Skip to main content

Assessment of source material for human intestinal organoid culture for research and clinical use



Human intestinal organoids (hIOs) have potential as a model for investigating intestinal diseases. The hIO system faces logistic challenges including limited access to biopsies or low expression of epithelial cell types. Previous research identified the feasibility of tissue from the transverse (TC) or sigmoid colon (SC), or from cryopreserved biopsies from regions of the gastrointestinal tract. We aimed to create a protocol for robust hIO generation that could be implemented across multiple centres, allowing for development of a consistent biobank of hIOs from diverse patients.


TC and SC hIOs were expanded from fresh or frozen biopsies with standard or refined media. The expression of epithelial cells was evaluated via PCR. Growth of TC and SC hIO from healthy donors was reproducible from freshly acquired and frozen biopsies. A refined media including insulin-like growth factor (IGF)-1 and fibroblast growth factor (FGF)-2 enabled the expression of epithelial cells, including higher expression of goblet cells and enterocytes compared to standard organoid media. We identified a consistent time point where hIOs generated from frozen biopsies reflect similar hIO composition from freshly acquired samples. Feasibility of hIOs as a tool for research and clinical use, including the use of frozen biopsies, was demonstrated.


Three dimensional human intestinal organoids (hIOs), derived from patient intestinal stem cells isolated from intestinal crypts, have considerable potential as a tool for investigating human intestinal diseases, such as inflammatory bowel diseases (IBD) [1,2,3,4,5,6,7,8,9,10,11,12]. Organoids have been used to investigate the function of the human intestinal epithelial barrier [13, 14]. The implementation of hIO technology in research and clinical settings faces many logistical challenges, including the need for sufficient samples from a limited number of patients and the invasiveness of the procedures required to collect biopsies for research purposes. The limitation of samples can also be explained by patient heterogeneity and individual research study criteria. We have combined research findings from three areas—site of biopsy [15], use of refined media [8] and cryopreservation [16], to design an optimised protocol for robust hIO generation under conditions that would facilitate collaborative research across multiple centres.

Main text


Biopsies derived from TC and SC of healthy people (n = 9, Additional file 1: Table S1) undergoing colonoscopy were collected at the Department of Gastroenterology, Southern District Health Board. From each patient, six biopsies (2.8 mm × 8.8 mm) were obtained either from the TC or SC using jumbo-biopsy forceps (Radial Jaw 4 Jumbo Forceps w/ Needle, Boston Scientific, MA, USA).

Isolation of intestinal crypts and generation of human intestinal organoids

Intestinal biopsies were collected with the following media: Advanced Dulbecco’s Modified Eagle Medium (DMEM) + F12 (Invitrogen, MA, USA), fungizone (2.5 µg/mL, Invitrogen), penicillin and streptavidin (1%, Thermo Fisher Scientific, MA, USA), fetal calf serum (FCS; 10%, Invitrogen), gentamicin (0.05 mg/mL, Invitrogen), normocin (0.1 mg/mL, Integrated Science, Sydney, Australia). For frozenhIOs, fresh biopsies were suspended in Recovery Cell Culture Freezing Medium (Invitrogen) and stored at -80 °C overnight, then transferred to liquid nitrogen and stored for up to 1 month. Both fresh or thawed biopsies were washed with phosphate buffered saline (PBS; Sigma-Aldrich, MS, USA) plus dithiothreitol (DTT;10 mM, Sigma-Aldrich), and suspended in PBS plus ethylenediaminetetraacetic acid (EDTA; 8 mM, BDH, Dubai, UAE) on ice for 60 min. EDTA was removed, and the biopsies were suspended on cold PBS. The biopsies were shaken and supernatant enriched with crypts isolated. Crypts were centrifuged at 40 g at 4 °C for 3 min to remove debris. Intestinal crypts were resuspended in Matrigel® (25 µL of crypts/Matrigel, Corning, NY, USA), pipetted on pre-warmed Nunclon Delta Surface 24 well flat-bottom plates (Thermo Fisher Scientific) and incubated for 15 min at 37 °C and 5% CO2 to allow polymerisation of Matrigel. 500 µL of standard media (SM) or refined media (RM) were added to wells and crypts incubated at 37 °C and 5% CO2 up to 20 days without passage. Culture media was refreshed every 2–3 days. Organoids derived from frozen biopsies were first incubated up to two days with RM plus Rock inhibitor (0.01 mM, Y-27632, Biogems, CA, USA).

