In vitro EBV Infection of Mononuclear Cells that Have Been Cryo-preserved

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Journal of Virology
Dec 2013



Epstein-Barr Virus (EBV) is a B-lymphotropic herpesvirus which the majority of adult human population is latently-infected with. Various immunological and molecular in vitro studies have been facilitated by the use of EBV’s ability to infect and transform B cells to immortalized polyclonal B cell lines. Many of these studies use freshly isolated cord-blood mononuclear cells (CBMC). Some experiments may, however, require EBV infection of samples that have been prospectively collected and cryo-preserved. Here we share a protocol that we used to successfully infect B cells from cryo-preserved CBMCs and peripheral-blood mononuclear cells (PBMC) (Sohlberg et al., 2013; Saghafian-Hedengren et al., 2013).

Materials and Reagents

  1. Cryo-preserved CBMC
  2. Cryo-preserved PBMC
  3. EBV strain B95-8 containing supernatant
    Note: The batch here had a titer of 2.5 x 105 Ramos infectious units. RaIU, which was determined by infection of the EBV negative Burkitts lymphoma B-cell line Ramos, followed by anti-complement immunoflourescent assay (ACIF) to detect the number of infected cells.
  4. RPMI 1640 (Life Technologies)
  5. 10% heat-inactivated fetal-calf serum (Hyclone)
  6. L-glutamine (2 mmol/l)
  7. Penicillin G-sodium (100 U/ml)
  8. Streptomycin sulfate (100 mg/ml) (Merck KGaA)
  9. Complete cell-culture medium (see Recipes)


  1. 48-well flat-bottomed tissue-culture treated plates (Sarstedt AG)
  2. Humified incubator with for adjustment of 37 °C and 5% CO2 for cell culture


Note about sample handling: Gentle and fast processing of samples during isolation of mononuclear cells contributes to higher B cell viability, which is central for successful in vitro infection.

  1. Thaw CBMCs or PBMCs quickly at room temperature and wash two times with incomplete RPMI-1640 at 350-400 x g and 5-10 min at room temperature.
  2. Determine cell numbers, and if applicable, divide cells to fractions that will be EBV infected and those that will serve as non-infected controls.
    Note: Remember that the cell numbers and concentrations in each well should be matched for the infected and non-infected fractions.
  3. Wash an additional time with RPMI 1640.
  4. For non-infected control cultures: Re-suspend cells in complete cell-culture medium to a concentration of 106 cells/ml and allocate to the appropriate wells in the plate. Incubate at 37 °C and 5% CO2.
  5. For EBV infection, discard supernatant completely and resuspend 106 cells per 100 μl B95-8 virus–containing supernatant in a small tube. Incubate at 37 °C and 5% CO2 for 90 min with gentle mixing by swirling the tube every 30 min.
  6. Wash the cells once with complete cell-culture medium, discard supernatant and re-suspended in complete cell-culture medium to a concentration of 106 cells/ml and then allocate to the appropriate wells in the plate.
  7. Feed cells with complete medium on a weekly basis. The proportion of EBV-transformed B cells will increase with time and eventually these cells take over the entire culture. As we assessed the dynamics of NK and T cell and their functional capacity (Sohlberg et al., 2013; Saghafian-Hedengren et al., 2013) in our previous experimental settings, we found a 1-2 week-period of time suitable for this purpose.
  8. The presence of EBV-infected cells can be confirmed by immunofluorescence staining for latent membrane protein-1 and EBV nuclear Ag 2 (Rasul et al., 2012).

Representative data

Refer to Figure 1 in Sohlberg et al. (2013) for representative data on B cell characteristics following in vitro EBV infection of B cells.


  1. Careful and fast processing of samples during isolation of mononuclear cells contributes to higher B cell viability, which is central for successful downstream in vitro EBV infection.
  2. This protocol uses cells from EBV seronegative donors. Deplete T cells (by for instance magnetic-based methods to remove CD3+ cells from StemCellTM or Miltenyi Biotech) prior to EBV in vitro infection in case samples are from EBV seropositive donors.


