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Si us plau utilitzeu sempre aquest identificador per citar o enllaçar aquest document: https://hdl.handle.net/2445/177843
Compartmentalised microfluidic culture systems for in vitro modelling of neurological and neuromuscular microenvironments
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[eng] Movement of skeletal-muscle fibres is generated by the locomotion circuit, in which many cells play a different role. Failures in any part of the circuit can cause or define the severity of neuromuscular diseases (NMD), such as amyotrophic lateral sclerosis (ALS). Conventional in vitro study models are based on cocultures of motoneurons and skeletal muscle cells from animal origin in 2D. These models have proved to be quite limited for the understanding of neuromuscular connection and NMD. They do not consider that: i) neural somas and muscles or peripheral glia are physically separated in vivo and have different microenvironment requirements; ii) both sensory and motor neurons can be altered in particular NMD; iii) glial cells are also affected and involved in several neuromuscular pathologies; iv) 2D cultures do not mimic physiological conditions; v) rodent models offer limited benefit translated into clinic research, as they do not carry human genetic background. Later progresses in neuromuscular-mimicking in vitro systems, have been achieved incorporating increasingly evolving technologies, such as 3D cell-culture techniques, human induced pluripotent stem cells (hiPSC) and compartmentalised microfluidic culture systems (cµFCS). The later ones are microfluidic devices for 2D or 3D cell- cultures, with several interconnected compartments, each mimicking different microenvironments or functional units in organ or tissue level. 3D cell culture techniques make cells acquire more in vivo like phenotype and genotype patterns. And finally, hiPSC serve to create study models that mimic the human physiology in both healthy and pathological conditions. This thesis, entitled “Compartmentalised microfluidic culture systems for in vitro modelling of neurological and neuromuscular microenvironments”, aims to study the neuromuscular context in vitro through cµFCS and to create physiologically relevant models. It offers an evolving prospective of in vitro models, moving from mice to human cells, from 2D to 3D cell cultures, from primary cells to hiPSC, and analysing both healthy and diseased cells. Chapter 1 reviews the state of the art in the neuromuscular circuit, amyotrophic lateral sclerosis, and the evolution of in vitro techniques available for their study. Chapter 2 presents the first approach of the neuromuscular in vitro connection model on a chip, showing the relevance of myelin in the peripheral nervous system and in the neuromuscular circuit. Chapter 3 moves to study the proprioception, the differentiation of human neural stem cells to proprioceptive sensory neurons, and their role in ALS. These concepts, together with the ones introduced in Chapter 2, are integrated in Chapter 4, presenting the development of a physiological human neuromuscular circuit on a microfluidic device, that integrates neuromuscular motor and sensory pathways in a 3D cell culture system. Lastly, in a context of neuromuscular vascularisation, Chapter 5 studies the blood-brain barrier and the techniques to monitor its permeability, known to be affected in some NMD such as ALS. This thesis presents the use of several cµFCS for different purposes, incorporating the study of several neuromuscular key role players and obtaining the following results. Myelination induction was successfully incorporated in a designed and fabricated compartmentalised microfluidic culture system (a PDMS device with two compartments connected through microchannels). This system was capable of a simplified mimicking of both peripheral nervous system and neuromuscular afferent or efferent pathways. To move onto human models, first proprioceptive sensory neuron (pSN) differentiation protocol was established. Genetic comparative analysis between healthy and ALS diseased samples revealed differences among pSN related genetic patterns and those involved in the communication between pSN and motoneurons (MN). Human pSN differentiation was combined with skeletal muscle cells to create sensorimotor units in a two-compartment commercial microfluidic device, showing for the first time the formation of synaptic bouton like structures in the contact points of an annulospiral wrapping. Then, a human neuromuscular circuit model was created, integrating for the first time human motor and sensory pathways in 3D cultures in tailored microfluidic devices. Finally, the blood-brain barrier was studied as an example of neural vascularisation, within the framework of potentially affected components in NMD. To that end, a new technology for the in vitro monitorisation of blood-brain barrier permeability was created and implemented in a device previously developed in the lab. This system could easily be translated for blood-spinal cord barrier studies. This thesis gathers many technological innovations from a Bioengineering point of view, paving the way for future studies in the neuromuscular field. It shows that the integration of the entire neuromuscular circuit components in the developed in vitro systems provides a wider view of the neuromuscular physiology and the pathological processes. These results show first steps towards future 3D physiological neuromuscular circuit models on a chip for NMD studies.
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BADIOLA MATEOS, Maider. Compartmentalised microfluidic culture systems for in vitro modelling of neurological and neuromuscular microenvironments. [consulta: 12 de desembre de 2025]. [Disponible a: https://hdl.handle.net/2445/177843]