<p>The ALI culture allows the simulation of the air-side differentiation of the epithelium, so that cell interactions can be simulated in an environment that mimics the natural barrier function of the airways. In contrast, the AOAO cultures represent a more complex, three-dimensional model that more closely mimics the cellular organisation and function of the airway epithelium. </p>
<p>The cell models generated should provide a reliable basis for reproducing the in vivo conditions of the human nasal and airway epithelium as closely as possible in vitro. </p>
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<p>The workflow of this experiment started with the collection of primary human nasal epithelial cells (hNECs) from nasopharyngeal swabs of human volunteers. This method allowed the non-invasive isolation of cells that represent a suitable model for the respiratory epithelium. After collection, the cells were isolated and cultured in a special medium to ensure their proliferation. This step was essential to generate a sufficient number of cells for the subsequent experiments. </p>
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<p>The workflow of this experiment started with the collection of primary human nasal epithelial cells (hNECs) from nasopharyngeal swabs of human volunteers. This method allowed the non-invasive isolation of cells that represent a suitable model for the respiratory epithelium. After collection, the cells were isolated and cultured in a special medium to ensure their proliferation. This step was essential to generate a sufficient number of cells for the subsequent experiments. </p>
<p>The next step was to culture the isolated cells in two different systems: the Air-Liquid Interface (ALI) culture as a 2D model and the Apical-Out Airway Organoid (AOAO) culture as a 3D model. In the ALI culture, the cells were grown on a porous membrane with the basal side of the cells in contact with a culture medium and the apical side exposed to air. This method allowed the cells to differentiate into a multilayered epithelium that reproduced different cell types, such as ciliated cells, goblet cells and basal cells, typical of the respiratory epithelium. </p>
<p>In parallel, a 3D model was established in AOAO culture in which the cells form spherical organoids with the apical side facing outwards. This 3D structure more closely mimicked the physiological conditions of the human airway epithelium and offered extended possibilities for studying cell-cell interactions in space. </p>
description="Schematic overview of the isolation and culture of primary human nasal epithelial cells (hNECs). First, nasopharyngeal swabs are taken to obtain the cells. The cells are then cultured in two different culture systems: Apical-Out-Airway-Organoids (AOAO) as a 3D culture (left) and Air-Liquid-Interface (ALI) as a 2D culture (right). In the 3D culture, the cells form a spherical organoid with an outward-facing apical surface, whereas in the 2D culture, the cells form a differentially structured epithelial barrier that mimics different cell types such as ciliated cells, goblet cells and basal cells."
description="Schematic overview of the isolation and culture of primary human nasal epithelial cells (hNECs). First, nasopharyngeal swabs are taken to obtain the cells. The cells are then cultured in two different culture systems: Apical-Out-Airway-Organoids (AOAO) as a 3D culture (left) and Air-Liquid-Interface (ALI) as a 2D culture (right). In the 3D culture, the cells form a spherical organoid with an outward-facing apical surface, whereas in the 2D culture, the cells form a differentially structured epithelial barrier that mimics different cell types such as ciliated cells, goblet cells and basal cells."
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<p>Characterisation of the cells in both culture systems was performed by a combination of microscopic examination and histological staining. In particular, haematoxylin-eosin (HE) and periodic acid-Schiff (PAS) staining was performed in the ALI culture to analyse cell differentiation and the formation of mucus-forming goblet cells. HE staining was used for general visualisation of cell morphology and cell stratification, while PAS staining was used specifically to identify the mucins in the goblet cells. These histological techniques allowed a detailed assessment of epithelial differentiation, in particular the maturation of ciliated and mucus-producing cells. </p>
<p>Once the cell cultures had been successfully established, a key objective of the project was pursued: To test gene our therapy approach in these cell models. The cells would be used to assess the efficacy and safety of new gene therapies before they could potentially be used in clinical applications. </p>
