Paediatric gastric organoids as a tool for disease modelling and clinical translation

Endoscopic mucosal and full-thickness surgical paediatric gastric biopsies were collected after an informed consent process with an independent research coordinator, in compliance with license 18DS02.

Figure 1 shows an overview of the gastric gland isolation and organoid culture process, as well as the potential applications of paediatric gastric organoids. Human paediatric gastric tissues, either endoscopic mucosal biopsies or full-thickness surgical specimens, were collected in ice-cold sterile Advanced DMEM F-12 (ADMEM/F12) basal medium (Thermo Fisher, 12634). Tissue was transported to laboratory on ice and processed within 1 hour of collection. Gastric gland stem cells were isolated from specimens using an adaptation of published dissociation protocols for adult tissues [9, 11].

Tissue was washed in cold Hank’s balanced salt solution (HBSS; Thermo Fisher, 88284) in a petri dish. The luminal mucus layer was then removed by scraping the mucosa with a glass coverslip. For full thickness specimens, the mucosa and muscle layers were separated by blunt dissection. Isolated mucosa was then cut into pieces < 5 mm in diameter. Mucosal pieces were washed vigorously with HBSS using a 10 mL pipette pre-coated with 1% bovine serum albumin (BSA; Sigma-Aldrich, A9418), discarding the supernatant after allowing the pieces to settle. This process was repeated until the supernatant was clear.

Mucosal pieces were then incubated in a chelating buffer containing 5.6 mmol/L Na₂HPO₄, 8.0 mmol/L KH₂PO₄, 96.2 mmol/L NaCl, 1.6 mmol/L KCl, 43.4 mmol/L sucrose, 54.9 mmol/L D-sorbitol, 0.5 mmol/L DL-dithiothreitol, and 2 mM EDTA (all from Sigma-Aldrich) dissolved in MilliQ water (18.2 mΩ/cm) for 30 min at 37 °C on a shaking platform. Pieces were transferred to a dry sterile petri dish on ice and glands were released from the mucosa using direct pressure with a glass coverslip. Glands were collected by washing the petri dish with ice cold ADMEM/F12 plus 1% penicillin–streptomycin (Thermo Fisher, 15140122), 10 mM HEPES (Thermo Fisher, 15630080), and 2 mM Glutamax (Thermo Fisher, 35050061) (called “ADEM/F12+++”). The gland-containing medium was passed through a 40 μm cell strainer and centrifuged at 300g for 5 min at 4 °C. After aspirating the supernatant, the cell pellet was resuspended in liquid growth factor reduced Matrigel® basement membrane matrix (Corning, 354230). Droplets of 30 μL were plated in warmed multi-well tissue culture plates. The plates were then inverted and incubated for 15 min at 37 °C to induce gelation of the Matrigel®, with gravity allowing the glands to be drawn towards the medium-facing surface of the inverted Matrigel® droplet during gelation. After gelation, a chemically defined, Good Manufacturing Practice (GMP)-compliant, human gastric organoid medium was added to the well (see below), along with Rho kinase inhibitor (Y-27632; Tocris, 1254) if single cells were visible in the droplet. Glands were cultured in normoxia with 5% CO₂ and medium was changed every 3–4 days. For small endoscopic biopsies, plating in a single 30 μL droplet was appropriate, while full thickness surgical specimens, such as gastrostomy closures, yielded enough glands to plate three or more 30 μL droplets of appropriate density.

Human paediatric gastric organoid medium includes “ADMEM/F12+++” (as above), 1X B-27 supplement without vitamin A (Thermo Fisher, 12587010), 1.25 mM N-acetylcysteine (Sigma Aldrich, A9165), 100 ng/mL Wnt-3A (Peprotech, 315–20), 500 ng/mL R-spondin 1 (Peprotech 120–38), 100 ng/mL Noggin (R&D Systems, 6057-NG), 50 ng/mL epidermal growth factor (EGF) (Thermo Fisher, PMG8043), 10 nM gastrin (Sigma Aldrich, G9020), 3 μM glycogen synthase kinase 3 (GSK-3) inhibitor (CHIR99021) (Tocris, 4423), 5 μM transforming growth factor beta (TGFβ) inhibitor (A83-01) (Sigma Aldrich, SML0788), and 200 ng/mL fibroblast growth factor 10 (FGF10) (Peprotech, 100–26) [12].

