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Method Article

Single Cell-Seeded Human Intestinal Organoids for Organoid Research

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DOI:

10.3791/69265

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February 6th, 2026

 ,  ,  ,  ,  , 
1Department of Anatomy, Graduate School of Medical Science, Brain Korea 21 Project, Yonsei University College of Medicine, 2Division of Gastroenterology and Hepatology, Department of Medicine, NYU Grossman School of Medicine, 3Division of Gastroenterology and Hepatology, Department of Medicine, University of Pennsylvania Perelman School of Medicine, 4Department of Pathobiology, University of Pennsylvania Perelman School of Veterinary Medicine

In This Article

Summary

We present a protocol to generate single cell-seeded patient-derived intestinal organoids, providing standardized cultures that reduce heterogeneity and improve reproducibility for downstream assays and precision medicine applications.

Abstract

Recent developments in organoid technology have enabled the creation of patient-derived intestinal organoids (PDIOs) that recapitulate the structural, functional, genetic, and epigenetic features of their original tissues. However, conventional passage-derived organoids inevitably yield heterogeneous populations in size and number, leading to inconsistent results even under identical conditions. To address this, a standardized approach, referred to here as "single cell-seeded PDIOs," was established. In this method, mature PDIOs were enzymatically dissociated into single cells and seeded at a defined number into individual wells of a 96-well plate. This controlled seeding normalized the size and number of PDIOs. Compared with passage-derived organoids, single cell-seeded PDIOs displayed reduced inter-well variability in organoid numbers and intra-well variability in organoid sizes, which enables the determination of generation efficiency and improves the reproducibility of viability assays. Moreover, this platform is compatible with downstream analysis, including transcriptomic analysis and protein expression profiling. Collectively, this approach may enhance experimental consistency and provide a practical foundation for reproducible PDIO-based studies.

Introduction

The human intestine is a highly complex ecosystem defined by finely tuned interactions among gut microbiota, intestinal epithelium, and the immune system1. These components maintain intestinal homeostasis through tightly regulated signaling networks. For instance, the gut microbiota, comprising over ten trillion microbial organisms, interacts with immune cells to modulate the activity of the immune system and maintains a balanced intestinal environment2. The intestinal epithelium is instrumental in mediating this microbiota-immune system crosstalk3,4. Intestinal stem cells (ISCs), the key driver of epithelial renewal, continuously regenerate and differentiate into specialized cell types, including secretory cell lineages (Paneth cells, goblet cells, enteroendocrine cells, and Tuft cells), absorptive enterocytes, and microfold cells5,6. Together, these cells form a vital physical barrier and functional interface that protects the host against inflammatory stimuli.

In recent years, PDIOs have emerged as a powerful platform in translational research7. These three-dimensional (3D) cultures are created from ISCs isolated from patient tissues and maintained in an extracellular matrix such as extracellular matrix (ECM), along with defined growth factors that recapitulate the ISC niche8,9. Notably, PDIOs retain the genetic and epigenetic characteristics of the donor tissue, allowing sustained expansion and reproducible experimentation. Since Sato et al. first developed PDIOs from leucine-rich repeat-containing G protein-coupled receptor 5+ (Lgr5⁺) ISCs10, subsequent studies have identified essential growth factors for the generation and differentiation of PDIOs, leading to widely adopted protocols across laboratories11,12,13. However, the experimental settings used to treat PDIOs with candidate drugs vary considerably across the studies and are often insufficiently described14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47. Furthermore, PDIOs are typically passaged by mechanical disruption, resulting in heterogeneity in organoid size, morphology, and spatial arrangement. This variability complicates the accurate assessment of drug responsiveness and underlying molecular mechanisms. To address this limitation, recent studies have developed PDIOs derived from single cell-suspension or sorted single cells45,48,49,50,51,52,53,54.

