Search Results (100)

Organoids allow to model healthy and diseased human tissues. and have applications in developmental biology, drug discovery, and cell therapy. Traditionally cultured in immersion/suspension, organoids face issues like lack of standardization, fusion, hypoxia-induced necrosis, continuous agitation, and high media volume requirements. To address these issues, we developed an air–liquid interface (ALi) technology for culturing organoids, termed AirLiwell. It uses non-adhesive microwells for generating and maintaining individualized organoids on an air–liquid interface. This method ensures high standardization, prevents organoid fusion, eliminates the need for agitation, simplifies media changes, reduces media volume, and is compatible with Good Manufacturing Practices. We compared the ALi method to standard immersion culture for midbrain organoids, detailing the process from human pluripotent stem cell (hPSC) culture to organoid maturation and analysis. Air–liquid interface organoids (3D-ALi) showed optimized size and shape standardization. RNA sequencing and immunostaining confirmed neural/dopaminergic specification. Single-cell RNA sequencing revealed that immersion organoids (3D-i) contained 16% fibroblast-like, 23% myeloid-like, and 61% neural cells (49% neurons), whereas 3D-ALi organoids comprised 99% neural cells (86% neurons). Functionally, 3D-ALi organoids showed a striking electrophysiological synchronization, unlike the heterogeneous activity of 3D-i organoids. This standardized organoid platform improves reproducibility and scalability, demonstrated here with midbrain organoids. The use of midbrain organoids is particularly relevant for neuroscience and neurodegenerative diseases, such as Parkinson’s disease, due to their high incidence, opening new perspectives in disease modeling and cell therapy. In addition to hPSC-derived organoids, the method’s versatility extends to cancer organoids and 3D cultures from primary human cells. Full article

Cystic fibrosis (CF), the most common hereditary lung disease in Caucasians, is caused by dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR). We evaluated CFTR function using a newly developed Ussing chamber system, the Multi Trans Epithelial Current Clamp (MTECC), in an in vitro model of human airway epithelia. Air–liquid interface (ALI) cultures were established from nasal brushings of healthy controls (HC) and CF patients with biallelic CFTR variants. ALI layer thickness was similar between groups (HC: 62 ± 13 µm; CF: 55 ± 9 µm). Immunofluorescence showed apical CFTR expression in HC, but reduced or absent signal in CF cultures. MTECC enabled continuous measurement of transepithelial resistance (Rt), potential difference (PD), and conductance (G_t). G_t was significantly reduced in CF cultures compared to HC (0.825 ± 0.024 vs. −0.054 ± 0.016 mS/cm²), indicating impaired cAMP-inducible ion transport by CFTR. Treatment of CF cultures with elexacaftor, tezacaftor, and ivacaftor (Trikafta^®) increased G_t, reflecting partial restoration of CFTR function. These findings demonstrate the utility of MTECC in detecting functional differences in CFTR activity and support its use as a platform for evaluating CFTR-modulating therapies. Our model may contribute to the development of personalized treatment strategies for CF patients. Full article

Cultures of primary human nasal epithelial cells (hNECs) differentiated at the air–liquid interface (ALI) represent a sophisticated and widely used model of the human upper respiratory epithelium. Despite the availability of various cell culture insert types and the well-established understanding that different culture media influence the cell culture characteristics, the possible impact of the insert brand remains rather underexplored. We cultured hNECs from nineteen healthy adult donors on three distinct brands of commercially available inserts—Corning^® Transwell^®, CELLTREAT^®, and ThinCert^®—and compared the ciliary activity and cellular composition of the cultures using high-speed video microscopy and flow cytometry, respectively. Additionally, we employed an alternative method of hNEC culture setup—the inverted condition—wherein the hNECs were seeded on the basal side of the insert with the idea to avoid mucus accumulation. Our results show that ciliary activity and cell type composition did not differ between insert types for both culture conditions. However, we found a higher ciliary beat frequency and a lower active (ciliated) area in the inverted setup compared to the conventional setup across all three insert brands. These findings indicate that all three mentioned insert types yield comparable cell cultures. Full article

Viral lung infections are a never-ending threat to public health due to the emergence of new variants and their seasonal nature. While vaccines offer some protection, the need for effective antiviral drugs remains high. The existing research methods using 2D cell culture and animal models have their limitations. Human cell-based tissue engineering approaches hold great promise for bridging this gap. Here, we describe a microextrusion bioprinting approach to generate three-dimensional (3D) lung models composed of four cell types: endothelial cells, primary fibroblasts, macrophage cells, and epithelial cells. A549 and Calu-3 cells were selected as epithelial cells to simulate the cells of the lower and upper respiratory tract, respectively. Cells were bioprinted in a hydrogel consisting of alginate, gelatin, hyaluronic acid, collagen, and laminin-521. The models were cultured under air–liquid interface (ALI) conditions to further enhance their physiological relevance as lung cells. Their viability, metabolic activity, and expression of specific cell markers were analyzed during long-term culture for 21 days. The constructs were successfully infected with both a seasonal influenza A virus (IAV) and the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) omicron variant, demonstrating their potential for studying diverse viral infections. Full article

