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)].

The authors have nothing to disclose.

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.remotexs.ntu.edu.sg/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 &amp;micro;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&#039;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 &amp;micro;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 &amp;ordm;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&#039;s balanced salt solution (HBSS)</td><td>Invitrogen</td><td>14170112</td><td>Store at 4 &amp;ordm;C.</td></tr><tr><td>0.25% Trypsin-EDTA</td><td>Invitrogen</td><td>25200056</td><td>Store at -20 &amp;ordm;C and warm at 37 &amp;ordm;C before use.</td></tr><tr><td>Bovine pancreatic deoxyribonuclease (Dnase) I</td><td>Sigma</td><td>10104159001</td><td>Store at -20 &amp;ordm;C and warm at 37 &amp;ordm;C before use.</td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium (DMEM) with Ham&#039;s F-12 Nutrient Mixture</td><td>Gibco</td><td>11330-032</td><td>Store at 4 &amp;ordm;C.</td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Atlanta Biologicals</td><td>S11195</td><td>Store at -20&amp;ordm;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&amp;ordm;C and warm at 37 &amp;ordm;C before use.</td></tr><tr><td>B-27 serum free supplement</td><td>Invitrogen</td><td>12587010</td><td>Store at -20 &amp;ordm;C and warm at 37 &amp;ordm;C before use.</td></tr><tr><td>L-glutamine</td><td>Invitrogen</td><td>35050061</td><td>Store at -20 &amp;ordm;C and warm at 37 &amp;ordm;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&amp;ordm;C.</td></tr><tr><td>Mouse anti-beta-tubulin III primary antibody</td><td>Sigma</td><td>T8578</td><td>Store at -20&amp;ordm;C.</td></tr><tr><td>Rabbit anti-collagen I primary antibody</td><td>Abcam</td><td>ab34710</td><td>Store at -20&amp;ordm;C.</td></tr><tr><td>Rabbit anti-vimentin</td><td>Millipore</td><td>AB3400</td><td>Store at -20&amp;ordm;C.</td></tr><tr><td>Mouse anti-nestin</td><td>Millipore</td><td>AB5326</td><td>Store at -20&amp;ordm;C.</td></tr><tr><td>Donkey anti-mouse 568 secondary antibody</td><td>Invitrogen</td><td>A10037</td><td>Store at 4&amp;ordm;C.</td></tr><tr><td>Donkey anti-rabbit 568 secondary antibody</td><td>Invitrogen</td><td>A10042</td><td>Store at 4&amp;ordm;C.</td></tr><tr><td>Donkey anti-rabbit 488 secondary antibody</td><td>Invitrogen</td><td>A21206</td><td>Store at 4&amp;ordm;C.</td></tr><tr><td>Hoechst 33342, Trihydrochloride</td><td>Invitrogen</td><td>H3570</td><td>Store at 4&amp;ordm;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.

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 | Bioengineering | Video

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 ...

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14.3K Views.  University of Pennsylvania. Wij demonstreren de ontwikkeling van zelf geassembleerde, driedimensionale bundels van lengterichting uitgelijnde astrocytic waarin en processen binnen het omhulsel van een roman biomaterial. Deze "levende steigers", het tentoonstellen van micron-schaal diameter nog uitbreiding centimeter lengte, kan dienen als proefnetwerken neurologische mechanismen bestuderen of vergemakkelijken van neuroregeneration door leiding neuronale migratie en/of axonale ontworpen vastloper.

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

Engineered 3D Silk-collagen-based Model of Polarized Neural Tissue

Despite huge efforts to decipher the anatomy, composition and function of the brain, it remains the least understood organ of the human body. To gain a deeper comprehension of the neural system scientists aim to simplistically reconstruct the tissue by assembling it in vitro from basic building blocks using a tissue engineering approach. Our group developed a tissue-engineered silk and collagen-based 3D brain-like model resembling the white and gray matter of the cortex. The model consists of silk porous sponge, which is pre-seeded with rat brain-derived neurons, immersed in soft collagen matrix. Polarized neuronal outgrowth and network formation is observed with separate axonal and cell body localization. This compartmental architecture allows for the unique development of niches mimicking native neural tissue, thus enabling research on neuronal network assembly, axonal guidance, cell-cell and cell-matrix interactions and electrical functions.

