Engineered biological neural networks on high density CMOS microelectrode arrays

Abstract

In bottom-up neuroscience, questions on neural information processing are addressed by engineering small but reproducible biological neural networks of defined network topology \textit{in vitro}. The network topology can be controlled by culturing neurons within polydimethylsiloxane (PDMS) microstructures that are combined with microelectrode arrays (MEAs) for electric access to the network. However, currently used glass MEAs are limited to 256 electrodes and pose a limitation to the spatial resolution as well as the design of more complex microstructures. The use of high density complementary metal-oxide-semiconductor (CMOS) MEAs greatly increases the spatiotemporal resolution, enabling sub-cellular readout and stimulation of neurons in defined neural networks. Unfortunately, the non-planar surface of CMOS MEAs complicates the attachment of PDMS microstructures. To overcome the problem of axons escaping the microstructures through the ridges of the CMOS MEA, we stamp-transferred a thin film of hexane-diluted PDMS onto the array such that the PDMS filled the ridges at the contact surface of the microstructures without clogging the axon guidance channels. Moreover, we provide an impedance-based method to visualize the exact location of the microstructures on the MEA and show that our method can confine axonal growth within the PDMS microstructures. Finally, the high spatiotemporal resolution of the CMOS MEA enabled us to show that we can guide action potentials using the unidirectional topology of our circular multi-node microstructure.

Engineering circuits of human iPSC-derived neurons and rat primary glia

“…In this work, we built upon the existing reports of a versatile and modular PDMS-based platform for bottom-up neuroscience research (Forro et al, 2018; Ihle et al, 2022; Girardin et al, 2022; Duru et al, 2022). We further demonstrate the versatility of the platform to build circuits of different ratios of human iNeurons and rat primary glial cells, initially seeded as either dissociated cells or spheroids, which can be cultured, imaged, and electrically monitored for more than 50 DIV.…”

“…Bottom-up neuroscience focuses on building and studying elementary circuits of neurons to infer the principles of more complex assemblies, with the main goal of getting a better understanding of the mechanisms behind information processing in the brain (Aebersold et al, 2016). Bottom-up neuroscience tools allow to build multiple, topologically-controlled circuits of tens to hundreds of neurons, which can be electrically stimulated and recorded from in parallel using microelectrode arrays (MEA) (Forro et al, 2018; Ihle et al, 2022; Duru et al, 2022; Girardin et al, 2022).…”

Microfluidic cell engineering on high-density microelectrode arrays for assessing structure-function relationships in living neuronal networks

“…The studies have revealed several non-random properties such as the modular architecture as a structure that is evolutionarily conserved in the nervous system (van den Heuvel et al, 2016) and provided mechanistic insights into how network structure defines system functions in both normal and pathological brains (Lynn and Bassett, 2019;van den Heuvel and Sporns, 2019;Suárez et al, 2021). While many studies have deciphered the structurefunction relationships in the nervous system in vivo (Meunier et al, 2010;Lee et al, 2016), recent advances in cell engineering technology using micropatterned proteins and microfluidic devices have enabled the use of cultured cells to study these relationships in a well-defined in vitro system (Feinerman et al, 2008;Lewandowska et al, 2015;Pan et al, 2015;Albers and Offenhäusser, 2016;Yamamoto et al, 2016bYamamoto et al, , 2018Forró et al, 2018;Nam, 2020, 2022;Takemuro et al, 2020;Duru et al, 2022;Ihle et al, 2022).…”

“…Recently, high-density MEA (HD-MEA) technology has been developed to mitigate the trade-off problem between spatial and temporal resolutions (Berdondini et al, 2009;Frey et al, 2010;Hierlemann et al, 2011;Bertotti et al, 2014;Obien et al, 2015;Yuan et al, 2020;Steinmetz et al, 2021). More precisely, recent HD-MEA devices offer spatial resolutions of over 3,000 electrodes mm −2 with the electrode pitch below 20 µm and a temporal resolution below 100 µs (Bertotti et al, 2014;Lewandowska et al, 2015;Kim et al, 2020;Yuan et al, 2020;Steinmetz et al, 2021;Duru et al, 2022;Shimba et al, 2022). The electrode pitch is comparable to the size of a neuronal cell body, and the temporal resolution is higher than a typical delay of synaptic transmission (∼0.6 ms) (Lisman et al, 2007).…”

A Scalable and Adaptive Ultra-high-density Fan-out Strategy for High-throughput Flexible Microelectrodes

“…There are two types of connections, direct and indirect, as shown in Fig.1A. The direct interconnection integrates electrodes and amplifiers through a CMOS process to achieve a high-density fan-out [16,17], but this method is currently not applicable to flexible electrodes. The indirect one is usually based on a welding process that first connects the electrodes to the chip or connector and then to the data acquisition system.…”