Functional Characterization of Three-Dimensional Cortical Cultures for In Vitro Modeling of Brain Networks

Abstract

Summary Three-dimensional (3D) in vitro cultures recapitulate key features of the brain including morphology, cell-cell and cell-extracellular matrix interactions, gradients of factors, and mechanical properties. However, there remains a need for experimental and computational tools to investigate network functions in these 3D models. To address this need, we present an experimental system based on 3D scaffold-based cortical neuron cultures in which we expressed the genetically encoded calcium indicator GCaMP6f to record neuronal activity at the millimeter-scale. Functional neural network descriptors were computed with graph-theory-based network analysis methods, showing the formation of functional networks at 3 weeks of culture. Changes to the functional network properties upon perturbations to glutamatergic neurotransmission or GABAergic neurotransmission were quantitatively characterized. The results illustrate the applicability of our 3D experimental system for the study of brain network development, function, and disruption in a biomimetic microenvironment.

Integrated functional neuronal network analysis of 3D silk-collagen scaffold-based mouse cortical culture

“…We have previously reported several in vitro brain models, with both primary rodent neurons and human induced pluripotent stem cells (iPSCs), using porous scaffolds fabricated with silk fibroin and extracellular matrix composites. Such models recapitulated many key features of the brain tissue ( Cairns et al., 2020 ; Cantley et al., 2018 ; Dingle et al., 2020 ; Liaudanskaya et al., 2020 ; Rouleau et al., 2020 ; Tang-Schomer et al., 2014 ).…”

“…This protocol includes instructions for fabricating protein-based composite scaffolds, 3D in vitro culture of embryonic mouse cortical neurons, virally induced expression of GCaMP6f, wide-field calcium imaging, and computational analysis with open-source software and custom MATLAB code. For complete details on the use and execution of this protocol, please refer to Dingle et al. (2020) .…”

“…For complete details on the use and execution of this protocol, please refer to Dingle et al. (2020) .…”

Electrophysiology Read-Out Tools for Brain-on-Chip Biotechnology

“…Generally, along with the advanced imaging tools, accurate and possibly automated analysis tools are developed ad hoc to dissect structural and functional information of the networks and for deriving connectivity maps [ 326 ]. Moreover, advanced computational and statistical methods show promise to enable inferring the correlation of calcium signals with the underlying cellular and network dynamics from the biophysical standpoint [ 327 , 328 , 329 ].…”

Neuronal Graphs: A Graph Theory Primer for Microscopic, Functional Networks of Neurons Recorded by Calcium Imaging

“…These graphs are known as weighted graphs and are used to embed a greater quantity of information within the structure of a graph (Barrat et al, 2004 ). Working with macroscopic, functional networks, i.e., graphs with nodes representing regions on interest (Dingle et al, 2020 ) used average edge weight as a measure of overall connectivity.…”