Financial support was provided by the National Institutes of Health [U01-NS094340 (Cullen) &#38; F31-NS090746 (Katiyar)], Michael J. Fox Foundation [Therapeutic Pipeline Program #9998 (Cullen)], Penn Medicine Neuroscience Center Pilot Award (Cullen), National Science Foundation [Graduate Research Fellowships DGE-1321851 (Struzyna)], Department of Veterans Affairs [RR&#38;D Merit Review #B1097-I (Cullen)], and the U.S. Army Medical Research and Materiel Command [#W81XWH-13-207004 (Cullen) &#38; W81XWH-15-1-0466 (Cullen)].

All procedures were approved by the Institutional Animal Care and Use Committees at the University of Pennsylvania and the Michael J. Crescenz Veterans Affairs Medical Center and adhered to the guidelines set forth in the NIH Public Health Service Policy on Humane Care and Use of Laboratory Animals (2015).
1. Development of the Agarose Hydrogel Micro-columns
<ol>
	<li>Make an agarose 3% weight/volume (w/v) solution by weighing 3 g of agarose and transferring it to a sterile beaker containing...

When compared to the more supportive environment of the PNS, the CNS is particularly limited in handling the detrimental consequences of neurotrauma and neurodegeneration. After a serious insult to the mammalian CNS, a glial scar is formed, consisting of a core of fibrotic and inflammatory cells surrounded by a dense meshwork of disorganized reactive astrocytes that secrete axon outgrowth-inhibiting proteoglycans14. This scar acts as a physical and biochemical obstruction against the regeneration ...

