Our research focuses on hereditary causes of gastric cancer. In particular, we're interested in hereditary diffused gastric cancer due to disease-causing variants in either the CDH1 or CTNNA1 gene. We also investigate the role of BRCA1 and BRCA2 in gastric cancer risk and carcinogenesis.
Organoids are increasingly being utilized in a variety of research domains, and gastroenterology is no exception, so with this, comes a need for standardization of these techniques to generate these organoids, in particular patient-derived gastric organoids. Our protocol offers a step-by-step method to reliably generate patient-derived gastric organoids from biopsies of both the antral and body regions of the stomach. In particular, we utilize a single-cell digest approach to allow for standardized seeding of cells in an effort to make reliable comparisons amongst organoids generated from different patients.
Our findings show that compared to patient-derived gastric organoids from the body region of the stomach, those from the antral region actually grow more numerous and larger in size over the course of 20 days post initial seeding. These findings should be taken into account by researchers looking to utilize gastric organoids derived from different regions of the stomach. To begin the isolation of single cells from the gastric biopsies collected during the upper endoscopy of the patient, first, allow the tissue to settle at the bottom of a 15 milliliter conical tube.
Once settled, use a pipette to aspirate the media, and wash the biopsies twice with one milliliter of antibiotic-supplemented PBS. Transfer a minimum of 20 milligrams of the tissue to a 1.5 milliliter tube containing one milliliter of PBS supplemented with DTT. Use fine dissection scissors to cut the tissue into pieces that are one to two millimeters or smaller.
Utilize a tabletop mini centrifuge for 15 seconds to aggregate the tissue pieces at the bottom of the tube, then aspirate as much supernatant as possible. Add five milliliters of freshly-warmed digestion buffer to a 50 milliliter conical tube. Transfer 500 microliters of the buffer from this tube to the 1.5 milliliter tube containing the tissue pieces.
Next, using scissors, cut the 1, 000 microliter pipette tip from the bottom, to increase the tip's diameter for minimizing tissue loss. Now, with the modified pipette tip, transfer the small tissue pieces from the 1.5 milliliter tube to the digestion buffer in the 50 milliliter tube. Incubate the digestion buffer and tissue mix at 37 degrees for 30 minutes with orbital shaking at 200 RPM.
Then, add five milliliters of warmed Trypsin with 0.25%EDTA to the tissue in the digestion buffer, and incubate. Neutralize the digestion buffer and Trypsin by adding an equal volume of advanced DMEM/F-12 media, and pass the suspension through a 70 micron cell strainer. To begin, remove the thawed basement membrane matrix from the ice.
Add the calculated volume of the basement membrane matrix to the gastric biopsy-derived single cells, and gently mix by pipetting up and down for approximately 10 seconds. Then using a pipette, rapidly transfer 50 microliter aliquots of the matrix and cell mixture to the center of individual wells in a 24-well tissue culture plate. Cover the plate immediately, and in a single smooth motion, flip the plate upside down.
Place the inverted plate in a 37 degrees Celsius tissue culture incubator for 35 minutes to allow the basement membrane matrix to polymerize. Then, add 500 microliters of pre-warmed gastric organoid media to each well, while ensuring the top of each dome is fully submerged in the media. Organoids are generally identifiable within 10 days post seeding of single cells.
By day 20, they are large and typically need to be passaged. The number of body and antral organoids peaked at day 10 post seeding before decreasing, although not significantly. Organoids generated from antral biopsy tissue displayed a greater growth rate compared to organoids generated from body biopsy tissue.
Across different patients, a diversity of organoid morphology was typically observed in any single dome. However, on average, organoid sphericity showed little variation. After the patient-derived gastric organoids are ready for passaging, begin routine passing of the organoids.
First, remove the media from each well containing the organoids embedded in the basement membrane matrix, done in the 24-well plate. Then, dispense one milliliter of ice-cold advanced DMEM/F-12 media directly onto a basement membrane matrix dome. Continue aspirating and dispensing the media until all fragments of the dome detach from the plate.
Then, transport both the media and the fragmented basement membrane matrix from the well to the next well, and repeat the process for all wells slated for passaging. After disassembling the last basement membrane matrix dome, dispense the media and the mixture into a 1.5 milliliter tube. Attach a 1, 000 microliter tip to a P1000 pipette and then insert this tip into a 200 microliter tip.
Using this pipette setup, vigorously pipette the organoid matrix mixture up and down approximately 25 times to fragment the organoids into small pieces. Centrifuge the fragmented organoid mixture using a tabletop centrifuge at four degrees Celsius for 30 seconds at 2, 000g. Using a pipette, aspirate the media and basement membrane matrix supernatant.
Add the calculated volume of fresh basement membrane matrix to the pelleted organoids in the tube. Gently pipette the content of the tube up and down to mix. Swiftly, aliquot 50 microliters of the organoid matrix mixture into individual wells of a 24-well plate.
Cover the plate immediately, and in a single smooth motion, flip the plate upside down. Place the inverted plate in a 37 degree Celsius tissue culture incubator for 35 minutes to allow the basement membrane matrix to polymerize. Finally, add 500 microliters of pre-warmed gastric organoid media to each well.
By day 20 post initiation, organoids were ready to be passaged, indicated by a large organoid size or a darkened interior. Organoids left to go beyond these points began to break down into 2D monolayers. After passaging and reseeding gastric, the domes contained many organoid fragments that reorganized themselves into many more organoids very quickly.
