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Neuroscience

Isolation and Culture of Mouse Cortical Astrocytes

Published: January 19th, 2013

DOI:

10.3791/50079

1Institute of Anatomy and Cell Biology, University of Freiburg , 2Centre of Chronic Immunodeficiency (CCI), University Medical Centre Freiburg, 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.

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.

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 informati....

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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.

  1. Before starting the dissection procedure, prewarm 30 ml of astrocyte culture media (DMEM, high glucose + 10% heat-inac.......

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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.

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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.......

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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.

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

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