Search Results (89)

Cystic fibrosis (CF) is a genetic disorder caused by dysfunction of the CFTR chloride ion channel. Progress in molecular understanding and therapy development relies on advanced cellular models and robust assays for evaluating CFTR function. This review traces the evolution of in vitro models, from primary and immortalized cell lines to patient-specific induced pluripotent stem cells (iPSCs) and complex three-dimensional systems. These advanced models, including air-liquid interface (ALI) cultures, organoids, and microfluidic organ-on-a-chip platforms, enable recapitulation of tissue architecture, cellular heterogeneity, and key pathological features such as impaired mucociliary clearance and chronic inflammation. A critical component of CF research is the accurate functional assessment of CFTR activity. We compare established high-resolution techniques (patch-clamp, Ussing chamber) with high-throughput screening assays, including fluorescence quenching of halide-sensitive YFP assay and organoid swelling tests. The article provides a framework for choosing the most appropriate CFTR functional assay tailored to specific research goals. Full article

Background/Objectives: Chronic rhinosinusitis with nasal polyps (CRSwNP) is associated with epithelial remodeling, impaired mucociliary clearance, and altered nitric oxide (NO) metabolism. However, cell type-specific mechanisms underlying epithelial NO signaling remain poorly understood. This study investigated NO-related signaling in differentiated human sinonasal epithelial cells. Methods: Human sinonasal tissues were obtained from patients with CRSwNP (n = 20) and control subjects (n = 20). Air–liquid interface (ALI) cultures were established from donor-derived epithelial cells. Ciliated and non-ciliated cells were identified by immunostaining for acetylated α-tubulin and BCAM. Expression of inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) was analyzed by quantitative RT-PCR. Intracellular NO-related fluorescence signals were evaluated using DAF-FM fluorescence imaging. Results: CRSwNP tissues exhibited significantly increased iNOS expression and elevated iNOS/eNOS ratios, whereas eNOS expression did not differ significantly from that in controls. ALI cultures reproduced differentiated sinonasal epithelium containing both ciliated and non-ciliated cell populations. DAF-FM fluorescence signals were significantly higher in ciliated cells than in non-ciliated cells (80.3 ± 25.3 vs. 49.3 ± 21.1). Non-selective NOS inhibition markedly reduced fluorescence signals in both cell types, whereas selective iNOS inhibition reduced but did not abolish signals in ciliated cells. Conclusions: NO-related signaling appears to differ among epithelial cell subtypes. Persistence of fluorescence signals after selective iNOS inhibition suggests a contribution of constitutive NOS activity in ciliated cells, whereas non-ciliated cells appear to rely more heavily on iNOS-dependent pathways. These findings support the hypothesis that altered epithelial NO signaling contributes to epithelial dysfunction and impaired mucociliary homeostasis in CRSwNP. Full article

Local pulmonary delivery offers a non-invasive application route for mRNA therapeutics with the potential for high bioavailability at the target-site of applications such as mucosal vaccination or the treatment of lung diseases. However, efficient delivery remains challenging due to major lung-specific barriers, particularly mucus. Herein, pH-responsive, amphiphilic xenopeptides comprising lipoamino fatty acids and oligoamino acids (OAAs) connected in distinct branched U-shape or bundle topologies were evaluated as mRNA polyplexes for delivery to A549 and Calu-3 lung cells under standard submerged or air–liquid interface (ALI) transfection conditions, and upon intratracheal application in BALB/c mice. Optionally, polyplexes were coated with negatively charged hyaluronic acid (HA) or colloidally stabilized with poly(ethylene glycol) (PEG). For U-shapes, hydrophobic modification of the OAA domain boosted their efficiency. Interestingly, best-performing formulations varied across transfection conditions. While the bundle topology showed the highest potential in submerged cell culture, U-shaped carriers were more efficient under ALI conditions. Polyplex surface modification with HA or PEG did not strongly alter in vitro transfections, whereas hydrophobized U-shape core polyplexes combined with surface modification enhanced their efficiency in vivo. Thus, the cationizable core and surface properties of mRNA nanoparticles require specific balancing in various lung cell models and lung. Full article

