Name: culturing in vivo-like murine astrocytes using the fast, simple, and inexpensive awesam protocol
Uploaded: 2018-01-10T13:00:00.000+00:00
Duration: 7 min 56 s
Description: Watch this Scientific Journal Video about Culturing In Vivo-like Murine Astrocytes Using the Fast, Simple, and Inexpensive AWESAM Protocol at JoVE.com

We acknowledge funding from the European Research Council (FP7/260916), the Alexander von Humboldt-Stiftung (Sofja Kovalevskaja), and the Deutsche Forschungsgemeinschaft (DE1951 and SFB889).

All animal experiments described here were performed in accordance with the guidelines for German animal welfare.
NOTE: The timeline and main steps of the protocol are illustrated in Figure 1.
1. Preparations
<ol>
	<li>Prepare the following solutions.
	<ol>
		<li>Prepare DMEM+ for culturing polygonal MD astrocytes: DMEM with 10% FCS, 100 U penicillin and 100 &#181;g/mL streptomycin.</li>
		<li>Prepare NB+H for culturing stellate astrocytes: neurobasal medium with&#160;5 ng/mL HBEGF, 1x B-27 supplement, 1x glutamine supplement, 100 U penicillin and 100 &#181;g/mL streptomycin. 
		NOTE: Aliquots of higher concentrations of HBEGF can be prepared and stored at -20 &#176;C to be added to the medium whenever needed.</li>
		<li>Prepare the dissection medium (10 mM HEPES in HBSS).</li>
		<li>Prepare aliquots of 6-7 mL of 0.05% trypsin-EDTA and 6-7 mL 0.25% trypsin-EDTA in 15 mL tubes. These can be stored at -20 &#176;C and thawed in a 37 &#176;C water bath.</li>
	</ol>
	</li>
	<li>The day before starting the protocol, coat 10 cm tissue culture-treated dishes with 5 mL of 0.04% polyethylenimine (PEI) each.</li>
	<li>The next day, remove the PEI and wash the dishes with deionized, distilled water twice. Add 12 mL of DMEM+ to each plate and pre-equilibrate in the incubator at 37 &#176;C and 5% CO2 for at least 30 min before plating cells.</li>
	<li>Prepare the dissection area: In a laminar flow dissection hood, place two 10 cm Petri dishes filled with dissection medium on ice. For each brain area to be isolated (e.g., cortex, hippocampus), prepare a 15-&#160; tube filled with 14 mL dissection medium and keep on ice. Spray dissection tools (fine scissors, Dumont #3 and #5 forceps) with 70% ethanol and place next to dissection microscope in the hood.</li>
</ol>
2. Rat or Mouse Brain Dissection
NOTE: Both mice and rats can be used. Animals older than P8 may also be used (we tested up to P12), but removing the meninges will become increasingly difficult. Further, the cell yield and health will diminish, since brain cells from older animals will have formed intricate processes and connections that may break during tissue dissociation, leading to increased cell death. Cortical preparations are described here, but other brain regions can also be used.
<ol>
	<li>Place 0.05% trypsin-EDTA in a 37 &#176;C water bath.</li>
	<li>Euthanize animals (pregnant rats for E19 pups, or P0-P8 animals) by slowly raising CO2 concentration, and isolate the brains. The following steps require sterile technique.</li>
	<li>If working with embryonic tissue, first remove the embryos from the uterus and open the embryonic sacs, then immediately cut off the heads with scissors. When all heads are cut off, transfer them into pre-cooled dissection medium.
	<ol>
		<li>Once the heads are submerged in the medium, open the skin and skull (both are very soft in young animals) with forceps and pinch off the cerebellum. Carefully lever the rest of the brain up and out of the skull. 
		NOTE: Two to four E19 embryos yield enough tissue for at least eight 10 cm dishes of cortical astrocytes plated at 500,000 cells per dish on the day of dissection.</li>
	</ol>
	</li>
