Abstract Microfluidic neuro-engineering design rules have been widely explored to create in vitro neural networks with the objective to replicate physiologically relevant structures of the brain. Several neurofluidic strategies have been reported to study the connectivity of neurons, either within a population or between two separated populations, through the control of the directionality of their neuronal projections. Yet, the in vitro regulation of the growth kinetics of those projections remains challenging. Here, we describe a new neurofluidic chip with a triangular design that allows the accurate monitoring of neurite growth kinetics in a neuronal culture. This device permits to measure the maximum achievable length of projecting neurites over time and to report variations in neurite length under several conditions. Our results show that, by applying positive or negative hydrostatic pressure to primary rat hippocampal neurons, neurite growth kinetics can be tuned. This work presents a pioneering approach for the precise characterization of neurite length dynamics within an in vitro minimalistic environment.
Abstract Compartmentalized microfluidic chips have demonstrated tremendous potential to create in vitro minimalistic environments for the reproduction of the neural circuitry of the brain. Although the protocol for seeding neural soma in these devices is well known and has been widely used in myriad studies, the accurate control of the number of neurites passing through the microchannels remains challenging. However, the regulation of axonal density among different groups of neurons is still a requirement to assess the inherent structural connectivity between neuronal populations. In this work, we report the effect of microchannel patterning strategies on the modulation of neuronal connectivity by applying dimensional modifications on microchannel-connected microfluidic chambers. Our results show that those strategies can modulate the direction and the number of neuronal projections of passage, therefore regulating the strength of the structural connections between two populations of neurons. With this approach, we provide innovative microfluidic design rules for the engineering of in vitro physiologically relevant neural networks.
In vitro modeling of human brain connectomes is key to explore the structure-function relationship of the centralnervous system. The comprehension of this intricate relationship will serve to better study the pathological mechanismsof neurodegeneration, and hence to perform improved drug screenings for complex neurological disorders, such asAlzheimer’s and Parkinson’s diseases. However, currently used in vitro modeling technologies lack potential to mimicphysiologically relevant neural structures, because they are unable to represent the concurrent interconnectivitybetween myriad subtypes of neurons across multiple brain regions. Here, we present an innovative microfluidic designthat allows the controlled and uniform deposition of various specialized neuronal populations within unique platingchambers of variable size and shape. By applying our design, we offer novel neuro-engineered microfluidic platforms,so called neurofluidic devices, which can be strategically used as organ-on-a-chip platforms for neuroscience research.Through the fine tuning of the hydrodynamic resistance and the cell deposition rate, the number of neurons seeded ineach plating chamber can be tailored from a thousand up to a million, creating multi-nodal circuits that representconnectomes existing within the intact brain. These advances provide essential enhancements to in vitro platforms inthe quest accurately model the brain for the investigation of human neurodegenerative diseases.
ABSTRACT The central nervous system is a dense, layered, 3D interconnected network of populations of neurons,and thus recapitulating that complexity for in vitro CNS models requires methods that can createdefined topologically-complex neuronal networks. Several three-dimensional patterning approacheshave been developed but none have demonstrated the ability to control the connections betweenpopulations of neurons. Here we report a method using AC electrokinetic forces that can guide,accelerate, slow down and push up neurites in un-modified collagen scaffolds. We present a means tocreate in vitro neural networks of arbitrary complexity by using such forces to create 3D intersectionsof primary neuronal populations that are plated in a 2D plane. We report for the first time in vitro basicbrain motifs that have been previously observed in vivo and show that their functional network is highlydecorrelated to their structure. This platform can provide building blocks to reproduce in vitro thecomplexity of neural circuits and provide a minimalistic environment to study the structure-functionrelationship of the brain circuitry. T. Honegger, M. I. Thielen, S. Feizi, N. E. Sanjana, J. Voldman, Sci. Rep. 2016, 6, 28384. Full text here
Abstract Axons in the developing nervous system are directed via guidance cues, whose expression varies both spatially and temporally, to create functional neural circuits. Existing methods to create patterns of neural connectivity in vitro use only static geometries, and are unable to dynamically alter the guidance cues imparted on the cells. We introduce the use of AC electrokinetics to dynamically control axonal growth in cultured rat hippocampal neurons. We find that the application of modest voltages at frequencies on the order of 105 Hz can cause developing axons to be stopped adjacent to the electrodes while axons away from the electric fields exhibit uninhibited growth. By switching electrodes on or off, we can reversibly inhibit or permit axon passage across the electrodes. Our models suggest that dielectrophoresis is the causative AC electrokinetic effect. We make use of our dynamic control over axon elongation to create an axon-diode via an axon-lock system that consists of a pair of electrode ‘gates’ that either permit or prevent axons from passing through. Finally, we developed a neural circuit consisting of three populations of neurons, separated by three axon-locks to demonstrate the assembly of a functional, engineered neural network. Action potential recordings demonstrate that the AC electrokinetic effect does not harm axons, and Ca2+ imaging demonstrated the unidirectional nature of the synaptic connections. AC electrokinetic confinement of axonal growth has potential for creating configurable, directional neural networks. T. Honegger, M. A. Scott, M. F. Yanik, J. Voldman, Lab Chip 2013, 13, 589. Full text here