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Microinjection intestinal organoids


I am attempting to microinject small intestinal organoids, using the Eppendorf FemtoJet 4i microinjector and Femtotip capillaries, and have come across several issues. This is a new set up in our lab, and I am first to attempt it so I apologise for the basic questions.

When attempting to fill the Femtotip capillaries using a needle or Western blot tip the liquid backs up and does not fill the capillary, leaving a large air bubble. Is this a common issue with the Femtotip capillaries? Are there other methods for filling (that do not require specialised microloader)?

When I attempted to use a tip where I had let the liquid be pulled to bottom by gravity, I had issues with leakage out of the tip bottom. Just to be absolutely clear, is it necessary for the entire tip to be filled with liquid prior to injection (no air bubbles)?

Finally I would be grateful for any advice on injection pressure and time used for organoids.

Thank you


I found that using the Femtotip loaders gives a continuous block of liquid, which can then be moved to the end of the tip by injecting several times. It is fine to have a air above the liquid at the tip.


Human intestinal models to study interactions between intestine and microbes

Instituto Mexicano del Seguro Social, Unidad de Investigación Médica en Medicina Reproductiva, Unidad Médica de Alta Especialidad en Ginecología y Obstetricia No. 4 ‘Dr. Luis Castelazo Ayala’, Av. Río Magdalena No. 289, Col. Tizapán San Ángel, C.P. 01090 Ciudad de México, México

Institut Pasteur, Unité d'Analyse d'Images Biologiques, 25 Rue du Dr Roux, 75015 Paris, France

Institut Pasteur, Unité d'Analyse d'Images Biologiques, 25 Rue du Dr Roux, 75015 Paris, France

Centre National de la Recherche Scientifique, UMR3691, 25 Rue du Dr Roux, 75015 Paris, France

Institut Pasteur, Unité d'Analyse d'Images Biologiques, 25 Rue du Dr Roux, 75015 Paris, France

Centre National de la Recherche Scientifique, ERL9195, 25 Rue du Dr Roux, 75015 Paris, France

Instituto Mexicano del Seguro Social, Unidad de Investigación Médica en Medicina Reproductiva, Unidad Médica de Alta Especialidad en Ginecología y Obstetricia No. 4 ‘Dr. Luis Castelazo Ayala’, Av. Río Magdalena No. 289, Col. Tizapán San Ángel, C.P. 01090 Ciudad de México, México

Institut Pasteur, Unité d'Analyse d'Images Biologiques, 25 Rue du Dr Roux, 75015 Paris, France

Institut Pasteur, Unité d'Analyse d'Images Biologiques, 25 Rue du Dr Roux, 75015 Paris, France

Centre National de la Recherche Scientifique, UMR3691, 25 Rue du Dr Roux, 75015 Paris, France

Institut Pasteur, Unité d'Analyse d'Images Biologiques, 25 Rue du Dr Roux, 75015 Paris, France

Centre National de la Recherche Scientifique, ERL9195, 25 Rue du Dr Roux, 75015 Paris, France


NOMENCLATURE FOR THREE-DIMENSIONAL INTESTINAL CULTURES

The term “organoid” was first used in 1987 to describe in vitro cultures derived from neuroblastomas (46) and lung (134). Short-term intestinal cultures were first described by Evans et al. (25), who defined an intestinal organoid as a structure of crypts and villi growing in vitro. Sato and Clevers described three-dimensional cultures of intestinal stem cells (ISCs) in 2009 as “organoids” and have continued to refer to purely epithelial cultures as organoids (95a). In the same year, Ootani and Kuo reported three-dimensional cultures of intestinal fragments, which they also referred to organoids (65). In 2012, the Intestinal Stem Cell Consortium published nomenclature guidelines to distinguish between types of intestinal cultures (109). The authors of this work suggest the term “organoid” for cultures that contain multiple cell types, including epithelial and mesenchyme, while cultures of pure epithelial populations are designated as “enteroids” or “colonoids,” if derived from the small intestine or colon, respectively. The term “spheroid” has also been used to denote epithelial three-dimensional cultures (76). In this review, all three-dimensional organ structures are referred to as “organoids”, and qualified by tissue location, species, and type of tissue (e.g., “colon tumor organoid”).


Approaches for promoting organoid maturation

Organoids exhibit restricted growth in vitro as they must rely on diffusion of oxygen and exogenous nutrients to survive. In addition, many hPSC-derived organoid models more closely resemble an immature tissue and fail to mature regardless of duration in culture. However, transplantation of organoids into various sites in mammalian hosts have resulted in organoid maturation and increased tissue functionality (Fig. 2). Organoids have been transplanted both orthotopically (i.e. into the analogous in vivo location) and ectopically (i.e. in a region outside of the native in vivo environment) and both transplantation methods are permissive for organoid maturation (Dye et al., 2016 Finkbeiner et al., 2015b Sugimoto et al., 2018 Takebe et al., 2013 van den Berg et al., 2018). These studies have demonstrated that organoid models are capable of maturing when provided with additional cues from the in vivo environment, thereby increasing their appeal to study human organogenesis and for use as pre-clinical models or in regenerative medicine. Here, we discuss methods to improve organoid complexity and maturation through transplantation in vivo.

Orthotopic and ectopic transplantation of human organoids. Both hPSC and primary tissue has been transplanted into immunocompromised mice. The colors represent different types of organoids, both primary and/or hPSC-derived, that have been transplanted into the indicated locations. Orthotopic transplantation refers to transplantation into the analogous in vivo location, whereas ectopic transplantation refers to transplantation into a region outside of the native in vivo environment (i.e. beneath kidney capsules, within fat pads).

Orthotopic and ectopic transplantation of human organoids. Both hPSC and primary tissue has been transplanted into immunocompromised mice. The colors represent different types of organoids, both primary and/or hPSC-derived, that have been transplanted into the indicated locations. Orthotopic transplantation refers to transplantation into the analogous in vivo location, whereas ectopic transplantation refers to transplantation into a region outside of the native in vivo environment (i.e. beneath kidney capsules, within fat pads).

Ectopic transplantation of organoids

Many experiments have transplanted organoids into immunocompromised mice for maturation. Typically, organoids were transplanted into highly vascularized sites that are amenable for organoid engraftment and growth for up to several months, without obstructing necessary murine organ function. Such sites include epididymal fat pads (Dye et al., 2016) and underneath kidney capsules (Finkbeiner et al., 2015a,b Múnera et al., 2017 Trisno et al., 2018 Tsai et al., 2017 Watson et al., 2014 Workman et al., 2016). Indeed, the complexity of co-cultures described earlier can be further enhanced by transplantation (Schlieve et al., 2017 Takebe et al., 2013 Workman et al., 2016).

