Micro-engineering the Cerebral Cortical Cell Niche:A new Cell Culture Tool for Neuroscience Research

A major problem in traditional cell culture methods, such as Petri dishes and culture flasks, is the very simplified artificial environment around the cells. Traditional cell culture methods lack features of the native cell niche, such as gradients and cell organization. This lack probably explains why pharmaceutics against the neurodegenerative Alzheimer's disease successfully stop the propagation of the disease in the Petri dish, but fail so far in clinical trials. This thesis intends to improve cell culture methods for neuroscience research related to neural developmental questions and neurodegenerative diseases. As the cortex is the main part in our brain, related to memory, emotions and perception, this thesis does focus on cell culture tools and protocols for primary cortical neurons. Currently, dissociated neurons are cultured in pure or co-culture of neural and non-neural cells, but structuring elements and controlled gradient formation is missing. The first part of this thesis discusses different studies that implicate environmental components for neural cells in their native neural cell niche. We will establish a generic neural cell niche, which consists of different neural and non-neural cells, a structured 3D environment, molecular gradients and oriented neurite networks, in a nutshell. Additionally, a simplified model of the generic neural cell niche is introduced that is implemented in a microfluidic base cell culture tool. This novel artificial neural cell niche will provide cell layer structure in 3D and local control of molecular gradients at the microscale. We will use microfluidic technology to integrate missing features in cell culture techniques for primary cortical neurons. The microfluidic device will consist of three parts: (1) a main cell culture channel that is used to organize neural cells in 3D hydrogel layers, side-by-side; (2) parallel perfusion channels to mimic nutrient supply and to control stable gradient formation, (3) interconnecting microchannels, called junction channels, that separate perfusion driven molecular transport from diffusive molecular transport. The perfusion channels are connected to device-incorporated reservoirs that allow maintenance of stable molecular gradients based on perfusion and diffusion without the use of peristaltic or syringe pumps. By injecting cortical neurons entrapped in an agarose-alginate solution in the microfluidic device, we generated 3D micropatterned neural cell layers with stable gradients perpendicular to the layer orientation. We demonstrated neurite outgrowth behavior until three weeks in culture. The application of different cell organization patterns revealed an influence of the pattern on the cell culture response. Using neurotrophic gradients of nerve growth factor (NGF) and nutrient supplements (B27), we showed that neurite guidance and synapse formation followed synergistic NGF/B27 gradients. We found that the gradient induced effects are very sensitive to changes in the environmental structure, such as the cell layer organization. Using a gradient of a phosphatase inhibitor, okadaic acid, we generated locally diseased states of the protein Tau in both 2D and 3D micropatterned neural cell cultures. The diseased form of Tau, hyper-phosphorylated Tau, is a major hallmark of Alzheimer's disease. For the first time, local formation of hyper-phosphorylated Tau was demonstrated to affect a defined cell population in a compartmentalized 2D or 3D neural cell culture. Our results revealed that the propagation mechanism of this Alzheimer's diseased lesion is determined through the formation of the 2D or 3D environment. We think that this new neural cell culture tool has the potential to answer biological questions related to environmental and structural parameters involved in the formation of the cerebral cortex and in the propagation of neurodegenerative diseases. Future studies in modern neuroscience research can now better investigate the effect of gradient parameters such as the slope, average concentration or gradient profile on cell culture and disease propagation in a controlled manner. Furthermore, the influence of cell patterns in 2D and 3D is addressed with the ability to modify cell position, density and type at the microscale. The local formation of neurodegenerative disease lesions in a micropatterned neural cell culture is generic, which makes the integration of other neurodegenerative disease models, such as Parkinson's disease, Amyotrophic lateral sclerosis or Huntington's disease, possible