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 CNS is insufficient due to the lack of directed guidance, the presence of outgrowth inhibitors, and reactive astrogliosis following damage to neural tissue2,8,9,10. Astrocytes typically have diverse functions in assisting neurons with ion homeostasis, neurotransmitter clearance, synapse formation, and neurovascular coupling11. Nevertheless, following even mild damage to neural tissue, astrocytes may undergo molecular, structural, and functional changes as they transition to a hypertrophic state11. In response to severe neurotrauma, these changes result in the formation of a scar with a penumbra containing migrating reactive astrocytes and a lesion core that includes leukocytes leaked from the ruptured blood-brain barrier (BBB), microglia, oligodendrocytes, and fibroblasts11,12,13. These reactive astrocytes attain a morphology of filamentous, disorganized processes and exhibit increased expression of intermediate filament proteins and chondroitin sulfate proteoglycans (CSPGs), which hinder neural regeneration12. Even though the glial scar initially helps restore BBB integrity and avoid transmission of the inflammatory response to surrounding healthy tissue, it serves as a physical and biochemical barrier against axon regeneration12,14,15,16. For instance, axons that encounter the glial scar display bulbous dystrophic growth cones and stunted growth12. Furthermore, the disorganization of astrocytic processes after injury impedes the extension of regenerating axons17. The outcome of these inhibitory characteristics is manifested in the often-permanent physical and neurological impairments that patients suffer after severe neurotrauma, including TBI and SCI.
Regardless of the extrinsic challenges facing functional regeneration in the CNS, axons have been shown to possess an intrinsic ability to regenerate. For instance, the dynamic nature of the dystrophic growth cones in contact with the glial scar suggests that these endings retain their capacity to extend12. Consequently, it is believed that a main hindrance to axonal re-growth is the inhibitory environment of the post-injury CNS and that providing a more permissive environment via reducing glial scarring and/or providing regenerative bridges across the scar would be advantageous. Indeed, previous studies have demonstrated that CNS neurons were capable of extending axons through a lesion using peripheral nerve grafts as bridges, which present a more favorable environment for axon regeneration12,18,19. Several other strategies have been pursued to exploit this vestigial regenerative capacity. For example, manipulation of cell growth signaling pathways in various injury models has resulted in axonal regeneration and glial scar reduction10,20,21. Additionally, studies have shown that treatment with chondroitinase ABC, which cleaves the majority of the sugar chains in CSPGs, lessens the inhibitory effect of CSPGs secreted by reactive astrocytes22. Despite encouraging results, these approaches do not provide directed guidance of growth cones, which can potentially result in aberrant regeneration12, and also do not account for the loss of neurons. Cell-based approaches have been utilized in attempts to surmount the effects of the glial scar and to replenish lost cells, particularly neurons. Some groups have dedifferentiated reactive astrocytes into neurons, while others have transplanted neural progenitor cells into CNS lesions to repopulate the injury area and promote axon regeneration23,24,25. However, stem cell transplantation alone is limited by low survival rates, poor integration, and modest retention in the damaged tissue5. Furthermore, these cell-based strategies fail to restore long-distance axonal tracts, especially in a controlled manner. Therefore, biomaterials in combination with other approaches are being explored as delivery vehicles for various neural and progenitor cells and growth factors26. Biomaterial-based approaches feature a high degree of design control to produce constructs that mimic the specific physical, haptotaxic, and chemotaxic cues present in the three-dimensional (3D) microenvironment of the target host tissue27,28,29,30,31,32,33,34. Reproduction of these environmental signals is paramount for transplanted cells to present native-like morphology, proliferation, migration, and signaling, among other neurobiological characteristics29. Despite these advantageous properties, advancement beyond traditional cell seeded biomaterial scaffolds is required to simultaneously promote directed long-distance axonal regeneration and replace lost neurons.
