==== Front Nat Commun Nat Commun Nature Communications 2041-1723 Nature Publishing Group UK London 20052 10.1038/s41467-020-20052-z Perspective A bioengineering perspective on modelling the intestinal epithelial physiology in vitro http://orcid.org/0000-0003-0129-5640Antfolk Maria maria.antfolk@bric.ku.dk 123 http://orcid.org/0000-0001-6569-1664Jensen Kim B. kim.jensen@bric.ku.dk 12 1 grid.5254.60000 0001 0674 042XBRIC – Biotech Research and Innovation Centre, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark 2 grid.5254.60000 0001 0674 042XNovo Nordisk Foundation Center for Stem Cell Biology (DanStem), Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark 3 grid.4514.40000 0001 0930 2361Department of Biomedical Engineering, Lund University, Lund, Sweden 7 12 2020 7 12 2020 2020 11 62442 4 2020 12 11 2020 © The Author(s) 2020Open 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 license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license 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 license, visit http://creativecommons.org/licenses/by/4.0/.The small intestine is a specialised organ, essential for nutrient digestion and absorption. It is lined with a complex epithelial cell layer. Intestinal epithelial cells can be cultured in three-dimensional (3D) scaffolds as self-organising entities with distinct domains containing stem cells and differentiated cells. Recent developments in bioengineering provide new possibilities for directing the organisation of cells in vitro. In this Perspective, focusing on the small intestine, we discuss how studies at the interface between bioengineering and intestinal biology provide new insights into organ function. Specifically, we focus on engineered biomaterials, complex 3D structures resembling the intestinal architecture, and micro-physiological systems. Maria Antfolk and Kim Jensen discuss how to model intestinal epithelial cell function in the dish and how various physiologically important environmental conditions, for example, extracellular matrix, pressure and flow, can be modelled and how this is applicable to clinical work. Subject terms Gastrointestinal modelsIntestinal stem cellsGastroenterologyTissueshttps://doi.org/10.13039/100010665EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 Marie Skłodowska-Curie Actions (H2020 Excellent Science - Marie Skłodowska-Curie Actions)746270/H2020-MSCA-IF-2016Antfolk Maria https://doi.org/10.13039/501100009708Novo Nordisk Fonden (Novo Nordisk Foundation)NNF16OC0020792NNF17OC0028730NNF18OC0034066NNF19OC0056411NNF17CC0027852Antfolk Maria Jensen Kim B. https://doi.org/10.13039/100008392Sundhed og Sygdom, Det Frie Forskningsråd (Medical Sciences, Danish Council for Independent Research)8020-00085BJensen Kim B. https://doi.org/10.13039/100010663EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council)ERCCoG682665Jensen Kim B. https://doi.org/10.13039/100010662EC | EU Framework Programme for Research and Innovation H2020 | H2020 Excellent Science (H2020 Priority Excellent Science)668294Jensen Kim B. issue-copyright-statement© The Author(s) 2020 ==== Body Introduction In the small intestine, the process of digestion occurs in symbiosis with billions of microbes residing in the lumen of the intestine. Here, the epithelial cell layer that faces the lumen forms a protective barrier that shields the body against direct exposure to microbes and food antigens. This layer is organised into finger-like protrusions (villi), which serve as the site of nutrient absorption and pockets (crypts) containing mostly proliferating cells. This outer layer of intestinal epithelial cells is supported by the underlying stroma containing mesenchymal cells, neurons and vasculature1. Throughout life, the epithelium is continuously replenished by intestinal epithelial stem cells that reside at the bottom of crypts. These stem cells are highly proliferative and will constantly give rise to progeny that move up along the crypt–villus axis, first transitioning into transit amplifying (TA) compartment, before they end up as differentiated post-mitotic cells on villi and eventually being shed into the lumen. These differentiated cells actively participate either in the absorption of nutrients (enterocytes) or in the conditioning of the environment as secretory cell types. Some of these cells, such as goblet, enteroendocrine and tuft cells, are primarily located on villi, while Paneth cells are found at the crypt bottom, where they are intercalated between stem cells and contribute to the intestinal stem cell niche (Fig. 1)2,3. It has, however, been very difficult to study the impact of tissue architecture, host–microbe interactions and general tissue replenishment in a refined manner using animal models or traditional cell culture techniques.Fig. 1 Structure of the small intestine. The intestinal topography includes villi, where the absorptive enterocytes, secretory enteroendocrine and goblet cells reside, and crypt domains harbouring the intestinal stem cell niche including stem cells and Paneth cells. Underlying mesenchymal cells like myofibroblasts support the epithelium by secreting important growth factors such as WNTs, R-spondins and BMP antagonists. Gradients are believed to exist for the factors promoting stem cell self-renewal, including WNT and ligands for the ErbB pathway, whereas opposing gradients exist for differentiation-promoting factors such as BMP. The gradient, indicated here for EGF, represents all the ligands of the family, including Nrg1, Areg, Hbegf and Ereg107–109, which support intestinal stem cell function. Many immune cells also reside in the intestine, constantly monitoring microbe and food antigens. Created with BioRender.com. Using classical cell culture methods primary intestinal epithelial cells could not be kept in culture for longer than a few days. Cancer cell lines cultured on plastic or on porous membranes have consequently been used for studying signalling in the intestinal epithelium4. Although these lines have provided us with instrumental knowledge, they have obvious limitations in addressing some of the unresolved questions in intestinal epithelial biology, such as how the natural heterogeneity is maintained in the intestine, which signalling mechanisms control cell fate decisions and how stem cells are maintained in an undifferentiated state for extended periods of time. This all changed with the advent of two complementary methods developed by the groups of Hans Clevers and Calvin Kuo. They reported that primary intestinal epithelial cells could be cultured long-term in three-dimensional (3D) scaffolds either as self-organising epithelial organoid units5 or as mixed cultures of epithelial and mesenchymal cells6. Human- and mouse-derived intestinal organoids can now be generated from single stem cells or crypts isolated from primary fetal or adult tissues5,7, embryonic stem cells or induced pluripotent stem cells (iPSCs)8 and can be propagated long-term as spherical or budding organoids5,9,10 (Box 1 and Fig. 2). As such these organoid models comprise near-physiological systems that provide the opportunity to complement studies using animal models, and address questions related to early human development of the intestine using stem cells and differentiated epithelial cells derived from a number of different sources (Table 1).Fig. 2 Different types of organoids. Intestinal organoid architecture comes in different flavours ranging from spherical structures to budding organoids (brown enterocytes, turquois intestinal stem cells, pink Paneth cells, purple enteroendocrine cells and green goblet cells) with distinct localisation of different cellular domains containing stem cells and differentiated cells, and organoids that include a supporting mesenchyme (illustrated with pink cells surrounding the organoid). The cells are arranged with the apical membrane inwards and with appropriate cellular junctions that normally support the formation of an intact barrier including tight junctions (purple). Created with BioRender.com. Table 1 Characteristics of different organoid models for the small intestine derived either from tissues or from pluripotent stem cells. Cell source Species Culture characteristics Matrix Supplementary requirements Special feature Disadvantages Advantages Reference Adult small intestine Mouse Budding and spherical Collagen R-spondin, EGF, Noggin Cultures at the air–liquid interface No evidence of passaging, reported for early neonatal tissue source Easier to culture, and source 6 Mouse Budding Matrigel R-spondin, EGF, Noggin Can be cultured from all stages 5 Mouse Spherical Collagen type I R-spondin, EGF, Noggin, WNT Induces a wound healing phenotype 53,61 Human Spherical Matrigel WNT, R-spondin, EGF, Noggin More challenging to culture, and source 9 Human Budding Matrigel WNT, R-spondin, EGF, Noggin, IGF-1, FGF-2 More challenging to culture, and source