Name: Author Spotlight: Establishing Mixed Neuronal and Glial Cell Cultures from Embryonic Mouse Brains to Study Infection and Innate Immunity
Uploaded: 2023-06-30T13:55:00
Description: Watch this Scientific Journal Video about Establishing Mixed Neuronal and Glial Cell Cultures from Embryonic Mouse Brains to Study Infection and Innate Immunity at JoVE.com

Vi vil gjerne takke medlemmer av Edgar og Linington laboratorier, spesielt Prof. Chris Linington, Dr Diana Arseni, og Dr Katja Muecklisch, for deres råd, nyttige kommentarer, og hjelp med å mate kulturer mens vi setter opp disse kulturer. Spesiell takk går til Dr Muecklisch for å gi utgangspunkt for Cell Profiler rørledninger. Dette arbeidet ble støttet av MS Society (stipend 122) og Yuri og Lorna Chernajovsky stiftelsen til MP; University of Glasgow finansiering til JC og MP; og Wellcome Trust (217093/Z/19/Z) og Medical Research Council (MRV0109721) til GJG.

Alle dyreforsøk fulgte lokale lover og retningslinjer for dyrebruk, og ble godkjent av den lokale etiske vurderingskomiteen ved University of Glasgow. Dyrene ble plassert under spesifikke patogenfrie forhold i samsvar med UK Animals Scientific Procedures Act 1986, i regi av en britisk Home Office Project License. For denne studien ble internavlet voksne C57BL/6J-mus brukt. Bruk av unge kvinner (8-12 uker) anbefales på grunn av graviditetens høyere suksessrate; Hanner kan gjenbrukes til flere runder med avl. <strong cl...

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

<ol>
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G., 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=Anti-ACSA-2+defines+a+novel+monoclonal+antibody+for+prospective+isolation+of+living+neonatal+and+adult+astrocytes.">Anti-ACSA-2 defines a novel monoclonal antibody for prospective isolation of living neonatal and adult astrocytes.</a> Glia. 65 (6), 990-1004 (2017).</li><li>Yin, J., Valin, K. L., Dixon, M. L., Leavenworth, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+microglia+and+macrophages+in+CNS+homeostasis,+autoimmunity,+and+cancer.">The role of microglia and macrophages in CNS homeostasis, autoimmunity, and cancer.</a> Journal of Immunology Research. 2017, (2017).</li><li>Gee, J. R., Keller, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocytes:+Regulation+of+brain+homeostasis+via+apolipoprotein+E.">Astrocytes: Regulation of brain homeostasis via apolipoprotein E.</a> The International Journal of Biochemistry and Cell Biology. 37 (6), 1145-1150 (2005).</li><li>Madeira, M. M., Hage, Z., Tsirka, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beyond+myelination:+possible+roles+of+the+immune+proteasome+in+oligodendroglial+homeostasis+and+dysfunction.">Beyond myelination: possible roles of the immune proteasome in oligodendroglial homeostasis and dysfunction.</a> Frontiers in Neuroscience. 16, 867357 (2022).</li><li>Keller, D., Er&#246;, C., Markram, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+densities+in+the+mouse+brain:+A+systematic+review.">Cell densities in the mouse brain: A systematic review.</a> Frontiers in Neuroanatomy. 12, 83 (2018).</li><li>Wang, Y. 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. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+brain+organoids+derived+from+pluripotent+stem+cells:+Promising+experimental+models+for+brain+development+and+neurodegenerative+disorders.">3D brain organoids derived from pluripotent stem cells: Promising experimental models for brain development and neurodegenerative disorders.</a> Journal of Biomedical Science. 24 (1), 59 (2017).</li><li>Asch, B. B., Medina, D., Brinkley, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtubules+and+actin-containing+filaments+of+normal,+preneoplastic,+and+neoplastic+mouse+mammary+epithelial+cells.">Microtubules and actin-containing filaments of normal, preneoplastic, and neoplastic mouse mammary epithelial cells.</a> Cancer Research. 39 (3), 893-907 (1979).</li><li>Koch, K. S., Leffertt, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+control+of+differentiated+adult+rat+hepatocytes+in+primary+culture.">Growth control of differentiated adult rat hepatocytes in primary culture.</a> Annals of the New York Academy of Sciences. 349, 111-127 (1980).</li><li>Nevalainen, T., Autio, A., Hurme, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Composition+of+the+infiltrating+immune+cells+in+the+brain+of+healthy+individuals:+effect+of+aging.">Composition of the infiltrating immune cells in the brain of healthy individuals: effect of aging.</a> Immunity and Ageing. 19 (1), 45 (2022).</li><li>Daneman, R., Prat, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+blood-brain+barrier.">The blood-brain barrier.</a> Cold Spring Harbor Perspectives in Biology. 7 (1), 020412 (2015).</li></ol>

