• breathing in-vitro
• lung in-vitro model
• spherical membrane fabrication

The growth of air pollution has led to an increase in cases of respiratory diseases accompanied by a more rapid spread and virulence of viruses. Therefore, the creation of human-relevant in vitro models for the pathophysiological study of the lung is of great interest to achieve a better understanding of how the apparatus works and how (nano)particles and viruses in the air interact with the body's cells.

Physiologically relevant alveolar models require an air-liquid interface (ALI) with an airflow, allowing gas exchange, and a fluid flow that mimics blood flow and provides nutrients and shear to the cells. They should also be capable of providing a radial deformation like that of the alveoli (around 5% at 0.2 Hz). Finally, a feature missing from most engineered models is the presence of a curved surface which recapitulates the 3D shape of alveoli, a feature known to modulate cell morpho-function.
In the literature, macro and microscale lung in vitro models are mainly based on flat membranes actuated with non-physiological pressures or motor driven strategies.

The aim of my thesis is to overcome this limitation through the design of a breathing acinus capable of cyclic stretching that can replicate the spherical structure and hierarchical architecture in vivo. The model will be composed of different spherical alveolar structures and an alveolar duct, constituting an alveolar sac. Thanks to the modularity of the systems, different sacs can be connected in parallel forming an acinus. I scaled the model from the in vivo dimension (225 µm for the alveolus, 240 µm for the duct in diameter) to 11.25 mm and 12 mm, respectively, to allow comparison with standard culture formats (e.g., 24 multi-well plates).

Two fabrication approaches were implemented: the first approach allowed the fabrication of a coupled duct and alveoli thanks to gel casting in fused deposition modelling (FDM) printed moulds and to the combination of a structural gel with a sacrificial gel to obtain the inner cavities of duct and alveoli. The advantages of this approach are the realisation of a single gel structure and the possibility of encapsulating the cells in the gel during casting. However, depending on the final application of the model, this approach may also have some limitations such as long-term stability under dynamic conditions. The second approach allowed the fabrication of spherical membranes which replicate alveolar curvature and are transparent, stretchable, biocompatible, and permeable. The membranes were characterised in terms of elastic and fatigue mechanical properties (tension tests at 0.2 Hz), permeability (methylene blue passage tests) and
biocompatibility (A549 cell adhesion tests). Finally, they were interfaced with an alveolar duct and liquid compartment obtained via stereolithographic printing. All the moulds and prototypes were designed using Autodesk Fusion 360.
Thanks to computational models (COMSOL Multiphysics), we verified the physiological oxygenation and shear stress level. In fact, oxygen concentration does not fall below the critical threshold at which cells become deficient and the shear stress does not exceed the maximum physiological value (<2 Pa). Breathing movements can be also obtained thanks to standard pressurisation methods, in which the airflow is controlled by a pressure regulator, or via muscle-like magnetic actuation obtained thanks to the integration
of a magneto-responsive gel in the liquid compartment.

In conclusion, the prototype developed in this work is a step further towards the design and engineering of a physiologically relevant lung model and can also contribute to the reduction of animal experiments in line with the principles of the 3Rs (replacement, reduction, and refinement).