A promising alternative approach is based on neural tissue engineered &#34;living scaffolds&#34;, which are distinct from other cell-based approaches due to the presence of living neural cells with a preformed cytoarchitecture that emulates native neuroanatomy and/or developmental mechanisms to facilitate targeted replacement, reconstruction, and regeneration of neural circuitry4,35. Considerations for the design of living scaffolds include the phenotypes and sources of neural cells, as well as the mechanical/physical properties and the biochemical signals dictated by the composition of any accompanying biomaterials35. After fabrication in vitro, these living scaffolds can be implanted in vivo to present cell-adhesion molecules and chemotactic and neurotrophic signals to actively regulate neural cell migration and axon outgrowth depending on the state and progression of regenerative processes35. Glial cells can serve as a basis for the engineered cytoarchitecture of living scaffolds since these cells mediate various developmental mechanisms in vivo. During brain development, new neurons rely on basal processes extended by radial glia from the ventricular zone towards the developing cortical plate as living scaffolds for directed migration36,37. Furthermore, extending growth cones are shown to orient themselves by sensing attractive and repellent signals elicited by glial guidepost cells, and so-called &#34;pioneering&#34; axons are suggested to reach the correct targets by extending along pre-patterned glial scaffolds35,38,39. Thus, glial cells are necessary for the guidance of pioneering axons, which later serve as axon-based &#34;living scaffolds&#34; to direct the projection of &#34;follower&#34; axons. Moreover, glia-mediated growth mechanisms have been shown to persist postnatally, as neuroblasts follow the rostral migratory stream (RMS) to navigate from the subventricular zone (SVZ), one of the few remaining areas of neurogenesis in the adult brain, to the olfactory bulb (OB)40. These neuroblasts in the RMS migrate within the glial tube (Figure 1A-1), which is comprised of longitudinally-aligned astrocytic processes, via direct cell-cell adhesions and localized soluble factors37,41. Finally, while CNS damage in mammals causes disrupted astrocytic process arrangement forming a glial scar that physically impedes axonal regeneration17, many non-mammalian systems lack the formation of a detrimental glial scar. Rather, glial cells of non-mammalian species maintain more organized, aligned patterns that are used as guides through the injured region17,42,43. For instance, in non-mammalian SCI models, axons are shown to grow in close association with glial bridges crossing the lesion, suggesting an important role for organized glial scaffolds as substrates facilitating axonal regeneration and functional recovery (Figure 1A-2)42,44,45. Recapitulation of the neuroanatomical features and the developmental/regenerative mechanisms described above may yield a new class of engineered glial-based living scaffolds that can concurrently drive immature neuronal migration and axonal pathfinding through otherwise non-permissive environments, thereby potentially mitigating the effects of neuronal and axon tract degeneration associated with CNS injury and disease.
Our research group has previously designed multiple types of living scaffolds for reconstruction and regeneration of axonal tracts in the CNS and the peripheral nervous system (PNS) via micro-tissue engineered neural networks (micro-TENNs) and tissue engineered nerve grafts (TENGs), respectively27,46,47,48. Both strategies are based inherently on biomimicry. Micro-TENNs are anatomically-inspired structures designed to structurally and functionally replace axonal tracts connecting distinct neuronal populations of the brain. TENGs exploit the developmental mechanism of axon-facilitated axonal regeneration, exemplified by &#34;follower&#34; axon growth along &#34;pioneer&#34; axons, to achieve targeted host axonal regeneration35,46,48. We recently capitalized on the versatility of the living scaffold technique using a similar encasement scheme as micro-TENNs and seeking inspiration from the glia-based mechanisms present throughout development. Here, we developed constructs consisting of aligned astrocytic bundles spanning the collagenous lumen of a hydrogel micro-column49. These astrocytic living scaffolds are developed by first filling a capillary tube-acupuncture needle assembly with liquid agarose to create a hollow cylindrical hydrogel with an outer diameter (OD) and inner diameter (ID) corresponding to the diameters of the tube and needle, respectively. Following agarose gelation and extraction of the hydrogel micro-column from the capillary tube, the hollow interior is coated with type I collagen to supply an environment permissive for astrocyte adhesion and aligned bundle formation (Figure 1B-1). Afterwards, the lumen is seeded with cerebral cortical astrocytes isolated from postnatal rat pups (Figure 1B-2). Contrary to two-dimensional (2D) alignment techniques that rely on the application of electric fields, micropatterned grooves, and extracellular matrix (ECM) protein patterning, astrocyte alignment in the living scaffold technique relies on self-assembly according to controllable variables such as substrate curvature (column ID), cell density, and collagen concentration50,51,52. The astrocytes contract and remodel the collagen, and acquire a bipolar, longitudinally-aligned morphology analogous to the natural scaffolds observed in vivo (Figure 1B-3). Indeed, we are actively pursuing the use of these cable-like structures as physical substrates for targeted guidance of migrating immature neurons as well as facilitating axonal regeneration through the unfavorable environment of the damaged CNS, particularly the mammalian glial scar (Figure 1C). This article will present the detailed fabrication method for the astrocytic micro-columns, phase contrast and immunofluorescence images of the expected cytoarchitecture, and a comprehensive discussion on the current limitations and future directions of the technique.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55848/55848fig1.jpg" /> 
