Abstract
Bottom-up neuroscience, which consists of building and studying controlled networks of neurons in vitro, is a promising method to investigate information processing at the neuronal level. However, in vitro studies tend to use cells of animal origin rather than human neurons, leading to conclusions that might not be generalizable to humans and limiting the possibilities for relevant studies on neurological disorders. Here we present a method to build arrays of topologically controlled circuits of human induced pluripotent stem cell (iPSC)-derived neurons. The circuits consist of 4 to 50 neurons with well-defined connections, confined by microfabricated polydimethylsiloxane (PDMS) membranes. Such circuits were characterized using optical imaging and microelectrode arrays (MEAs), suggesting the formation of functional connections between the neurons of a circuit. Electrophysiology recordings were performed on circuits of human iPSC-derived neurons for at least 4.5 months. We believe that the capacity to build small and controlled circuits of human iPSC-derived neurons holds great promise to better understand the fundamental principles of information processing and storing in the brain.
Highlights
In vitro networks of neurons can be engineered through two main approaches: surface patterning and physical confinement of the neurons
The PDMS microstructure is placed on a poly-D-lysine (PDL)coated glass coverslip or microelectrode array (MEA) with channels facing down
The PDMS microstructures are designed to be placed on top of a 60electrode MEA, aligning one electrode under each connecting microchannel of a 4-node circuit (Fig. 1c)
Summary
In vitro networks of neurons can be engineered through two main approaches: surface patterning and physical confinement of the neurons. Surface patterning consists of depositing specific molecules on a substrate to define cellattractive and cell-repellent areas. Neurons that connect together exert forces on each other leading to clustering and gradual changes in the network architecture.[13]. Coatings are degraded by the cells over time making it challenging to keep consistently patterned cultures over the long term. Since neurons typically take a week or more to become electrically active and functionally mature in vitro,[14,15] it is desirable to build networks that are stable over several weeks to be able to investigate their functional electrical activity. An alternative and more adopted method to engineer biological neuronal networks is the use of three-dimensional microfabricated structures to spatially confine cell bodies.[16].
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