Bリンパ芽球様細胞株の樹立のためのシンプルな赤血球溶解法
Summary
B-LCLを確立するための単純な赤血球溶解方法は、少量の血液を必要とし、凍結保存し、開始からの時間を節約し、高い不死化効率で開発されました。
Abstract
A number of methods exist for the transformation of B lymphocytes by the Epstein Barr virus in vitro into immortalized cell lines. We have developed a new method with a powerful and simple strategy for the establishment of B-LCLs, the red blood cell lysis method. This method simplified the PBMC separation procedure with red blood cell removal, and used as little as 0.5 mL of whole blood for establishing EBV-immortalized cell lines, which can proliferate to large cell numbers in a relatively short amount time with a 100% success rate. The method is simple, reliable, time saving, and applicable to treating a large number of the clinical samples.
Introduction
Resting B cells could be transformed by the Epstein-Barr virus (EBV) in vitro into actively proliferating B lymphoblastoid cell lines (B-LCLs). B-LCLs are similar to germ cells in differentiation and development, and the somatic mutation rate of B-LCLs was only 0.3%1, with negligible genetic and phenotypic alteration. Therefore, B-LCLs, as surrogates for peripheral blood mononuclear cells (PBMCs), have substantially accelerated the progress of genomics, transcriptomics and proteomics study of EBV associated diseases2,3. Moreover, B-LCLs also have important application in screening monoclonal antibodies4,5.
Several methods have been developed for the immortalization of B lymphocytes by EBV. The most major common procedures include the isolation of B lymphocytes and the use of B95-8 cell lines to produce infectious EBV. From isolation to transformation, these methods are divided into three strategies. Lymphocytes are separated from fresh blood by density gradient centrifugation and EBV transformation directly6-8, named density gradient separation method in this study. However, other techniques overcome the difficulty of shipping fresh blood and use a procedure for freezing purified lymphocytes and subsequent thawing and EBV infection9-11. Cryopreserved whole blood can also be used for the isolation of B lymphocytes and transformation12,13. In addition to density gradient centrifugation to separate B lymphocytes, some researchers use a magnetic cell sorter to select B lymphocytes12. However, magnetic separation is an expensive, complex and time consuming process. Although these methods are useful to immortalize B lymphocytes with a high success rate, the need to treat a large number of clinical samples and the relatively small volume of blood require a simpler method for the establishment of a permanent cell line.
Another less commonly used method is using whole blood as the source of nucleated cells for EBV infection and the establishment of B-LCLs. These methods simplify the technique by omitting the separation procedure. Only a small amount of whole blood, either fresh14 or frozen15,16, is sufficient to obtain the cell lines. Unfortunately, there is great variability in success from documents15,16 and several of our validation tests. Furthermore, transformed colonies are hardly recognized under a microscope because of the large contaminating impurities. Thus, a more reliable method for transformation is required.
Based on the above considerations, we have developed a novel method to establish B-LCLs without previous purification of the lymphocytes, named the red blood cell lysis method. This method is convenient, time saving and applicable to a large number of samples. As little as 0.5 mL of whole blood was enough to obtain B-LCLs that are easily obtainable from infants and the elderly without significantly harming their health.
Protocol
Representative Results
Discussion
我々は、赤血球を溶解することによって、全血からのヒトB細胞を不死化するための新規な方法の開発を報告し、そして確立されたB-LCLを、その細胞生存率をMTTアッセイにより評価しました。結果は、赤血球溶解法により確立B-LCLをの細胞生存率は、密度勾配分離法よりもはるかに高いことが示されました。赤血球溶解法の主な利点は、簡単であり、高い成功率と高い細胞生存率と永久細胞株?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
This work was supported by the key projects of Chinese Academy of Sciences (KFZD-SW-205), strategic biological resources technology support system of Chinese Academy of Sciences (CZBZX-1).
Materials
| Centrifuge | Techcomp | CT6T | Centrifugation |
| Automated Cell Counter | Countstar | IC 1000 | For cell counting |
| Epoch Microplate Spectrophotometer | BioTek Instruments | SN263839 | For measuring the absorbance |
| 96 well cell culture cluster | Corning Incorporated | 3599 | Polystyrene plates |
| 24 well cell culture cluster | Corning Incorporated | 3524 | Polystyrene plates |
| 25cm2 cell culture flask | Nest | 707001 | Polystyrene |
| FBS | Gibco | 10270 | Component of B-LCLs medium |
| RPMI Medium 1640 | Gibco | 31800-022 | For B-LCLs medium |
| L-Glutamine | Amresco | 0374 | Component of B-LCLs medium |
| 100 x streptomycin penicillin solution BioRoYeeBRY-2309 | BioRoYee | BRY-2309 | Component of B-LCLs medium |
| Ficoll paque plus | GE Healthcare | 17-1440-03 | For in vitro isolation of lymphocyte |
| PHA-M | Sigma | L8902 | For stimulating lymphocyte proliferation |
| Cyclosporin A | Cayman Chemical | 12088 | For inhibiting the cytotoxicity effect of T cells |
| Red cell lysis buffer | Tiangen | RT122-01 | For lysing red blood cell |
| MTT | Amresco | 0793 | For the detection of cell viability |
| DMSO | Sigma | D2650 | For freezing cells |
References
- Mohyuddin, A., et al. Genetic instability in EBV-transformed lymphoblastoid cell lines. Biochim Biophys Acta. 1670 (1), 81-83 (2004).
