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

Abstract

Source: Koss, K. M, et al. Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation. 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.

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

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 l....

Results

Figure 1: Dissection of day 1 Sprague Dawley rat pup brain. Images of whole brain (A), dissection of cortices and cerebellum (B), a single cortex with meninges (C), peeling of the meninges with forceps (

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
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