Closest to human in vivo 44 Fetal small intestine Mouse Spherical Matrigel (R-spondin), EGF, Noggin Does not mature spontaneously Easier to source than human fetal tissue 7,113 Human Spherical Matrigel WNT, R-spondin, EGF, Noggin, PGE2 More challenging to culture, and source 7 hPSC-derived Human Spherical, with mesenchyme EGF, Noggin, R-spondin1 More similar to human fetal primary cells Time consuming to establish, expert knowledge in PSC required Easier to source than human primary tissue 8 Human Spherical, without mesenchyme Matrigel EGF, Noggin, R-spondin, CHIR9902 More similar to human fetal primary cells Time consuming to establish, expert knowledge in PSC required Pure epithelial PSC-derived population 114 As a primary cell source, with a genome reflecting either healthy individuals or patients, and with multilineage differentiation potential in vitro, the organoid system is attractive for disease modelling. Furthermore, elegant studies have demonstrated that organoids can be genetically engineered in vitro to study the effect of individual genes11,12 or screen for genes with important specific functions13–15. In this way, organoids from patients have been used to model diseases such as cystic fibrosis16, inflammatory bowel disease17–19, enteropathies including congenital diarrhoeal disorders20, and for modelling host–pathogen interactions including infection with Cryptosporidium21, Salmonella22,23, Clostridium difficile24, and entero-35, noro-36 or coronaviruses25,26. Thus, the development of the organoid technology has already had great impact on our ability to study functional aspects of the physiology of intestinal epithelial cells from a range of different species including humans. Yet, we have to keep in mind that these approaches are reductionistic given that only one component of a complex tissue is studied in isolation and often in ECM derived from tumour tissues. It is consequently important to stress that there is significant room for improving the methodology, before it reaches a state where we should consider this as a replacement for animal models. Future studies are likely to guide us in this direction with more refined cellular models using defined components or enhanced structural guidance of the epithelium. Box 1 Definition of the organoid Historically, the word organoid has had different meanings. During the 1950s and 1960s the word was used predominately to describe intracellular organelles110, and used to describe tumour formation111. In a third case the word was used to describe spontaneous formation of organ-like structures from cultures of embryonic tissues112, closer to today’s use of the term organoid. The term organoid was reintroduced in 2009 to describe the structures that were formed when epithelial cells from the small intestine were cultured in Matrigel in the presence of epidermal growth factor (EGF), Noggin and R-spondin15. The term has subsequently been applied to cultured 3D structures from other organs including colon, pancreas, mammary gland, brain, retinal epithelium, cerebellum, stomach, lung, salivary gland, thyroid, liver and kidney10. Only a small subset of these tissue organoids recapitulates the in vivo morphology to the same extent as the mouse small intestine, where organoids form distinct crypt and villus domains. The definition of an organoid upon the reintroduction of the term in 2009 has since not reached a full consensus. It has been implied that organoids should develop from stem or progenitor cells, be constituted by multiple organ-specific cell types, self-organise into a 3D structure and recapitulate some organ-specific functions. Here, we have chosen a somewhat broader definition to include all different ways in which the word organoid is currently used. In this review we define an organoid as a self-organising 3D structure, grown and expanded in vitro, and consisting of organ-specific cells with a restricted lineage commitment and having organ-like functions. Our definition, therefore, also encompasses spheroids (or enteroids, as is a term used for spheroids derived from the small intestine and colon), where symmetry breaking events are less apparent. Our definition avoids restricting the origin of the organoid to stem cells and allows the inclusion of tumour organoids, where the debate about the existence of the cancer stem cell is not yet settled. Modelling the intestinal epithelial physiology in vitro The intestinal stem cell niche The intestinal stem cell niche represents a unique biophysical and biochemical microenvironment that supports stem cell self-renewal and maintains the epithelial cells in an undifferentiated yet proliferative state27–29. The conductive environment in the niche is provided by complex interactions with neighbouring epithelial cells including Paneth cells, different mesenchymal cell populations including the underlying muscle, enteric neurons, immune cells as well as the endothelium2,30–33. Within this environment stem cell behaviour is controlled by the local production of signalling molecules including Wingless-type MMTV integration site family members (WNTs), bone morphogenetic protein (BMP) inhibitors and members of the epidermal growth factors family34,35. In combination with an extracellular matrix (ECM), this bare minimum is sufficient to support stem cell cultures in vitro5. Interesting new observations also support that the gut microbiome via break-down of food into, e.g. short chain fatty acids, play a role in controlling stem cell behaviour and energy metabolism36–38 and that crypt structure, at the same time, provides a protective environment for exposure to other harmful microbial metabolites39. An additional essential component of the intestinal stem cell niche is the ECM that separates the epithelium from the underlying stroma, and which provides a structural component of the niche that influences cell fate choices. Careful characterisations of ECM components have revealed that although some components like collagen IV and fibronectin are present ubiquitously along the crypt–villus axis others show distinct expression patterns40,41. Laminins, which are a major constituent of the ECM, are composed of three subunits (α, β and γ) that via their α unit, form ligands for integrin cell surface receptors. Interestingly, laminins containing α2 and α4 subunits are detected at the bottom of crypts, whereas the α3 and α5 subunits are associated with the differentiated villus compartment and they could consequently have a functional role in controlling cell behaviour40–42. The exact roles of the different ECM components in the basement membrane are currently not known. In order to shed light on the role of individual ECM components, it will be advantageous to utilise in vitro systems where cells can be spatially organised in a controlled microenvironment. The basic requirements for intestinal epithelial cells The methods for growing organoids revealed that the recurrent mutations in colorectal cancer (loss of APC (WNT activation), activating mutations in Ras (growth factor signalling), and loss of SMAD4 (BMP signalling)) in combination with an ECM provide the minimum signalling requirements for stem cell maintenance of intestinal epithelial cells5,9,43. Here, R-spondin supports stem cell self-renewal as an agonist of the WNT pathway, epidermal growth factor (EGF) family members stimulate proliferation via growth factor signalling, and Noggin suppresses differentiation as an antagonist of bone morphogenetic protein (BMP)-induced differentiation5. Importantly, these minimal culture conditions (EGF, Noggin and R-spondin1 (ENR)) support cultures of epithelial cells derived from the mouse small intestine, where secretion of WNTs from Paneth cells is sufficient to support stem cell self-renewal2,5. For cultures of human small intestinal epithelial cells an exogenous source of WNT has to be provided9. These conditions provide the core signalling requirements for expansion of intestinal epithelial cells. Interestingly, recent work demonstrates that addition of Insulin-like Growth Factor 1 and Fibroblast Growth Factor 2 to this core growth factor cocktail enhances both clonal growth and lineage potential44. With the advances in the culture conditions the physiological relevance of the organoid systems is enhanced, yet, the influence of biophysical cues on lineage choices remains largely unexplored. This is what we will discuss in the following. Two-dimensional open monolayer cultures Even though organoid cultures have proven an extremely versatile tool, there is an interest in using primary intestinal epithelial cells in monolayer cultures for multiple reasons. Firstly, the small intestinal epithelium in vivo is constituted by a single cell layer supported by the submucosa. Here, the apical surface is exposed to the luminal content and the shear stresses from the movement of content along the