	<li>For newborn or older pups, cut off the heads with scissors, and isolate the brains by medially cutting the skin in a straight line from the back of the head to the tip of the nose. Pull the skin aside and carefully cut the skull left and right, then lift the skull bone up. Cut off the cerebellum, and lever the rest of the brain up and out of the skull. 
	NOTE: Two to three P0-P8 pups yield enough tissue for at least&#160;eight 10 cm dishes of cortical astrocytes plated at&#160;500,000 cells per dish on the day of dissection.</li>
	<li>Separate the cortex from the underlying midbrain structure. Place the forceps within the longitudinal fissure between the hemispheres, then move the forceps in between the cortex and midbrain structures of one hemisphere (perpendicular to the longitudinal fissure) to pinch each hemisphere free.</li>
	<li>Using the forceps, remove all meninges from each hemisphere (and its hippocampus), then separate the hippocampi. If hippocampal astrocytes are required, move each hippocampus into a 15 mL tube filled with 14 mL dissection medium on ice.</li>
	<li>Remove further areas from each hemisphere&#39;s cortex if specific regions are required (e.g., frontal cortex). Cut any cortical tissue to be used for generating astrocytes into pieces no larger than 1 mm3. Collect all pieces of cortical tissue in a separate 15-mL tube filled with dissection medium on ice.</li>
</ol>
3. Tissue Digestion and Dissociation
<ol>
	<li>Once all tissue pieces are collected, wait for them to settle to the bottom of the tube, then aspirate as much medium as possible.</li>
	<li>Add 2-3 mL of 0.05% trypsin-EDTA using a serological pipette, and incubate the tubes in a 37 &#176;C water bath for 20 min. 
	NOTE: Release all trypsin-EDTA from the pipette swiftly to mix the tissue pieces and allow to settle again.</li>
	<li>Carefully aspirate the trypsin-EDTA, leaving tissue pieces in as little liquid as possible, at the bottom of the tube.</li>
	<li>Add 5 mL of pre-cooled dissection medium to wash off the remaining trypsin-EDTA. Pipette to the side of the upper part of the tube to achieve mixing of the tissue pieces.</li>
	<li>Aspirate the dissection medium, and leave the tissue pieces in as little liquid as possible. Repeat this washing step with pre-cooled dissection medium twice more.</li>
	<li>After the final washing step, add 1 mL of DMEM+ (warmed to room temperature) using a 1,000 &#181;L pipette tip, and triturate the tissue by pipetting up and down without introducing bubbles. 
	NOTE: It is important to avoid producing bubbles, as this may change the pH of the solution, to which astrocytes are quite sensitive.</li>
	<li>Place a 100 &#181;m cell strainer in the opening of a 50-mL tube and pre-wet the filter with 4.5 mL of pre-cooled DMEM+.</li>
	<li>Pipette the 1 mL of dissociated tissue suspension onto the cell strainer and add another 4.5 mL pre-cooled DMEM+ to wash the cells through the cell strainer.</li>
</ol>
4. Cell Plating for Astrocyte Enrichment by Passaging
<ol>
	<li>Count the cells, e.g., by diluting 10 &#181;L of the cell suspension with 10 &#181;L trypan blue and pipetting the mix onto a hemocytometer.</li>
	<li>Plate 500,000 cells in 12 mL DMEM+ per 10-cm dish. 
	NOTE: DMEM+ is helpful for astrocytic growth, more so than for neurons or microglia. During shaking, the physical forces acting on glass coverslips within wells are much stronger than those acting on the 10-cm dishes (without coverslips). If cells are shaken on 24-well plates, they become unhealthy or die. Only plate astrocytes on 24-well plates after shaking.</li>
	<li>Keep cell cultures in the incubator at 37 &#176;C and 5% CO2 for seven days.</li>
