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 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 solution prior to dissection. Now pin the tissue on a silicone elastomer coated Petri dish in cold ringer 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 submucosal layer into two to five millimeter pieces. Place them into a 50 milliliter conical tube, containing five milliliters of orgono 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 orgono 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 3000 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 3000 G for three minutes at room temperature. After discarding the supernatant, resuspend the pellet in 10 milliliters of room temperature orgono 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 orgono 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 orgono 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 zero, 10, and 25 nanograms of interleukin-1 beta for 24 hours. Spindle-shaped cells consistent with enteric glial morphology were observed in the cultures, with pleomorphism and minimal contamination from other cell types.