Air–liquid interface (ALI) cultures recapitulate key features of the airway epithelium by driving basal cell differentiation into ciliated, club, and goblet cells and by generating a functional mucus barrier, thereby representing a highly relevant model of the human respiratory tract. Using a reduced factorial Design of Experiments (DoE) methodology, we simultaneously investigated the effects of seven variables on human coronavirus OC43 (HCoV-OC43) replication in air–liquid interface (ALI)-cultured primary human bronchial epithelial cells (pHBECs) to identify robust conditions that support infection and viral replication. Epithelial differentiation was monitored by measuring transepithelial electrical resistance and determining expression levels of marker genes for basal, goblet, club, and ciliated cells using reverse transcription quantitative PCR (RT-qPCR). HCoV-OC43 replication was monitored by quantifying genomic and subgenomic RNA by RT-qPCR. Viral RNA peaked three days post-infection in cell lysates and four days post-infection in apical washes. Initiation of ALI conditions induced epithelial differentiation, which was complete after 21 days and emerged as the strongest determinant of viral replication. Differentiated pHBEC cultures showed significantly reduced viral RNA compared with undifferentiated cultures, particularly following apical infection. In contrast, basal infection resulted in lower viral RNA levels in undifferentiated cultures than apical infection but was less dependent on epithelial differentiation. However, productive infection following basal exposure was less consistent and more strongly dependent on viral inoculum size. We further demonstrate that repeated mucus washes prior to infection increased HCoV-OC43 replication in mature cultures. In summary, our findings show that epithelial differentiation negatively affects HCoV-OC43 replication and we identify conditions that maximize viral replication in fully differentiated pHBEC cultures. Full article

For testing ocular irritation, 3D corneal models mimicking the corneal epithelium are considered reliable eye irritation tests and are detailed in regulatory guideline OECD Test Guideline (TG) 492. The aim of the present study was to develop and validate a Reconstructed human Cornea-like Epithelium (RhCE) in vitro irritation test method for ophthalmic medical devices according to OECD TG 492. Immortalized Human Primary Corneal Epithelium Cells (IM-HCEpiCs) were cultured on microporous inserts and exposed to an Air–Liquid Interface (ALI). Morphology was examined using standard (immuno-) histological techniques. Viability was quantified with MTT assay. Barrier integrity and function were monitored by trans-epithelial electrical resistance (TEER) and determination of IC₅₀ using MTT assay. Reproducibility was evaluated by calculating the inter-batch coefficient of variation (CV %) of the absorbance values of negative control-treated RhCE model replicates by MTT assay. Technical proficiency was verified using reference chemicals. Irritancy of ophthalmic medical devices was assessed. IM-HCEpiCs developed an epithelium-like barrier under the ALI. TEER increased after ALI introduction, and the obtained IC₅₀ value showed concordance with the guideline’s reference ranges. The developed RhCE test method demonstrated technical proficiency and correctly identified medical devices as non-irritants. A novel RhCE model was developed and validated according to OECD TG 492. Full article

Chronic exposure to benzo[a]pyrene (BaP), a Group 1 IARC carcinogen, is a major driver of lung carcinogenesis; however, how sustained subcytotoxic exposure reprograms bronchial epithelium toward preneoplastic states remains poorly defined. Here, we subjected BEAS-2B human bronchial epithelial cells to 12 weeks of continuous BaP at environmentally relevant concentrations (0.1 and 1.0 µM) and interrogated the resulting phenotypes using an integrated multi-scale framework encompassing functional toxicology, RT-qPCR, RNA-seq, phospho-kinase/NF-κB arrays, and organotypic air–liquid interface (ALI) cultures. Cells maintained metabolic competence throughout, evidenced by sustained CYP1A1 and CYP1B1 induction at both acute (4 h) and chronic (12-week) timepoints, while accumulating genotoxic stress as demonstrated by dose-dependent nuclear γ-H2AX foci formation and ATM phosphorylation (Ser1981). RNA-seq revealed a dose-dependent transcriptional shift: 0.1 µM BaP yielded 119 differentially expressed genes (DEGs; |log2FC| ≥ 1, FDR < 0.05), whereas 1.0 µM generated 255 DEGs. Downregulated transcripts were enriched for extracellular matrix and cell-adhesion programs (COL14A1, ADAMTS2, CSMD3, CADM3), while upregulated genes encompassed inflammatory, calcium-signaling, and vesicle-trafficking modules (NFATC4, CSF2RA, SYT1, PCLO). Phospho-kinase/NF-κB arrays confirmed a p53/NF-κB signaling nexus, with concurrent activation of MAPK/ERK (Thr202/Tyr204) and PI3K/Akt (Ser473) pathways. Despite persistent genotoxic stress, cells did not acquire anchorage-independent growth and remained non-tumorigenic in vivo. Critically, ALI organotypic cultures derived from BaP-exposed cells exhibited histological dysplasia, nuclear pleomorphism, and disrupted apical-basal polarity. These findings mechanistically link chronic BaP exposure to an initiation-like preneoplastic state and establish a validated 2D/3D multi-omics platform for PAH-driven lung carcinogenesis research. Full article

