Name: establishing mixed neuronal and glial cell cultures from embryonic mouse brains to study infection and innate immunity
Uploaded: 2023-06-30T13:55:00.000+00:00
Duration: 7 min 41 s
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

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 multiple rounds of breeding. Figure 1 represents a schematic overview of the described method to generate mixed neuronal and glial cultures.
1. Preparation of the tissue culture consumables
<ol>
	<li>Prepare the plates and/or dishes containing microscopy coverslips inside a Class 2 Safety cabinet. Sterilize all reagents or autoclave to ensure sterility during the culture period.</li>
	<li>Add the appropriate volume of BA-PLL (13.2 &#181;g/mL poly-L-lysinehydrobromide [PLL] in boric acid buffer [BA] [50 mM boric acid, 12.5 mM sodium tetraborate, pH 8.5]; see Table of Materials) to each well (1,000 &#181;L/well in a 6-well plate, 100 &#181;L/well in a 96-well plate; volumes are summarized in Table 1). For microscopy coverslips, add 20 mL of BA-PLL to a 9 cm diameter tissue culture dish containing 200 sterile coverslips, and swirl to distribute evenly.</li>
	<li>Incubate at 37 &#176;C for 1-2 h.</li>
	<li>Remove the BA-PLL solution from each well or dish containing microscopy coverslips, and wash by adding 20 mL of sterile water, swirling the coverslips, and then removing the water. Repeat this wash step three times. For a dish containing coverslips, leave sterile water in the tissue culture dish on the final wash to easily remove the coverslips.</li>
	<li>Remove as much liquid as possible with a sterile pipette and allow to dry for at least 2 h to&#160;overnight.</li>
	<li>Store the coated plates at 4 &#176;C for up to 2 months. 
	NOTE: The prepared boric acid with poly-l-lysine (BA-PLL) solution can be reused up to three time, adding new PLL each time. Store the BA-PLL at 4 &#176;C. Dishes are treated with BA-PLL, as the PLL allows the cells to stick down and grow. Without this treatment, the cells will lift after approximately 1 week of culture and no longer be able to differentiate.</li>
</ol>
2. Dissection of E17 embryonal brains
<ol>
	<li>Co-house one or multiple female mice with a male mouse. Check the females daily for a mucus plug, indicating mating has taken place. 
	NOTE: Any &#34;plugged&#34; female mice need to be separated from the male to ensure the correct start date of gestation. Mice can be weighed to confirm pregnancy or monitored visually.</li>
	<li>Cull the pregnant mouse at E17 using appropriate methods in compliance with local animal welfare guidance and laws, for example, by raising carbon dioxide (CO2) concentration, a lethal anesthetic overdose, or dislocation of the neck. 
	NOTE: The chosen method must not disrupt the embryos. For this study, exposure to a rising concentration of carbon dioxide gas followed by confirmation of death by severing the femoral artery was used to cull pregnant dams.</li>
	<li>Place the culled, pregnant mouse on its back on a dissection board; while pinning it down is not required, it might make it easier for inexperienced researchers. Pinch the midline of the abdomen using forceps. Using sharp scissors, cut open the abdomen through the skin and the peritoneum over the midline from the genitalia to the ribcage, being careful not to puncture the uterus.
	<ol>
		<li>The mouse uterus has two horns, each typically containing one to five embryos. Remove the uterus containing the embryos from the mother and immediately place it on ice.</li>
	</ol>
	</li>
	<li>Cut through the yolk sack on the side of the placentas, being careful not to damage the embryos, and remove the embryos from their yolk sack.</li>
	<li>Immediately decapitate the embryos. Add the decapitated heads into a dish with Hanks&#39; balanced salt solution (HBSS) without calcium (Ca2+) and magnesium (Mg2+) (HBSS-/-) on ice. 
	NOTE: If genotyping is required, one can remove the tail at this stage for genetic analysis. When multiple genotypes are expected, the heads of each embryo should be kept separately for culturing.</li>
	<li>Using angled forceps, position the head on its side facing left.</li>
	<li>Pierce the eye with one edge of the forceps, firmly holding the chin with the other.</li>
	<li>Starting at the nape, gently tear the skin of the scalp along the midline toward the tip of the snout.</li>
	<li>Entering through the spinal cord, noticeable as a white oval, use the angled forceps to crack open the skull along the midline, exposing the brain.</li>
	<li>Gently peel the skull away on the side facing upward, exposing the brain.</li>
	<li>Lift the brain out of the skull, disposing of the skull once the brain has been completely removed.</li>
	<li>Using the forceps, remove the meninges, which are noticeable as a thin membrane with dense blood vessels.</li>
	<li>Place the brains into a bijou (see Table of Materials) containing 2 mL of HBSS-/- on ice.</li>
	<li>Repeat steps 2.6 to 2.13 with the remaining brains, adding up to four brains per bijou.</li>
	<li>Add 250 &#181;L of 10x trypsin to the bijou and triturate the brains by shaking the bijou. Incubate for 15 min at 37 &#176;C. 
	NOTE: All steps from this point forward should be performed in a sterile tissue culture hood.</li>
	<li>Thaw 2 mL of soybean trypsin (SD) inhibitor (Leibovitz L-15, 0.52 mg/mL trypsin inhibitor from soybean, 40 &#181;g/mL DNase I, 3 mg/mL bovine serum albumin [BSA] fraction V; see Table of Materials) from -20 &#176;C by placing it at 37 &#176;C.</li>
	<li>Add 2 mL of SD inhibitor to each bijou containing brains (per bijou containing up to four brains), shaking the bijou again to disperse it evenly. 
	NOTE: SD inhibitor decreases the trypsin activity to prevent unnecessary digestion of the samples and preserve cell viability.</li>
	<li>Without centrifugation, remove 2 mL of the supernatant from each bijou and transfer into a 15 mL centrifuge tube, being careful not to transfer cell clumps.</li>
	<li>Triturate the remaining cells in the bijou with a 19 G needle attached to a 5 mL syringe by aspirating the suspension twice. This will create a thick mucus-like mixture.
	<ol>
		<li>Repeat twice more using a 21 G needle. If there are clumps remaining, triturate once more with the 21 G needle.</li>
	</ol>
	</li>
	<li>Transfer the cells from the bijou to the same 15 mL centrifuge tube (from step 2.18) using a 23 G needle.</li>
	<li>Centrifuge at 200 x g at room temperature (RT) for 5 min.</li>
	<li>Remove all the supernatant using a 5 mL serological pipette and transfer it to another 15 mL centrifuge tube, being careful not to disturb the loose pellet at the bottom containing the required cells.</li>
	<li>Centrifuge the supernatant again at 200 x g at RT for 5 min. 
	NOTE: This step is not essential, but if one requires many cells or has few embryos, one could perform this step to recover as many cells as possible from the supernatant.</li>
	<li>Using 10 mL of plating media (PM) (49% Dulbecco&#39;s modified eagle medium [DMEM], 1% penicillin/streptomycin [Pen/Strep], 25% horse serum, 25% HBSS with Ca2+ and Mg2+ [HBSS+/+]; see Table of Materials), combine and resuspend the two pellets together to create a whole cell suspension.</li>
	<li>Count the cells using trypan blue and either a hemocytometer or digital cell counter, and dilute the cell suspension with PM to a concentration of 1.8 x 106 cells/mL.</li>
</ol>
3. Plating the cells
<ol>
	<li>Add the required volume of the cell suspension to the required format as detailed in Table 1: 1,000 &#181;L per well in 6-well format, 50 &#181;L per well in 96-well format, or 100 &#181;L per coverslip.</li>
	<li>Incubate for 2-4 h at 37 &#176;C with 5%-7% CO2. Check the cells have adhered using an inverted microscope.</li>
	<li>Remove the media and top up with new differentiation media (DM+: DM- including 10 &#181;g/mL insulin. DM-: DMEM, 1% Pen/Strep, 50 nM hydrocortisone, 10 ng/mL biotin, 2.5 mL 100x N1 media supplement; see Table of Materials). Volumes are detailed in Table 1. Press down any floating coverslips using a sterile pipette tip.</li>
</ol>
4. Maintaining the cultures
NOTE: These cultures require feeding thrice weekly to support optimal growth and differentiation. The cultures will reach optimum health and maturity for experiments on DIV (days in vitro) 21. Cells can be kept in culture for up to 28 days, after which the cultures quickly degenerate.
<ol>
	<li>Three times per week until DIV12, replace part of the supernatant with fresh DM+ by removing 500 &#181;L per well in 6-well format, 50 &#181;L per well in 96-well format, or 500 &#181;L per dish containing three coverslips, and adding 600 &#181;L per well in 6-well format, 60 &#181;L per well in 96-well format, or 600 &#181;L per coverslip dish (Table 1).</li>
	<li>Three times per week from DIV13 onwards, replace part of the supernatant with fresh DM- by removing 500 &#181;L per well in 6-well format, 50 &#181;L per well in 96-well format, or 500 &#181;L per dish containing three coverslips, and adding 600 &#181;L per well in 6-well format, 60 &#181;L per well in 96-well format, or 600 &#181;L per coverslip dish (Table 1).</li>
</ol>

