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

Summary

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.

Abstract

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.

Introduction

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's, Parkinson's, and Huntington'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-interfaces^1,2,3, deep brain stimulation^4,5, intraspinal microstimulation^6,7,8,9,10) 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 site^13,14. 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 CNS^20,21,22,23. 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 acids^{32,33,34,35,36,37}. The bioactivity of these coatings has been further explored with growth factor release systems to emulate natural cell secretions^{30,38,39,40,41,42,43,44,45,46,47,48,49,50}. Simultaneously, some research groups have opted to remodel the electrode geometry, flexibility, and composition to decrease the mechanical mismatch between device and tissue^{51,52,53,54,55,56,57}. 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 failure^59,60.

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 pathways^63,64, 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 'tuned' 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.

Protocol

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 Isolations^67,68

NOTE: All media for isolation and cell culture is preheated to 37 °C in a water bath. Hank's balanced salt solution (HBSS) has 1% penicillin-streptomycin (PS). All Dulbecco's modified Eagle's media with Ham'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.

1. Per brain, pre-heat 15 mL of HBSS and 12.5 mL DMEM/F12 to 37 °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.
2. 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.
3. Place up to 2 brains in each 10 cm² dish (Figure 2A).
4. 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).
5. 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 'hanging off' the brain tissue. Finally remove this portion by gently tugging. See Figure 2C-2E for representative images of before, during, and after this step.
6. 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.
7. Incubate this tube for 25 min in a 37 °C bath to digest the tissue enzymatically.
8. Centrifuge the tube down at 500 x g for 2 min at room temperature, and pour out the supernatant.
9. 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.
10. Centrifuge the tube down at 500 x g for 2 min at room temperature, and pour out the supernatant.
11. 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.
12. Using a 10 mL syringe with an 18 gauge needle, triturate the solution 3 times.
13. Distribute this solution into increments of 12.5 mL of warm DMEM/F12 per brain.
14. Pipette 1 mL increments of the triturated solution into a 12 well plate (1 per brain).
15. Incubate at 37 °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.

2. Macromer Synthesis⁶⁹

1. Weigh 375 mg of hyaluronic acid (HA) salt in a 50 mL conical centrifuge tube and add 37.5 mL of distilled water.
2. 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.
3. Transfer solution to a 100 mL beaker with a magnetic stir bar (38 mm), and stir vigorously at room temperature.
4. Carefully titrate 5.0 M NaOH into the mixing HA and measure with pH paper until the pH stabilizes between 8 and 12.
5. 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.
6. Add 200 µL of MA into the mixing HA solution. The reaction lowers the pH of the solution below 8.
7. 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.
8. Allow the reaction to continue overnight at 4 °C, without mixing.
9. Transfer solution to 400 mL of cold ethanol (EtOH) in a 500 mL beaker and allow precipitation for 24 - 72 h at 4 °C.⁵⁶
10. 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.
11. Centrifuge the tubes at 200 x g in a centrifuge for 2 min at room temperature and dispose of the supernatant.
12. Top up the tube with cold EtOH, mix the solution with a vortexer for 1 min, and repeat step 2.10-2.11.
13. Add 25 mL of sterile deionized water, mix the solution with a vortexer for 1 min, sonicate (>20 kHz) for 1 h, and incubate at 4 °C overnight.
14. Place the solution in a -80 °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.
15. Freeze dry the tube (below 0.1 mBar and -30 °C) for 24 - 48 h until a snow-like powder is produced.
16. 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

