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Homeostatic mini-intestines through scaffold-guided organoid morphogenesis


Epithelial organoids, such as those derived from stem cells of the intestine, have great potential for modelling tissue and disease biology1,2,3,4. However, the approaches that are used at present to derive these organoids in three-dimensional matrices5,6 result in stochastically developing tissues with a closed, cystic architecture that restricts lifespan and size, limits experimental manipulation and prohibits homeostasis. Here, by using tissue engineering and the intrinsic self-organization properties of cells, we induce intestinal stem cells to form tube-shaped epithelia with an accessible lumen and a similar spatial arrangement of crypt- and villus-like domains to that in vivo. When connected to an external pumping system, the mini-gut tubes are perfusable; this allows the continuous removal of dead cells to prolong tissue lifespan by several weeks, and also enables the tubes to be colonized with microorganisms for modelling host–microorganism interactions. The mini-intestines include rare, specialized cell types that are seldom found in conventional organoids. They retain key physiological hallmarks of the intestine and have a notable capacity to regenerate. Our concept for extrinsically guiding the self-organization of stem cells into functional organoids-on-a-chip is broadly applicable and will enable the attainment of more physiologically relevant organoid shapes, sizes and functions.

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Fig. 1: Establishment of long-term homeostatic culture of tubular mini-guts.
Fig. 2: Cell-fate patterning and cellular diversity of tubular mini-guts.
Fig. 3: Perspectives for modelling intestine biology and disease.

Data availability

scRNA-seq data have been deposited to the Gene Expression Omnibus (GEO) public repository with the accession code GSE148366. Additional supporting data related to gene-expression analyses of mini-gut tubes infected with C. parvum have been deposited to data are provided with this paper.

Code availability

The code used for scRNA-seq data analysis is available at


  1. 1.

    Clevers, H. Modeling development and disease with organoids. Cell 165, 1586–1597 (2016).

    CAS  PubMed  Google Scholar 

  2. 2.

    Sato, T. & Clevers, H. Growing self-organizing mini-guts from a single intestinal stem cell: mechanism and applications. Science 340, 1190–1194 (2013).

    ADS  CAS  PubMed  Google Scholar 

  3. 3.

    Dekkers, J. F. et al. A functional CFTR assay using primary cystic fibrosis intestinal organoids. Nat. Med. 19, 939–945 (2013).

    CAS  PubMed  Google Scholar 

  4. 4.

    van de Wetering, M. et al. Prospective derivation of a living organoid biobank of colorectal cancer patients. Cell 161, 933–945 (2015).

    PubMed  PubMed Central  Google Scholar 

  5. 5.

    Sato, T. et al. Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature 459, 262–265 (2009).

    ADS  CAS  PubMed  Google Scholar 

  6. 6.

    Gjorevski, N. et al. Designer matrices for intestinal stem cell and organoid culture. Nature 539, 560–564 (2016).

    CAS  PubMed  Google Scholar 

  7. 7.

    Wang, Y. et al. Formation of human colonic crypt array by application of chemical gradients across a shaped epithelial monolayer. Cell. Mol. Gastroenterol. Hepatol. 5, 113–130 (2018).

    PubMed  Google Scholar 

  8. 8.

    Wang, Y. et al. A microengineered collagen scaffold for generating a polarized crypt-villus architecture of human small intestinal epithelium. Biomaterials 128, 44–55 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. 9.

    Haber, A. L. et al. A single-cell survey of the small intestinal epithelium. Nature 551, 333–339 (2017).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  10. 10.

    de Lau, W. et al. Peyer’s patch M cells derived from Lgr5+ stem cells require SpiB and are induced by RankL in cultured “miniguts”. Mol. Cell. Biol. 32, 3639–3647 (2012).

    PubMed  PubMed Central  Google Scholar 

  11. 11.

    Basak, O. et al. Induced quiescence of Lgr5+ stem cells in intestinal organoids enables differentiation of hormone-producing enteroendocrine cells. Cell Stem Cell 20, 177–190 (2017).

    CAS  PubMed  Google Scholar 

  12. 12.

    Hase, K. et al. Uptake through glycoprotein 2 of FimH+ bacteria by M cells initiates mucosal immune response. Nature 462, 226–230 (2009).

    ADS  CAS  Google Scholar 

  13. 13.