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

<ol>
	<li>Kovacs, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+reactions+of+the+central+nervous+system.">Cellular reactions of the central nervous system.</a> Handbook of Clinical Neurology. 145, 13-23 (2018).</li><li>Russell, W. M. S., Burch, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Principles of Humane Experimental Techniques. 1, (1959).</li><li>Hubrecht, R. C., Carter, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+3Rs+and+humane+experimental+technique:+Implementing+change.">The 3Rs and humane experimental technique: Implementing change.</a> Animals. 9 (10), 754 (2019).</li><li>Thomson, C. E., 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=synapsing+cultures+of+murine+spinal+cord-validation+as+an+in+vitro+model+of+the+central+nervous+system.">synapsing cultures of murine spinal cord-validation as an in vitro model of the central nervous system.</a> The European Journal of Neuroscience. 28 (8), 1518-1535 (2008).</li><li>Bijland, 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=An+in+vitro+model+for+studying+CNS+white+matter:+Functional+properties+and+experimental+approaches.">An in vitro model for studying CNS white matter: Functional properties and experimental approaches.</a> F1000Research. 8, 117 (2019).</li><li>Fragkoudis, R., 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=Neurons+and+oligodendrocytes+in+the+mouse+brain+differ+in+their+ability+to+replicate+Semliki+Forest+virus.">Neurons and oligodendrocytes in the mouse brain differ in their ability to replicate Semliki Forest virus.</a> Journal of Neurovirology. 15 (1), 57-70 (2009).</li><li>Hayden, 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=Lipid-specific+IgMs+induce+antiviral+responses+in+the+CNS:+Implications+for+progressive+multifocal+leukoencephalopathy+in+multiple+sclerosis.">Lipid-specific IgMs induce antiviral responses in the CNS: Implications for progressive multifocal leukoencephalopathy in multiple sclerosis.</a> Acta Neuropathologica Communications. 8 (1), 135 (2020).</li><li>Kantzer, C. 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>

The authors have nothing to disclose.

