Obtaining a Mixed Glial Cell Culture from a Neonatal Mouse Brain

Begin with a neonatal mouse brain and transfer it to a tube containing a proteolytic enzyme solution.

The enzymes degrade the tissue's extracellular matrix and initiate cell dissociation.

Repeatedly wash the digested tissue with a growth medium to remove the enzymes.

Use a larger-diameter pipette to move the tissue repeatedly through the pipette to dissociate it into smaller fragments.

Then, using a smaller-diameter pipette, continue to dissociate the fragments, creating a cell suspension.

Filter this suspension to remove cell clumps and obtain a single-cell suspension containing primary neurons and a mixed population of glial cells, including microglia and astrocytes.

Seed the cells in a culture flask containing a growth medium and incubate to promote their adherence. 

Over time, the absence of neuron growth factors in the media causes neuronal death.

Replace the spent media with fresh media. Glial cells utilize the nutrients and proliferate, generating a mixed glial culture of astrocytes and microglia.

Transfer the brain to a 50-milliliter tube containing 2 milliliters of 0.25% trypsin-EDTA, and incubate for 15 minutes in a 37 degrees Celsius water bath. Wash the brain with fresh growth medium, by adding and removing the medium. After completion of the washes, add 2 milliliters of growth medium per brain, and homogenize by triturating the brain.

When the brain tissue does not get smaller, transition to a pipette with a smaller aperture. At the end of the titration when the suspension is clear with no visible chunks, pass the suspension through a 70-micron cell strainer to create a single-cell culture.

Then, pipette 8 to 9 millimeters of growth medium into T75 flasks, two flasks per brain, and add 1 milliliter of brain homogenate to each flask. Grow the cells until isolation on the 16th day.