Supported by the Fazit Foundation Graduate fellowship to S.S., the Federal Ministry of Education and Research (BMBF 01 EO 0803) to K.B. and the European Commission FP7 Grant PIRG08-GA-2010-276989, NEUREX, and the German Research Foundation Grant SCHA 1442/3-1 to C.S. The authors have no conflicting financial interests.

1. Isolation and Plating of Mixed Cortical Cells
Mixed cortical cell isolation for astrocyte cultures can be performed using P1 to P4 mouse pups. In order to achieve proper astrocyte density it is necessary to use 4 mouse pup cortices per T75 tissue culture flask. Therefore, volumes in the following protocol are calculated for a cell preparation using 4 mouse pups.
<ol>
	<li>Before starting the dissection procedure, prewarm 30 ml of astrocyte culture media (DMEM, high glucose + 10% heat-inactivated fetal bovine serum + 1% Penicillin/Streptomycin; see table) to 37 &deg;C. Coat one T75 flask with 20 ml of poly-D-lysine (PDL) at a concentration of 50 &mu;g/ml in cell culture grade water for 1 hr at 37 &deg;C in the CO2 incubator.</li>
	<li>For the dissection procedure, prepare all necessary reagents and materials. You will need: surgical scissors, smooth fine forceps, flat tip forceps, paper towels, waste bag, 70% ethanol and 2 dissecting dishes (3.5 cm diameter) on ice filled with 2 ml HBSS each.</li>
	<li>Gently hold and spray the head and neck of the mouse pup with 70% ethanol. The animal is sacrificed by decapitation using the scissors.</li>
	<li>Perform a midline incision, posterior to anterior, along the scalp to reveal the skull.</li>
	<li>Cut the cranium carefully from the neck to the nose. Two additional cuts are performed to allow further access to the brain: The first cut is made anterior of the olfactory bulbs, the other one inferior of the cerebellum to disconnect the cranium from the skull base.</li>
	<li>Using the flat tip forceps, the cranial flaps are gently flipped to the side and the brain is taken out and placed into the first dissecting dish filled with HBSS. Place the dish back on ice and continue harvesting all 4 brains.</li>
	<li>The remainder of the dissection procedure is performed under a stereomicroscope. First, the olfactory bulbs and the cerebellum are removed using the fine dissecting forceps (Figure 1B).</li>
	<li>In order to retrieve the cortices, grab the posterior end of the brain with the fine forceps, perform a midline incision between the hemispheres, insert a second set of forceps to the created grove and peel away the plate-like structure of the cortex from the brain.</li>
	<li>Carefully dissect the meninges from the cortex hemispheres by pulling with the fine forceps (Figure 1D'). This step avoids contamination of the final astrocyte culture by meningeal cells and fibroblasts. Transfer the prepared cortex hemispheres into the second dish filled with HBSS and return it onto ice. Continue accordingly with all 4 cortices.</li>
	<li>Finally, cut each cortex hemisphere into small pieces using sharp blades (approximately 4 to 8 times).</li>
	<li>Under sterile conditions, transfer cortex pieces into one 50 ml Falcon tube and add HBSS to a total volume of 22.5 ml.</li>
	<li>Add 2.5 ml of 2.5 % trypsin, mix and incubate the tissue in the water bath at 37 &deg;C for 30 min. Mix by occasional shaking every 10 min.</li>
	<li>Centrifuge for 5 min at 300 x g to pellet cortex tissue pieces.</li>
	<li>Carefully remove supernatant by decantation. In order to avoid losing the tissue pellet you may mechanically retain it using a pipet. Dissociate the tissue into a single cell suspension by adding 10 ml astrocyte plating medium and vigorous pipetting using a 10 ml plastic pipette until tissue pieces are dissociated into single cells (20 to 30 times). Adjust volume to 20 ml using astrocyte plating medium. You can proof the dissociation of the cortex tissue into single cells by counting using a hematocytometer. One preparation of 4 mouse pup cortices should yield 10-15 x106 dissociated single cells.</li>
	<li>Aspirate PDL from the T75 culture flask, plate the dissociated single cell suspension and incubate at 37 &deg;C in the CO2 incubator.</li>
</ol>
2. Obtaining an Enriched Astrocyte Culture
<ol>
	<li>Change the medium 2 days after plating of the mixed cortical cells and all 3 days thereafter.</li>
	<li>After 7 to 8 days, when astrocytes are confluent and overlaying microglia sit exposed on the astrocyte layer or are already detached from the astrocyte layer (Figure 2), shake the T75 flask at 180 rpm for 30 min on an orbital shaker to remove microglia. Discard the supernatant containing microglia or if you wish to culture and examine microglia, spin it down and plate for culture 28,29.</li>
	<li>Add 20 ml fresh astrocyte culture medium and continue by shaking the flask at 240 rpm for 6 hr to remove oligodendrocyte precursor cells (OPC). Since some OPCs will not completely detach from the astrocyte layer, continue to shake vigorously by hand for 1 min in order to prevent OPC contamination. Discard the supernatant or spin it down and plate, if you wish to culture OPCs 29.</li>
	<li>Rinse the remaining confluent astrocyte layer twice with PBS, aspirate the PBS, add 5 ml trypsin-EDTA and incubate in the CO2 incubator at 37 &deg;C. Check detachment of astrocytes every 5 min and enforce detachment of astrocytes by hitting the flask against the palm of your hand (2-3 times).</li>
	<li>After astrocytes are detached from the culture flask, add 5 ml of astrocyte culture medium, spin cells at 180 x g for 5 min, aspirate supernatant and add 40 ml fresh astrocyte plating medium. One T75 tissue culture flask should yield around 1x106 cells after the first cell split. Plate cells in two T75 culture flasks and incubate at 37 &deg;C in the CO2 incubator. Change the medium every 2 to 3 days.</li>
	<li>12-14 days after the first split astrocytes are plated in the appropriate cell concentration 24-48 hr before performing the experiment. One T75 tissue culture flask should yield around 1.5-2 x106 cells after the second cell split.</li>
</ol>