Reverse transcription polymerase chain reaction

Organoids were released from Matrigel using Cell Recovery Solution (Corning). Total RNA was extracted using the RNeasy Plus Micro Kit (Qiagen, Hilden, Germany) followed by cDNA synthesis using the SuperScript III First-Strand Synthesis System and Oligo(dT)20 primer (Thermo Fisher Scientific). TaqMan™ Gene Expression Master Mix (Thermo Fisher Scientific), and gene expression assay were used for RT-PCR (Additional file 1: Table S2). The Relative Quantification app (Thermo Fisher Scientific cloud) was used for data analysis. RT-qPCR cycle values (CT) obtained for specific mRNA expression in each sample were normalised to CT values of human endogenous (housekeeping) gene HPRT1 (hypoxanthine phosphoribosyltransferase 1) resulting in ΔCT values (log ratio of the gene concentrations) and used to calculate relative gene expression [17]:

ΔCT = Mean CT of gene of interest – Mean CT housekeeping gene.

We performed an exponential conversion of ΔCT, namely 2−ΔCT, using:

2^(exponential) – ΔCT


Statistical analysis was performed using GraphPad Prism 8.0 (GraphPad Software, California, United States). For experiments with more than two groups of matched samples, ANOVA one-way followed by Sidak’s multiple comparisons was used (95% confidence interval).

Results and discussion

Sigmoid colon-derived organoids can be generated ex vivo and are similar to transverse colon-derived organoids

HIOs have been generated from the gastrointestinal tract including the stomach [18], small intestine [6], terminal ileum [19], transverse colon (TC), sigmoid colon (SC) [19, 20], and rectum [21]. A colonoscopy to collect TC biopsies is invasive, requires sedation and prolonged recovery time, and is costly. Collection of SC biopsies is less invasive, usually causes less discomfort and is performed without sedation. A flexible sigmoidoscopy is therefore a feasible alternative for research purposes.

We collected biopsies from both TC and SC of the same volunteers, grew TC- and SC-hIO for 12 days and harvested samples to test the expression of epithelial cell types via RT-PCR of cell-specific markers (Fig. 1A). We analysed crypt maturation via expression of cystic fibrosis transmembrane conductance regulator (CFTR), and surface epithelial maturation of TC-derived hIO via expression of sodium–hydrogen exchanger 3 (NHE3; encoded by SLC9A3 (solute carrier family 9 member A3)) and epithelial sodium channel (ENaC; encoded by SCNN1A (sodium channel epithelial 1 subunit alpha)) for SC-derived hIOs (Fig. 1A) [22, 23]. TC- and SC-derived hIO had similar frequencies of epithelial cells, defined by gene expression (Fig. 1B). However, we observed a higher expression of genes representing goblet cells (FCGBP, Fc-gamma binding protein) in TC-derived (0.004 ± 0.001) than SC-derived hIOs (0.001 ± 0.001) (Fig. 1C). Goblet cells produce mucin, a key component of intestinal mucus that serves as a barrier for the immune host defence against luminal microbiota but also allows constant nutrient absorption [24]. Fcgbp is the main binding protein of Muc2 [25]. Both SPIB (Transcription factor SpiB; M cells) and POU2F3 (Pit-Oct-Unc (POU) class 2 homeobox 3; Tuft cells) were detected at low expression in both TC- and SC-derived hIOs.

