Air-liquid interface bioreactor for skin tissue engineering applications: optimization, development and validation tests

In vivo, epithelial tissues are continuously in contact with the external environment. To mimic such condition in vitro the air-liquid interface (ALI) approach is widely adopted, which is traditionally obtained by using a transwell inserted in a well for separating the liquid and air compartments. However, such static environment lack in mimicking the in vivo nutrient and gas transport provided by the native microvasculature, leading to gradient formation within the culture. To overcome this limitation, culture medium recirculation can be adopted, knowing that it is crucial to avoid air bubbles stagnation under the ALI, to prevent sample drying. Indeed, the aim of this thesis was the optimization and validation of a biomimetic bioreactor for ALI culture with recirculating culture medium (RCM), to be used for skin tissue engineering investigations. The bioreactor is composed of a culture chamber (CC) and a RCM circuit. The CC has been designed (SolidWorks (Dassault Systèmes)) in four different models characterized by similar internal geometry. Each CC presents flow inlet and outlet aligned along the diameter with the outlet designed in a higher position than the inlet. Such models are: 1) Original: consisting of a single cylindrical CC (priming volume Vp = 0.3 ml); 2) Parallel (I): designed for biological test parallelization for statistical and reproducibility reasons (Vp = 1.25 ml); 3) Parallel (II): the output of 2) was tilted upwards to facilitate bubbles ejection (Vp = 1.3 ml); 4) Series: to perform influence tests on cells from different areas of the body (Vp = 1.95 ml). In each configuration, the CC is part of the closed-loop RCM circuit based on a multi-channel peristaltic pump, suitable to be used inside an incubator, with both inlet and outlet tubing connected to the peristaltic pump, for controlling the culture medium volume within the CC. For supporting the CCs geometry design and optimization, stationary computational fluid dynamic (CFD) simulations (COMSOL Multiphysics) were performed. The culture medium was modeled as an incompressible Newtonian’s fluid at 37°C and the transwell membrane as wall with no slip condition. Moreover, inlet and outlet flow rates of 0,506 - 0,552 ml/min respectively were imposed, for guaranteeing capillary-like flow velocity values within the CC. With the Original model, preliminary biological in both static and dynamic conditions (0.5 mL/min) were carried out for 7 days and live and dead assay was performed. Adopting an iterative process of design, modelling, prototyping, and testing, the bioreactor was optimized and validated. The CFD results showed that the fluid flows tangentially to the transwell membrane, avoiding recirculation regions. Preliminary in-house tests confirmed the bioreactor ease of use and reliability. It was assessed that a constant volume of culture medium was maintained, and, in case of air bubbles presence, these were easily expelled within the CC before reaching the transwell membrane. The biological tests highlighted that the system ensures sterility maintenance, indeed after both the static and dynamic culture a great amount of living cells was detected. Systematic tests on cells-laden hydrogel constructs cultured under dynamic conditions are ongoing. The proposed bioreactor provides a controlled 3D dynamic culture environment in which ALI culture and recirculating medium can be combined, with the final aim of in vitro producing three-dimensional skin tissue models.