Fast wide-volume functional imaging of engineered in vitro brain tissues

G. Palazzolo, M. Moroni, A. Soloperto, G. Aletti, G. Naldi, M. Vassalli, T. Nieus, F. Difato
2017 Scientific Reports  
The need for in vitro models that mimic the human brain to replace animal testing and allow highthroughput screening has driven scientists to develop new tools that reproduce tissue-like features on a chip. Three-dimensional (3D) in vitro cultures are emerging as an unmatched platform that preserves the complexity of cell-to-cell connections within a tissue, improves cell survival, and boosts neuronal differentiation. In this context, new and flexible imaging approaches are required to monitor
more » ... he functional states of 3D networks. Herein, we propose an experimental model based on 3D neuronal networks in an alginate hydrogel, a tunable wide-volume imaging approach, and an efficient denoising algorithm to resolve, down to single cell resolution, the 3D activity of hundreds of neurons expressing the calcium sensor GCaMP6s. Furthermore, we implemented a 3D co-culture system mimicking the contiguous interfaces of distinct brain tissues such as the cortical-hippocampal interface. The analysis of the network activity of single and layered neuronal co-cultures revealed cell-type-specific activities and an organization of neuronal subpopulations that changed in the two culture configurations. Overall, our experimental platform represents a simple, powerful and cost-effective platform for developing and monitoring living 3D layered brain tissue on chip structures with high resolution and high throughput. The design and development of realistic experimental models of the human brain remain major constraints in studying nervous system functions in health and disease, developing new treatment strategies, and testing prosthetic devices 1 . For these reasons, in vitro models represent an attractive and ethical tool since they allow high-throughput cost-accessible investigation 2 . Moreover, the development of cell reprogramming and differentiation protocols that allow investigators to recapitulate human diseases in a dish further discourages the use of animal models. The most common approach to building a brain on a chip is based on 2D cultures of defined neural cell types with a controlled network topology 3 . These models are highly reproducible and are compatible with standard monitoring systems 4 . However, growth of cells on a 2D support alters cellular physiology 5 and fails to reproduce the real properties of a tissue. Growth on stiff Petri dish surfaces induces aberrant branching of neuronal processes 6 , which, together with the lower degree of freedom of neurite navigation in a 2D setting, typically promotes overloading of interneuronal connections. This, in turn, may cause the observed amplification of the activity of 2D neuronal networks on stiff substrates 7 , which may lead to non-physiological states 8 . In this regard, it is widely recognized that mechanical and topographical cues influence cell survival, proliferation and differentiation and finally the development of functional neural networks 9 . Indeed, the architecture of a 3D scaffold allows isotropic distribution of adhesion contacts, thereby modifying the intracellular distribution of mechanical tension and consequently nuclear shape, chromatin arrangement and gene expression in cells 10 . Moreover, mechanotaxis influences the motility of neuronal processes 6 , and it has a critical role in axonal pathfinding in vivo 11 . For all of these reasons, there has been a growing demand for new technologies and materials (either natural or synthetic) Published: xx xx xxxx OPEN www.nature.com/scientificreports/ 2 SCIeNTIFIC RePoRTS | 7: 8499 |
doi:10.1038/s41598-017-08979-8 pmid:28819205 pmcid:PMC5561227 fatcat:twme6t7pzfd3ligmnaa2oa4wgm