HIOs are an example of an organoid model that has shown significant maturation after kidney capsule transplantation for several weeks (Finkbeiner et al., 2015b Watson et al., 2014). Both epithelium and mesenchyme of transplanted HIOs (tHIOs) displayed enhanced organization, with the emergence of a properly patterned villus-crypt architecture within the epithelium that was not apparent before transplantation. Further, smooth muscle actin (SMA)- and platelet derived growth factor receptor alpha (PDGFRA)-expressing mesenchymal lineages became organized in a manner that was indistinguishable from the native human intestine (Finkbeiner et al., 2015b). Vascularization by mouse blood vessels supported significant growth of transplanted HIOs up to 100-fold in size (Watson et al., 2014). Recently, HIOs were successfully transplanted into the intestinal mesentery and demonstrated similar growth and maturation hallmarks as those transplanted beneath the kidney capsule (Cortez et al., 2018 Finkbeiner et al., 2015a). The intestinal mesentery is therefore a complementary transplantation site that may be more physiologically relevant for future translational studies owing to its close proximity to the native gut.

hPSC-derived lung organoids (HLOs) are another in vitro model that has shown improved tissue architecture and maturation upon transplantation. Interestingly, unlike several other organoid models, HLOs did not survive transplantation beneath the kidney capsule (Dye et al., 2016). Rather, a microporous polylactide-co-glycolide (PLG) scaffold, which is commonly used to transplant pancreatic beta cells (Gibly et al., 2011 Hlavaty et al., 2014), was used to support HLO transplantation. The PLG scaffold served as a physical niche to support survival and maturation of transplanted HLOs, which displayed enhanced epithelial organization, morphology and differentiation into proximal airway (i.e. trachea/bronchi) lineages that more closely resembled the mature human proximal airways than HLOs grown in vitro. This result suggested that in vivo cues are required, along with the scaffold, to support HLO maturation. Although alveolar lineages were present in the in vitro gown HLOs, alveolar cell types and structures were not supported in transplanted HLOs (Dye et al., 2015, 2016). Notably, HLOs must be transplanted within the epididymal fat pad, because the kidney capsule compartment was not large enough to accommodate the scaffold. Despite the difference in transplantation location, similar to intestinal organoids, HLOs were extensively vascularized by host vessels and both the epithelial and mesenchymal components matured after in vivo growth (Dye et al., 2016 Finkbeiner et al., 2015b Watson et al., 2014). This study demonstrated the requirement of a physical niche to support survival, differentiation and maturation of transplanted HLOs.

These studies demonstrated that vascularization is key for engraftment, growth and survival of transplanted organoid tissues, but vascularization may be slow and/or inefficient for organoid systems that do not possess vasculature before transplantation. Liver bud organoids with pre-existing endothelial cells (HUVECs) engrafted and established blood flow within only a few days (Takebe et al., 2013). In addition, transplantation into a cranial window allowed for live imaging of organoids and demonstrated that HUVECs formed a stable vascular connection with the host.

Data suggests that most organoid models do not inherently contain vasculature however, endogenous vascular cells are present within hPSC-derived kidney organoids. This likely resulted from the directed differentiation approach, which pushes hPSCs into an intermediate mesoderm fate, generating nephrogenic lineages as well as endothelial cells (Freedman et al., 2015 Takasato et al., 2015, 2016). After transplantation beneath the kidney capsule, kidney organoids became extensively vascularized by both host and organoid endothelial cells that persisted for at least 2 weeks of transplantation (van den Berg et al., 2018). Moreover, use of an abdominal imaging window allowed for live imaging of the organoids throughout transplantation. Infusion of fluorescent dextran in the blood allowed the visualization of blood flow and helped to demonstrate that glomerular structures within the kidney organoid, which function to filter urine from the blood, became vascularized. This led to improved glomerular maturation, including deposition of glomerular basement membrane and a fenestrated endothelium within the transplanted organoids.

Orthotopic transplantation of organoids

Although ectopic transplantation is a valuable tool to allow for organoid maturation, there is interest in orthotopically transplanting organoids or organoid-derived cell types to advance our understanding of in vivo niches in regulating organoid maturation, as well as increasing the translational potential of organoid models for organ replacement therapies. Injury models have been used across various organ systems to create niches that are permissive for both primary and hPSC-derived organoid engraftment in physiologically relevant sites in adult mouse models (Huch et al., 2015 Miller et al., 2018 Sampaziotis et al., 2017 Sugimoto et al., 2018). For example, recent efforts have successfully orthotopically transplanted human primary tissue-derived colonic epithelial organoids into the murine colon after disruption of the endogenous epithelium (Sugimoto et al., 2018). Here, it was demonstrated that a significant area of injury was required to create a niche large enough to promote engraftment and persistence of human tissue over several months. Smaller injuries also allowed for engraftment, but ultimately engrafted human tissue was lost over time. Lineage tracing of the intestinal stem cells using a genome engineered intestinal stem cell-specific LGR5-CreER and a lineage reporter in human colonic organoids revealed important species-specific differences in cycling time between mouse and human colonic stem cells (Sugimoto et al., 2018). This insight into the intestinal stem cell niche would not have been possible with ectopic transplantation.

Similar injury-engraftment approaches have been used to introduce hPSC-derived human lung bud tip epithelial progenitor organoids into an injured adult mouse lung (Miller et al., 2018 Miller et al., 2019). Human lung bud tip progenitors can engraft into the trachea and proximal airways of the mouse lung following chemically induced bronchiolar injury. Moreover, these cells demonstrated multi-lineage differentiation potential into several proximal lineage phenotypes including mucus-producing cells, ciliated cells and neuroendocrine cells after 6 weeks post injury. Notably, these progenitors showed in vitro alveolar differentiation capabilities, but such lineages were not observed, likely because cells did not engraft in alveolar areas in which there was no injury in this particular injury model. This result highlights the importance of the in vivo microenvironment in mediating organoid progenitor differentiation (Miller et al., 2018). Finally, extrahepatic cholangiocyte primary tissue-derived organoids seeded on scaffolds were able to engraft into the epithelium of the gallbladder and common bile ducts to functionally rescue mouse models of extrahepatic biliary injury (Sampaziotis et al., 2017).

Human hPSC-derived cerebral organoids have also been successfully transplanted into the cerebrum of adult mice with robust organoid engraftment and host survival rates, despite the invasiveness of the procedure (Mansour et al., 2018). In comparison with age-matched in vitro-grown cerebral organoids, transplanted cerebral organoids contained a higher proportion of mature neuronal cell fates that included astrocytes and oligodendrocytes, compared with relatively immature neuronal precursors observed in vitro. In addition, axon projections were shown to invade various regions of the host brain, and marker analysis suggested that these human organoid-derived axons were forming synaptic connections with the murine neurons. Initial studies demonstrated that transplanted cerebral organoids responded to external calcium stimulation in a synchronized pattern most similar to a developing, rather than mature, brain. Therefore, although this was an impressive advancement in cerebral organoids, the current transplantation approaches result in only partial maturation (Mansour et al., 2018). Efforts have also derived brain organoids with endothelial cells from hPSCs that could be co-cultured in vitro for up to 5 weeks and transplanted into a murine host for at least 2 weeks, demonstrating proof-of-principle for generating human vasculature in transplanted brain organoids (Pham et al., 2018).