	<li>On day 6, the day before shaking and passaging cells, prepare the PEI-coated culture plates as described in step 5.</li>
</ol>
5. Passaging Cells
<ol>
	<li>The day before passaging, coat cell culture plates with 0.04% PEI.
	<ol>
		<li>For immunocytochemistry, viral transduction, or plasmid transfection, use 24-well or 6-well plates (each containing a sterilized 12 mm or 25 mm glass coverslip, respectively).</li>
		<li>Sterilize the glass coverslips by incubation in 70% ethanol for at least 1 h, then wash three times in deionized, distilled water before placing the coverslips in the wells.</li>
		<li>Add at least 70 &#181;L 0.04% PEI per 12-mm coverslip in 24-well plates, and 600 &#181;L 0.04% PEI per 25 mm coverslip in 6-well plates.</li>
		<li>For Western blot or RNA-Seq analysis, the 10 cm dishes already containing the cells after the dissection can continue to be used. Dishes only need to be shaken, then the cells are washed with 1x PBS and treated with NB+H (do not transfer to new dishes).</li>
	</ol>
	</li>
	<li>On day in vitro (DIV) 7 after the dissection, place the 10-cm dishes with cells in DMEM+ on a laboratory shaker in the incubator and shake the dishes at 110 rpm for 6 h.</li>
	<li>Wash off the PEI from coated coverslips with deionized, distilled water twice. Add NB+H medium to the plates (500 &#181;L per well for 24-well plates and 2 mL per well for 6-well plates). Pre-equilibrate the plates in the incubator at 37 &#176;C and 5% CO2 while the 10 cm dishes are shaking, or for at least 30 min.</li>
	<li>20-30 min before the end of the 6 h shaking, pre-warm 1x PBS, DMEM+, NB+H, and 0.25% trypsin-EDTA to 37 &#176;C in the water bath.</li>
	<li>After 6 h of shaking, take the culture dishes off the shaker and immediately replace the medium (that contains shaken-off neurons, oligodendrocytes, and microglia) with 10 mL pre-warmed 1x PBS per dish. 
	NOTE: It is important to immediately aspirate the old medium containing detached cells to avoid the non-astrocytic cells re-attaching.</li>
	<li>Remove the PBS and add 3 mL pre-warmed trypsin-EDTA per dish. Incubate each dish for 4 min in the incubator at 37 &#176;C and 5% CO2. 
	NOTE: If preparing samples for Western blot or RNA-Seq, do not add trypsin-EDTA. Instead, replace the PBS with 12 mL pre-warmed NB+H, after which cultures can be placed back in the incubator.</li>
	<li>Add 5 mL pre-warmed DMEM+ to the 3 mL trypsin-EDTA in each dish, pipette the cells off the dish using a 5 mL serological pipette, and collect the cell suspension in a 50 mL tube. 
	NOTE: Pipette directly from the bottom of the dish - the floor of the dish should become clearer as the cells detach. Only use serum-containing medium (in place of DMEM+), as serum inactivates trypsin.</li>
	<li>Centrifuge the cell suspension at 3,220 x g at 20 &#176;C for 4 min.</li>
	<li>Remove the supernatant and resuspend the cell pellet in 1 mL pre-warmed NB+H.</li>
</ol>
6. Cell Plating for Final Astrocyte Cultures
<ol>
	<li>Count the cells.</li>
	<li>Plate 20,000 cells per well in 6-well plates (in 2 mL NB+H medium per well) or 5,000 cells per well in 24-well plates (in 500 &#181;L NB+H medium per well).</li>
	<li>Keep the cell cultures in the incubator at 37 &#176;C and 5% CO2 until further treatment or analysis.</li>
	<li>Exchange half the medium once a week. 
	NOTE: In low-nutrient environments, astrocytes stop proliferating but any remaining microglia will proliferate (although in our experience, no microglia appeared in AWESAM cultures). Exchanging half the medium once a week prevents enrichment of any potentially remaining microglia8, and the monocultures remain healthy for at least three weeks.</li>
</ol>

