ENGINEERING THERMOPLASTIC ORGAN-ON CHIP PLATFORM FOR RELIABLE IN-VITRO MODELLING

The drug discovery pipeline remains one of the most resource-intensive and failure-prone processes in biomedical research. Despite unprecedented advances in genomics and highthroughput screening, more than 80% of drug candidates still fail during clinical development, predominantly due to lack of efficacy or unexpected toxicity. Traditional twodimensional (2D) cultures lack the physiological complexity of native tissues, while animal models are limited by interspecies differences, high costs, and ethical concerns. These limitations underscore the urgent need for predictive in vitro models that can recapitulate the cellular and microenvironmental dynamics of human organs. In this context, Organ-on-Chip (OoC) technologies are emerging as powerful alternatives, integrating microfluidics, biomaterials, and tissue engineering to provide dynamic, reproducible, and physiologically relevant platforms for drug testing and disease modelling. This doctoral research aimed to design, fabricate, and validate a new generation of OoC devices based on thermoplastic polymers, specifically polymethyl methacrylate (PMMA), combined with electrospun polylactic acid (PLA) scaffolds and hydrogels. The integration of scaffolds into microfluidic devices was conceived to mimic extracellular matrix–like properties, enabling long-term co-cultures, guiding cell morphology, and supporting physiologically relevant responses under flow. Importantly, the choice of thermoplastics—widely used in biomedical devices—addresses key translational challenges of PDMS-based devices, including small-molecule absorption, poor scalability, and lack of regulatory compatibility. The fabrication workflow was centered on rapid prototyping via CO₂ laser micromachining and multilayer thermal bonding. Process parameters were systematically optimized to preserve PMMA transparency, minimize thermal damage, and ensure robust sealing with commercial microfluidic connectors. Electrospun PLA scaffolds with either random or aligned architectures were integrated as culture substrates. Structural, thermal, and surface analyses demonstrated that scaffold morphology and porosity were largely preserved post-bonding, while fiber alignment provided a tunable handle on mechanical and biological properties. Plasma treatments further enhanced hydrophilicity and scaffolddevice integration. Three proof-of-concept applications were developed and validated: I. Liver-on-Chip (LoC): a dual-chamber device for studying chronic, low-dose exposure of hepatocytes to tumor-derived extracellular vesicles. The platform recapitulated key features of pre-metastatic niche formation, with hepatocytes acquiring mesenchymal-like traits under continuous vesicle perfusion, highlighting its potential for modelling tumor–organ cross-talk. II. III. IV. Tumor-on-Chip (ToC): a breast cancer model integrating stromal fibroblasts to investigate tumor–stroma interactions and therapeutic resistance. The system reproduced the protective role of CAFs against olaparib–dinaciclib treatment, consistent with in vivo xenograft data, thereby validating its translational relevance for preclinical oncology. Brain-on-Chip (BoC): a platform designed to study selective drug–cell interactions in neural co-cultures. By combining neuronal and microglial cells on scaffold substrates, the BoC demonstrated selective uptake of myelin-derived nanovesicles by microglia under physiologically relevant flow, providing a new tool for neuropharmacological research. ISO-compliant dual-scaffold platform – a multilayer thermoplastic microfluidic device designed according to ISO 22916:2022 standards for microfluidic interoperability. The system integrates an aligned electrospun PLA membrane and an in situ ionically crosslinked alginate hydrogel within a single architecture, enabling the co-culture of endothelial and tumor cells under controlled conditions. The device demonstrated excellent structural integrity, hydrogel confinement, and cellular viability over 72 hours, establishing a foundation for scalable, standardized OoC manufacturing and regulatory translation. Taken together, these projects establish thermoplastic, scaffold-integrated OoC platforms as versatile and reproducible systems for modelling organ-specific physiology and pathology. From an engineering perspective, the workflow—based on rapid prototyping, commercial connectors, and scalable materials—addresses critical challenges of standardization and industrial translation. From a biological standpoint, the integration of PLA scaffolds provided an extracellular matrix–like environment that preserved morphology, promoted co-culture interactions, and enabled physiologically relevant readouts. This work contributes to the broader effort of aligning OoC development with international standards (ISO 23494, ISO 10993, CEN/CENELEC FGOoC roadmap), fostering reproducibility, interoperability, and regulatory adoption. By bridging static 2D assays, animal models, and clinically relevant outcomes, the proposed platforms advance the case for OoCs as predictive preclinical tools. Future directions include the incorporation of primary and iPSC-derived cells, integration of vascular and immune compartments, coupling to multi-organ systems for PK/PD studies, and embedding real-time sensors for quantitative monitoring. In conclusion, this thesis demonstrates that scaffold-integrated thermoplastic Organ-onChip systems represent a significant step forward in preclinical research, offering scalable, ethical, and physiologically meaningful alternatives to animal testing. By combining engineering innovation with biological validation, these platforms pave the way for more predictive drug discovery pipelines and personalized therapeutic strategies.