Skip to main content
SpringerLink
Log in

Evaluation of Intestinal Epithelial Barrier Function in Inflammatory Bowel Diseases Using Murine Intestinal Organoids

  • Original Article
  • Published:
Tissue Engineering and Regenerative Medicine Aims and scope

Abstract

Background:

Intestinal organoids have evolved as potential molecular tools that could be used to study host-microbiome interactions, nutrient uptake, and drug screening. Gut epithelial barrier functions play a crucial role in health and diseases, especially in autoimmune diseases, such as inflammatory bowel diseases (IBDs), because they disrupt the epithelial mucosa and impair barrier function.

Methods:

In this study, we generated an in vitro IBD model based on dextran sodium sulfate (DSS) and intestinal organoids that could potentially be used to assess barrier integrity. Intestinal organoids were long-term cultivated and characterized with several specific markers, and the key functionality of paracellular permeability was determined using FITC-dextran 4 kDa. Intestinal organoids that had been treated with 2 µM DSS for 3 h were developed and the intestinal epithelial barrier function was sequentially evaluated.

Results:

The results indicated that the paracellular permeability represented epithelial characteristics and their barrier function had declined when they were exposed to FITC-dextran 4 kDa after DSS treatment. In addition, we analyzed the endogenous mRNA expression of pro-inflammatory cytokines and their downstream effector genes. The results demonstrated that the inflammatory cytokines genes significantly increased in inflamed organoids compared to the control, leading to epithelial barrier damage and dysfunction.

Conclusion:

The collective results showed that in vitro 3D organoids mimic in vivo tissue topology and functionality with minor limitations, and hence are helpful for testing disease models.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

References

  1. Chelakkot C, Ghim J, Ryu SH. Mechanisms regulating intestinal barrier integrity and its pathological implications. Exp Mol Med. 2018;50:103.

    Article  Google Scholar 

  2. Deuring JJ, de Haar C, Kuipers EJ, Peppelenbosch MP, van der Woude CJ. The cell biology of the intestinal epithelium and its relation to inflammatory bowel disease. Int J Biochem Cell Biol. 2013;45:798–806.

    Article  CAS  Google Scholar 

  3. Kraiczy J, Zilbauer M. Intestinal epithelial organoids as tools to study epigenetics in gut health and disease. Stem Cells Int. 2019;2019:7242415.

    Article  Google Scholar 

  4. Lancaster MA, Knoblich JA. Organogenesis in a dish: modeling development and disease using organoid technologies. Science. 2014;345:1247125.

    Article  Google Scholar 

  5. Sato T, Vries RG, Snippert HJ, van de Wetering M, Barker N, Stange DE, et al. Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature. 2009;459:262–5.

    Article  CAS  Google Scholar 

  6. Sato T, Stange DE, Ferrante M, Vries RG, Van Es JH, Van den Brink S, et al. Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett’s epithelium. Gastroenterology. 2011;141:1762–72.

    Article  CAS  Google Scholar 

  7. McCracken KW, Catá EM, Crawford CM, Sinagoga KL, Schumacher M, Rockich BE, et al. Modelling human development and disease in pluripotent stem-cell-derived gastric organoids. Nature. 2014;516:400–4.

    Article  CAS  Google Scholar 

  8. Jee JH, Lee DH, Ko J, Hahn S, Jeong SY, Kim HK, et al. Development of collagen-based 3D matrix for gastrointestinal tract-derived organoid culture. Stem Cells Int. 2019;2019:8472712.

    Article  Google Scholar 

  9. Ko JK, Auyeung KK. Inflammatory bowel disease: etiology, pathogenesis and current therapy. Curr Pharm Des. 2014;20:1082–96.

    Article  CAS  Google Scholar 

  10. Verstockt B, Smith KG, Lee JC. Genome-wide association studies in Crohn’s disease: past, present and future. Clin Transl Immunology. 2018;7:e1001.

    Article  Google Scholar 

  11. Zhang YZ, Li YY. Inflammatory bowel disease: pathogenesis. World J Gastroenterol. 2014;20:91–9.

    Article  Google Scholar 

  12. Westbrook AM, Szakmary A, Schiestl RH. Mechanisms of intestinal inflammation and development of associated cancers: lessons learned from mouse models. Mutat Res. 2010;705:40–59.

    Article  CAS  Google Scholar 

  13. Hibiya S, Tsuchiya K, Hayashi R, Fukushima K, Horita N, Watanabe S, et al. Long-term inflammation transforms intestinal epithelial cells of colonic organoids. J Crohns Colitis. 2017;11:621–30.

    PubMed  Google Scholar 

  14. DeVoss J, Diehl L. Murine models of inflammatory bowel disease (IBD): challenges of modeling human disease. Toxicol Pathol. 2014;42:99–110.

    Article  Google Scholar 

  15. Scheiffele F, Fuss IJ. Induction of TNBS colitis in mice. Curr Protoc Immunol. 2002;Chapter 15:Unit 15.19.

  16. Kiesler P, Fuss IJ, Strober W. Experimental models of inflammatory bowel diseases. Cell Mol Gastroenterol Hepatol. 2015;1:154–70.