Collectively, the establishment of orthotopic transplantation approaches enables the study of organoid maturation within the native tissue microenvironment, and at the same time brings researchers a step closer towards translational applications for organoid technologies. In both ectopic and orthotopic approaches, it has been challenging to identify the exact factors in the in vivo host environment that are responsible for organoid maturation. Moving forward it will be important to begin tackling these in vivo interactions in order to apply principles of maturation to create mature organoids in the dish. To address these questions, future experiments could generate organoids from a CRISPR knockout library or screen a small molecule library to independently assess factors in maturation.

Engineering-transplantation approaches to increase organoid maturity

Intestinal organoids are a promising source of replacement tissue for conditions such as short bowel syndrome, but clinical implementation will require increases in size and maturity to produce functional intestinal tissue. One approach to address this issue employed HIOs that were seeded on scaffolds to produce tissue-engineered small intestine (TESI) (Finkbeiner et al., 2015a). HIOs were seeded on acellular porcine intestinal matrices as well as synthetic scaffolds comprising polyglycolic/poly L lactic acid (PGA/PLLA). Using this approach, HIOs successfully reseeded the acellular matrices in vitro but did not persist or retain intestinal identity when transplanted into immunocompromised mice. The poor performance of acellular matrices could be due to removal of instructive cues during decellularization or blockage of vasculature infiltration to HIOs by the matrix. However, HIOs that were seeded on the synthetic matrices thrived in vivo and exhibited epithelial maturation, resulting in tissue that resembled the adult intestine. These tubular-shaped scaffolds are a promising approach to seed multiple HIOs and generate longer, more mature intestinal tissue in vitro. Although the TESI described here lacked key intestinal cell types such as an enteric nervous system, this can be overcome as described above (Schlieve et al., 2017 Workman et al., 2016).

A more recent approach to generate scalable, mature intestinal tissue from HIOs incorporated mechanical strain using lengthening springs (Poling et al., 2018). Mechanical strain has been shown to play a role in intestinal development (Kurpios et al., 2008 Savin et al., 2011 Shyer et al., 2015, 2013), and spring or stretch-based lengthening devices have been designed as treatment methods for short bowel syndrome (Demehri et al., 2015, 2014 Ralls et al., 2012 Rouch et al., 2016 Stark et al., 2012 Sueyoshi et al., 2013). In this approach, HIOs were transplanted into immunocompromised mice for 10 weeks. After this time, compressed nitinol springs were surgically inserted into the transplanted HIOs for an additional 14 days. The transplanted HIOs that had undergone strain exhibited increased intestinal length, larger villi and deeper crypts and appeared to be more mature based on global gene signatures, compared with transplanted HIOs without springs. These studies highlight bioengineering approaches to promote clinical applications of HIOs and demonstrate the importance of both biological and mechanical cues in promoting organoid maturation.


Microinjection intestinal organoids - Biology

This code is associated with the paper from Hill et al., "Bacterial colonization stimulates a complex physiological response in the immature human intestinal epithelium". eLife, 2017. http://dx.doi.org/10.7554/eLife.29132

Real-time measurement of epithelial barrier permeability in human intestinal organoids

David R. Hill 1# , Sha Huang 1 , Yu-Hwai Tsai 1 , Jason R. Spence 1,2 , and Vincent B. Young 1,3,4 ,

1 Department of Internal Medicine, Division of Gastroenterology, University of Michigan, Ann Arbor MI 48109 2 Department of Cell and Developmental Biology, University of Michigan, Ann Arbor MI 48109 3 Department of Internal Medicine, Division of Infectious Disease, University of Michigan, Ann Arbor MI 48109 4 Department of Microbiology and Immunology, University of Michigan, Ann Arbor MI 48109

# author for correspondence: David R. Hill ([email protected])

This protocol describes the measurement of epithelial barrier permeability in real-time following pharmacologic treatment in human intestinal organoids using fluorescent microscopy and live cell microscopy

Advances in 3D culture of intestinal tissues obtained through biopsy or generated from pluripotent stem cells via directed differentiation have resulted in sophisticated in vitro models of the intestinal mucosa. Leveraging these emerging model systems will require adaptation of tools and techniques developed for 2D culture systems and animals. Here, we describe a technique for measuring epithelial barrier permeability in human intestinal organoids in real-time. This is accomplished by microinjection of fluorescently-labeled dextran and imaging on an inverted microscope fitted with epifluorescent filters. Real-time measurement of the barrier permeability in intestinal organoids facilitates the generation of high-resolution temporal data in human intestinal epithelial tissue, although this technique is can also be applied to fixed timepoint imaging approaches. This protocol is readily adaptable for the measurement of epithelial barrier permeability following exposure to phamacologic agents, bacterial products or toxins, or live microorganisms. With minor modifications, this protocol can also serve as a general primer on microinjection of intestinal organoids and users may choose to supplement this protocol with additional or alternative downstream applications following microinjection.

A Stem-cell derived human intestinal organoids (HIO) microinjected with 2 mg/ml FITC-dextran (4 kDa) imaged for 20 hours. HIOs were also microinjected with PBS (control) or the Clostridium difficile toxin TcdA (12.8 ng/μl) or treated with 2 mM EGTA added to the external culture media. 4X Magnification. B Plot of mean normalized FITC intensity over time in HIOs treated with PBS (control), TcdA, or EGTA. Error bars represent S.E.M and N = 4-6 HIOs per group.

This protocol is on GitHub for a reason. We invite you to ask questions, make suggestions, and engage with the science presented here. Use the GitHub "Issues" feature to post questions and comments that you want to make available for public discussion. Contact the authors if you would like to be added as a contributor to this repository. Submit a pull request. Or fork this repository and make your own changes

We suspect that the majority of users will want to simply download the PDF version of the manuscript. Go for it!

If you are interested in understanding the inner workings of the code for conducting the analysis and generating the paper (have you heard of this thing called "Reproducible Research"?), you may want to try cloning this repository to your computer and compiling the paper.

To compile the paper from source, you will need the following tools:

Then navigate to your local clone of this repository and type the following in the command prompt:

Hill, D. R., Huang, S., Tsai, Y. H., Spence, J. R., Young, V. B. Real-time Measurement of Epithelial Barrier Permeability in Human Intestinal Organoids. J. Vis. Exp. (), e56960, doi:10.3791/56960 (2017).