Fig. 1
figure 1

Composition of hIOs derived from transverse and sigmoid colon. hIOs derived from transverse colon (TC) and sigmoid colon (SC) biopsies (n = 3) from non-IBD donors were expanded over 12 days under standard media (SM). Epithelial composition was evaluated on day 12 by RT-qPCR. A. Scheme depicting the overall experiment. B. Principal component analysis (PCA) from expression of epithelial cells of TC- and SC hIO. C. Relative gene expression (2−ΔCT) of epithelial surface marks, crypt markers, and epithelial cell marker between TC- and SC hIO

NHE3 (SLC9A3) is highly expressed in the surface epithelium of the proximal colon (including TC) where most fluid reabsorption occurs [22, 26]. We observed higher expression in TC-derived hIOs (0.014 ± 0.001) compared to SC-derived hIOs (0.003 ± 0.003; Fig. 1C). ENaC (SCNN1A) is normally expressed in the surface epithelium of the distal colon, which includes the SC, and can be used as a marker for epithelium maturation of SC-derived hIOs [23]. However we did not observe a high expression of EnaC (SCNN1A) in SC-derived hIOs (Fig. 1C).

TC- and SC-hIOs had similar expression of genes for differentiated epithelial cell types, with the exception of goblet cells. The difference in the transcript levels of the goblet cell marker in SC-hIOs compared to TC-hIOs is similar to the difference seen in the human colon, where SC has lower numbers of sulphated Muc2 + goblet cells compared to TC [27, 28]. We demonstrate that generation of SC-hIOs is feasible.

Human sigmoid colon derived intestinal organoids can be generated from frozen biopsies

A drawback of the hIO system is that organoids are usually generated from freshly acquired patient tissue. This practice is limited by the collection of biopsies from local centres with immediate access to research labs. We evaluated the generation of hIOs from frozen biopsies and the impact of cryopreservation on gene expression. We grew SC-hIOs derived from freshly acquired biopsies for 15 days, or SC-hIOs derived from frozen biopsies (cryopreserved for 1 month) from the same donor. After 15 and 20 days of growth, we measured the expression of genes related to cell markers via RT-PCR (Fig. 2A).

Fig. 2
figure 2

Comparison analysis of epithelial composition between organoids derived from fresh versus frozen biopsies. hIO were grown for 20 d from fresh and frozen SC biopsies (1 month at liquid nitrogen) (n = 6). Epithelial composition was evaluated on days 15 and 20 by RT-qPCR. A. Scheme depicting the overall experiment. B. PCA from RT-qPCR relative gene expression (2−ΔCT) data freshhIO (day 15) and frozenhIO (day 15,20). C. Relative gene expression between freshhIO (day 15) and frozenhIO (day 15,20). ANOVA one-way followed by Sidak’s multiple comparisons test were performed.* p < 0.05, *** p < 0.001, ns = not significant

frozenhIO (day 15) epithelial cell expression differed from that of freshhIO (day 15) and frozenhIO (day 20) (Fig. 2B). The frozenhIO (day 15) demonstrated high expression of CFTR (0.3193 ± 0.08732) compared to freshhIO (day 15) (0.1520 ± 0.06552) and frozenhIO (day 20) (0.1698 ± 0.05636) (Fig. 2C). We observed an increase in the stem cell marker, LGR5 (leucine rich repeat containing G protein-coupled receptor 5), in frozenhIO (day 15) (Fig. 2C), whereas expression of genes representing early enterocytes and goblet cells was less in frozenhIO (day 15) but fully or partially restored in frozenhIO (day 20) (Fig. 2C), indicating the immature status of the organoids. Although frozenhIO (day 15) and freshhIO (day 15) differed in respect to expression of cell markers related to epithelial cells, these differences were reduced in frozenhIO (day 20), indicating that cryopreservation of SC-derived biopsies likely does not impact generation of hIOs. These data demonstrate the feasibility of cryopreserved biopsies in the generation of hIOs, enhancing the potential for nationwide collaboration and cryopreservation of SC-derived biopsies for later clinical use.

