Summary

A Simple Red Blood Cell Lysis Methode voor de oprichting van B lymfoblastoïde cellijnen

Published: January 14, 2017
doi:

Summary

Een eenvoudige rode bloedcel lysis methode voor het vaststellen van B-LCL werd ontwikkeld met hoge efficiency immortalisatie, die een kleine hoeveelheid bloed en tijdsbesparing van initiatie tot cryopreservatie.

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

Deze studie werd goedgekeurd door de ethische commissie van het Instituut voor Genetica en Developmental Biology, en het protocol volgt de institutionele richtlijnen voor het menselijk welzijn. 1. Bereiding van EB Virus Op dag 1, ontdooien B95-8 cellen en kweek in 6 ml compleet RPMI 1640 medium, aangevuld met 10% foetaal runderserum, 2 mM L-glutamine en antibiotica (100 pg / ml streptomycine, 100 U / ml penicilline) in een T25 kolf. Plaats de kolf in 5% CO2 incubator bij 37 ° C. …

Representative Results

Tijdens het transformatieproces worden de morfologische veranderingen van de cellen gevisualiseerd door lichtmicroscopie (figuur 2). Kleine clusters van cellen waren duidelijk zichtbaar met de dichtheidsgradiënt scheidingsmethode 7 dagen na infectie, terwijl slechts een dikke laag celfragmenten zijn zichtbaar vanaf de rode bloedcellen lysismethode. Echter, lymfoblastoïde cel clusters zijn zichtbaar onder de dikke laag celfragmenten 30 dagen na infectie. Met frequentere…

Discussion

Wij rapporteren de ontwikkeling van een nieuwe werkwijze voor het onsterfelijk maken van humane B cellen uit bloed door lyseren van rode bloedcellen en de cellevensvatbaarheid van gevestigde B-LCL werd geëvalueerd door de MTT-bepaling. De resultaten toonden aan dat de cellevensvatbaarheid van de bij de rode bloedcel lysis methode B-LCL veel hoger dan die van de dichtheidsgradiënt scheidingswerkwijze. Het grote voordeel van de rode bloedcellen lysis methode is dat het eenvoudig en vereist een klein volume (slechts 0,5 …

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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Cite This Article
Liu, X., Xu, C., Duan, Z. A Simple Red Blood Cell Lysis Method for the Establishment of B Lymphoblastoid Cell Lines. J. Vis. Exp. (119), e55191, doi:10.3791/55191 (2017).

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