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

Summary

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.

Abstract

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.

Introduction

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 proces....

Protocol

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

1. Prepare the following solutions.
   1. Prepare DMEM+ for culturing polygonal MD astrocytes: DMEM with 10% FCS, 100 U penicillin and 100 µg/mL streptomycin.
   2. Prepare NB+H for culturing stellate astrocytes: neurobasal medium with 5 ng/mL HBEGF, 1x B-27 supplement, 1x glutamine supplement, 100 U penicillin and 100 µg/mL st....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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