老年人容易感染病毒，这在社会上已广为人知。但由于缺乏合适的病毒感染模型，在体外研究中还未获得适当的测试。在本报告中，我们研究了年龄对假多层平面空气液体界面(ALI)培养的支气管上皮细胞对呼吸道合胞病毒(RSV)的影响。这种细胞培养方式在形态学和生理上比沉浸在癌细胞系培养中更能模拟人体气道上皮。我们向来自 8 个年龄不同(28-72 岁)的捐赠者获取的支气管上皮细胞中部接种 RSV A2 病毒，分析病毒载量和炎症细胞因子的时间轴。结果表明，RSV A2在ALI培养的支气管上皮细胞中有良好的复制效果，而年龄≤60 岁(n=4)和> 65 岁(n=4，即老年组)的捐赠者的病毒高峰日和病毒载量峰值相似，但老年组的病毒清除能力较差。此外，从病毒载量峰值到采样结束(接种3-10天)计算的面积下曲线(AUC)分析显示，在老年组病毒活载量(PFU检测)和病毒基因组数目(PCR检测)均更高，并且病毒载量与年龄呈正相关。此外，RANTES、LDH和dsDNA(cell damage marker)的AUC在老年组中也显著更高，老年组表现出CXCL8、CXCL10和粘液产生AUC的趋势更高。而老年组在基线上的p21CDKN1A(细胞衰老标志物)基因表达也较高，基线p21表达与病毒载量或RANTES(AUC)之间呈良好的正相关。在ALI-culture模型中，年龄是影响病毒动力学和生物标志物的关键因素。目前，针对病毒研究，已经引入了新型或创新的体外细胞模型，但与处理其他临床样本时类似，年龄平衡对于获得更准确的结果非常重要。版权所有 © 2023 Ito、Daly 和 Coates。

Elderly people are known to be vulnerable to virus infection. However, this has not been appropriately tested in in vitro studies due to a lack of appropriate virus infection models. In this report, we investigated the impact of age on respiratory syncytial virus (RSV) in pseudostratified air-liquid-interface (ALI) culture bronchial epithelium, which more closely mimic human airway epithelium morphologically and physiologically, than submerged cancer cell line cultures.RSV A2 was inoculated apically to the bronchial epithelium obtained from 8 donors with different ages (28-72 years old), and time-profiles of viral load and inflammatory cytokines were analyzed.RSV A2 replicated well in ALI-culture bronchial epithelium. The viral peak day and peak viral load were similar between donors at ≤60 years old (n = 4) and  > 65 years old (n = 4; elderly group), but virus clearance was impaired in the elderly group. Furthermore, area under the curve (AUC) analysis, calculated from viral load peak to the end of sample collection (from Day 3 to 10 post inoculation), revealed statistically higher live viral load (PFU assay) and viral genome copies (PCR assay) in the elderly group, and a positive correlation between viral load and age was observed. In addition, the AUCs of RANTES, LDH, and dsDNA (cell damage marker) were statistically higher in the elderly group, and the elderly group showed a trend of higher AUC of CXCL8, CXCL10 and mucin production. The gene expression of p21CDKN1A (cellular senescence marker) at baseline was also higher in the elderly group, and there was a good positive correlation between basal p21 expression and viral load or RANTES (AUC).Age was found to be a key factor affecting viral kinetics and biomarkers post virus infection in an ALI-culture model. Currently, novel or innovative in vitro cell models are introduced for virus research, but when virus studies are conducted, similarly to working with other clinical samples, the age balance is important to obtain more accurate results.Copyright © 2023 Ito, Daly and Coates.