In this article, as one of the complementary and practical methods, "single cell-seeded PDIOs" is introduced to generate standardized PDIOs from a single cell suspension using conventional ECM and media, enabling accurate and reproducible assays. Briefly, single cells isolated from the mature PDIOs were embedded in ECM at defined cell densities, and the resulting cultures were monitored over 2 weeks while number, size, and morphology were quantified. Because each ECM dome is embedded with a known cell input, the organoid count per well is consistent, allowing generation efficiency to be calculated as a readout for regenerative capacity in each organoid line49,54. Although the dome-shaped Matrigel produces size heterogeneity due to the spatial gradient of Wnt3a concentration arising from its inherent instability and limited diffusion55, inter-well size variability in single cell-seeded PDIOs is markedly lower than in passage-derived organoids49,54. However, this approach still has practical limitations. Without fluorescence-activated cell sorting, some organoids may be generated from doublets or triplets, resulting in intrinsic biological heterogeneity within PDIOs. Also, the optimal number of single cells must be empirically determined for each organoid line due to varied generation efficiencies. Finally, fully differentiated single cell-seeded PDIOs were subjected to downstream analyses including quantitative reverse transcription PCR (qRT-PCR), Western blot, and immunohistochemistry.

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Protocol

All materials and reagents required for the experiment are listed in the Table of Materials.

1. Preparation of reagents and materials

  1. Pre-warm the organoids culture plate in a 37 °C incubator for more than 90 min.
    NOTE: Pre-warming the culture plate will help solidify the ECM and maintain dome shape. 90 min is the minimum required time for ECM polymerization. It is highly recommended to pre-warm the plate for more than 12 h.
  2. Use organoid growth medium (EXP) supplemented with 0.5% penicillin-streptomycin (PS) as the organoid culture medium.
  3. For differentiation of single cell-seeded PDIOs, use a differentiation medium (DIF) composed of a basal medium supplemented with growth factors (Table 1). The basal medium consists of advanced DMEM/F12 supplemented with 0.5 % PS, 0.25% gentamicin sulfate, and 1% glutamine, together with the following growth factors: Noggin (100 ng/mL), R-Spondin-1 (1 µg/mL), Wnt3a (100 ng/mL), epidermal growth factor (EGF; 50 ng/mL), N-acetyl-L-cysteine (1 mM), [Leu15]-gastrin I human (10 nM), A 83-01 (500 nM), insulin-like growth factor 1 (IGF-I; 200 ng/mL), fibroblast growth factor (FGF; 100 ng/mL), and B-27 supplement (1x) (Table 1).
    NOTE: PS and gentamicin are biohazardous and should be handled with gloves and eye protection.
  4. Freshly prepare the cell recovery solution (CRS), washing buffer (DMEM high glucose with 1% BSA), 1 mM Y-27632, EXP, and DIF, and keep on ice until use.
    NOTE: Allow EXP and DIF to equilibrate to room temperature for at least 20 min before adding to pre-warmed culture plates.
  5. Thaw the ECM on ice and ensure that it is always kept cold.
    NOTE: ECM requires thawing on ice and should always be kept cold to prevent premature polymerization.
  6. Equilibrate the enzymatic dissociation reagent to room temperature. Pre-warm the reagent in a 37 °C water bath before application.
    NOTE: Enzymatic dissociation reagent is an irritant and should be handled with appropriate personal protective equipment.

2. Thawing and recovery of cryopreserved PDIOs

  1. Retrieve the cryovial tube containing organoids from the liquid nitrogen tank. Loosen the tube cap slightly and thaw in a 37 °C water bath for 60-90 s.
    NOTE: Immediately transfer the tube to ice after thawing.
  2. Pre-wet a wide-bore pipette tip with washing buffer and transfer the organoids from the cryovial tube into a 50 mL tube.
  3. Rinse the cryovial with 1 mL of washing buffer and transfer the remaining contents to the 50 mL tube. Repeat this rinse step twice.
  4. Centrifuge at 290 × g for 7 min at 4 °C. Ensure that a visible pellet forms at the bottom of the tube.
  5. Aspirate the supernatant.
  6. Resuspend in ECM using a wide-bore pre-wetted pipette tip.
  7. Dot ECM onto a pre-warmed 24-well plate and incubate at 37 °C for 10 min.
    NOTE: Use 30 µL of ECM per well.
  8. Add EXP and surround the wells with sterile 1x phosphate-buffered saline (PBS) or distilled water.
    NOTE: Use 500 µL of EXP per well. Culture in EXP supplemented with 10 µM Y-27632 for the first 3 days, then changed to EXP without Y-27632.