The cystic fibrosis transmembrane conductance regulator (CFTR) is a member of the atypical ATP-binding cassette (ABC) family that functions as a phosphorylation-regulated epithelial anion channel. Cystic fibrosis (CF) is characterised by variants in the CFTR gene that lead to impaired epithelial chloride–ion transport and increased mucus viscosity. Although CFTR modulators such as Trikafta^® have transformed the care of many CF patients, individuals harbouring rare CFTR variants still have no effective treatment options. In this study, we used primary air–liquid interface (ALI) airway cultures obtained from 21 CF patients (pwCF) and 21 healthy controls (HC) to evaluate the therapeutic efficacy of CFTR restoration based on chitosan-mediated CFTR mRNA and modulators. While modulators restored CFTR channel function in most cultures derived from CF patients, those with class I or other rare variants showed no improvement. Chitosan-mediated CFTR mRNA delivery successfully restored CFTR function in ALI cultures of patients carrying rare CFTR variants with limited or no observed clinical response to modulator therapy, assessed by electrophysiology using our newly developed Multi Transepithelial Current Clamp (MTECC) Ussing chamber. This was then confirmed by morphological visualisation of CFTR protein expression in modulator-responsive patient samples using immunofluorescence (IF) staining. IF revealed an increase in CFTR signal and the restoration of epithelial barrier integrity following chitosan-mRNA and modulator treatment as a secondary outcome alongside CFTR functional measurements. Notably, MUC5AC expression, a major gel-forming mucin expressed by airway goblet cells and mucus viscosity were elevated in CF cultures, but were markedly reduced following successful intervention, approaching the levels seen in HCs. These findings establish the potential of chitosan-mRNA delivery as a therapeutic approach for CF patients, particularly those who do not respond to modulators. They also provide a practical, comparative evaluation of advanced mRNA-based treatments in patient-derived airway models. Full article

Background/Objectives: Individual responses to CFTR modulators vary widely among people with cystic fibrosis (pwCF), underscoring the need for functional approaches that provide biological context alongside genotype-based therapy selection. Nasal epithelial cultures provide an individual-specific model for theratyping, but most studies rely on freshly isolated cells, restricting repeated testing and long-term sample use. In this study, we tested whether CFTR modulator responses measured in biobanked nasal cells were associated with real-world clinical outcomes. Methods: Cryopreserved nasal epithelial cells from 23 pwCF were differentiated at the air–liquid interface and assessed for CFTR modulator-responsive ion transport using Ussing chambers. In vitro responses were correlated with 6-month changes in sweat chloride concentration (SCC), FEV₁, and BMI. Results: Cryopreserved cultures retained donor-specific CFTR modulator responsiveness. Modulator-induced forskolin/IBMX-stimulated currents correlated with changes in SCC (R = −0.512). CFTR inhibitor-sensitive currents correlated with FEV₁ (R = 0.564). Associations between forskolin/IBMX-stimulated currents and FEV₁ were positive but did not reach statistical significance using two-tailed analysis. BMI changes showed no significant association. Conclusions: Biobanked nasal epithelial cultures preserve clinically relevant CFTR modulator responses at the cohort level, supporting their use as functional assays for population-level assessment in cystic fibrosis. This cryopreservation-based strategy enables repeated testing and may expand access to theratyping beyond freshly obtained samples. Full article

Background/Objectives: Interactions between colorectal tumours and their immune microenvironment critically influence disease progression and response to immunotherapy. However, most organoid systems fail to preserve the complex architecture and immune composition of the original tissue. Here, we applied the air–liquid interface (ALI) organoid model to paired tumour and perilesional colon tissues from colorectal cancer patients to evaluate its ability to retain immune and genetic features and to reproduce responses to chemotherapy and immune checkpoint blockade. Methods: Fresh human tumour and matched healthy colon tissues were processed to generate ALI organoids. Their histological organization, immune cell composition (including CD45⁺ subsets), and genomic profiles were compared with those of the parental tissues and with conventional Matrigel organoids, either alone or co-cultured with peripheral blood mononuclear cells (PBMCs). Organoids were exposed to 5-FU and nivolumab (anti–PD-1) to assess local immune modulation. Results: ALI organoids faithfully preserved the three-dimensional architecture, native immune infiltrates, and somatic mutational landscape of the source tissues. Importantly, upon PD-1 blockade with nivolumab, ALI organoids consistently exhibited a local expansion of regulatory T cells (Tregs), a phenomenon that could contribute to adaptive immune resistance. This response was not reproduced in PBMC–Matrigel co-culture systems, highlighting the importance of preserving endogenous tumour–immune interactions. Conclusions: Patient-derived ALI organoids represent a physiologically relevant platform that conserves key structural, immunological, and genomic hallmarks of colorectal cancer. By capturing clinically relevant immune remodeling events, such as Treg expansion following PD-1 blockade, this model provides a powerful tool for dissecting tumour–immune interactions. Full article

