Gastric Fundic Glands Isolation: A Method to Isolate Fundic Glands from Human Stomach Tissue

All procedures involving human participants have been performed in compliance with the institutional, national and international guidelines for human welfare and have been reviewed by the local institutional review board.

1. Establishing Human-derived 3D Gastric Fundic Organoids

NOTE: To avoid contamination, perform protocol in its entirety within a sterile tissue culture hood. Human fundic tissue was collected from patients undergoing sleeve gastrectomy.

1. Collect the tissue from patients undergoing sleeve gastrectomy and place it in the ice-cold Ca²⁺/Mg²⁺-free Dulbecco's Phosphate Buffered Saline (DPBS) in a large plastic container.
2. Using forceps, wash the tissue vigorously in a sterile beaker containing 100 mL Ca²⁺/Mg²⁺-free DPBS. Wash for approximately 30 s or until the debris and the blood has been removed. Next, using sterile gauze, wipe away the mucous layer from the epithelium.
3. Using large forceps, firmly grasp the muscle layer of the tissue and, with force, scrape away the epithelium using the large curved hemostatic forceps. Collect the scraped epithelial fragments into a sterile, polystyrene Petri dish.
4. Mince the epithelial tissue into approximately 2.0 x 2.0 mm² sized fragments using razors to optimize tissue digestion. Wash the tissue fragments with Ca²⁺/Mg²⁺ -free DBPS supplemented with antibiotics (Ca²⁺/Mg²⁺ -free DPBS, 1% Penicillin/Streptomycin, 0.25 mg/mL Amphotericin B /10 mg/mL Gentamicin, 50 mg/mL Kanamycin), until the wash is free of blood. Perform multiple washes if needed. Discard off the wash by carefully filtering the wash through a sterile gauze into a waste beaker.
5. Collect washed fragments into a 125 mL glass round bottom flask with a 25 mm stir bar and pre-warmed incubation media (basal media supplemented with 2 mM L-glutamine, 1% Penicillin/Streptomycin, 10 mM HEPES Buffer, 1 mg/mL Collagenase Type 1, 2 mg/mL cell culture grade Bovine Serum Albumin). Seal the round bottom flask with rubber septa.
6. Insert a 20G spinal needle into the septa and connect it to an oxygen tank with rubber hosing. To filter out any contaminants, insert tissue paper into the tubing to create a filter. Turn on the oxygen outflow on low. Insert 10–15 outflow needles into the septa to avoid the rupture of septa.
7. Secure the set up to a ring stand using clamps and place it in a water bath calibrated to 37 °C with a stir plate for 30–45 min. After the tissue has been incubating for 15 min, remove 50 µL of incubation media and check visually for dissociated glands. If glands are not dense or have not separated from the tissue, leave for an additional 5–10 min.
8. Immediately following incubation, add 50 mL pre-heated DMEM/F-12 to incubation media using a sterile serological pipette or by pouring directly into the incubation mixture.
9. Filter the gland mixture through a sterile gauze into four 50 mL conical tubes. Keep the filtrate on ice for 15 min to allow extracted glands to settle to the bottom of the conical tube. Be careful not to disturb the settled glands; remove and discard off the top 40 mL of supernatant using a serological pipette. Resuspend the remaining 10 mL in 10 mL of DPBS supplemented with antibiotics.
10. Distribute the mixture evenly into 5 mL cell culture test tubes. Centrifuge for 5 min at 65 x g at 4 °C and remove the supernatant carefully using a pipette.
    NOTE: It is crucial to perform this step on the ice and to avoid polymerization of the basement membrane matrix while mixing. Additionally, it is important to visually inspect the gland density by sampling for 50 µL. The optimal density is ~70% confluency.
11. Next, plate the glands in 50 µL of the basement membrane matrix using a wide-tipped pipette. Avoid producing any bubbles while plating.
12. To allow basement membrane matrix to polymerize, incubate for 10 to 15 min at 37 °C. If plating in a 12-well culture plate, add 1 mL of hFGO media (Advanced Dulbecco's modified Eagle medium/F-12 medium supplemented with 2mM L-glutamine, 1% Penicillin/Streptomycin, 0.25 mg/mL Amphotericin B /10 mg/mL Gentamicin, 50 mg/mL Kanamycin, 10 mM HEPES Buffer, 1 mM n-Acteylcystine, 1 x N2, 1 x B27, 50% Wnt-conditioned medium, 20% R-spondin-conditioned medium supplemented with 100 ng/mL bone morphogenetic protein inhibitor, 1 nM gastrin, 50 ng/mL Epidermal Growth Factor, 200 ng/mL Fibroblast Growth Factor 10, 10 mM Nicotinamide, and 10 µM Y-27632 ROCK inhibitor) per well. If not using a 12-well culture plate, add enough media to submerge the basement membrane matrix bubble.
13. Culture the cells at 37 °C in a 5% CO₂ humidified cell culture incubator. Change media every 4-5 days.