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The authors have nothing to disclose.

Four steps within the protocol are critical: 1) preparing the HBEGF concentration to precisely 5 ng/mL, since small increases in the concentration can lead to immature cultures, e.g., 10 ng/mL HBEGF will de-differentiate astrocytes4; 2) avoiding bubbles in the solutions that contain tissue or cells, which can change the pH and impede astrocyte health; 3) pre-equilibrating media to achieve the optimal pH and temperature for growing healthy astrocytes; 4) exchanging only half the medium once a week because any remaining microglia are less likely to proliferate when sufficient nutrients are present and the medium is exchanged regularly8 (although no microglia were found in AWESAM cultures so far). For this last point, it is important to note that astrocyte-derived factors within the culture should not be entirely removed, as they may support astrocytic survival, differentiation, and proliferation.
It is important to use DIV14 or older cultures as the mature astrocyte source (but for transduction or transfection steps, earlier cultures can be used) because astrocytes younger than DIV14 still have neural stem cell potential9. For viral transduction, mixed cultures of neurons and astrocytes transduced on DIV7 (using adeno-associated viruses) efficiently express virally transduced GFP10. For plasmid transfection, DIV10-DIV24 astrocytes are often used and fluorophore-tagged proteins imaged 2-5 days later11. In our experience, both viral transduction and plasmid transfection work best if astrocytes are transduced between DIV9 and DIV14. The transfection and transduction efficiency depend on the construct and method, but we find that transfection efficiency is ~20% in astrocyte monocultures using lipofectamine transfection, and adeno-associated virus (AAV) transduction efficiency is ~90% (see our previous work4 for further details, e.g., construct used). When transducing astrocytes with AAV particles and transfecting by lipofectamine, we allowed at least 5 and 2 days, respectively, for expression of the construct. Before transduction or transfection, the astrocytes should also be allowed to recover for at least one day after passaging.
Moreover, when preparing cultures for comparing RNA/protein expression at different time points, cultures can be plated at different densities to achieve similar amounts of material, e.g., astrocyte monocultures to be harvested at DIV7 and DIV21 can be plated at densities of 1 x 106 and 500,000 cells per 10-cm dish, respectively. For example, the whole protein lysate yield will be around 0.5 mg/mL on DIV14 when harvested after initially plating 500,000 AWESAM astrocytes on a 10-cm dish.
The AWESAM protocol represents a fast, simple, and inexpensive way to culture astrocytes in isolation from neurons but with in vivo-like characteristics of RNA and protein expression, morphology, and Ca2+ signaling4, which thus far is provided by no other protocol. AWESAM astrocytes can be used for analyses that are typical for cell culture studies, including immunocytochemistry, immunoblotting, RNA expression analysis, TIRF microscopy, and Ca2+ imaging4. In particular, recent advances in genetically encoded Ca2+ indicators allow for visualizing fine astrocytic processes in greater detail than classical Ca2+ dyes14. Ca2+ signaling, vesicular uptake, and exocytosis in both thick and thin astrocytic processes is physiologically relevant, as these processes can influence brain function in terms of trophic support, blood flow, synaptic signaling and plasticity, and intercellular communication. The AWESAM protocol allows for the study of the fine astrocytic processes relevant for brain physiology15 and disease16,17, without neuronal interference and with the great accessibility that in vitro culture supplies.

Astrocytes influence brain function through trophic support, blood flow, synaptic signaling and plasticity, and intercellular communication - all of which are mechanisms that center around the thin astrocytic processes. The AWESAM protocol described here allows the study of these processes without interference from neurons and other glia, which is useful e.g., because the expression of many proteins and even Ca2+ signaling overlap across different brain cell types. Further, this method overcomes limitations of previously available techniques. In particular, our protocol provides in vivo-like morphology (stellate astrocytes with thin processes) and gene expression in a quick, easy, and cheap manner.
Cell morphology, gene expression, and many other regulatory processes are directly influenced by the environment. In a culture dish, this refers to factors released by surrounding cells, but also the medium in which the cells are grown. For astrocytes, HBEGF was previously reported to induce a stellate morphology1 but was also found to de-differentiate astrocytes2. However, lower concentrations of HBEGF were later used for generating more in vivo-like astrocytes as identified via RNA sequencing in two different protocols3,4. Moreover, astrocyte morphology changes with the medium composition, regardless of the protocol4: Neurobasal medium with a low concentration of HBEGF is optimal for growing stellate astrocytes, while other medium compositions (e.g., serum-containing DMEM) produce polygonal cells (even in astrocytes freshly isolated by immunopanning).
The AWESAM protocol overcomes disadvantages of previous techniques for growing astrocyte monocultures4. Previous techniques for growing astrocyte monocultures have the following disadvantages: polygonal morphology, which is uncharacteristic of stellate astrocytes in vivo (MD method)5; the length and cost of protocol (preparing astrocytes from induced pluripotent stem cells takes three months and requires many reagents, including expensive growth factors)6; the low amounts of material (immunopanning method)3; and the de-differentiation of astrocytes and requirement of a 3D matrix (Puschmann et al.)1,2. In contrast, the AWESAM protocol provides: in vivo-like morphology (stellate astrocytes with thin processes); a quick, easy, and cheap method; large quantities of material; and the most in vivo-like gene expression compared with immunopanned and MD astrocytes. Additionally, 2D culture allows for the study of Ca2+ signaling and vesicle recycling in thin processes, and in events close to the membrane (e.g., TIRF microscopy is not possible in 3D cultures).
The MD method has been extensively used since its publication in 19805, offering a simple and fast technique for polygonal astrocyte monocultures. In brief, the MD method entails growing mixed brain cells in fetal calf serum (FCS)-containing DMEM, followed by shaking steps that enrich for astrocytes (as all other brain cell types detach from the dish) and further culture in the same medium. DMEM supplemented with FCS is used for many other cell types, ranging from fibroblasts and adipocytes to different cancer cell lines, all of which share the polygonal morphology exhibited by MD astrocytes in culture. Until the early 2000s7, little thought had been given to media tailored to astrocytes, specifically to favor their typical stellate morphology found in vivo. One protocol published in 2011 does achieve such stellate morphology: Called the immunopanning method, it employs serum-free medium optimized for culturing astrocytes3. Using this method, freshly isolated brain cells are exposed to a series of dishes coated with antibodies that target cell type-specific cell surface proteins, to enrich for astrocytes. Despite the more in vivo-like morphology and expression profile of immunopanned astrocytes, the majority of in vitro studies still rely on the MD astrocyte method. The MD method is simple and fast, while the immunopanning method comprises more complex and time-consuming steps on the first day of culture (such as longer enzyme digestion periods, careful layering of solutions of different density, immunopanning itself, and several centrifugation steps - all before plating). However, by using more specialized medium, the AWESAM method offers both the speed of the MD method and the in vivo-like morphology of the immunopanning protocol.
Overall, the AWESAM protocol is useful for studying more in vivo-like astrocytes in 2D monocultures isolated from neurons and other glia (as previously characterized4 by immunostainings, immunoblots, and RNA sequencing). It allows for the study of thin astrocytic processes, and provides great accessibility for visualizing spontaneous Ca2+ signaling and events close to the membrane (e.g., imaged by TIRF microscopy).