Figure 1: Inspiration, Fabrication Protocol, and Proposed Applications for the Aligned Astrocytic Networks. (A) Neurobiological inspiration: (1) Neuroblasts originating from the neurogenic subventricular zone (SVZ) utilize the longitudinally aligned glial tube in the rostral migratory stream (RMS) for directed migration towards the olfactory bulb (OB); (2) Non-mammals such as amphibians and fish can sustain regeneration after neural tissue damage in part due to the formation of a glial bridge that connects the ends of a lesion (e.g. transected spinal cord) and serves as a scaffold for the guidance of regenerating axons. (B) Fabrication overview: (1) construction of a micron-sized, hollow hydrogel micro-column with the lumen coated with ECM, (2) seeding of primary cortical astrocytes isolated from postnatal rat pups, (3) self-assembly of the longitudinally-oriented bundles in culture, and (4) extraction of the bundle from the biomaterial encasement for future implantation studies. (C) In vivo applications: (1) These living scaffolds may serve as engineered glial tubes for directed neuron migration from neurogenic centers to repopulate neuron-deficient regions; (2) Recapitulation of the developmental mechanism of pioneering axon guidance and the regenerative mechanism of glial bridges in non-mammals may endow these astrocytic scaffolds with the capacity to direct axon regeneration across the non-permissive environment of the mammalian glial scar. <a href="//cloudflare.jove.com/files/ftp_upload/55848/55848fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table>
	<tbody>
		<tr>
			<td>Acupuncture needle (300 &#181;m diameter)</td>
			<td>Lhasa Medical</td>
			<td>HS.30x40</td>
			<td>The diameter may be varied according to the desired size for the micro-column lumen.</td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>Fisher</td>
			<td>08772B</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s phosphate buffered saline (DPBS)</td>
			<td>Invitrogen</td>
			<td>14200075</td>
			<td></td>
		</tr>
		<tr>
			<td>Polystyrene disposable serological pipet</td>
			<td>Fisher</td>
			<td>13-678-11D</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Sigma</td>
			<td>A9539-50G</td>
			<td></td>
		</tr>
		<tr>
			<td>Microliter glass capillary tube (701 &#181;m)</td>
			<td>Fisher</td>
			<td>21-170J</td>
			<td>The diameter may be varied according to the desired size for the micro-column shell.</td>
		</tr>
		<tr>
			<td>Microcap bulb dispenser</td>
			<td>Fisher</td>
			<td>21-170J</td>
			<td>Bulb comes with the microcap tubes.</td>
		</tr>
		<tr>
			<td>Hot plate</td>
			<td>Fisher</td>
			<td>SP88857200</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic bar</td>
			<td>Fisher</td>
			<td>1451352</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette</td>
			<td>Sigma</td>
			<td>Z683884-1EA</td>
			<td></td>
		</tr>
		<tr>
			<td>25 mm gauge needle</td>
			<td>Fisher</td>
			<td>14-826-49</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscalpel</td>
			<td>Roboz Surgical</td>
			<td>RS-6270</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>Fine Science Tools</td>
			<td>14081-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>World Precision Instruments</td>
			<td>501985</td>
			<td></td>
		</tr>
		<tr>
			<td>Hot bead sterilizer</td>
			<td>Sigma</td>
			<td>Z378550-1EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereoscope</td>
			<td>Nikon</td>
			<td>SMZ800N</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-spatula</td>
			<td>Fisher</td>
			<td>S50821</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat tail type I collagen</td>
			<td>Corning</td>
			<td>354236</td>
			<td>Maintain at 4 &#186;C and remove only when needed. Use ice to preserve its temperature when in use.</td>
		</tr>
		<tr>
			<td>Microcentrifuge tube</td>
			<td>Fisher</td>
			<td>02-681-256</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide (NaOH)</td>
			<td>Fisher</td>
			<td>SS2661</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid (HCl)</td>
			<td>Fisher</td>
			<td>SA48-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Litmus paper</td>
			<td>Fisher</td>
			<td>09-876-18</td>
			<td></td>
		</tr>
		<tr>
			<td>Hank&#39;s balanced salt solution (HBSS)</td>
			<td>Invitrogen</td>
			<td>14170112</td>
			<td>Store at 4 &#186;C.</td>
		</tr>
		<tr>
			<td>0.25% Trypsin-EDTA</td>