Insight into the complex actions of the brain requires advanced research tools. Here we demonstrate a novel silk-collagen-based 3D engineered model of neural tissue resembling brain-like architecture. The model can be used to study neuronal network assembly, axonal guidance, cell-cell interactions and electrical activity.

Despite huge efforts to decipher the anatomy, composition and function of the brain, it remains the least understood organ of the human body. To gain a ...

Synaptic Microcircuit Modeling with 3D Cocultures of Astrocytes and Neurons from Human Pluripotent Stem Cells

A barrier to our understanding of how various cell types and signals contribute to synaptic circuit function is the lack of relevant models for studying the human brain. One emerging technology to address this issue is the use of three dimensional (3D) neural cell cultures, termed &#39;organoids&#39; or &#39;spheroids&#39;, for long term preservation of intercellular interactions including extracellular adhesion molecules. However, these culture systems are time consuming and not systematically generated. Here, we detail a method to rapidly and consistently produce 3D cocultures of neurons and astrocytes from human pluripotent stem cells. First, pre-differentiated astrocytes and neuronal progenitors are dissociated and counted. Next, cells are combined in sphere-forming dishes with a Rho-Kinase inhibitor and at specific ratios to produce spheres of reproducible size. After several weeks of culture as floating spheres, cocultures (&#39;asteroids&#39;) are finally sectioned for immunostaining or plated upon multielectrode arrays to measure synaptic density and strength. In general, it is expected that this protocol will yield 3D neural spheres that display mature cell-type restricted markers, form functional synapses, and exhibit spontaneous synaptic network burst activity. Together, this system permits drug screening and investigations into mechanisms of disease in a more suitable model compared to monolayer cultures.

In this protocol, we aim to describe a reproducible method for combining dissociated human pluripotent stem cell derived neurons and astrocytes together into 3D sphere cocultures, maintaining these spheres in free floating conditions, and subsequently measuring synaptic circuit activity of the spheres with immunoanalysis and multielectrode array recordings.

A barrier to our understanding of how various cell types and signals contribute to synaptic circuit function is the lack of relevant models for studying ...

Developmental Biology

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation

In the central nervous system, numerous acute injuries and neurodegenerative disorders, as well as implanted devices or biomaterials engineered to enhance function result in the same outcome: excess inflammation leads to gliosis, cytotoxicity, and/or formation of a glial scar that collectively exacerbate injury or prevent healthy recovery. With the intent of creating a system to model glial scar formation and study inflammatory processes, we have generated a 3D cell scaffold capable of housing primary cultured glial cells: microglia that regulate the foreign body response and initiate the inflammatory event, astrocytes that respond to form a fibrous scar, and oligodendrocytes that are typically vulnerable to inflammatory injury. The present work provides a detailed step-by-step method for the fabrication, culture, and microscopic characterization of a hyaluronic acid-based 3D hydrogel scaffold with encapsulated rat brain-derived glial cells. Further, protocols for characterization of cell encapsulation and the hydrogel scaffold by confocal immunofluorescence and scanning electron microscopy are demonstrated, as well as the capacity to modify the scaffold with bioactive substrates, with incorporation of a commercial basal lamina mixture to improved cell integration.

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation

Herein, we present a protocol for the 3D culture of rat brain-derived glia cells, including astrocytes, microglia, and oligodendrocytes. We demonstrate primary cell culture, methacrylated hyaluronic acid (HAMA) hydrogel synthesis, HAMAphoto-polymerization and cell encapsulation, and sample processing for confocal and scanning electron microscopic imaging.

In the central nervous system, numerous acute injuries and neurodegenerative disorders, as well as implanted devices or biomaterials engineered to enhance ...

