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.