  1. Complete cell-culture medium
    10% heat-inactivated fetal-calf serum
    L-glutamine (2 mmol/l)
    Penicillin G-sodium (100 U/ml)
    Streptomycin sulfate (100 mg/ml)


This protocol has been adapted from the previously published paper Sohlberg et al., (2013). At the time of development and implementation of this protocol, the following sources of funding were used: Swedish Research Council Grants 57X-15160-07-03 and 57X-15160-10-4, the Swedish Association for Allergology, the Ragnar Söderberg Foundation, the Ellen, Walter and Lennart Hesselman Foundation, the Konsul Th. C. Bergh Foundation, the Golden Jubilee Memorial Foundation, the Petrus and Augusta Hedlund Foundation, the Crown Princess Lovisa’s/Axel Tielman’s Foundation, the Swedish Cancer Society, and the Karolinska Institute. E.R. and N.N. are recipients of cancer research fellowships from the Cancer Research Institute (New York)/Concern Foundation (Los Angeles). We thank Ehsan Rasul and Eva Klein (Department of Microbiology, Tumor and Cell Biology, KI, Stockholm, Sweden) for their contribution in optimizing the protocol.


  1. Rasul, A. E., Nagy, N., Sohlberg, E., Adori, M., Claesson, H. E., Klein, G. and Klein, E. (2012). Simultaneous detection of the two main proliferation driving EBV encoded proteins, EBNA-2 and LMP-1 in single B cells. J Immunol Methods 385(1-2): 60-70.
  2. Saghafian-Hedengren, S., Sohlberg, E., Theorell, J., Carvalho-Queiroz, C., Nagy, N., Persson, J. O., Nilsson, C., Bryceson, Y. T. and Sverremark-Ekström, E. (2013). Epstein-barr virus coinfection in children boosts cytomegalovirus-induced differentiation of natural killer cells. J Virol 87(24): 13446-13455.
  3. Sohlberg, E., Saghafian-Hedengren, S., Rasul, E., Marchini, G., Nilsson, C., Klein, E., Nagy, N., Sverremark-Ekström, E. (2013). Cytomegalovirus-seropositive children show inhibition of in vitro EBV infection that is associated with CD8+CD57+ T cell enrichment and IFN-γ. J Immunol 191(11): 5669-5676.


爱泼斯坦 - 巴尔病毒(EBV)是一种B淋巴细胞疱疹病毒,其大多数成年人群潜在感染。 通过使用EBV感染和将B细胞转化到永生化多克隆B细胞系的能力,已经促进了各种免疫学和分子外体外研究。 许多这些研究使用新鲜分离的脐带血单核细胞(CBMC)。 然而,一些实验可能需要EBV感染已经预期收集和冷冻保存的样品。 在这里我们共享一个协议,我们用于成功感染来自低温保存的CBMC和外周血单核细胞(PBMC)的B细胞(Sohlberg等人,2013; Saghafian-Hedengren等人 。,2013)。


  1. 低温保存的CBMC
  2. 冷冻保存的PBMC
  3. EBV菌株B95-8含有上清液
    注意:这里的批次的滴度为2.5 x 10 5 Ramos感染单位。 RaIU,其通过EBV阴性Burkitts淋巴瘤B细胞系Ramos的感染测定,随后是抗补体免疫荧光测定(ACIF)以检测感染细胞的数目。
  4. RPMI 1640(Life Technologies)
  5. 10%热灭活胎牛血清(Hyclone)
  6. L-谷氨酰胺(2mmol/l)
  7. 青霉素G钠(100U/ml)
  8. 链霉素硫酸盐(100mg/ml)(Merck KGaA)
  9. 完整的细胞培养基(参见配方)


