Next Generation In Vitro Models to Study Chronic Pulmonary Diseases - Volume II

Tony J.F. Guo^1,2Alen Faiz^3,4Emmanuel T. Osei^1,2Simon D. Pouwels^5,6*

• ¹University of British Columbia, Okanagan Campus, Kelowna, Canada
• ²The University of British Columbia Okanagan Department of Biology, Kelowna, Canada
• ³University of Technology Sydney, Sydney, Australia
• ⁴Respiratory Bioinformatics and Molecular Biology, University of Technology Sydney, Sydney, Australia
• ⁵Universitair Medisch Centrum Groningen, Groningen, Netherlands
• ⁶Universitair Medisch Centrum Groningen Groningen Research Institute for Asthma and COPD, Groningen, Netherlands

Editorial: Chronic respiratory diseases, such as asthma and chronic obstructive pulmonary disease (COPD), are leading causes of morbidity and mortality worldwide, and pose a significant burden on healthcare systems [1–3]. Improving our understanding of their pathophysiology is crucial for the development of better therapeutics with progress in this area increasingly reliant on data derived from in vitro experiments and models [4]. In vitro cell culture models provides a reliable and accessible method for conducting scientific experiments without using live animals or humans [4,5]. During recent years, these models have advanced rapidly, progressing from static and rigid 2D cell culture systems to advanced 3D models that incorporate features such as soft extracellular matrices, fluid flow, stretch, and co-culture of several different cell types [5–9]. These innovations have enhanced the physiological relevance of in vitro systems, making their findings more translatable to in vivo biology. A notable advancement in the field is the development of an affordable, customizable open-source alternative to commercial microfluidic models [10]. This large airway-on-chip model has a cell culture area comparable to a 24-well plate, enabling collection of sufficient sample volumes for assays such as multi-omics analyses. The model consists of two large cell culture chambers with independent medium or air flow, separated by a semi-permeable membrane, and supports up to 2 × 104 adherent structural lung cells per chamber. It allows for close contact co-culture of various lung cell types, including airway epithelial cells, fibroblasts, smooth muscle cells, and endothelial cells. Epithelial cells can be cultured at air-liquid interface (ALI) to promote differentiation into a pseudostratified epithelium and allow exposure to airborne particles. Requiring only a syringe or peristaltic pump and a 3D printer, this open-source model offers a practical entry point into advanced 3D lung cell culture. Another significant recent advancement is the development of a complex 3D vascularized tri-culture airway model [7]. In this system, bio-printed hydrogel composed of 80% polyethylene glycol diacrylate (PEGDA) and 20% gelatin methacrylate (GelMa) was embedded with lung fibroblasts, while epithelial and endothelial cells were cultured on the apical and luminal side of the hydrogel, respectively. This vascularized, bio-printed tri-culture model offers a more physiologically relevant representation of the human airway and is adaptable, allowing for incorporation of other cell types to increase complexity. In the special issue, titled 'Next Generation In Vitro Models to Study Chronic Pulmonary Diseases - Volume II', six articles highlight the latest developments in respiratory in vitro models (Figure.1). The first study introduces a novel chronic bronchitis ALI co-culture model comprising 16HBE bronchial epithelial cells and MRC-5 lung fibroblasts, optimized for the exposure to aerosolized palladium nanoparticles (Pd-NP), a component of vehicle exhaust [11]. To induce a chronic bronchitis phenotype, cells were stimulated basolaterally with recombinant human interleukin-13 over a two-week ALI differentiation period. Nanoparticle exposure was done using the XposeALI® system. The study developed both chronic and non-chronic bronchitis cell-line ALI models and reported PD-NP-induced inflammatory responses, oxidative stress, and altered markers associated with tissue injury and repair. The second study in this special issue presents the characterization of 2D and 3D co-culture, as well as an alveolar epithelial-fibroblast organoid model, to investigate the effects of mechanical strain [6]. A549 epithelial cells and MRC-5 fibroblasts were co-cultured and subjected to cyclic mechanical strain using the Flexcell cell stretching bioreactor. In the collagen I-embedded organoid model, the authors demonstrated that 