Name: culturing in vivo-like murine astrocytes using the fast, simple, and inexpensive awesam protocol
Uploaded: 2018-01-10T13:00:00
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 culturi...

<ol>
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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 onc...

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 border="0" cellpadding="0" cellspacing="0" width="898">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">0.05% trypsin-EDTA</td>
			<td style="width:171px;">Gibco</td>
			<td style="width:344px;">25300054</td>
			<td style="width:167px;">warm in 37 &#186;C waterbath before use</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">0.25% trypsin-EDTA</td>
			<td>Gibco</td>
			<td>25200-056</td>
			<td>warm in 37 &#186;C waterbath before use</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">B-27 supplement</td>
			<td>Gibco</td>
			<td>17504-044</td>
			<td>50X stock</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Glutamax</td>
			<td>Gibco</td>
			<td>35050-061</td>
			<td>100X stock</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Penicillin / streptomycin</td>
			<td>Gibco</td>
			<td>15070-063</td>
			<td>50 stock; penicillin: 5000 U/ml; streptomycin: 5000 &#181;g/ml</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Neurobasal medium</td>
			<td>Gibco</td>
			<td>21103049</td>
			<td>without L-glutamine</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">DMEM</td>
			<td>Gibco</td>
			<td>41966029</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">HBEGF</td>
			<td>Sigma</td>
			<td>4643</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">HEPES</td>
			<td>Gibco</td>
			<td>15630080</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">HBSS</td>
			<td>Gibco</td>
			<td>14170112</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">FCS</td>
			<td>Gibco</td>
			<td>10437028</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">PBS</td>
			<td>Gibco</td>
			<td>10010049</td>
			<td>1X</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">100 &#956;m nylon cell strainer</td>
			<td>BD</td>
			<td style="width:344px;">352360</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Trypan Blue</td>
			<td>Sigma</td>
			<td>T8154&#160;</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Cell culture incubator</td>
			<td>Thermo Fisher Scientific</td>
			<td>Hera Cell 240i cell culture incubator</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Laboratory shaker</td>
			<td>Heidolph</td>
			<td>Rotamax 120&#160;</td>
			<td>use inside cell culture incubator</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Centrifuge</td>
			<td>Eppendorf</td>
			<td>5810 R&#160;</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...

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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