E.R. acknowledges a Ph.D. fellowship from the Wellcome Trust (215027/Z/18/Z). G.M.J. acknowledges a Ph.D. fellowship from the Wellcome Trust (203757/Z/16/A). D.C. acknowledges a Ph.D. studentship from the NIHR GSTT BRC. J.F.N. acknowledges a Marie Sk&#322;odowska-Curie Fellowship, a King&#39;s Prize fellowship, an RCUK/UKRI Rutherford Fund fellowship (MR/R024812/1), and a Seed Award in Science from the Wellcome Trust (204394/Z/16/Z). We also thank the BRC flow cytometry core team based at Guy&#39;s Hospital. Rorc(&#947;t)-GfpTG C57BL/6 reporter mice were a generous gift from G. Eberl (Institut Pasteur, Paris, France). CD45.1 C57BL/6 mice were kindly given by T. Lawrence (King&#39;s College London, London) and P. Barral (King&#39;s College London, London).

<ol>
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The authors have no conflicts of interest.

This protocol describes the methods for establishing murine small intestine organoids, isolating rare ILC1 by minimizing the loss of lymphocytes during the intestinal dissociation protocol, and establishing co-cultures between these two compartments. There are many steps to this protocol, and while some are specific to ILC1s, this approach can be applied to other intestinal immune cell types, and co-culture setups can be modularly adapted to suit individual research questions. Several critical steps (that are recommended...

Communication between the intestinal epithelium and gut-resident immune system is central to the maintenance of intestinal homeostasis1. Disruptions to these interactions are associated with both local and systemic diseases, including Inflammatory Bowel Disease (IBD) and gastrointestinal cancers2. A notable example of one more recently described critical regulator of homeostasis comes from the study of innate lymphoid cells (ILCs), which have emerged as key players in the intestinal immune landscape3. ILCs are a group of heterogenous innate immune cells that regulate intestinal homeostasis and orchestrate inflammation largely through cytokine-mediated signalling4.
Murine ILCs are broadly divided into subtypes based on transcription factor, receptor, and cytokine expression profiles5. Type-1 ILCs, which include cytotoxic Natural Killer (NK) cells and helper-like type-1 ILCs (ILC1s), are defined by expression of the transcription factor (eomesodermin) Eomes and T-box protein expressed in T cells (T-bet)6, respectively, and secrete cytokines associated with T helper type-1 (TH1) immunity: interferon-&#947; (IFN&#947;) and tumor necrosis factor (TNF), in response to interleukin (IL)-12, IL-15 and IL-187. During homeostasis, tissue-resident ILC1s secrete Transforming Growth Factor &#946; (TGF-&#946;) to drive epithelial proliferation and matrix remodelling8. Type-2 ILCs (ILC2s) primarily respond to helminth infection via secretion of T helper type-2 (TH2) associated cytokines: IL-4, IL-5, and IL-13, and are characterized by the expression of retinoic acid-related orphan receptor (ROR) &#945; (ROR-&#945;)9 and GATA Binding Protein 3 (GATA-3)10,11,12. In mice, intestinal &#34;inflammatory&#34; ILC2s are further characterized by expression of Killer cell lectin-like receptor (subfamily G member 1, KLRG)13 where they respond to epithelial tuft-cell derived IL-2514,15. Finally, type-3 ILCs, which include lymphoid tissue inducer cells and helper-like type-3 ILCs (ILC3s), are dependent on the transcription factor ROR-&#947;t16, and cluster into groups that secrete either Granulocyte Macrophage Colony Stimulating Factor (GM-CSF), IL-17, or IL-22 in response to local IL-1&#946; and IL-23 signals17. Lymphoid tissue inducer cells cluster in Peyer&#39;s patches and are crucial for the development of these secondary lymphoid organs during development18, whereas ILC3s are the most abundant ILC subtype in the adult murine small intestine lamina propria. One of the earliest murine intestinal organoid co-culture systems with ILC3s was harnessed to tease apart the impact of the cytokine IL-22 on Signal Transducer And Activator Of Transcription 3 (STAT-3) mediated Leucine-Rich Repeat Containing G Protein Coupled Receptor 5 (Lgr5)+ intestinal stem cell proliferation19, a powerful example of a regenerative ILC-epithelial interaction. ILCs exhibit imprint-heterogeneity between organs20,21 and exhibit plasticity between subsets in response to polarizing cytokines22. What drives these tissue-specific imprints and plasticity differences, and what role they play in chronic diseases such as IBD23, remain exciting topics that could be addressed using organoid co-cultures.
Intestinal organoids have emerged as a successful and reliable model to study the intestinal epithelium24,25. These are generated by culturing intestinal epithelial Lgr5+ stem cells, or entire isolated crypts, which include Paneth cells as an endogenous source of Wnt Family Member 3A (Wnt3a). These 3D structures are maintained either in synthetic hydrogels26 or in biomaterials that mimic the basal lamina propria, for instance, Thermal-crosslinking Basal Extracellular Matrix (TBEM), and are further supplemented with growth factors that mimic the surrounding niche, most notably Epithelial Growth Factor (EGF), the Bone Morphogenetic Protein (BMP)-inhibitor Noggin, and an Lgr5-ligand and Wnt-agonist R-Spondin127. Under these conditions, organoids maintain epithelial apico-basal polarity and recapitulate the crypt-villi structure of the intestinal epithelium with budding stem cell crypts that terminally differentiate into absorptive and secretory cells in the center of the organoid, which then shed into the internal pseudolumen by anoikis28. Although intestinal organoids alone have been hugely advantageous as reductionist models of epithelial development and dynamics in isolation29,30, they hold tremendous future potential for understanding how these behaviors are regulated, influenced, or even disrupted by the immune compartment.
In the following protocol, a method of co-culture between murine small intestinal organoids and lamina propria derived ILC1s is described, which was recently used to identify how this population unexpectedly decreases intestinal signatures of inflammation and instead contributes to increased epithelial proliferation via TGF-&#946; in this system8.

