Gastric Fundic Glands Isolation: A Method to Isolate Fundic Glands from Human Stomach Tissue
Transcripción
To begin, collect the prepared tissue in a large plastic container filled with calcium/magnesium-free DPBS. Leave the plastic container on ice until use. Next, with the help of forceps wash the tissue vigorously in a sterile beaker, containing 100 milliliters of calcium/magnesium-free DPBS, for about 30 seconds. Then, use a sterile gauze to wipe away the mucus layer from the epithelium.
Next, use large forceps to hold the muscle layer of the tissue firmly. Then, use large curved hemostatic forceps and scrape the epithelium with force. Store the scraped epithelium in a sterile polystyrene Petri dish. To optimize the tissue digestion, mince the epithelial tissue into fragments. Then, wash the tissue with calcium/magnesium-free DPBS supplemented with antibiotics.
Wash multiple times to remove the blood. Discard the wash in a waste beaker. Then, collect the washed epithelial fragments in a 125-milliliter glass round bottom flask with a 25-milliliter stir bar and pre-warmed incubation medium. After collecting the tissue fragments, seal the round bottom flask with a rubber septa. Then, insert a 20G spinal needle in the septa. Connect the septa to an oxygen tank with rubber hosing.
Insert 10 to 15 outflow needles into the septa to prevent its rupture and then use clamps to secure the setup to a ring stand. Then, switch on the oxygen outflow on low. After this, calibrate the water bath at 37 degrees Celsius on a stir plate for 30 to 45 minutes. Once calibrated, place the setup in the water bath. After 15 minutes of incubation, check for dissociated glands.
After the desired incubation time, add 50 milliliters of preheated DMEM/F-12 to the incubation medium using a serological pipette. Next, filter the gland mixture obtained through a sterile gauze in four 50-milliliter conical tubes. Then, incubate the filtrate on ice for 15 minutes. This will allow the gland to settle to the bottom of the conical tube. Use a serological pipette to discard 40 milliliters of the supernatant from the top of the gland suspension.
Resuspend the remaining 10 milliliters of the gland mixture in 10 milliliters of DPBS supplemented with antibiotics. Then, distribute the entire mixture into 5-milliliter cell culture test tubes. Centrifuge the tubes at 65 x g for 5 minutes at 4 degrees Celsius. After centrifugation, carefully remove the supernatant.
