The authors are grateful for financial support from NSERC, CFI, AIHS, Alberta Health Services, and the Davey Endowment for Brain Research.

The protocol for the brain tissue extraction from day 1 Sprague Dawley rat pups, euthanized by decapitation, was approved by the Animal Care and Use Committee at the University of Alberta.
1. Microglia and Astrocyte Isolations67,68
NOTE: All media for isolation and cell culture is preheated to 37 &#176;C in a water bath. Hank&#39;s balanced salt solution (HBSS) has 1% penicillin-streptomycin (PS). All Dulbecco&#39;s modified Eagle&#39;s media with Ham&#39;s F12 nutrient mixture (DMEM/F12) is supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (PS). Only mixtures with trypsin lack FBS. All materials in this protocol are sterile (filter, alcohol, purchased, and autoclaved) and every step subsequent to step number 1.8 is performed in a biosafety cabinet with aseptic technique.
<ol>
	<li>Per brain, pre-heat 15 mL of HBSS and 12.5 mL DMEM/F12 to 37 &#176;C in 50 mL conical centrifuge tubes, and approximately 2 mL of 0.25% trypsin with 1 mM ethylenediaminetetraacetic acid (EDTA) in a 15 mL conical centrifuge tube. Heat an additional 25 mL of DMEM/F12. Warm trypsin about 10 min prior to use to prevent loss of enzymatic activity.</li>
	<li>Pour enough warm HBSS into sterile, 6 and 10 cm culture dishes to cover the surface. Prepare one 10 cm dish for every two brains plus an additional 6 cm dish.</li>
	<li>Place up to 2 brains in each 10 cm2 dish (Figure 2A).</li>
	<li>Under a dissection microscope, use curved and straight forceps to separate the cortices along the midline, and the cerebellum with brain stem. This yields three sections of tissue per brain (Figure 2B).</li>
	<li>Peel the thin, semi-transparent, layer (meninges) of tissue containing blood vessels from each section of tissue and discard, transferring the remaining tissue into a separate culture dish. Grasp the thin layers exposed by the tissue cuts, with the forceps, and carefully pull until major portions are &#39;hanging off&#39; the brain tissue. Finally remove this portion by gently tugging. See Figure 2C-2E for representative images of before, during, and after this step.</li>
	<li>In a biosafety cabinet, remove the remaining HBSS from the culture dish, carefully retaining the tissue. Macerate the tissue with a scalpel and transfer tissue to the pre-warmed 15 mL tube with trypsin.</li>
	<li>Incubate this tube for 25 min in a 37 &#176;C bath to digest the tissue enzymatically.</li>
	<li>Centrifuge the tube down at 500 x g for 2 min at room temperature, and pour out the supernatant.</li>
	<li>Using a 10 mL serological pipette, add 10 mL of warm DMEM/F12 to inactivate the trypsin and triturate the solution 5 times to break up the tissue.</li>
	<li>Centrifuge the tube down at 500 x g for 2 min at room temperature, and pour out the supernatant.</li>
	<li>Using a 10 mL serological pipette, re-suspend pellet into 10 mL of warm DMEM/F12 and transfer the solution to a 50 mL Conical centrifuge tube.</li>
	<li>Using a 10 mL syringe with an 18 gauge needle, triturate the solution 3 times.</li>
	<li>Distribute this solution into increments of 12.5 mL of warm DMEM/F12 per brain.</li>
	<li>Pipette 1 mL increments of the triturated solution into a 12 well plate (1 per brain).</li>
	<li>Incubate at 37 &#176;C and 5% CO2in a humidified cell culture incubator. Replace media after three days, and refresh media every subsequent three days. After 2 weeks, the cells can be collected for experiments.</li>
</ol>
2. Macromer Synthesis69
<ol>
	<li>Weigh 375 mg of hyaluronic acid (HA) salt in a 50 mL conical centrifuge tube and add 37.5 mL of distilled water.</li>
	<li>Flush the solution with a vortexer for 1 min and sonicate the mixture until it appears homogenous and loses its gel-like viscosity. This process may take 6 h.</li>
	<li>Transfer solution to a 100 mL beaker with a magnetic stir bar (38 mm), and stir vigorously at room temperature.</li>
	<li>Carefully titrate 5.0 M NaOH into the mixing HA and measure with pH paper until the pH stabilizes between 8 and 12.</li>
	<li>Collect 3 mL of fresh methacrylic anhydride (MA, 94%), blanketed by nitrogen during storage. 
	NOTE: MA is exposed to the atmosphere for long durations (days), it will lose its reactivity. 
	Caution: All use of MA should be done in a fume hood.</li>
	<li>Add 200 &#181;L of MA into the mixing HA solution. The reaction lowers the pH of the solution below 8.</li>
	<li>Repeat steps 2.4 to 2.6 until the 3 mL of MA have been added to the solution. This process may take up to 1-2 h.</li>
	<li>Allow the reaction to continue overnight at 4 &#176;C, without mixing.</li>
	<li>Transfer solution to 400 mL of cold ethanol (EtOH) in a 500 mL beaker and allow precipitation for 24 - 72 h at 4 &#176;C.56</li>
	<li>Carefully decant the major portion of the solution into a separate beaker for disposal, and collect the precipitate in a 50 mL conical centrifuge tube. This may be distributed in 2-4 tubes, if necessary.</li>
	<li>Centrifuge the tubes at 200 x g in a centrifuge for 2 min at room temperature and dispose of the supernatant.</li>
	<li>Top up the tube with cold EtOH, mix the solution with a vortexer for 1 min, and repeat step 2.10-2.11.</li>
	<li>Add 25 mL of sterile deionized water, mix the solution with a vortexer for 1 min, sonicate (&#62;20 kHz) for 1 h, and incubate at 4 &#176;C overnight.</li>