Addition of IGF-1 and FGF-2 increased expression of epithelial cell markers in human intestinal organoids

While standard organoid media promotes the generation of hIOs, it also reduces the expression of specific intestinal epithelial cells, such as goblet cells [8, 29, 30]. We evaluated the possibility of growing hIO with a refined media (RM; IGF-1, FGF-2, epidermal growth factor (EGF), without p38i; Additional file 1: Data S3) [31]. We compared hIOs generated using RM with those generated using standard media (SM; EGF, p38i) from both TC and SC biopsies (Fig. 3A).

Fig. 3
figure 3

Effect of refined organoid media in the epithelial composition of TC and SC organoids. Healthy organoids derived from TC and SC biopsies (n = 3) were expanded over 12 days with either SM or RM. The epithelial composition of hIO was evaluated by RT-qPCR on day 12. A. Scheme depicting the overall experiment. B. Relative gene expression (2−ΔCT) of epithelial composition of TC- and SC-hIO

hIOs generated in RM had higher expression of FCGBP in TC-hIOs (SM: 0.004 ± 0.001; RM: 0.037 ± 0.014) (Fig. 3B) but not SC-hIOs. Both SPIB and POU2F3 (representing M cells and Tuft cells, respectively) were detected at low expression in TC- and SC-derived hIOs cultured in either SM or RM. hIOs cultured in RM had higher expression of CFTR in both TC (SM: 0.004 ± 0.001; RM: 0.037 ± 0.014) and SC (SM: 0.040 ± 0.021; RM: 0.094 ± 0.007) derived hIOs. RM enhanced early enterocyte (FABP1, fatty acid binding protein 1) expression in both TC and SC-hIOs (Fig. 3B) compared to SM. RM increased expression of enteroendocrine (CHGA, chromogranin A) cells in SC-hIOs, but not in TC-hIOs (Fig. 3B), whereas in TC-hIOs, RM enhanced SLC9A3 (SM: 0.014 ± 0.001; RM: 0.075 ± 0.087). RM may suppress LGR5 expression in both TC and SC-hIOs (Fig. 3B). Taken together, despite the low sample size, RM appears superior to SM in terms of the gene expression level of epithelial cells; therefore our data is aligned with that of Fujii et al. [31].

Our data demonstrated the feasibility of cryopreserved biopsies, corroborating earlier findings of Tsai et al. [16]. They observed a delay in the initial growth of organoids from frozen samples compared to organoids derived from fresh biopsies, but the organoids were indistinguishable, even at transcript level [16]. Further, we have identified a time point (d20) where hIOs generated from frozen biopsies reflect similar hIO composition from freshly acquired samples.

Established by Sato et al. [29], hIOs can be expanded in vitro by recreating the stem cell niche through a combination of growth factors, hormones and other molecules, e.g., EGF, WNT, R-spondin-1, and noggin. However, the use of these molecules leads to rapid organoid growth inhibition over time. EGF, for instance, is crucial for organoid growth but the binding of EGF to the EGF receptor (EGFR) leads to activation of p38-MAPK and downregulation of EGFR, implicating p38-signalling pathway in growth inhibition of hIO system over time [4]. Therefore, inhibition of p38 is critical for stabilisation of EGFR and long-term maintenance of hIOs using the conventional media [29]. However, while this trade-off effect enables long-term maintenance of hIO, suppression of p38 reduces the ability of stem cells to differentiate into specific intestinal epithelial cells, such as goblet cells [8, 29]. We validated a new refined media proposed by Fujii et al. [8]. The new media substitutes suppression of p38 by adding IGF-1 and FGF-2. IGF-1 and FGF-2 play an important role in the differentiation of cells and intestinal epithelium regeneration [32, 33]. Both molecules can be used with or without EGF without p38 inhibition (p38i) for effective hIO expansion [8]. We used a non-differentiation media that favours expansion of hIOs and stem cells, characterised by the presence of WNT and R-spondin; for the proper maturation of hIOs for function studies, we recommended the removal of WNT, R-spondin, and EGF [29, 34].


