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...

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>1. Materials for HAMA synthesis and photopolymerization</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>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>2. Materials for glial cell isolation and cell culture</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&#39;s balanced salt solution (HBSS)</td>
			<td>Gibco</td>
			<td>14170-112</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s modified Eagle&#39;s medium and Ham&#39;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>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>3. Materials for microscopy (confocal and scanning electron microscopy)</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...

Watch this Scientific Journal Video about Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation at JoVE.com

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 ...

The overall goal of this 3D cell culture protocol is to model the glial scar event of the central nervous system using key indicator cells:microglia, astrocytes and oligodendrocytes.This method can help answer key questions in the neuro-inflammation field, such as the potential for glial cells to enhance regenerative strategies and unravel the glial scar.The main advantage of this technique is that glial biology can be rapidly characterized in a 3D micro-environment with systematic and high-through put manipulations.To prepare cover slips for seeding, begin by adding one milliliter of 3-propyl methacrylate to 49 milliliters of distilled water to make a 2%solution.Then place 18 millimeter glass cover slips into the solution and rock for one hour at room temperature.After one hour, rinse the cover slips by serially dipping each cover slip in three beakers of 100 milliliters of deionized water.Then dry the cover slips in a vacuum with a desiccator and oven at 40 degrees Celsius overnight.The next day, use forceps to quickly immerse individual cover slips in 70%ethanol.Then, without drying, drop each cover slip into a well of a 12-well plate.Next, wash each well with one milliliter of sterile deionized water.Then add one milliliter of sterile poly

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

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JoVE Journal

Bioengineering

14.1K Görüntüleme.  University of Alberta. The overall goal of this 3D cell culture protocol is to model the glial scar event of the central nervous system using key indicator cells:microglia, astrocytes and oligodendrocytes.This method can help answer key questions in the neuro-inflammation field, such as the potential for glial cells to enhance regenerative strategies and unravel the glial scar.The main advantage of this technique is that glial biology can be rapidly characterized in a 3D micro-environment with systematic and high-t...