Supplementary Material for: Establishment and Characterization of an SV40 Large T Antigen-Transduced Porcine Colonic Epithelial Cell Line

Continuous cell lines have become indispensable tools that have enabled investigations into cellular mechanisms by increasing experimental reproducibility and sample availability, and decreasing the use of experimental animals. To facilitate studies of epithelial barrier function of the porcine colon, we aimed to establish an epithelial cell line with an extended replicative capacity. Cells were isolated from the proximal colon of a 3-week-old piglet and transduced using a recombinant retroviral vector construct containing the simian virus 40 large T antigen (SV40 TAg). We established a clonal epithelial cell line, referred to as PoCo83-3, that stably expressed the SV40 TAg, verified at mRNA and protein levels. PoCo83-3 showed epithelial cell-specific features, such as cobblestone-like morphology, dome structure formation, the presence of apical microvilli, and the expression of keratin 18, E-cadherin and the tight junction-associated proteins zonula occludens-1, occludin, and claudin-1. To validate PoCo83-3 as an in vitro model in epithelial barrier research, proinflammatory cytokine-inducible alterations in barrier integrity were demonstrated by incubating the cells with TNF-α and IFN-γ for 48 h. These cytokine treatments promoted a decreased transepithelial electrical resistance. In summary, PoCo83-3 exhibited an extended life span and a differentiated phenotype while maintaining epithelial characteristics. Based on these results, we present this cell line as a valuable in vitro model for investigations of epithelial barrier function in the porcine colon.

连续细胞系（Continuous cell lines）已成为不可或缺的科研工具，通过提升实验可重复性、增加样本可获得性并减少实验动物使用量，为细胞机制研究提供了关键支撑。为推动猪结肠上皮屏障功能相关研究，本研究旨在构建一株具备延长复制能力的上皮细胞系。研究人员从一头3周龄仔猪的近端结肠中分离细胞，并使用携带猿猴病毒40大T抗原（SV40 TAg）的重组逆转录病毒载体构建体进行转导。我们成功构建了一株命名为PoCo83-3的克隆化上皮细胞系，该细胞系可稳定表达SV40 TAg，这一结果在mRNA和蛋白水平均得到验证。PoCo83-3具备上皮细胞特异性特征：呈现鹅卵石样形态、可形成穹窿结构、存在顶端微绒毛，且表达角蛋白18（keratin 18）、上皮钙黏蛋白（E-cadherin）以及紧密连接相关蛋白闭锁小带蛋白-1（zonula occludens-1）、闭合蛋白（occludin）和claudin-1。为验证PoCo83-3作为上皮屏障研究体外模型的适用性，我们将细胞与肿瘤坏死因子α（TNF-α）和干扰素γ（IFN-γ）共孵育48小时，证实促炎细胞因子可诱导上皮屏障完整性发生改变。该细胞因子处理可导致跨上皮电阻（transepithelial electrical resistance, TEER）显著降低。综上，PoCo83-3具备延长的体外增殖寿命与分化表型，同时保留上皮细胞特性。基于上述研究结果，我们推荐该细胞系作为研究猪结肠上皮屏障功能的优质体外模型。