    Moor, A. E. et al. Spatial reconstruction of single enterocytes uncovers broad zonation along the intestinal villus axis. Cell 175, 1156–1167 (2018).

    CAS  PubMed  Google Scholar 

  14. 14.

    Ayyaz, A. et al. Single-cell transcriptomes of the regenerating intestine reveal a revival stem cell. Nature 569, 121–125 (2019).

    ADS  CAS  PubMed  Google Scholar 

  15. 15.

    Yui, S. et al. YAP/TAZ-dependent reprogramming of colonic epithelium links ECM remodeling to tissue regeneration. Cell Stem Cell 22, 35–49 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. 16.

    Kiesler, P., Fuss, I. J. & Strober, W. Experimental models of inflammatory bowel diseases. Cell. Mol. Gastroenterol. Hepatol. 1, 154–170 (2015).

    PubMed  PubMed Central  Google Scholar 

  17. 17.

    Kotloff, K. L. The burden and etiology of diarrheal illness in developing countries. Pediatr. Clin. North Am. 64, 799–814 (2017).

    PubMed  Google Scholar 

  18. 18.

    Heo, I. et al. Modelling Cryptosporidium infection in human small intestinal and lung organoids. Nat. Microbiol. 3, 814–823 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. 19.

    Wilke, G. et al. A stem-cell-derived platform enables complete Cryptosporidium development in vitro and genetic tractability. Cell Host Microbe 26, 123–134 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. 20.

    Kim, H. J., Huh, D., Hamilton, G. & Ingber, D. E. Human gut-on-a-chip inhabited by microbial flora that experiences intestinal peristalsis-like motions and flow. Lab Chip 12, 2165–2174 (2012).

    CAS  PubMed  Google Scholar 

  21. 21.

    Kasendra, M. et al. Development of a primary human Small Intestine-on-a-Chip using biopsy-derived organoids. Sci. Rep. 8, 2871 (2018).

    ADS  PubMed  PubMed Central  Google Scholar 

  22. 22.

    Trietsch, S. J. et al. Membrane-free culture and real-time barrier integrity assessment of perfused intestinal epithelium tubes. Nat. Commun. 8, 262 (2017).

    ADS  PubMed  PubMed Central  Google Scholar 

  23. 23.

    Chen, Y., Zhou, W., Roh, T., Estes, M. K. & Kaplan, D. L. In vitro enteroid-derived three-dimensional tissue model of human small intestinal epithelium with innate immune responses. PLoS ONE 12, e0187880 (2017).

    PubMed  PubMed Central  Google Scholar 

  24. 24.

    Workman, M. J. et al. Enhanced utilization of induced pluripotent stem cell-derived human intestinal organoids using microengineered chips. Cell. Mol. Gastroenterol. Hepatol. 5, 669–677 (2018).

    PubMed  Google Scholar 

  25. 25.

    Wang, Y. et al. Long-term culture captures injury-repair cycles of colonic stem cells. Cell 179, 1144–1159 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. 26.

    Bhatia, S. N. & Ingber, D. E. Microfluidic organs-on-chips. Nat. Biotechnol. 32, 760–772 (2014).

    CAS  PubMed  Google Scholar 

  27. 27.

    Wang, Y. et al. Bioengineered systems and designer matrices that recapitulate the intestinal stem cell niche. Cell. Mol. Gastroenterol. Hepatol. 5, 440–453 (2018).

    PubMed  PubMed Central  Google Scholar 

  28. 28.

    Nikolaev, M. et al. Bioengineering microfluidic organoids-on-a-chip. Protoc. Exch. (2020).

  29. 29.

    Koliaraki, V. & Kollias, G. Isolation of intestinal mesenchymal cells from adult mice. Bio-protocol 6, e1940 (2016).

    Google Scholar 

  30. 30.

    Huch, M. et al. In vitro expansion of single Lgr5+ liver stem cells induced by Wnt-driven regeneration. Nature 494, 247–250 (2013).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  31. 31.

    Takata, K. et al. Induced-pluripotent-stem-cell-derived primitive macrophages provide a platform for modeling tissue-resident macrophage differentiation and function. Immunity 47, 183–198 (2017).

    CAS  PubMed  Google Scholar 

  32. 32.