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 investigate how the brain might react to a stimulus. The ability to study these cells, and their interactions, in an in vitro context means a great variety of techniques can be employed to investigate various experimental questions easily. We have outlined a quick and efficient method to obtain and culture the main CNS cell types from the prenatal brain, which can mimic individual cell populations in the adult mouse brain12.
Previously established protocols for generating CNS cultures use E13.5 spinal cords4,5. In addition to having a suitable model for the spinal cord, it is highly relevant to have a model that recapitulates the brain. Therefore, brains from these E13.5 mice were originally used to generate the CNS cultures described here using the same protocol. However, after DIV14, the cells started to form large aggregates with densely packed neuronal cell bodies. While astrocytes were present, it is not clear if they were evenly distributed. It is unclear if the cells in the center of these cell bodies would receive sufficient nutrients and oxygen from the culture medium as the cells outside would. When experiencing oxygen and nutrient deprivation, neurons are liable to go through apoptosis and release stress signals to glial cells13, which might lead to a proinflammatory phenotype14. Neurogenesis was thought to be a likely cause of these densely packed aggregates, and this phase subsides by E1715. When brains from E17 mouse embryos were used, the cells still formed networks but did not cluster into the large aggregates seen in E13.5, and were therefore regarded as a more suitable age for the brain cultures to be generated from.
The current technique also results in a greater number of cells obtained from one pregnancy (averaging 1 x 107 cells/brain compared to 3-4 x 106 cells/spinal cord)7. For an average pregnant mouse with 8-10 embryos, this correlates to up to 54 different experimental conditions in the 6-well plate format, or 150 experimental conditions for microscopy (compared to 19 and 64, respectively, from the spinal cord). As such, these CNS cultures are a great tool to reduce the required number of mice16. In particular, the 96-well format can be easily used for high throughput assays, such as enabling the screening of drug candidates prior to testing in animals, vastly reducing the number of required animals for such studies. This will hopefully improve the low success rate of testing CNS drugs in animals and improve translation to clinical trials in humans17,18.
The cultures reach peak maturity by DIV21. Cell counts increase until this timepoint, after which the cells start degenerating with decreasing numbers of each type of cell. This was measured both by looking at the quantified cell numbers (Figure 2N) as well as the number of pyknotic nuclei (dense DAPI stains) (Figure 2M). While the developmental markers nestin and NG2 are still expressed on DIV14 (Figure 2G) and DIV21 (Figure 2J), this is due to some of the astrocytes going through astrogliosis, which upregulates nestin19, while some of the oligodendrocytes are not fully mature and therefore still express some NG220. Based on the microscopy validation, only a small portion of the cells still express developmental markers compared to the fully mature cells by DIV21 (Figure 2I,L); therefore, the cultures are largely mature by this time. Of note, in vivo, the density of microglia is highly variable across the murine CNS. The level of microglia, as defined by Iba1 expression using microscopy, is at the high end of this density in our cultures12. The high percentage of microglia that survive the flow cytometry procedure is likely due to microglia being more robust than other CNS cells, which generally have delicate extensions, and are therefore more likely to be damaged during flow cytometry preparation.
It only takes 3 weeks following dissection until the cells are ready for experimentation, which is shorter than most human-based in vitro methods, such as organoids or induced pluripotent stem cell-derived models21. As long as in vivo research is required to ensure the safety of new therapeutics, using in vitro cell culturing methods such as ours will aid in testing toxicity and efficacy efficiently before testing in animals, reducing the requirement for animals and enhancing the translation of research from in vitro to in vivo. Hopefully, this will eventually lead to more efficient translation to human-based clinical trials as well. Ultimately, these cultures were created to allow the study of the CNS. While in vitro systems cannot fully express the complexity of the CNS, primary cell-derived cultures more accurately represent in vivo CNS properties22,23. In vitro approaches can allow for a more reductive approach to investigate the CNS in the absence of infiltrating immune cells24 and the blood-brain barrier integrity25. Removing this layer of complexity can make it easier to unravel mechanisms. As such, the present culturing system is a useful tool to answer research questions that complement in vivo animal research.
The ability to harvest, isolate, and culture CNS cells has already greatly advanced our understanding of the innate CNS16. This article demonstrates the dissection of E17 mouse brains and the resulting trituration and culturing of the cells to generate a cell culture system containing all the main cell types of the CNS. Multiple molecular techniques have been employed on these cultures, showcasing the effectiveness these cultures have for investigating the CNS.

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><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.