Screening of Intestinal Crypt Organoids: A Simple Readout for Complex Biology

Oral and intestinal mucositis is a debilitating side effect of radiation treatment. A mouse model of radiation-induced mucositis leads to weight loss and tissue damage, reflecting the human ailment as it responds to keratinocyte growth factor (KGF), the standard-of-care treatment. Cultured intestinal crypt organoids allowed the development of an assay monitoring the effect of treatments of intestinal epithelium to radiation-induced damage. This in vitro assay resembles the mouse model as KGF and roof plate-specific spondin-1 (RSPO1) enhanced crypt organoid recovery following radiation. Screening identified compounds that increased the survival of organoids postradiation. Testing of these compounds revealed that the organoids changed their responses over time. Unbiased transcriptome analysis was performed on crypt organoid cultures at various time points in culture to investigate this adaptive behavior. A number of genes and pathways were found to be modulated over time, providing a rationale for the altered sensitivity of the organoid cultures. This report describes an in vitro assay that reflects aspects of human disease. The assay was used to identify bioactive compounds, which served as probes to interrogate the biology of crypt organoids over prolonged culture. The pathways that are changing over time may offer potential targets for treatment of mucositis.

Keywords: KGF RSPO mucositis primary 3D cell cultures small intestinal crypt organoids.


Standardisation Remains the Major Challenge for In Vitro Inflammation Models

As outlined, enormous advances in the stem cell and organoid field have emerged and their potential in translational research, and even healthcare, is obvious however, current limitations of organoids remain an important caveat (Figure 2B).

A general limitation of organoid derivation is the high variability of the phenotypes that they can produce. Organoid and stem cell culture systems and their molecular outcome are critically dependent on the quality and properties of the sampled tissue. Variations in the pre-processing phase heavily interfere with immune signatures of these samples. Moreover, inflamed tissue is more likely to undergo cell death, might have different nutrient and culturing needs and thus the recovery rate of vital organoids from inflamed tissue is significantly lower (60). Whilst inflammatory signatures ex vivo are similar to their in vivo origin (9), particularly complex diseases such as IBD are characterised by a diversity of inflammatory flavours. Furthermore, biopsies are reflecting the biology of the sampled region, and therefore stem cells/organoids should be harvested from different regions. When using iPSCs, variations exist in organoids depending on the genetic background of the individual and the culture protocol used by the lab. Isogenic lines generated via gene-editing approaches can limit this variability in regard to genetic differences.

Because organoids from iPSCs are formed through differentiation of a homogeneous population, tissue-specific cell types and their microenvironment must be newly created. This challenges the use of iPSCs and despite significant similarities in structure and function between organoid and adult tissue, organoids often retain immature characteristics, making them more similar to foetal tissue. In an example of iPSC-derived intestinal organoid, this limitation could be resolved by either co-culturing it with T cells providing a more realistic growth niche (61), engineering of more sophisticated 3D scaffolds to improve organoid architecture and thus increase the similarity to the original tissue in vivo (62, 63), or in vivo transplantation, which resulted in mature intestinal epithelium with preserved intestinal stem cell niches, crypt/villus architecture and a laminated human mesenchyme, both supported by mouse vascular ingrowth (64). Apart from immature characteristics, organoids often show a limited lifespan once a certain size is reached. Due to the lack of diffusion, cells are not supplied with sufficient nutrients to support continued development. The use of bioreactors could improve the nutrient supply. Ideally, organoids are engineered towards the induction of endothelium development resulting in vascularization as shown recently (41). Another strategy would be to integrate endothelial cells, or their progenitors, during organoid development, and to include bioprinting methods to design 3D-scaffolds for the endothelial cells (65, 66). However, the original material tissue of iPSC-derived organoids is usually not inflamed, e.g. skin fibroblasts, and have been reprogrammed and cultured for many weeks to months in the absence of an inflammatory milieu. For the study of complex, multifactorial inflammation, iPSC-derived systems are therefore not the first choice in terms of a model. Vice versa, iPSC-derived organoids might be excellent tools to study mainly genetically driven inflammatory disorders, such as monogenic IBD.

Another important challenge is the magnitude of effort, time and expenses spent on organoid cultures. Whilst rather simple protocols are available for conventional organoid cultures at manageable costs, differentiation protocols include either expensive factors or culturing media or require conditioned media, which need to be produced by feeder cell lines. Co-culturing systems often include 2D models, which require much higher culturing efforts and time. Adding to this challenge, the generation of iPSC and their differentiation into specific tissue often require several weeks of intense culturing effort, which increases the risk of adverse events, e.g. contamination or undesired differentiation.

To combat the aforementioned issues, close collaboration of academic research and high-tech industrial partners may be a promising strategy to overcome the infrastructural challenges. Whilst academia can provide problem-derived ideas and a hypothesis-driven vision of novel disease models, industry can deliver the necessary technology, capacities for large-scale production and standardisation. Setting up these collaborative infrastructures are necessary and can foster future advances of stem cell technology.


Intestinal organoids for modelling intestinal development and disease

Gastrointestinal diseases are becoming increasingly prevalent in developed countries. Immortalized cells and animal models have delivered important but limited insight into the mechanisms that initiate and propagate these diseases. Human-specific models of intestinal development and disease are desperately needed that can recapitulate structure and function of the gut in vitro. Advances in pluripotent stem cells and primary tissue culture techniques have made it possible to culture intestinal epithelial cells in three dimensions that self-assemble to form ‘intestinal organoids'. These organoids allow for new, human-specific models that can be used to gain insight into gastrointestinal disease and potentially deliver new therapies to treat them. Here we review current in vitro models of intestinal development and disease, considering where improvements could be made and potential future applications in the fields of developmental modelling, drug/toxicity testing and therapeutic uses.

This article is part of the theme issue ‘Designer human tissue: coming to a lab near you'.

1. Intestinal structure and function

The intestines are a vital organ derived from definitive endoderm and can be subdivided into the small and large intestines [1]. Collectively, the small and large intestines perform the vital function of digestion, nutrient absorption and waste elimination [2]. The small intestine contains several functionally distinct areas including the duodenum, jejunum and ilium, and its surface comprises a highly folded epithelium of villi, microvilli and intestinal crypts [3]. This intricate folding of villi and microvilli serves to dramatically increase the total surface area of the small intestine, thereby facilitating greater nutrient absorption. The intestinal crypt functions as the niche in which the LGR5 + stem cells reside [4]. LGR5 + cells are relatively slow cycling cells and give rise to a highly proliferative and multipotent progenitor population known as the transit-amplifying (TA) cells, which differentiate as they migrate from the crypt towards the villi. By the time TA cells have moved out of the crypt, they are fully differentiated into one of the many cell types required for normal functionality of the epithelium [5].

The large intestine comprises four main regions: the ascending, transverse, descending and sigmoid colon [6]. While most digestion occurs in the small intestine, the large intestine functions to absorb water and ions, in addition to vitamins and short chain fatty acids (SCFA) synthesized by commensal bacteria. Production of SCFA by commensal bacteria in the large intestine is recognized as an important feature of maintaining normal gut health and has been shown to have several protective effects including creating an environment hostile to pathogenic microbes, providing an energy source for intestinal epithelial cells and enhancing mucus production [7].

