A Human Vocal Fold Organ-On-Chip for Studying Platform-Dependent Mucosal Responses to Particulate Matter

Coarse particulate matter (PM₁₀) deposits at the vocal fold (VF) mucosa, yet upper airway responses remain poorly characterized. Existing in vitro VF models use monocultures that lack the stratified epithelium, lamina propria, and physiological perfusion. We developed a chip-based co-culture model of human VF mucosa and applied it to acute PM₁₀ exposure.

Vocal fold organ-on-chip (VF-OOC) paired primary VF fibroblasts with either immortalized laryngeal epithelial cells (iLEC) or induced pluripotent stem cells (iPSC)-derived VF epithelial cells. Transwell and 2D chip cultures were controls. Epithelia matured at an air-liquid interface on fibroblast-embedded collagen over a perfused microchannel. PM₁₀ urban dust (0 to 400 μg/mL) was applied for 24 hours. Responses were assessed by histology, immunofluorescence, transmission electron microscopy, qPCR, and ELISA.

VF-OOC produced a thicker stratified epithelium with upregulated barrier, mucin, and extracellular matrix genes versus transwell controls. Intercellular junctions and basement membrane matched adult human VF mucosa. PM₁₀ remained at the epithelial surface across all doses. Transwell_iLEC downregulated basal markers (TP63, KRT5, KRT14). VF-OOC_iLEC upregulated MUC1 and HAS3, consistent with an adaptive mucosal response. VF-OOC_iPSC additionally induced suprabasal, junctional, extracellular matrix, and cytokine genes largely absent in iLEC and transwell formats.

VF-OOC reproduced key features of native human VF mucosa and captured distinct, platform-specific responses to PM₁₀ that differed by epithelial cell source. By introducing new approach methodologies (NAMs) into laryngology, this platform extends respiratory toxicology to the upper airway beyond bronchial and alveolar compartments and allows mechanistic studies of exposure-linked diseases such as laryngitis.