<table><tbody><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>2-Mercaptoethanol</td><td>Gibco</td><td>21985023</td><td></td></tr><tr><td>Anti-mouse CD45 (BV510)</td><td>BioLegend</td><td>103137</td><td></td></tr><tr><td>Anti-mouse NK1.1 (PE)</td><td>Thermo Fisher Scientific</td><td>12-5941-83</td><td></td></tr><tr><td>B-27 Supplement (50X), serum free</td><td>Gibco</td><td>17504044</td><td></td></tr><tr><td>CD127 Monoclonal Antibody (APC)</td><td>Thermo Fisher Scientific</td><td>17-1271-82</td><td></td></tr><tr><td>CD19 Monoclonal Antibody (eFluor 450)</td><td>Thermo Fisher Scientific</td><td>48-0193-82</td><td></td></tr><tr><td>CD3e Monoclonal Antibody (eFluor 450)</td><td>Thermo Fisher Scientific</td><td>48-0051-82</td><td></td></tr><tr><td>CD5 Monoclonal Antibody (eFluor 450)</td><td>Thermo Fisher Scientific</td><td>48-0031-82</td><td></td></tr><tr><td>CHIR99021</td><td>Tocris</td><td>4423/10</td><td></td></tr><tr><td>COLLAGENASE D, 500MG</td><td>Merck</td><td>11088866001</td><td></td></tr><tr><td>Cultrex HA- RSpondin1-Fc HEK293T Cells</td><td>Cell line was used to harvest conditioned RSpondin1 supernatant, the cell line and Materials Transfer Agreement was provided by the Board of Trustees of the Lelands Stanford Junior University (Calvin Kuo, MD,PhD, Stanford University)</td><td></td><td></td></tr><tr><td>DISPASE II (NEUTRAL PROTEASE, GRADE II)</td><td>Merck</td><td>4942078001</td><td></td></tr><tr><td>DMEM/F12 (1:1) (1X) Dulbecco&#039;s Modified Eagle Medium Nutrient Mixture F-12 (Advanced DMEM/F12)</td><td>Gibco</td><td>11320033</td><td></td></tr><tr><td>DNASE I, GRADE II</td><td>Merck</td><td>10104159001</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium (1X)</td><td>Gibco</td><td>21969-035</td><td></td></tr><tr><td>Ethilenediamine Tetraacetate Acid</td><td>Thermo Fisher Scientific</td><td>BP2482-100</td><td></td></tr><tr><td>FC block</td><td>2B Scientific</td><td>BE0307</td><td></td></tr><tr><td>Fetal Bovine Serum, qualified, hear inactivated</td><td>Gibco</td><td>10500064</td><td></td></tr><tr><td>GlutaMAX (100X)</td><td>Gibco</td><td>3050-038</td><td></td></tr><tr><td>Hanks&#039; Balanced Salt Solution (10X)</td><td>Gibco</td><td>14065056</td><td></td></tr><tr><td>HBSS (1X)</td><td>Gibco</td><td>12549069</td><td></td></tr><tr><td>HEK-293T- mNoggin-Fc Cells</td><td>Cell line was used to harvest conditioned Noggin supernatant, cell line acquired through Materials Transfer Agreement with the Hubrecth Institute, Uppsalalaan8, 3584 CT Utrecht, The Netherlands, and is based on the publication by Farin, Van Es, and Clevers Gastroenterology (2012).