	<li>Place the solution in a -80 &#176;C freezer for 15 min or until frozen or flash freeze in liquid nitrogen. 
	CAUTION: When using liquid nitrogen, use protective gloves, a face shield, and an apron.</li>
	<li>Freeze dry the tube (below 0.1 mBar and -30 &#176;C) for 24 - 48 h until a snow-like powder is produced.</li>
	<li>At this point, test the sample for purity via 1H nuclear magnetic resonance, where the amount of methacrylate protons (peaks at 6.1 and 5.6 ppm), relative to methyl protons (1.9 ppm), on the HA backbone can be confirmed. A minimum ratio of 95% methacrylate to methyl protons is acceptable.69</li>
</ol>
3. Gel Formation and 3D Encapsulation65
<ol>
	<li>Coverslip and Mold Preparation
	<ol>
		<li>Make a 50 mL distilled water solution of 2% 3-(Trimethoxysilyl)propyl methacrylate in a 50 mL conical centrifuge tube.</li>
		<li>Place 18 mm glass coverslips in solution and rock for 1 h at room temperature. Up to 50 coverslips can be added at once.</li>
		<li>Rinse coverslips by serially dipping each in 3 beakers of 100 mL deionized water.</li>
		<li>Dry the coverslips in a vacuum at 40 &#176;C overnight with a desiccator and oven.</li>
		<li>Quickly immerse individual coverslips in 70% EtOH with forceps.</li>
		<li>Without drying, drop each coverslip into a well of a 12 well plate.</li>
		<li>Wash and aspirate each well with sterile deionized water (1 mL per well).</li>
		<li>Add 1 mL of sterile 2 &#181;g /mL poly-L-lysine (PLL) to each well and incubate for &#62;2 h.</li>
		<li>Aspirate PLL solution and allow the coverslips to air dry.</li>
		<li>Immerse polydimethyl siloxane (PDMS) molds (see Figure 1) in 70% EtOH, and place one upon the center of each coverslip, and allow to air dry and create a seal between the mold and glass.
		<ol>
			<li>Prepare PDMS molds by casting PDMS premix reagents in a 10:1 ratio in a flat-bottomed polystyrene dish. The quantity of PDMS prepared is selected to yield a sheet approximately 1 mm thick. To form wells in the PDMS, cut the sheets with a circle punch with an inner diameter of 10.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Cell Preparation 
	NOTE: All steps in the protocol are performed with sterile technique in a biosafety cabinet. The phosphate buffered saline (PBS) solution is adjusted to pH 7.4 for all steps.
	<ol>
		<li>24 h prior to cell encapsulation in HAMA, refresh the medium for the 2 week primary cultures (from Step 1.15) in the 12 well plates. Add 1 mL of DMEM/F12 (10% FBS and 1% PS) medium per well.</li>
		<li>For each 12-well plate of cells prepare 12.5 mL of dilute trypsin (0.25% trypsin-EDTA diluted 30% with DMEM/F12 media), and 25 mL DMEM/F12 (10% FBS and 1% PS) and warm in at 37 &#176;C in a water bath.</li>
		<li>Collect conditioned medium from plates using a 10 mL serological pipette (&#62; 0.5 mL per well), thoroughly aspirate remaining liquid, and replace with 1 mL per well of dilute trypsin (0.075% trypsin-EDTA) for 20-30 min in an incubator (37 &#176;C, 5% CO2) until the confluent cell layer detaches from the plate.</li>
		<li>Recover suspended cell material with a 1 mL pipette (appears as a single floating piece) and collect in a 15 mL conical centrifuge tube. Dilute with an equivalent volume of DMEM/F12 (10% FBS and 1% PS), and centrifuge at 200 x g for 2 min at room temperature, discarding the supernatant.</li>
		<li>Resuspend the cell pellet in 10 mL warm DMEM/F12 (10% FBS and 1% PS), triturate the cells 5 times with a 10 mL pipette, and centrifuge at 200 x g for 2 min.</li>
		<li>Decant the supernatant, re-suspend the cells in 5 mL warm DMEM/F12, and transfer the cells to a 50 mL conical centrifuge tube.</li>
		<li>Using a 10 mL syringe with a 18 gauge needle, triturate the cell solution 3 times, and filter the suspension through a 40 &#181;m cell sieve into a new conical centrifuge tube.</li>
		<li>Collect 10 &#181;L of cell suspension, dilute 1:100 in warm DMEM/F12, and count cells with an automated cell counter</li>
		<li>Incubate in a 37 &#176;C water bath until encapsulation step.</li>
	</ol>
	</li>
	<li>Cell Encapsulation 
	NOTE: All steps in the protocol are performed with sterile technique in a biosafety cabinet.
	<ol>
		<li>For each 12-well plate of 3D hydrogels warm 12.5 mL of DMEM/F12 and 12.5 mL of the conditioned DMEM/F12 media collected during cell preparation.</li>
		<li>Weigh the quantity of methacrylated hyaluronic acid (HAMA) required for a final concentration of 0.5% wt/vol. Dissolve this HAMA in sterile filtered PBS at a concentration of (2% wt/vol); a concentration 4 times higher than the final concentration is utilized to accommodate the addition of cells and other reagents. One 12 well plate of hydrogels requires ~350 &#181;L PBS with 7 mg HAMA.</li>
		<li>Sonicate (&#62;20 kHz) solutions until HAMA is fully dissolved for 60 min.</li>
		<li>Prepare individual 10% solutions of both triethanolamine (TEA) and 1-vinyl-2-pyrrolidinone (NVP), and a 1 mM solution of Eosin Y (EY) in 1 mL aliquots of sterile PBS pH 7.4.</li>
		<li>For one 12 well plate, make a final 1.4 mL mixture of 1 x 107 cells, 0.5% wt/vol HAMA (350 &#181;L of 2% wt/vol), 0.1% TEA (14 &#181;L of 10%), 0.1% NVP (14 &#181;L of 10%), and 0.01 mM EY (14 &#181;L of 1 mM), and 20% basal lamina mixture (280 &#181;L of stock). The remaining volume is PBS.</li>
		<li>Gently mix the solution and pipette 100 &#181;L into each PDMS mold with a 1 mL tip.</li>
		<li>Expose samples to a high intensity green LED light (~520 nm, 60 mW, in an enclosed 20 cm x 20 cm x 20 cm box) for 5 min at room temperature.</li>