We have demonstrated that hIOs can be grown from easily accessible SC biopsies and from frozen biopsies, and we propose that the methodology and bedside-to-bench pipeline described here provide opportunities for nationwide collaborative research using hIOs to address a variety of research and clinical questions.


This work was designed to create robust protocols to facilitate standardised clinical research across multiple centres. The research most likely to benefit from these protocols is the study of IBD. hIOs have been used to study epithelial barrier function in IBD [14, 35], bacterial infection [36, 37] and intestinal epithelial inflammasomes [38]; to predict chemotherapy response in colorectal cancer [39, 40] and ovarian cancer [41], and as a drug screening system for cystic fibrosis patients [42]. However, our protocols have been designed using data acquired from healthy individuals, and not those with tumours, inflammation or other potential defects in intestinal permeability.

Availability of data and materials

The datasets generated and/or analysed during the current study are not publicly available due to ethical regulation constraints but are available from the corresponding author on reasonable request.



Chromogranin A


Dulbecco’s Modified Eagle Medium


Ethylenediaminetetraacetic acid


Epidermal growth factor


Epithelial sodium channel


Fatty acid binding protein 1


Fetal calf serum


Fibroblast growth factor


Human intestinal organoid


Hypoxanthine phosphoribosyltransferase 1


Inflammatory bowel diseases


Insulin-like growth factor


Leucine rich repeat containing G protein-coupled receptor 5


Sodium hydrogen exchanger 3


Phosphate buffered saline




POU class 2 homeobox 3


Refined media


Sigmoid colon


Sodium channel epithelial 1 subunit alpha


Solute carrier family 9 member A3


Standard media


Transcription factor SpiB


Transverse colon


  1. Liu F, Huang J, Ning B, Liu Z, Chen S, Zhao W. Drug discovery via human-derived stem cell organoids. Front Pharmacol. 2016;7:334.

    PubMed  PubMed Central  Google Scholar 

  2. Angus HCK, Butt AG, Schultz M, Kemp RA. Intestinal organoids as a tool for inflammatory bowel disease research. Front Med (Lausanne). 2019;6:334.

    Article  Google Scholar 

  3. Fujii M, Matano M, Nanki K, Sato T. Author Correction: Efficient genetic engineering of human intestinal organoids using electroporation. Nat Protoc. 2019;14(8):2595.

    CAS  Article  Google Scholar 

  4. Fujii M, Shimokawa M, Date S, Takano A, Matano M, Nanki K, Ohta Y, Toshimitsu K, Nakazato Y, Kawasaki K, et al. A colorectal tumor organoid library demonstrates progressive loss of niche factor requirements during tumorigenesis. Cell Stem Cell. 2016;18(6):827–38.

    CAS  Article  Google Scholar 

  5. Sato T, van Es JH, Snippert HJ, Stange DE, Vries RG, van den Born M, Barker N, Shroyer NF, van de Wetering M, Clevers H. Paneth cells constitute the niche for Lgr5 stem cells in intestinal crypts. Nature. 2011;469(7330):415–8.

    CAS  Article  Google Scholar 

  6. Sato T, Vries RG, Snippert HJ, van de Wetering M, Barker N, Stange DE, van Es JH, Abo A, Kujala P, Peters PJ, et al. Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature. 2009;459(7244):262–5.

    CAS  Article  Google Scholar 

  7. Broguiere N, Isenmann L, Hirt C, Ringel T, Placzek S, Cavalli E, Ringnalda F, Villiger L, Züllig R, Lehmann R, et al. Growth of epithelial organoids in a defined hydrogel. Adv Mater. 2018;30:1801621.

    Article  Google Scholar 

  8. Fujii M, Matano M, Toshimitsu K, Takano A, Mikami Y, Nishikori S, Sugimoto S, Sato T. Human intestinal organoids maintain self-renewal capacity and cellular diversity in niche-inspired culture condition. Cell Stem Cell. 2018;23:787-793.e786.