<ol>
	<li>Nedergaard, M., Ransom, B., Goldman, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+roles+for+astrocytes:+redefining+the+functional+architecture+of+the+brain.">New roles for astrocytes: redefining the functional architecture of the brain.</a> Trends Neurosci. 26, 523-530 (2003).</li><li>Belanger, M., Allaman, I., Magistretti, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+energy+metabolism:+focus+on+astrocyte-neuron+metabolic+cooperation.">Brain energy metabolism: focus on astrocyte-neuron metabolic cooperation.</a> Cell Metab. 14, 724-738 (2011).</li><li>Allen, N. J., Barres, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroscience:+Glia+-+more+than+just+brain+glue.">Neuroscience: Glia - more than just brain glue.</a> Nature. 457, 675-677 (2009).</li><li>Ballas, N., Lioy, D. T., Grunseich, C., Mandel, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-cell+autonomous+influence+of+MeCP2-deficient+glia+on+neuronal+dendritic+morphology.">Non-cell autonomous influence of MeCP2-deficient glia on neuronal dendritic morphology.</a> Nat. Neurosci. 12, 311-317 (2009).</li><li>Jacobs, S., Nathwani, M., Doering, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fragile+X+astrocytes+induce+developmental+delays+in+dendrite+maturation+and+synaptic+protein+expression.">Fragile X astrocytes induce developmental delays in dendrite maturation and synaptic protein expression.</a> BMC Neurosci. 11, 132 (2010).</li><li>Kaneko, N., 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=New+neurons+clear+the+path+of+astrocytic+processes+for+their+rapid+migration+in+the+adult+brain.">New neurons clear the path of astrocytic processes for their rapid migration in the adult brain.</a> Neuron. 67, 213-223 (2010).</li><li>Min, R., Nevian, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocyte+signaling+controls+spike+timing-dependent+depression+at+neocortical+synapses.">Astrocyte signaling controls spike timing-dependent depression at neocortical synapses.</a> Nat. Neurosci. , (2012).</li><li>Eroglu, C., Barres, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+synaptic+connectivity+by+glia.">Regulation of synaptic connectivity by glia.</a> Nature. 468, 223-231 (2010).</li><li>Sasaki, T., Matsuki, N., Ikegaya, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Action-potential+modulation+during+axonal+conduction.">Action-potential modulation during axonal conduction.</a> Science. 331, 599-601 (2011).</li><li>Bozoyan, L., Khlghatyan, J., Saghatelyan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocytes+control+the+development+of+the+migration-promoting+vasculature+scaffold+in+the+postnatal+brain+via+VEGF+signaling.">Astrocytes control the development of the migration-promoting vasculature scaffold in the postnatal brain via VEGF signaling.</a> J. Neurosci. 32, 1687-1704 .</li><li>Alvarez, J. I., 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=The+Hedgehog+pathway+promotes+blood-brain+barrier+integrity+and+CNS+immune+quiescence.">The Hedgehog pathway promotes blood-brain barrier integrity and CNS immune quiescence.</a> Science. 334, 1727-1731 (2011).</li><li>Abbott, N. J., Ronnback, L., Hansson, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocyte-endothelial+interactions+at+the+blood-brain+barrier.">Astrocyte-endothelial interactions at the blood-brain barrier.</a> Nat. Rev. Neurosci. 7, 41-53 (2006).</li><li>Tao-Cheng, J. H., Nagy, Z., Brightman, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tight+junctions+of+brain+endothelium+in+vitro+are+enhanced+by+astroglia.">Tight junctions of brain endothelium in vitro are enhanced by astroglia.</a> J. Neurosci. 7, 3293-3299 (1987).</li><li>Gordon, G. R., Choi, H. B., Rungta, R. L., Ellis-Davies, G. C., MacVicar, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+metabolism+dictates+the+polarity+of+astrocyte+control+over+arterioles.">Brain metabolism dictates the polarity of astrocyte control over arterioles.</a> Nature. 456, 745-749 (2008).</li><li>Silver, J., Miller, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+beyond+the+glial+scar.">Regeneration beyond the glial scar.</a> Nat. Rev. Neurosci. 5, 146-156 (2004).</li><li>Schachtrup, C., Moan, N. L. e., Passino, M. A., Akassoglou, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatic+stellate+cells+and+astrocytes:+Stars+of+scar+formation+and+tissue+repair.">Hepatic stellate cells and astrocytes: Stars of scar formation and tissue repair.</a> Cell Cycle. 10, 1764-1771 (2011).</li><li>Schachtrup, C., 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=Fibrinogen+triggers+astrocyte+scar+formation+by+promoting+the+availability+of+active+TGF-beta+after+vascular+damage.">Fibrinogen triggers astrocyte scar formation by promoting the availability of active TGF-beta after vascular damage.</a> J. Neurosci. 30, 5843-5854 (2010).</li><li>Bushong, E. A., Martone, M. E., Jones, Y. Z., Ellisman, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protoplasmic+astrocytes+in+CA1+stratum+radiatum+occupy+separate+anatomical+domains.">Protoplasmic astrocytes in CA1 stratum radiatum occupy separate anatomical domains.</a> J. Neurosci. 22, 183-192 (2002).</li><li>Ogata, K., Kosaka, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+and+quantitative+analysis+of+astrocytes+in+the+mouse+hippocampus.">Structural and quantitative analysis of astrocytes in the mouse hippocampus.</a> Neuroscience. 113, 221-233 (2002).</li><li>Dabir, D. V., 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=Impaired+glutamate+transport+in+a+mouse+model+of+tau+pathology+in+astrocytes.">Impaired glutamate transport in a mouse model of tau pathology in astrocytes.</a> J. Neuroscience. 26, 644-654 (2006).</li><li>Wisniewski, H. M., Wegiel, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+relationships+between+astrocytes+and+classical+plaque+components.">Spatial relationships between astrocytes and classical plaque components.</a> Neurobiol. Aging. 12, 593-600 (1991).</li><li>Shin, J. Y., 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=Expression+of+mutant+huntingtin+in+glial+cells+contributes+to+neuronal+excitotoxicity.">Expression of mutant huntingtin in glial cells contributes to neuronal excitotoxicity.</a> J. Cell Biol. 171, 1001-1012 (2005).</li><li>Wakabayashi, K., Hayashi, S., Yoshimoto, M., Kudo, H., Takahashi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NACP/alpha-synuclein-positive+filamentous+inclusions+in+astrocytes+and+oligodendrocytes+of+Parkinson's+disease+brains.">NACP/alpha-synuclein-positive filamentous inclusions in astrocytes and oligodendrocytes of Parkinson's disease brains.</a> Acta Neuropathol. 99, 14-20 (2000).</li><li>Lioy, D. T., 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=A+role+for+glia+in+the+progression+of+Rett's+syndrome.">A role for glia in the progression of Rett's syndrome.</a> Nature. 475, 497-500 (2011).</li><li>Quinlan, R. A., Brenner, M., Goldman, J. E., Messing, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GFAP+and+its+role+in+Alexander+disease.">GFAP and its role in Alexander disease.</a> Exp. Cell Res. 313, 2077-2087 (2007).</li><li>Beck, K., Schachtrup, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+damage+in+the+central+nervous+system:+a+multifaceted+role+for+vascular-derived+TGF-beta.">Vascular damage in the central nervous system: a multifaceted role for vascular-derived TGF-beta.</a> Cell Tissue Res. 347, 187-201 (2012).</li><li>McCarthy, K. D., de Vellis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+separate+astroglial+and+oligodendroglial+cell+cultures+from+rat+cerebral+tissue.">Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue.</a> J. Cell Biol. 85, 890-902 (1980).</li><li>Siao, C. J., 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=Tissue+plasminogen+activator+mediates+microglial+activation+via+its+finger+domain+through+annexin+II.">Tissue plasminogen activator mediates microglial activation via its finger domain through annexin II.</a> J. Neurosci. 22, 3352-3358 (2002).</li><li>Armstrong, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+immature+oligodendrocyte+lineage+cells.">Isolation and characterization of immature oligodendrocyte lineage cells.</a> Methods. 16, 282-292 (1998).</li><li>Cahoy, J. D., 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=A+transcriptome+database+for+astrocytes,+neurons,+and+oligodendrocytes:+a+new+resource+for+understanding+brain+development+and+function.">A transcriptome database for astrocytes, neurons, and oligodendrocytes: a new resource for understanding brain development and function.</a> J. Neurosci. 28, 264-278 (2008).</li><li>Anthony, T. E., Heintz, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+folate+metabolic+enzyme+ALDH1L1+is+restricted+to+the+midline+of+the+early+CNS,+suggesting+a+role+in+human+neural+tube+defects.">The folate metabolic enzyme ALDH1L1 is restricted to the midline of the early CNS, suggesting a role in human neural tube defects.</a> J. Comp. Neurol. 500, 368-383 (2007).</li><li>Skoff, R. P., Knapp, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Division+of+astroblasts+and+oligodendroblasts+in+postnatal+rodent+brain:+evidence+for+separate+astrocyte+and+oligodendrocyte+lineages.">Division of astroblasts and oligodendroblasts in postnatal rodent brain: evidence for separate astrocyte and oligodendrocyte lineages.</a> Glia. 4, 165-174 (1991).</li><li>Molofsky, A. V., 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=Astrocytes+and+disease:+a+neurodevelopmental+perspective.">Astrocytes and disease: a neurodevelopmental perspective.</a> Genes Dev. 26, 891-907 (2012).</li><li>Zhang, Y., Barres, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocyte+heterogeneity:+an+underappreciated+topic+in+neurobiology.">Astrocyte heterogeneity: an underappreciated topic in neurobiology.</a> Curr. Opin. Neurobiol. 20, 588-594 (2010).</li><li>Foo, L. C., 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=Development+of+a+method+for+the+purification+and+culture+of+rodent+astrocytes.">Development of a method for the purification and culture of rodent astrocytes.</a> Neuron. 71, 799-811 (2011).</li><li>Jungblut, M., 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=Isolation+and+characterization+of+living+primary+astroglial+cells+using+the+new+GLAST-specific+monoclonal+antibody+ACSA-1.">Isolation and characterization of living primary astroglial cells using the new GLAST-specific monoclonal antibody ACSA-1.</a> Glia. 60, 894-907 (2012).</li></ol>