    Blokzijl, F. et al. Tissue-specific mutation accumulation in human adult stem cells during life. Nature 538, 260–264 (2016).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  33. 33.

    Miyoshi, H. & Stappenbeck, T. S. In vitro expansion and genetic modification of gastrointestinal stem cells in spheroid culture. Nat. Protocols 8, 2471–2482 (2013).

    CAS  PubMed  Google Scholar 

  34. 34.

    Sachs, N. et al. Long-term expanding human airway organoids for disease modeling. EMBO J. 38, e100300 (2019).

    PubMed  PubMed Central  Google Scholar 

  35. 35.

    Brandenberg, N. & Lutolf, M. P. In situ patterning of microfluidic networks in 3D cell-laden hydrogels. Adv. Mater. 28, 7450–7456 (2016).

    CAS  PubMed  Google Scholar 

  36. 36.

    Chen, C. et al. Bioengineered bile ducts recapitulate key cholangiocyte functions. Biofabrication 10, 034103 (2018).

    ADS  PubMed  Google Scholar 

  37. 37.

    Butler, A., Hoffman, P., Smibert, P., Papalexi, E. & Satija, R. Integrating single-cell transcriptomic data across different conditions, technologies, and species. Nat. Biotechnol. 36, 411–420 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. 38.

    Becht, E. et al. Dimensionality reduction for visualizing single-cell data using UMAP. Nat. Biotechnol. 37, 38–44 (2019).

    CAS  Google Scholar 

  39. 39.

    Kowalczyk, M. S. et al. Single-cell RNA-seq reveals changes in cell cycle and differentiation programs upon aging of hematopoietic stem cells. Genome Res. 25, 1860–1872 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. 40.

    Wickham, H. ggplot2: Elegant Graphics for Data Analysis (Springer, 2016).

  41. 41.

    Subramanian, A. et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc. Natl Acad. Sci. USA 102, 15545–15550 (2005).

    ADS  CAS  PubMed  Google Scholar 

  42. 42.

    Korsunsky, I. et al. Fast, sensitive and accurate integration of single-cell data with Harmony. Nat. Methods 16, 1289–1296 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. 43.

    Kolotuev, I. Positional correlative anatomy of invertebrate model organisms increases efficiency of TEM data production. Microsc. Microanal. 20, 1392–1403 (2014).

    ADS  CAS  PubMed  Google Scholar 

  44. 44.

    Burel, A. et al. A targeted 3D EM and correlative microscopy method using SEM array tomography. Development 145, dev160879 (2018).

    PubMed  Google Scholar 

  45. 45.

    Mabbott, N. A., Donaldson, D. S., Ohno, H., Williams, I. R. & Mahajan, A. Microfold (M) cells: important immunosurveillance posts in the intestinal epithelium. Mucosal Immunol. 6, 666–677 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. 46.

    Nakato, G. et al. New approach for M-cell-specific molecules screening by comprehensive transcriptome analysis. DNA Res. 16, 227–235 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. 47.

    Hartl, M. & Schneider, R. A unique family of neuronal signaling proteins implicated in oncogenesis and tumor suppression. Front. Oncol. 9, 289 (2019).

    PubMed  PubMed Central  Google Scholar 

  48. 48.

    Funda, D. P. et al. CD14 is expressed and released as soluble CD14 by human intestinal epithelial cells in vitro: lipopolysaccharide activation of epithelial cells revisited. Infect. Immun. 69, 3772–3781 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. 49.

    Nakamura, Y., Kimura, S. & Hase, K. M cell-dependent antigen uptake on follicle-associated epithelium for mucosal immune surveillance. Inflamm. Regen. 38, 15 (2018).

    PubMed  PubMed Central  Google Scholar 

  50. 50.

    Hase, K. et al. Distinct gene expression profiles characterize cellular phenotypes of follicle-associated epithelium and M cells. DNA Res. 12, 127–137 (2005).

    CAS  PubMed  Google Scholar 

  51. 51.

    Dillon, A. & Lo, D. D. M cells: intelligent engineering of mucosal immune surveillance. Front. Immunol. 10, 1499 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  52. 52.

    Lim, J. S. et al. Caveolae-mediated entry of Salmonella typhimurium in a human M-cell model. Biochem. Biophys. Res. Commun. 390, 1322–1327 (2009).