</td><td></td><td></td></tr><tr><td>HEPES Buffer Solution (1M)</td><td>Gibco</td><td>15630-056</td><td></td></tr><tr><td>KLRG1 Monoclonal Antibody (PerCP eFluor-710)</td><td>Thermo Fisher Scientific</td><td>46-5893-82</td><td></td></tr><tr><td>Live/Dead Fixable Blue Dead Cell Stain Kit, for UV excitation</td><td>Thermo Fisher Scientific</td><td>L23105</td><td></td></tr><tr><td>Ly-6G/Ly-6C Monoclonal Antibody (eFluor 450)</td><td>Thermo Fisher Scientific</td><td>48-5931-82</td><td></td></tr><tr><td>Matrigel Growth Factor Reduced Basement Membrane Matrix, Phenol Red-free, LDEV-free</td><td>Corning</td><td>356231</td><td></td></tr><tr><td>N-2 Supplement (100X)</td><td>Gibco</td><td>17502048</td><td></td></tr><tr><td>N-acetylcysteine (500mM)</td><td>Merck</td><td>A9165</td><td></td></tr><tr><td>NKp46 Monoclonal Antibody (PE Cyanine7)</td><td>Thermo Fisher</td><td>25-3351-82</td><td></td></tr><tr><td>PBS (1 X) 7.2 pH</td><td>Thermo Fisher Scientific</td><td>12549079</td><td></td></tr><tr><td>PBS (10X)</td><td>Gibco</td><td>70013032</td><td></td></tr><tr><td>Percoll</td><td>Cytiva</td><td>17089101</td><td></td></tr><tr><td>Recombinant Human EGF, Animal-Free Protein</td><td>R&amp;amp;D Systems</td><td>AFL236</td><td></td></tr><tr><td>Recombinant Human IL-15 GMP Protein, CF</td><td>R&amp;amp;D Systems</td><td>247-GMP</td><td></td></tr><tr><td>Recombinant Human IL-2 (carrier free)</td><td>BioLegend</td><td>589106</td><td></td></tr><tr><td>Recombinant Mouse IL-7 (carrier free)</td><td>R&amp;amp;D Systems</td><td>407-ML-005/CF</td><td></td></tr><tr><td>UltraComp eBeads</td><td>Thermo Fisher Scientific</td><td>01-2222-42</td><td></td></tr><tr><td>Y-27632 dihydrochloride (ROCK inhibitor)</td><td>Bio-techne</td><td>1254</td><td></td></tr><tr><td>&lt;strong&gt;Plastics&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>50 mL tube</td><td>Falcon</td><td>10788561</td><td></td></tr><tr><td>1.5 mL tube</td><td>Eppendorf</td><td>30121023</td><td></td></tr><tr><td>10 mL pippette</td><td>StarLab</td><td>E4860-0010</td><td></td></tr><tr><td>15 mL tube</td><td>Falcon</td><td>11507411</td><td></td></tr><tr><td>25 mL pippette</td><td>StarLab</td><td>E4860-0025</td><td></td></tr><tr><td>p10 pippette tips</td><td>StarLab</td><td>S1121-3810-C</td><td></td></tr><tr><td>p1000 pippette tips</td><td>StarLab</td><td>I1026-7810</td><td></td></tr><tr><td>p200 pippette tips</td><td>StarLab</td><td>E1011-0921</td><td></td></tr><tr><td>Standard tissue culture treated 24-well plate</td><td>Falcon</td><td>353047</td><td></td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5810 R</td><td></td></tr><tr><td>CO2 and temperature controled incubator</td><td>Eppendorf</td><td>Galaxy 170 R/S</td><td></td></tr><tr><td>Flow Assisted Cellular Sorter</td><td>BD equipment</td><td>FACS Aria II</td><td></td></tr><tr><td>Heated shaker</td><td>Stuart Equipment</td><td>SI500</td><td></td></tr><tr><td>Ice box</td><td>-</td><td>-</td><td></td></tr><tr><td>Inverted light microscope</td><td>Thermo Fisher Scientific</td><td>EVOS XL Core Imaging System (AMEX1000)</td><td></td></tr><tr><td>p10 pippette</td><td>Eppendorf</td><td>3124000016</td><td></td></tr><tr><td>p1000 pippette</td><td>Eppendorf</td><td>3124000063</td><td></td></tr><tr><td>p200 pippette</td><td>Eppendorf</td><td>3124000032</td><td></td></tr><tr><td>Pippette gun</td><td>Eppendorf</td><td>4430000018</td><td></td></tr><tr><td>Wet ice</td><td>-</td><td>-</td><td></td></tr></tbody></table>