No conflicts of interest declared.

The method outlined here is based on the astrocyte culture preparation from rodent neonatal brains, originally described by McCarthy and de Vellis in 1980 27. The modified method of the isolation and culture of cortical astrocytes from postnatal P1 to P4 mouse brain presented here is fast, yields pure primary astrocytes and is highly reproducible. This technique can easily be transferred to isolate astrocytes from other species, such as from rat or pig and from other brain regions, such as the spinal cord. Whereas astrocyte progenitor cell isolation from neonatal brain by the McCarthy and deVellis method generates highly proliferative cells, the cell proliferation and propagation of isolated astrocytes of postnatal P1-P4 mouse pups is limited. After splitting astrocytes once at 7 days in vitro (DIV), they will grow to confluency and mature. In vivo, most astrocyte proliferation is largely complete by P14 32. Here, we suggest to use astrocytes for experiments at day 21 to 28 DIV (Figure 2F) to ensure the mature phenotype of isolated astrocytes. Due to the intrinsic restraint to proliferate astrocyte cultures should not be splitted more than 3 times.
	Critical steps in the described isolation method are the digestion of cortices and the following trituration of the digested tissue for obtaining the single cell suspension. Therefore, it is necessary to optimize trypsin concentration and digestion time in order to obtain a single cell suspension after trituration of the brain cortex tissue for 20-30 times. In order to minimize variation trypsin should be aliquoted and freeze-thaw cycles should be avoided. The described procedure relies on plating the mixed cortical cells on PDL-coated culture flasks, which assures the binding of astrocytes and promotes a confluent astrocyte layer several days after plating. While PDL-coating is not necessary for astrocyte maintenance after their separation from microglia and oligodendrocytes, it may be performed for certain downstream applications such as immunofluorescene staining. Good timing of the first cell split is important for cell integrity and yield. If astrocytes are cultured beyond they reached confluency, you may lose the majority of cells due to insufficient detachment during the first cell split. This cannot be overcome by increasing the time for detachment, since extensive incubation time with trypsin negatively influences cell integrity. In contrast, plating the cortical cell suspension too scarcely will result in insufficient formation of a confluent astrocyte cell layer. The best time for the first cell split is at 7 to 8 days after plating of mixed cortical cells, when astrocytes are confluent and microglia cells sit on the topmost position of the astrocyte layer.
	In the described cortical cell culture, astrocytes show cellular heterogeneity (Figure 3), as it has been described for astrocytes in vivo 33,34. However, defining diverse astrocyte morphology and functionality has been hampered by the limited number of markers to identify and distinguish potentially heterogeneous astrocyte subtypes. A well characterized marker of mature fibrous and reactive astrocytes is GFAP. However, GFAP is barely expressed by mature protoplasmic astrocytes, limiting its use as a marker for all astrocytes and it is also expressed by RG cells during development and by B cells in the adult, restricting its use as a stage-specific marker. Other markers of astrocytes, including GLAST, ALDH1L1 or BLBP, are also expressed by immature astrocytes and therefore do not exclusively mark mature astrocytes. Finally, mature astrocyte markers such as GFAP, Aquaporin-4, and S100B (Figure 3) are increasingly up-regulated during postnatal maturation.
	In our hands, astrocyte cultures at an age of 4 weeks have characteristics of mature astrocytes in vivo. Using primary astrocyte cultures we could identify the molecular mechanism how the blood born protein fibrinogen induces astrocyte activation 17. Our studies revealed that fibrinogen is a carrier of latent TGF-&beta;. Treatment of primary astrocytes with fibrinogen led to active TGF-&beta; formation and the activation of the TGF-&beta;/Smad signaling pathway in astrocytes 17,26. These results have been confirmed using fibrinogen injections in vivo. Furthermore, astrocytes control other cell types by the secretion of substances, which can be analyzed by harvesting astrocyte-conditioned medium and applying this conditioned medium to other cell types. We previously used astrocyte-conditioned medium to analyze in a functional assay how conditioned medium of fibrinogen-treated astrocytes affects neurite outgrowth. Reactive astrocytes express and secrete proteins of the CSPG family, which inhibit neurite outgrowth 16. Indeed, conditioned medium from fibrinogen-treated astrocytes significantly decreased both neurite length and the percentage of cells showing neurite outgrowth 17.
	The isolation and culture of cortical astrocytes described in this protocol provides a powerful tool for investigating astrocyte biology, since their manageability in diverse applications can greatly complete their investigation in vivo. However, it should be kept in mind that the obtained astrocytes have been cultured in vitro and while they reflect many astrocyte characteristics, they also differ from in vivo astrocytes. Therefore, other methods of direct selection and isolation of astrocytes by immunopanning 35 or antibody-based FACS isolation 36 represent new avenues to further investigate the fundamental properties of astrocytes.

