Characterization of Air-Liquid Interface Culture from Human-Derived Airway Cells on Electrospun Polycaprolactone

Abstract

In vitro airway epithelial models play a crucial role in respiratory research, particularly for studying barrier function, drug delivery, and host-pathogen interactions. This thesis aimed to characterize electrospun polycaprolactone (e-PCL) in a 96-well plate format as a potential platform for supporting the attachment, proliferation, and differentiation of human airway epithelial cells (Calu-3) under air-liquid interface (ALI) conditions. Surface coatings using polydopamine (PDA), collagen type I, or both combined were compared by evaluating their effects on cell behaviour and epithelial function and compared to no pre-coated e-PCL as control. Initial optimization assessed metabolic activity via the WST-1 assay and confirmed that polydopamine-coated surfaces did not inhibit metabolic activity. As shown by fluorescence imaging and area coverage quantification, Calu-3 cells demonstrated improved adhesion and confluency on polydopamine-based coatings. A-FITC-Dextran permeability assay was used to evaluate epithelial barrier formation. Results indicated a progressive decrease in permeability under ALI conditions on coated e-PCL. WST-1 assay confirmed sustained metabolic activity across the culture period. Tissue processing methods were also optimized using low-melting paraffin and custom 3D-printed moulds to enable successful sectioning of e-PCL membranes, which had previously not been possible. Histological analysis demonstrated morphological changes over 28 days of ALI culture, with evidence of mucin production and epithelial maturation. Immunofluorescence staining revealed expression of MUC5AC and E-cadherin but very low acetylated α-tubulin, likely due to the limited ciliogenesis potential of Calu-3 cells and possibly influenced by DMEM/F12 media. Overall, this study demonstrates that polydopamine-based coatings enhance the performance of e-PCL as a miniaturized airway model in a 96-well plate format. These results support the potential of this model for scalable, reproducible, and ethical in vitro applications in respiratory research and highlight future directions, including marker quantification and the use of primary human airway epithelial cells.

Popular Abstract

Our lungs are lined by a thin layer of cells that are important in keeping us healthy. These cells, called airway epithelial cells, act as a shield to block harmful particles, help trap dust and germs and produce mucus that protects our airways. Scientists study these cells in the laboratory to learn more about lung diseases, test new drugs, and explore how the lungs respond to different conditions. For this study to be useful, the laboratory setup needs to be as close as possible to how things work in the body. In this thesis, we explored a new way for growing these lung cells using a special material called electrospun polycaprolactone (e-PCL), which forms a thin scaffold comprised of thin fibres. This material was placed into a 96-well plate using an insert, a standard tool in laboratories that allows many samples to be tested at once, making the method scalable and efficient. We worked with a specific type of human airway cell called Calu-3 and tested how well they could grow, survive, and differentiate (or mature) on e-PCL under air-liquid interface (ALI) conditions, where the top of the cells is exposed to air, like how they live in the lung. To help cell stickiness and growth, we coated the surface of the e-PCL with polydopamine, a substance inspired by how mussels attach to rocks, which helps cells attach, and tested it alone and in combination with collagen type I, a protein found in the body. The results showed that these coatings improved how well the cells adhered and formed a barrier. We used several tests to measure whether the cells were alive, how strong their barrier was, and mucus production, a feature of healthy lungs. Additionally, we improved the method of preparing these samples to be clearly seen under a microscope. This helped us better understand how the cells were growing and working. These findings suggest that polydopamine-coated e-PCL in a 96-well plate provides a promising mini lung model for future research and drug testing. It could also help reduce the need for animal testing and support more ethical and effective lung studies.

• author: Kispa, Tera Dria
• supervisor: Darcy Wagner
• organization: Degree Projects in Molecular Biology
• course: MOBN03 20242
• year: 2025
• type: H2 - Master's Degree (Two Years)
• subject: Biology and Life Sciences
• language: English
• id: 9215000
• date added to LUP: 2025-11-06 13:43:24
• date last changed: 2025-11-06 13:43:24