The goal of the project was to develop a microfluidic platform that would enable to design neural circuits mimicking for instance Hutchinson’s disease, and making it possible to measure neural electric activity at eh cell scale. While many researches focus on creating individual cell monitoring, the approach in Connexio consists in an interdisciplinary network level approach. First, rules to engineer in-vitro physiological relevant neural networks using rat primary neurons were designed. These rules had to be adapted to microfluidic chips, where gas exchange differ from standard culture conditions. Second, the microfluidic chips were equipped with electric sensors and oxygen control system. These developments required the collaboration of interns with distinct background: electrical engineering, biotechnology and micro and nanotechnology.
The platform was planned to enable the injection of pharmaceutical compounds as well as the control of the oxygen concentration. The protocols for the fabrication of microfluidic chips for neuron culture were set up. These chips were designed to control axon orientation. In particular, the design of geometries allowing the establishment of stationary chemical gradients in the microfluidic chambers and the design of micrometric constrictions leading to the directed growth of neurites were performed. A cell culture device that allows continuous renewal of the culture medium was implemented on culture microplates (96 wells), making it possible to conduct 8 experiments in parallel. Then these devices were used to study directed neuronal growth in order to reconstitute a neural network of controlled geometry, to reproduce in vitro the connection defects identified in Hutchinson's disease. These experiments required primary neurons. These were extracted from rat embryos and immediately used in the experiments. The culture of these neurons in the presence of a parallel network of micrometric constrictions was optimised, with a view to obtaining an electrical flow oriented in a precise direction.
Holographic imaging was developed to enable the analysis at once of a large population of neurons. Nevertheless, this technique has a spatial resolution of the order of 10 µm, which is not sufficient to visualize neurites or axons. Efforts were directed toward improving the resolution, for instance by developing a reconstruction algorithm that exploits the diffraction pattern capture at various heights above the cell culture.
In parallel, preliminary studies were conducted to design microfluidic chips making it possible to measure action potential of a neuronal population at the single cell level. A liquid crystal-based electro-optical device was designed, that have shown promising results. A microfluidic device was also developed to model ischemia by controlling (and measuring) oxygen tension in the chip. The functionality of the chip was demonstrated.