    CAS  PubMed  Google Scholar 

  53. 53.

    Terahara, K. et al. Comprehensive gene expression profiling of Peyer’s patch M cells, villous M-like cells, and intestinal epithelial cells. J. Immunol. 180, 7840–7846 (2008).

    CAS  PubMed  Google Scholar 

  54. 54.

    Hase, K. et al. The membrane-bound chemokine CXCL16 expressed on follicle-associated epithelium and M cells mediates lympho-epithelial interaction in GALT. J. Immunol. 176, 43–51 (2006).

    CAS  PubMed  Google Scholar 

  55. 55.

    Kanaya, T. & Ohno, H. The mechanisms of M-cell differentiation. Biosci. Microbiota Food Health 33, 91–97 (2014).

    PubMed  PubMed Central  Google Scholar 

  56. 56.

    Roulis, M. et al. Paracrine orchestration of intestinal tumorigenesis by a mesenchymal niche. Nature 580, 524–529 (2020).

    ADS  CAS  PubMed  Google Scholar 

Download references


We thank M. Juhas for the generation of stem-cell-derived macrophages and help with co-culture experiments; R. Guiet and O. Burri for programming image-processing plug-ins; J. Dorsaz, J. Pernollet and all engineers of the Center of Micronanotechnology (CMi, EPFL) for support in microfabrication; S. Rezakhani, G. Sorrentino and K. Schoonjans for help with cholangiocyte isolation; D. Schaefer and M. Riggs for providing oocysts; R. O’Connor for expertise in analysing C. parvum epicellular stages; F. Gorostidi and S. Kishore for providing trachea tissue samples; and A. Manfrin, S. Höhnel, G. Rossi, M. Knobloch and A. Persat for inputs on the manuscript. We acknowledge support from the following EPFL core facilities: CMi, Histology, BIOP, CryoEM and CECF. This work was funded by the Swiss National Science Foundation (SNSF) research grant 310030_179447; the National Center of Competence in Research (NCCR) ‘Bio-Inspired Materials’ (; the EU Horizon 2020 research programme INTENS (; the Personalized Health and Related Technologies (PHRT) Initiative from the ETH Board; and EPFL. S.G. was supported in part by a fellowship from the Novartis Foundation for Medical-Biological Research. N.G. was supported in part by an EMBO Long-Term Postdoctoral Fellowship.

Author information




M.N. and M.P.L. conceived the study, designed experiments, analysed data and wrote the manuscript. N. Broguiere analysed all scRNA-seq data. S.G. developed an early version of the organoid culture technology and contributed to study design. M.N. and Y.T. designed the microdevice. M.N and O.M. conducted experiments on modelling damage and regeneration. O.M. performed human mini-gut and airway tube experiments. M.N. and D.D. performed and analysed C. parvum infection experiments. I.K. performed electron microscopy imaging of the C.-parvum-infected samples. B.E. performed bile duct tube experiments. N. Brandenberg and N.G. conducted preliminary experiments on matrix microstructuring and contributed to study design. H.C. proposed and designed C. parvum infection experiments and provided feedback on the manuscript.

Corresponding author

Correspondence to Matthias P. Lutolf.

Ethics declarations

Competing interests

The EPFL has filed for patent protection (EP16199677.2, PCT/EP2017/079651, US20190367872A1) on the scaffold-guided organoid technology described herein, and M.P.L., M.N., S.G., Y.T., N. Brandenberg and N.G. are named as inventors on those patents. M.P.L. and N. Brandenberg are shareholders in SUN bioscience SA, which is commercializing those patents. H.C. is an inventor on several patents related to organoid technology. The other authors declare no competing interests.

Additional information

Peer review information Nature thanks Dominic Grun, Thomas F. Meyer, Honorine Ward and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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

Extended data figures and tables

Extended Data Fig. 1 Bioengineering intestinal stem cell epithelia with a tubular, in-vivo-like architecture.

a, Photograph of the fully assembled microchip system. b, Schematic cross-sectional view of laser ablation using a nanosecond-pulsed laser. c, Hybrid collagen I/Matrigel scaffold in the central chamber before and after microchannel ablation. Scale bars, 200 μm. d, Fluorescence confocal images of a representative three-day old epithelial tube. Cells are labelled with DAPI (blue, nuclei) and E-cadherin (green). Images correspond to the maximal intensity projection of a z-stack of 100 μm. Scale bars, 50 μm. Data are representative of at least two independent experiments.

