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 therapeuti....

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-s....

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