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 markers, SMI31 (axons), MBP (myelin), and NeuN (neuron cell body) as neuronal markers, or CNP (oligodendrocytes), GFAP (astrocytes), and Iba1 (microglia) as glial markers (Figure 2).
The various cell types were quantified using CellProfiler pipelines (based on &#8242;,6-diamidino-2-phenylindole-positive (https://github.com/muecs/cp/tree/v1.1). Each individual data point was generated from an average of 10 images taken from three coverslips per timepoint. For microglia, neurons, and oligodendrocytes, the percentage of cell type per 4&#8242;,6-diamidino-2-phenylindole-positive (DAPI+) cell was calculated. The percentage field of view was used instead of the number of cells for quantifying the number of astrocytes. This was due to difficulties differentiating between the individual cells as they frequently overlapped (Figure 2M,N). The cultures reached peak maturity and cell density at DIV21, after which the cultures started to degrade (Figure 2N).
Importantly, these cultures can easily be treated with drugs, such as potential therapeutics, or used to trace in vitro infections. In this example, cultures on coverslips were transferred to a 24-well plate and infected with the highly neurotropic virus Semliki Forest virus (SFV) (strain SFV6)6, which expresses zsGreen in infected cells. A multiplicity of infection (MOI-the number of virus particles added per cell) of 0.05, as titrated on baby hamster kidney (BHK) cells, was used to ensure low-level infection. After 0-72 h post-infection (hpi), cultures were fixed in 4% PFA and stained for analysis by immunofluorescent imaging. Figure 3 illustrates that, in line with in vivo infection, SFV mainly infects oligodendrocytes and neurons6.
qRT-PCR 
In addition to microscopy, these CNS cultures can be used for analysis by molecular methods, such as RT-qPCR of mRNA responses to treatment. To further investigate the innate antiviral response, 6-well plate cultures were treated with a range of doses of the potent antiviral cytokine interferon beta (IFN-&#946;) for 24 h. Cultures were lysed with guanidium-thiocyanate/phenol, and the RNA was isolated, converted to cDNA, and analyzed by qPCR as previously described7. Using this method, differential expression of many genes can be measured. Here, the upregulation of Ccl5 was quantified against the housekeeping gene 18s. CCL5 is a chemotactic cytokine (chemokine) involved in the inflammatory response. IFN-&#946; treatment resulted in an upregulation of Ccl5 mRNA in the cultures (Figure 4A).
Enzyme-linked immunosorbent assay (ELISA) 
The 96-well format of cultures is an excellent tool for high throughput screenings of treatments. To investigate whether the upregulation of Ccl5 mRNA results in the expression of CCL5 protein, CCL5 was measured in the supernatant of the cultures by ELISA, according to the manufacturer&#39;s instructions (see Table of Materials). In this example, 96-well cultures were treated in duplicate with 27 incremental doses of IFN-&#946; to generate a dose-response curve (Figure 4B). In line with the increased mRNA expression (Figure 4A), there was an increase in CCL5 released in the supernatant. As expected, Figure 4C demonstrates a clear correlation between mRNA and protein expression.
Flow cytometry 
Flow cytometry is a powerful tool for simultaneously investigating the expression of many intra- and/or extracellular markers. However, analyzing complex, highly interactive cultures by flow cytometry can be challenging due to cell damage and death when taking cells from culture to a single-cell suspension for processing and analysis. Comparing a variety of protocols using 0.05%-0.5% trypsin-ethylenediaminetetraacetic acid (EDTA), 1x-10x trypsin without EDTA, and gentle dissociation reagent, it was found that after treating the 6-well plate cultures with 0.05% trypsin-EDTA for 10 min at 37 &#176;C with gentle agitation, the cells were lifted with gentle trituration to create a single-cell suspension. Especially for flow cytometric analysis of CNS cells, it is critical to be gentle during cell preparation, minimize wash steps, and take great care to keep the cells at 4 &#176;C or on ice at all times.
To assess the viability and cell types in this single-cell suspension, cells were stained with a viability dye and fluorescently labeled antibodies against CD45, CD11b, O4, ACSA-2, and CD24 to allow visualization of each individual cell population in the cultures8 (Figure 5). Around 70% cell viability was obtained from this method, averaging approximately 2 x 106 viable, single cells per 6-well plate (Figure 5C). In line with the microscopy results (Figure 2N), there were large numbers of microglia, neurons, and astrocytes, while oligodendrocytes were the least abundant.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65331/65331fig01.jpg" /> 