  1. 48孔平底组织培养处理的平板(Sarstedt AG)
  2. 具有用于调节37℃和5%CO 2的细胞培养的湿润培养箱



  1. 在室温下迅速解冻CBMC或PBMC,并用350-400×g的不完全RPMI-1640洗涤两次,室温下5-10分钟。
  2. 确定细胞数量,如果适用,将细胞分为将被EBV感染的分数和将作为未感染对照的分数。
  3. 用RPMI 1640再冲洗一次。
  4. 对于未感染的对照培养物:将完全细胞培养基中的细胞重悬浮至10 6个细胞/ml的浓度,并分配至板中的合适孔中。在37℃和5%CO 2下孵育
  5. 对于EBV感染,完全丢弃上清液,并在小管中将每100μl含B95-8病毒的上清液重悬106个细胞。在37℃和5%CO 2下孵育90分钟,伴随轻柔混合,每30分钟旋转管。
  6. 用完全细胞培养基洗涤细胞一次,弃去上清液并重悬于完全细胞培养基中至10 6个细胞/ml的浓度,然后分配到板中的合适孔中。
  7. 每周用完全培养基饲养细胞。 EBV转化的B细胞的比例将随时间增加,并且最终这些细胞接管整个培养物。当我们评估NK和T细胞的动力学 和它们的功能能力(Sohlberg等人,2013; Saghafian-Hedengren等人,2013)在我们先前的实验设置中,我们发现1-2周期 的时间适合此目的。
  8. EBV感染的细胞的存在可以通过对潜伏膜蛋白-1和EBV核Ag 2的免疫荧光染色来证实(Rasul等人,2012)。


参见图1在Sohlberg等人 (2013)关于B细胞体外EB病毒感染的B细胞特征的代表性数据。


  1. 在单核细胞分离期间仔细和快速处理样品有助于更高的B细胞活力,这是成功的下游体外EB病毒感染的中心。
  2. 该协议使用来自EBV血清阴性供体的细胞。 在EBV体外感染之前消耗T细胞(通过例如基于磁性的方法从StemCell TM或Miltenyi Biotech去除CD3 +细胞) 如果样品来自EBV血清阳性供体


  1. 完成细胞培养基
    L-谷氨酰胺(2mmol/l) 青霉素G钠(100U/ml) 链霉素硫酸盐(100mg/ml)


该协议已经从先前发表的论文Sohlberg等人,(2013)改编而来。在制定和实施本议定书时,使用了以下资金来源:瑞典研究委员会批准57X-15160-07-03和57X-15160-10-4,瑞典过敏协会,RagnarSöderberg基金会,艾伦,沃尔特和伦纳特·赫塞尔曼基金会,Konsul Th。 C. Bergh基金会,金禧纪念基金会,Petrus和Augusta Hedlund基金会,皇冠公主Lovisa的/Axel Tielman的基金会,瑞典癌症协会和Karolinska研究所。 E.R.和N.N.是来自癌症研究所(纽约)/关注基金会(洛杉矶)的癌症研究奖学金的接受者。我们感谢Ehsan Rasul和Eva Klein(微生物学,肿瘤和细胞生物学系,KI,斯德哥尔摩,瑞典)对优化方案的贡献。


  1. Rasul,A.E.,Nagy,N.,Sohlberg,E.,Adori,M.,Claesson,H.E.,Klein,G.and Klein,E。(2012)。 同时检测两种主要增殖驱动EBV编码的蛋白质,EBNA-2和LMP-1单一B细胞。 J Immunol Methods 385(1-2):60-70。
  2. Saghafian-Hedengren,S.,Sohlberg,E.,Theorell,J.,Carvalho-Queiroz,C.,Nagy,N.,Persson,JO,Nilsson,C.,Bryceson,YT and Sverremark-Ekström, )。 爱泼斯坦 - 巴尔病毒在儿童中的合并感染促进巨细胞病毒诱导的自然杀伤细胞分化。 J Virol 87(24):13446-13455。
  3. Sohlberg,E.,Saghafian-Hedengren,S.,Rasul,E.,Marchini,G.,Nilsson,C.,Klein,E.,Nagy,N.,Sverremark-Ekström, 巨细胞病毒 - 血清反应阳性的儿童显示出体外EB病毒感染的抑制作用 与CD8 + sup/+ CD57 + sup + T细胞富集和IFN-γ。 191(11):5669-5676 。
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引用:Saghafian-Hedengren, S., Sohlberg, E., Theorell, J., Carvalho-Queiroz, C., Nagy, N., Persson, J., Nilsson, C., Bryceson, Y. T. and Sverremark-Ekström, E. (2014). In vitro EBV Infection of Mononuclear Cells that Have Been Cryo-preserved. Bio-protocol 4(14): e1177. DOI: 10.21769/BioProtoc.1177.