Three-dimensional Tissue Engineered Aligned Astrocyte Networks to Recapitulate Developmental Mechanisms and Facilitate Nervous System Regeneration

Kritika S. Katiyar; Carla C. Winter; Wisberty J. Gordián-Vélez; John C. O'Donnell; Yeri J. Song; Nicole S. Hernandez; Laura A. Struzyna; D. Kacy Cullen

doi:10.3791/55848

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Method Article

Three-dimensional Tissue Engineered Aligned Astrocyte Networks to Recapitulate Developmental Mechanisms and Facilitate Nervous System Regeneration

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DOI:

10.3791/55848

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January 10th, 2018

Kritika S. Katiyar^*¹^,²^,³ , Carla C. Winter^*¹^,²^,⁴ , Wisberty J. Gordián-Vélez¹^,²^,⁴ , John C. O'Donnell¹^,² , Yeri J. Song¹^,⁵ , Nicole S. Hernandez¹^,⁵ , Laura A. Struzyna¹^,²^,⁴ , D. Kacy Cullen¹^,²^,⁵

¹Center for Brain Injury & Repair, Department of Neurosurgery, Perelman School of Medicine, University of Pennsylvania, ²Center for Neurotrauma, Neurodegeneration & Restoration, Michael J. Crescenz Veterans Affairs Medical Center, ³School of Biomedical Engineering, Drexel University, ⁴Department of Bioengineering, School of Engineering and Applied Sciences, University of Pennsylvania, ⁵Neuroscience Graduate Group, Perelman School of Medicine, University of Pennsylvania

^* These authors contributed equally

Summary

We showcase the development of self-assembled, three-dimensional bundles of longitudinally aligned astrocytic somata and processes within a novel biomaterial encasement. These engineered "living scaffolds", exhibiting micron-scale diameter yet extending centimeters in length, may serve as test-beds to study neurodevelopmental mechanisms or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding.

Abstract

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to replace lost neurons and regenerate axonal pathways. However, during nervous system development, neuronal migration and axonal extension often occur along pathways formed by other cells, referred to as "living scaffolds". Seeking to emulate these mechanisms and to design a strategy that circumvents the inhibitory environment of the CNS, this manuscript presents a protocol to fabricate tissue engineered astrocyte-based "living scaffolds". To create these constructs, we employed a novel biomaterial encasement scheme to induce astrocytes to self-assemble into dense three-dimensional bundles of bipolar longitudinally-aligned somata and processes. First, hollow hydrogel micro-columns were assembled, and the inner lumen was coated with collagen extracellular-matrix. Dissociated cerebral cortical astrocytes were then delivered into the lumen of the cylindrical micro-column and, at a critical inner diameter of <350 µm, spontaneously self-aligned and contracted to produce long fiber-like cables consisting of dense bundles of astrocyte processes and collagen fibrils measuring <150 µm in diameter yet extending several cm in length. These engineered living scaffolds exhibited >97% cell viability and were virtually exclusively comprised of astrocytes expressing a combination of the intermediate filament proteins glial-fibrillary acidic protein (GFAP), vimentin, and nestin. These aligned astrocyte networks were found to provide a permissive substrate for neuronal attachment and aligned neurite extension. Moreover, these constructs maintain integrity and alignment when extracted from the hydrogel encasement, making them suitable for CNS implantation. These preformed constructs structurally emulate key cytoarchitectural elements of naturally occurring glial-based "living scaffolds" in vivo. As such, these engineered living scaffolds may serve as test-beds to study neurodevelopmental mechanisms in vitro or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding following CNS degeneration in vivo.

Introduction

The central nervous system (CNS) has a limited capacity to counteract the loss and/or dysfunction of neurons and axonal pathways that accompany conditions such as traumatic brain injury (TBI), stroke, spinal cord injury (SCI), and neurodegenerative disease¹^,²^,³^,⁴^,⁵. Neurogenesis in the CNS is restricted to a limited number of areas in the brain, hampering the restoration of lost neurons⁶^,⁷. Additionally, regeneration of lost axonal pathways in the C....