Isolation and Culture of Mouse Cortical Astrocytes

Summary

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.

Abstract

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.

Introduction

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 development^4-6, 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.

Protocol

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-inactivated fetal bovine serum + 1% Penicillin/Streptomycin; see table) to 37 °C. Coat one T75 flask with 20 ml of poly-D-lysine (PDL) at a concentration of 50 μg/ml in cell culture grade water for 1 hr at 37 °C in the CO₂ incubator.
2. 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.
3. Gently hold and spray the head and neck of the mouse pup with 70% ethanol. The animal is sacrificed by decapitation using the scissors.
4. Perform a midline incision, posterior to anterior, along the scalp to reveal the skull.
5. 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.
6. 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.
7. 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).
8. 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.
9. 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.
10. Finally, cut each cortex hemisphere into small pieces using sharp blades (approximately 4 to 8 times).
11. Under sterile conditions, transfer cortex pieces into one 50 ml Falcon tube and add HBSS to a total volume of 22.5 ml.
12. Add 2.5 ml of 2.5 % trypsin, mix and incubate the tissue in the water bath at 37 °C for 30 min. Mix by occasional shaking every 10 min.
13. Centrifuge for 5 min at 300 x g to pellet cortex tissue pieces.
14. 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.
15. Aspirate PDL from the T75 culture flask, plate the dissociated single cell suspension and incubate at 37 °C in the CO₂ incubator.

2. Obtaining an Enriched Astrocyte Culture

1. Change the medium 2 days after plating of the mixed cortical cells and all 3 days thereafter.
2. 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.
3. 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.
4. 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 °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).
5. 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 °C in the CO₂ incubator. Change the medium every 2 to 3 days.
6. 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.

Results

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.

Discussion

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...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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