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.

To remove the epithelial mucosa, first, use fine scissors to cut the intestines into approximately 0.5-centimeter pieces and collect the pieces in 30 milliliters of ice-cold EDTA/HEPES/DPBS solution. Rock the tissue-containing solution at 4 degrees Celsius for 10 minutes at approximately 60 tilts per minute.

Pre-moisten a 5-milliliter plastic pipette by pipetting the EDTA/HEPES/DPBS buffer up and down one time, and then use the pre-moistened pipette to triturate the tissue and buffer suspension 20 times to dislodge the epithelial mucosa.

Collect the tissue by pouring the mixture through a 100-micron nylon cell strainer, and use needlenose forceps to place the tissue retained in the strainer into a new 50-milliliter conical centrifuge tube containing 30 milliliters of ice-cold EDTA/HEPES/DPBS. Repeat the EDTA/HEPES/DPBS incubation a second time by rocking the tissue at 4 degrees Celsius for 10 minutes at approximately 60 tilts per minute to strip off additional epithelial mucosa.

Perform a gentle trituration using a pre-moistened 5-milliliter pipette. The tissue will tend to stick to the inside of the pipette as more of the epithelium is removed.

Gentle trituration is required at this point, since the enteric glial cells are more fragile.

Collect the tissue after the second incubation by pouring the mixture through a 100-micron nylon cell strainer, and discard the flow through. The second flow-through should be noticeably less turbid than the first. Repeat the 10-minute EDTA incubations until the solution is nearly clear.

Transfer the tissue from the nylon strainer to a 15-milliliter conical centrifuge tube containing 5 milliliters of the commercially available cell recovery solution. Then, rock for 25 to 30 minutes at 4 degrees Celsius. Gently triturate 10 times to dissociate the enteric glial cells from the lamina propria, then filter through a 40-micron filter and collect the filtrate into a clean 50-milliliter tube.

Rinse the tissue on the nylon filter with 1 milliliter of DPBS. Discard the tissue and transfer the 5 to 6 milliliters of filtrate to a clean 15-milliliter conical centrifuge tube. Spin the filtrate at 2,000 times g in a swinging bucket centrifuge for five minutes at 4 degrees Celsius.

Resuspend the glial cell-containing pellet in at least 1 milliliter of resuspension buffer by gently pipetting the pellet up and down with the 500-microliter pipette tip without introducing bubbles. Finally, plate the enteric glial cells by adding 200 microliters of the cell suspension into each well of a laminin-coated 6-well plate or 100 microliters to each well of a 12-well plate containing glial growth media.