We would like to thank members of the Edgar and Linington labs, particularly Prof. Chris Linington, Dr&#160;Diana Arseni, and Dr&#160;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.

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...

Embryonic Mouse Brain Mixed Cell Cultures to Study Infection and Innate Immunity

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X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxygenglucose+deprivation+enhancement+of+cell+death/apoptosis+in+PC12+cells+and+hippocampal+neurons+correlates+with+changes+in+neuronal+excitatory+amino+acid+neurotransmitter+signaling+and+potassium+currents.">Oxygenglucose deprivation enhancement of cell death/apoptosis in PC12 cells and hippocampal neurons correlates with changes in neuronal excitatory amino acid neurotransmitter signaling and potassium currents.</a> Neuroreport. 27 (8), 617-626 (2016).</li><li>Rock, K. L., Kono, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+inflammatory+response+to+cell+death.">The inflammatory response to cell death.</a> Annual Review of Pathology: Mechanisms of Disease. 3, 99-126 (2008).</li><li>Chen, V. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histology+atlas+of+the+developing+prenatal+and+postnatal+mouse+central+nervous+system,+with+emphasis+on+prenatal+days+E7.5+to+E18.5.">Histology atlas of the developing prenatal and postnatal mouse central nervous system, with emphasis on prenatal days E7.5 to E18.5.</a> Toxicologic Pathology. 45 (6), 705-744 (2017).</li><li>Gordon, J., Amini, S., White, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=General+overview+of+neuronal+cell+culture.">General overview of neuronal cell culture.</a> Methods in Molecular Biology. 1078, 1-8 (2013).</li><li>Kesselheim, A. S., Hwang, T. J., Franklin, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+decades+of+new+drug+development+for+central+nervous+system+disorders.">Two decades of new drug development for central nervous system disorders.</a> Nature Reviews Drug Discovery. 14 (12), 815-816 (2015).</li><li>Gribkoff, V. K., Kaczmarek, L. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+need+for+new+approaches+in+CNS+drug+discovery:+Why+drugs+have+failed,+and+what+can+be+done+to+improve+outcomes.">The need for new approaches in CNS drug discovery: Why drugs have failed, and what can be done to improve outcomes.</a> Neuropharmacology. 120, 11-19 (2017).</li><li>Zamanian, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+analysis+of+reactive+astrogliosis.">Genomic analysis of reactive astrogliosis.</a> Journal of Neuroscience. 32 (18), 6391-6410 (2012).</li><li>Nishiyama, A., Suzuki, R., Zhu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NG2+cells+(polydendrocytes)+in+brain+physiology+and+repair.">NG2 cells (polydendrocytes) in brain physiology and repair.</a> Frontiers in Neuroscience. 8, 133 (2014).</li><li>Lee, C. T., Bendriem, R. M., Wu, W. W., Shen, R. 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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...

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 valuable research tool to investigate neuro(immuno)logy questions in a relevant model, in line with the Replacement, Reduction and Refinement (3Rs) principles2,3.
Thomson et al. described a cell culture method using prenatal spinal cord cells that differentiate into all the aforementioned main CNS cell types4. This system also has synapse formation, myelinated axons, and nodes of Ranvier. The main limitation of this culturing method is that, being spinal cord, it does not usefully model the brain, and the cell yields from embryonic day 13 (E13) spinal cords are constricting. Thus, this limits the number of experimental conditions that can be investigated. Therefore, this study aimed to develop a new cell culture system that recapitulates the characteristics of the brain with increased cell yield to reduce the requirements for animals.
Using Thomson et al. as a starting point, we developed a cell culture model derived purely from prenatal mouse brains. These cultures have the same cell populations, interconnectivity, and treatment options as the spinal cord cultures, except there is less myelination by comparison. However, having a CNS in vitro model with an approximately threefold higher cell yield is more efficient, requiring fewer mice and less time processing embryos. We optimized this unique culture system for multiple downstream applications and scales, including using glass coverslips for microscopy analysis and various sizes of plastic well plates, including 96-well plates for high throughput research.

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

establishing mixed neuronal and glial cell cultures from embryonic mouse brains to study infection and innate immunity

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.

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

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

The ultimate research question of my project is to figure out how we can manipulate the innate immune responses of the central nervous system to protect against viral infections. Currently, we research their response to the profoundly antiviral protein interferon beta. An experimental challenge is generating enough cells to allow for detailed investigation.Each result needs to be validated using multiple techniques and this can, unfortunately, result in many animals being culled. Due to the large number of cells generated by this novel culture technique, many different experimental conditions can be investigated from one pregnant mouse. Additionally, using in vitro alternatives, where possible, also greatly decreases animal suffering.This method reduces and refines animal use, closely following the principles of the three Rs.This culture method can be used to address many different research questions using a variety of readout methods such as ELISA, qPCR, microscopy and flow cytometry. This technique could help other researchers to reduce and refine their animal usage. We hope to improve our understanding of the innate antiviral response, modulating it to protect against opportunistic viruses such as human polyomavirus 2, or John Cunningham virus, as it is commonly known, the primary agent of PML which can be a severe side effect of immune suppressive drugs used to treat MS and other diseases.

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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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 ...

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