Dynamical and Mechanical Characterisation of Bi- and Tri-dimensional Neuronal Cultures: from Health to Disease

Abstract

[eng] In this PhD thesis, we leverage the well established foundations of complex systems physics and integrate insights from medicine and materials engineering to advance our understanding of neuroscience. Our primary focus revolves around neuronal cultures, se rving as the central protagonists in our exploration. We endeavor to establish a robust connection between neurons, the underlying physics dictating their interactions, and the materials shaping their environment. The document is divided into three different lines of investigation: the application of primary neuronal cultures to model neurodegenerative diseases (ND), the characterization of a hydrogel scaffold that facilitates the study of 3D neuronal cultures, and a combination of both mentioned lines, in which we establish a relationship between the stiffness of the hydrogel scaffold and the activity behavior of the neuronal network. Within the scope of this research, we push the boundaries of primary neuronal cultures to model ND, with a specific focus on tauopathies an array of devastating disorders characterized by abnormal forms of the microtubule associated tau. Conducted as part of the 'La Caixa Health Research 2019' initiative, our research involved probing the impact of extracellular seed competent tau on the neuronal activity of primary cortical cultures derived from wild type mice. Utilizing calcium fluorescence imaging and ad vanced analytical tools, we examined dynamic and functional features of tau treated neuronal networks. Surprisingly, no discernible changes in cultured neurons were observed after treatment, challenging assumptions about tau's role in the disease. This out come prompts a re evaluation of the in vitro model's suitability or suggests a more complex disease mechanism. Recognizing the limitations of 2D neuronal cultures, our focus shifted to the evolving field of 3D cultures. We introduced a semi synthetic hydrogel for neuronal cultures, PEGylated fibrin, and characterized its mechanical behavior over a three week period using a rheometer an essential step to understanding the neuron matrix relationship and determining the lifespan of cultures. This investigation encompassed hydrogels seeded with neurons, providing valuable insights into the impact of neuronal network dev elopment within the 3D hydrogel structure. Our findings reaffirmed the suitability of the selected hydrogel for cultivating neuronal cultures. Its stiffness closely mimicked that of the actual brain, and it demonstrated an optimal lifespan for studying the development of neuronal cultures. In an effort to bridge the two aforementioned lines of research, we investigated the relationship between the scaffold's stiffness and the functional organization of neuronal networks cultivated within these structures. The microstructure of PEGylated fibr in hydrogels can be finely tuned by adjusting the concentration of thrombin, one of its main components. Therefore, we focused on creating three distinct variants of PEGylated fibrin hydrogel with different concentrations of thrombin. Employing our establi shed protocol, we characterized the mechanical properties of these hydrogel variants. Simultaneously, through calcium imaging, we recorded the spontaneous activity of neuronal networks developed within these structures and conducted a comprehensive functio nal analysis. While our preliminary results indicated an intriguing correlation between the initial neuronal development and the stiffness of the hydrogel, deciphering this effect proved challenging due to the myriad factors at play and the limitations of available microscopy techniques in this study. This investigation serves as a pioneering step, laying the groundwork for future research in this exciting multidisciplinary field.

[spa] Esta tesis doctoral fusiona la de física de sistemas complejos con conceptos de medicina y ciencia de materiales para avanzar en nuestra concepción de los cultivos neuronales primarios y su papel dentro de la neurociencia. La investigación está dividida en tres ramas diferentes que abarcan (i) el uso de cultivos neuronales para modelar enfermedades neurodegenerativas (NDs) in vitro, (ii) la presentación de una matriz de hidrogel para el desarrollo de cultivos neuronales en 3D, y (iii) un estudio que intenta establecer una relación entre las propiedades mecánicas de la matriz de hidrogel previamente mencionada y las características de la actividad neuronales desarrollada en su interior. Nuestra investigación intenta arrojar algo de luz en el estudio de las taupatías, un tipo de NDs caracterizada por la aparición de agregados de proteína tau anormal. Para ello tratamos cultivos neuronales primarios con tau ‘patológica’ y aplicamos técnicas de fluorescencia de calcio y herramientas de análisis complejo, para caracterizar las redes neuronales resultantes. Los resultados no mostraron cambios perceptibles en las neuronas tras el tratamiento, lo que cuestiona las suposiciones sobre el papel de tau en la enfermedad o sobre el modelo seleccionado para su estudio. Reconociendo las limitaciones de los cultivos neuronales en 2D, nuestro interés se centró en el desarrollo de cultivos 3D. Nuestro estudio se basa en la caracterización mecánica de un hidrogel semisintético, la fibrina PEGilada, para comprender la relación neurona. Con la ayuda de un reómetro seguimos la rigidez del hidrogel, con y sin neuronas, durante un periodo de tres semanas. Nuestros resultados reafirmaron la idoneidad del hidrogel seleccionado para el cultivo de neuronas. Para conectar las dos líneas de investigación presentadas, quisimos estudiar la relación entre la rigidez del hidrogel y la organización funcional de las redes neuronales cultivadas en su interior. Para ello creamos tres variantes de fibrina PEGilada con diferente rigidez y caracterizamos la evolución de las propiedades mecánicas de las tres variantes del hidrogel a lo largo de tres semanas. En paralelo, realizamos un análisis funcional exhaustivo de la actividad neuronal dentro de estas estructuras. Esta investigación constituye las bases para futuras investigaciones en este apasionante campo multidisciplinar.