation revealed. Immunol Today 1982;3:217-22.  Back to cited text no. 7
[PUBMED]    
8.
Miller G, Shope T, Lisco H, Stitt D, Lipman M. Epstein-Barr virus: Transformation, cytopathic changes, and viral antigens in squirrel monkey and marmoset leukocytes. Proc Natl Acad Sci U S A 1972;69:383-7.  Back to cited text no. 8
[PUBMED]    
9.
Traggiai E, Becker S, Subbarao K, Kolesnikova L, Uematsu Y, Gismondo MR, et al. An efficient method to make human monoclonal antibodies from memory B cells: Potent neutralization of SARS coronavirus. Nat Med 2004;10:871-5.  Back to cited text no. 9
[PUBMED]    
10.
Yang ZY, Kong WP, Huang Y, Roberts A, Murphy BR, Subbarao K, et al. A DNA vaccine induces SARS coronavirus neutralization and protective immunity in mice. Nature 2004;428:561-4.  Back to cited text no. 10
[PUBMED]    
11.
Bernasconi NL, Traggiai E, Lanzavecchia A. Maintenance of serological memory by polyclonal activation of human memory B cells. Science 2002;298:2199-202.  Back to cited text no. 11
[PUBMED]    
12.
Raab-Traub N, Dambaugh T, Kieff E. DNA of Epstein-Barr virus VIII: B95-8, the previous prototype, is an unusual deletion derivative. Cell 1980;22:257-67.  Back to cited text no. 12
[PUBMED]    
13.
Monks A, Scudiero D, Skehan P, Shoemaker R, Paull K, Vistica D, et al. Feasibility of a high-flux anticancer drug screen using a diverse panel of cultured human tumor cell lines. J Natl Cancer Inst 1991;83:757-66.  Back to cited text no. 13
[PUBMED]    
14.
Ormerod MG. Flow Cytometry: A Practical Approach. Great Britain, Wiltshire: Oxford University Press; 2000.  Back to cited text no. 14
    
15.
Okano M, Thiele GM, Davis JR, Grierson HL, Purtilo DT. Epstein-Barr virus and human diseases: Recent advances in diagnosis. Clin Microbiol Rev 1988;1:300-12.  Back to cited text no. 15
[PUBMED]    
16.
Beck JC, Beiswanger CM, John EM, Satariano E, West D. Successful transformation of cryopreserved lymphocytes: A resource for epidemiological studies. Cancer Epidemiol Biomarkers Prev 2001;10:551-4.  Back to cited text no. 16
[PUBMED]    
17.
Thorley-Lawson DA, Gross A. Persistence of the Epstein-Barr virus and the origins of associated lymphomas. N Engl J Med 2004;350:1328-37.  Back to cited text no. 17
[PUBMED]    
18.
Gordon J, Guy G, Walker L. Autocrine models of B-lymphocyte growth. I. Role of cell contact and soluble factors in T-independent B-cell responses. Immunology 1985;56:329-35.  Back to cited text no. 18
    
19.
Young LS, Rickinson AB. Epstein-Barr virus: 40 years on. Nat Rev Cancer 2004;4:757-68.  Back to cited text no. 19
[PUBMED]    
20.
Moffatt MF, Kabesch M, Liang L, Dixon AL, Strachan D, Heath S, et al. Genetic variants regulating ORMDL3 expression contribute to the risk of childhood asthma. Nature 2007;448:470-3.  Back to cited text no. 20
[PUBMED]    
Advanced Biomedical Research
Volume 2017, july
July 2017
Pages 1-5