2. Cells of the small and large intestine

The intestinal epithelium comprises several different cell types including enterocytes, goblet cells, Paneth cells and enteroendocrine cells, all of which are derived from the resident LGR5 + stem cells found at the base of intestinal crypts [8] (figure 1). Epithelial cells are critical in maintaining immune homeostasis within intestinal tissue, closely regulating the interplay between the intestinal mesenchyme, and commensal and pathogenic bacteria. They maintain a barrier formed with tight junctions, essential to the control of macromolecular transport and immune homeostasis. Enterocytes are the most common cell type found in the epithelium, and they mainly function in nutrient absorption. Secretory epithelial cells consist of Paneth and goblet cells and multiple enteroendocrine cell types, with functions ranging from cytokine to hormone production [9].

Figure 1. Anatomy of the small and large intestine. The small intestine is composed of repetitive villus and crypt structures. LGR5 + stem cells and Paneth cells are located at the base of the crypts, followed by the TA cells, and then mature epithelium composed of goblet cells, enteroendocrine cells and enterocytes (a). The large intestine lacks the villi structures of the small intestine and instead is composed of colonic crypts. At the base of colonic crypts are LGR5 + stem cells and Reg4 + cells, followed by TA cells and mature epithelium that contains a high proportion of goblet cells (b).

Goblet cells play a critical role in the functioning and protection of the intestinal tract. There are nearly twice as many goblet cells in the colon as in the small intestine, which is in proportion to the increase in bacteria [10]. Goblet cells function by producing glycoproteins known as mucins, which are vital constituents of the mucus that lines the epithelium. This mucosal layer acts as the first line of defence, preventing the bacteria present in the gut from being in direct contact with the intestinal epithelium [11]. Mucus production is increased in response to inflammatory cytokines including interferon-γ and interleukins-9, -10 and -13 [12]. The glycoproteins that comprise this mucus are toxic to many strains of bacteria. Additionally, this layer acts as a matrix to which defensins and antibodies adhere that target specific pathogenic bacteria adhere. When mucus production is hindered, this can cause commensal bacteria in the gut microbiota to penetrate the epithelial barrier, triggering an immune response [13]. Goblet cells also produce other constituents of the mucus layer, including trefoil factors that stabilize the mucus layer by forming cross-links between different components of the mucus [14]. This cross-linking creates a unique property in that the innermost layer is thicker and more viscous while the outer layer is more aqueous, allowing commensal bacteria to reside there.

The small and large intestine is a high turnover organ, with complete epithelial renewal approximately every 7 days. This process is driven by LGR5 + intestinal stem cells (figure 1a,b). The pioneering work of Hans Clevers demonstrated that LGR5 + stem cells reside at the base of the intestinal crypts that form the intestinal stem-cell niche [8,15]. LGR5 + cells are crucial for the continual renewal of the intestinal epithelium and undergo asymmetric division approximately every 24 h, giving rise to a daughter stem cell and a TA cell. These rapidly proliferating TA cells begin in the TA zone and migrate up the intestinal crypt, while dividing four to five times along the way, before terminally differentiating into any of the intestinal cell types [16]. Once differentiated, these cells continue to migrate upwards towards the top of the crypts and have a lifespan of approximately 7 days. Once these cells have reached the top of the villi, they typically undergo anoikis and are shed into the intestinal lumen.

Paneth are also generated from the asymmetrical division of the LGR5 + stem cell [16]. However, instead of migrating upwards, Paneth cells migrate downwards into the base of the crypt. Paneth cells are a longer-lived cell type, surviving for approximately 30 days at the base of intestinal crypts where they play an integral role in producing and maintaining the stem-cell niche [17,18]. In addition to maintaining the niche, Paneth cells secrete a substantial amount of antimicrobrial peptides such as defensins that help regulate intestinal microbiota and protect against invading pathogens.

Paneth cells are absent in the majority of colonic crypts. However, secretory cells expressing typical Paneth cell markers, including CD24 are present. Reg4 has been found to be a reliable marker of these cells, residing adjacent to LGR5 + stem cells in all colonic crypts [19]. These cells have been shown to secrete epidermal growth factor (EGF) and the Notch ligands DII1 and DII4, while LGR5 + stem cells were found to express the corresponding receptors [19]. These secretory Reg4 + cells were found to more closely resemble Paneth cells, rather than goblet cells found in either the small or large intestine. Reg4 + cells are integral to controlling stem-cell positioning in the crypt and for maintaining stemness of LGR5 + colonic stem cells. These cells have many distinct similarities with Paneth cells of the small intestine, with the exception that Reg4 + cells do not produce Wnt ligands, while colonic LGR5 + stem cells do express Wnt receptors [19]. Therefore, mesenchymal tissue surrounding the colonic crypts, known to produce Wnt ligands, may provide the necessary Wnt required for stem-cell maintenance in the intestinal crypt in vivo [20].

3. Cell signalling in the intestinal epithelium

Self-renewal and differentiation of LGR5 + stem cells are achieved via tight regulation of several signalling pathways within the intestinal crypts. Wnt signalling is considered the central signalling pathway maintaining LGR5 + stem-cell proliferation, controlling cell positioning within the crypt and activating terminal differentiation of Paneth cells. Dysregulation of this pathway has been shown to be important in the development of some colon cancers [21].

Higher concentrations of Wnt3a and EGF are present at the base of the crypts and are required for stem-cell maintenance and proliferation, respectively [22] (figure 2a). A concentration gradient of Wnt3a is established in the intestinal crypt, with highest levels found in the base of the crypt that then decrease towards the top of the crypt [23]. This concentration gradient along with a decrease in Notch signalling and increased BMP signalling at the top of the crypts facilitates the initial differentiation of the LGR5 + stem cells along with the proliferation and terminal differentiation of the TA cells [24]. Paneth cells are integral in producing the crypt environment via secretion of several factors, including Wnt3a, EGF, transforming growth factor α (TGFα) and the Notch ligand D114 [18].

Figure 2. Regulation of stemness in the intestinal stem-cell niche. During normal maintenance of the stem-cell niche several signalling gradients are established that either promote stemness (Wnt) or differentiation (BMP) (a). Multiple signal pathway agonists and antagonists are active in the intestinal crypts that are also used during in vitro culture to simulate the crypt niche environment (b).

4. Intestinal organoids

The identification of LGR5 + stem cells and the characterization of the intestinal stem-cell niche has led to the development of three-dimensional (3D) organoid cultures and the ability to amplify intestinal epithelium in vitro. These primary tissue cultures can be maintained long term without any substantial changes to genetic integrity or tissue physiology.