			<td>Invitrogen</td>
			<td>25200056</td>
			<td>Store at -20 &#186;C and warm at 37 &#186;C before use.</td>
		</tr>
		<tr>
			<td>Bovine pancreatic deoxyribonuclease (Dnase) I</td>
			<td>Sigma</td>
			<td>10104159001</td>
			<td>Store at -20 &#186;C and warm at 37 &#186;C before use.</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM) with Ham&#39;s F-12 Nutrient Mixture</td>
			<td>Gibco</td>
			<td>11330-032</td>
			<td>Store at 4 &#186;C.</td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Atlanta Biologicals</td>
			<td>S11195</td>
			<td>Store at -20&#186;C.</td>
		</tr>
		<tr>
			<td>Postnatal day 0 or day 1 Sprague Dawley rat pups</td>
			<td>Charles River</td>
			<td>Strain 001</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal embryonic neuron basal medium</td>
			<td>Invitrogen</td>
			<td>21103049</td>
			<td>Store at 4&#186;C and warm at 37 &#186;C before use.</td>
		</tr>
		<tr>
			<td>B-27 serum free supplement</td>
			<td>Invitrogen</td>
			<td>12587010</td>
			<td>Store at -20 &#186;C and warm at 37 &#186;C before use.</td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Invitrogen</td>
			<td>35050061</td>
			<td>Store at -20 &#186;C and warm at 37 &#186;C before use.</td>
		</tr>
		<tr>
			<td>G5 astrocytic supplement</td>
			<td>Invitrogen</td>
			<td>17503012</td>
			<td></td>
		</tr>
		<tr>
			<td>Sprague Dawley embryonic day 18 rats</td>
			<td>Charles River</td>
			<td>Strain 001</td>
			<td></td>
		</tr>
		<tr>
			<td>Pasteur pipette</td>
			<td>Fisher</td>
			<td>22-042816</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL centrifuge tube</td>
			<td>EMESCO</td>
			<td>1194-352099</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex</td>
			<td>Fisher</td>
			<td>02-215-414</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Fisher</td>
			<td>05-413-115</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>Fisher</td>
			<td>02-671-6</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Fisher</td>
			<td>13 998 076</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde 40%</td>
			<td>Fisher</td>
			<td>F77P-4</td>
			<td>Formaldehyde is a toxic compound known to be carcinogenic, and must be disposed of in a separate container.</td>
		</tr>
		<tr>
			<td>Glass cover slip</td>
			<td>Fisher</td>
			<td>12-548-5M</td>
			<td></td>
		</tr>
		<tr>
			<td>Nail polish</td>
			<td>Electron Microscopy Sciences (EMS)</td>
			<td>72180</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluoromont mounting medium</td>
			<td>Southern Biotech</td>
			<td>0100-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-lysine</td>
			<td>Sigma</td>
			<td>P4707</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Fisher</td>
			<td>BP3994</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal horse serum</td>
			<td>Gibco</td>
			<td>16050-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit anti-glial acidic fibrillary protein (GFAP) primary antibody</td>
			<td>Millipore</td>
			<td>AB5804</td>
			<td>Store at -20&#186;C.</td>
		</tr>
		<tr>
			<td>Mouse anti-beta-tubulin III primary antibody</td>
			<td>Sigma</td>
			<td>T8578</td>
			<td>Store at -20&#186;C.</td>
		</tr>
		<tr>
			<td>Rabbit anti-collagen I primary antibody</td>
			<td>Abcam</td>
			<td>ab34710</td>
			<td>Store at -20&#186;C.</td>
		</tr>
		<tr>
			<td>Rabbit anti-vimentin</td>
			<td>Millipore</td>
			<td>AB3400</td>
			<td>Store at -20&#186;C.</td>
		</tr>
		<tr>
			<td>Mouse anti-nestin</td>
			<td>Millipore</td>
			<td>AB5326</td>
			<td>Store at -20&#186;C.</td>
		</tr>
		<tr>
			<td>Donkey anti-mouse 568 secondary antibody</td>
			<td>Invitrogen</td>
			<td>A10037</td>
			<td>Store at 4&#186;C.</td>
		</tr>
		<tr>
			<td>Donkey anti-rabbit 568 secondary antibody</td>
			<td>Invitrogen</td>
			<td>A10042</td>
			<td>Store at 4&#186;C.</td>
		</tr>
		<tr>
			<td>Donkey anti-rabbit 488 secondary antibody</td>
			<td>Invitrogen</td>
			<td>A21206</td>
			<td>Store at 4&#186;C.</td>
		</tr>
		<tr>
			<td>Hoechst 33342, Trihydrochloride</td>
			<td>Invitrogen</td>
			<td>H3570</td>
			<td>Store at 4&#186;C. Hoechst is a known mutagen that should be treated as a carcinogen. Therefore, it must be disposed of in a separate container.</td>
		</tr>
		<tr>
			<td>Calcein AM</td>
			<td>Sigma</td>
			<td>C1359</td>
			<td>4 mM in anhydrous DMSO</td>
		</tr>
		<tr>
			<td>Ethidium homodimer-1</td>
			<td>Life Technologies</td>
			<td>E1169</td>
			<td>2 mM in DMSO/H2O 1:4 (v/v)</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxane (DMSO)</td>
			<td>Sigma</td>
			<td>276855</td>
			<td></td>
		</tr>
		<tr>
			<td>A1RSI Laser Scanning Confocal Microscope</td>
			<td>Nikon</td>
			<td>--------------</td>
			<td>Used for taking the confocal reconstructions of immunolabeled constructs.</td>
		</tr>
		<tr>
			<td>Eclipse Ti-S Microscope</td>
			<td>Nikon</td>
			<td>--------------</td>
			<td>Used for taking the phase-contrast images. With digital image acquisition using a QiClick camera interfaced with Nikon Elements Basic Research software (4.10.01).</td>
		</tr>
	</tbody>
</table>