length of the gastrointestinal tract. In organoids, the epithelium is polarised with the basolateral membrane facing outwards and the apical membrane facing towards the hollow lumen (Fig. 2). This complicates studies that involve natural barrier translocation or microbial–epithelial interaction studies, where the initial interaction occurs at the luminal cell surface. This caveat can be bypassed by microinjecting microbes or compounds into the organoid lumen, however, this is a cumbersome process and difficult to automate23. Two-dimensional culture systems are consequently tractable either as classical adherent cultures or placed on top of a supporting scaffold providing access to the apical membrane. Alternatively, this type of topology could also be achieved using organoids with an inversed morphology45. Secondly, the majority of methodologies for imaging and manipulating epithelial cells have been developed for 2D cultures, although recent advances in light sheet microscopy are enhancing the imaging of 3D structures46. It has, however, proven challenging to establish 2D culture systems supporting both stem cell self-renewal and differentiation of epithelial cells. Most described monolayer cultures consequently constitute a means to an end, where cells seeded in 2D lose their ability to self-renew and eventually become terminally differentiated47. Despite these limitations, short-term monolayer cultures of intestinal epithelial cells have proven useful for assessing the function of growth factors/morphogens on the behaviour of intestinal epithelial cells including lineage choices48. Moreover, refined monolayer-culture methods provide strong indications that new medium compositions might facilitate long-term culturing of primary intestinal epithelial cells in 2D from both human and mouse49–51. Interestingly, recent studies assessing stem cell functions in 2D report that cells, even under these conditions, self-organise into proliferative regions expressing markers of stem cell and Paneth cells, and domains of differentiated enterocytes, goblet, tuft and enteroendocrine cells48–51. These are important observations, which for now provide testable models for assessing whether the mechanism that allow cells to self-organise is the same in 2D, 3D and in vivo, and also how topography might influence cell fate choices, patterns of differentiation and cellular maturation. The importance of the matrix for growth ECM components represent an important element of the stem cell niche and are instrumental for providing the specific biophysical environment that controls cell fate. Using refined models such as organoids, it is now possible to modulate the properties of the ECM and assess how this influences cell fate choice. Such refined questions could not be addressed with cancer cell lines given that the natural mechanisms regulating cell fate choices are compromised. There are, however, a number of aspects that should be considered. Firstly, it is necessary to characterise the cellular system used. The mesenchyme naturally represents a major source of ECM components. As an elegant example illustrating the guiding properties of the matrix produced by the mesenchyme, organoids that are co-derived with mesenchymal populations from PSCs can be cultured in gels completely lacking adhesive support52. It is consequently difficult to perform a refined study of how specific matrix components affects cell fate, if the epithelial cells are sheltered from the matrix by mesenchymal subpopulations producing their own ECM components. Secondly, one needs to consider the composition of the matrix. Matrigel or basement-membrane extracts are the most widely used ECM to support the growth of intestinal epithelial organoids and are composed of laminin (Lam111 ~60%), type IV collagen (~30%) and nidogen (~8%), as well as a number of growth factors. Type I collagen has also been widely used as a matrix scaffold; however, here results have been somewhat diverging. Unlike Matrigel, type I collagen does not by itself support the growth of murine intestinal epithelial organoids in the presence of EGF, Noggin and R-spondin1, unless WNT is supplied as a purified component53. However, epithelial organoids can be maintained in type I collagen in conditions that include mesenchymal cells6,54. Yet, it remains to be shown whether the mesenchyme under these conditions is shielding the epithelium from the type I collagen via de novo matrix production, secreting high levels of WNT thereby bypassing the need for an exogenous source, or that the matrices used for the different studies differ in their biophysical properties. Apart from these two more widely used matrices, mixtures of fibronectin and laminin have been explored as a more defined matrix55, and decellularised small intestinal submucosa as a natural scaffold56. Despite the partial successes of these matrices, engineered protein gels provide clear advantages for addressing how the specific properties of the ECM support the intestinal epithelium. Thirdly, bioengineering methods now allow the generation of artificial matrices based on individual components that confer specific properties. It is consequently possible to decouple biophysical properties from biochemical cues, so that one variable can be studied at a time. Using these methods, it is possible to fine-tune, e.g. the stiffness of the matrix, pore sizes and degree of cross linking of different components, and introduce various linker elements that can be cleaved by the cells in the organoids upon secretion of, e.g. matrix metalloproteinases (MMP)57–60. Bioengineering new stem cell niches Much of the research performed in vitro has until now relied on the self-organisation of cellular structures using either cell lines or primary cells to recapitulate the intestinal epithelium. The development of advanced bioengineering approaches provides new and exciting possibilities for directing the organisation of cells in controlled environments. The tools facilitate engineering 3D matrices and materials with defined sizes, structures, porosities, stiffnesses and biochemical profiles, and can be complemented with dynamic features such as controlled shear stress, mechanical stimulation and motion. By controlling the microenvironment through bioengineering approaches, it is consequently possible to obtain greater insights into the mechanisms that ultimately control cell fate. Engineered 3D matrices Tumour‐derived basement-membrane extracts are the most widely used matrix for cultures of intestinal epithelial organoids5. The composition of these matrices is, however, poorly defined and the production is associated with batch-to-batch variability, making it difficult to completely standardise studies that focus on the specific interactions between the microenvironment and the cells. The issues of variation between batches and the fact that it is extracted from animal tissue also complicate the direct translation of current organoid technologies into regenerative therapies1. Collagen gels have been proposed as an alternative to these poorly defined matrices61. It is, however, clear that the specific properties of the matrix will have significant impact on cell fate decisions, and the matrix used therefore needs to be tailored to specific needs and that it is difficult to extrapolate findings from one matrix to the other53. As a complement to natural matrices, artificial materials have been utilised for supporting the growth of intestinal organoids62,63. These synthetic matrices provide a new and refined context for assessing how cell fate choices are influenced by extracellular components and thereby the environment. Their synthetic nature offers parallels to toy building blocks where different pieces can be combined independently and in a patterned manner, using click-chemistry, thereby allowing control of both the biochemical environment, with adhesion opportunities and growth factor densities, and the biophysical properties including stiffness and porosity (Fig. 3).Fig. 3 Synthetic matrices for stem cell research. Synthetic matrix assembly using, e.g. click-chemistry, resembling the use of toy building blocks where cell-instructive building blocks of choice, e.g. adhesion peptides or growth factors, are combined with a polymer backbone to form a functionalised scaffold where cells can adhere and be cultured. Created with BioRender.com. Cell adhesion to the biomaterial, proliferation and differentiation can be stimulated by incorporating adhesion ligands such as the Arg-Gly-Asp (RGD) peptide or full-length proteins such as laminins. By varying the cross-linker concentration the biophysical properties including the material stiffness and nano-porosity can be controlled64. Importantly, ligand display and material stiffness can be varied independently in synthetic biomaterials, allowing for a greater control of the experimental