Various tissues and cells, such as blood vessels, muscles, nerve fibers, and white blood cells, are present near the intestinal crypts. Therefore, it was impossible to eliminate the possibility of introducing unwanted cells during the tissue collection. The cells that may be introduced during the collection of intestinal tissues, include fibroblasts, mesenchymal cells, vascular cells, and blood cells. These cells are designated as “unwanted cells,” and their purity is verified by selecting appropriate antibody markers (negative markers, see Table 1) and using methods, such as flow cytometry.

To confirm pluripotency, specific stem cell markers were assessed using immunostaining or qPCR (Andrews and Gokhale, 2024) (see Table 2).

To confirm differentiation, markers specific to the ectoderm, endoderm, and mesoderm were assessed using immunostaining or qPCR (see Table 3). Additionally, differentiation into the three germ layers was assessed using teratoma formation in vivo, which served as an assessment method for confirming the differentiation process (Son et al., 2017).

Recombinant human R-Spondin1 (Rspo1), recombinant human noggin, recombinant human EGF, N-acetylcysteine, nicotinamide, p38 MAPK inhibitor (SB202190), ALK5 inhibitor (A83-01), and prostaglandin E2 (PGE2).

Matrigel or other extracellular matrix (ECM) basement membrane extracts (BME) (Pleguezuelos-Manzano et al., 2020), advanced DMEM/F12, B-27 supplement, collagenase type II, Y-27632 Rho kinase inhibitor, HEPES, red blood cell lysis solution, and antibiotics (penicillin/streptomycin, gentamicin, and Primocin).

Crypts were extracted from human biopsy tissues of the small intestine and colon. Subsequently, they were cultured in a dome shape using an expansion medium containing growth factors to facilitate organoid growth.

The culture was maintained at 37°C in a 5% CO₂ incubator at saturated humidity.

All culture steps were performed in a sterile environment to prevent contamination.

The recombinant human growth factors used in the generation of DE, HG, hIOs, and Mat-hIOs were Activin A; FGF4 and WNT3A or CHIR99021; Rspo1, Noggin, and EGF; and IL-2, Rspo1, noggin, and EGF, respectively.

For the generation of hPSCs, DE, HG, and hIOs the reagents used were mTeSR™1 complete kit, Y-27632 Rho kinase inhibitor, dispase or accutase, Matrigel or other ECM; RPMI 1640 medium supplemented with dFBS, L-glutamine, and penicillin/streptomycin; DMEM/F12, dFBS, L-glutamine, and penicillin/streptomycin; and Matrigel or other ECM (Kim et al., 2022; Li et al., 2024), advanced DMEM/F12, B-27 and N2 supplements, L-glutamine, HEPES, and penicillin/streptomycin, respectively.

The culture was maintained at 37°C in a 5% CO₂ incubator with saturating humidity. All culture steps were performed in a sterile environment to prevent contamination. Additionally, after the HG stage, it was essential to culture hPSC-derived organoids for a minimum of 28 days to obtain ISC characteristics.

Note: At 28 days, the organoids exhibited the absence of G-protein-coupled receptor 5 (LGR5) expression and broad expression of Achaete-scute complex homolog 2 (ASCL2). They were not restricted to SOX9+ proliferative zones. However, organoids cultured for up to 56 days exhibited the co-expression of ASCL2 and LGR5 in a limited epithelial domain overlapping with SOX9+ zones.

The hASC-IOs exhibited a bud-like morphology with a central lumen and retained a circumferential epithelial cell structure (Sato et al., 2009; Sato et al., 2011a; Wang R. et al., 2022).

The size of the hPSC-IOs ranged from 0.5 to 2 mm. They exhibited bud-like structures and featured a central lumen that closely interacted with circumferential epithelial cells. The formation of highly intricate epithelial structures was facilitated by the surrounding primitive mesenchyme (intestinal stromal cells) (Spence et al., 2011).

Matured hPSC-IOs were larger (1–2 mm) than that of the hPSC-Ios, exhibiting numerous and complex bud-like structures. They formed an intricate epithelial structure surrounded by the primitive mesenchyme (intestinal stromal cells) (Jung et al., 2018).

The hASC-IOs consisted of ISCs, TA cells, enterocytes, goblet cells, and enteroendocrine cells (Tetteh et al., 2016).

The hPSC-IOs consisted of ISCs, TA cells, enterocytes, goblet cells, Paneth cells, and enteroendocrine cells. They were enveloped by the primitive mesenchyme (intestinal stromal cells) (Spence et al., 2011).

Olfactomedin-4 (OFLM4+) is expressed in ISCs, Defensin Alpha 5 (DEFA5+) in Paneth cells, and mucin 13 (MUC13+) in goblet cells. Additionally, Keratin 20 (KRT20) was expressed in the mature epithelium of the crypt (Jung et al., 2018).

