Journal: Cell Reports Methods

Article Title: Rapid expansion of primary human vocal fold epithelial cells via targeted pathway inhibition and anchorage-independent sphere culture

doi: 10.1016/j.crmeth.2026.101310

Figure Lengend Snippet: Simultaneous TGF-β, ROCK, and Notch pathway inhibition promotes VFE proliferation while maintaining core epithelial phenotype (A) Effect of candidate small-molecule inhibitors on VFE proliferation. Cells (5 × 10 4 ) were incubated with 1 μM A-83-01 (TGF-β inhibitor), 10 μM Y-27632 (ROCK inhibitor), and 5 μM DAPT (Notch inhibitor), as indicated. Counts were performed at day 9; data are plotted as mean ± SEM ( n = 6); p values were obtained using mixed-model ANOVA with planned pairwise comparisons shown. Additional dose-response data for DAPT are presented in . (B) Experimental conditions used for subsequent experiments. Cells in the 3i-VFE condition were incubated with a three-molecule cocktail of 1 μM A-83-01, 10 μM Y-27632, and 5 μM DAPT; cells in the VFE condition were incubated with DMSO vehicle. (C) Bright-field images of live cells at passage 3; all cells exhibited cuboidal morphology over serial passages. Scale bar, 20 μm. (D) Single-passage growth curves and population doubling times. Data are plotted as mean ± SEM ( n = 4); p values were obtained using mixed-model ANOVA. (E) Flow cytometry data showing KRT14 and KRT19 expression. Positive/negative gates (versus unstained controls) are shown in gray. Data are plotted as mean ± SEM ( n = 3); p values were obtained using a paired t test. (F) RT-qPCR data showing TP63 , PROM1 , KIT , CDH1 , and MUC1 transcription; TUBB is reported as an additional reference standard. Data are presented as fold change relative to VFE (mean ± SEM; n = 3); p values were obtained using a paired t test. (G) Transepithelial electrical resistance. Data are plotted as mean ± SEM ( n = 6); the p value was obtained using a paired t test. (H) Flow cytometry data showing CD90 and MUC1 (also known as CD227) expression; VFFs are included as a non-epithelial cell control. High/low gates are shown in black. Data are plotted as mean ± SEM ( n = 3); p values were obtained using paired and unpaired t tests. Note that the 3i-VFE data (light blue) are largely superimposed on the VFE data (dark blue) in the dot plot.

Article Snippet: VFE were seeded in 24-well plates (Corning) at a density of 5 × 10 4 cells per well, cultured in VFE-orientated medium until ∼30% confluent, serum starved for 12 h, then incubated with various combinations of 1 μM TGF-β inhibitor A-83-01, 10 μM ROCK inhibitor Y-27632, and 5 μM Notch inhibitor DAPT (Selleck Chemicals), each prepared from a 1,000× stock solution in DMSO (Thermo Fisher).

Techniques: Inhibition, Incubation, Flow Cytometry, Expressing, Quantitative RT-PCR, Control