Astrocytes are a very abundant cell type in the central nervous system (CNS). The ratio of astrocytes to neurons is 1:3 in the cortex of mice and rats, whereas there are 1.4 astrocytes per neuron in the human cortex 1. Interest in astrocyte function has increased dramatically in recent years. A key function of astrocytes is their role in providing structural and metabolic support to neurons 2,3. Newly discovered roles for astrocytes cover a broad spectrum of functions. These include guiding the migration of developing axons and certain neuroblasts during development4-6, functions in synaptic transmission, synapse strength and information processing by neural circuits 7-9, roles in blood-brain barrier (BBB) formation 10 and integrity 11-13 and regulation of the cerebrovascular tone 14. Another major feature of astrocytes is their response to injury. Under pathological conditions astrocytes become reactive and further upregulate the expression of the intermediate filament glial fibrillary acidic protein (GFAP) and inhibitory extracellular matrix (ECM) proteins 15,16. Reactive astrocytes demarcate the injury site from healthy tissue by forming a glial scar, which consists mainly of astrocyte secreted ECM proteins of the chondroitin sulfate proteoglycan (CSPG) family, the major factors that inhibit axonal regeneration after CNS injury 15-17.
Astrocytes originate from radial glial (RG) cells during late embryogenesis and early postnatal life. After astrocyte specification has occurred, astrocyte precursors migrate to their final positions, where they begin the process of terminal differentiation. In vivo, astrocytes appear to be mature three to four weeks after birth as indicated by their typical morphology 18,19. A subpopulation of RG cells convert into subventricular zone astrocytes (type B cells). Both, RG and type B cells function as astrocyte-like neural stem cells (NSCs) during development and in the adult, respectively. Like astrocytes, RG and type B cells also express the astrocyte-specific glutamate transporter (GLAST), brain lipid-binding protein (BLBP), and GFAP, indicating that these markers cannot be exclusively used to specifically label adult astrocytes. In contrast to adult parenchymal astrocytes, which do not divide in the healthy brain, RG and type B cells exhibit stem cell potential such as the capacity to self-renew. Dysregulation of astrocytes has been implicated in numerous pathologies, including Alzheimer's disease 20,21, Huntington's disease 22, Parkinson's disease 23, Rett syndrome 24 and Alexander's disease 25. Moreover, astrocytes react to all insults of the CNS, leading to astrocyte activation and astrocytic glial scar formation 16,26. The astrocytic glial scar that forms following brain trauma or spinal cord injury is thought to be the prime barrier preventing neuronal regeneration 15.
The development of reliable methods to isolate and maintain purified populations of cells has been essential to our understanding of the nervous system. Pioneering work by McCarthy and de Vellis enables investigators to date to prepare nearly pure cultures of astrocytes from neonatal rat tissue 27. Much has been learned about astrocyte biology using this method, which is presented here in a slightly modified form for isolating mouse cortical astrocytes. Complementing in vivo studies, astrocytes as well as conditioned medium obtained using the described in vitro culture, are valuable tools to further gain insights into astrocyte functions.

