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

Summary

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.

Abstract

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.

Introduction

Improving our understanding of the central nervous system (CNS) is critical to improve therapeutic options for many neuroinflammatory and neurodegenerative diseases. The CNS, a complex network of interconnected cells within the brain, spinal cord, and optic nerves, comprises neurons, oligodendrocytes, astrocytes, and their innate immune cells, the microglia¹. An in vitro approach can often drastically reduce the number of mice required to perform meaningful research; however, the complex nature of the CNS makes it impossible to recapitulate the in vivo situation using cell lines. Mixed neural cell cultures provide an extremely....

Protocol

All animal experiments complied with local laws and guidelines for animal use, and were approved by the local Ethical Review Committee at the University of Glasgow. Animals were housed in specific pathogen-free conditions in accordance with the UK Animals Scientific Procedures Act 1986, under the auspices of a UK Home Office Project License. For this study, in-house bred adult C57BL/6J mice were used. The use of young females (8-12 weeks) is recommended due to the higher success rate of pregnancy; males can be reused for.......

Discussion

The CNS is a complex network that spans from the brain down to the spinal cord and consists of many cell types, predominantly neurons, oligodendrocytes, astrocytes, and microglia¹. As each cell has an important role in maintaining homeostasis and generating appropriate responses to challenges in the CNS^9,10,11, a culture system that contains all these cell types is a useful and versatile tool to investiga.......

Materials

Name	Company	Catalog Number	Comments
10x Trypsin	Sigma	T4549-100ML	To digest tissue
140 mm TC Dish	Fisher	11339283	Put 8 35 mm dishes per 1 140 mm dish
15 mL Falcon	Sarstedt	62554502	To collect cells into pellet for resuspension in plating media
18 G needle	Henke Sass Wolf	4710012040	For trituration of sample
21 G needle	BD	304432	For trituration of sample
23 G needle	Henke Sass Wolf	4710006030	For trituration of sample
35 mm TC Dish	Corning	430165	Plate out 3 PLL coated coverslips per 1 35 mm dish
5 mL syringe	Fisher	15869152	For trituration of sample
6 well plate	Corning	3516	To plate out cells for RT-qPCR, and flow cytometry
7 mL Bijoux	Fisher	DIS080010R	To put brains intp
96 well plate	Corning	3596	To plate out cells for high-throughput testing
ACSA-2 Antibody, anti-mouse, PE	Miltenyi	130-123-284	For flow cytometry staining of astrocytes
Angled forceps	Dumont	0108-5/45-PO	For dissection
Biotin	Sigma	B4501	For DM+/-
Boric Acid	Sigma	B6768-500G	For boric acid buffer
Brilliant Violet 421 anti-mouse CD24 Antibody, clone M1-69	Biolegend	101825	For flow cytometry staining of neurons and astrocytes
Brilliant Violet 605 anti-mouse CD45 Antibody, clone 30-F11	Biolegend	103139	For flow cytometry staining of microglia
Brilliant Violet 785 anti-mouse/human CD11b Antibody, clone M1/70	Biolegend	101243	For flow cytometry staining of microglia
BSA Fraction V	Sigma	A3059-10G	For SD Inhibitor
CNP	Abcam	AB6319	Mature oligodendrocytes
Coverslip	VWR	631-0149	To plate out cells for microscopy
Dissection Scissors	Sigma	S3146-1EA	For dissection
DMEM High glucose, sodium pyruvate, L-Glutamine	Gibco	21969-035	For DM+/-, and for plating media
DNase I	Thermofisher	18047019	For SD Inhibitor, can use this or the other Dnase from sigma
DNase I	Sigma	D4263	For SD Inhibitor, can use this or the other Dnase from thermofisher
eBioscience Fixable Viability Dye eFluor 780	Thermofisher	65-0865-14	Live / Dead stain
Fine forceps	Dumont	0102-SS135-PO	For dissection
GFAP	Invitrogen	13-0300	Astrocytes
HBSS w Ca Mg	Sigma	H9269-500ML	For plating media
HBSS w/o Ca Mg	Sigma	H9394-500ML	For brains to be added to
Horse Serum	Gibco	26050-070	For plating media
Hydrocortisone	Sigma	H0396	For DM+/-
Iba1	Alpha-Laboratories	019-1971	Microglia
Insulin	Sigma	I1882	For DM+
Leibovitz L-15	GIbco	11415-049	For SD Inhibitor
MBP	Bio-Rad	MCA409S	Myelin
Mouse CCL5/RANTES DuoSet ELISA Kit	BioTechne	DY478-05	ELISA kit for quantifying concentration of CCL5 in supernatants of 96 well plate
N1 media supplement	Sigma	N6530-5ML	For DM+/-
Nestin	Merck	MAB353	Neuronal stem/progenitor cells
NeuN	Thermofisher	PA578499	Neuronal cell body
NG2	Sigma	AB5320	Immature oligodendrocytes
O4 Antibody, anti-human/mouse/rat, APC	Miltenyi	130-119-155	For flow cytometry staining of oligodendrocytes
Pen/Strep	Sigma	P0781-100ML	For DM+/-, and for plating media
Poly-L-Lysinehydrobromide	Sigma	P1274	For Boric acid / poly-L-lysine solution to coat coverslips
SMI31	BioLegend	801601	Axons
Sodium Tetraborate	Sigma	221732-100G	For boric acid buffer
Trizol	Thermofisher	15596026	For lysing cells for RT-qPCR
Trypsin inhibitor from soybean	Sigma	T9003-100MG	For SD Inhibitor