parameters. The biocompatibility of the material can also be improved by incorporating elements improving biodegradability through MMP-sensitive cross linkers57. Within the 3D space of a biomaterial such elements promote cell spreading, which is especially important for larger cellular structures such as organoids to be able to form and grow63. In addition, microporosity can be introduced during the scaffold formation, allowing the cells to instantly spread through the material without having to degrade the biomaterial first65. To dynamically modulate the stiffness of the biomaterial and introduce a gradual softening, e.g. acrylate groups can be introduced, where the ester bonds of this group will undergo hydrolysis and gradually soften the biomaterial over a course of days62. Instead, by introducing allyl sulphide groups, the biomaterial is rendered photodegradable, which allows for instant softening or even full degradation in a matter of seconds when exposing the biomaterial to UV light66. Using this method local spatial softening of the biomaterial can also be introduced, directing the budding or crypt formation of an intestinal organoid. Using synthetic matrices Gjorevski et al. elegantly illustrated how these matrices can be applied to address biophysical requirements for mouse organoidsʼ formation starting from single intestinal stem cells. Studying the process from formation and growth into fully mature structures, the authors assessed how this is dynamically supported by the environment. The authors revealed that this is a multistep process with phases that have discrete requirements62. By adding a dynamic aspect to a semisynthetic polyethylene glycol (PEG)-based matrix, including the RGD-peptide and laminin-111 for enhanced cell adhesion, they provided a degradable matrix that initially had the optimal stiffness for organoid formation (G = 1.3 kPa) and in the second growth phase became softer allowing organoids to expand as budding structures (G = 190 Pa)62. The healthy small intestine has been measured to have Young’s modulus of E = 2.9 kPa67. With the relationship in biological tissues being E = 3G68, this would correspond to G = 967 Pa, indeed in the range observed to be optimal for culturing intestinal epithelial organoids. Interestingly, a subsequent study found that a PEG-based matrix functionalised with the cell adhesion domains from type I collagen (GFOGER) on its own was sufficient as a matrix in supporting the growth of primary human intestinal epithelial cells as spheroids69. This again points to the important role of biomechanical cues in controlling cell fate and is supported by the observation that organoid ‘crypt’ formation depends on local mechanical signals as the length and the number of crypts per organoid can be modulated by softening the matrix70. A complementing study using high-resolution live-imaging reported that organoid formation in basement-membrane extracts go through two distinct phases similar to organoids grown in synthetic matrices. Here, the first phase is characterised by expansion as a spheroid, followed by budding and differentiation. Mechanistically, the transition between the two states is, both in the synthetic matrix and the basement-membrane extract, driven by a reduction in the mechanosensory YAP signalling pathway46,62. Synthetic matrices have also been applied to hPSC-derived intestinal organoids illustrating that these can also be grown in a PEG-based matrix. Here, the matrices functionalised with the RGD peptide, the cell adhesion domains from type I collagen (GFOGER) or laminin α1 subunit (IKVAV or AG73) all supported survival of the organoids, although the scaffold containing the RGD peptide had the greatest functionality. The synthetic matrix also contained a protease-cleavable peptide to allow for modulation of the matrix, but the importance of this modality was not reported71. Notably, the requirements for mouse intestinal epithelial organoids were surprisingly more complex than those described for hPSC-derived organoids. However, here it is important to keep in mind that the hPSC-derived organoids used in these studies also contain mesenchymal cells. This will consequently provide an additional source of ECM components. It is therefore difficult to perform direct comparisons between the two systems. In fact, it has been shown that these hPSC-derived organoids can be cultured in matrices completely lacking adhesive support, illustrating that one important function of the associated mesenchyme is to provide a functional extracellular scaffold52. It will be exciting to follow how bioengineering can be exploited to reveal the impact of the biochemical and biophysical environment on intestinal stem cell behaviour. Here, the PEG-based matrix without adhesive properties could provide instrumental insight into deciphering how the supporting mesenchyme naturally supports epithelial stem cell properties. However, to pinpoint the requirements for the epithelial cells, it is essential to work in much more reductionist models such as pure epithelial cultures from either mouse or human tissues. Complex 3D strategies for structural guidance Although organoids have proven a versatile tool for addressing the key questions in intestinal epithelial biology, their form is not optimal for studies focusing on barrier function and interactions with the luminal surface of the epithelium. Moreover, topographic constraints that will exert divergent mechanical forces on the tissues as well as provide a basis for growth factor gradients are difficult to recapitulate when cells are cultured in a 3D matrix. A range of 2D topologies have consequently been investigated to overcome not only the closed nature of the organoid structures but also to assess how additional architectural features such as tube structures and scaffolds shaped with villi and crypts affect cell behaviour72–75 (Fig. 4 and Box 2). Although the application of structural guidance based on scaffolds mimicking the stromal surface of the intestine is still in its infancy, such technologies might provide a framework for advanced studies of complex interactions between microbes and the intestinal surface76. Here an important study recently revealed that an intestinal epithelium can be maintained long-term in hydrogel-based microchannels formed to include crypts and support laminar flow through the device. In this patterned structure, stem cells were located at the bottom of the crypts, whereas differentiated cells were facing the lumen facilitating studies of regeneration and host–microbe interactions77. It will be central for the field to follow how this technology further develops.Fig. 4 Establishing complex 3D structures for organ mimicry and dynamic micro-physiological systems. a Either single cells or organoid fragments can be used to seed these systems. b Complex 3D structures, e.g. crypt-like or villus-like, have been used to guide the cellular organisation. These structures can be fabricated from, e.g. biomaterials, PDMS, silicon or plastics such as polystyrene. c Intestine-on-a-chip devices have been used to create dynamic culture conditions including shear stress from fluid flow as well as cyclic mechanical deformation that resemble the natural movements associated with peristalsis. Intestine-on-a-chip device seen from above with an upper (purple) and a lower (blue) channel. Cross section of the device which is commonly composed of an upper (purple) and a lower (blue) channel separated by a porous membrane. Epithelial cells can be seeded into the upper channel, e.g. as a flat 2D layer or on a 3D construct that provides topographic features such as villi (as shown in the figure). These devices are most often made of PDMS, a transparent polymeric organosilicon, also making them ideal for live-cell imaging. d The intestinal barrier can be assessed by introducing various compounds to the upper surface of the 3D constructs, or through the inlet of the intestine-on-a-chip with access to the apical side of the epithelium. Medium can subsequently be collected from below the 3D construct or through both channel outlets of the intestine-on-a-chip for further analysis. In addition, cells seeded on these structures can later be isolated for, e.g. gene or protein expression analysis. Moreover, cells can be directly monitored to follow cell behaviour, using live-cell imaging. Created with BioRender.com. From a physiological perspective the architecture of villi is extremely important, as they increase the surface area of the small intestine and thereby the nutrient absorptive capacity by more than 30-fold. Villi are finger-like protrusions extending 0.5–1.6 mm into the lumen of the small intestine, and are composed of an epithelial surface on top of an underlying stroma that includes myofibroblasts, blood vessels and lymphatics. Studies of the fetal intestine suggest that the topology of the adult