一个简单的红细胞溶解法对B淋巴母细胞系的建立
Summary
建立B-LCLS一个简单的红细胞裂解法与高效率的永生化开发，需要的少量血液，并节省了时间，从开始到冷冻保存。
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
我们报告的新方法的发展，为通过裂解红血细胞永生化从全血中的人类B细胞，和建立的B-LCLS通过MTT法测定其细胞生存力。结果表明，由红血细胞裂解法建立的B-LCLS的细胞存活率比密度梯度分离方法的高得多。红血细胞裂解方法的主要优点是，它是简单的，并且需要的血液小体积（低至0.5毫升）建立一个永久的细胞系具有高成功率和高细胞生存力。
只有EBV感染之前，还需要一?…
Disclosures
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 |
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