Figure 1: Schematic overview of the described method to generate mixed neuronal and glial cultures. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/65331/65331fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65331/65331fig02.jpg" /> 
Figure 2: E17 CNS cultures stained for immunofluorescence imaging cell markers over time. Cells were fixed with 4% PFA before being permeabilized and stained to visualize different populations. Representative images of (A,D,G,J,M) developmental markers NG2 (oligodendrocyte precursor cells) and nestin (neuronal and glial precursor cells), (B,E,H,K,N) neuronal markers SMI31 (axons), MBP (myelin), and NeuN (neuron cell body), and (C,F,I,L,O) glial markers CNP (oligodendrocytes), GFAP (astrocytes), and Iba1 (microglia). Scale bars = 50 &#181;m. (P) Counts of DAPI per mm2, n = 3, each n from technical triplicates of 10 images per triplicate. (Q) Cell counts as a percentage of DAPI+ cells (microglia, oligodendrocytes, and neurons) and percentage of the field of view (astrocytes), n = 3, each n from technical triplicates of 10 images per triplicate. Error bars are &#177; standard error of the mean (SEM). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/65331/65331fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65331/65331fig03.jpg" /> 
Figure 3: Semliki Forest virus (SFV) infection of E17 CNS cultures over time. Representative images of uninfected control cultures (A,C,E,G) and cultures infected with 0.05 MOI SFV (B,D,F,H). Cells were infected for 0-48 h and stained with CNP (oligodendrocyte marker) and NeuN (neuron marker) (A-D) or GFAP (astrocyte marker) and Iba1 (microglial marker) (E-H). White arrows indicate an infected cell. This strain of SFV expresses the green fluorescent protein zsGreen to enable the tracing of viral infection. Scale bars = 20 &#181;m. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/65331/65331fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65331/65331fig04.jpg" /> 
Figure 4: CCL5 mRNA and protein expression of E17 CNS cultures following IFN-&#946; treatment. Cells were treated in 6-well plate format (mRNA) or 96-well plate format (protein) for 24 h with a range of doses of IFN-&#946;. (A) Expression of Ccl5 mRNA after treatment with 0-100 ng/mL IFN-&#946;, biological n = 2, each taken from technical triplicates. (B) CCL5 concentration in the supernatant following 0-100 ng/mL IFN&#946; treatment, biological n = 1, taken from technical triplicates. (C) Cellular mRNA Ccl5 upregulation versus CCL5 protein concentration in the supernatant with log trendline. Error bars are &#177; SEM. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/65331/65331fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/65331/65331fig05.jpg" /> 
Figure 5: Flow cytometry gating strategy to generate single-cell populations from E17 CNS cultures. Cells were dissociated using 0.05% trypsin-EDTA, stained with appropriate antibodies, and sorted into individual cell populations. (A-C) Gating strategy to sort for live, single cells. (D) Strategy to select for microglia (CD45+&#160;CD11b+). (E) Strategy to select oligodendrocytes (CD45- CD11b- O4+). (F) Strategy to sort for astrocytes (CD45- CD11b- O4- ACSA-2+ CD24+) and neurons (CD45- CD11b- O4- ACSA-2- CD24+). (G) Individual cell populations overlaid on each other, showing discrete populations. (H) Individual cell types as a percentage of the total cells sorted, n = 4. Error bars are &#177; SEM. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/65331/65331fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Required format</td>
			<td>BA-PLL/well&#160;</td>
			<td>Water for wash</td>
			<td>Volume cells in Plating Media (PM) to plate out</td>
			<td>Topping up Media</td>
			<td>Remove/add when feeding cultures (3x/week)</td>
		</tr>
		<tr>
			<td rowspan="2">6 well format (9.6cm2)</td>
			<td rowspan="2">1000 &#181;L</td>
			<td rowspan="2">1000 &#181;L</td>
			<td rowspan="2">1000 &#181;L</td>
			<td>-400 &#181;L</td>
			<td>-500 &#181;L Media</td>
		</tr>
		<tr>
			<td>+600 &#181;L DM+</td>
			<td>+600 &#181;L DM+/-</td>
		</tr>
		<tr>
			<td rowspan="2">96 well format (0.32 cm2)</td>
			<td rowspan="2">100&#181;L</td>
			<td rowspan="2">100 &#181;L</td>
			<td rowspan="2">50 &#181;L</td>
			<td rowspan="2">+60 &#181;L DM+</td>
			<td>-50 &#181;L Media</td>
		</tr>
		<tr>
			<td>+60 &#181;L DM+/-</td>
		</tr>
		<tr>
			<td rowspan="2">Coverslip in dish format&#160;</td>
			<td rowspan="2">20 mL per 200 coverslips</td>
			<td rowspan="2">20 mL</td>
			<td rowspan="2">100 &#181;L per coverslip</td>
			<td>+600 &#181;L DM+</td>
			<td>-500 &#181;L Media</td>
		</tr>
		<tr>
			<td>+300 &#181;L PM</td>
			<td>+600 &#181;L DM+/-</td>
		</tr>
	</tbody>
</table>
Table 1: Required volumes for preparation of coated tissue culture plastics.

