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>

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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><tbody><tr><td colspan="4">&lt;strong&gt;&lt;em&gt;Astrocyte culture media&lt;/em&gt;&lt;/strong&gt;</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 colspan="4">&lt;strong&gt;&lt;em&gt;Solution for brain tissue digestion&lt;/em&gt;&lt;/strong&gt;</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 colspan="4">&lt;strong&gt;&lt;em&gt;Other&lt;/em&gt;&lt;/strong&gt;</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 &amp;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 &amp;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 cm&lt;sup&gt;2&lt;/sup&gt; 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 &amp;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="Brain section analysis, fluorescence microscopy images, highlighting specific tissue regions with arrows." src="/files/ftp_upload/50079/50079fig1.jpg" data-altupdatedat="1747911167000" data-alt="Figure 1"> 
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="Cell differentiation stages; microscope image; Div 1-21 visual comparisons; cellular development." src="/files/ftp_upload/50079/50079fig2.jpg" data-altupdatedat="1747911167000" data-alt="Figure 2"> 
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 μm.
<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 μm.

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

Nanyang Technological University

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Neuroscience

91.5K Views.  University of Freiburg. Astrocyten zijn erkend als veelzijdige cellen die deelnemen in de fundamentele biologische processen die essentieel zijn voor de normale ontwikkeling van de hersenen en functies binnen het centraal zenuwstelsel repareren. Hier presenteren we een snelle procedure voor pure muis astrocyten culturen te verkrijgen om de biologie van deze belangrijke klasse van het centrale zenuwstelsel cellen te bestuderen.

Video: Isolation and Culture of Mouse Cortical Astrocytes

Isolation of Cortical Microglia with Preserved Immunophenotype and Functionality From Murine Neonates

Isolation of microglia from CNS tissue is a powerful investigative tool used to study microglial biology ex vivo. The present method details a procedure for isolation of microglia from neonatal murine cortices by mechanical agitation with a rotary shaker. This microglia isolation method yields highly pure cortical microglia that exhibit morphological and functional characteristics indicative of quiescent microglia in normal, nonpathological conditions in vivo. This procedure also preserves the microglial immunophenotype and biochemical functionality as demonstrated by the induction of morphological changes, nuclear translocation of the p65 subunit of NF-&#954;B (p65), and secretion of the hallmark proinflammatory cytokine, tumor necrosis factor-&#945; (TNF-&#945;), upon lipopolysaccharide (LPS) and Pam3CSK4 (Pam) challenges. Therefore, the present isolation procedure preserves the immunophenotype of both quiescent and activated microglia, providing an experimental method of investigating microglia biology in ex vivo conditions.

One key to successful investigation of microglial biology is the preservation of microglial immunofunction ex vivo during isolation from CNS tissue. Isolating microglia via rotary shaking results in highly pure and immunofunctional cell cultures as assessed by fluorescent imaging, immunocytochemistry, and ELISA following microglia activation with the proinflammatory stimuli lipopolysaccharide (LPS) and Pam3CSK4 (Pam).

Isolation of microglia from CNS tissue is a powerful investigative tool used to study microglial biology ex vivo. The present method details a procedure ...

Immunology and Infection

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 ...

The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

Proper neuronal development and function is the prerequisite of the developing and the adult brain. However, the mechanisms underlying the highly controlled formation and maintenance of complex neuronal networks are not completely understood thus far. The open questions concerning neurons in health and disease are diverse and reaching from understanding the basic development to investigating human related pathologies, e.g.,&#160;Alzheimer&#39;s disease and&#160;Schizophrenia. The most detailed analysis of neurons can be performed in vitro. However, neurons are demanding cells and need the additional support of astrocytes for their long-term survival. This cellular heterogeneity is in conflict with the aim to dissect the analysis of neurons and astrocytes. We present here a cell-culture assay that allows for the long-term cocultivation of pure primary neurons and astrocytes, which share the same chemically defined medium, while being physically separated. In this setup, the cultures survive for up to four weeks and the assay is suitable for a diversity of investigations concerning neuron-glia interaction.

The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

This protocol describes the indirect neuron-astrocyte coculture for compartmentalized analysis of neuron-glia interactions.

Proper neuronal development and function is the prerequisite of the developing and the adult brain. However, the mechanisms underlying the highly ...

Visualizing and Analyzing Intracellular Transport of Organelles and Other Cargos in Astrocytes

Astrocytes are among the most abundant cell types in the adult brain, where they play key roles in a multiplicity of functions. As a central player in brain homeostasis, astrocytes supply neurons with vital metabolites and buffer extracellular water, ions, and glutamate. An integral component of the &#8220;tri-partite&#8221; synapse, astrocytes are also critical in the formation, pruning, maintenance, and modulation of synapses. To enable these highly interactive functions, astrocytes communicate among themselves and with other glial cells, neurons, the brain vasculature, and the extracellular environment through a multitude of specialized membrane proteins that include cell adhesion molecules, aquaporins, ion channels, neurotransmitter transporters, and gap junction molecules. To support this dynamic flux, astrocytes, like neurons, rely on tightly coordinated and efficient intracellular transport. Unlike neurons,&#160;where intracellular trafficking has been extensively delineated, microtubule-based transport in astrocytes has been less studied. Nonetheless, exo- and endocytic trafficking of cell membrane proteins and intracellular organelle transport orchestrates astrocytes&#8217; normal biology, and these processes are often affected in disease or in response to injury. Here we present a straightforward protocol to culture high quality murine astrocytes, to fluorescently label astrocytic proteins and organelles of interest, and to record their intracellular transport dynamics using time-lapse confocal microscopy. We also demonstrate how to extract and quantify relevant transport parameters from the acquired movies using available image analysis software (i.e., ImageJ/FIJI) plugins.