Extended Data Fig. 2 Establishment of shape-controlled organoid culture from a variety of epithelial stem and progenitor cells.

a, Development of bile duct tube composed of mouse cholangiocytes. b, Fluorescence confocal images of representative 3-day-old bile duct tubes, showing an entire tissue (top) stained for EpCAM (orange) and a higher magnification view (bottom) stained for actin filaments (green) and tight junction protein ZO-1 (red). Nuclei stained with DAPI (blue). Scale bars, 50 μm. c, Development of tubular mini-guts composed of human ISCs. d, Fluorescent confocal image showing formation of tightly packed single-layered epithelium in 15-day-old human mini-gut. e, Fluorescent confocal image showing proliferating Ki67+ cells predominantly localized to the crypts in 10-day-old human mini-gut. Nuclei and actin filaments stained with DAPI (blue) and Phalloidin (green), respectively. Images correspond to the maximal intensity projection of a z-stack of 80 μm. f, Formation of a human mini-airway epithelial tube and establishment of air–liquid interface (ALI) culture from day 5 onwards. Scale bars, 100 μm. g, Fluorescence confocal image of a representative 7-day-old human mini-airway epithelial tube, showing an entire tissue (top) and a higher magnification view (bottom) stained for nuclei (DAPI, blue), E-cadherin (green) and Ki67+ proliferating cells (pink). Scale bars, 50 μm. All data are representative of at least two independent experiments.

Extended Data Fig. 3 Establishment of long-term culture and in vitro tissue homeostasis.

a, Bright-field (middle) and LGR5–eGFP fluorescence (right) images of mini-gut progression on days 7 and 10, compared to organoids (left). b, Long-term propagation of mini-guts (up to 30 days) shown in bright-field (left) and LGR5–eGFP fluorescence (right). Data are representative of at least four independent experiments. EDF of bright-field images, calculated for a z-stack of 80 μm; fluorescence confocal images correspond to a maximal intensity projection of a z-stack of around 60 μm. Scale bars, 50 μm. c, Bright-field and LGR5–eGFP fluorescence of mini-gut deterioration due to the massive accumulation of dead cells within the lumen in the absence of perfusion. Scale bars, 100 μm. d, Tubular mini-guts maintain epithelial integrity and morphology in different cell culture media used for lumina perfusion. No difference in epithelium morphology and stability was detected when tissues were apically exposed to organoid culture medium (ENR) or minimal media lacking growth factors (BMGF, BM). Similar results were obtained in at least two independent experiments with n = 2 samples per each condition. Scale bars, 50 μm. e, Frequency map showing the localization of LGR5–eGFP-expressing ISCs in 7-10-day-old tissues (left) and 30-day-old tissues (right). Average of the maximal intensity projection of a z-stack of around 60 μm for n = 20 tissues (7–10-day-old) and n = 8 tissues (30-day-old).

Extended Data Fig. 4 Mini-gut tubes undergo rapid cell turnover and comprise key functional intestinal cell types.

a, Epithelial tissue turnover assessed through EdU pulse-chase experiments. Ten-day-old mini-guts were treated with EdU for 12 h basally and apically, followed by a chase period of four days. At 0 h after EdU removal, the majority of EdU+ cells resided within the crypts and adjacent regions. A 24-h EdU pulse-chase revealed distinct regions of cell proliferation that were to a large extent restricted to the crypts. Two days after the EdU pulse, numerous EdU+ cells were found in the lumen, suggesting the occurrence of intestinal epithelial cell migration from the crypts to the villus-like domains. Labelled cells were virtually absent four days after the EdU pulse, suggesting that tubular mini-guts underwent full turnover of the epithelium. Data are representative of one EdU labelling experiment with n = 2 replicates per condition. Scale bars, 50 μm. b, Micrograph of a mini-gut tube removed from the microchip for downstream histological sectioning and analysis. Scale bar, 200 μm. c, Histological cross-sections of 7-day-old mini-gut tubes stained with Alcian Blue showing acidic polysaccharides of the mucus layer (blue) counterstained with Nuclear Fast Red. The entire perpendicular section (left) and a higher magnification view of the goblet cells (right) are shown. Similar results were obtained for 10 sections from two biologically independent samples. Scale bars, 20 μm. d, Transmission electron microscopy cross-sectional views of 7-day-old mini-gut tube. Goblet cell (left; scale bar, 2 μm) and enterocyte brush border (right; scale bar, 0.3 μm). Data are representative of two samples. e, Gradual increase in aminopeptidase activity after induction of differentiation in mini-gut tubes. Mean ± s.d. from n = 3 biologically independent experiments.