three-dimensional tissue engineered aligned astrocyte networks to recapitulate developmental mechanisms and facilitate nervous system regeneration

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 &#34;living scaffolds&#34;. 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 &#34;living scaffolds&#34;. 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 &#60;350 &#181;m, spontaneously self-aligned and contracted to produce long fiber-like cables consisting of dense bundles of astrocyte processes and collagen fibrils measuring &#60;150 &#181;m in diameter yet extending several cm in length. These engineered living scaffolds exhibited &#62;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 &#34;living scaffolds&#34; 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.

Initially, phase-contrast microscopy was used to monitor the progression of astrocyte adhesion and bundle formation and the overall stability of the cytoarchitecture as a function of time. At 1 h after plating, astrocytes were found throughout the lumen of the micro-columns with a spherical morphology (Figure 2A). At 5 h, astrocytes started extending processes and contracting, while by 8 h cells exhibited a complete process-bearing morphology and formed cable...

Watch this Scientific Journal Video about Three-dimensional Tissue Engineered Aligned Astrocyte Networks to Recapitulate Developmental Mechanisms and Facilitate Nervous System Regeneration at JoVE.com

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

We showcase the development of self-assembled, three-dimensional bundles of longitudinally aligned astrocytic somata and processes within a novel biomaterial encasement. These engineered &#34;living scaffolds&#34;, 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.

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to ...