24 hours of cyclic strain with amplitudes and frequencies, which mimics pathological breathing patterns, altered cellular morphology, proliferation, inflammation and cytotoxicity. These multicellular dynamic models provide a valuable platform for investigating strain-induced inflammatory and fibrotic responses relevant to lung diseases. Next, ALI-cultured primary bronchial epithelial cells from COPD patients and a cigarette smoke–exposed murine model, were used to investigate the effects of senolytic therapy on markers of senescence [12]. The study reported that the senescent phenotype of COPD-derived epithelial cells was maintained following ALI differentiation. Treatment with a combination of the senolytics dasatinib and quercetin reduced H2O2-induced senescence in vitro. In the cigarette smoke-exposed murine model, the same senolytic cocktail treatment reduced markers of senescence and alveolar inflammatory cell burden. These findings suggest that senolytic therapy may offer a strategy to reduce senescence-associated inflammation in COPD. Another study in this special issue investigated the role of the NFIL3/TIM3 pathway in Th1 imbalance in COPD [13]. Using Havcr2 (encoding TIM3) and Nfil3 knockout mice exposed to cigarette smoke for 24 weeks to induce emphysema, along with isolated CD4+ T cells, the authors demonstrated that disruption of this pathway increased Th1 cell infiltration, elevated IFN-γ expression, and caused a more severe emphysematous phenotype. These findings suggest that the Tim3 expression in CD4+ T cells is regulated by NFIL3 and that TIM3 has anti-inflammatory effects, counteracting cigarette smoke-induced inflammation and providing protection against the development of emphysema. Advanced co-culture models incorporating T cells, epithelial cells, and stromal cells could help elucidate the mechanisms of TIM3 signaling and how its modulation may influence tissue remodeling. Another study conducted a retrospective analysis to identify risk factors for pneumothorax following ¹²⁵I particle implantation in advanced lung cancer patients [14]. A needle-pleura angle of less than 50°, pre-operative CT evidence of emphysema, presence of atelectasis, or lesions in the left lung fissure were identified as independent risk factors. Advanced 3D models that recapitulate alveolar structures and pleural layers could help simulate needle puncture and particle implantation, enabling assessment of cellular and matrix responses to mechanical injury. Lastly, a study used Mendelian randomization and publicly available genome-wide association study data to explore causal relationships between immune cells, COPD and metabolic mediators [15]. The analysis identified eight immune cell phenotypes influenced by eight specific metabolites, which may serve as biomarkers for early COPD detection. Together, this special issue highlights the value of advanced in vitro models in respiratory research (Figure.1). These physiologically relevant systems support the investigation of disease mechanisms, environmental exposures, and therapeutic responses, and offer a practical way to build on insights from patient data and in vivo studies. As they evolve, they are well positioned to connect basic and translational research. This Special Issue includes six articles that collectively highlight the diverse research applications of advanced in vitro respiratory models. The development and optimization of these systems remain critical, as demonstrated by Ji et al., who showcased the utility of air–liquid interface cultures to study metal nanoparticle exposure, and Al Yazeedi et al., who characterized multiple models, including alveolar organoids, under mechanical strain. In vitro platforms are also powerful tools for elucidating disease mechanisms and pathophysiology: Ke et al. reported that cigarette smoke exposure upregulated NFIL3/Tim3, validating this in a knockout model that revealed Th1 imbalance; Baker et al. showed that COPD epithelial cells maintained a senescent phenotype in air–liquid interface culture, with senolytic treatment reducing senescence markers. Furthermore, advanced models can bridge clinical and epidemiological findings with translational and predictive applications. For example, they could be used to study cellular and matrix responses to injury in pneumothorax, as informed by the retrospective risk-factor analysis of Ding et al., or to explore biochemical responses and pathways identified by Cao et al., whose Mendelian randomization analysis linked immune cell phenotypes to COPD via plasma metabolites.