Organoids are now becoming an increasingly popular option for exploring an array of diseases currently lacking suitable treatments. Multiple organoid culture platforms have now been described including liver and pancreatic organoids [25–28], kidney organoids [29], central nervous system organoids [30] and intestinal organoids [31–33]. An organoid is often defined as ‘an in vitro 3D cellular cluster derived exclusively from primary tissue, ESCs or IPSCs, capable of self-renewal and self-organization, and exhibiting similar organ functionality as the tissue of origin' [34]. Indeed, intestinal organoids are clusters of cells that self-organize in 3D structures that recapitulate major features of their native tissue. Intestinal organoids have been derived from both human stem cells and direct biopsy of adult intestinal tissue. In each case, the resulting intestinal organoids share many features, including a highly folded epithelium structure consisting of crypts and villi similar to native intestinal epithelium. Once embedded in Matrigel™, they self-assemble so that the luminal surface of epithelium is directed towards the centre of the organoid and the basolateral side is in contact with the Matrigel™ and surrounding medium. Analysis of the different cell types present within intestinal organoids has shown that all cell types usually found in vivo are present, and are therefore useful for studying the complexities of interplay between cell types during homeostasis and disease states.

Intestinal organoids have been shown to exhibit the same functions as those that occur in vivo, including mucus production, and absorptive and secretory functions [24]. Intestinal organoids mimic in vivo epithelial regenerative capacity, with apoptotic cells being continually released into the lumen of the organoid as new cells are differentiated from the LGR5 + cells within the crypts to replenish the epithelium.

5. Isolation and culture of intestinal organoids

There are two approaches to creating intestinal organoids: either through isolation of intestinal crypts from patient donors or via in vitro differentiation of human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hIPSCs). Both methods result in organoids comprising all intestinal epithelial cell types found in vivo, in similar proportions and arrangements.

Culture conditions for both primary tissue and stem-cell-derived organoids are essentially the same, requiring a basal media comprising Advanced DMEM/F12, supplemented with N2 and B27, nicotinamide and N-acetylcysteine. To the basal medium additional growth factors are added to support the growth of the organoids including EGF, Noggin and gastrin, which are essential for regulation of gut mucosal growth and proliferation of intestinal epithelial cells. The presence of Wnt3a is crucial to regulate self-renewal, proliferation and differentiation, as expression of LGR5 in stem cells found in intestinal crypts is dependent upon the canonical Wnt signalling pathway, regulated by R-Spondin-1 and BMP4 inhibition by Noggin [35,36] (figure 2b).

Intestinal crypts can be isolated from intestinal biopsies during endoscopy or surgical resection [18,37] (figure 3). Crypts are manually dissected from the epithelium and embedded into a Matrigel™ containing Wnt, R-Spondin, EGF and Noggin. Once Matrigel™ has solidified and encapsulated the isolated crypts, the Matrigel™ is overlaid with tissue culture medium containing all additives and growth factors. Over the course of 7–10 days intestinal crypts elongate and expand to form the first organoid structures, which can then be manually dissociated and expanded. In addition to LGR5 + stem cells, mature enteroids and colonoids comprised enterocytes, enteroendocrine cells, goblets cells and Paneth cells.

Figure 3. Schematic of human intestinal organoid creation. Intestinal organoids can be generated directly from intestinal biopsy or tissue from surgical resection. Isolated crypts can be placed directly into 3D Matrigel™ cultures along with Wnt, Noggin, EGF and R-Spondin to establish stable cell lines. An alternative approach to generating patient-specific intestinal organoids requires a skin biopsy, isolation and amplification of skin fibroblasts. Skin fibroblasts can then be reprogrammed to hIPSCs using the Yamanaka factors and differentiated into endoermal cells and finally, intestinal epithelium before long-term culture in 3D Matrigel™ conditions.

Using a stem-cell-based approach, hESCs or hIPSCs can be differentiated following normal developmental stages to generate intestinal epithelium (figure 3). This includes differentiation of cells via the major developmental milestones of definitive endoderm, hindgut endoderm and intestinal progenitor cell stages. Once differentiated into sufficiently committed intestinal progenitor cells, they can be transitioned from what is normally a two-dimensional (2D) differentiation platform into the 3D organoid platform, where cells then spontaneously rearrange themselves into intestinal organoids.

6. Functional analysis of organoids

A variety of assays have been used to assess functionality of intestinal organoids. A primary function of enterocytes is transport of water and electrolytes across the intestinal barrier. In vitro, organoids have been shown to maintain this function via derivation of organoids lacking the apical transporters SGLT-1 or PEPT1. These organoids were shown to have inhibited d -glucose, d -fructose and peptide transport across the epithelial membrane, validating their potential use as a model of nutrient and drug transport mechanisms [38].

Permeability of the intestinal epithelium is an important factor in both health and disease, affecting the diffusion of small molecules across the intestinal barrier and preventing bacterial translocation via the bloodstream. Recently, it has been demonstrated that intestinal organoids can be used to model epithelial permeability and changes can be recorded in real time [39]. Hill et al. [39] demonstrated this technique by injecting fluorescently labelled dextran into the lumen of organoids while automated microscopy was used to capture, in real time, live cells and quantify breaks in the luminal surface. Using this technique, alterations to intestinal permeability can be studied under chronic inflammatory conditions and during bacterial invasions.

Forskolin-induced organoid swelling assay has also been used to demonstrate functionality of the cystic fibrosis transmembrane conductance regulator (CFTR) ion channel. Organoids with a functional CFTR swell in response to forskolin treatment, while any changes to the function of CFTR due to inhibition or genetic mutation can be detected due to lack of swelling [40]. This assay has the potential to be adapted to a drug-screening platform for cystic fibrosis (CF).

7. Applications of organoid technology

Owing to its differing physiology and distinct functions, the gastrointestinal tract is affected by an array of disorders. The intestinal epithelium is continuously exposed to antigens from commensal bacteria and consumed food, hence it has an extensive immune system, inclusive of adjacent lymphoid tissue, heavily populated with lymphocytes. The mucosa is the main site at which the immunological functions take place however, loss of this carefully maintained immune homeostasis can cause chronic inflammation and multiple disorders. Intestinal organoids have now become increasingly popular as a platform to model many intestinal diseases caused by chronic inflammation or physical injury.

Inflammatory bowel disease (IBD) is a chronic, progressive and relapsing disease affecting the entire gastrointestinal tract. It is a result of dysregulated innate and adaptive immune responses against antigens present in the gastrointestinal tract. IBD is categorized into two main subtypes: ulcerative colitis (UC), which is restricted to the colon, and Crohn's disease (CD), which can affect any area of the digestive tract [41]. During active IBD, the small and large intestine can become highly inflamed, leading to destruction of the epithelium and deterioration in digestive capacity. Intestinal organoids derived from IBD patients present an opportunity to understand how innate immunity is regulated in IBD patients and to identify novel therapeutics to reduce the severity of the disease.