3. Passaging of PDIOs

  1. Aspirate the spent EXP.
  2. Pre-wet a wide-bore pipette tip with washing buffer, add 1 mL of cell dissociation reagent to detach the ECM, and transfer the contents to a 15 mL tube.
  3. Incubate the tube on a rocker for 10 min at room temperature.
  4. Centrifuge at 300 × g for 7 min at 4 °C.
  5. Decant the supernatant and add 1 mL of washing buffer. Resuspend 20 times.
  6. Add another 1 mL of washing buffer, resuspend 20 times, and transfer to a 50 mL tube.
  7. Centrifuge at 300 × g for 7 min at 4 °C.
  8. Aspirate the supernatant.
  9. Resuspend in ECM using a wide-bore pre-wetted pipette tip.
  10. Dot ECM onto a pre-warmed 24-well plate and incubate at 37 °C for 10 min.
    NOTE: Use 30 µL of ECM per well.
  11. Add EXP and surround the wells with sterile 1x PBS or distilled water.
    NOTE: Use 500 µL of EXP per well. Culture in EXP supplemented with 10 µM Y-27632 for the first 3 days, then changed to EXP without Y-27632.

4. Single cell-seeded PDIO culture

  1. Aspirate the spent EXP.
    NOTE: Waste containing antibiotics (e.g., spent medium with PS or gentamicin) should be autoclaved prior to disposal.
  2. Wash with 1 mL of sterile 1x PBS.
  3. Pre-wet a wide-bore pipette tip with washing buffer, add 300 µL of CRS to detach ECM, and transfer the contents to a 50 mL tube.
  4. Centrifuge at 300 × g for 5 min at 4 °C (Figure 1A).
  5. Aspirate the supernatant, add 3 mL of washing buffer, and resuspend 20 times.
    NOTE: This step mechanically disrupts the organoids.
  6. Centrifuge at 300 × g for 5 min at 4 °C (Figure 1B).
  7. Aspirate the supernatant and add 5 mL of pre-warmed enzymatic dissociation reagent containing 10 µM Y-27632. Swirl 2-3 times and incubate in 37 °C water bath for 20 min, shaking the tube every 5 min.
    NOTE: Debris aggregates typically appear within 10 min of incubation. Limit the total treatment of the enzymatic dissociation reagent to under 20 min. Do not exceed 25 min, as over-digestion markedly compromises the yield and viability of single cells.
  8. Add 10 mL of DMEM high glucose.
  9. Centrifuge at 300 × g for 5 min at 4 °C (Figure 1C).
  10. Aspirate the supernatant and add 1 mL of washing buffer. Resuspend 20 times.
    NOTE: Waste containing enzymatic dissociation reagent should be collected separately and inactivated with 0.5-1% bleach for at least 30 min before disposal according to institutional biosafety guidelines.
  11. Filter through a 40 µm cell strainer into a new 50 mL tube (Figure 1D).
    NOTE: This step is critical for isolating single cells from the mature organoids. A 40 µm cell strainer should be used to isolate single cells.
  12. Count cells using trypan blue. Transfer the calculated volume to a 1.5 mL tube.
    NOTE: Plate 3,000-4,000 cells per well in a pre-warmed 96-well plate.
  13. Centrifuge at 300 × g for 5 min at 4 °C (Figure 1E).
  14. Remove the supernatant using a pipette.
  15. Resuspend the cells in ECM (100-500 cells/µL) using a wide-bore pre-wetted pipette tip.
  16. Dot ECM into a pre-warmed 96-well plate and incubate at 37 °C for 10 min.
    NOTE: Use 10 µL of ECM per well.
  17. Add DIF to the wells. Surround the wells with sterile 1x PBS or distilled water.
    NOTE: Use 150 µL of DIF per well. Culture in DIF supplemented with 10 µM Y-27632 for 3 days, then changed to DIF without Y-27632. Change medium every 3 days and maintain for a total of 10 days.