Interfacing 3D Engineered Neuronal Cultures to Micro-Electrode Arrays: An Innovative In Vitro Experimental Model

Currently, large-scale networks derived from dissociated neurons growing and developing in vitro on extracellular micro-transducer devices are the gold-standard experimental model to study basic neurophysiological mechanisms involved in the formation and maintenance of neuronal cell assemblies. However, in vitro studies have been limited to the recording of the electrophysiological activity generated by bi-dimensional (2D) neural networks. Nonetheless, given the intricate relationship between structure and dynamics, a significant improvement is necessary to investigate the formation and the developing dynamics of three-dimensional (3D) networks. In this work, a novel experimental platform in which 3D hippocampal or cortical networks are coupled to planar Micro-Electrode Arrays (MEAs) is presented. 3D networks are realized by seeding neurons in a scaffold constituted of glass microbeads (30-40 &#181;m in diameter) on which neurons are able to grow and form complex interconnected 3D assemblies. In this way, it is possible to design engineered 3D networks made up of 5-8 layers with an expected final cell density. The increasing complexity in the morphological organization of the 3D assembly induces an enhancement of the electrophysiological patterns displayed by this type of networks. Compared with the standard 2D networks, where highly stereotyped bursting activity emerges, the 3D structure alters the bursting activity in terms of duration and frequency, as well as it allows observation of more random spiking activity. In this sense, the developed 3D model more closely resembles in vivo neural networks.

Interfacing 3D Engineered Neuronal Cultures to Micro-Electrode Arrays: An Innovative In Vitro Experimental Model

In this work, a novel experimental model in which 3D neuronal cultures are coupled to planar Micro-Electrode Arrays (MEAs) is presented. 3D networks are built by seeding neurons in a scaffold made up of glass microbeads on which neurons grow and form interconnected 3D structures.

Currently, large-scale networks derived from dissociated neurons growing and developing in vitro on extracellular micro-transducer devices are the ...

Neuroscience

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

Functional recovery rarely occurs&#160;following injury or disease-induced degeneration within the central nervous system (CNS)&#160;due to the inhibitory environment and the limited capacity for neurogenesis. We are developing&#160;a strategy to simultaneously address&#160;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&#160;&#34;living scaffolds&#34; 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.

This manuscript details the fabrication of micro-tissue engineered neural networks: three-dimensional micron-sized constructs comprised of long aligned axonal tracts spanning aggregated neuronal population(s) encased in a tubular hydrogel. These living scaffolds can serve as functional relays to reconstruct or modulate neural circuitry or as biofidelic test-beds mimicking gray-white matter neuroanatomy.

Functional recovery rarely occurs&#160;following injury or disease-induced degeneration within the central nervous system (CNS)&#160;due to the inhibitory ...

3D Bioprinting of Murine Cortical Astrocytes for Engineering Neural-Like Tissue

Astrocytes are glial cells with an essential role in the central nervous system (CNS), including neuronal support and functionality. These cells also respond to neural injuries and act to protect the tissue from degenerative events. In vitro studies of astrocytes&#39; functionality are important to elucidate the mechanisms involved in such events and contribute to developing therapies to treat neurological disorders. This protocol describes a method to biofabricate a neural-like tissue structure rich in astrocytes by 3D bioprinting astrocytes-laden bioink. An extrusion-based 3D bioprinter was used in this work, and astrocytes were extracted from C57Bl/6 mice pups&#39; brain cortices. The bioink was prepared by mixing cortical astrocytes from up to passage 3 to a biomaterial solution composed of gelatin, gelatin-methacryloyl (GelMA), and fibrinogen, supplemented with laminin, which presented optimal bioprinting conditions. The 3D bioprinting conditions minimized cell stress, contributing to the high viability of the astrocytes during the process, in which 74.08% &#177; 1.33% of cells were viable right after bioprinting. After 1 week of incubation, the viability of astrocytes significantly increased to 83.54% &#177; 3.00%, indicating that the 3D construct represents a suitable microenvironment for cell growth. The biomaterial composition allowed cell attachment and stimulated astrocytic behavior, with cells expressing the specific astrocytes marker glial fibrillary acidic protein (GFAP) and possessing typical astrocytic morphology. This&#160;reproducible protocol provides a valuable method to biofabricate 3D neural-like tissue rich in astrocytes that resembles cells&#39; native microenvironment, useful to researchers that aim to understand astrocytes&#39; functionality and their relation to the mechanisms involved in neurological diseases.

Here we report a method of 3D bioprinting murine cortical astrocytes for biofabricating neural-like tissues to study the functionality of astrocytes in the central nervous system and the mechanisms involving glial cells in neurological diseases and treatments.

Astrocytes are glial cells with an essential role in the central nervous system (CNS), including neuronal support and functionality. These cells also ...