<table><tbody><tr><td>0.05% trypsin-EDTA</td><td>Gibco</td><td>25300054</td><td>warm in 37 &amp;ordm;C waterbath before use</td></tr><tr><td>0.25% trypsin-EDTA</td><td>Gibco</td><td>25200-056</td><td>warm in 37 &amp;ordm;C waterbath before use</td></tr><tr><td>B-27 supplement</td><td>Gibco</td><td>17504-044</td><td>50X stock</td></tr><tr><td>Glutamax</td><td>Gibco</td><td>35050-061</td><td>100X stock</td></tr><tr><td>Penicillin / streptomycin</td><td>Gibco</td><td>15070-063</td><td>50 stock; penicillin: 5000 U/ml; streptomycin: 5000 &amp;micro;g/ml</td></tr><tr><td>Neurobasal medium</td><td>Gibco</td><td>21103049</td><td>without L-glutamine</td></tr><tr><td>DMEM</td><td>Gibco</td><td>41966029</td><td></td></tr><tr><td>HBEGF</td><td>Sigma</td><td>4643</td><td></td></tr><tr><td>HEPES</td><td>Gibco</td><td>15630080</td><td></td></tr><tr><td>HBSS</td><td>Gibco</td><td>14170112</td><td></td></tr><tr><td>FCS</td><td>Gibco</td><td>10437028</td><td></td></tr><tr><td>PBS</td><td>Gibco</td><td>10010049</td><td>1X</td></tr><tr><td>100 &amp;mu;m nylon cell strainer</td><td>BD</td><td>352360</td><td></td></tr><tr><td>Trypan Blue</td><td>Sigma</td><td>T8154&amp;nbsp;</td><td></td></tr><tr><td>Cell culture incubator</td><td>Thermo Fisher Scientific</td><td>Hera Cell 240i cell culture incubator</td><td></td></tr><tr><td>Laboratory shaker</td><td>Heidolph</td><td>Rotamax 120&amp;nbsp;</td><td>use inside cell culture incubator</td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5810 R&amp;nbsp;</td><td></td></tr></tbody></table>

culturing in vivo-like murine astrocytes using the fast, simple, and inexpensive awesam protocol

The AWESAM (a low-cost easy stellate astrocyte method) protocol entails a fast, simple, and inexpensive way to generate large quantities of in vivo-like mouse and rat astrocyte monocultures: Brain cells can be isolated from different brain regions, and after a week of cell culture, non-astrocytic cells are shaken off by placing the culture dishes on a shaker for 6 h in the incubator. The remaining astrocytes are then passaged into new plates with an astrocyte-specific medium (termed NB+H). NB+H contains low concentrations of heparin-binding EGF-like growth factor (HBEGF), which is used in place of serum in medium. After growing in NB+H, AWESAM astrocytes have a stellate morphology and feature fine processes. Moreover, these astrocytes have more in vivo-like gene expression than astrocytes generated by previously published methods. Ca2+ imaging, vesicle dynamics, and other events close to the membrane can thus be studied in the fine astrocytic processes in vitro, e.g., using live cell confocal or TIRF microscopy. Notably, AWESAM astrocytes also exhibit spontaneous Ca2+ signaling similar to astrocytes in vivo.

