Publication
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 chang