<table border="1">
 <tbody>
 <tr>
 <td>Name of working solution</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Final concentration</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Astrocyte culture media</td>
 </tr>
 <tr>
 <td>DMEM, high glucose</td>
 <td>Life Technologies</td>
 <td>31966-021</td>
 <td></td>
 </tr>
 <tr>
 <td>FBS, heat-inactivated</td>
 <td>Life Technologies</td>
 <td>10082-147</td>
 <td>Final Concentration: 10%</td>
 </tr>
 <tr>
 <td>Penicillin-Streptomycin</td>
 <td>Life Technologies</td>
 <td>15140-122</td>
 <td>Final Concentration: 1%</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Solution for brain tissue digestion</td>
 </tr>
 <tr>
 <td>HBSS</td>
 <td>Life Technologies</td>
 <td>14170-088</td>
 <td></td>
 </tr>
 <tr>
 <td>2.5% Trypsin</td>
 <td>Life Technologies</td>
 <td>15090-046</td>
 <td>Final Concentration: 0.25%</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Other</td>
 </tr>
 <tr>
 <td>70% (vol/vol) ethanol</td>
 <td>Roth</td>
 <td>9065.2</td>
 <td></td>
 </tr>
 <tr>
 <td>Poly-D-Lysine </td>
 <td>Millipore</td>
 <td>A-003-E</td>
 <td>50 &mu;g/ml </td>
 </tr>
 <tr>
 <td>Water</td>
 <td>PAA</td>
 <td>S15-012</td>
 <td>cell culture grade</td>
 </tr>
 <tr>
 <td>PBS</td>
 <td>PAA</td>
 <td>H15-002</td>
 <td>cell culture grade</td>
 </tr>
 <tr>
 <td>0.05% Trypsin-EDTA </td>
 <td>Life Technologies</td>
 <td>25300-062 </td>
 <td></td>
 </tr>
 <tr>
 <td>0.45 &mu;m Sterile filter </td>
 <td>Sartorius </td>
 <td>16555 </td>
 <td></td>
 </tr>
 <tr>
 <td>3.5 cm petri dish</td>
 <td>BD Falcon</td>
 <td>353001</td>
 <td></td>
 </tr>
 <tr>
 <td>15 ml Falcon tube</td>
 <td>BD Falcon</td>
 <td>352096</td>
 <td></td>
 </tr>
 <tr>
 <td>50 ml Falcon tube</td>
 <td>BD Falcon</td>
 <td>352070</td>
 <td></td>
 </tr>
 <tr>
 <td>75 cm2 Tissue culture flask</td>
 <td>BD Falcon</td>
 <td>353136</td>
 <td></td>
 </tr>
 <tr>
 <td>Forceps, fine </td>
 <td>Dumont</td>
 <td>2-1032; 2-1033</td>
 <td># 3c; # 5</td>
 </tr>
 <tr>
 <td>Forceps, flat tip</td>
 <td>KLS Martin</td>
 <td>12-120-11</td>
 <td></td>
 </tr>
 <tr>
 <td>13 cm surgical scissors</td>
 <td>Aesculap</td>
 <td>BC-140-R</td>
 <td></td>
 </tr>
 <tr>
 <td>Stereomicroscope</td>
 <td>Leica</td>
 <td></td>
 <td>MZ7.5</td>
 </tr>
 <tr>
 <td>Stereomicroscope + Camera</td>
 <td>Leica</td>
 <td></td>
 <td>MZ16F; DFC320</td>
 </tr>
 <tr>
 <td>Microscope + Camera</td>
 <td>Zeiss; Canon</td>
 <td></td>
 <td>Primo Vert; PowerShot A650 IS</td>
 </tr>
 <tr>
 <td>Centrifuge</td>
 <td>Eppendorf</td>
 <td>5805000.017</td>
 <td>Centrifuge5804R</td>
 </tr>
 <tr>
 <td>Orbital Shaker</td>
 <td>Thermo Scientific</td>
 <td>SHKE 4450-1CE</td>
 <td>MaxQ 4450 </td>
 </tr>
 <tr>
 <td>Water bath</td>
 <td>Julabo</td>
 <td></td>
 <td>SW20; 37 &deg;C</td>
 </tr>
 </tbody>
 </table>

isolation and culture of mouse cortical astrocytes

Astrocytes are an abundant cell type in the mammalian brain, yet much remains to be learned about their molecular and functional characteristics. In vitro astrocyte cell culture systems can be used to study the biological functions of these glial cells in detail. This video protocol shows how to obtain pure astrocytes by isolation and culture of mixed cortical cells of mouse pups. The method is based on the absence of viable neurons and the separation of astrocytes, oligodendrocytes and microglia, the three main glial cell populations of the central nervous system, in culture. Representative images during the first days of culture demonstrate the presence of a mixed cell population and indicate the timepoint, when astrocytes become confluent and should be separated from microglia and oligodendrocytes. Moreover, we demonstrate purity and astrocytic morphology of cultured astrocytes using immunocytochemical stainings for well established and newly described astrocyte markers. This culture system can be easily used to obtain pure mouse astrocytes and astrocyte-conditioned medium for studying various aspects of astrocyte biology.

Upon isolation of the complete mouse brain (Figure 1A), the cerebellum and the olfactory bulbs have to be removed (Figure 1B). The cortices are peeled of the mouse brain stem (Figure 1C) and meninges of the individual cortex (Figure 1D') are carefully removed (Figure 1E). Meninges are obvious by the meningeal artery system and incomplete removal results in contamination of the final astrocyte culture by meningeal cells and fibroblasts.
After plating of the mixed cortical cell suspension, some astrocytes already attach to the culture flask within 24 hr (Figure 2A). However, the cell culture supernatant will contain cell debris and dying neurons for the first 3 to 5 days (Figure 2A-C), as the culture medium favors survival and growth of glial cells. Once astrocytes are confluent and microglia sit exposed on the astrocyte layer, usually at day 7 after isolation of mixed cortical cells, astrocytes should be separated from microglia and OPCs by shaking (Figure 2D). 2 days after the first split cells show astrocyte morphology. At this point astrocyte density is low and astrocytes are voluminous (Figure 2E, black arrows mark one cell). The expected yield at this point is low with about 4-6 x105 cells per T75 tissue culture flask. However, the cells still proliferate and mature in culture at this timepoint. Astrocytes are usually used for experiments at day 21 to 28 (Figure 2F) to ensure a mature phenotype of isolated astrocytes. At this point the expected yield is about 1.5-2 x106 cells per T75 tissue culture flask.
The astrocyte culture purity has been characterized by electron microscopy morphological studies, cell marker expression and pharmacological responsiveness 27. Contamination of the astrocyte culture can be examined by using marker for microglia (IBA-1) and OPCs (O4). The obtained astrocyte culture purity and maturity should be examined by immunocytochemical examination using an antibody against the GFAP protein. It is expected that the majority of the cells show an intensive, filamentous signal (Figure 3). Our isolation and culture method of cortical astrocytes resulted in an astrocyte purity of greater than 98%, revealed by using an antibody against the GFAP protein (Figure 3). If the procedure was successful, contaminating cells are rare (&lt;2%) and contain microglia and OPCs, which can be stained by using IBA-1 and O4, respectively. If desired, further analysis of other astrocyte markers, such as Aquaporin-4, BLBP, S100B and GLAST may be used. In addition to GFAP, these markers were all expressed by the 28 days old astrocyte cultures. Recent gene expression profiling of purified astrocyte populations revealed new potential astrocyte markers, such as the folate metabolic enzyme ALDH1L1 30,31, which is also expressed by the majority of the isolated cortical astrocytes (Figure 3).
<img alt="Figure 1" src="/files/ftp_upload/50079/50079fig1.jpg" /> 
Figure 1. Dissection of postnatal (P3) mouse cortex. A) Whole brain. B) Brain after removal of olfactory bulbs and cerebellum. C) Isolation of cortices by peeling off the plate-like structure of the cortex from the brain. D, D') Cortex from ventral and dorsal site with meninges (black arrows indicate meningeal arteries). E) Cortex without meninges. Scale bar, 1.5 mm.
<img alt="Figure 2" src="/files/ftp_upload/50079/50079fig2.jpg" /> 
Figure 2. Morphological overview of isolated mixed cortical cells and pure astrocyte culture at different timepoints after isolation. A) 1 day after plating of mixed cortical cells. First astrocytes are attached to the bottom of the flask (black arrows) and dying neurons are in the supernatant. B) 3 days after plating of mixed cortical cells. Astrocyte layer is forming (black arrows). Neurons are almost absent. C) 5 days after plating of mixed cortical cells. First microglia and OPCs on top of a astrocyte layer (black arrows). D) 7 days after plating of mixed cortical cells. Astrocyte layer is completely confluent. E) After removing microglia and OPCs by vigorous shaking and 2 days after splitting, attached cells show astrocyte morphology with low density (arrows indicate one cell). F) Astrocyte layer shows high density 2 weeks after the first split. Scale bar, 10 &mu;m.
<img alt="Figure 3" src="/files/ftp_upload/50079/50079fig3.jpg" /> 
Figure 3. Purity of primary astrocyte culture. Immunolabeling of primary mouse astrocyte cultures with the markers GFAP, GLAST, S100B, Aquaporin-4, ALDH1L1 and BLBP (all green) revealed pure primary astrocyte culture. Nuclei are stained with 4',6'-diamidino-2-phenylindole (DAPI) (blue). Scale bar: 10 &mu;m.

