Equine Enteric Glial Culture and Application to the Study of a Neural Inflammatory Mechanism in Equine Colic

Horses face devastating inflammatory postoperative conditions following colic surgery, In human research, enteric glial cells are gaining notoriety as key sensors of the intestinal environment, as well as influencers of inflammation. We aimed to isolate enteric glial cells from equine small intestine in order to assess their role in understanding and preventing postoperative complications. We have developed a reproducible protocol for isolating submucosal enteric glial cells from equine intestine.

We were also able to demonstrate its application to understanding the effect of inflammatory enteric glia on intestinal barrier function, which may have important implications for future colic, postoperative complication, and intestinal disease research. Our lab will continue to focus on the relationship of enteric glia in inflammatory intestinal environments, as well as how the prevention of the loss of barrier function by anti-inflammatory biologics may be used to prevent postoperative complications. To begin, coat the 24-well plates with the coating solution and incubate for two hours at 37 degrees Celsius or overnight at room temperature.

Wash the plates two times with sterile water and store them at room temperature until the cells are ready for plating. Now, take a section of the horse jejunum and place it in cold Ringer's solution on ice. Remove a 10 centimeter portion from the jejunum and open it at the anti-mesenteric border, positioning non-lymphoid regions at the center.

Then trim the portion into an approximately seven by seven centimeter section and rinse the tissue well in fresh Ringer's solution prior to dissection. Now, pin the tissue on a silicone elastomer-coated Petri dish in cold Ringer's solution or PBS with the mucosal side up while stretching it as much as possible. Using curved forceps, remove the intestinal villi and then remove the lamina propria layer from approximately five by five centimeter non-lymphoid region.

Next, with microdissection scissors, remove the submucosa in one sheet by freeing it at one corner. Observe the natural separation while peeling away the submucosa from the inner circular muscle layer. Then mince the collected submucosa layer into two to five millimeter pieces.

Place them into a 50 milliliter conical tube containing five milliliters of Organo without FBS, collagenase, protease, and BSA. Incubate the tissue for two to three hours at 37 degrees Celsius for enzymatic digestion. After incubation, add 10 milliliters of room temperature Organo with FBS to stop the enzyme action.

Pipette the tissue mixture up and down 15 times using a 10 milliliter serological pipette to dissociate the cells from the tissue. Then centrifuge the mixture at 3, 000 G for three minutes at room temperature. Afterward, discard the supernatant and resuspend the pellet in 10 milliliters of room temperature PBS by pipetting the solution up and down 15 times with a 10 milliliter serological pipette.

Centrifuge the conical tube at 3, 000 G for three minutes at room temperature. After discarding the supernatant, resuspend the pellet in 10 milliliters of room temperature Organo with FBS by pipetting up and down 15 times. Next, filter the cells sequentially through a 100 micrometer, 70 micrometer, and 40 micrometer pore size cell strainer.

After centrifuging the cells and discarding the supernatant, resuspend the pellet in one milliliter of Organo with FBS. Then stain the cells with trypan blue to identify live cells and count them using a hemocytometer. Now, seed approximately 400, 000 cells in 300 microliters of Organo with FBS in each well of a 24-well plate.

Add five microliters of N2, five microliters of G5, and 10 microliters of B27 to each well. Place the plate in a 37 degrees Celsius incubator with 5%carbon dioxide to allow cell adhesion. When cells from the first passage reach 70 to 80%confluence, expose the wells to 0, 10, and 25 nanograms of interleukin-1 beta for 24 hours.

Spindle-shaped cells consistent with enteric glia morphology were observed in the cultures, with pleomorphism and minimal contamination from other cell types. To begin, coat the Transwell inserts with 42 microliters of 0.05%Matrigel. Incubate for one hour at 37 degrees Celsius to allow Matrigel to set.

Aspirate the extra medium and dry the Transwells for 30 minutes uncovered. Then add 200 microliters of DMEM F12 medium to each insert and store at 37 degrees Celsius until use. Wash the Matrigel patties containing the organoids with PBS.

Expose them to 500 microliters of cell recovery while on ice and pipette the mixture repeatedly to ensure a full collection of cells in a 15 milliliter tube. Then centrifuge the tube at 300 G for five minutes at four degrees Celsius. Remove the supernatant and resuspend the cell pellet in the target volume of intestinal epithelial stem cell or IESC medium.

Now, pre-coat a one milliliter syringe in IESC medium. aspirate the cell suspension through a 16 gauge needle. Then replace the 16 gauge needle with a 28 gauge needle and eject the suspension to promote separation of the organoids.

Next, place 200 microliters of the resulting cell suspension into the apical side of the coated Transwells. Add 500 microliters of IESC medium and growth factors to the basal side. Use a dual electrode TEER measuring chamber to quantify the TEER across the monolayers.

Fill the culture cup with 1.5 milliliters of IESC media and ensure exactly 200 microliters of media is present in the Transwell insert so that the fluid levels match during measurement. Expose the basolateral aspect of the Transwell inserts to either inflammatory cytokines of interleukin-1 beta or glial products from cultures exposed to interleukin-1 beta. Measure the TEER every 10 to 15 minutes for 45 minutes by placing each Transwell into the culture cup.

Basolateral exposure to media from glial cultures treated with 10 and 25 nanograms of interleukin-1 beta significantly increased epithelial barrier permeability. No significant increase in epithelial permeability was observed after exposure to equine interleukin-6 across various concentrations.