Short bowel syndrome (SBS) develops often due to partial resection of large intestine as a result of IBD, colorectal cancer (CRC) or ischaemic disease, or can be present at birth. Owing to a significantly reduced surface area of the intestinal epithelium, malabsorption and malnutrition are major issues. Congenital SBS has a genetic basis linked with the CLMP gene. Patient-derived small intestinal organoids offer a chance to study the genetic susceptibility in greater detail, including the molecular mechanisms involved in impaired intestinal elongation during development that are currently not well understood [42]. Furthermore, the combination of intestinal organoids with tissue-engineering techniques for the lengthening of the small intestine, offer a potential method for the restoration of normal functioning of the bowel irrespective of the cause of SBS.

Coeliac disease is an example of an autoimmune disease mainly affecting the small intestine upon consumption of gluten. The resulting inflammation causes malabsorption, leading to a range of other successive symptoms. Without exclusion of all gluten products from the diet, prolonged inflammation can lead to osteoporosis and cancer. Organoids cultured from patients suffering from coeliac disease can offer insight into genetic predispositions and mechanisms driving the disease forward. Alterations to intestinal barrier integrity, which are known to occur in coeliac patients, can be explored using organoids [43]. Similarly, patient-derived organoids could be used as a drug-screening platform for the identification of novel therapies.

Diverticular disease occurs typically in the sigmoid colon and is the result of physical damage to the intestines leading to loss of function. Diverticula are sacs that form from the herniation of the intestinal epithelium, erupting through the surrounding muscular layer. They are suspected to form due to excessive pressure throughout the large intestine, due, in part, to a low-fibre diet, however, aetiology remains largely unknown. These diverticula can become infected and inflamed, leading to the patient developing fever, pain, nausea and diarrhoea. A genetic element is suspected that predisposes individuals to developing diverticulitis. Therefore, genetic factors involved in the pathogenesis of diverticulitis can be studied using patient tissue-derived organoids from a relatively non-invasive intestinal biopsy. These can then be compared to healthy intestinal tissue to determine genetic and functional differences. Owing to the mechanical nature of the development of diverticular disease, mechanical stress could be applied to the individual organoids to examine the response of the intestinal epithelium under differing levels of stretch stress. However, a tissue-engineering approach is required in combination with organoid technology to model the development of diverticula. Intestinal organoids alone would not be suitable to model diverticula development due to the involvement of the mesenchyme and muscular layer surrounding the intestinal epithelium in vivo.

Cystic fibrosis is perhaps best known for its effect in the lung but also causes complications in other organs, including the pancreas and intestines. The most serious acute complication of the intestine in CF is obstruction of the terminal ileum or proximal large intestine, which if untreated can result in rupture and sepsis. Intestinal organoids are a suitable model of CF due to their expression of CFTR and by using a combination of forskolin-induced swelling and voltage-gate measurements, fluid and ion transport can be accurately measured. This assay has been performed on human intestinal organoids, demonstrating physiologically accurate CFTR function [44]. Drug responsiveness can, therefore, be measured using the current organoid model to identify the most effective treatment for CF or as a diagnostic tool [45].

CRCs are among the most commonly diagnosed cancers in the developed world. There are many contributing factors to development of CRC, but importantly long-term CD or UC significantly increases lifetime risk. Organoids are increasingly being used as models of CRC. Current models of CRC are unable to reproduce the progression of the disease at the early stages and are not representative of the heterogeneous nature of tumours. Genome-editing techniques, such as CRISPR/Cas9, however, can be used as a means to introduce genetic mutations into genes of interest. Following genetic manipulation organoids can then be transplanted into mice, in which the in vivo mechanisms of tumour progression and invasion can be measured [46].

8. Host–pathogen interactions

Different methods are employed to expose intestinal organoids to bacteria. Microinjection of live bacteria or bacterial proteins is a common approach to study intestinal infections, including Salmonella and Clostridium difficile infections. For example, Forbester et al. [31] used hIPSCs to generate intestinal organoids that were then microinjected with Salmonella. mRNA sequencing was used to create a global profile of changes in gene expression in response to Salmonella infection [31]. Similarly, Leslie et al. [35] used a microinjection methodology to deliver C. difficile into the lumen of hIPSC-derived intestinal organoids. They then observed that C. difficile remained in the lumen for a prolonged duration, suggesting that organoids possess suitable conditions for the survival of C. difficile and hence other obligate anaerobes. Microinjection of C. difficile toxins has also been shown to exhibit expected effects on epithelial integrity and changes to the expression of certain tight junctions [35].

9. Limitations of organoids

Despite increasing interest in organoid platforms to model intestinal development and disease, organoids used in today's research lack certain elements of the complete organ found in vivo (table 1). This includes a lack of mesenchymal tissue, immune and neural cells that in vivo contribute to the overall structure and functioning of the intestines. Organoids currently used in research are comprised mainly of epithelium, including the niche that enables self-renewal of intestinal stem cells.

Table 1. Advantages and disadvantages for the use of intestinal organoids in the study of disease.

Organoids differentiated from induced pluripotent stem cells (IPSCs) into intestinal epithelial structures are known as induced intestinal organoids [47]. The differentiation protocols promoting the generation of such organoids also generates mesenchyme that, when transplanted under the renal capsule of mice, differentiate into smooth muscle and myofibroblasts [24]. These, therefore, are more representative of the intestines in vivo. However, they require a longer period of time to generate and are still devoid of immune cells which are necessary to address the most common intestinal disease IBD.

Other limitations of intestinal organoid include practical limitations due to the fact that cells must be embedded in Matrigel™ and grown in 3D. This creates additional complications when manipulating cells for simple procedures such as isolating mRNA, DNA and performing immuncytochemistry because organoids will require removal from Matrigel™ prior to processing. 3D culture also creates problems when attempting genetic manipulation via transfection and CRISPR/Cas9 gene editing approaches. Again, cells require removal of Matrigel™ prior to manipulation, which can create suboptimal conditions for organoid maintenance and growth.

It is clear that intestinal organoids can be used as an effective model of host–pathogen interactions that occur within the intestinal tract. However, further study is required to incorporate additional immune elements into the model to give a more complete illustration of inflammatory response. Thus far, microinjection has been used effectively to deliver the bacteria into the lumen of the organoid. However, this is a time-consuming technique. Therefore, other techniques have been trialled as more efficient alternatives. Treating a monolayer, for example, is much quicker to execute, however, this method is less indicative of how the 3D in vivo intestine would respond due to the lack of 3D architecture and the stem-cell niche. Without this native architecture it cannot be determined how intestinal stem cells would respond to such an infection.

10. Future challenges

Intestinal organoids remain a promising, tunable model for developmental and disease modelling, drug and toxicity testing, and host–pathogen interaction studies. In the future, in conjunction with CRISPR/Cas9 technology intestinal organoids hold great potential for gene therapy and transplantation applications in humans to treat chronic inflammatory disorders, such as CD, UC and CRCs. However, there are many aspects of this technology that still require significant investment to create a model that is more representative of in vivo tissue and their translational use in humans to become a reality.

