All primary human tissues and nonblood cells were procured or purchased commercially with approval of the University of Wisconsin Health Sciences Institutional Review Board. Normal VFFs and epithelial cells were isolated from three donors (58-year-old Caucasian female, 61-year-old Caucasian male, and 66-year-old Caucasian male) undergoing total laryngectomy with no clinical evidence of VF pathology, as previously described (6). Normal DFs were purchased from the American Type Culture Collection (ATCC) (40-year-old Asian female) and Lonza (42-year-old African American female and 59-year-old Caucasian male).

Normal bone marrow–derived MSCs were purchased from ATCC or isolated from leftover clinical specimens as previously described (64). Briefly, cells were isolated from filters retained after bone marrow harvest from donors (with no known hematopoietic or immunologic disorders) to human leukocyte antigen-matched sibling recipients. Specimens were rinsed in phosphate-buffered saline (PBS), and mononuclear cells were isolated using Ficoll-Hypaque (1.073 g/ml; GE Healthcare) and Leucosep tubes (Greiner Bio-One), according to manufacturer protocols. Following ACK lysis of red blood cells (3-min incubation), mononuclear cells were suspended in α-MEM containing 10% FBS (HyClone), 1× nonessential amino acids, and 4 mM l-glutamine (Sigma-Aldrich). Cells were cultured on plastic at 37°C in 5% CO2 with medium change every 72 hours. Nonadherent cells were removed at the first medium change; adherent cells were passaged before confluence and characterized as MSCs according to criteria of the International Society for Cellular Therapy (59).

Before experimental use, primary VFFs, DFs, and MSCs were seeded at a density of 0.5 × 104 to 1.0 × 104 cells/cm2 on plastic and cultured in complete medium at 37°C in 5% CO2. Medium was changed every 48 to 72 hours. Primary VF epithelial cells were seeded at a density of 0.5 × 104 to 1.0 × 104 cells/cm2 on a matrix substrate [EBSS containing COL1 (30 μg/ml), fibronectin (10 μg/ml), and BSA (10 μg/ml)] (63) and cultured in epithelial medium at 37°C in 5% CO2. Medium was changed every 24 to 48 hours.

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