    Article  Google Scholar 

  17. Bredenoord AL, Clevers H, Knoblich JA. Human tissues in a dish: the research and ethical implications of organoid technology. Science. 2017;355:eaaf9414.

    Article  Google Scholar 

  18. Sato T, Clevers H. Growing self-organizing mini-guts from a single intestinal stem cell: mechanism and applications. Science. 2013;340:1190–4.

    Article  CAS  Google Scholar 

  19. Lee BR, Kim H, Park TS, Moon S, Cho S, Park T, et al. A set of stage-specific gene transcripts identified in EK stage X and HH stage 3 chick embryos. BMC Dev Biol. 2007;7:60.

    Article  Google Scholar 

  20. Rallabandi HR, Yang H, Jo YJ, Lee HC, Byun SJ, Lee BR. Identification of female specific genes in the W chromosome that are expressed during gonadal differentiation in the chicken. Korean J Poult Sci. 2019;46:287–96.

    Article  Google Scholar 

  21. Pearce SC, Al-Jawadi A, Kishida K, Yu S, Hu M, Fritzky LF, et al. Marked differences in tight junction composition and macromolecular permeability among different intestinal cell types. BMC Biol. 2018;16:19.

    Article  Google Scholar 

  22. Sala FG, Kunisaki SM, Ochoa ER, Vacanti J, Grikscheit TC. Tissue-engineered small intestine and stomach form from autologous tissue in a preclinical large animal model. J Surg Res. 2009;156:205–12.

    Article  Google Scholar 

  23. Yan KS, Chia LA, Li X, Ootani A, Su J, Lee JY, et al. The intestinal stem cell markers Bmi1 and Lgr5 identify two functionally distinct populations. Proc Natl Acad Sci U S A. 2012;109:466–71.

    Article  CAS  Google Scholar 

  24. Pastula A, Middelhoff M, Brandtner A, Tobiasch M, Höhl B, Nuber AH, et al. Three-dimensional gastrointestinal organoid culture in combination with nerves or fibroblasts: a method to characterize the gastrointestinal stem cell niche. Stem Cells Int. 2016;2016:3710836.

    Article  Google Scholar 

  25. Nagatake T, Fujita H, Minato N, Hamazaki Y. Enteroendocrine cells are specifically marked by cell surface expression of claudin-4 in mouse small intestine. PLoS One. 2014;9:e90638.

    Article  Google Scholar 

  26. Altay G, Larrañaga E, Tosi S, Barriga FM, Batlle E, Fernández-Majada V, et al. Self-organized intestinal epithelial monolayers in crypt and villus-like domains show effective barrier function. Sci Rep. 2019;9:10140.

    Article  CAS  Google Scholar 

  27. Schmitt M, Schewe M, Sacchetti A, Feijtel D, van de Geer WS, Teeuwssen M, et al. Paneth cells respond to inflammation and contribute to tissue regeneration by acquiring stem-like features through SCF/c-Kit signaling. Cell Rep. 2018;24:2312–28.e7.

  28. Dotti I, Salas A. Potential use of human stem cell-derived intestinal organoids to study inflammatory bowel diseases. Inflamm Bowel Dis. 2018;24:2501–9.

    PubMed  PubMed Central  Google Scholar 

  29. Kitajima S, Takuma S, Morimoto M. Histological analysis of murine colitis induced by dextran sulfate sodium of different molecular weights. Exp Anim. 2000;49:9–15.

    Article  CAS  Google Scholar 

  30. Okayasu I, Hatakeyama S, Yamada M, Ohkusa T, Inagaki Y, Nakaya R. A novel method in the induction of reliable experimental acute and chronic ulcerative colitis in mice. Gastroenterology. 1990;98:694–702.

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  32. Michielan A, D’Incà R. Intestinal permeability in inflammatory bowel disease: pathogenesis, clinical evaluation, and therapy of leaky gut. Mediators Inflamm. 2015;2015:628157.

    Article  Google Scholar 

  33. Jaworska K, Konop M, Bielinska K, Hutsch T, Dziekiewicz M, Banaszkiewicz A, et al. Inflammatory bowel disease is associated with increased gut-to-blood penetration of short-chain fatty acids: a new, non-invasive marker of a functional intestinal lesion. Exp Physiol. 2019;104:1226–36.

    Article  CAS  Google Scholar 

  34. Dobre M, Milanesi E, Mănuc TE, Arsene DE, Ţieranu CG, Maj C, et al. Differential intestinal mucosa transcriptomic biomarkers for Crohn’s disease and ulcerative colitis. J Immunol Res. 2018;2018:9208274.

    Article  Google Scholar 

  35. Kobayashi K, Arimura Y, Goto A, Okahara S, Endo T, Shinomura Y, et al. Therapeutic implications of the specific inhibition of causative matrix metalloproteinases in experimental colitis induced by dextran sulphate sodium. J Pathol. 2006;209:376–83.