co-culture of murine small intestine epithelial organoids with innate lymphoid cells

Complex co-cultures of organoids with immune cells provide a versatile tool for interrogating the bi-directional interactions that underpin the delicate balance of mucosal homeostasis. These 3D, multi-cellular systems offer a reductionist model for addressing multi-factorial diseases and resolving technical difficulties that arise when studying rare cell types such as tissue-resident innate lymphoid cells (ILCs). This article describes a murine system that combines small intestine organoids and small intestine lamina propria derived helper-like type-1 ILCs (ILC1s), which can be readily extended to other ILC or immune populations. ILCs are a tissue-resident population that is particularly enriched in the mucosa, where they promote homeostasis and rapidly respond to damage or infection. Organoid co-cultures with ILCs have already begun shedding light on new epithelial-immune signaling modules in the gut, revealing how different ILC subsets impact intestinal epithelial barrier integrity and regeneration. This protocol will enable further investigations into reciprocal interactions between epithelial and immune cells, which hold the potential to provide new insights into the mechanisms of mucosal homeostasis and inflammation.

When successfully completed, freshly isolated crypts should form budding crypt structures within 2-4 days (Figure 1A). Healthy and robust organoid cultures should be actively growing and can be passaged and expanded as detailed in the protocol.
This protocol describes the isolation of small intestinal ILC1 from the ROR&#947;tGFP murine transgenic reporter line, which allows the isolation of live ILC1 by FACS (Figure 2). Usi...

Watch this Scientific Journal Video about Co-Culture of Murine Small Intestine Epithelial Organoids with Innate Lymphoid Cells at JoVE.com

Co-Culture of Murine Small Intestine Epithelial Organoids with Innate Lymphoid Cells

This protocol offers detailed instructions for establishing murine small intestine organoids, isolating type-1 innate lymphoid cells from the murine small intestine lamina propria, and establishing 3-dimensional (3D) co-cultures between both cell types to study bi-directional interactions between intestinal epithelial cells and type-1 innate lymphoid cells.

Complex co-cultures of organoids with immune cells provide a versatile tool for interrogating the bi-directional interactions that underpin the delicate ...

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Research

JoVE Journal

Immunology and Infection

5.4K Views.  King’s College London. Dit protocol biedt een belangrijk hulpmiddel om de interacties tussen intestinale immuuncellen en het epitheel te bestuderen en om te ontleden hoe deze interacties kunnen reguleren in intestinale homeostase en ontsteking. Het belangrijkste voordeel van deze techniek is de voortreffelijke hoeveelheid experimentele controle die wordt vergemakkelijkt door het reductie-in vitromodel, dat kan worden gebruikt om vragen in epitheliale en immuunbiologie aan te pakken. Dit systeem is al gebruikt om de...

