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.
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 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).
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
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.
3. Tissue Digestion and Dissociation
4. Cell Plating for Astrocyte Enrichment by Passaging
5. Passaging Cells
6. Cell Plating for Final Astrocyte Cultures
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.
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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.
The authors have nothing to disclose.
We acknowledge funding from the European Research Council (FP7/260916), the Alexander von Humboldt-Stiftung (Sofja Kovalevskaja), and the Deutsche Forschungsgemeinschaft (DE1951 and SFB889).
0.05% trypsin-EDTA | Gibco | 25300054 | warm in 37 ºC waterbath before use |
0.25% trypsin-EDTA | Gibco | 25200-056 | warm in 37 ºC waterbath before use |
B-27 supplement | Gibco | 17504-044 | 50X stock |
Glutamax | Gibco | 35050-061 | 100X stock |
Penicillin / streptomycin | Gibco | 15070-063 | 50 stock; penicillin: 5000 U/ml; streptomycin: 5000 µg/ml |
Neurobasal medium | Gibco | 21103049 | without L-glutamine |
DMEM | Gibco | 41966029 | |
HBEGF | Sigma | 4643 | |
HEPES | Gibco | 15630080 | |
HBSS | Gibco | 14170112 | |
FCS | Gibco | 10437028 | |
PBS | Gibco | 10010049 | 1X |
100 μm nylon cell strainer | BD | 352360 | |
Trypan Blue | Sigma | T8154 | |
Cell culture incubator | Thermo Fisher Scientific | Hera Cell 240i cell culture incubator | |
Laboratory shaker | Heidolph | Rotamax 120 | use inside cell culture incubator |
Centrifuge | Eppendorf | 5810 R |