Search Results (171)

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous disorder characterized by impaired mucociliary clearance due to defects in motile cilia. This study investigates the impact of loss-of-function mutations in the CFAP300 gene on the ciliary structure and function in three PCD patients. Using a multimodal approach, we integrated molecular genetic testing, transmission electron microscopy, the high-speed video microscopy assay and immunofluorescence staining to analyze ciliary motility and protein expression in both ex vivo and in vitro-obtained ciliary cells. Our results revealed that the pathogenic variant c.198_200delinsCC (p.Phe67ProfsTer10) in CFAP300 led to the absence of the functional CFAP300 protein, the complete loss of outer and inner dynein arms and immotile cilia. Air–liquid interface (ALI)-cultured cells from patients exhibited no ciliary beating, contrasting with healthy controls. Immunostaining confirmed the absence of CFAP300 in patient-derived cilia, underscoring its critical role in dynein arm assembly. These findings highlight the diagnostic utility of ALI cultures combined with functional and protein analyses for PCD, offering a clinically actionable framework that can be readily incorporated into standard diagnostic workflows. Full article

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

Male-factor infertility accounts for approxiamately half of all infertility cases globally, yet therapeutic options remain limited for individuals with no retrievable spermatozoa, such as those with non-obstructive azoospermia (NOA). In recent years, artificial gametogenesis has emerged as a promising avenue for fertility restoration, driven by advances in two complementary strategies: organotypic in vitro spermatogenesis (IVS), which aims to complete spermatogenesis ex vivo using native testicular tissue, and in vitro gametogenesis (IVG), which seeks to generate male gametes de novo from pluripotent or reprogrammed somatic stem cells. To evaluate the current landscape and future potential of these approaches, a narrative, semi-systematic literature search was conducted in PubMed and Scopus for the period January 2010 to February 2025. Additionally, landmark studies published prior to 2010 that contributed foundational knowledge in spermatogenesis and testicular tissue modeling were reviewed to provide historical context. This narrative review synthesizes multidisciplinary evidence from cell biology, tissue engineering, and translational medicine to benchmark IVS and IVG technologies against species-specific developmental milestones, ranging from rodent models to non-human primates and emerging human systems. Key challenges—such as the reconstitution of the blood–testis barrier, stage-specific endocrine signaling, and epigenetic reprogramming—are discussed alongside critical performance metrics of various platforms, including air–liquid interface slice cultures, three-dimensional organoids, microfluidic “testis-on-chip” devices, and stem cell-derived gametogenic protocols. Particular attention is given to clinical applicability in contexts such as NOA, oncofertility preservation in prepubertal patients, genetic syndromes, and reprocutive scenarios involving same-sex or unpartnered individuals. Safety, regulatory, and ethical considerations are critically appraised, and a translational framework is outlined that emphasizes biomimetic scaffold design, multi-omics-guided media optimization, and rigorous genomic and epigenomic quality control. While the generation of functionally mature sperm in vitro remains unachieved, converging progress in animal models and early human systems suggests that clinically revelant IVS and IVG applications are approaching feasibility, offering a paradigm shift in reproductive medicine. 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

Microorganisms metabolising low-value carbon sources can produce a diverse range of bio-based and biodegradable materials compatible with circular economy principles. One such material is bacterial cellulose (BC), which can be obtained in high purity through the fermentation of sweetened tea by a Symbiotic Culture of Bacteria and Yeast (SCOBY). In recent years, there has been a growing research interest in SCOBYs as a promising solution for sustainable material design. In this work, we have explored a novel method to grow SCOBYs vertically using a waste-based scaffold system. Waste sheep wool and cotton fabric were soaked in a SCOBY infusion to serve as scaffolds, carrying the infusion and facilitating vertical growth through capillary forces. Remarkably, vertical membrane growth up to 5 cm above the liquid–air interface (LAI) was observed after just one week. Membranes with different microstructures were found in sheep wool and cotton, randomly oriented between the scaffold fibre, resulting in a high surface area. This study demonstrated that vertical growth in scaffolds is possible, proving the concept of a new method of growing composite materials with potential high-value applications in biomedicine, energy storage, or filtration. 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

The development of alternatives to animal models and traditional cell cultures has led to the emergence of organ-on-chip (OoC) systems, which replicate organ functions under both physiological and pathological conditions. These microfluidic platforms simulate key tissue interfaces—such as tissue–air, tissue–liquid, and tissue–tissue interactions—while incorporating biomechanical stimuli to closely resemble in vivo environments. This makes OoC systems particularly suitable for modeling biological barriers such as the skin, the placenta, and the blood–brain barrier, which play essential roles in maintaining homeostasis. This review explores various biological barrier models that can be replicated using the OoC technology, discussing the integration of induced pluripotent stem cells (iPSCs) to advance personalized medicine. Additionally, we examine the methods for assessing barrier formation, including real-time monitoring through integrated sensors, and discuss the advantages and challenges associated with these technologies. The potential of OoC systems in disease modeling, drug discovery, and personalized therapeutic strategies is also highlighted. 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