Establishment of novel immortalized middle ear cell lines as models for otitis media

Objective: Otitis media (OM) is among the most frequently diagnosed pediatric diseases in the US. Despite the significant public health burden of OM and the contribution research in culture models has made to understanding its pathobiology, a singular immortalized human middle ear epithelial (MEE) cell line exists (HMEEC-1, adult-derived). We previously developed MEE cultures from pediatric patients with non-inflamed MEE (PCI), recurrent OM (ROM), or OM with effusion (OME) and demonstrated differences in their baseline inflammatory cytokine expression and response to stimulation with an OM-relevant pathogen lysate and cytokines. Herein, we sought to immortalize these cultures and assess retention of their phenotypes.Methods: MEE cultures were immortalized via lentivirus encoding temperature-sensitive SV40 T antigen. Immortalized MEE lines and HMEEC-1 grown in monolayer were stimulated with non-typeable Haemophilus influenzae (NTHi) lysate. Gene expression (TNFA, IL1B, IL6, IL8, MUC5AC, and MUC5B) was assessed by qPCR.Results: Similar to parental cultures, baseline cytokine expressions were higher in pediatric OM lines than in HMEEC-1 and PCI, and HMEEC-1 cells were less responsive to stimulation than pediatric lines.Conclusion: Immortalized MEE lines retained the inflammatory expression and responsiveness of their tissues of origin and differences between non-OM versus OM and pediatric versus adult cultures, supporting their value as novel in vitro culture models for OM.