Compartmentalised microfluidic culture systems for in vitro modelling of neurological and neuromuscular microenvironments

[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.[spa] El movimiento de las fibras musculoesqueléticas está generado por el circuito locomotor. Los fallos en cualquier parte del circuito pueden originar la aparición de enfermedades neuromusculares (ENM) — como la esclerosis lateral amiotrófica (ELA) — o determinar su gravedad. Los modelos de estudio in vitro convencionales, basados en cocultivos de motoneuronas con células de músculo esquelético, no son representativos de la fisiología humana. Además, el estudio de fragmentos del circuito no puede aportar una visión global y exhaustiva del proceso patológico. Esta tesis tiene como objetivo estudiar el contexto neuromuscular in vitro a través de sistemas microfluídicos de cultivo compartimentado (cµFCS) y crear modelos de estudio fisiológicamente relevantes. Ofrece una perspectiva evolutiva de modelos in vitro, pasando de células murinas a humanas, de cultivos celulares 2D a 3D, de células primarias a células madre humanas pluripotentes inducidas (hiPSC), y analizando tanto células sanas como enfermas. El Capítulo 1 hace una revisión bibliográfica del estado del arte en el circuito neuromuscular, la ELA, y la evolución de las técnicas in vitro disponibles para su estudio. El Capítulo 2 presenta la optimización inicial del modelo de conexión neuromuscular in vitro en un chip, junto con el estudio de la relevancia de la mielina en el sistema nervioso periférico y en el circuito neuromuscular. El Capítulo 3 pasa a estudiar la propiocepción, la diferenciación de células madre neurales humanas a neuronas sensoriales propioceptivas, y su papel en el desarrollo de la ELA. Estos conceptos, junto con los introducidos en el Capítulo 2, se integran en el Capítulo 4, presentando el desarrollo de un circuito neuromuscular humano fisiológico en un dispositivo microfluídico, que integra las vías sensoriales y motoras neuromusculares en un sistema de cultivo celular en 3D. Finalmente, en el contexto de la vascularización neuromuscular, el Capítulo 5 estudia la barrera hematoencefálica y las técnicas para monitorizar su permeabilidad, afectada en ciertas ENM como la ELA. Esta tesis recoge muchas innovaciones tecnológicas desde el punto de vista de la Bioingeniería, abriendo nuevas puertas para futuros estudios en el campo neuromuscular. Muestra que la integración de todos los componentes del circuito neuromuscular en los sistemas in vitro aquí desarrollados aporta una visión más amplia de la fisiología neuromuscular y los procesos patológicos. Estos resultados representan un primer paso hacia futuros modelos de circuitos neuromusculares 3D fisiológicos en un chip para estudios de ENM