Note: The proportion of LGR5+ or ASCL2+ ISCs; KI67+ and LGR5− TA cells; alkaline phosphatase inhibitor (ALPI+), Villin+, or intestinal fatty acid binding protein (IFABP+) enterocytes; MUC2+ goblet cells; lysozyme (LYZ+) or matrix metallopeptidase 7 (MMP7+) Paneth cells; and chromogranin A (CHGA+) enteroendocrine cells in the intestinal epithelial cells should be a minimum of 30%.

• LGR5+ or ASCL2+: ISCs (Barker et al., 2007)

• LGR5+, ASCL2+ or OLFM4+: ISCs

IAP is an intrinsically expressed protein in the intestinal epithelium that plays a crucial role in maintaining intestinal homeostasis (Sensoy and Oznurlu, 2019; Wang et al., 2020).

Mucin is a highly glycosylated protein that protects epithelial cells. Muc2, secreted by goblet cells in the small intestine and colon, is a significant component of the loose mucus layer that entraps luminal material (Chang et al., 1994).

Karyotype analysis was used to assess the size, shape, and number of chromosomes in the cell samples from the organoids, ensuring the presence of a complete set of 46 chromosomes (Sato et al., 2011a).

Short Tandem Repeat (STR) loci are highly informative human genome markers. STR profiling ensures the quality and integrity of intestinal organoids (Lee et al., 2015).

Copy Number Variations (CNVs) identify genetic variations in intestinal organoids and reflect structural changes that result in abnormal copy numbers of one or more genes.

• Cell type marker gene analysis (Real-time Fluorescence Quantitative PCR Method) compared target protein with glyceraldehyde 3-phosphate dehydrogenase (GAPDH)

Note: Cell type markers: LGR5 and ASCL2 for ISCs; MUC2 for goblet cells; ALPI, Villin, and IFABP for enterocytes; CHGA for enteroendocrine cells; MKI67 for proliferating cells. Paneth cell marker (LYZ) was detected in colonic organoids. Additionally, hPSC-derived intestinal organoids expressed CDX2, KLF5, and SOX9.

• Goblet cell function assessment:Mucin test (Mucin staining method)

The medium was removed from the cultured organoids, rinsed liquid nitrogen twice with an equal volume of PBS, and fixed with 4% PFA. ALPI staining was performed following to the manufacturer instructions. Detection was assessed based on staining intensity.

The medium was removed from the cultured organoids, rinsed liquid nitrogen twice with an equal volume of PBS, and fixed with 4% PFA. AB-PAS or mucin staining was performed following the manufacturer’s instructions. Detection was assessed based on the staining intensity.

A culture medium containing forskolin (20 mM) and DMSO was prepared, each added at 0.1% (v/v). The culture medium containing forskolin or DMSO was added to the organoids and treated for approximately 10–12 h at 37°C in a CO₂ incubator. Organoid swelling was observed under a microscope by comparing the test group (forskolin-treated) with the negative control group (DMSO-treated) (Boj et al., 2017; Vonk et al., 2020).

Organoids were isolated from the Matrigel using a pipette, washed with PBS, and collected through centrifugation.

Chromosomes were isolated from cell nuclei, stained using specific methods, and mounted on slides. Subsequently, the chromosomes were photographed under a microscope and arranged in pairs using the jigsaw puzzle method. They were organized by size from 1 to 22, followed by the sex chromosomes as the 23rd pair. Abnormalities were analyzed using chromosome pair analysis.

CNV testing revealed fine-scale DNA copy number variations that were not detected through chromosomal karyotype analysis. It was commonly analyzed using chromosomal microarrays or next-generation sequencing (NGS).

STR analysis was performed at ten different loci on different chromosomes to confirm the presence of organoid samples. The target DNA was amplified through PCR using fluorescent-labeled primers, and the resulting DNA was assessed using a DNA sequencer.

Viability testing was performed using calcein-AM. Organoid size, shape, and conditions were observed under a microscope. Organoids that met the final requirements (diameter ≥200 μm) were counted. The solution was incubated with calcein-AM stock solution at a final concentration of 0.2 μmol/L for 60 min at 37°C. Live organoids (diameter ≥ 200 μm) exhibiting fluorescence (green) were counted under a fluorescence microscope (excitation: 490 nm, emission: 515 nm).

Cell viability was assessed using a 3D cell viability assay. Organoids were cultured in an opaque multi-well plate and treated with compounds following to the manufacturer’s instructions. The experimental conditions were optimized based on sample volume and microtissue characteristics. Following staining, live organoids emitting fluorescence were assessed using a fluorescence microscope.

1. Intestinal organoids were separated from the Matrigel or BME domes and collected in a 60 mm cell culture dish.

For hASC-IOs, 10 mL of DMSO (Sigma-Aldrich, Catalog Number D2650) and 40 mL of FBS (Sigma-Aldrich, Catalog Number F7524) were used. For organoids derived from hPSCs, a commercially available freezing medium without FBS was used.

1. A warm (37°C) basic medium was prepared for intestinal organoids and proliferation medium.