Anatomically Inspired Three-dimensional Micro-tissue Engineered Neural Networks for Nervous System Reconstruction, Modulation, and Modeling

Abstract

Functional recovery rarely occurs following injury or disease-induced degeneration within the central nervous system (CNS) due to the inhibitory environment and the limited capacity for neurogenesis. We are developing a strategy to simultaneously address neuronal and axonal pathway loss within the damaged CNS. This manuscript presents the fabrication protocol for micro-tissue engineered neural networks (micro-TENNs), implantable constructs consisting of neurons and aligned axonal tracts spanning the extracellular matrix (ECM) lumen of a preformed hydrogel cylinder hundreds of microns in diameter that may extend centimeters in length. Neuronal aggregates are delimited to the extremes of the three-dimensional encasement and are spanned by axonal projections. Micro-TENNs are uniquely poised as a strategy for CNS reconstruction, emulating aspects of brain connectome cytoarchitecture and potentially providing means for network replacement. The neuronal aggregates may synapse with host tissue to form new functional relays to restore and/or modulate missing or damaged circuitry. These constructs may also act as pro-regenerative "living scaffolds" capable of exploiting developmental mechanisms for cell migration and axonal pathfinding, providing synergistic structural and soluble cues based on the state of regeneration. Micro-TENNs are fabricated by pouring liquid hydrogel into a cylindrical mold containing a longitudinally centered needle. Once the hydrogel has gelled, the needle is removed, leaving a hollow micro-column. An ECM solution is added to the lumen to provide an environment suitable for neuronal adhesion and axonal outgrowth. Dissociated neurons are mechanically aggregated for precise seeding within one or both ends of the micro-column. This methodology reliably produces self-contained miniature constructs with long-projecting axonal tracts that may recapitulate features of brain neuroanatomy. Synaptic immunolabeling and genetically encoded calcium indicators suggest that micro-TENNs possess extensive synaptic distribution and intrinsic electrical activity. Consequently, micro-TENNs represent a promising strategy for targeted neurosurgical reconstruction of brain pathways and may also be applied as biofidelic models to study neurobiological phenomena in vitro.

Biomimetic neural scaffolds: a crucial step towards optimal peripheral nerve regeneration

“…98,99 Various studies have investigated the efficacy of introducing ECM proteins as NGC luminal wall coating. Laminin is the most frequently used ECM protein for the surface modification of neural scaffolds, and has ultimately led to improved Schwann cell viability in in vitro assays and nerve regeneration in vivo using a 10–12 mm rat sciatic gap injury model.…”

Emerging regenerative medicine and tissue engineering strategies for Parkinson’s disease

“…15,19 To date, micro-TENNs have been fabricated using dorsal root ganglia neurons, cerebral cortical neurons (e.g., mixed glutamatergic and GABAergic), embryonic rodent ventral mesencephalic dopaminergic neurons, and human ESC-derived dopaminergic neurons. 5,125,126,129,[141][142][143][144] As our understanding of PD pathophysiology expands, it is possible that multiple micro-TENNs could be transplanted to reconnect different damaged regions, comprised of alternative neuronal phenotypes, such as noradrenergic, serotoninergic, and cholinergic cell types. However, it is difficult to predict how transplant therapies, including either transplanted cells or pathways, such as micro-TENNs, could be used to recapitulate widely dispersed innervation of cerebral cortex from cholinergic, serotonergic, or noradrenergic brainstem nuclei.…”

When Bio Meets Technology: Biohybrid Neural Interfaces

“…Building on their previous technology, Struzyna et al [54] developed microtissue engineered neural networks (micro-TENNs) which are essentially a form of "living scaffold." [46,54,55] These microTENNS comprise of neural bodies at one end of an agarose-collagen hydrogel microcolumn structure and their axonal projections growing unidirectionally toward the target tissue these multiple microstructures form a neural network that could act as a replacement for CNS reconstruction. [37] These microTENNS are fabricated in a similar fashion to the living electrodes by pouring a hydrogel into cylindrical moulds that have a needle in the center.…”