Fig. 2
From: A human relevant in vitro alveolar epithelial barrier model to assess inhaled pollutant hazard
Cell count, viability and barrier function of epithelial co-cultures and monocultures in different media types. Total cell counts (A), viability (B) and barrier function (C) of hAELVi monocultures, NCI-H441 monocultures, or hAELVi/NCI-H441 co-cultures (black, dark grey and light bar bars, respectively) at 96 h post seeding using LUNA II automated cell counter under submerged conditions for 72 h before being exposed to the ALI for 24 h. The barrier integrity (C) was measured using blue dextran assay where the absorbances have been normalised to a negative control (cell culture insert containing no cells for each media type) each media type possess varying background absorbances. The cells were cultured in either RPMI 1640 (NCI-H441 media), huAEC media (hAELVI media), or a 50/50 mixture of the two media types (50/50 media). The mean reading of three biological replicates is plotted ± SEM. Difference was assessed for via a two-way ANOVA utilising a Tukeys post hoc test. Seeding density seeded at 2.5 × 105 or 5 × 105 cell/mL (2.5 × 105–2.25 × 105 hAELVi & 2.5 × 104 NCI-H441. 5 × 105–4.5 × 105 hAELVi and 5 × 105 NCI-H441) were compared for total cell count (D), viability (E) and blue dextran (F) for co-cultures seeded at 2.5 × 105 or 5 × 105 cell/mL (2.5 × 105 – 2.25 × 105 hAELVi & 2.5 × 104 NCI-H441. 5 × 105 – 4.5 × 105 hAELVi and 5 × 105 NCI-H441) (submerged conditions for 72 h before being exposed to ALI for 24 h). NCI-H441 monocultures were assessed for their ability to grow on surfaces coated with huAEC coating solution shown through viability (G), total cell count (H), membrane integrity (I), IL-6 release (J), IL-8 release (K) and DAPI/phalloidin staining (L). * = p ≤ 0.05 ** = p ≤ 0.01, *** = p ≤ 0.001, shown through unpaired t-test. Bars show mean ± SEM across three biological replicates (n = 3).
