The overall goal of this culture system is to generate a primary mouse or human-derived gastric monolayer for the study of regeneration in a wound healing assay. This method can help answer key questions in the gastric field such as the mechanism of gastric epithelial cell regeneration in response to injury. The main advantage of this technique is first, the study of cell migration and regeneration in a wound healing assay, generated from primary mouse or human-derived gastric cells, and second generation of human derived gastric organoids for large scale experiments.
Though this method can provide insight into regeneration of the gastric epithelium, it can also be applied to other systems such as helicobacter pylori infection and epithelial immune cell co-cultures. Demonstrating the procedure will be Emma Teal, A grad student from my laboratory. 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 scrap the epithelium with force. Store the scraped epithelium in a steril 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 millimeter stir bar and pre-warmed incubation medium. After collecting the tissue fragments seal the round-bottom flask with a septa. And then use clamps to secure the setup to a ring stand.
Then insert a 20 gauge 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 it's rupture, the 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 15 milliliters of preheated DMEMF12 to the incubation medium using a serological pipet. Next, filter the gland mixture obtained through a sterile gauze in four 15 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 pipet to discard 40 milliliters of the supernatant from the top of the gland suspension. Re-suspend the remaining 10 milliliters of the glad mixture in 10 milliliters of DPBS supplemented with antibiotics.
Then distribute the entire mixture into five milliliter cell culture test tubes. Centrifuge the tubes at 65 times G for five minutes at four degrees celsius. After centrifugation carefully remove the supernatant.
Dissolve the obtained glandular pellet with thought basement membrane matrix using a wide 200 microliter pipet tip. Mix the suspension until homogenous. Then, plate the glands in 15 microliters of the basement membrane matrix with the help of a wide tipped pipet.
For the basement membrane matrix to polymerize, incubate at 37 degrees celsius for 10 to 15 minutes. Then add one milliliter of hFGO medium in each well if cultured in a 12 well culture plate. Then culture the cells at 37 degrees celsius in a humidified cell culture incubator.
Change the medium every 45 days. After six to seven days of 3D mouse or human derived FGO culture, start transferring the 3D organoids to the 2D monolayer. To do so, add one milliliter of calcium magnesium free DPBS on the center of the bubble to dislodge the 3D basement membrane bubble from the plate.
Then collect the basement membrane matrix and organoid mixture in the cell culture tube. Then centrifuge at 40 times G for five minutes at four degrees celsius. After centrifugation, use a vacuum system to remove the supernatant and as much basement membrane matrix as possible.
Then, dissolve the pellet in four milliliters of calcium magnesium free DPBS. Be sure to remove as much of the basement matrix as possible after harvesting the organoids in preparation for the monolayer. Next, add one milliliter of pre-warmed cell detachment solution at 37 degrees celsius to each test tube.
Then mix the contents of the tube by inverting several times. Finally, incubate the tube at 37 degrees celsius for 10 minutes. To obtain a single cell suspension add the two milliliters of calcium magnesium free DPBS directly to each of the test tubes.
Then use a 26 gauge needle to mix the suspension two to three times. After mixing, centrifuge the cell suspension, then dissolve the cell pellet in 2D FGO medium. To obtain the whole organoid, plate the cell suspension on coated plates.
Then incubate the plates at 37 degrees celsius for about four days. Change the medium if the color turns yellow. As soon at the culture reaches 100 percent confluency, scratch the monolayer using a razor blade.
Then fix the monolayers using 3.7 percent paraformaldehyde at room temperature for 15 minutes. Next, permeablize the monolayers with Triton X-100 for 20 minutes at room temperature. After permeablization, block monolayers with two percent donkey serum for an hour.
After one hour of blocking, incubate the monolayer with primary antibody solution at four degrees celsius overnight. Wrap the monolayer in parafilm to avoid evaporation and drying out of the culture overnight. The next day, wash with 0.1 percent of non-ionic detergent in PBS.
Then, incubate with secondary antibody and 10 micrograms per milliliter of host cell nuclei stain for an hour at room temperature. Next, stain the cells. Then, identify the surface mucous pit cells in the 2D human-derived gastric monolayer culture under the confocal microscope.
Finally, capture images of the wound every few hours. These images from time lapse analysis show the formation of human FGOs. These organoids are transferred to a gastric epithelial monolayer on basement membrane matrix coded chamber slides.
The gastral epithelial monolayer is analyzed every eight hours for a day. The image shows that the wound area keeps decreasing and completely disappears in the culture in just one day. This immunol florescence staining shows the presents of E-cadherin positive monolayer that consists of the surface mucous cells.
This indicates the regeneration of the gastric epithelial monolayer. The nuclei present is stained with hoechst. Then, RTPCR analysis is done using RNA extracted from the human gastric epithelial monolayer to confirm the expression of parietal surface mucous, chief, and mucous neck cells.
Presence of ATPa and PgC, shows the presence of parietal and chief cells respectively. Presence of MUC5AC and MUC6 shows the presence of surface mucous and mucous neck cells respectively. In this immunol florescence image, proliferating cells in the human gastric epithelial monolayer culture is shown by the ready uptake of EdU.
These immunol florescence images show the regeneration of the gastric epithelium and expression of surface mucous and parietal cells at zero, eight, 16 and 24 hours post scratch wound. Once mastered, the 3D to 2D transfer can be done within one hour if it is performed properly. While attempting this procedure, it's important to remember to wash away as much of the basement membrane matrix from the harvested organoids prior to 2D transfer.
Following this procedure, other methods like helicobacter pylori infection in monolayers and monolayer immune cell co-cultures can be performed in order to answer additional questions, like host pathogen interactions. After its development, this technique paved the way for researchers in the field of gastroenterology to explore diseases and regeneration in the stomach. Don't forget that working with human tissue can be extremely hazardous, and precautions such as sterile technique and personal protective measures should always be taken while performing this procedure.