Cystic Fibrosis (CF) is a common genetic disease in the United States, resulting from mutations in the Cystic Fibrosis transmembrane conductance regulator (cftr) gene. CFTR modulators, particularly Elexacaftor/Tezacaftor/Ivacaftor (ETI), have significantly improved clinical outcomes for patients with CF. However, many CFTR mutations are not eligible for CFTR modulator therapy due to their rarity. In this study, we report that a patient carrying rare complex CFTR mutations, c.1680-877G>T and c.3067_3072delATAGTG, showed positive clinical outcomes after ETI treatment. We demonstrate that ETI was able to increase the expression of CFTR harboring c.3067_3072delATAGTG in a heterologous system. Importantly, patient-derived nasal epithelial cells in an air–liquid interface (ALI) culture showed improved CFTR function following ETI treatment. These findings supported the initiation of ETI with the patient. Retrospective studies have suggested that the patient has shown small but steady improvement over the past two years in several clinical metrics, including lung function, body mass index (BMI), and sweat chloride levels. Our studies suggest that ETI could be beneficial for patients carrying c.3067_3072delATAGTG. Full article

Cancer organoids have emerged as transformative models for studying tumor biology and therapeutic responses due to the ability to replicate the complexity of the tumor microenvironment (TME). Tumor organoids recapitulate the genetic and phenotypic diversity of cancers, making them invaluable for investigating mechanisms of resistance and identifying novel therapeutic targets. Patient-derived organoids (PDOs) allow specific treatment methods to be designed based on the properties of each individual tumor in vitro. Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with an immunosuppressive nature. PDAC has a poor prognosis, with the survival rates of metastatic PDAC being improved only minimally over the last few decades. In this study, we demonstrate the antitumor effects of an IL-1 receptor antagonist (IL-1RA) in murine and human PDAC organoids. By reducing the burden of suppressive tumor elements like CAFs, IL-1RA treatment facilitates better immune cell access and response. Full article

Current animal and in vitro cell culture models do not fully recapitulate the physiological and pathophysiological characteristics of the human lung. As a result, the translation of these models to clinical practice is very limited, and clinical trials initiated on the extrapolation of such data fail. Although current models are beneficial in fundamental research, there is a need to constantly improve models to more accurately predict outcomes in clinical trials and personalized medicine. Here, we report important strategies to develop a 3D lung model with human primary lung cells. Starting from the well-established air-liquid interface (ALI) culture system, we describe a gradual increase in the complexity of the system by co-culturing different primary cell types, by testing different coatings, and by adding a three-dimensional matrix. As a result, we have established a reproducible 3D in vitro model of the airway consisting of human primary cells representing a differentiated mucociliary airway epithelium, an underlying submucosa with fibroblasts, and an endothelial interface. Full article

Congenital diaphragmatic hernia (CDH) is a complex disorder whereby improper formation of the diaphragm allows herniation of the internal organs into the thoracic cavity, resulting in pulmonary hypoplasia among other complications. Although epithelial dysfunction is central to CDH pathology, relatively little attention has been paid to the underlying mechanisms orchestrating epithelial malfunction. Proinflammatory signaling downstream of impaired mechanotransduction due to in utero lung compression has been elucidated to drive epithelial cell phenotypes. This has been illustrated by a reduction in nuclear YAP and the upregulation of NF-kB in CDH models. In this review, we draw from recent findings using emerging technologies to examine epithelial cell mechanisms in CDH and discuss the role of compression as a central and, crucially, sufficient driver of CDH phenotypes. In recognition of the limitations of using genetic knockout models to recapitulate such a heterogenic and etiologically complicated disease, we discuss alternative models such as the established nitrofen rat model, air–liquid interface (ALI) cultures, organoids and ex vivo lung explants. Throughout, we acknowledge the importance of involving mechanical compression in the modeling of CDH in order to faithfully recapitulate the disease. Finally, we explore novel therapeutic strategies from stem cell and regenerative therapies to precision medicine and the importance of defining CDH endotypes in order to guide treatments. Full article