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

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

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.

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 ...

establishing-mixed-neuronal-glial-cell-cultures-from-embryonic-mouse

Universidad San Sebastian

Research

JoVE Journal

Neuroscience

2.6K Views.  University of Glasgow. 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.

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

Derivation of Enriched Oligodendrocyte Cultures and Oligodendrocyte/Neuron Myelinating Co-cultures from Post-natal Murine Tissues

Identifying the molecular mechanisms underlying OL development is not only critical to furthering our knowledge of OL biology, but also has implications for understanding the pathogenesis of demyelinating diseases such as Multiple Sclerosis (MS). Cellular development is commonly studied with primary cell culture models. Primary cell culture facilitates the evaluation of a given cell type by providing a controlled environment, free of the extraneous variables that are present in vivo. While OL cultures derived from rats have provided a vast amount of insight into OL biology, similar efforts at establishing OL cultures from mice has been met with major obstacles. Developing methods to culture murine primary OLs is imperative in order to take advantage of the available transgenic mouse lines. 
Multiple methods for extraction of OPCs from rodent tissue have been described, ranging from neurosphere derivation, differential adhesion purification and immunopurification 1-3. While many methods offer success, most require extensive culture times and/or costly equipment/reagents. To circumvent this, purifying OPCs from murine tissue with an adaptation of the method originally described by McCarthy &amp; 
de Vellis 2 is preferred. This method involves physically separating OPCs from a mixed glial culture derived from neonatal rodent cortices. The result is a purified OPC population that can be differentiated into an OL-enriched culture. This approach is appealing due to its relatively short culture time and the unnecessary requirement for growth factors or immunopanning antibodies. 
While exploring the mechanisms of OL development in a purified culture is informative, it does not provide the most physiologically relevant environment for assessing myelin sheath formation. Co-culturing OLs with neurons would lend insight into the molecular underpinnings regulating OL-mediated myelination of axons. For many OL/neuron co-culture studies, dorsal root ganglion neurons (DRGNs) have proven to be the neuron type of choice. They are ideal for co-culture with OLs due to their ease of extraction, minimal amount of contaminating cells, and formation of dense neurite beds. While studies using rat/mouse myelinating xenocultures have been published 4-6, a method for the derivation of such OL/DRGN myelinating co-cultures from post-natal murine tissue has not been described. Here we present detailed methods on how to effectively produce such cultures, along with examples of expected results. These methods are useful for addressing questions relevant to OL development/myelinating function, and are useful tools in the field of neuroscience.

This article describes methods to derive enriched populations of murine oligodendrocyte precursor cells (OPCs) in primary culture, which differentiate to produce mature oligodendrocytes (OLs). In addition, this report describes techniques to produce murine myelinating co-cultures by seeding mouse OPCs onto a neurite bed of mouse dorsal root ganglion neurons (DRGNs).

Identifying the molecular mechanisms underlying OL development is not only critical to furthering our knowledge of OL biology, but also has implications ...

Isolation and Culture of Hippocampal Neurons from Prenatal Mice

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual neurons, researchers are able to analyze properties related to cellular trafficking, cellular structure and individual protein localization using a variety of biochemical techniques. Results from such experiments are critical for testing theories addressing the neural basis of memory and learning. However, unambiguous results from these forms of experiments are predicated on the ability to grow neuronal cultures with minimum contamination by other brain cell types. In this protocol, we use specific media designed for neuron growth and careful dissection of embryonic hippocampal tissue to optimize growth of healthy neurons while minimizing contaminating cell types (i.e. astrocytes). Embryonic mouse hippocampal tissue can be more difficult to isolate than similar rodent tissue due to the size of the sample for dissection. We show detailed dissection techniques of hippocampus from embryonic day 19 (E19) mouse pups. Once hippocampal tissue is isolated, gentle dissociation of neuronal cells is achieved with a dilute concentration of trypsin and mechanical disruption designed to separate cells from connective tissue while providing minimum damage to individual cells. A detailed description of how to prepare pipettes to be used in the disruption is included. Optimal plating densities are provided for immuno-fluorescence protocols to maximize successful cell culture. The protocol provides a fast (approximately 2 hr) and efficient technique for the culture of neuronal cells from mouse hippocampal tissue.

We provide a protocol for the culture of highly purified hippocampal neurons from prenatal mouse brains without the use of a feeder glial cell layer.

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual ...

An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

The enteric nervous system is a vast network of neurons and glia running the length of the gastrointestinal tract that functionally controls gastrointestinal motility. A procedure for the isolation and culture of a mixed population of neurons and glia from the myenteric plexus is described. The primary cultures can be maintained for over 7 days, with connections developing among the neurons and glia. The longitudinal muscle strip with the attached myenteric plexus is stripped from the underlying circular muscle of the mouse ileum or colon and subjected to enzymatic digestion. In sterile conditions, the isolated neuronal and glia population are preserved within the pellet following centrifugation and plated on coverslips. Within 24-48 hr, neurite outgrowth occurs and neurons can be identified by pan-neuronal markers. After two days in culture, isolated neurons fire action potentials as observed by patch clamp studies. Furthermore, enteric glia can also be identified by GFAP staining. A network of neurons and glia in close apposition forms within 5 - 7 days. Enteric neurons can be individually and directly studied using methods such as immunohistochemistry, electrophysiology, calcium imaging, and single-cell PCR. Furthermore, this procedure can be performed in genetically modified animals. This methodology is simple to perform and inexpensive. Overall, this protocol exposes the components of the enteric nervous system in an easily manipulated manner so that we may better discover the functionality of the ENS in normal and disease states.

An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

We demonstrate a cell culture protocol for the direct study of neuronal and glial components of the enteric nervous system. A neuron/glia mixed culture on coverslips is prepared from the myenteric plexus of adult mouse providing the ability to examine individual neuron and glia function by electrophysiology, immunohistochemical, etc.

The enteric nervous system is a vast network of neurons and glia running the length of the gastrointestinal tract that functionally controls ...