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Results

To generate single cell-seeded PDIOs, mucosal pinch biopsies of the terminal ileum and the ascending colon were collected from consenting healthy subjects during surveillance colonoscopy using 2.8 mm standard biopsy forceps, under the NYU Grossman School of Medicine Institutional Review Board (Mucosal Immune Profiling in Patients with Inflammatory Bowel Disease; S12-01137). Then, single cell-seeded PDIOs were generated by dissociating 6,000 cells isolated from mature PDIOs into the ECM, followed by culturing in DIF. The ...

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Discussion

PDIOs hold immense promise for precision disease modeling, drug screening, and regenerative medicine, as they faithfully preserve the genetic and epigenetic landscape of individual patients. However, their practical applications are often hampered by difficulties in standardizing organoid generation and characterization. To overcome these challenges, an approach using PDIOs derived from single cell suspension or single cells has been introduced in recent studies45,

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Disclosures

Jordan E. Axelrad has received research grants from BioFire Diagnostics, Genentech, Janssen, and Takeda; consultancy fees, honorarium, or advisory board fees from AbbVie, Abivax, Adiso, Biomerieux, Bristol-Myers Squibb, Celltrion, Ferring, Fresenius, Janssen, Merck, Pfizer, Sanofi, Takeda, and Vedanta. K.C. is an inventor on US patent 10,722,600 and provisional patents 62/935,035 and 63/157,225.

Acknowledgements

This work was supported in part by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (RS-2025-00557588, RS-2024-00411768, RS-2025-18362970, and RS-2025-02214844, Kyung Ku Jang); a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI) funded by the Ministry of Health & Welfare, Republic of Korea (RS-2024-00406488, Kyung Ku Jang); National Institutes of Health (NIH) grant DK093668 (Ken Cadwell); K23DK124570 (Jordan E. Axelrad); Crohn's & Colitis Foundation (Jordan E. Axelrad). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
[Leu15]-Gastrin I humanSigma-AldrichG91450-0.1 mg
15 mL conical tubeSPL50015
20X PBS, pH 7.4BiosesangP2007-7.4
50 mL conical tubeSPL50050
A 83-01Tocris2939-10 mg
Advanced DMEM/F-12Gibco12634010
Axygen 1000 µL pipet tips, wide-bore, clear, nonsterileAxygenT-1005-WB-C
Axygen 200 µL pipet tips, wide-bore, clear, nonsterileAxygenT-205-WB-C
B-27 supplement (50X) serum freeGibco17504044
Bovine serum albuminRoche10735078001
Corning 96-well clear polystyrene microplatesCorning3599
Corning cell recovery solutionCorning354253
Corning ECM Matrix (Corning Matrigel) Corning356231
Costar 24-well clear TC-treated multiple well platesCorning3526
DMEM high glucose w/ stable glutamine w/ sodium pyruvateBiowestL0103-500
GentamicinGibco15750060
Gentle cell dissociation reagentStemcell100-0485
Human FGF-basic (FGF-2/bFGF) (154 aa) recombinant proteinGibco100-18B-100UG
Human IGF-I, animal-free recombinant proteinGibcoAF-100-11-500UG
Human Noggin proteinAcrobiosystemNON-H5219-1mg
Human R-Spondin 1 / RSPO1 (21-146) proteinAcrobiosystemRS6-H4220-1mg
IntestiCult Organoid Growth Medium (Human)Stemcell6010
L-glutamine 100XBiowestX0550-100
Mouse EGF recombinant proteinGibco315-09-100UG
N-acetyl-L-cysteineSigma-AldrichA9165-5 g
Penicillin-streptomycin solution 100XBiowestl0022-100
Recombinant human Wnt3a surrogateAcrobiosystemWNE-W5253-50ug
TrypLE (enzymatic dissociation reagent) express enzyme (1X), no phenol red Gibco12604013
Y-27632 dihydrochlorideTocris1254-10MG

