A Simple Technique to Isolate and Culture Murine Astrocytes

Cut a mouse cortex into small pieces and transfer to a cold dissection medium containing a buffered salt solution.

Add digestive enzymes to dissociate the extracellular matrix.

To remove residual enzymes, wash with the cold dissection medium.

Replace this medium with an astrocyte-enrichment medium.

Next, use a pipette to dissociate the tissue pieces.

Pass the suspension through a strainer to remove undissociated tissue and obtain single cells.

Transfer the cells to a positively charged polymer-coated plate. Incubate with shaking.

Astrocytes adhere firmly, while the loosely bound neurons, microglia, and oligodendrocytes detach.

The enrichment medium converts mature astrocytes into dividing polygonal astrocytes.

Remove the detached cells and wash astrocytes with a phosphate buffer.

Add digestive enzymes to detach astrocytes.

Add a medium containing serum to stop the enzymatic activity. Collect the astrocytes in a tube and centrifuge.

Discard the supernatant. Resuspend cells in an astrocyte-maturation medium to promote a typical star-shaped mature astrocyte structure.

Cut any cortical tissue to be used for generating astrocytes into pieces no larger than 1 cubic millimeter. Once all tissue pieces are collected, wait for them to settle to the bottom of the tube. Then, aspirate as much medium as possible. Following that, add 2 to 3 milliliters of 0.05% trypsin EDTA, and incubate the samples in a 37-degree Celsius water bath for 20 minutes.

Afterward, carefully aspirate the trypsin EDTA, leaving tissue pieces in a minimum amount of liquid at the bottom of the tube. Next, add 5 milliliters of precooled dissection medium to wash off the remaining trypsin EDTA, and pipette to the side of the upper tube to achieve mixing of the tissue pieces. Then aspirate the dissection medium and leave the tissue pieces in as little liquid as possible.

Repeat this washing step with precooled dissection medium twice. After the final washing step, add 1 milliliter of room temperature DMEM PLUS and titrate the tissue by pipetting up and down without introducing bubbles.

Astrocytes are quite sensitive to pH, so it is important to avoid producing bubbles as this may change the pH of the solution.

Next, place a 100-micron cell strainer in the opening of a 50-milliliter tube and pre-wet the filter with 4.5 milliliters of precooled DMEM PLUS. Pipette 1 milliliter of the dissociated tissue suspension onto the cell strainer, and add another 4.5 milliliters of precooled DMEM PLUS to wash the cells through it. On day in vitro 7 after the dissection, place the 10-centimeter dishes with cells in DMEM PLUS on a shaker in the incubator and shake the dishes at 110 RPM for six hours.

20 to 30 minutes before the end of the six-hour shaking, pre-warm 1X PBS, DMEM PLUS, NB+H, and 0.25% trypsin EDTA to 37 degrees in the water bath. After six hours of shaking, take the culture dishes off the shaker, and immediately replace the medium with 10 milliliters of pre-warmed 1X PBS per dish. Subsequently, remove PBS and add 3 milliliters of pre-warmed trypsin EDTA per dish.

Then, incubate the samples for four minutes at 37 degrees Celsius. Next, add 5 milliliters of pre-warmed DMEM PLUS to 3 milliliters of trypsin EDTA in each dish. Pipette the cells off the dish and collect the cell suspension in a 50-milliliter tube. After that, centrifuge the cell suspension at 3,220 times g at 20 degrees Celsius for four minutes. Then remove the supernatant, then resuspend the cell pellet in 1 milliliter of pre-warmed NB+H.