Astrocytes that are co-cultured with neurons and grown in NB+ medium appear stellate after 2 weeks of culture (Figure 2). In addition to NB+ medium, the co-cultured astrocytes are also exposed to unknown neuron-derived factors that likely contribute to their survival and morphology. In contrast, MD astrocytes grown in DMEM+ as monocultures (after shaking off other cell types before passage) appear polygonal after 2 weeks. AWESAM astrocytes grown in NB+H after shaking, appear stellate with many fine processes after 2 weeks of culture.
We previously showed that the RNA expression profile of AWESAM astrocytes is closer to that of astrocytes in vivo when compared to MD and immunopanned astrocytes4. In terms of astrocyte-specific protein expression, cortical astrocytes express high levels of ALDH1L1 and low levels of GFAP in vivo12. This also applies to AWESAM astrocytes, and the immunostaining of ALDH1L1, as a cytoplasmic enzyme, reveals finer detail than the cytoskeletal protein GFAP (Figure 2B).
Aside from the RNA and protein expression, astrocytes in vivo show spontaneous Ca2+ signaling (i.e., without external stimulation), but polygonal MD astrocytes in vitro often require stimulation to elicit Ca2+ signaling, e.g., by adding ATP or glutamate. AWESAM astrocytes exhibit spontaneous Ca2+ signaling in vivo, in both cell bodies and fine processes, which can be visualized by virally transducing cells with the membrane-targeted Lck-GCaMP3 (Figure 3). Spontaneous waves of Ca2+ throughout adjoining astrocytes also occur (Figure 3A). Astrocytic Ca2+ signaling (Figure 3B) in fine processes and microdomains can be analyzed using freeware specific to astrocytes13.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56092/56092fig1.jpg" /> 
Figure 1: Protocol timeline. A timeline showing the main steps of the AWESAM protocol, including the time it takes to perform these steps on DIV0 (day of dissection), DIV7 (day of passage), and what to expect at later time points. The green box illustrates when cells can be harvested as stellate astrocytes, where lighter versus darker shades represent less versus more complex stellate morphology and maturity, respectively. <a href="https://cloudflare.jove.com/files/ftp_upload/56092/56092fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56092/56092fig2v3.jpg" /> 
Figure 2: Medium determines morphology. (A) Schematic showing how astrocyte morphology changes when co-cultured with neurons (as previously described4) vs. grown as monocultures, and depending on the medium. Astrocyte-specific staining with GFAP outlines the stellate morphology in co-cultured and AWESAM astrocytes, and the polygonal morphology in MD astrocytes. (B) GFAP labels MD astrocytes more strongly than ALDH1L1, and ALDH1L1 (but not GFAP) outlines the fine processes of AWESAM astrocytes. Neither astrocyte monoculture exhibits the neuronal marker MAP2. <a href="https://cloudflare.jove.com/files/ftp_upload/56092/56092fig2v3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56092/56092fig3.jpg" /> 
Figure 3: Spontaneous Ca2+ signaling in astrocytes. (A) GCaMP-transduced AWESAM astrocytes exhibit spontaneous Ca2+ events throughout astrocyte networks, including in the thin processes (arrowheads). A Ca2+ wave travels through several astrocytes from left to right in images from distinct time points, where light color depicts areas of high Ca2+ concentrations, and black areas represent extracellular regions. Images were acquired using live cell confocal microscopy. (B) Ca2+ concentrations change in the color-coded regions in (A) over time, shown as GCaMP fluorescence intensity traces that illustrate how a Ca2+ signaling wave spreads across several astrocytes. <a href="https://cloudflare.jove.com/files/ftp_upload/56092/56092fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Culturing In Vivo-like Murine Astrocytes Using the Fast, Simple, and Inexpensive AWESAM Protocol at JoVE.com

Culturing In Vivo-like Murine Astrocytes Using the Fast, Simple, and Inexpensive AWESAM Protocol

The AWESAM protocol described here is optimal for culturing murine astrocytes in isolation from other brain cells in a fast, simple, and inexpensive manner. AWESAM astrocytes exhibit spontaneous Ca2+ signaling, morphology, and gene expression profiles similar to astrocytes in vivo.

