Optimization and validation of the ALIce bioreactor for dynamic air–liquid interface skin models

In vivo, epithelial tissues are directly exposed to the external environment. To replicate this condition in vitro, the air–liquid interface (ALI) approach is widely used. Traditionally, ALI is obtained using a transwell insert with a semipermeable membrane placed in a well to separate the liquid and the air compartments. However, this static culture environment does not mimic the nutrient and gas transport provided by the native microvasculature, leading to nutrient gradients. To overcome this limitation, culture medium recirculation can be adopted. In this context, this thesis aims to optimize, prototype, characterize and validate an ALI culture environment (ALIce) bioreactor with a recirculating medium system for skin tissue engineering applications. In detail, the ALIce bioreactor was designed to: i) guarantee ALI culture; ii) allow culture medium recirculation; iii) ensure a continuous nutrient and oxygen transport across the ALI while avoiding air bubble stagnation under the membrane; iv) be easy-to-use with standard lab equipment. To fulfil these requirements, the ALIce bioreactor comprises: i) a culture chamber (CC) designed to house a commercial transwell insert, which separates the liquid and the air compartments; ii) a recirculation circuit enabling continuous culture medium flow; iii) a custom-made support base for parallelized experiments with up to four ALIce CCs. The CC was designed in SolidWorks and 3D printed via stereolithography using a biocompatible and autoclavable material. It consists of a lid and a cylindrical base with two barbed channels for medium inlet and outlet, to connect tubing. Moreover, the lid was designed with a bayonet closure to ensure both secure sealing and the maintenance of atmospheric pressure within the CC through an air filter connected to a female luer-lock port on the lid. The CC is part of a closed-loop recirculation circuit based on a multi-channel peristaltic pump, a medium reservoir, oxygen-permeable tubing and luer-lock connectors. In particular, a custom lid for the reservoir was designed to ensure full integration with the bioreactor. It was 3D printed using BioMed Clear Resin and features four barbed channels: two connected to the inlet and outlet of the CC, one for an air filter, and the last allowing medium sampling without opening the reservoir, while preserving sterility via an injection site. Finally, the support base was 3D printed via fused deposition modeling. In-house tests confirmed the functionality of the ALIce bioreactor, particularly the maintenance of constant liquid volume inside the CC, the expulsion of air bubbles before reaching the transwell, and the tight sealing of both chamber and reservoir lids. Preliminary biological tests were performed using 3D in vitro skin models embedding HFF-1 human fibroblasts in GelMA hydrogel (dermis) and HaCaT keratinocytes seeded on top (epidermis). The models were cultured in dynamic conditions (0.5 mL/min) for 14 days (n = 2), with static culture as control (n = 2). Immunofluorescence and ddPCR analyses demonstrated that dynamic culture improved skin model development, showing a thicker epidermis and a younger dermal profile. The optimized ALIce bioreactor represents a powerful parallelizable tool for investigating the complexity of skin tissue, overcoming the limitation of conventional methods, for skin tissue engineering applications.