small intestine does have a function. In contrast to adult villi that are covered by post-mitotic and terminally differentiated epithelial cells, the smaller rudimentary villi found in the fetus are linked with an immature phenotype of the epithelial component. Importantly, the transition into the mature adult phenotypes coincides with emergence of the crypt–villus axis78. It remains an open question how the villi could play an active role in the cellular organisation and directing cell fate choices within the epithelium. There could be multiple explanations for why the structure would guide cell fate choice either directly or indirectly. At one level it is worth considering cell shapes. As cells translocate from crypt bottom to the villus top, there is a gradual decrease in the fraction of the cell membrane that faces the ECM, when compared to the apical counterpart. At the hinge that forms, where cells exit the crypt and enter the villus, this aspect changes dramatically79, and such changes could have significant consequences on mechanosensory pathways and thereby cell fate decisions. In addition to form, it is clear that the crypt–villus axis in vivo serves an important function in establishing gradients of factors that are maintaining stem cell self-renewal, e.g. WNTs and pro-differentiation signals such as BMP. This supports the spatial confinement of stem cells and differentiated cells, and elegant genetic studies have demonstrated that interfering with these gradients is sufficient to interfere with the cellular boundaries80,81. Similarly, flow across a cell membrane can act not only via shear stress on the cell surface but also by removing or transferring substances from cells nearby. Structural guidance via bioengineered solutions represent elegant tools for specifically addressing the importance of these individual components and mechanisms. In line with the spatial confinement of differentiated and non-differentiated cells in vivo along the crypt–villus axis, studies of Caco-2 cells on various scaffolds, mimicking the morphology of the small intestine with crypts and villi, indicate that 3D architecture affects the expression and organisation of cells into proliferating and differentiating domains82,83. Interestingly, a complementing study using primary human intestinal epithelial cells cultured on a similar 3D scaffold did not reveal any type of confinement of cells with a particular phenotype. Instead, proliferating cells were found along the entire crypt–villus axis. Mimicking the natural system, growth factor gradients had to be applied along the crypt–villus axis to confine undifferentiated cells in the crypt domain and differentiated cells on the villi. Here, the authors supplemented the medium under the device with WNT3a, R-spondin3 and Noggin, and the medium above the device with the Notch-inhibitor (DAPT), which is known to induce secretory differentiation. Using a nano-porous biomaterial allowed for the formation of stable linear gradients across the cellularised device84. The 3D architecture, as observed during fetal intestine development, is consequently not in itself sufficient to impose strict cell fate decision, but can provide the basis for changes in growth factor availability and potentially also the probability to respond to specific cellular signals. The future will reveal how the implementation of new technologies from the bioengineering field such as 3D printing of different matrix components as well as different cell types will change studies of the intestinal epithelium. Here, one area of interest will be to address how topography instructs cells via structure and also how growth factor gradients are first established within the tissues. Box 2 Microfabrication of biomimetic small intestinal models Complex micropatterned 3D structures resembling the small intestinal architecture are commonly fabricated using microfabrication processes and micromoulding. A master mould for the biomaterial scaffold can be fabricated by utilising either photo lithography84, micromilling, laser ablation115 or 3D printing. Using these master moulds, a more flexible mould of PDMS is usually casted84,115. Sometimes another moulding step is also utilised to improve the detachability115 of the biomaterial scaffold or work as a sacrificial layer74 to facilitate the fabrication of softer or more brittle scaffolds. An exciting new opportunity for sculpting scaffolds as an alternative to micromoulding is 3D bioprinting and it will be interesting to follow how this technology will impact the bioengineering arena116. Irrespective of the method, the scaffolds need to possess a high enough stiffness to not break during the fabrication process or deform under its own weight or due to cellular traction forces. Consequently, these scaffolds are limited in the stiffness that can be used. As previous work on organoids showed that softer scaffolds are required for more complex growth patterns when working with primary cells62, it will be interesting to further monitor the development of additional biomaterials that support not only spheroid growth but also budding into functional domains of organoids derived from different cell sources. It does, however, still remain unclear whether the observations related to stiffness and cell fate are directly translatable from organoid studies, but future studies will hopefully help elucidate this. Organ-on-a-chip The emerging organ-on-a-chip technology has enabled new and innovative ways to study organ level functions, and provides an alternative technology for complementing studies on health, tissue development and disease85. The technology utilises microfluidics and microtechnology in combination with cell culture models to recapitulate tissue- and organ-level physiology in a dynamic mode that has been difficult to emulate using classical static cell culture methods. The organ-on-a-chip device represents a minimal functional unit, such as a barrier or tissue interface, required to model selected in vivo functions of the organ. The device is built using electronic microchip manufacturing methods, whereby micro-sized cell culture chambers can be fabricated with high precision. This allows for media perfusion and mechanical stimulation. These systems recapitulate aspects of the native tissue to a higher degree than conventional 2D or 3D culture systems, and have the capacity to model tissue–tissue interfaces, shear stresses induced by fluid flow, tension and compression, which are part of the mechanical motion of tissues. Importantly, despite the name, these devices are not meant to recapitulate whole organ physiology. In a conventional macrofluidic system such as a traditional bioreactor the fluid regime is characterised by a turbulent flow, inducing mixing, whereas the flow in a microfluidic system is laminar. The microfluidic system consequently offers greater control over the fluid flow, and it is easier to control the impact on cells within the device. Furthermore, microfluidic devices provide the opportunity for high-resolution on-line monitoring, via measurements leading to minimal sample and reagents consumption and real-time imaging86,87. Importantly, these systems also offer the opportunity to simulate relevant interactions between different cell types. One example is the addition of immune cells to tubes covered with endothelial or epithelial cells and the ability to remove these cells from the system through flow, thereby emulating the body’s blood or lymph system. Organ-on-a-chip devices are generally made up of an upper and a lower microfluidic channels separated by an ECM-coated porous membrane on which cells are seeded creating a surface, e.g. for studies of barrier function88 (Fig. 4). Polydimethylsiloxane (PDMS) is commonly used as a fabrication material for these devices. This silicone elastomer has a limited ability to support cell adhesion, but can be modified with ECM proteins to allow cells to adhere89. The first studies using these principles in combination with Caco-2 cells aimed at reconstructing normal tissue architecture and barrier function via the application of fluid flow-induced shear stress87. Since then, an increasing number of organ-on-a-chip devices have been reported, including advancements such as the incorporation of stretchable membranes for cell support to simulate aspects of lung function via a breathing motion90. These principles have been adapted to an intestinal device based on Caco-2 cells. Here cyclic peristalsis-like motions in combination with low shear stress via fluid flow resulted in the formation of a highly polarised epithelium with a buckling surface91. The authors of the study argue that this mimics intestinal villi architecture; however, buckling phenomena is often incurred in cell culture models, as a consequence of over-proliferation, and it remains to be shown whether the observed structures