    CAS  Article  Google Scholar 

  9. Gadeock S, Schultz M, Butt G. An inherent defect in tight junction structure and permeability is apparent in colonoids from Crohn’s disease patients. FASEB J. 2017;31:1043.

    Google Scholar 

  10. Kozuka K, He Y, Koo-McCoy S, Kumaraswamy P, Nie B, Shaw K, Chan P, Leadbetter M, He L, Lewis JG, et al. Development and characterization of a human and mouse intestinal epithelial cell monolayer platform. Stem cell reports. 2017;9:1976–90.

    CAS  Article  Google Scholar 

  11. Noben ME, Hendriks N, Vermeire S, Van Assche GA, Verfaillie C, Ferrante M. Intestinal organoids derived from inflamed tissues reach transcription levels comparable to non-inflamed tissues and healthy controls. Gastroenterology. 2017;152:S411–2.

    Article  Google Scholar 

  12. Samuel G, Gadeock S, Schultz M, Butt G. The differential response of human epithelial derived colonic organoids to TLR agonists. FASEB J. 2017;31:1049.

    Google Scholar 

  13. Min S, Kim S, Cho S-W. Gastrointestinal tract modeling using organoids engineered with cellular and microbiota niches. Exp Mol Med. 2020;52(2):227–37.

    CAS  Article  Google Scholar 

  14. Sayoc-Becerra A, Krishnan M, Fan S, Jimenez J, Hernandez R, Gibson K, Preciado R, Butt G, McCole DF. The JAK-inhibitor tofacitinib rescues human intestinal epithelial cells and colonoids from cytokine-induced barrier dysfunction. Inflamm Bowel Dis. 2020;26(3):407–22.

    Article  Google Scholar 

  15. Lukonin I, Serra D, Challet Meylan L, Volkmann K, Baaten J, Zhao R, Meeusen S, Colman K, Maurer F, Stadler MB, et al. Phenotypic landscape of intestinal organoid regeneration. Nature. 2020;586(7828):275–80.

    CAS  Article  Google Scholar 

  16. Tsai Y-H, Czerwinski M, Wu A, Dame MK, Attili D, Hill E, Colacino JA, Nowacki LM, Shroyer NF, Higgins PDR, et al. A method for cryogenic preservation of human biopsy specimens and subsequent organoid culture. Cell Mol Gastroenterol Hepatol. 2018;6:218-222.e217.

    Article  Google Scholar 

  17. Pfaffl MW, Tichopad A, Prgomet C, Neuvians TP. Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper–Excel-based tool using pair-wise correlations. Biotechnol Lett. 2004;26(6):509–15.

    CAS  Article  Google Scholar 

  18. Seidlitz T, Merker SR, Rothe A, Zakrzewski F, von Neubeck C, Grützmann K, Sommer U, Schweitzer C, Schölch S, Uhlemann H, et al. Human gastric cancer modelling using organoids. Gut. 2019;68:207–17.

    CAS  Article  Google Scholar 

  19. Kraiczy J, Nayak KM, Howell KJ, Ross A, Forbester J, Salvestrini C, Mustata R, Perkins S, Andersson-Rolf A, Leenen E, et al. DNA methylation defines regional identity of human intestinal epithelial organoids and undergoes dynamic changes during development. Gut. 2019;68:49–61.

    CAS  Article  Google Scholar 

  20. Takahashi Y, Sato S, Kurashima Y, Yamamoto T, Kurokawa S, Yuki Y, Takemura N, Uematsu S, Lai C-Y, Otsu M, et al. A refined culture system for human induced pluripotent stem cell-derived intestinal epithelial organoids. Stem Cell Rep. 2018;10(1):314–28.

    CAS  Article  Google Scholar 

  21. Kolahi KS, Nakano M, Kuo CJ. Organoids as oracles for precision medicine in rectal cancer. Cell Stem Cell. 2020;26(1):4–6.

