Department of Medical Microbiology School of Health, Namik Kemal University, Takirdağ, Turkey.
Int Immunopharmacol. 2012 May;13(1):61-8. doi: 10.1016/j.intimp.2012.03.009. Epub 2012 Mar 27.
In this study, we examined the effects of cryoprotectant, freezing and thawing, and adenovirus (Adv) transduction on the viability, transgene expression, phenotype, and function of human dendritic cells (DCs). DCs were differentiated from cultured peripheral blood (PB) monocytes following Elutra isolation using granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) for 6 days and then transduced using an Adv vector with an IL-12 transgene. Fresh, cryopreserved, and thawed transduced immature DCs were examined for their: 1) cellular concentration and viability; 2) antigenicity using an allogeneic mixed lymphocyte reaction (MLR); 3) phenotype (HLA-DR and CD11c) and activation (CD83); and 4) transgene expression based on IL-12 secretion. Stability studies revealed that transduced DCs could be held in cryoprotectant for as long as 75 min at 2-8°C prior to freezing with little effect on their viability and cellularity. Further, cryopreservation, storage, and thawing reduced the viability of the transduced DCs by an average of 7.7%; and had no significant impact on DC phenotype and activation. In summary, cryopreservation, storage, and thawing had no significant effect on DC viability, function, and transgene expression by Adv-transduced DCs.
在这项研究中，我们研究了冷冻保护剂、冷冻和解冻以及腺病毒（Adv）转导对人树突状细胞（DC）的活力、转基因表达、表型和功能的影响。使用粒细胞巨噬细胞集落刺激因子（GM-CSF）和白细胞介素-4（IL-4）从培养的外周血（PB）单核细胞中分离出 Elutra 后，将 DC 分化 6 天，然后使用带有 IL-12 转基因的 Adv 载体进行转导。新鲜、冷冻保存和解冻的转导未成熟 DC 用于检查：1）细胞浓度和活力；2）同种异体混合淋巴细胞反应（MLR）的抗原性；3）表型（HLA-DR 和 CD11c）和激活（CD83）；4）基于 IL-12 分泌的转基因表达。稳定性研究表明，在 2-8°C 下，转导的 DC 可以在冷冻保护剂中保存长达 75 分钟，而对其活力和细胞数量几乎没有影响。此外，冷冻保存、储存和解冻会降低转导 DC 的活力，平均降低 7.7%；对 DC 表型和激活没有显著影响。总之，冷冻保存、储存和解冻对 Adv 转导的 DC 的活力、功能和转基因表达没有显著影响。