This protocol aims to guide the fabrication of self-assembled, three-dimensional bundles of longitudinally aligned astrocytic somata and processes within a micron-sized hydrogel micro-column for use as an in vitro neuro-biological model or as a neuro-regenerative therapy. Trauma and neurodegenerative disease are particularly detrimental to patients due to the restricted neurogenic capacity in inhibitory environment that limit regeneration in the central nervous system. Engineered astrocytic bundles address the limitations of current treatment strategies by attempting to replicate key neuroanatomical features and glia-mediated developmental mechanisms to promoted targeted cell migration and axon pathfinding.The architecture of these astrocytic scaffolds is primarily inspired by the aligned astrocytic processes in the glial tubes of the rostral migratory stream. Begin by preparing a 3%weight per volume agarose solution and DPBS in a sterile beaker. Maintain the heating and stirring to prevent the solution from solidifying prematurely.Insert an acupuncture needle into the bottom opening of the bulb dispenser. Place a capillary tube over the exposed needle and secure the two by entering part of it into the rubber section of the bulb dispenser cylinder. Cap the cylinder to complete the assembly.Transfer one milliliter of liquid agarose to the surface of an empty petri dish. While pinching the rubber cap of the bulb dispenser, insert one end of the capillary tube into the agarose, and release the pressure on the cap to draw agarose into the tube. Place the bulb, tube, needle assemblies on a petri dish and let the agarose solidify inside the capillary tubes for 5 minutes.Then, pull the needle out of the capillary tube, leaving the micro-column still surrounding the needle. Use the tips of foreceps to nudge the micro-column to the end of the needle, then hold the needle over an open DPBS-containing petri dish and push the micro-column into the DPBS. To fabricate micro-column boats, which facilitate later handling of these constructs, begin by making a 4%weight per volume agarose solution, and keeping the solution heated and stirred.Use forceps to move a micro-column to an empty petri dish. Then, while working under a stereoscope, use a micro scalpel to trim the micro-columns to the desired length, with the ends cut at a 45 degree angle. After trimming three more micro-columns, line up the four columns in parallel.Load a pipet with 50 microliters of 4%agarose solution, and dispense a line of liquid over the micro-column array to connect and bundle the constructs into a boat. Allow to cool for 1 to 2 minutes to permit gelation of the 4%agarose. Then use fine forceps to pick up the micro-column boat by the connecting agarose bridge.And move it to a petri dish containing DPBS. Transfer the micro-column boat to a biosafety cabinet and sterilize with exposure to ultraviolet light for thirty minutes. Inside a biosafety cabinet, prepare a 1 milligram per milliliter solution of rat tail type 1 collagen in co-culture medium.And then place it on ice. Adjust the pH of the ECM solution with acid or base as necessary until the pH is stable in the 7.2 to 7.4 range. Transfer the micro-column boat to a 35 or 60 mL petri dish with forceps.Using the stereoscope for guidance, situate the 10 mL tip of a micro-pipette at one end of each micro-column and suction to empty the lumen of DPBS and air bubbles. Charge a P10 micropipette with the collagen solution. Place the 10 mL tip against one end of each micro-column, and deliver enough solution to fill the lumen.Then pipette a reservoir of ECM on either end of the micro-column. Once all columns have been filled, pipette coculture medium in a ring around the petri dish to prevent the columns from drying out. Incubate the dish containing the micro-columns at 37 degrees Celsius and 5%carbon dioxide for at least one hour to promote polymerization of collagen before adding cells.Afterwards, place the tip of a P10 micropipette at one end of a micro-column and transfer approximately 5 mL of cell solution into the lumen to fill it. After seating all of the micro-columns in a boat, incubate it at 37 degrees Celsius and 5%carbon dioxide for 1 hour to promote the attachment of astrocytes to the ECM. After the incubation period, carefully fill the dishes containing the seated micro-columns with 3 or 6 mLs of coculture media, for 35 or 60 mL petri dishes respectively.Maintain the plated micro-columns and culture at 37 degrees Celsius and 5%carbon dioxide to promote the self assembly of the aligned of the astrocytic bundles, which should form a bundled, cable-like structure after six to ten hours. One hour after plating, astrocytes are spherical in shape and disperse throughout the construct. By five hours after plating, astrocytes initiate process extension and contraction.At eight hours after plating, astrocytes have aligned and contracted. By 24 hours after plating, a dense bundle of astrocytic processes has formed in this example. This fully-formed astrocytic bundle, 24 hours after plating, shows the zippering effect, where the ends of the cable attach to distinct ends of the wall of the lumen.This video shows bundling in action, 3 to 10 hours after plating. Immuno-labeling of the astrocytic scaffolds demonstrates the longitudinal alignment of astrocytic processes. In this confocal image of an astrocytic bundle, the astrocytic marker GFAP is read.And the nuclei are seen in blue. This higher magnification of the bundle permits visualization of the cyto-architecture of the astrocytes within the micro-columns. After watching this video, you should be able to fabricate living scaffolds consisting of longitudinally aligned astrocytic bundles with a biologically inspired cytoarchitecture within a biomaterial encasement.Following this procedure, a transplantation strategy can be designed to deliver the astrocytic bundles with or without the biomaterial encasement to injured brain tissue to promote directed cell migration from neurogenic regions and acts on guidance to the limiting CNS microenvironment. In addition, this technique can be expanded as biofidelic modeling platform glia-mediated neurobiological phenomenon benefited by the mimicry of the native three dimensional environment and the inherent experimental control of in vitro systems.

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14.2K Views.  University of Pennsylvania. This protocol aims to guide the fabrication of self-assembled, three-dimensional bundles of longitudinally aligned astrocytic somata and processes within a micron-sized hydrogel micro-column for use as an in vitro neuro-biological model or as a neuro-regenerative therapy. Trauma and neurodegenerative disease are particularly detrimental to patients due to the restricted neurogenic capacity in inhibitory environment that limit regeneration in the central nervous system. Engineered astrocytic b...

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