References

  1. Kaiko, G. E., Stappenbeck, T. S. Host-microbe interactions shaping the gastrointestinal environment. Trends Immunol. 35 (11), 538-548 (2014).
  2. Zheng, D., Liwinski, T., Elinav, E. Interaction between microbiota and immunity in health and disease. Cell Res. 30 (6), 492-506 (2020).
  3. Liebing, E., Krug, S. M., Neurath, M. F., Siegmund, B., Becker, C. Wall of Resilience: How the Intestinal Epithelium Prevents Inflammatory Onslaught in the Gut. Cell Mol Gastroenterol Hepatol. 19 (2), 101423(2025).
  4. Soderholm, A. T., Pedicord, V. A. Intestinal epithelial cells: at the interface of the microbiota and mucosal immunity. Immunology. 158 (4), 267-280 (2019).
  5. Peterson, L. W., Artis, D. Intestinal epithelial cells: Regulators of barrier function and immune homeostasis. Nat Rev Immunol. 14 (3), 141-153 (2014).
  6. van der Flier, L. G., Clevers, H. Stem cells, self-renewal, and differentiation in the intestinal epithelium. Annu Rev Physiol. 71, 241-260 (2009).
  7. Lucafò, M., et al. Patient-derived organoids for therapy personalization in inflammatory bowel diseases. World J Gastroenterol. 28 (24), 2636-2653 (2022).
  8. Zhao, Z., et al. Organoids. Nat Rev Methods Primers. 2, (2022).
  9. Parente, I. A., Chiara, L., Bertoni, S. Exploring the potential of human intestinal organoids: Applications, challenges, and future directions. Life Sci. 352, 122875(2024).
  10. Sato, T., et al. Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature. 459 (7244), 262-265 (2009).
  11. Sato, T., et al. Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett's epithelium. Gastroenterology. 141 (5), 1762-1772 (2011).
  12. Miyoshi, H., Stappenbeck, T. S. In vitro expansion and genetic modification of gastrointestinal stem cells in spheroid culture. Nat Protoc. 8 (12), 2471-2482 (2013).
  13. Fujii, M., et al. Human intestinal organoids maintain self-renewal capacity and cellular diversity in niche-inspired culture condition. Cell Stem Cell. 23 (6), 787-793.e6 (2018).
  14. An, S., Huh, H., Ko, J. S., Moon, J. S., Cho, K. Y. Establishment and characterization of patient-derived intestinal organoids from pediatric Crohn's disease patients. Pediatr Gastroenterol Hepatol Nutr. 27 (6), 355-363 (2024).
  15. d'Aldebert, E., et al. Characterization of human colon organoids from inflammatory bowel disease patients. Front Cell Dev Biol. 8, 363(2020).
  16. Arnauts, K., et al. Ex vivo mimicking of inflammation in organoids derived from patients with ulcerative colitis. Gastroenterology. 159 (4), 1564-1567 (2020).
  17. Deleu, S., et al. High acetate concentration protects intestinal barrier and exerts anti-inflammatory effects in organoid-derived epithelial monolayer cultures from patients with ulcerative colitis. Int J Mol Sci. 24 (1), (2023).
  18. Dennison, T. W., et al. Patient-derived organoid biobank identifies epigenetic dysregulation of intestinal epithelial MHC-I as a novel mechanism in severe Crohn's Disease. Gut. 73 (9), 1464-1477 (2024).
  19. Ding, W., Marx, O. M., Mankarious, M. M., Koltun, W. A., Yochum, G. S. Disease severity impairs generation of intestinal organoid cultures from inflammatory bowel disease patients. J Surg Res. 293, 187-195 (2024).
  20. Dotti, I., Mayorgas, A., Salas, A. Generation of human colon organoids from healthy and inflammatory bowel disease mucosa. PLoS One. 17 (10), e0276195(2022).
  21. Ferrer-Picón, E., et al. Intestinal inflammation modulates the epithelial response to butyrate in patients with inflammatory bowel disease. Inflamm Bowel Dis. 26 (1), 43-55 (2020).
  22. Gopalakrishnan, S., et al. Comprehensive protocols for culturing and molecular biological analysis of IBD patient-derived colon epithelial organoids. Front Immunol. 14, 1097383(2023).
