Periodic Reporting for period 3 - CONNEXIO (Physiologically relevant microfluidic neuro-engineering)
Periodo di rendicontazione: 2019-11-01 al 2021-04-30
Developing minimalistic biological neural networks and observing their functional activity is crucial to decipher the information processing in the brain. This project aims to address two major challenges: to design and fabricate in vitro biological neural networks that are organized in physiological relevant ways and to provide a label-free monitoring platform capable of observing neural activity both at the neuron resolution and at large fields of view. To do so, the project has developed a unique microfluidic compartmentalized chips where populations of primary neurons are seeded in deposition chambers with physiological relevant number and densities. Chambers are connected by microgrooves in which neurites only can grow and whose dimensions will be tuned according to the connectivity pattern to reproduce. To observe the activity of such complex neural networks, we developed a disruptive observation technique that transduces the electrical activity of spiking neurons into optical differences observed on a lens-free platform, without calcium labelling and constantly in-incubo. By combining neuro-engineering patterning and the lens-free platform, we compared individual spiking to global oscillators in basic neural networks under localized external stimulations. Such results provide experimental insight into computational neuroscience current approaches.
The first months of the project were dedicated to the implementation of his team and the setting up of the necessary environment to fulfil the first experiments. The dissemination of the project was developed, as well as various discussions with experts in the microfluidic field.
The protocols were set up for the fabrication of microfluidic chips for neuron culture. 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 was implemented. A cell culture device was developed that allows continuous renewal of the culture medium. This device has been implemented on culture microplates (96 wells), making it possible to conduct 8 experiments in parallel.
The devices developed were used to study directed neuronal growth. The aim was 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. During the first year of the project, a focus on the optimisation of the culture of these neurons has been set up in the presence of a parallel network of micrometric constrictions, with a view to obtaining an electrical flow oriented in a precise direction.
The protocols were set up for the fabrication of microfluidic chips for neuron culture. 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 was implemented. A cell culture device was developed that allows continuous renewal of the culture medium. This device has been implemented on culture microplates (96 wells), making it possible to conduct 8 experiments in parallel.
The devices developed were used to study directed neuronal growth. The aim was 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. During the first year of the project, a focus on the optimisation of the culture of these neurons has been set up in the presence of a parallel network of micrometric constrictions, with a view to obtaining an electrical flow oriented in a precise direction.
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.
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.