The presence of micro- and nano-plastics in the atmosphere has become evident, necessitating risk assessments for humans. Although submerged culture systems are often used to evaluate the safety of fine particles, some plastics float in culture media owing to their low density. Therefore, developing an air–liquid interface (ALI) system capable of assessing plastic exposure is essential. In this study, we developed a chamber for exposing nanoplastic aerosols to ALI cultures and evaluated their toxicological effects. A glass exposure chamber integrated with a donut-shaped culture plate was constructed. The aerosols were introduced through four upper inlets and discharged through five lower outlets. The culture temperature was controlled by circulating water through the inside space of the plate. A nano-polystyrene (PS) suspension was nebulized and introduced into the chamber. Exposure of co-culture of Calu-3 and U937 cells to nano-PS aerosols resulted in a spatial mass concentration-dependent increase in hydrogen peroxide concentration in the culture medium, elevated expression of inflammatory cytokines and chemokines (including IL-6 and IL-8) in Calu-3 cells and decreased trans-epithelial electrical resistance. These findings indicate that nano-PS aerosol exposure induces oxidative stress and inflammatory responses, leading to alveolar barrier dysfunction. Overall, the developed ALI exposure system provides a useful in vitro culture system for evaluating the safety of nanomaterials, including nanoplastics, and highlights the importance of aerosol-based approaches in assessing the toxicity of respirable particles. Full article

Background/Objectives: Developing effective therapies for patients with triple-negative breast cancer (TNBC) remains an urgent clinical priority. Compared with other subtypes, the basal B type of TNBC exhibits a less differentiated and mesenchymal-like phenotype that models highly invasive and metastatic breast malignancies. To target metastatic TNBC, our current study sought to identify effective therapeutic drugs and the underlying mechanisms. Methods: A systematic screening of 140 FDA-approved drugs was conducted for repurposing using live-cell imaging-based wound-healing assays. Candidate efficacy was validated by in vitro transwell invasion assays, in vivo allograft/xenograft models, and ex vivo three-dimensional air–liquid interface (ALI) and patient-derived organoid (PDO) cultures. Results: Dasatinib emerged as a promising anti-cancer agent in aggressive TNBC, particularly in the basal B type, with high ETS proto-oncogene 1 (ETS1) expression. Mechanistically, dasatinib disrupts the actin cytoskeleton, impairing cell motility and migration while concurrently suppressing the expression of ETS1 and matrix metalloproteinase-3 (MMP3) to remodel the extracellular matrix (ECM) and inhibit invasion. Moreover, the combination of dasatinib with an anti-programmed cell death protein-1 (PD-1) antibody represents a potential therapeutic strategy. Conclusions: These findings highlight dasatinib as a potential therapeutic option for metastatic TNBC and suggest that selecting patients with high ETS1 expression may optimize treatment response. Full article

Air–liquid interface (ALI) cultures offer a physiologically relevant in vitro model of the airway epithelium (AE), capable of recapitulating key structural and functional features observed in vivo. In this study, we established and validated a murine ALI culture system comprising pseudostratified epithelia with functional tight junctions, ciliated cells and goblet cells. To assess their innate immune functions, we designed and 3D-printed an autoclavable aerosol deposition chamber, which allowed us to expose differentiated AE cultures to house dust mite (HDM) allergen. Upon HDM exposure, AE cells mounted a time-dependent innate immune response characterized by the secretion of complement component C3, the generation of its active cleavage products C3a and increased expression of C3aR and C5aR1. This was associated with increased intracellular TSLP and IL-25 production and TSLP release in AE cells. Progressive loss of tight junction integrity and reduced transepithelial electrical resistance (TEER) demonstrated epithelial susceptibility to allergen protease-induced cell damage. Together, we established a murine ALI system preserving airway epithelial architecture and a nebulization system to study innate immune activation of AE cells in response to HDM mimicking the initial phase of allergen sensitization. More generally, we described a powerful and accessible platform for studying epithelial-driven mechanisms in murine airway immune responses. Full article

