Isolating Enteric Glial Cells From the Submucosa and Lamina Propria of a Mouse

Take a mouse small intestine segment previously stripped of the longitudinal muscle and myenteric plexus, layers containing enteric neurons, and glial cells.

Cut up the tissue and place it in a buffer containing EDTA. Incubate with rocking.

EDTA disrupts cell-cell junctions, detaching the epithelial mucosa from the underlying lamina propria and submucosa, connective tissue layers containing enteric glial cells.

Pipette repeatedly to dislodge the epithelial cells.

Filter through a cell strainer and discard the flow-through.

Transfer the tissue to a non-enzymatic dissociation buffer and incubate it with rocking. The dissociation buffer loosens the lamina propria and submucosal extracellular matrix, detaching the cells.

Pipette repeatedly to further dissociate the cells.

Filter through a cell strainer to collect the cells.

Centrifuge and resuspend the cells in a buffer.

Take adhesion protein-coated wells containing a glial growth medium and plate the cells.

The coating facilitates cell adhesion, while the growth factors selectively promote glial cell proliferation, establishing a lamina propria and submucosal glial cell culture.