are true villi. Follow-up studies suggested that basolateral flow below the membrane facilitates continuous removal of the WNT antagonist Dickkopf-1 and that elevated WNT-mediated proliferation facilitates the epithelial folding92. It remains to be elucidated whether this is via a canonical or non-canonical WNT signalling pathway, given that Caco-2 cells have mutations in the APC protein that normally regulates canonical WNT signalling. Importantly, results from other groups support that fluid flow is sufficient for epithelium proliferation and folding of the epithelial layer93,94. Devices like these consequently provide a tool whereby it is possible, in a reductionistic manner, to study dynamic processes mimicking the consequences of flow above and below the epithelium. Microfluidic devices have not only been used for studying established cell lines but also for primary intestinal epithelial cells. New findings suggest that primary intestinal epithelial cells cultured in these devices, at the transcriptional level, more closely resemble the in vivo epithelium than the organoids cultured in Matrigel95,96. Moreover, the technology can also be applied to intestinal epithelial cells derived from hPSCs, although a comparison to their in vivo counterpart is still missing97. The fact that the technology can be applied to human intestinal epithelial cells makes it a very attractive technology for applications addressing the effects of microbial overgrowth and intestinal inflammation98,99, co-cultures with anaerobic bacteria100,101 and viruses102, and for modelling, e.g. radiation exposure103. Moreover, based on the observation from cancer cell lines that flow affects autocrine and paracrine signalling, this also opens up opportunities to address how cells signal to each other within the epithelium. Fluid flow both over and under the epithelial surface has also been important for the primary applications for intestines-on-chips focusing on bacterial–epithelial interactions98–101. Here, direct access to the apical surface allows bacteria exposure to the epithelial surface without compromising the epithelial barrier integrity. The fluid flow across the device here simulates the continuous removal of material observed in vivo and peristalsis-like mechanical motion of the epithelium promotes mixing of the luminal content, thereby reducing the risk for bacterial colonisation and overgrowth commonly seen in static culture systems. Many diseases, such as diabetes104 and IBD105 have been correlated with changes in gut microbiota composition. However, it remains unknown whether these observed correlations between specific diseases and microbial composition is driven by an underlying disease mechanism or disease-associated patient behaviour, and whether microbe composition precedes and triggers the disease. Here, the opportunity to provide more physiologically relevant culture conditions for the intestinal epithelial cells in the microfluidic devices is a clear advantage. Despite the successes reported for organ-on-a-chip devices, they still require complex fluidic and pneumatic setups. In addition, the physiological relevance of the supporting porous membrane for cell support is questionable in terms of stiffness, thickness and adhesion opportunities, although of course this can be optimised. Attempts to circumvent these potential drawbacks of organ-on-a-chip devices include membrane- and pump-free models enabling the simultaneous analysis of up to 40 separate cell-covered tubes in parallel106. Although there are obvious advantages due to the simplicity of the system and the generation of fluid flow-induced shear stress over the cells, the system does not deplete secreted factors to the same degree as traditional microfluidics systems. Perspectives We have come a long way from first focusing on cancer cell lines for studying the normal physiology to a phase looking at self-organising epithelium in more or less defined matrices. Currently, we can control the composition of the matrix and the biophysical properties, and we have added a dynamic setting where the culture systems can be controlled in 4D. Although significant advances have been made in the past decade, especially with the introduction of organoid technology, there are still major challenges to overcome to develop more in vivo-like models. Presently, there is a distance between the technological and the biological developments within the field. Technological advances in biomaterials and micro-engineering fields are complemented by simpler biological studies most often using cell lines, e.g. Caco-2 cells as a first phase, whereas more advances in biological systems studying primary epithelial cells or hPSC-derived cells are performed in simpler microenvironmental systems often involving Matrigel. More advanced studies are required to address physiological questions that go beyond simple cell behaviour and represent an arena where there will be tremendous potential in combining the advances in micro-engineered devices with primary cell derivatives. A further combination of advanced topographic 3D biomaterial models with the microfluidic field is also expected. We anticipate that the future will merge the organoid field even more with the organ-on-a-chip technology. This merge may contribute towards controlling the growth and self-renewal properties of stem cells by establishing perfusion or gradient cultures, thereby providing a more controlled biochemical microenvironment, or to provide spatial control facilitating e.g. imaging and high throughput screening. Moreover, the next step in studies of intestinal biology is to go beyond only the epithelium. Here, micro-engineered culture systems provide excellent platforms to determine the relevance in the context of defined structural confinements. This will be a first step towards generating a fully functional small intestine in vitro. With this we look forward to an exciting future and are eagerly awaiting what will come. Peer review information Nature Communications thanks Toshiro Sato 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. Acknowledgements The authors thank members of the Jensen lab for insightful discussions. Work in the Jensen lab is supported by Marie Curie fellowship programme (746270/H2020-MSCA-IF-2016 to M.A.), the Novo Nordisk Foundation (NNF16OC0020792 to M.A.; NNF17OC0028730 to K.B.J.; NNF18OC0034066 to K.B.J.; NNF19OC0056411 to K.B.J.), the Danish Medical Research Council (8020-00085B to K.B.J.), the European Union’s Horizon 2020 research and innovation programme (grant agreements STEMHEALTH ERCCoG682665 K.B.J. and INTENS 668294). The Novo Nordisk Foundation Center for Stem Cell Biology is supported by Novo Nordisk Foundation grant (NNF17CC0027852). Author contributions M.A. and K.B.J. wrote the manuscript. Competing interests The authors declare no competing interests. ==== Refs References 1. Clevers H Tissue-engineering the inestine: the trials before the trials Cell Stem Cell 2019 24 855 859 10.1016/j.stem.2019.04.018 31173715 2. Sato T Paneth cells constitute the niche for LGR5 stem cells in intestinal crypts Nature 2011 469 415 418 10.1038/nature09637 21113151 3. Beumer, J. & Clevers, H. Cell fate specification and differentiation in the adult mammalian intestine. Nat. Rev. Mol. Cell Biol. 10.1038/s41580-020-0278-0 (2020). 4. Fogh J One hundred and twenty-seven cultured human tumor cell lines producing tumors in nude mice J. Natl. Cancer Inst. 1977 59 221 226 10.1093/jnci/59.1.221 327080 5. Sato T Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche Nature 2009 459 262 265 10.1038/nature07935 19329995 6. Ootani A Sustained in vitro intestinal epithelial culture within a Wnt-dependent stem cell niche Nat. Med. 2009 15 701 706 10.1038/nm.1951 19398967 7. Fordham RP Transplantation of expanded fetal intestinal progenitors contributes to colon regeneration after injury Cell Stem Cell 2013 13 734 744 10.1016/j.stem.2013.09.015 24139758 8. Spence JR Directed differentiation of human pluripotent stem cells into intestinal tissue in vitro Nature 2011 470 105 109 10.1038/nature09691 21151107 9. Sato T Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett’s epithelium Gastroenterology 2011 141 1762 1772 10.1053/j.gastro.2011.07.050 21889923 10. Schweiger PJ Jensen KB Modeling human disease using organotypic cultures Curr. Opin. Cell Biol. 2016 43 22 29 10.1016/j.ceb.2016.07.003 27474805 11. Drost J Sequential cancer mutations in cultured human intestinal stem cells Nature 2015 521 43 47 10.1038/nature14415 25924068 12. Schwank G Functional repair of CFTR by CRISPR/Cas9 in intestinal stem cell organoids of cystic fibrosis patients Cell Stem Cell 2013 13 653 658 10.1016/j.stem.2013.11.002 24315439 13. Ringel T Genome-scale CRISPR screening in human intestinal organoids identifies drivers of TGF-β resistance Cell Stem Cell 2020 26 431 440 10.1016/j.stem.2020.02.007 32142663 