		<li>Add 1 mL per well of warm conditioned DMEM/F12 to a 12 well plate.</li>
		<li>Grasp each coverslip between thumb and forefinger, slowly peel the PDMS mold off with curved tweezers being careful not to displace the gel, and place it into a well with conditioned medium.</li>
		<li>Add an additional 1 mL warm DMEM/F12 to each well.</li>
		<li>Incubate plates at 37 &#176;C with 5% CO2 for 2 weeks, refreshing cell media every week by pipetting 1 mL of media from each wells and replacing with 1 mL DMEM/F12.</li>
	</ol>
	</li>
</ol>
4. Microscopy
<ol>
	<li>Immunocytochemistry 
	NOTE: An inverted confocal microscope was used to image these samples and ImageJ was used to process and display the images. Microglia, astrocytes, oligodendrocytes, and nuclei, will be labeled after 3 weeks in the HAMA culture.
	<ol>
		<li>Preheat 10% PBS-buffered formalin (pH 7.0) in a 37 &#176;C bath. 
		Caution: Although formalin can be used outside a fume hood, this material is reactive with tissue, and should be handle with care and gloves always. It should be dispose separately.</li>
		<li>Carefully aspirate media from the plates and pipette 1 mL of 10% formalin to each well.</li>
		<li>Incubate the plates at 37 &#176;C in a 5% CO2 for 20 min.</li>
		<li>Aspirate liquid from each plate, pipette 2 mL of PBS (pH 7.4) into each well, and incubate for 15 min.</li>
		<li>Repeat step 4.1.4 twice.</li>
		<li>Aspirate liquid from the plates, add 1 mL per well of PBS with 10% normal horse serum (NHS) and 0.5% triton X-100, and gently rock at minimum speed) for 1 h.</li>
		<li>Repeat step 4.1.4 three times.</li>
		<li>Aspirate liquid and add 1 mL per well of the following mixture in PBS: rabbit ionized calcium-binding adapter molecule (Iba1) 1:1,000, chicken glial fibrillary protein (GFAP) 1:5,000, mouse 2&#39;,3&#39;-cyclic-nucelotide 3&#39;-phophodiesterase (CNPase) 1:1,000, 1% NHS, and 0.1% cell perforating surfactant. Incubate at 4 &#176;C overnight, rocking the plate as gently as possible.</li>
		<li>Repeat step 4.1.4 three times with 1 h incubations in between.</li>
		<li>Aspirate liquid and add 1 mL per well of the following mixture in PBS: 647 nm excited donkey anti-rabbit antibody 1:200, 546 nm excited goat anti-chicken antibody 1:200, 488 nm excited donkey anti-mouse antibody 1:200, 1% NHS, 1:1,000 blue nuclear stain. Incubate at room temperature for 1 h, gently rocking the plate.</li>
		<li>Repeat step 4 three times with 1 h incubations in between.</li>
		<li>To preserve the hydrogels, aspirate buffer, and immerse them in 0.5 mL of cell mounting medium for 1 min. Pipette out the mounting medium and gently place a coverslip on top of the gel, creating a layer between the glass. Add a small amount of mounting medium to the uncovered side of the coverslips to prevent drying of the hydrogel. 
		NOTE: For troubleshooting purposes, gels can be imaged at step 4.1.11. to see proper labeling before further processing.</li>
	</ol>
	</li>
	<li>Scanning Electron Microscopy (SEM) 
	NOTE: To visualize the hydrogel, a scanning electron microscope was used to image these samples.
	<ol>
		<li>Pre-heat a fixative mixture of 2.5% glutaraldehyde and 2% paraformaldehyde in PBS (0.1 M) in a 37 &#176;C water bath. 
		Caution: These reagents are highly reactive with tissues, and should be handled with care and gloves. They should be handled in a fume hood as much as possible and disposed separately.</li>
		<li>Aspirate medium from the cell culture plates and add 1 mL of the pre-heated fixative mixture.</li>
		<li>Incubate the plates at 37 &#176;C in a 5% CO2 for 20 min.</li>
		<li>Place the plates in 4 &#176;C (fridge/cooler) overnight.</li>
		<li>Aspirate the fixative solution from the wells and add 1 mL of PBS.</li>
		<li>Aspirate liquid from each plate, add 2 mL of PBS into each well, and incubate for 15 min.</li>
		<li>Repeat step 4.2.6 two more times.</li>
		<li>Aspirate the PBS and add 1 mL of 1% osmium tetraoxide buffered in PBS to each well. 
		NOTE: Until samples are dried, this step and all subsequent steps are performed in a fume hood.</li>
		<li>Allow fixation to occur for 30 min. Osmium vapor from the liquid can fix other samples in the same plate; therefore, partition samples accordingly before this step. 
		CAUTION: Osmium tetraoxide is highly reactive with tissue and volatile. It is dangerous and should be handled in the fume hood with gloves. It and any immediate washed should be disposed separately.</li>
		<li>Repeat step 4.2.6 three times.</li>
		<li>Aspirate the liquid and add the dehydrating mixtures in the list below, in order, and incubate (room temperature) for the indicated time. Start by gradually adding ethanol (EtOH) followed then hexamethyldisilazane (HMDS). See Table 2 for each increment. 
		NOTE: When adding HMDS, the coverslips can strongly adhere to the plastic and become very difficult to remove. This can be prevented by placing a small plastic platform under the glass slip after the first application of HMDS. After the second EtOH incubation, the sample can be taken out and quenched in liquid nitrogen for few seconds to freeze fracture the sample.</li>
		<li>Carefully remove the dried samples and mount the samples on scanning electron microscope stubs with double sided conductive tape.</li>
		<li>Sputter coat the samples with a minimum amount of gold. Place the samples in a holder and operate the machine for 2 min at 15 mA.</li>
	</ol>
	</li>
</ol>