Source data

Extended Data Fig. 5 Canonical markers from the various intestinal cell types are accurately reproduced in vitro.

Heat map of unbiased top markers (Methods) associated with the various cell type clusters found in vitro. Canonical signature genes of stem cells (Olmf4, Lgr5, Axin2) form a continuous gradient towards enterocytes (Fabp1, Apoa1) and villus-top enterocytes (Ada, Krt20). Paneth cells express an array of antibacterial markers (defensins, lysozyme), enteroendocrine cells are marked by chromogranins (Chga, Chgb) and subpopulation-specific hormones. Tuft cells selectively express the landmark gene Dclk1, as well as eicosanoid biosynthesis pathway enzymes (Alox5ap, Hpgds, Ltc4s) and receptors implicated in taste transduction (Trpm5, Gng13).

Source data

Extended Data Fig. 6 Cell types identified in vitro closely resemble their in vivo counterparts.

af, h, Overlay of canonical cell type markers expression (a, d), cell-cycle phase (c, f) and corresponding attributed cell types (b, e, h) found in vitro (ac) and in vivo (df). g, i, Combined aligned in vitro and in vivo datasets showing good match between the cell types identified in the separate analyses. The in vivo versus in vitro datasets (g, i) are generated with different protocols, and gene expression values are therefore not directly comparable between the two.

Extended Data Fig. 7 Identification of rare cell types in the mini-guts.

a, Dot plot highlighting genes relevant to the identification of the small cluster designated as microfold-like (M-like) cells that share similarities with M cells residing in follicle associated epithelia (FAE) in vivo. M-like cells express the canonical immature M-cell markers Anxa5 and Marcksl145, involved in gram-negative bacteria binding/endocytic transport45,46 and regulation of cytoskeleton/adhesion, respectively47. Other genes related to bacterial sampling are also expressed, including Prnp46, Cd1448 and Aif1l49. M-like cells also selectively express additional phagocytosis-related markers such as Myadm and Cyba. Notably, transcripts marking mucus secretion (sum of Muc1, Muc2, Muc3, Muc3a, Muc4 and Muc13, here referred to as ‘Mucins’) and IgA transcytosis (Pigr) are missing, which is another trait of FAE47. Several other genes related to cytoskeleton and adhesion are also strongly upregulated in this population, including the FAE/M-cell markers Actn112,50 and Itgb149. Additional similarities to transcripts marking M-cells include the tight junction marker Cldn4 (Claudin 4) involved in antigen sampling/endocytosis45,51, the caveolae marker Cav152 and the cytokine Cxcl1653 that mediates lympho-epithelial interaction in gut associated lymphoid tissue54, as well as several upregulated NFκB target genes51. Several other known FAE and M-cell markers are missing in these M-like cells, including Spib, the master controller of M-cell differentiation acting downstream of RANKL signalling55. This suggested that M-like cells in mini-gut tubes are only partially analogous to M-cells. We noted that our M-like cell population also shared many transcriptional similarities with two recently described, rare cell populations in the intestine, namely ‘revival stem cells’ (RSCs)14,56 and regenerative fetal-like stem cell15,56. In particular, M-like cells in mini-guts were found to selective express the RSC markers Clu and Msln14,56, previously reported as FAE/M-cell markers9,46,53, and Ly6a (Sca1), that also defines regenerative fetal-like epithelial cells15,56. A characteristic feature of both RSCs and fetal-like stem cells is the activation of the YAP pathway, mediated by focal adhesions, inflammation or prostaglandin E2. Both YAP target genes and prostaglandin-related genes were found to be strongly and selectively expressed in our M-like cell population as well. b, Fluorescence confocal images of representative 15 days-old mini-gut tube, showing an entire tissue (left column) and a higher magnification view (right column) containing GP2+ (red) M-like cells. Data are representative of two replicates. Scale bar, 100 μm. c, Expression of the key enteroendocrine genes in the mini-guts tubes. Neurog3, a marker of immature enteroendocrine cells, forms a gradient towards Chga, Chgb and Neurod1, marking mature enteroendocrine cells. Furthermore, a subpopulation of the enterochromaffin cells defined by hormone substance P (Tac1) and Tph1, encoding the rate-limiting enzyme in serotonin synthesis, can be detected. A subpopulation of cholecystokinin producing I-cells (Cck) was found, co-expressing proglucagon products (Gcg), and varying levels of peptide YY (Pyy), ghrelin (Ghrl) and gastrin (Gast). Enteroendocrine cells were also found to highly express Wnt3, which may partially contribute to the observed higher number of stem cells in mini-guts.