    Article  CAS  Google Scholar 

  36. Kawamoto A, Nagata S, Anzai S, Takahashi J, Kawai M, Hama M, 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. 2019;13:495–509.

    Article  Google Scholar 

  37. Dhillon SS, Mastropaolo LA, Murchie R, Griffiths C, Thöni C, Elkadri A, et al. Higher activity of the inducible nitric oxide synthase contributes to very early onset inflammatory bowel disease. Clin Transl Gastroenterol. 2014;5:e46.

    Article  CAS  Google Scholar 

  38. Giles EM, Sanders TJ, McCarthy NE, Lung J, Pathak M, MacDonald TT, et al. Regulation of human intestinal T-cell responses by type 1 interferon-STAT1 signaling is disrupted in inflammatory bowel disease. Mucosal Immunol. 2017;10:184–93.

    Article  CAS  Google Scholar 

  39. Armaka M, Apostolaki M, Jacques P, Kontoyiannis DL, Elewaut D, Kollias G. Mesenchymal cell targeting by TNF as a common pathogenic principle in chronic inflammatory joint and intestinal diseases. J Exp Med. 2008;205:331–7.

    Article  CAS  Google Scholar 

  40. Sandborn WJ, Hanauer SB. Antitumor necrosis factor therapy for inflammatory bowel disease: a review of agents, pharmacology, clinical results, and safety. Inflamm Bowel Dis. 1999;5:119–33.

    Article  CAS  Google Scholar 

Download references

Acknowledgement

This work was supported with the National Institute of Animal Sciences (Grant No. PJ01422201), Rural Development Administration (RDA), Korea.

Author information

Authors and Affiliations

  1. Animal Biotechnology Division, National Institute of Animal Science, Rural Development Administration, 1500 Kongjwipatjwi-ro, Iseo-myeon, Wanju-gun, Jeollabuk-do, 55365, Republic of Korea

    Harikrishna Reddy Rallabandi, Hyeon Yang, Keon Bong Oh, Hwi Cheul Lee, Sung June Byun & Bo Ram Lee

Authors
  1. Harikrishna Reddy Rallabandi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Hyeon Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Keon Bong Oh
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Hwi Cheul Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Sung June Byun
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Bo Ram Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Bo Ram Lee.

Ethics declarations

Conflicts of interest

The authors declare no conflicts of interest.

Ethical statement

The experimental use of mouse was performed after receiving approval of the Institutional Animal Care and Use Committee (IACUC) of National Institute of Animal Science (NIAS-2019-366), Korea.

Additional information

Publisher's Note

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

Electronic supplementary material

Below is the link to the electronic supplementary material.

Figure S1.

Isolation of intestinal crypts. A ~5-week-old ICR mouse was used to extract the intestinal stem cells (left upper panel). A section of the small intestine was obtained from the euthanized mouse and washed thoroughly with PBS under aseptic conditions (left lower panel). Histological H and E staining of vertical and horizontal sections from the small intestine showing the presence of crypts (oval structures at the bottom) and villi (finger like structures). The purple spots show nuclei and the epithelium is stained pink. Scale bar: 100 μm (right upper panel) and 50 μm (right middle and lower panels). (PPT 6462 kb)

Figure S2. In vitro intestinal organoid IBD model generation using DSS. (A) The organoids were treated with the chemical agent DSS at 1 µM, 2 µM, or 5 µM per well (upper panel) to identify and optimize the lethal dose needed to induce inflammation. After incubation for 1 h, inflammation was observed in the 2 µM and 5 µM wells, but not in 1 µM wells. (B) Time course data showing the effects of the 2 µM lethal dose at 30 min, 60 min, and 180 min post-treatment (lower panel). There was minimal damage at 30 min post-treatment, but the organoids were highly inflamed and had begun to disintegrate at 180 min. There was also damage to the basement membrane, magnitude of inflammation was indicated in circles using designated colors yellow (damage) and red (lethal) in the figure. Scale bar: 100 μm (PPT 6462 kb)

Figure S3. Culturing and measuring the growth rate of mouse intestinal organoids in long-term cultures. (A) Long term culturing of intestinal organoids for up to 10 generations (P2-P10). Middle and lower panels show various developmental stages. The organoids can be spheroidal (round shaped), show budding (spheroids with extension), and have mature villus and crypt like structures (branched structures). (B) Growth rate graph showing the number of organoids/well (mean n = 3 wells) growing in a 100 µL matrigel dome on a 24 well plate. Scale bar: 100 μm (PPT 6462 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Rallabandi, H.R., Yang, H., Oh, K.B. et al. Evaluation of Intestinal Epithelial Barrier Function in Inflammatory Bowel Diseases Using Murine Intestinal Organoids. Tissue Eng Regen Med 17, 641–650 (2020). https://doi.org/10.1007/s13770-020-00278-0

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s13770-020-00278-0

Keywords

Search

Navigation