CNS er et komplekst nettverk som spenner fra hjernen ned til ryggmargen og består av mange celletyper, hovedsakelig nevroner, oligodendrocytter, astrocytter og mikroglia1. Siden hver celle har en viktig rolle i å opprettholde homeostase og generere passende svar på utfordringer i CNS 9,10,11, er et kultursystem som inneholder alle disse celletypene et nyttig og allsidig verktøy for å undersøke hvordan hjernen kan reagere ...

Å forbedre vår forståelse av sentralnervesystemet (CNS) er avgjørende for å forbedre terapeutiske muligheter for mange nevroinflammatoriske og nevrodegenerative sykdommer. CNS, et komplekst nettverk av sammenkoblede celler i hjernen, ryggmargen og optiske nerver, består av nevroner, oligodendrocytter, astrocytter og deres medfødte immunceller, mikroglia1. En in vitro-tilnærming kan ofte drastisk redusere antall mus som kreves for å utføre meningsfull forskning; Den komplekse naturen til CNS gjør det imidlertid umulig å rekapitulere in vivo-situasjonen ved hjelp av cellelinjer. Blandede nevrale cellekulturer gir et ekstremt verdifullt forskningsverktøy for å undersøke nevrologiske spørsmål i en relevant modell, i trådmed prinsippene Replacement, Reduction and Refinement (3Rs) 2,3.
Thomson et al. beskrev en cellekulturmetode ved bruk av prenatale ryggmargsceller som skiller seg ut i alle de nevnte CNS-celletypene4. Dette systemet har også synapsedannelse, myeliniserte aksoner og noder av Ranvier. Hovedbegrensningen til denne dyrkingsmetoden er at den, som ryggmarg, ikke er nyttig modell av hjernen, og celleutbyttet fra embryonale dag 13 (E13) ryggmargene er sammentrekkende. Dermed begrenser dette antall eksperimentelle forhold som kan undersøkes. Derfor hadde denne studien som mål å utvikle et nytt cellekultursystem som rekapitulerer hjernens egenskaper med økt celleutbytte for å redusere kravene til dyr.
Ved å bruke Thomson et al. som utgangspunkt, utviklet vi en cellekulturmodell avledet rent fra prenatale musehjerner. Disse kulturene har samme cellepopulasjoner, sammenkobling og behandlingsmuligheter som ryggmargskulturer, bortsett fra at det er mindre myelinisering til sammenligning. Imidlertid er det mer effektivt å ha en CNS in vitro-modell med omtrent tre ganger høyere celleutbytte, noe som krever færre mus og mindre tid på å behandle embryoer. Vi optimaliserte dette unike kultursystemet for flere nedstrøms applikasjoner og vekter, inkludert bruk av glassdeksel for mikroskopianalyse og forskjellige størrelser av plastbrønnplater, inkludert 96-brønnplater for forskning med høy gjennomstrømning.