Video: Co-Culture of Murine Small Intestine Epithelial Organoids with Innate Lymphoid Cells

Three-Dimensional Culture of Murine Colonic Crypts to Study Intestinal Stem Cell Function Ex Vivo

The intestinal epithelium regenerates every 5-7 days, and is controlled by the intestinal epithelial stem cell (IESC) population located at the bottom of the crypt region. IESCs include active stem cells, which self-renew and differentiate into various epithelial cell types, and quiescent stem cells, which serve as the reserve stem cells in the case of injury. Regeneration of the intestinal epithelium is controlled by the self-renewing and differentiating capabilities of these active IESCs. In addition, the balance of the crypt stem cell population and maintenance of the stem cell niche are essential for intestinal regeneration. Organoid culture is an important and attractive approach to studying proteins, signaling molecules, and environmental cues that regulate stem cell survival and functions. This model is less expensive, less time-consuming, and more manipulatable than animal models. Organoids also mimic the tissue microenvironment, providing in vivo relevance. The present protocol describes the isolation of colonic crypts, embedding these isolated crypt cells into a three-dimensional gel matrix system and culturing crypt cells to form colonic organoids capable of self-organization, proliferation, self-renewal, and differentiation. This model allows one to manipulate the environment-knocking out specific proteins such as claudin-7, activating/deactivating signaling pathways, etc.-to study how these effects influence the functioning of colonic stem cells. Specifically, the role of tight junction protein claudin-7 in colonic stem cell function was examined. Claudin-7 is vital for maintaining intestinal homeostasis and barrier function and integrity. Knockout of claudin-7 in mice induces an inflammatory bowel disease-like phenotype exhibiting intestinal inflammation, epithelial hyperplasia, weight loss, mucosal ulcerations, epithelial cell sloughing, and adenomas. Previously, it was reported that claudin-7 is required for intestinal epithelial stem cell functions in the small intestine. In this protocol, a colonic organoid culture system is established to study the role of claudin-7 in the large intestine.

Three-Dimensional Culture of Murine Colonic Crypts to Study Intestinal Stem Cell Function Ex Vivo

The present protocol describes establishing a murine colonic organoid system to study the activity and functioning of colonic stem cells in a claudin-7 knockout model.

The intestinal epithelium regenerates every 5-7 days, and is controlled by the intestinal epithelial stem cell (IESC) population located at the bottom of ...

Cancer Research

Organoids as Model for Infectious Diseases: Culture of Human and Murine Stomach Organoids and Microinjection of Helicobacter Pylori

Recently infection biologists have employed stem cell derived cultures to answer the need for new and better models to study host-pathogen interactions. Three cellular sources have been used: Embryonic stem cells (ESC), induced pluripotent stem cells (iPSC) or adult stem cells. Here, culture of mouse and human gastric organoids derived from adult stem cells is described and used for infection with the gastric pathogen Helicobacter pylori. Human gastric glands are isolated from resection material, seeded in a basement matrix and embedded in medium containing growth factors epidermal growth factor (EGF), R-spondin, Noggin, Wnt, fibroblast growth factor (FGF) 10, gastrin and transforming growth factor (TGF) beta inhibitor. In these conditions, gastric glands grow into 3-dimensional organoids containing 4 lineages of the stomach. The organoids expand indefinitely and can be frozen and thawed similarly as cell lines. For infection studies, bacteria are microinjected into the lumen of the organoids. Infected organoids are processed for imaging. The described methods can be adapted to other organoids and infections with other bacteria, viruses or parasites. This allows the study of infection-induced changes in primary cells.

Stem cell derived cultures harbor tremendous potential to model infectious diseases. Here, the culture of mouse and human gastric organoids derived from adult stem cells is described. The organoids are microinjected with the gastric pathogen Helicobacter pylori.

Recently infection biologists have employed stem cell derived cultures to answer the need for new and better models to study host-pathogen interactions. ...

The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids

Primary intestinal organoids are a valuable model system that has the potential to significantly impact the field of mucosal immunology. However, the complexities of the organoid growth characteristics carry significant caveats for the investigator. Specifically, the growth patterns of each individual organoid are highly variable and create a heterogeneous population of epithelial cells in culture. With such caveats, common tissue culture practices cannot be simply applied to the organoid system due to the complexity of the cellular structure. Counting and plating based solely on cell number, which is common for individually separated cells, such as cell lines, is not a reliable method for organoids unless some normalization technique is applied. Normalizing to total protein content is made complex due to the resident protein matrix. These characteristics in terms of cell number, shape and cell type should be taken into consideration when evaluating secreted contents from the organoid mass. This protocol has been generated to outline a simple procedure to culture and treat small intestinal organoids with microbial pathogens and pathogen associated molecular patterns (PAMPs). It also emphasizes the normalization techniques that should be applied when protein analysis are conducted after such a challenge.