The authors have nothing to disclose.

Towards the goal of generating a 3D culture system to model glial bioreactivity and the glial scarring process, we have developed a system that can support primary cultured microglia, astrocytes and oligodendrocytes and enables robust characterization of cell morphology and cell-cell interactions. From the micrographs shown, the morphology of each cell type was distinctly different with 2D, 3D-HAMA, and 3D HAMA-Basal lamina mixture platforms. In the 2D system, morphology was distinctly biased along the plane of the surfa...

Inflammation of the central nervous system (CNS) has long been considered a hallmark of acute (e.g., ischemic stroke, traumatic brain and spinal cord injury) and chronic (e.g. Alzheimer&#39;s, Parkinson&#39;s, and Huntington&#39;s diseases) CNS injury, but is increasingly recognized as a causal contributor to neurodegenerative and neuropsychiatric disorders. Sustained or inappropriate inflammation can cause neural injury and demyelination (e.g. multiple sclerosis), and negatively influence brain development (e.g., schizophrenia, autism) and mood states (e.g., depression, anxiety, bipolar disorder). Further, novel therapeutic strategies using implantable devices (e.g.,brain-computer-interfaces1,2,3, deep brain stimulation4,5, intraspinal microstimulation6,7,8,9,10) generate a predictable inflammatory response at the interface between the device and the CNS resulting in a protective tissue response that can cause loss of efficacy or device failure over the lifetime of the implant11. Inflammation in the CNS is typically initiated by microglia, which function as the resident immune cells of the CNS responsible for tissue surveillance and mounting the foreign body response (reviewed12). Depending on the severity of an insult, the microglia signal and recruit additional cell types to an injury site. Specifically, the microglia activate the astrocytes, which in turn act as secondary inflammatory cells and form a dense protective barrier to contain an injury site13,14. Microglia can also initiate an activity cascade in the cells of the peripheral immune system, which can result in the breakdown of the BBB to permit immune infiltration (reviewed in reference15).
In the case of devices implanted into the CNS, tissue damage resulting from device insertion as well as the continued presence of the foreign device may initiate a process termed glial scarring. In this process, the microglia migrate to and proliferate at the site of injury. They also initiate the release of inflammatory factors to neutralize potential threats and recruit additional glial cells. Subsequently, activated astrocytes become hypertrophic and begin encapsulating the implanted device to form a continuous fibrous barrier16. Inflammatory signalling also serves to promote withdrawal of neuronal processes from the vicinity of the implant and eventually recruits fibroblasts to reinforce the developing glial scar17. The oligodendrocytes, responsible for sheathing neurons in myelin to enhance conductance, do not survive this process and distant cells are partitioned from the implant by the scar18. Glial scarring greatly reduces the function and lifetime of implanted devices, particularly for recording electrodes, and ultimately serves to limit the functionality of neural interfaces19.
Several approaches have been exploited to increase the biocompatibility and interface activity of implanted devices in the CNS20,21,22,23. An extensive review is available on the biocompatible design of these neural interfaces24.The most prominent strategies include surrounding the electrode with compatible coatings such as polyelthyleneglycol (PEG), polylactic-co-glycolic acids (PLGA)25, or enhancing the electrode with conductive polymers such as poly(ethylene dioxythiophene) (PEDOT), and polypyrrole (PPy)26,27,28,29,30,31. Bioactive coatings have also been employed to provide cues for neural tissue growth using ligands derived from extracellular matrices including collagens, fibronectins, and hyaluronic acids32,33,34,35,36,37. The bioactivity of these coatings has been further explored with growth factor release systems to emulate natural cell secretions30,38,39,40,41,42,43,44,45,46,47,48,49,50. Simultaneously, some research groups have opted to remodel the electrode geometry, flexibility, and composition to decrease the mechanical mismatch between device and tissue51,52,53,54,55,56,57. Altogether, these strategies have lead to many promising improvements in next-generation neural interfacial devices, however the long-term compatibility is an on-going issue and progress may be hampered by complex and time-consuming in vivo models.
Animal model-based approaches can limit the experimental throughput and increase costs of testing electrode biocompatibility. In vitro approaches using conventional cell culture techniques offer a more cost-effective alternative but fail to recapitulate much of the complexity of the interaction between device and tissue58. In particular, testing of surface coatings using 2D cell culture limits the modeling of electrode geometry and the influence of mechanical mismatch and micromotion thought to contribute to generating a host response contributing to device failure59,60.
To overcome problems associated with 2D cell culture, hydrogel cultures of neural cells have been developed for a wide variety of applications, pharmacological studies61, to direct neural cell differentiation62, to understand disease pathways63,64, or layered in co-culture with other cell types to model cell migration, neuroprotection, or to model tissue microenvironments61. Hydrogels are readily formed at different sizes and geometries can incorporate numerous types of primary or immortalized cell cultures, and are highly amenable to analysis by commonly used techniques such as confocal fluorescence microscopy. To create a model of the glial scarring process, we have recently developed and characterised a hyaluronic-acid based 3D hydrogel system for high-throughput testing of the glial response to implanted electrodes (Figure 1)65. This system has several distinct advantages: 1) primary glial cells (microglia, astrocytes, and oligodendrocytes) are encapsulated in a 3D matrix composed of polymers of hyaluronic acid, which is an endogenous extracellular matrix component; 2) the matrix stiffness can be &#39;tuned&#39; to recreate the mechanical properties of brain or spinal cord tissue; and 3) cells can be encapsulated in the matrix in a rapid bench-top approach using photopolymerization with green light, limiting toxicity during encapsulation. This system enables key features of in vivo biocompatibility: devices are inserted into the hydrogel in a comparable manner to tissue, and the cellular response to implanted devices are monitored for a wide range of parameters65. These include mechanical mismatch between devices and the hydrogel coatings of various structures and electrical stimulation pulses. This system also includes oligodendrocyte and related precursors, which are often present and recruited in glial scars. Their damage, death, and phagocytosis by microglia are highly indicative of inflammatory injury and as a model reduced scarring or recovery, they have the capacity to demonstrate re-myelination of neurons66.
Herein we describe a method for synthesis and formation of hybrid hyaluronic acid hydrogels combined with commercially available basement membrane formulations to improve cell incorporation. Further, we will demonstrate the incorporation of primary cultured glial cells (microglia, astrocytes, and oligodendrocytes) and analysis of culture growth using immunocytochemistry and confocal microscopy.