(1) Background: Respiratory allergens, particularly ragweed (RW) pollen and house dust mites (HDMs), are major triggers of respiratory inflammation and allergic diseases. This study investigated the impact of single- versus combined-allergen exposure on the barrier function of normal human bronchial epithelial (NHBE) cells cultured at the air–liquid interface (ALI). (2) Methods: NHBE cells were exposed to RW pollen extract (200 µg/mL), HDM extract (200 µg/mL) and their combination at varying concentrations (200 µg/mL, 100 µg/mL, 50 µg/mL, 25 µg/mL). Additional groups included a mixture of Amb a 1, Amb a 11 and Amb a 12 (100 mg/mL) and combinations of Der p 1 with the ragweed allergens (50 mg/mL, 100 µg/mL). Transepithelial electrical resistance (TEER) was recorded over 72 hours to assess barrier integrity, and immunofluorescence (IF) staining for zonula occludens-1 (ZO-1) was performed to evaluate tight junction alterations. (3) Results: TEER measurements showed a significant reduction in epithelial barrier integrity following allergen exposure, with the most pronounced disruption observed with the combined exposure to RW and HDM groups. IF staining confirmed extensive tight junction damage, highlighting their synergistic impact. (4) Conclusions: These findings emphasize the importance of assessing cumulative allergen effects, as combined exposure may exacerbate epithelial dysfunction and represent a key aspect in the management of allergic rhinitis and asthma. Full article

Background/Objectives: In vitro models play a crucial role in preclinical respiratory research, enabling the testing and screening of mucosal formulations, dosage forms, and inhaled drugs. Mucociliary clearance (MCC) is an essential defense mechanism in mucosal drug delivery but is often impaired in respiratory diseases. Despite its importance, standardized in vitro MCC assays are rarely reported. Furthermore, many published methods primarily measure cilia beat frequency (CBF), which requires high-speed cameras that are not accessible to all laboratories. Therefore, this study aimed to develop a physiologically relevant, differentiated in vitro model of the respiratory epithelium that incorporates both beating cilia and functional MCC. We chose porcine airway mucosa as an alternative to human tissue due to ethical considerations and limited availability. The established model is designed to provide a reproducible and accessible method for a broad range of research laboratories. Methods: The previously published tracheal mucosal primary cell (TMPC DS) model, derived from porcine tissue, lacked the presence of beating cilia, which are crucial for effective MCC analysis. For accurate MCC assessment, beating cilia are essential as they play a key role in mucus clearance. To address this limitation, the here-described ciliated tracheal mucosal primary cell (cTMPC) model was developed. cTMPCs were isolated from porcine tissue and cultured under air–liquid interface (ALI) conditions for 21 days to promote differentiation. This model was evaluated for cell morphology, tight junction formation, ciliated and mucus-producing cells, barrier function, gene expression, and tracer/IgG transport. MCC and the model’s suitability for standardized MCC assays were assessed using an inverted microscope. In contrast to the TMPC DS model, which lacked beating cilia and thus could not support MCC analysis, the cTMPC model allows for comprehensive MCC studies. Results: The developed differentiated in vitro model demonstrated key structural and functional features of the respiratory epithelium, including well-differentiated cell morphology, tight junction integrity, ciliated and mucus-producing cells, and effective barrier function. Functional MCC was observed, confirming the model’s potential for standardized clearance assays. Conclusions: This differentiated in vitro model closely replicates the structural and functional characteristics of in vivo airways. It provides a valuable platform for studying mucociliary clearance, toxicology, drug uptake, and evaluating mucosal formulations and dosage forms in respiratory research. Full article

Human metapneumovirus (HMPV) and respiratory syncytial virus (RSV) are pneumoviruses causing lower respiratory tract infections, primarily in infants and children rather than in healthy adults. Human bronchial epithelial cells serve as a viral replication target and source of the innate immune response to these viruses. To better understand the immune responses induced by RSV and HMPV in the pediatric airway epithelium, we comparatively studied pediatric and adult epithelial responses. We used normal human bronchial epithelial (NHBE) cells cultured in an air–liquid interface culture system (ALI), which helps to mimic the architecture of the human lower respiratory tract epithelium. Our results demonstrate differential viral replication patterns and reduced interferons; and inflammatory cytokines’ expression in pediatric cells compared to adult cells. However, pediatric epithelial cells expressed an increased mucus response and induced a stronger pro-inflammatory response in monocyte-derived dendritic cells. These findings reveal age-dependent immune epithelial responses that may contribute to more severe infections by HMPV and RSV. Full article

