Development of a Hydrogel-Based Microfluidic Platform to Guide and Control Morphogen Gradients in 3D Neural Tube Organoid Models

The development of three dimensional (3D) in vitro models, capable of recapitulating the physical and biochemical microenvironment of human embryonic development, is essential for investigating neural tube (NT) morphogenesis and associated pathologies. This thesis work adopts a bioengineering approach to overcome the limitations of conventional organoid cultures, implementing a hydrogel-based microfluidic platform that enables precise control of morphogen gradients in 3D Neural Tube Organoid (NTO) models. The main objective was to develop and validate a microfluidic platform, based on coumarin-derivative-functionalized polyethylene glycol (PEG) hydrogel which contains functional groups capable of undergoing photo-triggered [2+2] cycloaddition upon exposure to specific wavelengths of light, enabling polymer crosslinking. By using a multi-photon laser with specific wavelength, it was possible to decrosslink the hydrogel to generate high-resolution microchannel within it. These microchannels facilitate controlled morphogen flow, establishing spatiotemporally controlled gradients. The platform was characterized through durability tests performed by dextran perfusion over multiple days and closed-circuit pressure assays, which quantified the mechanical behaviour of the hydrogel. These experiments demonstrated the robustness of the system for prolonged applications under dynamic flow conditions. A key focus of the platform development was the design of a precise niche to host the organoid within the hydrogel, achieved by using 3D-printed optimized pillars with specific dimensions. Subsequent integration of NTOs into the platform aimed at enabling cell differentiation along the dorso-ventral (D-V) axis, to faithfully recapitulate in vivo morphogenic signals and overcoming the limitations of static culture methods. The establishment of morphogen gradients was assessed through confocal microscopy. The developed microfluidic platform represents a significant advancement in tissue bioengineering, providing a powerful tool for studies on neurulation and modeling of congenital diseases, such as caudal regression syndrome.