<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

Modeller av sentralnervesystemet (CNS) må rekapitulere det komplekse nettverket av sammenkoblede celler som finnes in vivo. CNS består hovedsakelig av nevroner, astrocytter, oligodendrocytter og mikroglia. På grunn av økende innsats for å erstatte og redusere bruk av dyr, har en rekke in vitro cellekultursystemer blitt utviklet for å utforske medfødte celleegenskaper, som tillater utvikling av terapeutiske midler for CNS-infeksjoner og patologier. Mens visse forskningsspørsmål kan løses av menneskebaserte cellekultursystemer, for eksempel (induserte) pluripotente stamceller, har arbeid med humane celler sine egne begrensninger med hensyn til tilgjengelighet, kostnader og etikk. Her beskriver vi en unik protokoll for isolering og dyrking av celler fra embryonale musehjerner. De resulterende blandede nevrale cellekulturer etterligner flere cellepopulasjoner og interaksjoner funnet i hjernen in vivo. Sammenlignet med dagens ekvivalente metoder, etterligner denne protokollen nærmere hjernens egenskaper og får også flere celler, slik at flere eksperimentelle forhold kan undersøkes fra en gravid mus. Videre er protokollen relativt enkel og svært reproduserbar. Disse kulturene er optimalisert for bruk på forskjellige skalaer, inkludert 96-brønnbaserte skjermer med høy gjennomstrømning, 24-brønnsmikroskopianalyse og 6-brønnskulturer for flowcytometri og revers transkripsjon-kvantitativ polymerasekjedereaksjon (RT-qPCR) analyse. Denne kulturmetoden er et kraftig verktøy for å undersøke infeksjon og immunitet i sammenheng med noe av kompleksiteten til CNS med bekvemmeligheten av in vitro-metoder .

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.

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

Etablering av blandede nevron- og gliacellekulturer fra embryonale musehjerner for å studere infeksjon og medfødt immunitet

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.

Mikroskopi Kulturer dyrket på glassdeksel er ideelle for å analysere ved mikroskopi. For å visualisere utviklingen av kulturene ble dekkene festet i 4% paraformaldehyd (PFA) på flere tidspunkter fra DIV0 (når celler var festet) til DIV28. Dyrkningene ble farget for immunfluorescensavbildning som tidligere beskrevet5 ved hjelp av tre ulike fargekombinasjoner: NG2 (umodne oligodendrocytter) og nestin (nevrale stam-/stamceller) som utviklingsmarkører, SMI31 (aksoner), MBP (m...

Watch this Scientific Journal Video about Establishing Mixed Neuronal and Glial Cell Cultures from Embryonic Mouse Brains to Study Infection and Innate Immunity at JoVE.com

Denne protokollen presenterer en unik måte å generere cellekulturer i sentralnervesystemet fra dag 17 musehjerner for nevro (immun) logi forskning. Denne modellen kan analyseres ved hjelp av ulike eksperimentelle teknikker, inkludert RT-qPCR, mikroskopi, ELISA og flowcytometri.

Models of the central nervous system (CNS) must recapitulate the complex network of interconnected cells found in vivo. The CNS consists primarily of ...

Det ultimate forskningsspørsmålet i prosjektet mitt er å finne ut hvordan vi kan manipulere de medfødte immunresponsene i sentralnervesystemet for å beskytte mot virusinfeksjoner. For tiden undersøker vi deres respons på det dypt antivirale proteininterferon beta. En eksperimentell utfordring er å generere nok celler til å tillate detaljert undersøkelse.Hvert resultat må valideres ved hjelp av flere teknikker, og dette kan dessverre føre til at mange dyr blir avlivet. På grunn av det store antallet celler som genereres av denne nye kulturteknikken, kan mange forskjellige eksperimentelle forhold undersøkes fra en gravid mus. I tillegg reduserer bruk av in vitro-alternativer, der det er mulig, også dyrs lidelse.Denne metoden reduserer og foredler dyrebruk, og følger nøye prinsippene i de tre Rs.Denne kulturmetoden kan brukes til å løse mange forskjellige forskningsspørsmål ved hjelp av en rekke avlesningsmetoder som ELISA, qPCR, mikroskopi og flowcytometri. Denne teknikken kan hjelpe andre forskere til å redusere og forfine bruken av dyr. Vi håper å forbedre vår forståelse av den medfødte antivirale responsen, modulere den for å beskytte mot opportunistiske virus som humant polyomavirus 2, eller John Cunningham-virus, som det er kjent, det primære middelet til PML som kan være en alvorlig bivirkning av immundempende legemidler som brukes til å behandle MS og andre sykdommer.