Name: isolation and culture of mouse cortical astrocytes
Uploaded: 2013-01-19T14:00:00
Description: Watch this Scientific Journal Video about Isolation and Culture of Mouse Cortical Astrocytes at JoVE.com

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-inac...

<ol>
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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 cor...

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.</...

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 ...

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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 ...

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 ...

Isolation of Cerebrospinal Fluid from Rodent Embryos for use with Dissected Cerebral Cortical Explants

The CSF is a complex fluid with a dynamically varying proteome throughout development and in adulthood. During embryonic development, the nascent CSF ...

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

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 ...

Sex Differences in Mouse Hippocampal Astrocytes after In-Vitro Ischemia

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 ...

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 ...