Here we describe an in vitro live-imaging method to visualize intracellular transport of organelles and trafficking of plasma membrane proteins in murine astrocytes. This protocol also presents an image analysis methodology to determine cargo transport itineraries and kinetics.

Astrocytes are among the most abundant cell types in the adult brain, where they play key roles in a multiplicity of functions. As a central player in ...

Isolation and Direct Neuronal Reprogramming of Mouse Astrocytes

Direct neuronal reprogramming is a powerful approach to generate functional neurons from different starter cell populations without passing through multipotent intermediates. This technique not only holds great promises in the field of disease modeling, as it allows to convert, for example, fibroblasts for patients suffering neurodegenerative diseases into neurons, but also represents a promising alternative for cell-based replacement therapies. In this context, a major scientific breakthrough was the demonstration that differentiated non-neural cells within the central nervous system, such as astrocytes, could be converted into functional neurons in vitro. Since then, in vitro direct reprogramming of astrocytes into neurons has provided substantial insights into the molecular mechanisms underlying forced identity conversion and the hurdles that prevent efficient reprogramming. However, results from in vitro&#160;experiments performed in different labs are difficult to compare due to differences in the methods used to isolate, culture, and reprogram astrocytes. Here, we describe a detailed protocol to reliably isolate and culture astrocytes with high purity from different regions of the central nervous system of mice at postnatal ages via magnetic cell sorting. Furthermore, we provide protocols to reprogram cultured astrocytes into neurons via viral transduction or DNA transfection. This streamlined and standardized protocol can be used to investigate the molecular mechanisms underlying cell identity maintenance, the establishment of a new neuronal identity, as well as the generation of specific neuronal subtypes and their functional properties.

Here we describe a detailed protocol to generate highly enriched cultures of astrocytes derived from different regions of the central nervous system of postnatal mice and their direct conversion into functional neurons by the forced expression of transcription factors.

Direct neuronal reprogramming is a powerful approach to generate functional neurons from different starter cell populations without passing through ...

Efficient Extraction of Astrocytes and Microglia from the Adult Mouse Spinal Cord

Isolation of Pure Astrocytes and Microglia from the Adult Mouse Spinal Cord For In Vitro Assays and Transcriptomic Studies

Astrocytes and microglia play pivotal roles in central nervous system development, injury responses, and neurodegenerative diseases. These highly dynamic cells exhibit rapid responses to environmental changes and display significant heterogeneity in terms of morphology, transcriptional profiles, and functions. While our understanding of the functions of glial cells in health and disease has advanced substantially, there remains a need for in vitro, cell-specific analyses conducted in the context of insults or injuries to comprehensively characterize distinct cell populations. Isolating cells from the adult mouse offers several advantages over cell lines or neonatal animals, as it allows for the analysis of cells under pathological conditions and at specific time points. Furthermore, focusing on spinal cord-specific isolation, excluding brain involvement, enables research into spinal cord pathologies, including experimental autoimmune encephalomyelitis, spinal cord injury, and amyotrophic lateral sclerosis. This protocol presents an efficient method for isolating astrocytes and microglia from the adult mouse spinal cord, facilitating immediate or future analysis with potential applications in functional, molecular, or proteomic downstream studies.

Author Spotlight: Isolation of Glial Cells from the Adult Mouse Spinal Cord 

This protocol outlines the isolation of purified astrocytes and microglia from the adult mouse spinal cord, facilitating subsequent applications such as RNA analysis and cell culture. It includes detailed cell dissociation methods and procedures designed to enhance both the quality and yield of isolated cells.

Astrocytes and microglia play pivotal roles in central nervous system development, injury responses, and neurodegenerative diseases. These highly dynamic ...

Isolation of Microglia from the Cortex of an Adult Mouse Brain

Source: Zhou, L., et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/60347">Isolation of Region-specific Microglia from One Adult Mouse Brain Hemisphere for Deep Single-cell RNA Sequencing.</a> J. Vis. Exp. (2019)
The video demonstrates a technique for isolating microglia from the cortex of an adult mouse brain. First, the cortex undergoes segmentation and homogenization to remove neurons and other glial cells, preserving microglia. Following that, the cell suspension is exposed to anti-myelin magnetic microbeads and a magnetic field, effectively separating microglia and myeloid cells from myelin debris and cells containing myelin.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Mechanical Tissue Dissociation
...

JoVE Encyclopedia of Experiments

Neuronal Culture Techniques

Primary Neuronal Culture

A Simple Technique to Isolate and Culture Murine Astrocytes

Source: Wolfes, A. C., et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/56092">Culturing In Vivo-like Murine Astrocytes Using the Fast, Simple, and Inexpensive AWESAM Protocol.</a> J. Vis. Exp. (2018)
The video demonstrates a simple method to culture murine astrocytes in isolation from other brain cells. During culturing, by using astrocyte-specific media and inhibiting the adherence of non-astrocytic cells, the method selectively cultures astrocytes. These cultured astrocytes exhibit stellate morphology with fine processes.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Rat or Mouse Brain Dissection
...

Astrocyte Isolation: A Method to Obtain Pure Preparation of Mouse Cortical Astrocytes

Source: Schildge, S. et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/v/50079/isolation-and-culture-of-mouse-cortical-astrocytes">Isolation and Culture of Mouse Cortical Astrocytes.</a> J. Vis. Exp. (2013)
In this video, we describe the method to obtain a pure preparation of astrocytes from newly born mice.

Source: Schildge, S. et al. Isolation and Culture of Mouse Cortical Astrocytes. J. Vis. Exp. (2013)
In this video, we describe the method to obtain a pure ...

Isolation and Culture of Mouse Cortical Astrocytes

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