Culturing intestinal organoids in 3D creates additional layers of complexity when attempting manipulations involving gene editing, transfection or when studies require access to the apical surface of intestinal epithelium. Previously, processing organoids has required specialized and time-consuming procedures such as the use of micromanipulator and microinjection platforms. More recently, several studies have addressed this problem and have begun developing methodologies to use 2D culture of dissociated intestinal organoids as a platform for high-throughput drug testing, migration assays and host–pathogen interaction studies. By using transwells with permeable membranes, experimentation can be performed with ease of access to both the apical and basolateral sides of the newly formed epithelial membrane [24]. However, this approach also disrupts the stem-cell compartment, meaning that using current culture conditions cells cannot be propagated long term using this 2D culture approach. It is feasible, however, to maintain organoids in 3D while performing manipulations in transient 2D cultures.

Current organoid platforms require the spontaneous self-assembly of epithelial tissue following dissociation and then embedding in Matrigel™. This approach creates organoids of differing shapes and sizes that lack the gross anatomical features of the intestine. Accuracy in developmental and disease modelling will be dramatically enhanced by the use of 3D scaffolds constructed from either decellularization and then recellularization of an ex vivo extracellular matrix or a cellularized synthetic scaffold. This approach offers a base on which to culture the cells into the tubular structure with more precise patterning of epithelium and consistent numbers of cells. This technique is potentially tunable, enabling the cell composition to be altered depending on the region of intestinal tract being studied or disease process being modelled. This model, as well as being a closer approximation of the intestine, is likely to be easier to manipulate by providing easier access to apical and basolateral sides of cells. Studies carried out into the development of this model have shown that it can accumulate a mucus layer on the luminal surface, as with organoids, with a thickness of 20 µm [48]. This can, therefore, recapitulate the host–pathogen interactions, occurring in vivo, that require an intact mucosal layer. So far, these models lack neural and immune cells to form a complete organ and require many months of set-up until the platform is suitable for experimental use. A notable disadvantage to this system is the deterioration in the model after only a few weeks following the seeding of cells to the 3D scaffold, which could be due to nutrient and oxygen starvation of cells, further reinforcing the need for models that recapitulate the complete organ including vascularization. This, therefore, must be overcome before it can be used for longer-term studies, including that of chronic inflammation.

Several studies have demonstrated the potential for direct transplantation of intestinal organoids into mice and rodents [32,49]. Fordham et al. successfully transplanted fetal intestinal progenitors, which had not yet been differentiated into intestinal organoids, into a colonic injury mouse model [32]. They found that immature enterospheres have regenerative capacity when transplanted into adult damaged colonic epithelium. Additionally, they demonstrated the ability of these immature cells to mature in vivo, appropriate for the region of engraftment, expressing Mucin-2-positive goblet cells and lacking markers of small intestine. Whether the same approach could be used to treat human intestinal injury from both acute and chronic IBD is yet to be determined. When combined with decellularization/recellularization techniques, 3D printing and development of synthetic and biodegradable scaffold technologies, stem-cell-derived intestinal tissue or patient-derived intestinal tissue could be used to replace large segments of the small and large intestine.

One of the more significant drawbacks to modelling disease using intestinal organoids is the absence of an immune element that can be used to understand mechanisms driving the autoimmune destruction of the intestinal mucosa that occurs during CD and UC in vivo. Thus far, intestinal organoids have been able to model the immune response at an innate level, determining the effects of gene expression and cytokine signalling in the development of CD and in host–pathogen interaction studies. CD is known to be triggered by a combination of genetic susceptibility and environmental factors, resulting in dysregulation of immune homeostasis. Studies have shown that human intestinal organoids generated from hIPSCs are a suitable model for studying the interactions between intestinal epithelium and the enteric pathogen Salmonella typhimurium to dissect elements of the innate immune response [31]. The model, however, needs to be further developed to include elements of systemic immune regulation including cells such as macrophages, neutrophils, T cells, B lymphocytes, NK cells and dendritic cells. Addition of these cell types will create a more complete model but will be a significant challenge requiring all cell types to be from the same donor.

Intestinal organoids, whether derived from primary tissue biopsy or stem-cell differentiation techniques, have great potential in the future of disease modelling, drug discovery and personalized medicine. They have been shown to have stable phenotype, are relatively simple to create and manipulate while at the same time able to be maintained in long-term culture. However, current organoid derivation and culture techniques do not allow for the assembly of complex multi-cell-type organoids that can model the complete complexity of a multifactoral disease such as IBD. Furthermore, as our need to modify and manipulate organoids and organoid culture conditions advances, the practice of culturing intestinal organoids may pose technical limitations on what is achievable. Developing our understanding of the initiation and development of complex intestinal disease, such as IBD and CRC, will require continued investment and research into the development of more complex intestinal organoid platforms.


Abstract

Oral and intestinal mucositis is a debilitating, often dose limiting side effect of radiation treatment. A mouse model of mucositis, induced by gamma irradiation, leads to weight loss and tissue damage, similar to that observed in patients. This model reflects the human ailment as it responds to keratinocyte growth factor (KGF), the standard of care treatment.
Culturing of intestinal crypt organoids derived from primary cells allowed the development of a 3D assay to monitor the effect of treatments of intestinal epithelium to radiation-induced damage. This in vitro assay closely resembles the mouse model as KGF and Roof Plate-Specific Spondin-1 (RSPO1) enhanced the recovery of crypt organoids following radiation. Screening identified tool compounds that increased the survival of organoids post radiation. Repeated testing of these compounds revealed that the organoids changed their response over time. To investigate this adaptive behavior, intestinal organoid cultures were studied over time. Samples of organoids at various time points were used to prepare mRNA for unbiased transcriptome analyses. This expression profiling revealed a number of genes and pathways that were modulated over time, providing a rationale for the altered sensitivity of the intestinal crypt organoid cultures.
This report describes the development of an in vitro assay that reflects the response of disease to therapeutic treatment. The assay was miniaturized and used to identify bioactive tool compounds, which served as probes to interrogate the patho-physiology of organoids over prolonged culture conditions. In vitro disease models based on primary 3D cell cultures represent valuable tools to identify potential drug targets and bioactive hits.


Affiliations

Broad Institute of Massachusetts Institute of Technology and Harvard University, Cambridge, MA, USA

Benjamin E. Mead & Jeffrey M. Karp

Institute for Medical Engineering and Science (IMES), Massachusetts Institute of Technology, Cambridge, MA, USA

Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA, USA

Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA

Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology and Harvard University, Cambridge, MA, USA

Engineering in Medicine, Department of Medicine, Center for Regenerative Therapeutics, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA

Harvard−Massachusetts Institute of Technology Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA, USA


Watch the video: How to Culture Mouse Intestinal Organoids: Isolating Intestinal Crypts and Establishing Organoids (January 2022).