Extended Data Fig. 8 Capacity of mini-gut tubes to regenerate after radiation-induced damage.

a, Mini-gut tubes fail to regenerate and rapidly deteriorate upon exposure to 8 Gy radiation dose. Overlaid bright-field and LGR5–eGFP fluorescence time-course images of epithelial damage in mini-gut tubes induced by exposure to 8 Gy radiation dose are shown. Scale bars, 60 μm. b, Rapid replenishment of LGR5–eGFP+ stem cells initially eliminated by exposure to 2 Gy radiation dose. A graph showing normalized fluorescence intensity of eGFP measured in the crypts before and after exposure to 2 Gy and 8 Gy radiation dose. Mean ± s.d. for n = 4 samples. c, Time-course of mini-gut tubes regeneration upon 2 Gy radiation dose-induced damage shown in bright-field and LGR5–eGFP fluorescence. EDF of bright-field images, calculated for a z-stack of 80 μm; fluorescence confocal images correspond to a maximal intensity projection of a z-stack of around 60 μm. Scale bars, 60 μm. All data are representative of at least two independent experiments with n = 3 replicates per each condition.

Source data

Extended Data Fig. 9 Modelling C. parvum infection in mini-gut tubes.

a, Schematic representation of the C. parvum life cycle and how it can be assessed in mini-gut tube cultures. b, Bright-field live imaging of C. parvum infection in mini-guts with major epicellular stages. After about 24 h of infection, floating half-empty oocysts, broken shells and freshly excysted sporozoites were observed; on the following day, 6–8-merozoite-containing type I meronts and 4-merozoite-containing type II meronts could be detected; 3 days post infection microgamonts containing 12–16 microgametes were detected and starting from day 5 oocysts containing 1–4 sporozoites were again observed. Identity of the observed epicellular stages was confirmed by specific immunostaining (Fig. 3d). Scale bars, 3 μm. c, d, Scanning electron microscopy of distinct stages of C. parvum life cycle with c, different epicellular stages observed in a single cross-section, including microgamont, early zygote and developing oocyst in the mini-gut tubes 96 h after infection. Scale bar, 20 μm. d, All major epicellular stages of the C. parvum were observed in other samples, including trophozoite (24 h post infection), type II meront (48 h post infection) and macrogamont, early zygote and developing oocyst (72–96 h post infection). Scale bars, 1 μm. Data are representative of independent observations from one experiment. e, Quantification of oocysts produced in mini-gut tubes over four weeks. Mean ± s.d. for n = 3 replicates analysed. Data are representative of independent observations from one experiment. f, Hallmark pathways from the molecular signature database (MSigDB) enriched in C.-parvum-infected mini-guts compared to control tissues, as estimated from GSEA. The epithelium responds to the infection through interferon-α, with a family wise error rate (FWER). P value <0.001 (one-sided, empirical P value accounting for multiple testing for 50 signatures, found by integration of the tail of the null-distribution histogram generated from 10,000 phenotype permutations, n = 961 versus 611 cells in the comparison, from at least 3 tubes per condition, merged for RNA library construction and sequencing). g, Volcano plots showing differential gene expression in single cell types of infected versus control mini-guts, with interferon response genes highlighted.

