Here, we describe the isolation of enteric-glial cells from the intestinal-submucosa using sequential EDTA incubations to chelate divalent cations and then incubation in non-enzymatic cell recovery solution. Plating the resultant cell suspension on poly-D-lysine and laminin results in a highly enriched culture of submucosal glial cells for functional analysis.
This method can help answer key questions in the neuroscience field, specifically the function of the enteric nervous system. The main advantage of the technique is that it is a rapid, non-enzymatic method to isolate enteric cells from the lamina propria and submucosa rather than the smooth muscle. Begin by identifying the distal stomach and pylorus.
Hold the pylorus with forceps while snipping away the mesentery. Then use blunt dissection with the back of the scissors to scrape away adherent pancreas. Then use scissors to remove seven centimeters of the proximal intestine without tearing.
Place the intestinal segment into ice cold DPBS without calcium or magnesium. Next, use a five or 10 milliliter syringe attached to a blunt end 18 to 20 gauge needle to flush the fecal contents with ice cold DPBS. Divide the intestine into three centimeter segments to remove the longitudinal muscle.
Rapid removal of the mesentery and longitudinal muscle is essential to isolating healthy enteric glia from the lamina propria and submucosa. Next, take a cotton swab and break off about four centimeters to separate the wooden stick from the cotton tip. Soak the wooden stick in DPBS.
Slip one of the intestinal segments smoothly onto the wetted stick and then use needle nose forceps to remove any remaining adherent mesentery. Then use a clean razor blade or scalpel to make two longitudinal nicks along the intestine where the mesentery was attached. Hold the tip of the stick between the thumb and forefinger while stabilizing the segment with the middle and fourth finger.
Wet the cotton tip with DPBS. Rub the wetted cotton tip longitudinally along the muscle to loosen the Longitudinal Muscle Mesenteric Plexus or LMMP. Then move the cotton tip horizontally to tease away the LMMP from the circular muscle.
When complete, the LMMP will easily peel off the intestinal segment. Discard the LMMP unless harvesting EGCs from the mesenteric plexus is desired. Then use a razor blade to flay open the intestinal segment longitudinally to remove the wooden stick.
Keep the stripped intestinal tissue consisting predominantly of mucosa and submucosa plus circular muscle in DPBS on ice. Repeat the dissection for other samples until all intestinal segments are prepared. 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 four degrees Celsius for 10 minutes at approximately 60 tilts per minute. Pre-moisten a five 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.
Then use needle nose 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 four 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 five 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 glia 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 incubation until the solution is nearly clear. Transfer the tissue from the nylon strainer to a 15 milliliter conical centrifuge tube containing five milliliters of the commercially available cell recovery solution.
Then rock for 25 to 30 minutes at four 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 one milliliter of DPBS. Discard the tissue and transfer the five to six 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 four degrees Celsius.
Resuspend the glial cell containing pellet in at least one 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 six-well plate or 100 microliters to each well of a 12-well plate containing glial growth media. Prep should be considered unsuccessful if enteric glial cells do not adhere and spread within 24 hours as seen here.
Here is a representative example of an excellent prep 24 hours after cell isolation and resuspension where enteric glial cells have adhered in patches to the PDL laminin-coated plate. The number of glial cells can be determined after 24 hours when the cells adhere and display evidence of spreading into flat aggregates. Cells at the edge of the clusters tend to extend long processes and expressed classic glial markers such as GFAP, S100B, and p75NTR.
Following this procedure, other methods like immunohistochemistry, western blots, flow cytometry, and imaging calcium flux can be performed in order to answer additional questions on enteric glial cell heterogeneity. After its development, this technique has paved the way for researchers studying the enteric nervous system to explore glial cell biology and its impact on the adjacent mucosa and microbiota in mice and by extension in humans.