Culturing In Vivo-like Murine Astrocytes Using the Fast, Simple, and Inexpensive AWESAM Protocol

The AWESAM (a low-cost easy stellate astrocyte method) protocol entails a fast, simple, and inexpensive way to generate large quantities of in vivo-like ...

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Nanyang Technological University

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8.4K Views.  University of Oxford. The AWESAM protocol described here is optimal for culturing murine astrocytes in isolation from other brain cells in a fast, simple, and inexpensive manner. AWESAM astrocytes exhibit spontaneous Ca2+ signaling, morphology, and gene expression profiles similar to astrocytes in vivo.

Video: Culturing In Vivo-like Murine Astrocytes Using the Fast, Simple, and Inexpensive AWESAM Protocol

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.

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 ...

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 ...

Monitoring Astrocyte Reactivity and Proliferation in Vitro Under Ischemic-Like Conditions

Ischemic stroke is a complex brain injury caused by a thrombus or embolus obstructing blood flow to parts of the brain. This leads to deprivation of oxygen and glucose, which causes energy failure and neuronal death. After an ischemic stroke insult, astrocytes become reactive and proliferate around the injury site as it develops. Under this scenario, it is difficult to study the specific contribution of astrocytes to the brain region exposed to ischemia. Therefore, this article introduces a methodology to study primary astrocyte reactivity and proliferation under an in vitro model of an ischemia-like environment, called oxygen glucose deprivation (OGD). Astrocytes were isolated from 1-4 day-old neonatal rats and the number of non-specific astrocytic cells was assessed using astrocyte selective marker Glial Fibrillary Acidic Protein (GFAP) and nuclear staining. The period in which astrocytes are subjected to the OGD condition can be customized, as well as the percentage of oxygen they are exposed to. This flexibility allows scientists to characterize the duration of the ischemic-like condition in different groups of cells in vitro. This article discusses the timeframes of OGD that induce astrocyte reactivity, hypertrophic morphology, and proliferation as measured by immunofluorescence using Proliferating Cell Nuclear Antigen (PCNA). Besides proliferation, astrocytes undergo energy and oxidative stress, and respond to OGD by releasing soluble factors into the cell medium. This medium can be collected and used to analyze the effects of molecules released by astrocytes in primary neuronal cultures without cell-to-cell interaction. In summary, this primary cell culture model can be efficiently used to understand the role of isolated astrocytes upon injury.

Monitoring Astrocyte Reactivity and Proliferation in Vitro Under Ischemic-Like Conditions

Ischemic stroke is a complex event in which the specific contribution of astrocytes to the affected brain region exposed to oxygen glucose deprivation (OGD) is difficult to study. This article introduces a methodology to obtain isolated astrocytes and study their reactivity and proliferation under OGD conditions.

Ischemic stroke is a complex brain injury caused by a thrombus or embolus obstructing blood flow to parts of the brain. This leads to deprivation of ...

Primary Cultures of Rat Astrocytes and Microglia and Their Use in the Study of Amyotrophic Lateral Sclerosis

This protocol demonstrates how to prepare primary cultures of glial cells, astrocytes, and microglia from the cortices of Sprague Dawley rats and how to use these cells for the purpose of studying the pathophysiology of amyotrophic lateral sclerosis (ALS) in the rat hSOD1G93A model. First, the protocol shows how to isolate and culture astrocytes and microglia from postnatal rat cortices, and then how to characterize and test these cultures for purity by immunocytochemistry using the glial fibrillary acidic protein (GFAP) marker of astrocytes and the ionized calcium-binding adaptor molecule 1 (Iba1) microglial marker. In the next stage, methods are described for dye-loading (calcium-sensitive Fluo 4-AM) of cultured cells and the recordings of Ca2+ changes in video imaging experiments on live cells.
The examples of video recordings consist of: (1) cases of Ca2+ imaging of cultured astrocytes acutely exposed to immunoglobulin G (IgG) isolated from ALS patients, showing a characteristic and specific response compared to the response to ATP as demonstrated in the same experiment. Examples also show a more pronounced transient rise in intracellular calcium concentration evoked by ALS IgG in hSOD1G93A astrocytes compared to non-transgenic controls; (2) Ca2+ imaging of cultured astrocytes during a depletion of calcium stores by thapsigargin (Thg), a non-competitive inhibitor of the endoplasmic reticulum Ca2+ ATPase, followed by store-operated calcium entry elicited by the addition of calcium in the recording solution, which demonstrates the difference between Ca2+ store operation in hSOD1G93A and in non-transgenic astrocytes; (3) Ca2+ imaging of the cultured microglia showing predominantly a lack of response to ALS IgG, whereas ATP application elicited a Ca2+ change. This paper also emphasizes possible caveats and cautions regarding critical cell density and purity of cultures, choosing the correct concentration of the Ca2+ dye and dye-loading techniques.

