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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Neuroscience

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

Published: June 30th, 2023

DOI:

10.3791/65331

1School of Infection and Immunity, University of Glasgow, 2Faculty of Science and Technology, Institute of Technology, University of Tartu

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.

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.

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....

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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.......

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Microscopy
Cultures grown on glass coverslips are ideal to analyze by microscopy. To visualize the development of the cultures, the coverslips were fixed in 4% paraformaldehyde (PFA) at multiple timepoints from DIV0 (once cells were attached) until DIV28. The cultures were stained for immunofluorescence imaging as previously described5 using three different staining combinations: NG2 (immature oligodendrocytes) and nestin (neuronal stem/progenitor cells) as developmental mar.......

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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 CNS9,10,11, a culture system that contains all these cell types is a useful and versatile tool to investiga.......

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We would like to thank members of the Edgar and Linington labs, particularly Prof. Chris Linington, Dr Diana Arseni, and Dr Katja Muecklisch, for their advice, helpful comments, and assistance with feeding the cultures while we set up these cultures. Particular thanks go to Dr Muecklisch for providing the starting points for the Cell Profiler pipelines. This work was supported by the MS Society (grant 122) and the Yuri and Lorna Chernajovsky foundation to MP; University of Glasgow funding to JC and MP; and Wellcome Trust (217093/Z/19/Z) and Medical Research Council (MRV0109721) to GJG.

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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

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