Watch this Scientific Journal Video about Isolation and Culture of Mouse Cortical Astrocytes at JoVE.com

Isolation and Culture of Mouse Cortical Astrocytes

Astrocytes have been recognized to be versatile cells participating in fundamental biological processes that are essential for normal brain development and function, and central nervous system repair. Here we present a rapid procedure to obtain pure mouse astrocyte cultures to study the biology of this major class of central nervous system cells.

Astrocytes  are an abundant cell type in the mammalian brain, yet much remains to be  learned about their molecular and functional characteristics. In ...

isolation-and-culture-of-mouse-cortical-astrocytes

University of Wisconsin - Madison

Research

JoVE Journal

Neuroscience

90.8K Views.  University of Freiburg. Astrocytes have been recognized to be versatile cells participating in fundamental biological processes that are essential for normal brain development and function, and central nervous system repair. Here we present a rapid procedure to obtain pure mouse astrocyte cultures to study the biology of this major class of central nervous system cells.

Video: Isolation and Culture of Mouse Cortical Astrocytes

Nucleofection and Primary Culture of Embryonic Mouse Hippocampal and Cortical Neurons

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and synapse formation and function. An advantage of culturing these neurons is that they readily polarize, forming distinctive axons and dendrites, on a two dimensional substrate at very low densities. This property has made them extremely useful for determining many aspects of neuronal development. Furthermore, by providing glial conditioning for these neurons they will continue to develop, forming functional synaptic connections and surviving for several months in culture. In this protocol we outline a technique to dissect, culture and transfect embryonic mouse hippocampal and cortical neurons. Transfection is accomplished by electroporating DNA into the neurons before plating via nucleofection. This protocol has the advantage of expressing fluorescently-tagged fusion proteins early in development (~4-8hrs after plating) to study the dynamics and function of proteins during polarization, axon outgrowth and branching. We have also discovered that this single transfection before plating maintains fluorescently-tagged fusion protein expression at levels appropriate for imaging throughout the lifetime of the neuron (&gt; 2 months in culture). Thus, this methodology is useful for studying protein localization and function throughout CNS development with little or no disruption of neuronal function.

This protocol outlines the steps required to dissect, transfect via electroporation and culture mouse hippocampal and cortical neurons. Short-term cultures may be used for studies of axon outgrowth and guidance, while long-term cultures can be used for studies of synaptogenesis and dendritic spine analysis.

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and ...

Lectin-based Isolation and Culture of Mouse Embryonic Motoneurons

Spinal motoneurons develop towards postmitotic stages through early embryonic nervous system development and subsequently grow out dendrites and axons. Neuroepithelial cells of the neural tube that express Nkx6.1 are the unique precursor cells for spinal motoneurons1. Though postmitotic motoneurons move towards their final position and organize themselves into columns along the spinal tract2,3. More than 90% of all these differentiated and positioned motoneurons express the transcription factors Islet 1/2. They innervate the muscles of the limbs as well as those of the body and the inner organs. Among others, motoneurons typically express the high affinity receptors for brain derived neurotrophic factor (BDNF) and Neurotrophin-3 (NT-3), the tropomyosin-related kinase B and C (TrkB, TrkC). They do not express the tropomyosin-related kinase A (TrkA)4. Beside the two high affinity receptors, motoneurons do express the low affinity neurotrophin receptor p75NTR. The p75NTR can bind all neurotrophins with similar but lower affinity to all neurotrophins than the high affinity receptors would bind the mature neurotrophins. Within the embryonic spinal cord, the p75NTR is exclusively expressed by the spinal motoneurons5. This has been used to develop motoneuron isolation techniques to purify the cells from the vast majority of surrounding cells6. Isolating motoneurons with the help of specific antibodies (panning) against the extracellular domains of p75NTR has turned out to be an expensive method as the amount of antibody used for a single experiment is high due to the size of the plate used for panning. A much more economical alternative is the use of lectin. Lectin has been shown to specifically bind to p75NTR as well7. The following method describes an alternative technique using wheat germ agglutinin for a preplating procedure instead of the p75NTR antibody. The lectin is an extremely inexpensive alternative to the p75NTR antibody and the purification grades using lectin are comparable to that of the p75NTR antibody. Motoneurons from the embryonic spinal cord can be isolated by this method, survive and grow out neurites.

An alternative way of isolating mouse embryonic motoneurons from the spinal cord is described. The method takes into account the fact that lectin can bind to the low affinity nerve growth factor receptor p75NTR. This lectin-based preplating allows a purification similar to that with a specific antibody against the p75NTR.

Spinal motoneurons develop towards postmitotic stages through early embryonic nervous system development and subsequently grow out dendrites and axons. ...