<table><tbody><tr><td>&lt;strong&gt;1. Materials for HAMA synthesis and photopolymerization&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Hyaluronic acid (HA)</td><td>Sigma-Aldrich</td><td>53747-10G</td><td>Streptococcus equi, MW: 1.5 - 1.8 X 10^6</td></tr><tr><td>Methacrylic anhydride (MA)</td><td>Sigma-Aldrich</td><td>275585-100ML</td><td></td></tr><tr><td>Sodium hydroxide (NaOH)</td><td>Sigma-Aldrich</td><td>221465-25G</td><td></td></tr><tr><td>Ethanol (EtOH)</td><td>Commerical Alcohols Inc.</td><td></td><td>Anhydrous</td></tr><tr><td>Phosphate buffered saline (pH 7.4) tablets</td><td>Fisher Scientific</td><td>18912014</td><td></td></tr><tr><td>Triethanolamine (TEA)</td><td>Sigma-Aldrich</td><td>90279-100ML</td><td></td></tr><tr><td>1-Vinyl-2pyrrolidinone (NVP)</td><td>Sigma-Aldrich</td><td>V3409-5G</td><td></td></tr><tr><td>EosinY (EY)</td><td>Sigma-Aldrich</td><td>E6003-25G</td><td></td></tr><tr><td>Polydimethylsiloxane (PDMS) Sylgard 184 Silicone Elastomer Kit</td><td>Dow Corning</td><td></td><td></td></tr><tr><td>3-(Trimethoxysilyl)propyl methacrylate</td><td>Sigma-Aldrich</td><td>440159-100ML</td><td></td></tr><tr><td>Beaker (100 mL)</td><td>Corning</td><td>1000-100</td><td></td></tr><tr><td>Beaker (500 mL)</td><td>Corning</td><td>1000-600</td><td></td></tr><tr><td>pH paper (Labstick)</td><td>Sigma-Aldrich</td><td>9580</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;2. Materials for glial cell isolation and cell culture&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>P1-2 Sprague Dawley rat pups</td><td>Charles River</td><td>CD Sprague Dawley rat strain code 001</td><td></td></tr><tr><td>Dissector scissors - slim blades (small)</td><td>Fine Science Tools</td><td>14081-09</td><td></td></tr><tr><td>Surgical scissors - Toughcut (large)</td><td>Fine Science Tools</td><td>14130-17</td><td></td></tr><tr><td>Fine forceps (Dumont #5)</td><td>Fine Science Tools</td><td>11521-10</td><td></td></tr><tr><td>Curved fine forceps (Dumont #7)</td><td>Fine Science Tools</td><td>11271-30</td><td></td></tr><tr><td>Hank&#039;s balanced salt solution (HBSS)</td><td>Gibco</td><td>14170-112</td><td></td></tr><tr><td>Dulbecco&#039;s modified Eagle&#039;s medium and Ham&#039;s nutrient mixture F-12 (DMEM/F12)</td><td>Gibco</td><td>11320-033</td><td></td></tr><tr><td>Penicillin-streptomycin (PS)</td><td>Gibco</td><td>15140-122</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Gibco</td><td>12483-020</td><td></td></tr><tr><td>0.25% Trypsin-ethylenediaminetetraacetic acid (EDTA)</td><td>Gibco</td><td>25200-072</td><td></td></tr><tr><td>Poly-L-lysine (PLL)</td><td>Sigma-Aldrich</td><td>P-6282</td><td></td></tr><tr><td>50 mL conical centrifuge tube</td><td>Fisher Scientific</td><td>05-539-13</td><td></td></tr><tr><td>15 mL conical centrifuge tube</td><td>Fisher Scientific</td><td>05-539-5</td><td></td></tr><tr><td>12 well Tissue culture treated plates (Cellstar)</td><td>Greiner Bio-One</td><td>665 108</td><td></td></tr><tr><td>10 mL serological pipette</td><td>Fisher Scientific</td><td>13-676-10F</td><td></td></tr><tr><td>25 mL serological pipette</td><td>Fisher Scientific</td><td>12-676-10K</td><td></td></tr><tr><td>Petri dish (60 mm X 15 mm)</td><td>Fisher Scientific</td><td>FB0875713A</td><td></td></tr><tr><td>Petri dish (100 mm X 15 mm)</td><td>Fisher Scientific</td><td>FB0875712</td><td></td></tr><tr><td>Microscope Coverslip (18 mm)</td><td>Fisher Scientific</td><td>12-545-100 18CIR</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;3. Materials for microscopy (confocal and scanning electron microscopy)&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Mouse monoclonal anti-CNPase</td><td>abcam</td><td>ab6319</td><td></td></tr><tr><td>Rabbit anti-Iba1</td><td>Wako Laboratory Chemicals</td><td>019-17741</td><td></td></tr><tr><td>Chicken anti-GFAP</td><td>abcam</td><td>ab4674</td><td></td></tr><tr><td>Hoechst 33342</td><td>Fisher Scientific</td><td>62249</td><td></td></tr><tr><td>Fluoromount-G</td><td>Fisher Scientific</td><td>00-4958-02</td><td></td></tr><tr><td>Formalin</td><td>Sigma Aldrich</td><td>HT501128-4L</td><td>Buffered (10%)</td></tr><tr><td>Triton X-100</td><td>Fisher Scientific</td><td>BP151-500</td><td></td></tr><tr><td>Horse Serum</td><td>Gibco</td><td>16050-122</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Electon Microscopy Sciences</td><td>157-8</td><td>Buffered (8%)</td></tr><tr><td>Guteraldehyde</td><td>Electon Microscopy Sciences</td><td>16019</td><td>Buffered (8%)</td></tr><tr><td>Osmium tetraoxide</td><td>Electon Microscopy Sciences</td><td>19152</td><td>Buffered (2%)</td></tr><tr><td>Hexamethyldilazane (HMDS)</td><td>Electon Microscopy Sciences</td><td>16700</td><td></td></tr><tr><td>Ethanol (EtOH)</td><td>Electon Microscopy Sciences</td><td>15055</td><td>Anhydrous</td></tr><tr><td>Microscope Slide (25 X 75 X 1 mm)</td><td>VWR International</td><td>48311-703</td><td></td></tr></tbody></table>