- Hu, V. W., Frank, B. C., Heine, S., Lee, N. H., Quackenbush, J. Gene expression profiling of lymphoblastoid cell lines from monozygotic twins discordant in severity of autism reveals differential regulation of neurologically relevant genes. BMC Genomics. 7, 118 (2006).
- Toda, T., Sugimoto, M. Proteome analysis of Epstein-Barr virus-transformed B-lymphoblasts and the proteome database. J Chromatogr B Analyt Technol Biomed Life Sci. 787 (1), 197-206 (2003).
- Chiorazzi, N., Wasserman, R. L., Kunkel, H. G. Use of Epstein-Barr virus-transformed B cell lines for the generation of immunoglobulin-producing human B cell hybridomas. J Exp Med. 156 (3), 930-935 (1982).
- Traggiai, E., et al. An efficient method to make human monoclonal antibodies from memory B cells: potent neutralization of SARS coronavirus. Nat Med. 10 (8), 871-875 (2004).
- Hussain, T., Kotnis, A., Sarin, R., Mulherkar, R. Establishment & characterization of lymphoblastoid cell lines from patients with multiple primary neoplasms in the upper aero-digestive tract & healthy individuals. Indian J Med Res. 135 (6), 820-829 (2012).
- Neitzel, H. A Routine Method for the Establishment of Permanent Growing Lymphoblastoid Cell-Lines. Hum Genet. 73 (4), 320-326 (1986).
- Oh, H. M., et al. An efficient method for the rapid establishment of Epstein-Barr virus immortalization of human B lymphocytes. Cell Prolif. 36 (4), 191-197 (2003).
- Louie, L. G., King, M. C. A Novel-Approach to Establishing Permanent Lymphoblastoid Cell-Lines – Epstein-Barr-Virus Transformation of Cryopreserved Lymphocytes. Am J Hum Genet. 48 (3), 637-638 (1991).
- Tremblay, S., Khandjian, E. V. Successful use of long-term frozen lymphocytes for the establishment of lymphoblastoid cell lines. Clin Biochem. 31 (7), 555-556 (1998).
- Reidy, J. A., Wheeler, V. A. Sample age and Epstein-Barr virus transformation of cryopreserved lymphocytes. In Vitro Cell Dev Biol. 28 (6), 383-384 (1992).
- Amoli, M. M., Carthy, D., Platt, H., Ollier, W. E. R. EBV immortalization of human B lymphocytes separated from small volumes of cryo-preserved whole blood. Int J Epidemiol. 37, 41-45 (2008).
- Lacelle, C., Wang, E. Establishing lymphoblastoid cell lines from frozen blood of extremely old individuals. Mech Ageing Dev. 123 (10), 1415-1418 (2002).
- Tohda, H., Oikawa, A., Kudo, T., Tachibana, T. A greatly simplified method of establishing B-lymphoblastoid cell lines. Cancer Res. 38 (10), 3560-3562 (1978).
- Ventura, M., et al. Use of a Simple Method for the Epstein-Barr Virus Transformation of Lymphocytes from Members of Large Families of Reunion Island. Hum Hered. 38 (1), 36-43 (1988).
- Chenevixtrench, G., Kerr, B., Walters, M. Lymphoblastoid Cell-Lines from Frozen Whole-Blood – a Quick and Economical Safeguard for Linkage Analysis. Am J Hum Genet. 46 (1), 635-636 (1990).
- Hansen, M. B., Nielsen, S. E., Berg, K. Re-Examination and Further Development of a Precise and Rapid Dye Method for Measuring Cell-Growth Cell Kill. J Immunol Methods. 119 (2), 203-210 (1989).
- Bogoslovsky, T., et al. Preservation and enumeration of endothelial progenitor and endothelial cells from peripheral blood for clinical trials. Biomark Med. 9 (7), 625-637 (2015).
- Ambach, A., Bonnekoh, B., Gollnick, H. Routine flow cytometric immuno-staining of T-cell perforin is preserved using diethylene glycol for erythrocyte-lysis but lost by the use of ammonium chloride. Exp Dermatol. 12 (6), 825-831 (2003).
- Beck, J. C., Beiswanger, C. M., John, E. M., Satariano, E., West, D. Successful transformation of cryopreserved lymphocytes: A resource for epidemiological studies. Cancer Epidemiol Biomarkers Prev. 10 (5), 551-554 (2001).
- Tosato, G., Cohen, J. I. Generation of Epstein-Barr Virus (EBV)-immortalized B cell lines. Curr Protoc Immunol. , 22 (2007).
