Establishment and Characterization of an Immortalized Porcine Gastric Epithelial Cell Line

Epithelial cell lines are crucial for studying the molecular mechanisms, diseases, and digestive functions of the pig stomach. However, primary cell cultures have limitations, such as isolation challenges, short lifespan, and limited proliferation, highlighting the need for an improved in vitro model. In this study, we developed methods to isolated highly pure and viable porcine gastric epithelial cells (PGECs) using stepwise digestion and established an immortalized PGECs (i-PGECs) by transducing them with lentiviral vectors expressing simian virus 40 large T antigen (SV40T) and human telomerase reverse transcriptase (hTERT). Both SV40T and hTERT were successfully integrated into the i-PGECs. These cells retained the structure and function of PGECs, with positive expression of epithelial markers, including cytokeratin 18, EpCAM, and E-cadherin. The i-PGECs exhibited normal cell structure, growth rate, and karyotype. RNA-seq indicated that i-PGECs maintained their immortal properties by upregulating genes associated with cell proliferation pathways. Evaluation of the immune response of i-PGECs to aflatoxin B1 exposure revealed a rapid activation of immune-related factors, such as NPC1 and PLAUR. The i-PGECs, having exceeded 50 passages, confirm the reliability and potential of this model for in vitro investigations into the mechanisms of pathogen infection in the porcine gastric epithelium.