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.

To model the neural tissue host response and the glial scar in a high throughput, in vitro system requires a 3D cell scaffold with a matrix material that is biocompatible, does not incur a cytotoxic event during in situ formation, and is modifiable with bioactive components to guide benevolent response. To this end, we have created a 3D cell scaffold system based on hyaluronic acid, and encapsulated a primary mixed glial cell population to study cell-cell interactions an...

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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.

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 ...

improved-3d-hydrogel-cultures-primary-glial-cells-for-vitro-modelling

Research

JoVE Journal

Bioengineering

14.4K Views.  University of Alberta. Hierin presenteren wij een protocol voor de 3D cultuur van rat hersenen-afgeleide glia cellen, met inbegrip van astrocyten, oligodendrocyten en microglia. We tonen primaire celculturen, methacrylated hyaluronzuur (HAMA) hydrogel synthese, HAMAphoto-polymerisatie en cel inkapseling en monster verwerking voor confocale en scanning elektronen microscopische beeldvorming.

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

Co-culture of Glioblastoma Stem-like Cells on Patterned Neurons to Study Migration and Cellular Interactions

Glioblastomas (GBMs), grade IV malignant gliomas, are one of the deadliest types of human cancer because of their aggressive characteristics. Despite significant advances in the genetics of these tumors, how GBM cells invade the healthy brain parenchyma is not well understood. Notably, it has been shown that GBM cells invade the peritumoral space via different routes; the main interest of this paper is the route along white matter tracts (WMTs). The interactions of tumor cells with the peritumoral nervous cell components are not well characterized. Herein, a method has been described that evaluates the impact of neurons on GBM cell invasion. This paper presents an advanced co-culture in vitro assay that mimics WMT invasion by analyzing the migration of GBM stem-like cells on neurons. The behavior of GBM cells in the presence of neurons is monitored by using an automated tracking procedure with open-source and free-access software. This method is useful for many applications, in particular, for functional and mechanistic studies as well as for analyzing the effects of pharmacological agents that can block GBM cell migration on neurons.

Here, we present an easy-to-use co-culture assay to analyze glioblastoma (GBM) migration on patterned neurons. We developed a macro in FiJi software for easy quantification of GBM cell migration on neurons, and observed that neurons modify GBM cell invasive capacity.

Glioblastomas (GBMs), grade IV malignant gliomas, are one of the deadliest types of human cancer because of their aggressive characteristics. Despite ...