  23. Hammoudi, N., et al. Autologous organoid co-culture model reveals T cell-driven epithelial cell death in Crohn's Disease. Front Immunol. 13, 1008456(2022).
  24. Jurickova, I., et al. Eicosatetraynoic acid and butyrate regulate human intestinal organoid mitochondrial and extracellular matrix pathways implicated in Crohn's disease strictures. Inflamm Bowel Dis. 28 (7), 988-1003 (2022).
  25. Kakni, P., Truckenmüller, R., Habibović, P., van Griensven, M., Giselbrecht, S. A Microwell-based intestinal organoid-macrophage co-culture system to study intestinal inflammation. Int J Mol Sci. 23 (23), (2022).
  26. Karakasheva, T. A., et al. Patient-derived colonoids from disease-spared tissue retain inflammatory bowel disease-specific transcriptomic signatures. Gastro Hep Adv. 2 (6), 830-842 (2023).
  27. Kawamoto, A., et al. Ubiquitin D is upregulated by synergy of notch signalling and TNF-α in the inflamed intestinal epithelia of IBD patients. J Crohns Colitis. 13 (4), 495-509 (2019).
  28. Kelsen, J. R., et al. Colonoids from patients with pediatric inflammatory bowel disease exhibit decreased growth associated with inflammation severity and durable upregulation of antigen presentation genes. Inflamm Bowel Dis. 27 (2), 256-267 (2021).
  29. Lee, C., et al. Intestinal epithelial responses to IL-17 in adult stem cell-derived human intestinal organoids. J Crohns Colitis. 16 (12), 1911-1923 (2022).
  30. Matsuzawa-Ishimoto, Y., et al. An intestinal organoid-based platform that recreates susceptibility to T-cell-mediated tissue injury. Blood. 135 (26), 2388-2401 (2020).
  31. Matsuzawa-Ishimoto, Y., et al. The γδ IEL effector API5 masks genetic susceptibility to Paneth cell death. Nature. 610 (7932), 547-554 (2022).
  32. Murthy, S., et al. Single-cell transcriptomics of rectal organoids from individuals with perianal fistulizing Crohn's disease reveals patient-specific signatures. Sci Rep. 14 (1), 26142(2024).
  33. Nanki, K., et al. Somatic inflammatory gene mutations in human ulcerative colitis epithelium. Nature. 577 (7789), 254-259 (2020).
  34. Niklinska-Schirtz, B. J., et al. Ileal derived organoids from Crohn's disease patients show unique transcriptomic and secretomic signatures. Cell Mol Gastroenterol Hepatol. 12 (4), 1267-1280 (2021).
  35. Nishimura, R., et al. Establishment of a system to evaluate the therapeutic effect and the dynamics of an investigational drug on ulcerative colitis using human colonic organoids. J Gastroenterol. 54 (7), 608-620 (2019).
  36. Rees, W. D., et al. Enteroids derived from inflammatory bowel disease patients display dysregulated endoplasmic reticulum stress pathways, leading to differential inflammatory responses and dendritic cell maturation. J Crohns Colitis. 14 (7), 948-961 (2020).
  37. Sarvestani, S. K., et al. Induced organoids derived from patients with ulcerative colitis recapitulate colitic reactivity. Nat Commun. 12 (1), 262(2021).
  38. Skovdahl, H. K., et al. Patient derived colonoids as drug testing platforms-critical importance of oxygen concentration. Front Pharmacol. 12, 679741(2021).
  39. Suzuki, K., et al. Single cell analysis of Crohn's disease patient-derived small intestinal organoids reveals disease activity-dependent modification of stem cell properties. J Gastroenterol. 53 (9), 1035-1047 (2018).
  40. Tindle, C., et al. A living organoid biobank of patients with Crohn's disease reveals molecular subtypes for personalized therapeutics. Cell Rep Med. 5 (10), 101748(2024).
  41. Tsuchiya, M., et al. Functional analysis of isoflavones using patient-derived human colonic organoids. Biochem Biophys Res Commun. 542, 40-47 (2021).
  42. Vanhove, W., et al. Biopsy-derived intestinal epithelial cell cultures for pathway-based stratification of patients with inflammatory bowel disease. J Crohns Colitis. 12 (2), 178-187 (2018).
