Entwicklung von in vitro Modellen der oberen und unteren Atemwege zum Studium der Aerosolentstehung und der Virusverpackung

Abstract

Establishing 3D in vitro models for human airway systems exhibits significant challenges, due to the intrinsic heterogeneity and complex cellular composition of respiratory tissues. Different regions in the human respiratory system have diverse cell types, differences in layered structures, and varied regenerative capacities across the upper, lower, and especially alveolar lung. This complicates the creation of biomimetic in vitro models. This work is aiming towards addressing these complexities by developing region-specific, co-culture-based models that provide possibilities to replicate better cell-cell and cell- extracellular matrix (ECM) interactions, essential for mimicking the behaviour of distinct areas within the lung, such as the upper airway, lower airway, and alveolar regions. In this work, one critical challenge was the selection of appropriate scaffold material for biomimetic ECM modelling. Following recent literature reviews, electrospun membranes composed of biocompatible polymers like PCL, PTMC (blended into varied proportions, namely 50:50 and 70:30), and PA were fabricated and characterised with respect to me- chanical properties to assess their suitability as ECM-like scaffolds for each respiratory region. Characterisation of the membrane variants for mechanical properties like tensile strength and Young’s modulus measurements revealed that the PCL:PTMC 50:50 as well as PA promise to provide a balance of flexibility and tensile strength closely matching the elasticity of native lung tissue, revealing them promising candidates for the dynamic environment of alveolar models. Conversely, standard and commercialised PET-based models exhibit higher structural rigidity, which proved advantageous for static or short- term applications where a stable barrier was essential but inappropriate for alveolar tissue models. The second vital challenge was to optimise cell seeding strategies to improve barrier function within the models. Tissue region-specific cellular components were selected, in- cluding standardised cell lines such as Calu-3 and A549 for upper airway regions, as well as primary human-derived cells, human endothelial cells (hEC), and human airway epithelial cells, including alveolar epithelium type 2 (huAEC), which were used to produce complex co-cultures. Additionally, human lung biopsies were used to isolate native human lung biopsy-derived fibroblasts. The integration of fibroblasts derived from lung biopsies fur- ther enhanced these models by promoting layered cell organisation and ECM remodelling, yielding a tissue structure closer to native lung architecture. Two cell seeding sequences were evaluated to examine how cellular arrangement influences permeability (Papp) and tight junction formation. The huAEC-first configuration demonstrated improved early barrier integrity across multiple membrane types, with faster reductions in Papp indicating effective tight junction establishment. A further critical component of this work involved characterizing the mucus layer gener- ated within these models. Mucus characterisation of in vitro airway models is necessary to ensure functional mimicry of respiratory tissue. As an application in further analytical studies such as viral interaction or aerosol formation and encapsulation studies, critically depends on physical properties like surface tension and viscosity of the mucus layer. De- tailed rheological assessments, including surface tension and viscoelasticity analyses, were conducted using a rheometer to evaluate the mucus’s stability and behaviour under strain. This study evaluated surface tension (ST) measurement methodologies for mucus samples in in vitro airway models, demonstrating that repetitive cyclic measurements yield stable and reliable ST values. Variations in ST across different environmental settings and prepa- ration methods underline the importance of humidity control and measurement protocol customisation to capture accurate mucus properties. Relative viscosity measurements explored across varied settings for amplitude and frequency sweep analyses confirmed that the mucus layer exhibited shear-thinning properties, transitioning from elastic to viscous behaviour under higher strains, a characteristic behaviour of native respiratory mucus that supports its function in protecting epithelial layers. In essence, this thesis will demonstrate ways through which scaffold material selections can be optimised by means of essential mechanical characteristics of elasticity modulus and tensile strength. Secondly, the critical selection of cellular components and cell seeding strategies are presented to accomplish region-specific in vitro model generation. And thirdly, the steps of functional characterisation of in vitro models as well as, most importantly, mucus using standardised protocols when establishing high-fidelity 3D in vitro models of the lung. These models represent a significant step forward in the recreation of structural and functional complexities of human respiratory system regions, based on the mechanical properties of the ECM and cellular heterogeneity. Presented region-specific in vitro models based on novel synthetic electrospun polymeric scaffold membranes and selective cell co-culture provide a potential platform for applications, especially to study aerosol production as a basis for virus encapsulation in respiratory research, drug testing, and extended application in Tissue Engineering.