Cancer Research

Hyaluronic-Acid Based Hydrogels for 3-Dimensional Culture of Patient-Derived Glioblastoma Cells

Glioblastoma (GBM) is the most common, yet most lethal, central nervous system cancer. In recent years, many studies have focused on how the extracellular matrix (ECM) of the unique brain environment, such as hyaluronic acid (HA), facilitates GBM progression and invasion. However, most in vitro culture models include GBM cells outside of the context of an ECM. Murine xenografts of GBM cells are used commonly as well. However, in vivo models make it difficult to isolate the contributions of individual features of the complex tumor microenvironment to tumor behavior. Here, we describe an HA hydrogel-based, three-dimensional (3D) culture platform that allows researchers to independently alter HA concentration and stiffness. High molecular weight HA and polyethylene glycol (PEG) comprise hydrogels, which are crosslinked via Michael-type addition in the presence of live cells. 3D hydrogel cultures of patient-derived GBM cells exhibit viability and proliferation rates as good as, or better than, when cultured as standard gliomaspheres. The hydrogel system also enables incorporation of ECM-mimetic peptides to isolate effects of specific cell-ECM interactions. Hydrogels are optically transparent so that live cells can be imaged in 3D culture. Finally, HA hydrogel cultures are compatible with standard techniques for molecular and cellular analyses, including PCR, Western blotting and cryosectioning followed by immunofluorescence staining.

Here, we present a protocol for three-dimensional culture of patient-derived glioblastoma cells within orthogonally tunable biomaterials designed to mimic the brain matrix. This approach provides an in vitro, experimental platform that maintains many characteristics of in vivo glioblastoma cells typically lost in culture.

Glioblastoma (GBM) is the most common, yet most lethal, central nervous system cancer. In recent years, many studies have focused on how the extracellular ...

Preparation of Primary Mixed Glial Cultures from Adult Mouse Spinal Cord Tissue

It has been well-accepted that spinal cord glial responses contribute significantly to the development of neuropathic pain. Tremendous information regarding glial activities at the cellular and molecular levels has been obtained through in vitro cell culture systems. The in vitro systems utilized, mainly include primary glia derived from neonatal brain cortical tissue and immortalized cell lines. However, these systems may not reflect the characteristics of spinal cord glial cells in vivo. In order to further investigate the roles of spinal cord glial cells in the development of peripheral nerve injury-induced neuropathic pain using a culture system that better reflects the in vivo condition, our laboratory has developed a method to establish primary spinal cord mixed glial cultures from adult mice. Briefly, spinal cords are collected from adult mice and processed through papain digestion followed by myelin removal with a density-gradient medium. Single cell suspensions are cultured in complete Dulbecco&#39;s modified Eagle media (cDMEM) supplemented with 2-mercaptoethanol (2-ME) at 35.9 oC. These culture conditions were optimized specifically for the growth of mixed glial cells. Under these conditions, cells are ready to be used for experimentation between 12 - 14 d&#160;(cells are usually in log phase during this time) after the establishment of the culture (D 0) and can be kept in culture conditions up to D 21. This culture system can be used to investigate the responses of spinal cord glial cells upon stimulation with various substances and agents. Besides neuropathic pain, this system can be used to study glial responses in other diseases that involve pathological changes of spinal cord glial cells.

The development of neuropathic pain involves pathological changes of spinal cord glial cells. A reliable glial culture system derived from adult spinal cord tissue and designed for studying these cells in vitro is lacking. Therefore, we show here how&#160;to establish primary mixed glial cultures from adult mouse spinal cord tissue.

It has been well-accepted that spinal cord glial responses contribute significantly to the development of neuropathic pain. Tremendous information ...

Neuroscience

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 ...

A Cell Culture Model for Studying the Role of Neuron-Glia Interactions in Ischemia

Ischemic stroke is a clinical condition characterized by hypoperfusion of brain tissue, leading to oxygen and glucose deprivation, and the consequent neuronal loss. Numerous evidence suggests that the interaction between glial and neuronal cells exert beneficial effects after an ischemic event. Therefore, to explore potential protective mechanisms, it is important to develop models that allow studying neuron-glia interactions in an ischemic environment. Herein we present a simple approach to isolate astrocytes and neurons from the rat embryonic cortex, and that by using specific culture media, allows the establishment of neuron- or astrocyte-enriched cultures or neuron-glia cultures with high yield and reproducibility.
To study the crosstalk between astrocytes and neurons, we propose an approach based on a co-culture system in which neurons cultured in coverslips are maintained in contact with a monolayer of astrocytes plated in multiwell plates. The two cultures are maintained apart by small paraffin spheres. This approach allows the independent manipulation and the application of specific treatments to each cell type, which represents an advantage in many studies.
To simulate what occurs during an ischemic stroke, the cultures are subjected to an oxygen and glucose deprivation protocol. This protocol represents a useful tool to study the role of neuron-glia interactions in ischemic stroke.

Here, we present a simple approach using specific culture media that allows the establishment of neuron- and astrocyte-enriched cultures, or neuron-glia cultures from the embryonic cortex, with high yield and reproducibility.

Ischemic stroke is a clinical condition characterized by hypoperfusion of brain tissue, leading to oxygen and glucose deprivation, and the consequent ...