One Mouse, Two Cultures: Isolation and Culture of Adult Neural Stem Cells from the Two Neurogenic Zones of Individual Mice

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult neural stem cells in vitro. These assays can be used to compare the precursor potential of cells isolated from genetically different or differentially treated animals&#160;to determine the effects of exogenous factors on neural precursor cell proliferation and differentiation and to generate neural precursor cell lines that can be assayed over continuous passages. The neurosphere assay is traditionally used for the post-hoc identification of stem cells, primarily due to the lack of definitive markers with which they can be isolated from primary tissue&#160;and has the major advantage of giving a quick estimate of precursor cell numbers in brain tissue derived from individual animals. Adherent monolayer cultures, in contrast, are not traditionally used to compare proliferation between individual animals, as each culture is generally initiated from the combined tissue of between 5-8 animals. However, they have the major advantage that, unlike neurospheres, they consist of a mostly homogeneous population of precursor cells and are useful for following the differentiation process in single cells. Here, we describe, in detail, the generation of neurosphere cultures and, for the first time, adherent cultures from individual animals. This has many important implications including paired analysis of proliferation and/or differentiation potential in both the subventricular zone (SVZ) and dentate gyrus (DG) of treated or genetically different mouse lines, as well as a significant reduction in animal usage.

Here we describe a detailed protocol for the simultaneous generation of neural precursor cell cultures, as either adherent monolayers or neurospheres, from the subventricular zone and dentate gyrus of individual adult mice.

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult ...

Preparation of Primary Mixed Glial Cultures from Adult Mouse Spinal Cord Tissue

It has been well-accepted that spinal cord glial responses contribute significantly to the development of neuropathic pain. Tremendous information regarding glial activities at the cellular and molecular levels has been obtained through in vitro cell culture systems. The in vitro systems utilized, mainly include primary glia derived from neonatal brain cortical tissue and immortalized cell lines. However, these systems may not reflect the characteristics of spinal cord glial cells in vivo. In order to further investigate the roles of spinal cord glial cells in the development of peripheral nerve injury-induced neuropathic pain using a culture system that better reflects the in vivo condition, our laboratory has developed a method to establish primary spinal cord mixed glial cultures from adult mice. Briefly, spinal cords are collected from adult mice and processed through papain digestion followed by myelin removal with a density-gradient medium. Single cell suspensions are cultured in complete Dulbecco&#39;s modified Eagle media (cDMEM) supplemented with 2-mercaptoethanol (2-ME) at 35.9 oC. These culture conditions were optimized specifically for the growth of mixed glial cells. Under these conditions, cells are ready to be used for experimentation between 12 - 14 d&#160;(cells are usually in log phase during this time) after the establishment of the culture (D 0) and can be kept in culture conditions up to D 21. This culture system can be used to investigate the responses of spinal cord glial cells upon stimulation with various substances and agents. Besides neuropathic pain, this system can be used to study glial responses in other diseases that involve pathological changes of spinal cord glial cells.

The development of neuropathic pain involves pathological changes of spinal cord glial cells. A reliable glial culture system derived from adult spinal cord tissue and designed for studying these cells in vitro is lacking. Therefore, we show here how&#160;to establish primary mixed glial cultures from adult mouse spinal cord tissue.

It has been well-accepted that spinal cord glial responses contribute significantly to the development of neuropathic pain. Tremendous information ...

Primary Culture of Neurons Isolated from Embryonic Mouse Cerebellum

The use of primary cell cultures has become one of the major tools to study the nervous system in vitro. The ultimate goal of using this simplified model system is to provide a controlled microenvironment and maintain the high survival rate and the natural features of dissociated neuronal and nonneuronal cells as much as possible under in vitro conditions. In this article, we demonstrate a method of isolating primary neurons from the developing mouse cerebellum, placing them in an in vitro environment, establishing their growth, and monitoring their viability and differentiation for several weeks. This method is applicable to embryonic neurons dissociated from cerebellum between embryonic days 12&ndash;18.

Conducting in vitro experiments to reflect in vivo conditions as adequately as possible is not an easy task. The use of primary cell cultures is an important step toward understanding cell biology in a whole organism. The provided protocol outlines how to successfully grow and culture embryonic mouse cerebellar neurons.

The use of primary cell cultures has become one of the major tools to study the nervous system in vitro. The ultimate goal of using this simplified model ...

Dissection and Isolation of Murine Glia from Multiple Central Nervous System Regions

The methods presented here demonstrate laboratory procedures for the dissection of four different regions of the central nervous system (CNS) from murine neonates for the isolation of glial subpopulations. The purpose of the procedure is to dissociate microglia, oligodendrocyte progenitor cells (OPCs), and astrocytes from cortical, cerebellar, brainstem, and spinal cord tissue to facilitate further in vitro analysis. The CNS region isolation procedures allow for the determination of regional heterogeneity among glia in multiple cell culture systems. Rapid CNS region isolation is performed, followed by the mechanical removal of meninges to prevent meningeal cell contamination of glia. This protocol combines gentle tissue dissociation and plating on a specified matrix designed to preserve cell integrity and adherence. Isolating mixed glia from multiple CNS regions provides a comprehensive analysis of potentially heterogenous glia while maximizing the use of individual experimental animals. Additionally, following dissociation of regional tissue, mixed glia are further divided into multiple cell types including microglia, OPCs, and astrocytes for use in either single cell type, cell culture plate inserts, or co-culture systems. Overall, the demonstrated techniques provide a comprehensive protocol of broad applicability for careful dissection of four individual CNS regions from murine neonates and includes methods for the isolation of three individual glia cell types to examine regional heterogeneity in any number of in vitro cell culture systems or assays.