Bilaminar Co-culture of Primary Rat Cortical Neurons and Glia

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or co-culture model. This system is suitable for a variety of experimental needs requiring either a glass or plastic growth substrate and can also be used for culture of other types of neurons.
Rat cortical neurons obtained from the late embryonic stage (E17) are plated on glass coverslips or tissue culture dishes facing a feeder layer of glia grown on dishes or plastic coverslips (known as Thermanox), respectively. The choice between the two configurations depends on the specific experimental technique used, which may require, or not, that neurons are grown on glass (e.g. calcium imaging versus Western blot). The glial feeder layer, an astroglia-enriched secondary culture of mixed glia, is separately prepared from the cortices of newborn rat pups (P2-4) prior to the neuronal dissection.
A major advantage of this culture system as compared to a culture of neurons only is the support of neuronal growth, survival, and differentiation provided by trophic factors secreted from the glial feeder layer, which more accurately resembles the brain environment in vivo. Furthermore, the co-culture can be used to study neuronal-glial interactions1.
At the same time, glia contamination in the neuronal layer is prevented by different means (low density culture, addition of mitotic inhibitors, lack of serum and use of optimized culture medium) leading to a virtually pure neuronal layer, comparable to other established methods1-3. Neurons can be easily separated from the glial layer at any time during culture and used for different experimental applications ranging from electrophysiology4, cellular and molecular biology5-8, biochemistry5, imaging and microscopy4,6,7,9,10. The primary neurons extend axons and dendrites to form functional synapses11, a process which is not observed in neuronal cell lines, although some cell lines do extend processes.
A detailed protocol of culturing rat hippocampal neurons using this co-culture system has been described previously4,12,13. Here we detail a modified protocol suited for cortical neurons. As approximately 20x106 cells are recovered from each rat embryo, this method is particularly useful for experiments requiring large numbers of neurons (but not concerned about a highly homogenous neuronal population). The preparation of neurons and glia needs to be planned in a time-specific manner. We will provide the step-by-step protocol for culturing rat cortical neurons as well as culturing glial cells to support the neurons.

Here we provide a protocol for culturing rat cortical neurons in the presence of a glial feeder layer. The cultured neurons establish polarity and create synapses, and can be separated from the glia for use in various applications, such as electrophysiology, calcium imaging, cell survival assays, immunocytochemistry, and RNA/DNA/protein isolation.

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or ...

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 ...

Isolation and Culture of Human Fungiform Taste Papillae Cells

Taste cells are highly specialized, with unique histological, molecular and physiological characteristics that permit detection of a wide range of simple stimuli and complex chemical molecules contained in foods. In human, individual fungiform papillae contain from zero to as many as 20 taste buds. There is no established protocol for culturing human taste cells, although the ability to maintain taste papillae cells in culture for multiple cell cycles would be of considerable utility for characterizing the molecular, regenerative, and functional properties of these unique sensory cells. Earlier studies of taste cells have been done using freshly isolated cells in primary culture, explant cultures from rodents, or semi-intact taste buds in tissue slices1,2,3,4. Although each of these preparations has advantages, the development of long-term cultures would have provided significant benefits, particularly for studies of taste cell proliferation and differentiation. Several groups, including ours, have been interested in the development and establishment of taste cell culture models. Most attempts to culture taste cells have reported limited viability, with cells typically not lasting beyond 3-5 d5,6,7,8. We recently reported on a successful method for the extended culture of rodent taste cells9. We here report for the first time the establishment of an in vitro culture system for isolated human fungiform taste papillae cells. Cells from human fungiform papillae obtained by biopsy were successfully maintained in culture for more than eight passages (12 months) without loss of viability. Cells displayed many molecular and physiological features characteristic of mature taste cells. Gustducin and phospholipase C &beta;2, (PLC-&beta;2) mRNA were detected in many cells by reverse transcriptase-polymerase chain reaction and confirmed by sequencing. Immunocytochemistry analysis demonstrated the presence of gustducin and PLC-&beta;2 expression in cultured taste cells. Cultured human fungiform cells also exhibited increases in intracellular calcium in response to appropriate concentrations of several taste stimuli indicating that taste receptors and at least some of the signalling pathways were present. These results sufficient indicate that taste cells from adult humans can be generated and maintained for at least eight passages. Many of the cells retain physiological and biochemical characteristics of acutely isolated cells from the adult taste epithelium to support their use as a model taste system. This system will enable further studies of the processes involved in proliferation, differentiation and function of mammalian taste receptor cells in an in vitro preparation.
Human fungiform taste papillae used for establishing human fungiform cell culture were donated for research following proper informed consent under research protocols that were reviewed and approved by the IRB committee. The protocol (#0934) was approved by Schulman Associates Institutional Review Board Inc., Cincinnati, OH. Written protocol below is based on published parameters reported by Ozdener et al. 201110.

We aimed to develop a reproducible protocol for isolating and maintaining long-term cultures of human fungiform taste papillae cells. Cells from human fungiform papillae obtained by biopsy were successfully maintained in culture for more than eight passages (12 months) without loss of viability.

Taste cells are highly specialized, with unique histological, molecular and physiological characteristics that permit detection of a wide range of simple ...

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease pathogenesis and their utility for preclinical drug development. In order to design a better model system for these applications, phenotypically wild-type cortical astrocytes and neural stem cells (NSC) from conditional, genetically engineered mice (GEM) that harbor various combinations of floxed oncogenic alleles were harvested and grown in culture. Genetic recombination was induced in vitro using adenoviral Cre-mediated recombination, resulting in expression of mutated oncogenes and deletion of tumor suppressor genes. The phenotypic consequences of these mutations were defined by measuring proliferation, transformation, and drug response in vitro. Orthotopic allograft models, whereby transformed cells are stereotactically injected into the brains of immune-competent, syngeneic littermates, were developed to define the role of oncogenic mutations and cell type on tumorigenesis in vivo. Unlike most established human glioblastoma cell line xenografts, injection of transformed GEM-derived cortical astrocytes into the brains of immune-competent littermates produced astrocytomas, including the most aggressive subtype, glioblastoma, that recapitulated the histopathological hallmarks of human astrocytomas, including diffuse invasion of normal brain parenchyma. Bioluminescence imaging of orthotopic allografts from transformed astrocytes engineered to express luciferase was utilized to monitor in vivo tumor growth over time. Thus, astrocytoma models using astrocytes and NSC harvested from GEM with conditional oncogenic alleles provide an integrated system to study the genetics and cell biology of astrocytoma pathogenesis in vitro and in vivo and may be useful in preclinical drug development for these devastating diseases.