14. Kakiuchi N Frequent mutations that converge on the NFKBIZ pathway in ulcerative colitis Nature 2020 577 260 265 10.1038/s41586-019-1856-1 31853061 15. Michels BE Pooled in vitro and in vivo CRISPR-Cas9 screening identifies tumor suppressors in human colon organoids Cell Stem Cell 2020 26 782 792.e7 10.1016/j.stem.2020.04.003 32348727 16. Dekkers J A functional CFTR assay using primary cystic fibrosis intestinal organoids Nat. Med. 2013 19 939 945 10.1038/nm.3201 23727931 17. Grabinger T Inhibitor of apoptosis protein-1 regulates tumor necrosis factor - mediated destruction of intestinal epithelial cells Gastroenterology 2017 152 867 879 10.1053/j.gastro.2016.11.019 27889570 18. Noben M Epithelial organoid cultures from patients with ulcerative colitis and Crohn’s disease: a truly long-term model to study the molecular basis for inflammatory bowel disease? Gut 2017 66 2193 2195 10.1136/gutjnl-2016-313667 28159838 19. Arnauts K Ex vivo mimicking of inflammation in organoids derived from patients with ulcerative colitis Gastroenterology 2020 159 1564 1567 10.1053/j.gastro.2020.05.064 32474118 20. van Rijn J Intestinal failure and aberrant lipid metabolism in patients with DGAT1 deficiency Gastroenterology 2018 155 130 143 10.1053/j.gastro.2018.03.040 29604290 21. Heo I Modelling Cryptosporidium infection in human small intestinal and lung organoids Nat. Microbiol. 2018 3 814 823 10.1038/s41564-018-0177-8 29946163 22. Forbester JL Interaction of Salmonella enterica Serovar Typhimurium with intestinal organoids derived from human induced pluripotent stem cells Infect. Immun. 2015 83 2926 2934 10.1128/IAI.00161-15 25964470 23. Wilson SS A small intestinal organoid model of non-invasive enteric pathogen–epithelial cell interactions Mucosal Immunol. 2015 8 352 361 10.1038/mi.2014.72 25118165 24. Leslie JL Persistence and toxin production by Clostridium difficile within human intestinal organoids result in disruption of epithelial paracellular barrier function Infect. Immun. 2015 83 138 145 10.1128/IAI.02561-14 25312952 25. Lamers MM SARS-CoV-2 productively infects human gut enterocytes Science 2020 369 50 54 10.1126/science.abc1669 32358202 26. Zhou J Infection of bat and human intestinal organoids by SARS-CoV-2 Nat. Med. 2020 26 1077 1083 10.1038/s41591-020-0912-6 32405028 27. Yeung TM Regulation of self-renewal and differentiation by the intestinal stem cell niche Cell. Mol. Life Sci. 2011 68 2513 2523 10.1007/s00018-011-0687-5 21509540 28. Mills JC Gordon JI The intestinal stem cell niche: there grows the neighborhood Proc. Natl. Acad. Sci. USA 2001 98 12334 12336 10.1073/pnas.231487198 11675485 29. Biswas S Microenvironmental control of stem cell fate in intestinal homeostasis and disease J. Pathol. 2015 237 135 145 10.1002/path.4563 25974319 30. McCarthy N Distinct mesenchyma cell populations generate the essential intestinal BMP signaling gradient Cell Stem Cell 2020 26 391 402 10.1016/j.stem.2020.01.008 32084389 31. Shoshkes-Carmel M Subepithelial teolcytes are an important source of Wnts that supports intestinal crypts Nature 2018 557 242 246 10.1038/s41586-018-0084-4 29720649 32. Stzepourginski I Cd34+ mesenchymal cells are a major component of the intestinal stem cell niche at homeostasis and after injury Proc. Natl. Acad. Sci. USA 2017 114 E506 E513 10.1073/pnas.1620059114 28074039 33. Farin H Redundant sources of Wnt regulate intestinal stem cells and promote formation of Paneth cells Gastroenterology 2012 143 1518 1529 10.1053/j.gastro.2012.08.031 22922422 34. Powell DW Mesenchymal cells of the intestinal lamina propria Annu. Rev. Physiol. 2011 73 213 237 10.1146/annurev.physiol.70.113006.100646 21054163 35. Gehart H Clevers H Tales from the crypt: new insights into intestinal stem cells Nat. Rev. Gastroenterol. Hepatol. 2019 16 19 34 10.1038/s41575-018-0081-y 30429586 36. Beyaz S High-fat diet enhances stemness and tumorigenicity of intestinal progenitors Nature 2016 531 53 58 10.1038/nature17173 26935695 37. Chen L HNF4 regulates fatty acid oxidation and is required for renewal of intestinal stem cells in mice Gastroenterology 2020 158 985 999.e9 10.1053/j.gastro.2019.11.031 31759926 38. Mihaylova MM Fasting activates fatty acid oxidation to enhance intestinal stem cell function during homeostasis and aging Cell Stem Cell 2018 22 769 778.e4 10.1016/j.stem.2018.04.001 29727683 39. Kaiko GE The colonic crypt protects stem cells from microbiota-derived matabolites Cell 2016 165 1708 1720 10.1016/j.cell.2016.05.018 27264604 40. Benoit Y RGD-dependent epithelial cell–matrix interactions in the human intestinal crypt J. Signal Transduct. 2012 2012 248759 10.1155/2012/248759 22988499 41. Wang Y Bioengineered systems and designer matrices that recapitulate the intestinal stem cell niche Cell. Mol. Gastroenterol. Hepatol. 2018 5 440 453 10.1016/j.jcmgh.2018.01.008 29675459 42. Meran L Intestinal stem cell niche: the extracellular matrix and cellular components Stem Cells Int. 2017 2017 7970385 10.1155/2017/7970385 28835755 43. Jung P Isolation and in vitro expansion of human colonic stem cells Nat. Med. 2011 17 1225 1227 10.1038/nm.2470 21892181 44. Fujii M Human intestinal organoids maintain self-renewal capacity and cellular diversity in niche-inspired culture condition Cell Stem Cell 2018 23 787 793 10.1016/j.stem.2018.11.016 30526881 45. Co JY Controlling epithelial polarity: a human enteroid model for host–pathogen interactions Cell Rep. 2019 26 2509 2520 10.1016/j.celrep.2019.01.108 30811997 46. Serra D Self-organization and symmetry breaking in intestinal organoid development Nature 2019 569 66 72 10.1038/s41586-019-1146-y 31019299 47. Moon C Development of a primary mouse intestinal epithelial cell monolayer culture system to evaluate factors that modulate IgA transcytosis Mucosal Immunol. 2014 7 818 828 10.1038/mi.2013.98 24220295 48. Thorne CA Enteroid monolayers reveal an autonomous WNT and BMP circiut controlling intestinal epithelial growth and organization Dev. Cell 2018 44 624 633 10.1016/j.devcel.2018.01.024 29503158 49. Altay G Self-organized intestinal epithelial monolayers in crypt and cillus-like domains show effective barrier function Sci. Rep. 2019 9 10140 10.1038/s41598-019-46497-x 31300688 50. Liu Y Monolayer culture of intestinal epithelium sustains Lgr5+ intestinal stem cells Cell Discov. 2018 4 32 10.1038/s41421-018-0036-z 29928510 51. Scott A Long-term renewable human intestinal epithelial stem cells as monolayers: a potential for clinical use J. Pediatr. Surg. 2016 51 995 1000 10.1016/j.jpedsurg.2016.02.074 26995514 52. Capeling MM Nonadhesive alginate hydrogels support growth of pluripotent stem cell-derived intestinal organoids Stem Cell Rep. 2019 12 381 394 10.1016/j.stemcr.2018.12.001 53. Yui S YAP/TAZ-dependent reprogramming of colonic epithelium links ECM remodeling to tissue regeneration Cell Stem Cell 2018 22 35 49.e7 10.1016/j.stem.2017.11.001 29249464 54. DiMarco RL Engineering of three-dimensional microenvironments to promote contractile behavior in primary intestinal organoids Integr. Biol. 2014 6 127 142 10.1039/C3IB40188J 55. Broguiere N Growth of epithelial organoids in a defined hydrogel Adv. Mater. 2018 30 1801621 10.1002/adma.201801621 56. Giobbe GG Extracellular matrix hydrogel derived from decellularized tissues enables endodermal organoid culture Nat. Commun. 2019 10 5658 10.1038/s41467-019-13605-4 31827102 57. Lutolf MP Synthetic matrix metalloproteinase-sensitive hydrogels for the conduction of tissue regeneration: engineering cell-invasion characteristics Proc. Natl Acad. Sci. USA 2003 100 5413 5418 10.1073/pnas.0737381100 12686696 58. Lutolf MP Repair of bone defects using synthetic mimetics of collagenous extracellular matrices Nat. Biotechnol. 2003 21 513 518 10.1038/nbt818 12704396 59. Lutolf MP Hubbell JA Synthesis and physicochemical characterization of end-linked poly(ethylene glycol)-co-peptide hydrogels formed by Michael-type addition Biomacromolecules 2003 4 713 722 10.1021/bm025744e 12741789 60. Lutolf MP Cell-responsive synthetic hydrogels Adv. Mater. 2003 15 888 892 10.1002/adma.200304621 61. Yui S Functional engraftment of colon epithelium expanded in vitro from a single adult Lgr5+ stem cell Nat. Med. 2012 18 618 623 10.1038/nm.2695 22406745 62. Gjorevski N Designer matrices for intestinal stem cell and organoid culture Nature 2016 539 560 564 10.1038/nature20168 27851739 63. DiMarco RL Protein-engineered scaffolds for in vitro 3D culture of primary adult intestinal organoids Biomater. Sci. 2015 3 1376 1385 10.1039/C5BM00108K 26371971 64. Kowalski PS Smart biomaterials: recent advances and future directions ACS Biomater. Sci. Eng. 2018 4 3809 3817 10.1021/acsbiomaterials.8b00889 65. Griffin DR Accelerated wound healing by injectable microporous gel scaffolds assembled from annealed building blocks Nat. Mater. 