3D Bioprinted Cocultures of iPSC-Derived Neurons and Astrocytes

Three-Dimensional Bioprinting of Human iPSC-Derived Neuron-Astrocyte Cocultures for Screening Applications

For a cell model to be viable for drug screening, the system must meet throughput and homogeneity requirements alongside having an efficient development time. However, many published 3D models&#160;do not satisfy these criteria. This therefore, limits their usefulness in early drug discovery applications. Three-dimensional (3D) bioprinting is a novel technology that can be applied to the development of 3D models to expedite development time, increase standardization, and increase throughput. Here, we present a protocol to develop 3D bioprinted coculture models of human induced pluripotent stem cell (iPSC)-derived glutamatergic neurons and astrocytes. These cocultures are embedded within a hydrogel matrix of bioactive peptides, full-length extracellular matrix (ECM) proteins, and with a physiological stiffness of 1.1 kPa. The model can be rapidly established in 96-well and 384-well formats and produces an average post-print viability of 72%. The astrocyte-to-neuron ratio in this model is shown to be 1:1.5, which is within the physiological range for the human brain. These 3D bioprinted cell populations also show expression of mature neural cell type markers and growth of neurite and astrocyte projections within 7 days of culture. As a result, this model is suitable for analysis using cell dyes and immunostaining techniques alongside neurite outgrowth assays. The ability to produce these physiologically representative models at scale makes them ideal for use in medium-to-high throughput screening assays for neuroscience targets.

Author Spotlight: Advancing 3D Cell Modeling &#8211; A High-Throughput Approach for Neural Cocultures 

Here, we present a protocol to produce 3D-bioprinted cocultures of iPSC-derived neurons and astrocytes. This coculture model, generated within a hydrogel scaffold in 96- or 384-well formats, demonstrates high post-print viability and neurite outgrowth within 7 days and shows the expression of maturity markers for both cell types.

For a cell model to be viable for drug screening, the system must meet throughput and homogeneity requirements alongside having an efficient development ...

Generating 3D Co-culture Spheres of Astrocytes and Neurons to Induce Synapse Formation

Source: Cvetkovic, C., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/58034">Synaptic Microcircuit Modeling with 3D Cocultures of Astrocytes and Neurons from Human Pluripotent Stem Cells.</a> J. Vis. Exp. (2018).
This video demonstrates a procedure for co-culturing the astrocytes and neurons to form 3D spheres. These 3D spheres provide a distinct platform for examining synapse formation and neural network integration, thereby serving as a potential model for research in developmental neuroscience, neurodegenerative diseases, and neuropharmacological interventions.

1. Preparation and Maintenance of 3D Sphere Cocultures

	Dissociate hAstros from 3D aggregates in suspension.&#8203;
	
		Gently dissociate human astrocyte ...

JoVE Encyclopedia of Experiments

Neuronal Culture Techniques

Co-culture and Interaction Studies

An In Vitro Method to Generate Astrocyte Scaffolds within Hydrogel Micro-columns

Source: Katiyar, K. S., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/55848">Three-dimensional Tissue Engineered Aligned Astrocyte Networks to Recapitulate Developmental Mechanisms and Facilitate Nervous System Regeneration.</a> J. Vis. Exp. (2018).
This video demonstrates a method to generate three-dimensional astrocyte scaffolds within hydrogel micro-columns. The inside of the microcolumns are coated with collagen. Astrocytes are introduced, which adhere to collagen and, with further incubation, self-assemble into a three-dimensional scaffold.

1. Development of the Agarose Hydrogel Micro-columns

	Make an agarose 3% weight/volume (w/v) solution by weighing 3 g of agarose and transferring it to a ...

Advanced Neuronal Models

A Method for 3D Bioprinting Murine Cortical Astrocytes

Source: de Melo, B. A. G., et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/62691">3D Bioprinting of Murine Cortical Astrocytes for Engineering Neural-Like Tissue.</a> J. Vis. Exp. (2021)
The video demonstrates the process of creating a 3D bioprinted construct using astrocytes suspended in a bioink solution. The bioprinted construct, after crosslinking, facilitates astrocyte adhesion and their spreading, forming neural-like tissue.

1. Synthesis of gelatin methacryloyl (GelMA)

	Weigh 10 g of gelatin obtained from porcine skin and dissolve in 100 mL of PBS by letting the solution stir ...

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

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