    CAS  Article  Google Scholar 

  22. Sandle GI. Salt and water absorption in the human colon: a modern appraisal. Gut. 1998;43(2):294–9.

    CAS  Article  Google Scholar 

  23. Zeissig S, Bergann T, Fromm A, Bojarski C, Heller F, Guenther U, Zeitz M, Fromm M, Schulzke JD. Altered ENaC expression leads to impaired sodium absorption in the noninflamed intestine in Crohn’s disease. Gastroenterology. 2008;134(5):1436–47.

    CAS  Article  Google Scholar 

  24. Kim JJ, Khan WI. Goblet cells and mucins: role in innate defense in enteric infections. Pathogens. 2013;2(1):55–70.

    Article  Google Scholar 

  25. Johansson MEV, Thomsson KA, Hansson GC. Proteomic Analyses of the two mucus layers of the colon barrier reveal that their main component, the Muc2 Mucin, is strongly bound to the Fcgbp protein. J Proteome Res. 2009;8(7):3549–57.

    CAS  Article  Google Scholar 

  26. van der Post S, Hansson GC. Membrane protein profiling of human colon reveals distinct regional differences. Mol Cell Proteomics. 2014;13(9):2277–87.

    Article  Google Scholar 

  27. Akiyama S, Mochizuki W, Nibe Y, Matsumoto Y, Sakamoto K, Oshima S, Watanabe M, Nakamura T. CCN3 expression marks a sulfomucin-nonproducing unique subset of colonic goblet cells in mice. Acta Histochem Cytochem. 2017;50(6):159–68.

    CAS  Article  Google Scholar 

  28. Thiagarajah JR, Yildiz H, Carlson T, Thomas AR, Steiger C, Pieretti A, Zukerberg LR, Carrier RL, Goldstein AM. Altered goblet cell differentiation and surface mucus properties in Hirschsprung disease. PLoS ONE. 2014;9(6):e99944.

    Article  Google Scholar 

  29. Sato T, Stange DE, Ferrante M, Vries RG, Van Es JH, Van den Brink S, Van Houdt WJ, Pronk A, Van Gorp J, Siersema PD, et al. Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett’s epithelium. Gastroenterology. 2011;141(5):1762–72.

    CAS  Article  Google Scholar 

  30. Otsuka M, Kang YJ, Ren J, Jiang H, Wang Y, Omata M, Han J. Distinct effects of p38alpha deletion in myeloid lineage and gut epithelia in mouse models of inflammatory bowel disease. Gastroenterology. 2010;138(4):1255–65.

    CAS  Article  Google Scholar 

  31. Fujii M, Matano M, Toshimitsu K, Takano A, Mikami Y, Nishikori S, Sugimoto S, Sato T. Human intestinal organoids maintain self-renewal capacity and cellular diversity in niche-inspired culture condition. Cell Stem Cell 2018;23(6):787–793 e786.

  32. Zheng Y, Song Y, Han Q, Liu W, Xu J, Yu Z, Zhang R, Li N. Intestinal epithelial cell-specific IGF1 promotes the expansion of intestinal stem cells during epithelial regeneration and functions on the intestinal immune homeostasis. Am J Physiol Endocrinol Metab. 2018;315(4):E638–49.

    CAS  Article  Google Scholar 

  33. Song X, Dai D, He X, Zhu S, Yao Y, Gao H, Wang J, Qu F, Qiu J, Wang H, et al. Growth factor FGF2 cooperates with interleukin-17 to repair intestinal epithelial damage. Immunity. 2015;43(3):488–501.

    CAS  Article  Google Scholar 

  34. Merenda A, Fenderico N, Maurice MM. Wnt signaling in 3D: recent advances in the applications of intestinal organoids. Trends Cell Biol. 2020;30(1):60–73.

    CAS  Article  Google Scholar 

  35. Bardenbacher M, Ruder B, Britzen-Laurent N, Schmid B, Waldner M, Naschberger E, Scharl M, Muller W, Gunther C, Becker C, et al. Permeability analyses and three dimensional imaging of interferon gamma-induced barrier disintegration in intestinal organoids. Stem Cell Res. 2019;35:101383.