After formation of organoids using the protocol described above, organoids were passaged in culture every 6–8 days by one of two methods: (1) manual disaggregation in a narrowed glass pipette, (2) enzymatic dissociation to single cells.

Human gastric tissues were fixed in 4% paraformaldehyde (PFA; Sigma-Aldrich, 100496) for 30 min to 2 h, depending on the size of the sample, and then washed extensively in PBS. Samples were dehydrated overnight in 30% sucrose and then embedded in PolyFreeze tissue freezing medium (Polysciences, 25113) over dry ice. Sections were cut at 7 μm on a Bright Instruments cryostat and stored at − 20 °C until staining.

Organoids at 7 days from last passage were removed from Matrigel® by incubation in Cell Recovery Solution (Corning, 354253) for 45 min at 4 °C. This treatment released the organoids from the Matrigel® without damaging the structure of the organoids allowing whole mount immunofluorescence staining. Organoids were collected, washed in PBS, and transferred to 1% BSA pre-coated 1.5 mL Eppendorf tubes, and resuspended in 4% PFA for 20 min in rotation. PFA was removed and quenched with 0.1 M NH₄Cl (Sigma-Aldrich, 254134) for 1 h in rotation to decrease aldehyde-related autofluorescence. Quenched organoids were stored in PBS with 1% penicillin–streptomycin until staining.

Native gastric mucosal sections were blocked and permeabilised with 0.5% Triton X-100 (Sigma-Aldrich, 100496) with 1% BSA in PBS for 2 h at room temperature. Organoid whole mounts were treated the same way but were maintained in rotation during this time. Primary antibodies were diluted in blocking buffer and incubated with sections or organoids for 24 h at 4 °C (static for sections, in rotation for organoids). Samples were then extensively washed in 0.5% Triton X-100 in PBS. Secondary antibodies were diluted and applied to samples as for primary antibodies and incubated overnight at 4 °C. Organoids in suspension were transferred to a glass-bottomed Petri dish immediately prior to imaging.

Primary antibodies used in this study were chromogranin A (Abcam, ab15160), mucin 5AC (Invitrogen, ma5-12178), mucin 6 (Abcam, ab216017), pepsinogen C (Abcam, ab9013-1), gastrin (Leica, PA0681), all at 1 in 100 dilution, except gastrin and pepsinogen C which were used at 1 in 500. Secondary antibodies were AlexaFluor® anti-rabbit 488 (Thermo Fisher, A11008), AlexaFluor® anti-rabbit 568 (Thermo Fisher, A11011), AlexaFluor® anti-mouse 488 (Thermo Fisher, A11001), and AlexaFluor® anti-sheep 488 (Thermo Fisher, A11015) all at 1 in 200 dilution. AlexaFluor® Phalloidin 647 (Thermo Fisher, A22287) was used at 1 in 100 dilution and Hoechst 33342 (Thermo Fisher, H1399) at 10 μg/mL.

Freshly isolated gastric glands and organoids were imaged during cell culture in bright field using a Zeiss Axio Observer A1. Immunofluorescence images of native gastric mucosal sections and organoid whole mounts were acquired on a Zeiss LSM710 confocal microscope.

Gastric tissue samples were obtained from six patients ranging in age from 4 months to 16 years. No patients had primary diseases of the stomach at final diagnosis. Two of the samples were endoscopic mucosal biopsies (Fig. 2a) and the other four were full-thickness surgical specimens. As expected, paediatric gastric mucosa contained mature surface (pit) mucin producing cells (mucin 5AC, MUC5AC), gland (neck) mucin producing cells (mucin 6, MUC6), enteroendocrine cells (chromogranin A, CHGA), and chief cells (pepsinogen C, PGC) (Fig. 2b, c).