We present here a protocol on how to prepare primary cultures of glial cells, astrocytes, and microglia from rat cortices for time-lapse video imaging of intracellular Ca2+ for research on pathophysiology of amyotrophic lateral sclerosis in the hSOD1G93A rat model.

This protocol demonstrates how to prepare primary cultures of glial cells, astrocytes, and microglia from the cortices of Sprague Dawley rats and how to ...

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 ...

Generating a Primary Culture of Astrocytes from a Rat Pup Brain

Source: Mili&#263;evi&#263;, K., et al., <a href="https://app.jove.com/v/63483">Primary Cultures of Rat Astrocytes and Microglia and Their Use in the Study of Amyotrophic Lateral Sclerosis.</a> J. Vis. Exp. (2022)
The video demonstrates a technique for culturing primary astrocytes from the rat cortex. The cortex is mechanically dissociated to obtain a homogeneous glial cell suspension, which is then plated on polypeptide-coated plates to promote selective proliferation of astrocytes. Subsequently, the astrocytes are enzymatically dissociated from the plates and transferred to fresh media, allowing the culture of pure astrocytes.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Primary cell culture preparation

...

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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/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
...

Direct Neuronal Reprogramming of Mouse Astrocytes into Neurons

Source: Hersbach, B. A. et al., <a href="https://app.jove.com/v/64175">Isolation and Direct Neuronal Reprogramming of Mouse Astrocytes.</a> J. Vis. Exp. (2022) 
This video showcases the direct reprogramming of astrocytes expressing transforming factors into neurons. Transformation factors trigger a cascade of molecular events facilitating astrocyte-to-neuron conversion. The process is further supported by supplying specific agents and supplements through a neuronal differentiation medium.

1. Seeding of astrocytes for reprogramming
NOTE: The following steps have to be performed under a biological safety cabinet with a safety level 1 (SL1).

	...

Advanced Neuronal Models

An In Vitro Method to Generate Astrocyte Scaffolds within Hydrogel Micro-columns

Source: Katiyar, K. S., et al. <a href="https://app.jove.com/v/55848">Three-dimensional Tissue Engineered Aligned Astrocyte Networks to Recapitulate Developmental Mechanisms and Facilitate Nervous System Regeneration.</a> J. Vis. Exp. (2018).
This video demonstrates a method to generate three-dimensional astrocyte scaffolds within hydrogel micro-columns. The inside of the microcolumns are coated with collagen. Astrocytes are introduced, which adhere to collagen and, with further incubation, self-assemble into a three-dimensional scaffold.

1. Development of the Agarose Hydrogel Micro-columns

	Make an agarose 3% weight/volume (w/v) solution by weighing 3 g of agarose and transferring it to a ...

Culturing In Vivo-like Murine Astrocytes Using the Fast, Simple, and Inexpensive AWESAM Protocol

Summary

Explore More Videos

Chapters in this video

Isolation and Culture of Mouse Cortical Astrocytes

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

Monitoring Astrocyte Reactivity and Proliferation in Vitro Under Ischemic-Like Conditions

Primary Cultures of Rat Astrocytes and Microglia and Their Use in the Study of Amyotrophic Lateral Sclerosis

Isolation and Direct Neuronal Reprogramming of Mouse Astrocytes

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

Generating a Primary Culture of Astrocytes from a Rat Pup Brain

A Simple Technique to Isolate and Culture Murine Astrocytes

Direct Neuronal Reprogramming of Mouse Astrocytes into Neurons

An In Vitro Method to Generate Astrocyte Scaffolds within Hydrogel Micro-columns