Development of engineered human-derived brain-on-a-chip models for electrophysiological recording

The study of the central nervous system represents a great challenge in the field of neuroscience. For years, various techniques have been developed to study neuronal cells in-vitro as it is difficult to conduct in-vivo experiments due to ethical problems deriving from its anatomical location. Consequently, both in-vivo and in-vitro animal models have been used extensively to gain new insights into basic functioning principles of neuronal tissue and therapeutic approaches for brain diseases. Over time, we have seen that there is a poor correlation between the clinical diagnosis and the underlying pathological mechanisms. In fact, some symptoms that may occur in the patient are not replicated in the animal, making many promising approaches in animal studies not translatable in the clinic. With the advent of human-induced pluripotent stem cells (h-iPSC) several protocols for the generation of human-neuronal cells are becoming available for all laboratories. The importance of this technique lies in the opportunity to develop a human model derived directly from the patient: the patient's in-vitro cells will exhibit the same genetic and epigenetic modifications as the in-vivo cells. This has raised hopes for the generation of engineered brain models that can be coupled to sensors / actuators in order to better investigate their functional properties in-vitro (i.e. brain-on-a-chip). A reliable method for evaluating the functionality of neuronal cultures is the study of the spontaneous electrophysiological activity using microelectrode arrays (MEA). There are numerous studies in the literature that used h-iPSC on MEAs, showing the characterization of neuronal patterns of patient-derived cultures, demonstrating how this platform is valid for disease phenotyping, drug discovery and translational medicine. Although these models helped to shed light on fundamental biological mechanisms, the majority is based on two-dimensional neuronal cultures, which lack some key features to mimic in-vivo behavior. Three-dimensional h-iPSC-derived models possess a microenvironment, tissue architecture and potential to model network activity with greater complexity than two-dimensional models. Depending on the purpose of the study, we can choose different approaches to recreate 3D in-vitro brain, from those that aim to reproduce the trajectories of neurodevelopment (i.e. brain-organoids) to the use of synthetic materials that reproduce the functionalities of the extracellular matrix (ECM) (i.e. scaffold-based) (Chiaradia and Lancaster, 2020, Tang et al., 2006). Although h-iPSC-derived brain models summarize many aspects of network function in the human brain, they are subject to variability and still do not perfectly mimic behavior in-vivo. Therefore, to reach the full potential of this model we need improvements in differentiation methods and bioengineering, making these models engineered and reproducible. The aim of this PhD thesis was to implement different 3D neuronal culture generation methodologies that can be integrated on MEA devices to offer robust engineered platforms for functional studies.