Here we present a protocol for in vitro isolation of multiple glial cell populations from a mouse CNS. This method allows for the segregation of regional microglia, oligodendrocyte precursor cells, and astrocytes to study the phenotypes of each in a variety of culture systems.

The methods presented here demonstrate laboratory procedures for the dissection of four different regions of the central nervous system (CNS) from murine ...

Primary Microglia Isolation from Postnatal Mouse Brains

Microglia are the mononuclear phagocytes in the central nervous system (CNS), which play key roles in maintaining homeostasis and regulating the inflammatory process in the CNS. To study the microglial biology in vitro, primary microglia show great advantages compared to immortalized microglial cell lines. However, microglia isolation from the postnatal mouse brain is relatively less efficient and time-consuming. In this protocol, we provide a quick and easy-to-follow method to isolate primary microglia from the neonatal mouse brain. The overall steps of this protocol include brain dissection, primary brain cell culture, and microglia isolation. Using this approach, researchers can obtain primary microglia with high purity. In addition, the harvested primary microglia were able to respond to the lipopolysaccharides challenge, indicating they retained their immune function. Collectively, we developed a simplified approach to efficiently isolate primary microglia with high purity, which facilitates a wide range of microglial biology investigations&#160;in vitro.

Primary cell culture is one of the primarily used approaches for studying microglial biology in vitro. Here, we developed a method for simple and rapid microglia isolation from the mouse postnatal day 1 (P1) to P4.

Microglia are the mononuclear phagocytes in the central nervous system (CNS), which play key roles in maintaining homeostasis and regulating the ...

 Primary Microglia and Neuron Co-Culture Protocol for CNS Interaction Studies

Generating and Co-culturing Murine Primary Microglia and Cortical Neurons

Microglia are tissue-resident macrophages of the central nervous system (CNS), performing numerous functions that support neuronal health and CNS homeostasis. They are a major population of immune cells associated with CNS disease activity, adopting&#160;reactive phenotypes that potentially contribute to neuronal injury during chronic neurodegenerative diseases such as multiple sclerosis (MS). The distinct mechanisms by which microglia regulate neuronal function and survival during health and disease remain limited due to challenges in resolving the complex in vivo interactions between microglia, neurons, and other CNS environmental factors. Thus, the in vitro approach of co-culturing microglia and neurons remains a valuable tool for studying microglia-neuronal interactions. Here, we present a protocol to generate and co-culture primary microglia and neurons from mice. Specifically, microglia were isolated after 9-10 days in vitro from a mixed glia culture established from brain homogenates derived from neonatal mice between post-natal days 0-2. Neuronal cells were isolated from brain cortices of mouse embryos between embryonic days 16-18. After 4-5 days in vitro, neuronal cells were seeded in 96-well plates, followed by the addition of microglia to form the co-culture. Careful timing is critical for this protocol as both cell types need to reach experimental maturity to establish the co-culture. Overall, this co-culture can be useful for studying microglia-neuron interactions and can provide multiple readouts, including immunofluorescence microscopy, live imaging, as well as RNA and protein assays.

Author Spotlight: In Vitro Co-Culture Model for Studying Microglia-Neuronal Interactions in Disease Conditions

This protocol describes a microglia-neuronal co-culture established from primary neuronal cells isolated from mouse embryos at embryonic days 15-16 and primary microglia generated from the brains of neonatal mice at post-natal days 1-2.

Microglia are tissue-resident macrophages of the central nervous system (CNS), performing numerous functions that support neuronal health and CNS ...

Obtaining a Mixed Glial Cell Culture from a Neonatal Mouse Brain

Source: Sarkar, S. et al., <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/55364">Rapid and Refined CD11b Magnetic Isolation of Primary Microglia with Enhanced Purity and Versatility.</a> J. Vis. Exp. (2017)
The video demonstrated the method of isolating and culturing cells from a neonatal mouse brain. It involved enzymatic and mechanical dissociation of the brain, followed by culturing in growth media lacking neuronal growth factor to obtain a mixed glial population.

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee. 
1. Growing of Mixed Glial ...

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

Summary

Explore More Videos

Chapters in this video

Derivation of Enriched Oligodendrocyte Cultures and Oligodendrocyte/Neuron Myelinating Co-cultures from Post-natal Murine Tissues

Isolation and Culture of Hippocampal Neurons from Prenatal Mice

An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

One Mouse, Two Cultures: Isolation and Culture of Adult Neural Stem Cells from the Two Neurogenic Zones of Individual Mice

Preparation of Primary Mixed Glial Cultures from Adult Mouse Spinal Cord Tissue

Primary Culture of Neurons Isolated from Embryonic Mouse Cerebellum

Dissection and Isolation of Murine Glia from Multiple Central Nervous System Regions

Primary Microglia Isolation from Postnatal Mouse Brains

Generating and Co-culturing Murine Primary Microglia and Cortical Neurons

Obtaining a Mixed Glial Cell Culture from a Neonatal Mouse Brain