The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids

Here, a protocol to harvest, maintain, and treat mouse small intestinal organoids with pathogen associated molecular patterns (PAMPs) and Listeria monocytogenes is described, as well as emphasis on gene expression and proper normalization techniques for protein.

Primary intestinal organoids are a valuable model system that has the potential to significantly impact the field of mucosal immunology. However, the ...

Isolating Lymphocytes from the Mouse Small Intestinal Immune System

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to dietary antigens and commensal bacteria while mounting effective immune responses to enteropathogenic microbes. In addition, it has become clear that local intestinal immunity has a profound impact on distant and systemic immunity. Therefore, it is important to study how an intestinal immune response is induced and what the immunologic outcome of the response is. Here, a detailed protocol is described for the isolation of lymphocytes from small intestine inductive sites like the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes and effector sites like the lamina propria and the intestinal epithelium. This technique ensures isolation of a large numbers of lymphocytes from small intestinal tissues with optimal purity and viability and minimal cross compartmental contamination within acceptable time constraints. The technical capability to isolate lymphocytes and other immune cells from intestinal tissues enables the understanding of immune responses to gastrointestinal infections, cancers, and inflammatory diseases.

Here we describe a detailed protocol for the isolation of lymphocytes from the inductive sites including the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes, and the effector sites including the lamina propria and the intestinal epithelium of the small intestinal immune system.

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to ...

Using Human Induced Pluripotent Stem Cell-derived Intestinal Organoids to Study and Modify Epithelial Cell Protection Against Salmonella and Other Pathogens

The intestinal &#8216;organoid&#8217; (iHO) system, wherein 3-D structures representative of the epithelial lining of the human gut can be produced from human induced pluripotent stem cells (hiPSCs) and maintained in culture, provides an exciting opportunity to facilitate the modeling of the epithelial response to enteric infections. In vivo, intestinal epithelial cells (IECs) play a key role in regulating intestinal homeostasis and may directly inhibit pathogens, although the mechanisms by which this occurs are not fully elucidated. The cytokine interleukin-22 (IL-22) has been shown to play a role in the maintenance and defense of the gut epithelial barrier, including inducing a release of antimicrobial peptides and chemokines in response to infection.
We describe the differentiation of healthy control hiPSCs into iHOs via the addition of specific cytokine combinations to their culture medium before embedding them into a basement membrane matrix-based prointestinal culture system. Once embedded, the iHOs are grown in media supplemented with Noggin, R-spondin-1, epidermal growth factor (EGF), CHIR99021, prostaglandin E2, and Y-27632 dihydrochloride monohydrate. Weekly passages by manual disruption of the iHO ultrastructure lead to the formation of budded iHOs, with some exhibiting a crypt/villus structure. All iHOs demonstrate a differentiated epithelium consisting of goblet cells, enteroendocrine cells, Paneth cells, and polarized enterocytes, which can be confirmed via immunostaining for specific markers of each cell subset, transmission electron microscopy (TEM), and quantitative PCR (qPCR). To model infection, Salmonella enterica serovar Typhimurium SL1344 are microinjected into the lumen of the iHOs and incubated for 90 min at 37 &#176;C, and a modified gentamicin protection assay is performed to identify the levels of intracellular bacterial invasion. Some iHOs are also pretreated with recombinant human IL-22 (rhIL-22) prior to infection to establish whether this cytokine is protective against Salmonella infection.

Using Human Induced Pluripotent Stem Cell-derived Intestinal Organoids to Study and Modify Epithelial Cell Protection Against Salmonella and Other Pathogens

Human induced pluripotent stem cell (hiPSC)-derived intestinal organoids offer exciting opportunities to model enteric diseases in vitro. We demonstrate the differentiation of hiPSCs into intestinal organoids (iHOs), the stimulation of these iHOs with cytokines, and the microinjection of Salmonella Typhimurium into the iHO lumen, enabling the study of an epithelial invasion by this pathogen.

The intestinal &#8216;organoid&#8217; (iHO) system, wherein 3-D structures representative of the epithelial lining of the human gut can be produced from ...