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Phenotypically wild-type astrocytes and neural stem cells harvested from mice engineered with floxed, conditional oncogenic alleles and transformed via viral Cre-mediated recombination can be used to model astrocytoma pathogenesis in vitro and in vivo by orthotopic injection of transformed cells into brains of syngeneic, immune-competent littermates.

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease ...

Sex Differences in Mouse Hippocampal Astrocytes after In-Vitro Ischemia

Astrogliosis following hypoxia/ischemia (HI)-related brain injury plays a role in increased morbidity and mortality in neonates. Recent clinical studies indicate that the severity of brain injury appear to be sex dependent, and that the male neonates are more susceptible to the effects of HI-related brain injury, resulting in more severe neurological outcomes as compared to females with comparable brain injuries. The development of reliable methods to isolate and maintain highly enriched populations of sexed hippocampal astrocytes is essential to understand the cellular basis of sex differences in the pathological consequences of neonatal HI. In this study, we describe a method for creating sex specific hippocampal astrocyte cultures that are subjected to a model of in-vitro ischemia, oxygen-glucose deprivation, followed by reoxygenation. Subsequent reactive astrogliosis was examined by immunostaining for the Glial Fibrillary Acidic Protein (GFAP) and S100B. This method provides a useful tool to study the role of male and female hippocampal astrocytes following neonatal HI, separately.

Sex Differences in Mouse Hippocampal Astrocytes after In-Vitro Ischemia

Astrocytes are one of the most important key players in the central nervous system (CNS). Here, we are reporting a practical method of sexed hippocampal astrocyte culture protocol in order to study the mechanisms underlying the astrocyte function in male and female neonate pups after in-vitro ischemia.

Astrogliosis following hypoxia/ischemia (HI)-related brain injury plays a role in increased morbidity and mortality in neonates. Recent clinical studies ...

Isolation and Culture of Adult Neural Stem Cells from the Mouse Subcallosal Zone

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation into three types of cells: neurons, astrocytes, and oligodendrocytes. Identifying aNSC-derived regions and characterizing the aNSC properties are critical for the potential use of aNSCs and for the elucidation of their role in neural regeneration. The subcallosal zone (SCZ), located between white matter and the hippocampus, has recently been reported to contain aNSCs and continuously give rise to neuroblasts. A low percentage of aNSCs from the SCZ is differentiated into neurons; most cells are differentiated into glial cells, such as oligodendrocytes and astrocytes. These cells are suggested to have a therapeutic potential for traumatic cortical injury. This protocol describes in detail the process to generate SCZ-aNSCs from an adult mouse brain. A brain matrix with intervals of 1 mm is used to obtain the SCZ-containing coronal slices and to precisely dissect the SCZ from the whole brain. The SCZ sections are initially subjected to a neurosphere culture. A well-developed culture system allows for the verification of their characteristics and can increase research on NSCs. A neurosphere culture system provides a useful tool for determining proliferation and collecting the genuine NSCs. A monolayer culture is also an in vitro system to assay proliferation and differentiation. Significantly, this culture system provides a more homogenous environment for NSCs than the neurosphere culture system. Thus, using a discrete brain region, these culture systems will be helpful for expanding our knowledge about aNSCs and their applications for therapeutic uses.

Establishing culture systems for the expansion of adult neural stem cells (aNSCs) allows for the examination and application of aNSCs for therapy. The subcallosal zone (SCZ) has recently been recognized as a novel neuroblast-forming region in adult mice. Here, methods for the isolation, expansion, and differentiation of SCZ-aNSCs are described.

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation ...

Differentiation of Mouse Embryonic Stem Cells into Cortical Interneuron Precursors

GABAergic cortical interneurons are a heterogeneous population of cells that play critical roles in regulating the output of excitatory pyramidal neurons as well as synchronizing the outputs of pyramidal neuron ensembles. Deficits in interneuron function have been implicated in a variety of neuropsychiatric disorders, including schizophrenia, autism, and epilepsy. The derivation of cortical interneurons from embryonic stem cells not only allows for the study of their development and function, but provides insight into the molecular mechanisms underlying the pathogenesis of cortical interneuron-related disorders. Interneurons also have the remarkable capacity to survive, migrate, and integrate into host cortical circuitry post-transplantation, making them ideal candidates for use in cell-based therapies. Here, we present a scalable, highly efficient, modified embryoid body-to-monolayer method for the derivation of Nkx2.1-expressing interneuron progenitors and their progeny from mouse embryonic stem cells (mESCs). Using a Nkx2.1::mCherry:Lhx6::GFP dual reporter mESC line, Nkx2.1 progenitors or their Lhx6-expressing post-mitotic progeny can be isolated via fluorescence-activated cell sorting (FACS) and subsequently used in a number of downstream applications. We also provide methods to enrich for parvalbumin (PV) or somatostatin (SST) interneuron subgroups, which may be helpful for studying aspects of fate determination or for use in therapeutic applications that would benefit from interneuron subgroup-enriched transplantations.

This protocol describes a method for generating cortical interneuron progenitors and post-mitotic interneuron precursors from mouse embryonic stem cells using a modified embryoid body-to-monolayer method. These progenitors/precursors can be used in vitro or fluorescently sorted and transplanted into neonatal neocortex for studying fate determination, or used in therapeutic applications.

GABAergic cortical interneurons are a heterogeneous population of cells that play critical roles in regulating the output of excitatory pyramidal neurons ...

Isolation and Culture of Embryonic Mouse Neural Stem Cells

Neural stem cells (NSCs) are multipotent and can give rise to the three major cell types of the central nervous system (CNS). In vitro culture and expansion of NSCs provide a suitable source of cells for neuroscientists to study the function of neurons and glial cells along with their interactions. There are several reported techniques for the isolation of neural stem cells from adult or embryo mammalian brains. During the microsurgical operation to isolate NSCs from different regions of the embryonic CNS, it is very important to reduce the damage to the brain cells to obtain the highest ratio of live and expandable stem cells. A possible technique for stress reduction during isolation of these cells from the mouse embryo brain is the reduction of surgical time. Here, we demonstrate a developed technique for rapid isolation of these cells from the E13 mouse embryo ganglionic eminence. Surgical procedures include harvesting E13 mouse embryos from the uterus, cutting the frontal fontanelle of the embryo with a bent needle tip, extracting the brain from the skull, microdissection of the isolated brain to harvest the ganglionic eminence, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in suspension culture to generate neurospheres.

Here, we introduce a novel microsurgical technique for the isolation of neural stem cells from the E13 mouse embryo ganglionic eminence.