On-chip microfluidics are advanced in vitro models that simulate lung tissue’s native 3D environment more closely than static 2D models to investigate the complex lung architecture and multifactorial processes that lead to pulmonary disease. Current microfluidic systems can be restrictive in the quantities of biological sample that can be retrieved from a single micro-channel, such as RNA, protein, and supernatant. Here, we describe a newly developed large-scale airway-on-chip model that employs a surface area for a cell culture wider than that in currently available systems. This enables the collection of samples comparable in volume to traditional cell culture systems, making the device applicable to any workflow utilizing these static systems (RNA isolation, ELISA, etc.). With our construction method, this larger culture area allows for easier handling, the potential for a wide range of exposures, as well as the collection of low-quantity samples (e.g., volatiles or mitochondrial RNA). The model consists of two large polydimethylsiloxane (PDMS) cell culture chambers under an independent flow of medium or air, separated by a semi-permeable polyethylene (PET) cell culture membrane (23 μm thick, 0.4 μm pore size). Each chamber carries a 5 × 18 mm, 90 mm² (92 mm² with tapered chamber inlets) surface area that can contain up to 1–2 × 10⁴ adherent structural lung cells and can be utilized for close contact co-culture studies of different lung cell types, including airway epithelial cells, fibroblasts, smooth muscle cells, and endothelial cells. The parallel bi-chambered design of the chip allows for epithelial cells to be cultured at the air–liquid interface (ALI) and differentiation into a dense, multi-layered, pseudostratified epithelium under biological flow rates. This millifluidic airway-on-chip advances the field by providing a readily reproducible, easily adjustable, and cost-effective large-scale fluidic 3D airway cell culture platform. Full article

Bovine viral diarrhea virus (BVDV), a pestivirus in the family Flaviviridae, is a major livestock pathogen. Horizontal transmission leads to acute transient infections via the oronasal route, whereas vertical transmission might lead to the birth of immunotolerant, persistently infected animals. In both cases, BVDV exerts an immunosuppressive effect, predisposing infected animals to secondary infections. E^rns, an immunomodulatory viral protein, is present on the envelope of the virus and is released as a soluble protein. In this form, it is taken up by cells and, with its RNase activity, degrades single- and double-stranded (ds) RNA, thus preventing activation of the host’s interferon system. Here, we show that E^rns of the pestiviruses BVDV and Bungowannah virus effectively inhibit dsRNA-induced IFN synthesis in well-differentiated airway epithelial cells cultured at the air–liquid interface. This activity was observed independently of the side of entry, apical or basolateral, of the pseudostratified, polarized cell layer. Virus infection was successful from both surfaces but was inefficient, requiring several days of incubation. Virus release was almost exclusively restricted to the apical side. This confirms that primary, well-differentiated respiratory epithelial cells cultured at the air–liquid interface are an appropriate model to study viral infection and innate immunotolerance in the bovine respiratory tract. Furthermore, evidence is presented that E^rns might contribute to the immunosuppressive effect observed after BVDV infections, especially in persistently infected animals. Full article

To develop in vitro respiratory models, it is crucial to identify the factors involved in epithelial cell differentiation. In this study, we comprehensively analyzed the effects of air–liquid interface (ALI) culture on epithelial cell differentiation using single-cell RNA sequencing (scRNA-seq). ALI culture induced a pronounced shift in cell composition, marked by a fivefold increase in ciliated cells and a reduction of more than half in basal cells. Transcriptional signatures associated with epithelial cell differentiation, analyzed using iPathwayGuide software, revealed the downregulation of VEGFA and upregulation of CDKN1A as key signals for epithelial differentiation. Our findings highlight the efficacy of the ALI culture for replicating the human lung airway epithelium and provide valuable insights into the crucial factors that influence human ciliated cell differentiation. Full article

This study represents an attempt toward the standardization of pulmonary NAMs and the development of a novel approach for toxicity testing of nanomaterials. Laboratory comparisons are challenging yet essential for identifying existing limitations and proposing potential solutions. Lung cells cultivated and exposed at the air-liquid interface (ALI) more accurately represent the physiology of human lungs and pulmonary exposure scenarios than submerged cell and exposure models. A triculture cell model system was used, consisting of human A549 lung epithelial cells and differentiated THP-1 macrophages on the apical side, with EA.hy926 endothelial cells on the basolateral side. The cells were exposed to silver nanoparticles NM-300K for 24 h. The model used here showed to be applicable for assessing the hazards of nanomaterials and chemicals, albeit with some limitations. Cellular viability was measured using the alamarBlue assay, DNA damage was assessed with the enzyme-modified comet assay, and the expression of 40 genes related to cell viability, inflammation, and DNA damage response was evaluated through RT² gene expression profiling. Despite harmonized protocols used in the two independent laboratories, however, some methodological challenges could affect the results, including sensitivity and reproducibility of the model. Full article