Induced Differentiation of M Cell-like Cells in Human Stem Cell-derived Ileal Enteroid Monolayers

M (microfold) cells of the intestine function to transport antigen from the apical lumen to the underlying Peyer&#8217;s patches and lamina propria where immune cells reside and therefore contribute to mucosal immunity in the intestine. A complete understanding of how M cells differentiate in the intestine as well as the molecular mechanisms of antigen uptake by M cells is lacking. This is because M cells are a rare population of cells in the intestine and because in vitro models for M cells are not robust. The discovery of a self-renewing stem cell culture system of the intestine, termed enteroids, has provided new possibilities for culturing M cells. Enteroids are advantageous over standard cultured cell lines because they can be differentiated into several major cell types found in the intestine, including goblet cells, Paneth cells, enteroendocrine cells and enterocytes. The cytokine RANKL is essential in M cell development, and addition of RANKL and TNF-&#945; to culture media promotes a subset of cells from ileal enteroids to differentiate into M cells. The following protocol describes a method for the differentiation of M cells in a transwell epithelial polarized monolayer system of the intestine using human ileal enteroids. This method can be applied to the study of M cell development and function.

This protocol describes how to induce the differentiation of M cells in human stem cell-derived ileal monolayers and methods to assess their development.

M (microfold) cells of the intestine function to transport antigen from the apical lumen to the underlying Peyer&#8217;s patches and lamina propria where ...

A Protocol for Lentiviral Transduction and Downstream Analysis of Intestinal Organoids

Intestinal crypt-villus structures termed organoids, can be kept in sustained culture three dimensionally when supplemented with the appropriate growth factors. Since organoids are highly similar to the original tissue in terms of homeostatic stem cell differentiation, cell polarity and presence of all terminally differentiated cell types known to the adult intestinal epithelium, they serve as an essential resource in experimental research on the epithelium. The possibility to express transgenes or interfering RNA using lentiviral or retroviral vectors in organoids has increased opportunities for functional analysis of the intestinal epithelium and intestinal stem cells, surpassing traditional mouse transgenics in speed and cost. In the current video protocol we show how to utilize transduction of small intestinal organoids with lentiviral vectors illustrated by use of doxycylin inducible transgenes, or IPTG inducible short hairpin RNA for overexpression or gene knockdown. Furthermore, considering organoid culture yields minute cell counts that may even be reduced by experimental treatment, we explain how to process organoids for downstream analysis aimed at quantitative RT-PCR, RNA-microarray and immunohistochemistry. Techniques that enable transgene expression and gene knock down in intestinal organoids contribute to the research potential that these intestinal epithelial structures hold, establishing organoid culture as a new standard in cell culture.

In this video protocol we give a step by step explanation of lentiviral transduction in organoids of primary intestinal epithelium and of processing and downstream analysis of these cultures by quantitative RT-PCR, RNA-microarray and immunohistochemistry.

Intestinal crypt-villus structures termed organoids, can be kept in sustained culture three dimensionally when supplemented with the appropriate growth ...

Biology

Culture Methods to Study Apical-Specific Interactions using Intestinal Organoid Models

The lining of the gut epithelium is made up of a simple layer of specialized epithelial cells that expose their apical side to the lumen and respond to external cues. Recent optimization of in vitro culture conditions allows for the re-creation of the intestinal stem cell niche and the development of advanced 3-dimensional (3D) culture systems that recapitulate the cell composition and the organization of the epithelium. Intestinal organoids embedded in an extracellular matrix (ECM) can be maintained for long-term and self-organize to generate a well-defined, polarized epithelium that encompasses an internal lumen and an external exposed basal side. This restrictive nature of the intestinal organoids presents challenges in accessing the apical surface of the epithelium in vitro and limits the investigation of biological mechanisms such as nutrient uptake and host-microbiota/host-pathogen interactions. Here, we describe two methods that facilitate access to the apical side of the organoid epithelium and support the differentiation of specific intestinal cell types. First, we show how ECM removal induces an inversion of the epithelial cell polarity and allows for the generation of apical-out 3D organoids. Second, we describe how to generate 2-dimensional (2D) monolayers from single cell suspensions derived from intestinal organoids, comprised of&#160;mature and differentiated cell types. These techniques provide novel tools to study apical-specific interactions of the epithelium with external cues in vitro and promote the use of organoids as a platform to facilitate precision medicine.