    CAS  Article  Google Scholar 

  36. Yin Y, Zhou D. Organoid and enteroid modeling of salmonella infection. Front Cell Infect Microbiol. 2018;8:102.

    Article  Google Scholar 

  37. Fattinger SA, Bock D, Di Martino ML, Deuring S, Samperio Ventayol P, Ek V, Furter M, Kreibich S, Bosia F, Muller-Hauser AA, et al. Salmonella Typhimurium discreet-invasion of the murine gut absorptive epithelium. PLoS Pathog. 2020;16(5):e1008503.

    CAS  Article  Google Scholar 

  38. Hausmann A, Russo G, Grossmann J, Zund M, Schwank G, Aebersold R, Liu Y, Sellin ME, Hardt WD. Germ-free and microbiota-associated mice yield small intestinal epithelial organoids with equivalent and robust transcriptome/proteome expression phenotypes. Cell Microbiol. 2020;22(6):e13191.

    CAS  Article  Google Scholar 

  39. van de Wetering M, Francies HE, Francis JM, Bounova G, Iorio F, Pronk A, van Houdt W, van Gorp J, Taylor-Weiner A, Kester L, et al. Prospective derivation of a living organoid biobank of colorectal cancer patients. Cell. 2015;161(4):933–45.

    Article  Google Scholar 

  40. Ooft SN, Weeber F, Dijkstra KK, McLean CM, Kaing S, van Werkhoven E, Schipper L, Hoes L, Vis DJ, van de Haar J, et al. Patient-derived organoids can predict response to chemotherapy in metastatic colorectal cancer patients. Sci Transl Med. 2019;11(513):eaay2574.

    CAS  Article  Google Scholar 

  41. de Witte CJ, Espejo Valle-Inclan J, Hami N, Lõhmussaar K, Kopper O, Vreuls CPH, Jonges GN, van Diest P, Nguyen L, Clevers H, et al. Patient-derived ovarian cancer organoids mimic clinical response and exhibit heterogeneous inter- and intrapatient drug responses. Cell Rep. 2020;31(11):107762.

    Article  Google Scholar 

  42. Berkers G, van Mourik P, Vonk AM, Kruisselbrink E, Dekkers JF, de Winter-de Groot KM, Arets HGM, Marck-van der Wilt REP, Dijkema JS, Vanderschuren MM et al: Rectal organoids enable personalized treatment of cystic fibrosis. Cell Rep 2019;26(7):1701–1708 e1703.

Download references


We thank Grant Butt and Lisa Fan for reagents and advice on hIO culture.


The research was supported by New Zealand Society of Gastroenterology, Otago Medical Research Foundation and the Healthcare Otago Charitable Trust.

Author information

Authors and Affiliations



PCMU conceived the idea, performed all experiments and wrote the manuscript. HCKA provided experimental data. SG provided experimental data and wrote the manuscript. MS provided all clinical samples and wrote the manuscript. RAK designed the project and wrote the manuscript. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Roslyn A. Kemp.

Ethics declarations

Ethics approval and consent to participate

This research proposal was approved by the New Zealand Health and Disability Ethics committee (registration code: 13/STH/155/AM02) and was performed according to the principles of the Declaration of Helsinki. Biopsies were collected after informed consent had been obtained. Written informed consent was obtained from all participants.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Additional file 1: Table S1.

Donors. Table S2. PCR primers. Data S3. Media.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit The Creative Commons Public Domain Dedication waiver ( applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Urbano, P.C.M., Angus, H.C.K., Gadeock, S. et al. Assessment of source material for human intestinal organoid culture for research and clinical use. BMC Res Notes 15, 35 (2022).

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI:


  • Organoids
  • IBD
  • Intestine
  • Inflammation
  • Development
  • Epithelium
  • Cryopreservation