Background/Objectives: Local delivery of gene therapy products through the airways shows great promise for the treatment of a number of serious lung diseases, but its effectiveness is hampered by the mucus layer protecting the lung epithelium in the trachea and bronchi. Methods: To overcome this barrier, we engineered carbon dots (CDs) with mucus penetrating properties. Results: The CDs were synthesized by solvothermal treatment of citric acid and branched polyethyleneimine, and functionalized with maleamic acid groups to create cationic mucoinert nanoparticles with tunable charge. We characterized their interactions with a mucus model through turbidity and transport measurements, and assessed their impact on the viscoelastic properties of the biopolymer. We then demonstrated that the carriers are effective at delivering pDNA to a variety of cell models in vitro. In particular, mucus-producing Calu-3 cells cultured at the air–liquid interface (ALI) were used as a discriminating model to evaluate intracellular delivery of the genetic cargo through a thick layer of mucus at the cell surface. Conclusions: The functionalization of CDs with maleamic acid groups resulted in a 1000- to 10,000-fold increase in transfection efficiency in the mucus-producing model, offering new opportunities for lung gene therapy. Full article

SARS-CoV-2 variants have demonstrated distinct epidemiological patterns and clinical presentations throughout the COVID-19 pandemic. Understanding variant-specific differences at the respiratory epithelium is crucial for understanding their pathogenesis. Here, we utilized human nasal organoid air–liquid interface (HNO-ALI) cell cultures to compare the viral replication kinetics, innate immune response, and epithelial damage of six different strains of SARS-CoV-2 (B.1.2, WA, Alpha, Beta, Delta, and Omicron). All variants replicated efficiently in HNO-ALIs, but with distinct replication kinetic patterns. The Delta variant exhibited delayed replication kinetics, achieving a steady state at 6 days post-infection compared to 3 days for other variants. Cytokine analysis revealed robust pro-inflammatory and chemoattractant responses (IL-6, IL-8, IP-10, CXCL9, and CXCL11) in WA1, Alpha, Beta, and Omicron infections, while Delta significantly dampened the innate immune response, with no significant induction of IL-6, IP-10, CXCL9, or CXCL11. Immunofluorescence and H&E analysis showed that all variants caused significant ciliary damage, though WA1 and Delta demonstrated less destruction at early time points (3 days post-infection). Together, these data show that, in our HNO-ALI model, the Delta variant employs a distinct “stealth” strategy characterized by delayed replication kinetics and epithelial cell innate immune evasion when compared to other variants of SARS-CoV-2, potentially explaining a mechanism that the Delta variant can use for its enhanced transmissibility and virulence observed clinically. Our findings demonstrate that variant-specific differences at the respiratory epithelium could explain some of the distinct clinical presentations and highlight the utility of the HNO-ALI system for the rapid assessment of emerging variants. Full article

COVID-19 has triggered the rapid adoption of human organoid-based tissue culture models to overcome the limitations of the commonly used Vero cell line that did not fully recapitulate SARS-CoV-2 infection of human tissues. As the primary site of SARS-CoV-2 infection, the human nasal epithelium (HNE) cultivated in vitro and differentiated at air–liquid interface (ALI) is an ideal model to study infection processes and for testing anti-viral antibodies and drugs. However, the need for primary basal cells to establish the ALI-HNE limits the scalability of this model system. To try and bypass this bottleneck, we devised an ALI-differentiated form of the human adenocarcinoma cell line Calu-3, reported to model most aspects of authentic SARS-CoV-2 infection, including viral entry. The ALI-Calu-3 were tested for infection by a panel of SARS-CoV-2 variants, including ancestral (VIC01) and early pandemic lineages (VIC2089, Beta, Delta), and Omicron subvariants (BA2.75, BA4, BA5, XBB1.5). All tested lineages infected the ALI-HNE. In stark contrast, infection of the ALI-Calu-3 by Omicron subvariants BA4 and XBB1.5 was reduced. These data support the use of ALI-Calu-3 as a complementary, intermediary model for most but not all SARS-CoV-2 lineages, and places the ALI-HNE as the benchmark culture model for SARS-CoV-2 infection. Full article