3. Gel Formation and 3D Encapsulation⁶⁵

1. Coverslip and Mold Preparation
   1. Make a 50 mL distilled water solution of 2% 3-(Trimethoxysilyl)propyl methacrylate in a 50 mL conical centrifuge tube.
   2. Place 18 mm glass coverslips in solution and rock for 1 h at room temperature. Up to 50 coverslips can be added at once.
   3. Rinse coverslips by serially dipping each in 3 beakers of 100 mL deionized water.
   4. Dry the coverslips in a vacuum at 40 °C overnight with a desiccator and oven.
   5. Quickly immerse individual coverslips in 70% EtOH with forceps.
   6. Without drying, drop each coverslip into a well of a 12 well plate.
   7. Wash and aspirate each well with sterile deionized water (1 mL per well).
   8. Add 1 mL of sterile 2 µg /mL poly-L-lysine (PLL) to each well and incubate for >2 h.
   9. Aspirate PLL solution and allow the coverslips to air dry.
   10. 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.
       1. 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.
2. 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.
   1. 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.
   2. 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 °C in a water bath.
   3. Collect conditioned medium from plates using a 10 mL serological pipette (> 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 °C, 5% CO₂) until the confluent cell layer detaches from the plate.
   4. 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.
   5. 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.
   6. Decant the supernatant, re-suspend the cells in 5 mL warm DMEM/F12, and transfer the cells to a 50 mL conical centrifuge tube.
   7. Using a 10 mL syringe with a 18 gauge needle, triturate the cell solution 3 times, and filter the suspension through a 40 µm cell sieve into a new conical centrifuge tube.
   8. Collect 10 µL of cell suspension, dilute 1:100 in warm DMEM/F12, and count cells with an automated cell counter
   9. Incubate in a 37 °C water bath until encapsulation step.
3. Cell Encapsulation
   NOTE: All steps in the protocol are performed with sterile technique in a biosafety cabinet.
   1. 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.
   2. 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 µL PBS with 7 mg HAMA.
   3. Sonicate (>20 kHz) solutions until HAMA is fully dissolved for 60 min.
   4. 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.
   5. For one 12 well plate, make a final 1.4 mL mixture of 1 x 10⁷ cells, 0.5% wt/vol HAMA (350 µL of 2% wt/vol), 0.1% TEA (14 µL of 10%), 0.1% NVP (14 µL of 10%), and 0.01 mM EY (14 µL of 1 mM), and 20% basal lamina mixture (280 µL of stock). The remaining volume is PBS.
   6. Gently mix the solution and pipette 100 µL into each PDMS mold with a 1 mL tip.
   7. 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.
   8. Add 1 mL per well of warm conditioned DMEM/F12 to a 12 well plate.
   9. 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.
   10. Add an additional 1 mL warm DMEM/F12 to each well.
   11. Incubate plates at 37 °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.

4. Microscopy

1. 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.
   1. Preheat 10% PBS-buffered formalin (pH 7.0) in a 37 °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.
   2. Carefully aspirate media from the plates and pipette 1 mL of 10% formalin to each well.
   3. Incubate the plates at 37 °C in a 5% CO₂ for 20 min.
   4. Aspirate liquid from each plate, pipette 2 mL of PBS (pH 7.4) into each well, and incubate for 15 min.
   5. Repeat step 4.1.4 twice.
   6. 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.
   7. Repeat step 4.1.4 three times.
   8. 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',3'-cyclic-nucelotide 3'-phophodiesterase (CNPase) 1:1,000, 1% NHS, and 0.1% cell perforating surfactant. Incubate at 4 °C overnight, rocking the plate as gently as possible.
   9. Repeat step 4.1.4 three times with 1 h incubations in between.
   10. 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.
   11. Repeat step 4 three times with 1 h incubations in between.
   12. 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.
2. Scanning Electron Microscopy (SEM)
   NOTE: To visualize the hydrogel, a scanning electron microscope was used to image these samples.
   1. Pre-heat a fixative mixture of 2.5% glutaraldehyde and 2% paraformaldehyde in PBS (0.1 M) in a 37 °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.
   2. Aspirate medium from the cell culture plates and add 1 mL of the pre-heated fixative mixture.
   3. Incubate the plates at 37 °C in a 5% CO₂ for 20 min.
   4. Place the plates in 4 °C (fridge/cooler) overnight.
   5. Aspirate the fixative solution from the wells and add 1 mL of PBS.
   6. Aspirate liquid from each plate, add 2 mL of PBS into each well, and incubate for 15 min.
   7. Repeat step 4.2.6 two more times.
   8. 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.
   9. 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.
   10. Repeat step 4.2.6 three times.
   11. 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.
   12. Carefully remove the dried samples and mount the samples on scanning electron microscope stubs with double sided conductive tape.
   13. 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.

Results

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...

Discussion

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...

Materials

List of materials used in this article

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