Gastric glands were successfully isolated from all six patient samples and encapsulated in Matrigel® (Fig. 2d, top panel). By day 2–4 in culture, elongated gastric glands sealed and formed cystic structures with a central cavity lined by a single layer of cells (Fig. 2d, middle panel). Organoids formed from each of the six patients. These spherical structures closely resemble the morphology of adult epithelial gastric organoids [9]. The organoids continued to proliferate and enlarge over the subsequent days, with some evidence of gland-like budding after day 7 in culture (Fig. 2d, bottom panel). Non-gland cells encapsulated in the Matrigel® died during the first 7 days in culture. These cells were likely either non-epithelial or non-progenitor epithelial cells, and as such were not supported by the gastric organoid-specific medium.

Following derivation from gastric glands, gastric organoids could be passaged using manual disaggregation or by dissociation to single cells. Figure 3a shows a typical passage following single cell dissociation and culture with rho kinase inhibitor, which was crucial to the formation of organoids from single cells (data not shown). By day 4 from dissociation to single cells, spherical organoids reliably formed with a central lumen. As the culture progressed, sloughed cells appeared in the lumen of the organoid consistent with epithelial turnover (Fig. 3a, middle panel). At day 7, organoids were well formed and had continued to grow in size compared to day 4, while remaining healthy and organised in appearance (Fig. 3a, bottom panel). Morphology of the gastric organoids is similar to that observed in freshly isolated gastric glands and remains unchanged up to and including passage 45 (52 weeks in culture), when the experiments were electively stopped. Single cells derived from the organoids in one 30 μL Matrigel® droplet were sufficient to form new organoids in six 30 μL droplets (1:6 split ratio) at similar density to the original droplet, demonstrating the expansion potential of this organoid culture system.

Organoids passaged using the manual disaggregation technique also re-formed organoids reliably after passaging. However, instead of being able to replate at a 1:6 split ratio as for single cell dissociation, manually disaggregated organoids were usually plated at a 1:3–1:5 ratio to preserve optimal density. Passaging in this manner avoided the need to use the anti-apoptotic rho kinase inhibitor that is required in single cell passaging.

Having demonstrated that gastric organoids have a morphology consistent with an epithelium-lined structure with a central lumen, we next examined cell polarity and the cellular subsets retained in the organoids. Paediatric gastric organoids showed appropriate epithelial polarity with apical surface of cells orientated towards a central lumen, as shown by localisation of f-actin to the luminal surface of the cells (Fig. 3b, top panel). The spherical morphology of the organoids suggested a substantial surface (or pit) domain and indeed there was prominent expression of the surface mucin 5AC on the luminal surface of the cells, consistent with secretion of the mucin into the central lumen of the organoids (Fig. 3b, top panel). Chief cells, marked by pepsinogen C (PGC), and gastrin-secreting enteroendocrine cells were present in the gastric organoids (Fig. 3b, middle and bottom panels). Pepsinogen C localised to the cytoplasm of chief cells in a speckled pattern, consistent with the secretory granules known to be present in chief cells in native tissue (Fig. 3b, bottom panel). Similarly, gastrin was appropriately localised to the cytoplasm of G cells (Fig. 3b, bottom panel). Chief and G cells were shown to be less common in the organoids compared to surface mucin cells, consistent with proportions observed in native stomach tissue. Parietal cells, marked by proton pump subunit ATP4B, were not observed in the organoids. However, neck mucus cells (MUC6), stem cells (Lgr5), and other enteroendocrine cell types (for example, somatostatin) were also present in the gastric organoids (data not shown). This suggests gastric organoids to be a representative in vitro model of paediatric gastric epithelium.