Differentiated Primary Human Bronchial Epithelial Cell-Fibroblast Co-Culture Model

pHBEC were obtained via bronchial brushing from healthy, donors ages 18–40 that were not currently smoking and had no more than a one pack-year of lifetime smoking history. Donors gave their consent after being informed of risks and procedures. The consent and collection protocol were approved by the UNC School of Medicine Committee on the Protection of the Rights of Human Subjects and by the US EPA. Collection of pHBEC from volunteers was performed in accordance with relevant guidelines and regulations. pHBEC were isolated from brush biopsy (“passage 0”) and expanded to passage 3 prior to being plated on 12 mm Transwell^® inserts (Corning #3460; 0.4 μm pore polyester membranes), becoming confluent, and differentiating for 24 days under ALI conditions. Detailed descriptions of the techniques, reagents, and materials used for the culture/expansion of pHBEC and differentiation of pHBEC at ALI used in this study are available as open access methods documents¹³ . Day 24 ALI cultures were visually evaluated for the presence of beating cilia and the production of mucus as indicators of differentiation status to qualify their use in experiments. Donor demographics are listed in Supplementary Table 1. All cultures were at ALI day 24 at the beginning of experiments. The human lung fibroblast cell line IMR90^{14 (link)} was obtained from the American Type Culture Collection (ATCC, No. CCL-186, Batch #64155514), and cultured as described in detail in the open-access methods document¹⁵ . Short tandem repeat (STR) service provided by ATCC (cat #135-XV) was used to authenticate IMR90 cells (Supplementary Fig. 1). As described in Fig. 1A, IMR90 fibroblasts were seeded onto a collagen-coated 12-well plate (Corning, #3513) at a density of 1×10⁵ cells/mL in 800 μL of Pneumacult ALI medium (StemCell Technologies, #05001). The following day, day 23 dpHBEC-ALI inserts were added into the fibroblast-seeded wells (dpHBEC-IMR90 ALI model). The dpHBEC-IMR90 ALI model was used for experiments on the following day. The dpHBEC-IMR90 ALI model was maintained at ALI or subjected to the addition of 250 μL (223 μL/cm²; equal to 73.5 μL in a 6.5 mm Transwell insert) of ALI medium to the apical surface of the culture (“liquid application”). This was the smallest volume of ALI medium that resulted in the most uniform coverage of the dpHBEC cultures during the 24-hour treatment duration as determined by a titration of apical volumes of a 0.5% Crystal Violet (Sigma, #C6158–50G; dissolved in Dulbecco’s Phosphate Buffered Saline (Gibco #14190–144)) stain solution to 12 mm Transwell inserts (Fig. 1B). Inserts were placed on a white light transilluminator (FUJIFILM #IPE4046) and photographed with an iPhone SE (Apple, Model #MX9M2LL/A). The cells were kept submerged in ALI medium for either 6 or 24 hours to represent “early” and “late” effects, respectively. Additionally, the 24-hour treatment is consistent with the liquid application treatment duration utilized in the recent OECD case study on the use of an ALI-differentiated in vitro NAM for an IATA to refine inhalation risk assessment for point of contact toxicity^{12 (link),16 –18} . The effect of liquid application on global gene expression, stress-responsive signaling protein phosphorylation, growth factor secretion, pro-inflammatory cytokine secretion, trans-epithelial electrical resistance (TEER), and permeability to a 20 kDa fluorescent dextran was then determined immediately after 6 and 24 hours of liquid application (Fig. 1A).