A micro-fabricated in vitro complex neuronal circuit platform

Abstract

Developments in micro-manufacture as well as biofabrication technologies are driving our ability to create complex tissue models such as ‘ organ-on-a-chip ’ devices. The complexity of neural tissue, however, requires precisely specific cellular connectivity across many neuronal populations, and thus there have been limited reports of complex ‘ brain-on-a-chip ’ technologies modelling specific cellular circuit function. Here we describe the development of a model of in vitro brain circuitry designed to accurately reproduce part of the complex circuitry involved in neurodegenerative diseases; using segregated co-culture of specific basal ganglia (BG) neuronal subtypes to model central nervous system circuitry. Lithographic methods and chemical modification were used to form structured micro-channels, which were populated by specifically cultured neuronal sub-types to represent parts of the inter-communicating neural circuit. Cell morphological assessment and immunostaining showed connectivity, which was supported by electrophysiology measurements. Electrical activity of cells was measured using patch-clamp, showing voltage dependant Na + and K + currents, and blocking of Na + current by TTX, and calcium imaging showing TTX-sensitive slow Ca 2+ oscillations resulting from action potentials. Monitoring cells across connected ports post-TTX addition demonstrated both upstream and downstream changes in activity, indicating network connectivity. The model developed herein provides a platform technology that could be used to better understand neurological function and dysfunction, contributing to a growing urgency for better treatments of neurodegenerative disease. We anticipate the use of this advancing technology for the assessment of pharmaceutical and cellular therapies as a means of pre-clinical assessment, and further for the advancement of neural engineering approaches for tissue engineering.

Mathematical Modeling of Neuronal Logic, Memory and Clocking Circuits

“…There are several ways in which these ideas may be implemented, however, in the short term it appears that the use of fluidic chambers and tunnels may provide the most robust, reliable and durable assays using current technology. Dr Roach and his group are currently fabricating platforms enabling the segregated co-culture of neurons to be controlled, with connectivity between neuronal populations being possible only by direction of neurites through microchannels [Kamudzandu, 2019]. Such devices are becoming more commonplace in the literature, although very few address the issues of multiple different neuronal types within a single device.…”

Advances in microfluidic in vitro systems for neurological disease modeling

“…Therefore, in vitro models where different cell populations can be grown and connected “ad hoc” present simplified but valuable tools to investigate network function as well as neuropathological conditions. Microfluidic technologies have been employed to create human brain‐mimicking neuronal circuits for the study of cortico‐striatal networks using calcium imaging (Lassus et al., 2018), to model neuroprotective mechanisms (Samson et al., 2016), to study structure–function relationship by precise neurite guidance achieved by electric fields [77]), to highlight brain region‐specific cell identities (Kamudzandu et al., 2019), physiology and function (Dauth et al., 2017), to create 3D structured circuits (van de Wijdeven et al., 2018) and for drug screening (S. R. Lee et al., 2019) (Figure 3). Kajtez et al, have also recently used a hybrid fabrication technique by integrating 3D printing with soft lithography to provide rapid prototyping at both micro‐ and macroscale, enabling open‐well compartmentalized devices with greater freedom of device design (Figure 3a) (Kajtez et al, 2020).…”

“…Similarly Hernández et al, used glutamate‐induced excitotoxicity in a compartmentalized microfluidic neuronal circuit to study axonal degeneration, showing co‐activation of two independent degenerative mechanisms in axon and soma, with axonal degeneration progressing via necroptotic kinases RIPK1 and RIPK3, while apoptotic events predominate in the soma (Hernandez et al., 2018). The use of microfluidic neuronal circuits consisting of multiple neuronal types, such as that presented by Kamudzandu et al which mimics the circuitry of the basal ganglia, may further shed light on the influence of excitotoxity on network activity (Kamudzandu et al., 2019).…”

“…Soscia et al use a removable insert to pattern primary rodent hippocampal and cortical neurons onto an MEA which showed characteristic bursting and communication between neuronal compartments (modified from Soscia et al, 2017 under the Creative Commons Attribution License) [Color figure can be viewed at wileyonlinelibrary.com]cell populations can be grown and connected "ad hoc" present simplified but valuable tools to investigate network function as well as neuropathological conditions. Microfluidic technologies have been employed to create human brain-mimicking neuronal circuits for the study of cortico-striatal networks using calcium imaging(Lassus et al, 2018), to model neuroprotective mechanisms(Samson et al, 2016), to study structure-function relationship by precise neurite guidance achieved by electric fields [77]), to highlight brain region-specific cell identities(Kamudzandu et al, 2019), physiology and function(Dauth et al, 2017), to create 3D structured circuits(van de Wijdeven et al, 2018) and for drug screening (S. R. (Figure 3).Kajtez et al, have also recently used a hybrid fabrication technique by integrating 3D printing with soft lithography to provide rapid prototyping at both micro-and macroscale, enabling open-well compartmentalized devices with greater freedom of device design(Figure 3a)(Kajtez et al, 2020). Using this system, they demonstrate a proofof-principle human in vitro model of the nigrostriatal pathway using human stem cell-derived neurons(Kajtez et al, 2020).…”

Development of two-photon polymerised scaffolds for optical interrogation and neurite guidance of human iPSC-derived cortical neuronal networks

“…Previous studies in microfluidics and micropatterning have shown that constrictive funnel motifs may be employed to gate directional neurite extension. 29,[63][64][65] Building upon these findings, we used 2PP to fabricate a matrix of 2.5D triangles in a repeated pattern, with the aim of guiding directional neurite extension over relatively large distances (>500 μm). Progenitors cultured upon patterned and planar unpatterned platforms were synchronously differentiated and analysed to determine their distribution and orientation (Fig.…”