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• W2990540908 abstract "The human bronchial epithelium acts as a protective barrier preventing microorganisms from entering the airways and has a great impact on biological mechanisms that causes airway diseases, such as asthma. Established in vitro airway models often consist of an epithelial layer cultured in two-dimensional (2D) monolayer in an air-liquid interface (ALI); however, great attention has to be paid to the composition of the growth medium in order to achieve the system to be sufficiently differentiated and functional.Studies have shown that fibroblasts regulate proliferation and differentiation of the epithelial cells while epithelial cells stimulate fibroblast migration and proliferation. In this work the aim was to engineer an in vitro bronchial mucosa model consisting of an epithelium and an underlying fibroblast layer separated by a biodegradable electrospun poly-L-lactic acid (PLLA) membrane. Electrospinning is a widely used scaffold fabrication technique and was chosen due to the ability of achieving highly porous (up to 90%) scaffolds with high surface area to volume ratio and due to its cost-effectiveness. Electrospun nanofibre PLLA membranes were used as as a substitute for the basement membrane of the bronchial mucosa and were coated with fibronectin (FN) to improve cell adhesion.Human bronchial epithelial cells (HBEpiCs) were seeded on the apical side of the membrane and fibroblasts on the basal side. The two cell types were co-cultured in ALI for 2 weeks and scanning electron microscopy (SEM) and immunostaining techniques were used to observe and analyse cellular behaviour of the cells and protein secretion of collagen type IV (Col IV) and laminin (LM). PLLA membranes consisting of non-beaded nano-fibres with a diameter of around 240nm were produced and coated with FN to improve cell attachment.When HBEpiCs were cultured on these membranes in ALI, the cells maintained their cellular polarity as well as the morphological and functional properties of bronchial epithelial cells. Both cell types attached and grew on FN coated PLLA surfaces compared to non-FN coated surfaces. After 2 weeks of co-culture tight junctions between the HBEpiCs were detected with E-cadherin staining and ciliated cells were observed with SEM. Fibroblasts seeded on the basal side of the PLLA membrane brought to the system a substantial improvement in epithelial cells differentiation in comparison to the HBEpiC cultured without a fibroblast layer. Col IV and LM secretion from both HBEpiCs and fibroblasts was confirmed with immunostaining after 1 week of culture. Additionally, this research aimed to investigate the possibilities of converting the bronchial mucosa model into an in vitro asthma model. This was done by introducing the cytokine Interleukin 13 (IL-13) to the cells as IL-13 is known to play a key role in asthma. IL-13 plays an important role in the pathogenesis of asthma by increasing the differentiation of mucus secreting epithelial cells and causing mucus hypersecretion leading to poor mucociliary clearance in the airways. The gold standard for treating asthmatic attacks currently includes inhaled glucocorticosteroids, which help the inflammatory reaction such as by reducing mucus hypersecretion. Dexamethasone is a type of corticosteroid and inhibits MUC2 and MUC5AC protein expression in some airway epithelial cell lines. However, the various airway epithelial cell lines and primary animal airway epithelial cells respond differently to dexamethasone, hence, the effect of dexamethasone on human bronchial epithelial cell mucin secretion was studied in this research.IL-13 was introduced to the in vitro bronchial mucosa model to develop an in vitro asthma model. ELISA was used to quantify MUC5AC expression and SEM was used to assess the ciliation of HBEpiCs. Poor ciliation of the HBEpiC was observed while the mucus secretion of the cells increased after 2 and 4 weeks of ALI co-culture. This suggest that IL-13 induces mucus hypersecretion and causes airway remodelling by reducing ciliation of epithelial cells.The ELISA showed low MUC5AC levels for epithelial cells treated with dexamethasone in both the in vitro asthma model and bronchial model. This reduction in MUC5AC secretion observed in the dexamethasone-treated HBEpiCs was similar to the reaction to inhaled glucocorticosteroids used for treating asthma, however, the dexamethasone did not improve the re-ciliation of the HBEpiCs.With this research, an in vitro bronchial mucosa model mimicking the in vivo cell morphology and the microenvironment in the basement membrane was established successfully by using ALI co-culture system and FN coated electrospun PLLA membranes. This work suggests an ALI co-culture system that would be suitable for acting as an in vitro disease model for studying airway diseases such as asthma and could be a possible model for asthma drug screening. The established in vitro bronchial mucosa model could be suitable for investigating and understanding key mechanisms involved in airway diseases such as asthma, Chronic Obstructive Pulmonary Disease (COPD), and airway repair." @default.
• W2990540908 created "2019-12-05" @default.
• W2990540908 creator A5079223606 @default.
• W2990540908 date "2019-01-01" @default.
• W2990540908 modified "2023-09-22" @default.
• W2990540908 title "In vitro bronchial mucosa model using air-liquid interface culture on PLLA electrospun membrane" @default.
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