  43. Xu, P., et al. Interleukin-28A induces epithelial barrier dysfunction in CD patient-derived intestinal organoids. Am J Physiol Gastrointest Liver Physiol. 320 (5), G689-G699 (2021).
  44. Ko, C. Y., et al. Alleviative Effect of alpha-lipoic acid on cognitive impairment in high-fat diet and streptozotocin-induced type 2 diabetic rats. Front Aging Neurosci. 13, 774477(2021).
  45. Jang, K. K., et al. Tofacitinib uptake by patient-derived intestinal organoids predicts individual clinical responsiveness. Gastroenterology. 167 (7), 1453-1456.e5 (2024).
  46. Lee, C., Hong, S. N., Kim, E. R., Chang, D. K., Kim, Y. H. Epithelial regeneration ability of Crohn's disease assessed using patient-derived intestinal organoids. Int J Mol Sci. 22 (11), (2021).
  47. Laudadio, I., et al. Characterization of patient-derived intestinal organoids for modelling fibrosis in Inflammatory Bowel Disease. Inflamm Res. 73 (8), 1359-1370 (2024).
  48. Marinucci, M., et al. Standardizing patient-derived organoid generation workflow to avoid microbial contamination from colorectal cancer tissues. Front Oncol. 11, 781833(2021).
  49. Lin, X., et al. IL-17RA-signaling in Lgr5(+) intestinal stem cells induces expression of transcription factor ATOH1 to promote secretory cell lineage commitment. Immunity. 55 (2), 237-253.e8 (2022).
  50. Miller, D. S., Talbot, C. A., Simpson, W., Korey, A. A comparison of naproxen sodium, acetaminophen and placebo in the treatment of muscle contraction headache. Headache. 27 (7), 392-396 (1987).
  51. Yang, L., et al. A tunable human intestinal organoid system achieves controlled balance between self-renewal and differentiation. Nat Commun. 16 (1), 315(2025).
  52. Abdulla, N., Aronson, R., Plessis, T. D., Bebington, B., Kaur, M. Protocol for the establishment and characterization of South African patient-derived intestinal organoids. STAR Protoc. 6 (3), 103970(2025).
  53. Kang, S., Lee, M. R., Choi, W., Kong, S. Y., Kim, Y. H. Protocol for generation and utilization of patient-derived organoids from multimodal specimen. STAR Protoc. 6 (3), 104039(2025).
  54. Zhou, C., et al. Epigenetic reprogramming alters intestinal stem cell fate in pouchitis. bioRxiv. , (2025).
  55. Shin, W., et al. Spatiotemporal gradient and instability of Wnt induce heterogeneous growth and differentiation of human intestinal organoids. iScience. 23 (8), 101372(2020).
  56. Jang, K. K., et al. Variable susceptibility of intestinal organoid-derived monolayers to SARS-CoV-2 infection. PLoS Biol. 20 (3), e3001592(2022).
  57. He, G. W., et al. Optimized human intestinal organoid model reveals interleukin-22-dependency of paneth cell formation. Cell Stem Cell. 29 (9), 1333-1345.e6 (2022).
  58. Watanabe, K., et al. A ROCK inhibitor permits survival of dissociated human embryonic stem cells. Nat Biotechnol. 25 (6), 681-686 (2007).
  59. Sockell, A., et al. A microwell platform for high-throughput longitudinal phenotyping and selective retrieval of organoids. Cell Syst. 14 (9), 764-776.e6 (2023).
  60. Saito, T., Amako, J., Watanabe, T., Shiraki, N., Kume, S. Human pluripotent stem cell-derived intestinal organoids for pharmacokinetic studies. Eur J Cell Biol. 104 (2), 151489(2025).
  61. Velasco, V., Shariati, S. A., Esfandyarpour, R. Microtechnology-based methods for organoid models. Microsyst Nanoeng. 6, 76(2020).
  62. Kakni, P., et al. Intestinal organoid culture in polymer film-based microwell arrays. Adv Biosyst. 4 (10), e2000126(2020).

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Single Cell SeedingPatient Derived OrganoidsOrganoid VariabilityOrganoid ReproducibilityOrganoid Viability AssayOrganoid Generation EfficiencyTranscriptomic AnalysisProtein Expression Profiling
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