2015 14 737 744 10.1038/nmat4294 26030305 66. Yavitt FM The effect of thiol structure on allyl sulfide photodegradable hydrogels and their application as a degradable scaffold for organoid passaging Adv. Mater. 2020 32 1905366 10.1002/adma.201905366 67. Johnson LA Matrix stiffness corresponding to strictured bowel induces a fibrogenic response in human colonic fibroblasts Inflamm. Bowel Dis. 2013 19 891 903 10.1097/MIB.0b013e3182813297 23502354 68. Hyland LL Using small-angle scattering techniques to understand mechanical properties of biopolymer-based biomaterials Soft Matter 2013 9 10218 10228 10.1039/c3sm51209f 69. Hernandez-Gordillo V Fully synthetic matrices for in vitro culture of primary human intestinal enteroids and endometrial organoids Biomaterials 2020 254 120125 10.1016/j.biomaterials.2020.120125 32502894 70. Hushka EA Relaxation of extracellular matrix forces directs crypt formation and architecture in intestinal organoids Adv. Healthcare Mater. 2020 9 1901214 10.1002/adhm.201901214 71. Cruz-Acuña R Synthetic hydrogels for human intestinal organoid generation and colonic wound repair Nat. Cell Biol. 2017 19 1326 1335 10.1038/ncb3632 29058719 72. Wang L Influence of micro-well biomimetic topography on intestinal epithelial Caco-2 cell phenotype Biomaterials 2009 30 6825 6834 10.1016/j.biomaterials.2009.08.046 19766306 73. Shaffiey SA Intestinal stem cell growth and differentiation on a tubular scaffold with evaluation in small and large animals Regen. Med. 2016 11 45 61 10.2217/rme.15.70 26395928 74. Sung JH Microscale 3-D hydrogel scaffold for biomimetic gastrointestinal (GI) tract model Lab Chip 2011 11 389 392 10.1039/C0LC00273A 21157619 75. Chen Y In vitro enteroid-derived three-dimensional tissue model of human small intestinal epithelium with innate immune responses PLoS One 2017 12 e0187880 10.1371/journal.pone.0187880 29186150 76. Costello CM 3-D Intestinal scaffolds for evaluating the therapeutic potential of probiotics Mol. Pharm. 2014 11 2030 2039 10.1021/mp5001422 24798584 77. Nikolaev M Homeostatic mini-intestines through scaffold-guided organoid morphogenesis Nature 2020 585 574 578 10.1038/s41586-020-2724-8 32939089 78. Guiu J Tracing the origin of adult intestinal stem cells Nature 2019 570 107 111 10.1038/s41586-019-1212-5 31092921 79. Sumigray K Morphogenesis and compartmentalization of the intestinal crypt Dev. Cell 2018 45 183 197 10.1016/j.devcel.2018.03.024 29689194 80. Davis H Aberrant epithelial GREM1 expression initiates colonic tumorigenesis from cells outside the stem cell niche Nat. Med. 2015 21 62 70 10.1038/nm.3750 25419707 81. Haramis A-PG De novo crypt formation and juvenile polyposis on BMP inhibition in mouse intestine Science 2004 303 1684 1686 10.1126/science.1093587 15017003 82. Creff J Fabrication of 3D scaffolds reproducing intestinal epithelium topography by high-resolution 3D stereolithography Biomaterials 2019 221 119404 10.1016/j.biomaterials.2019.119404 31419651 83. Kim SH Three-dimensional intestinal villi epithelium enhances protection of human intestinal cells from bacterial infection by inducing mucin expression Integr. Biol. 2014 6 1122 1131 10.1039/c4ib00157e 84. Wang Y A microengineered collagen scaffold for generating a polarized crypt-villus architecture of human small intestinal epithelium Biomaterials 2017 128 44 55 10.1016/j.biomaterials.2017.03.005 28288348 85. Bhatia SN Ingber DE Microfluidic organs-on-chips Nat. Biotechnol. 2014 32 760 772 10.1038/nbt.2989 25093883 86. Henry OYF Organs-on-chips with integrated electrodes for trans-epithelial electrical resistance (TEER) measurements of human epithelial barrier function Lab Chip 2017 17 2264 2271 10.1039/C7LC00155J 28598479 87. Kimura H An integrated microfluidic system for long-term perfusion culture and on-line monitoring of intestinal tissue models Lab Chip 2008 8 741 746 10.1039/b717091b 18432344 88. Thuenauer R Microfluidic approaches for epithelial cell layer culture and characterisation Analyst 2014 139 3206 3218 10.1039/C4AN00056K 24668405 89. Wang L Chemical and physical modifications to poly(dimethylsiloxane) surfaces affect adhesion of Caco-2 cells J. Biomed. Mater. Res. A 2009 93 1260 1271 90. Huh D Reconstituting organ-level lung functions on a chip Science 2010 328 1662 1668 10.1126/science.1188302 20576885 91. Kim HJ Human gut-on-a-chip inhabited by microbial flora that experiences intestinal peristalsis-like motions and flow Lab Chip 2012 12 2165 2174 10.1039/c2lc40074j 22434367 92. Shin W Human intestinal morphogenesis controlled by transepithelial morphogen gradient and flow-dependent physical cues in a microengineered gut-on-a-chip iScience 2019 15 391 406 10.1016/j.isci.2019.04.037 31108394 93. Chi M A microfluidic cell culture device (μFCCD) to culture epithelial cells with physiological and morphological properties that mimic those of the human intestine Biomed. Microdevices 2015 17 9966 10.1007/s10544-015-9966-5 26002774 94. Pocock K Intestine-on-a-chip microfluidic model for efficient in vitro screening of oral chemotherapeutic uptake ACS Biomater. Sci. Eng. 2017 3 951 959 10.1021/acsbiomaterials.7b00023 95. Kasendra M Development of a primary human small intestine-on-a-chip using biopsy-derived organoids Sci. Rep. 2018 8 2871 10.1038/s41598-018-21201-7 29440725 96. Kasendra M Duodenum intestine-chip for preclinical drug assessment in a human relevant model Elife 2020 9 e50135 10.7554/eLife.50135 31933478 97. Workman MJ Enhanced utilization of induced pluripotent stem cell–derived human intestinal organoids using microengineered chips Cell. Mol. Gastroenterol. Hepatol. 2018 5 669 677 10.1016/j.jcmgh.2017.12.008 29930984 98. Kim HJ Contributions of microbiome and mechanical deformation to intestinal bacterial overgrowth and inflammation in a human gut-on-a-chip Proc. Natl Acad. Sci. USA 2016 113 E7 E15 10.1073/pnas.1522193112 26668389 99. Shin W Kim HJ Intestinal barrier dysfunction orchestrates the onset of inflammatory host-microbiome cross-talk in a human gut inflammation-on-a-chip Proc. Natl Acad. Sci. USA 2018 115 E10539 E10547 10.1073/pnas.1810819115 30348765 100. Jalili-Firoozinezhad S A complex human gut microbiome cultured in an anaerobic intestine-on-a-chip Nat. Biomed. Eng. 2019 3 520 531 10.1038/s41551-019-0397-0 31086325 101. Shin W A robust longitudinal co-culture of obligate anaerobic gut microbiome with human intestinal epithelium in an anoxic-oxic interface-on-a-chip Front. Bioeng. Biotechnol. 2019 7 13 10.3389/fbioe.2019.00013 30792981 102. Villenave R Human gut-on-a-chip supports polarized infection of Coxsackie B1 virus in vitro PLoS One 2017 12 e0169412 10.1371/journal.pone.0169412 28146569 103. Jalili-Firoozinezhad S Modeling radiation injury-induced cell death and countermeasure drug responses in a human gut-on-a-chip Cell Death Dis. 2018 9 223 10.1038/s41419-018-0304-8 29445080 104. Knip M Siljander H The role of the intestinal microbiota in type 1 diabetes mellitus Nat. Rev. Endocrinol. 2016 12 154 167 10.1038/nrendo.2015.218 26729037 105. Ni J Gut microbiota and IBD: causation or correlation? Nat. Rev. Gastroenterol. Hepatol. 2017 14 573 584 10.1038/nrgastro.2017.88 28743984 106. Trietsch SJ Membrane-free culture and real-time barrier integrity assessment of perfused intestinal epithelium tubes Nat. Commun. 2017 8 262 10.1038/s41467-017-00259-3 28811479 107. Czerwinski, M. et al. In vitro and in vivo development of the human intestinal niche at single cell resolution. Preprint at http://bioRxiv.org2020.01.31.928788. 108. Jardé T Mesenchymal niche-derived Neuregulin-1 drives intestinal stem cell proliferation and regeneration of damaged epithelium. Cell Stem Cell 2020 27 646 662 10.1016/j.stem.2020.06.021 32693086 109. Gregorieff A Yap-dependent reprogramming of Lgr5+ stem cells drives intestinal regeneration and cancer Nature 2015 526 715 718 10.1038/nature15382 26503053 110. Pomerat C Quantitative cine analysis of cell organoid activity Ann. NY Acad. Sci. 1954 58 1311 1321 10.1111/j.1749-6632.1954.tb45911.x 14350520 111. Foulds L The histologic analysis of mammary tumors of mice. III. Organoid tumors J. Natl. Cancer Inst. 1956 17 755 781 112. Moscona A Development of heterotypic combinations of dissociated embryonic chick cells Proc. Soc. Exp. Biol. Med. 1956 92 410 416 10.3181/00379727-92-22495 13350365 113. Mustata R Identification of Lgr5-independent spheroid-generating progenitors of the mouse fetal intestinal epithelium Cell Rep. 2013 5 421 432 10.1016/j.celrep.2013.09.005 24139799 114. Hannan NRF Generation of multipotent foregut stem cells from human pluripotent stem cells Stem Cell Rep. 2013 1 293 306 10.1016/j.stemcr.2013.09.003 115. Costello CM Synthetic small intestinal scaffolds for improved studies of intestinal differentiation Biotechnol. Bioeng. 2014 111 1222 1232 10.1002/bit.25180 24390638 116. Kim W Kim GH Intestinal villi model with blood capillaries fabricated using collagen-based bioink and dual-cell-printing process ACS Appl. Mater. Interfaces 2018 10 41185 41196 10.1021/acsami.8b17410 30419164