Co-Culturing Microglia and Cortical Neurons Differentiated from Human Induced Pluripotent Stem Cells

The ability to generate microglia from human induced pluripotent stem cells (iPSCs) provides new tools and avenues for investigating the role of microglia in health and disease. Furthermore, iPSC-derived microglia can be maintained in co-culture with iPSC-derived cortical neurons, which enable investigations of microglia-neuron interactions that are hypothesized to be dysregulated in a number of neuropsychiatric disorders. Human iPSCs were differentiated to generate microglia using an adapted version of a protocol developed by the Fossati group, and the iPSC-derived microglia were validated with marker analysis and real-time PCR. Human microglia generated using this protocol were positive for the markers CD11C, IBA1, P2RY12, and TMEM119, and expressed the microglial-related genes AIF1, CX3CR1, ITGAM, ITGAX, P2RY12, and TMEM119. Human iPSC-derived cortical neurons that had been differentiated for 30 days were plated with microglia and maintained in co-culture until day 60, when experiments were undertaken. The density of dendritic spines in cortical neurons in co-culture with microglia was quantified under baseline conditions and in the presence of pro-inflammatory cytokines. In order to examine how microglia modulate neuronal function, calcium imaging experiments of the cortical neurons were undertaken using the calcium indicator Fluo-4 AM. Live calcium activity of cortical neurons was obtained using a confocal microscope, and fluorescence intensity was quantified using ImageJ. This report describes how co-culturing human iPSC-derived microglia and cortical neurons provide new approaches to interrogate the effects of microglia on cortical neurons.

This protocol describes a methodology to differentiate microglia from human iPSCs and maintain them in co-culture with iPSC-derived cortical neurons in order to study mechanistic underpinnings of neuroimmune interactions using human neurons and microglia.

The ability to generate microglia from human induced pluripotent stem cells (iPSCs) provides new tools and avenues for investigating the role of microglia ...

Replicating Glioblastoma Invasion with Hyaluronan-Collagen I Hydrogel System

A Photopolymerizable Hyaluronic Acid-Collagen Model of the Invasive Glioma Microenvironment with Interstitial Flow

Glioblastoma recurrence is a major hindrance to treatment success and is driven by the invasion of glioma stem cells (GSCs) into healthy tissue that are inaccessible to surgical resection and are resistant to existing chemotherapies. Tissue-level fluid movement, or interstitial fluid flow (IFF), regulates GSC invasion in a manner dependent on the tumor microenvironment (TME), highlighting the need for model systems that incorporate both IFF and the TME. We present an accessible method for replicating the invasive TME in glioblastoma: a hyaluronan-collagen I hydrogel composed of human GSCs, astrocytes, and microglia seeded in a tissue culture insert. Elevated IFF can be represented by applying a fluid pressure head to the hydrogel. Additionally, this model can be tuned to replicate inter- or intra-patient differences in cellular ratios, flow rates, or matrix stiffnesses. Invasion can be quantified, while gels can be harvested for a variety of outcomes, including GSC invasion, flow cytometry, protein or RNA extraction, or imaging.

Author Spotlight: Patient-Informed 3D Model for Studying Glioblastoma Invasion via Interstitial Fluid Flow

We present a method for replicating the glioma tumor microenvironment at the invasive front that incorporates interstitial fluid flow. This model is a hyaluronan-collagen I hydrogel in a tissue culture insert where a fluid pressure head can be applied. Invasion can be quantified, and cells can be isolated or lysed.

Glioblastoma recurrence is a major hindrance to treatment success and is driven by the invasion of glioma stem cells (GSCs) into healthy tissue that are ...

Embryonic Mouse Brain Mixed Cell Cultures to Study Infection and Innate Immunity

Establishing Mixed Neuronal and Glial Cell Cultures from Embryonic Mouse Brains to Study Infection and Innate Immunity

Models of the central nervous system (CNS) must recapitulate the complex network of interconnected cells found in vivo. The CNS consists primarily of neurons, astrocytes, oligodendrocytes, and microglia. Due to increasing efforts to replace and reduce animal use, a variety of in vitro cell culture systems have been developed to explore innate cell properties, which allow the development of therapeutics for CNS infections and pathologies. Whilst certain research questions can be addressed by human-based cell culture systems, such as (induced) pluripotent stem cells, working with human cells has its own limitations with regard to availability, costs, and ethics. Here, we describe a unique protocol for isolating and culturing cells from embryonic mouse brains. The resulting mixed neural cell cultures mimic several cell populations and interactions found in the brain in vivo. Compared to current equivalent methods, this protocol more closely mimics the characteristics of the brain and also garners more cells, thus allowing for more experimental conditions to be investigated from one pregnant mouse. Further, the protocol is relatively easy and highly reproducible. These cultures have been optimized for use at various scales, including 96-well based high throughput screens, 24-well microscopy analysis, and 6-well cultures for flow cytometry and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis. This culture method is a powerful tool to investigate infection and immunity within the context of some of the complexity of the CNS with the convenience of in vitro methods.

Author Spotlight: Establishing Mixed Neuronal and Glial Cell Cultures from Embryonic Mouse Brains to Study Infection and Innate Immunity  

This protocol presents a unique way of generating central nervous system cell cultures from embryonic day 17 mouse brains for neuro(immuno)logy research. This model can be analyzed using various experimental techniques, including RT-qPCR, microscopy, ELISA, and flow cytometry.

Models of the central nervous system (CNS) must recapitulate the complex network of interconnected cells found in vivo. The CNS consists primarily of ...

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 ...

Establishing a Three-Dimensional Culture of Rat Brain-Derived Glial Cells

Source: Koss, K. M, et al. <a href="https://app.jove.com/v/56615">Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation.</a> J. Vis. Exp. (2017).
This video demonstrates a technique to establish a three-dimensional culture of glial cells. Rat-brain-derived glial cells are encapsulated within a photocrosslinkable polymer matrix containing extracellular matrix components. Upon incubating in a growth medium, the glial cells proliferate within the supporting matrix to create a three-dimensional culture.

The protocol for the brain tissue extraction from day 1 Sprague Dawley rat pups, euthanized by decapitation, was approved by the Animal Care and Use ...

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

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