Source data

Extended Data Fig. 10 Perspectives for mimicking organ-level complexity in mini-gut tubes through spatially controlled co-cultures.

a, A schematic representation of the 3D hydrogel-containing mini-gut chip (left). A cross-sectional view (right) highlights the solid membrane-free co-culture, in a biomimetic 3D environment, of an epithelium and various non-parenchymal cell types seeded in the matrix surrounding the epithelium. b, Development of engineered blood vessel-like networks composed of human endothelial cells. Scale bars, 100 μm. c, Co-culture experiment of mini-gut tubes with macrophages embedded into the surrounding hydrogel. Time-lapse imaging revealed a direct interaction of highly motile macrophages with intestinal epithelia in long-term co-cultures (here: 20 days). From day 2 onwards, the macrophages acquire a distinct elongated morphology and migrate towards the epithelium. Scale bars, 50 μm. d, Macrophages perform their phagocytic function in a co-culture with mini-guts. Higher magnification views showing a macrophage ingesting a particle from the basal side of the epithelium by phagocytosis. Scale bars, 10 μm. e, Mini-gut tubes co-cultured with mouse intestinal myofibroblasts incorporated in the surrounding matrix. Myofibroblasts extensively migrate through the gel, extend processes and directly interact with the epithelium. Scale bars, 50 μm. f, A co-culture experiment of mini-guts epithelium with myofibroblasts initially co-seeded together with ISCs directly into the lumen. Myofibroblasts rapidly attach in the microchannel and remain incorporated into the continuous monolayer generated by ISCs. From day 3 onwards, myofibroblasts, localized predominantly in crypt regions, extended pseudopodia into the surrounding matrix (arrows), recapitulating the in vivo tissue architecture. Scale bars, 50 μm. All data are representative of at least two independent experiments. EDF of bright-field images (cf), calculated for a z-stack of 80 μm.

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Reporting Summary

Video 1

: Mini-gut tube development. Time-lapse video of the first 72 hours of the development of a representative mini-gut tube. This video is related to Fig. 1c. Data are representative of at least three independent experiments.

Video 2

: Tubular stem/progenitor cell epithelium. This video shows a coherent and tightly-packed epithelial sheet composed of cells expressing high levels of E-cadherin at cell-cell junctions. The video is related to Extended Data Fig. 1d. Data are representative of at least two independent experiments.

Video 3

: Epithelial cell turnover and tube perfusion. This video exemplifies how dead cells that are accumulated within the intestinal lumen can be readily removed using a microfluidic perfusion system. The video is related to Fig. 1e.

Video 4

: Long-term homeostatic mini-gut tube culture. This video shows the culture of mini-gut tubes over one month. The video is related to Extended Data Fig. 3a,b. Data are representative of at least four independent experiments.

Video 5

: Cell fate patterning in mini-gut tubes. This video shows the distribution of proliferative and differentiated cell types in spatially organized crypt and villus domains of a representative mini-gut tube. The video is related to Fig. 2a-f. Data are representative of at least two independent experiments.

Video 6

: Model of intestinal epithelial damage and regeneration after laser ablation. This video demonstrates the re-epithelialization of mini-gut tubes upon damage induced via laser ablation. The video is related to Fig. 3a. Data are representative of at least three independent experiments.

Video 7

: Model of intestinal epithelial damage and regeneration after DSS treatment. This video demonstrates a pronounced regenerative response of the mini-gut tubes upon exposure to DSS, as compared to classical organoids. The video is related to Fig. 3b,c. Data are representative of at least three independent experiments.

Video 8

: Model of intestinal epithelial damage and regeneration after gamma-radiation. This video demonstrates the pronounced regenerative response of the mini-gut tubes upon exposure to radiation. The video is related to Extended Data Fig. 8. Data are representative of at least two independent experiments.

Video 9

: Modelling Cryptosporidium parvum infection in mini-guts. This video shows live bright-field imaging of Cryptosporidium parvum life cycle progression in infected mini-guts. The video is related to Extended Data Fig. 9b. Data are representative of independent observations from one experiment.

Video 10

: Increasing tissue complexity of the mini-gut system. This video shows time-lapse imaging of the mini-guts co-cultured with macrophages. The video is related to Extended Data Fig. 10 c,d.

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Nikolaev, M., Mitrofanova, O., Broguiere, N. et al. Homeostatic mini-intestines through scaffold-guided organoid morphogenesis. Nature 585, 574–578 (2020).

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