Here we present two protocols that allow the modeling of intestinal apical-specific interactions. Organoid-derived intestinal monolayers and Air-Liquid Interface (ALI) cultures facilitate the generation of well-differentiated epithelia accessible from both luminal and basolateral sides, whereas polarity-inverted intestinal organoids expose their apical side and are amenable to high throughput assays.

The lining of the gut epithelium is made up of a simple layer of specialized epithelial cells that expose their apical side to the lumen and respond to ...

Developing 2D Monolayer Cultures from Bovine Intestinal Organoids

Advancements in Bovine Organoid Technology Using Small and Large Intestinal Monolayer Interfaces

Advancing knowledge of gastrointestinal physiology and its diseases critically depends on the development of precise, species-specific in vitro models that faithfully mimic in vivo intestinal tissues. This is particularly vital for investigating host-pathogen interactions in bovines, which are significant reservoirs for pathogens that pose serious public health risks. Traditional 3D organoids offer limited access to the intestinal epithelium's apical surface, a hurdle overcome by the advent of 2D monolayer cultures. These cultures, derived from organoid cells, provide an exposed luminal surface for more accessible study. In this research, a detailed protocol is introduced for creating and sustaining 2D monolayer cultures from cells of bovine small and large intestinal organoids. This method includes protocols for assessing membrane integrity through transepithelial electrical resistance and paracellular permeability alongside immunocytochemistry staining techniques. These protocols lay the groundwork for establishing and characterizing a 2D bovine monolayer culture system, pushing the boundaries of these method applications in biomedical and translational research of public health importance. Employing this innovative approach enables the development of physiologically pertinent in vitro models for exploring both normal and diseased states of cattle intestinal physiology. The implications for biomedical and agricultural advancements are profound, paving the way for more effective treatments for intestinal ailments in cattle, thereby enhancing both animal welfare and food safety.

Author Spotlight: Development and Characterization of 2D Intestinal Monolayer Models from Bovine Organoids for Pathogen Interaction Studies

This study presents a protocol for generating bovine intestinal 2D monolayers from organoids, offering improved access for studying host-pathogen interactions. It includes methods for assessing membrane integrity and functionality, advancing in vitro models that mimic cattle's gastrointestinal physiology. This approach promises significant biomedical and agricultural benefits, including enhanced treatment strategies.

Advancing knowledge of gastrointestinal physiology and its diseases critically depends on the development of precise, species-specific in vitro models ...

Medicine

Generation and Characterization of Murine Oral Mucosal Organoid Cultures

The mucous lining covering the inside of our mouth, the oral mucosa, is a highly compartmentalized tissue and can be subdivided into the buccal mucosa, gingiva, lips, palate, and tongue. Its uppermost layer, the oral epithelium, is maintained by adult stem cells throughout life. Proliferation and differentiation of adult epithelial stem cells have been intensively studied using in vivo mouse models as well as two-dimensional (2D) feeder-cell based in vitro models. Complementary to these methods is organoid technology, where adult stem cells are embedded into an extracellular matrix (ECM)-rich hydrogel and provided with a culture medium containing a defined cocktail of growth factors. Under these conditions, adult stem cells proliferate and spontaneously form three-dimensional (3D) cell clusters, the so-called organoids. Organoid cultures were initially established from murine small intestinal epithelial stem cells. However, the method has since been adapted for other epithelial stem cell types. Here, we describe a protocol for the generation and characterization of murine oral mucosal organoid cultures. Primary epithelial cells are isolated from murine tongue tissue, embedded into an ECM hydrogel, and cultured in a medium containing: epidermal growth factor (EGF), R-spondin, and fibroblast growth factor (FGF) 10. Within 7 to 14 days of initial seeding, the resulting organoids can be passaged for further expansion and cryopreservation. We additionally present strategies for the characterization of established organoid cultures via 3D whole-mount imaging and gene-expression analysis. This protocol may serve as a tool to investigate oral epithelial stem cell behavior ex vivo in a reductionist manner.

We present a method for the generation and characterization of oral mucosal organoid cultures derived from the tongue epithelium of adult mice.

The mucous lining covering the inside of our mouth, the oral mucosa, is a highly compartmentalized tissue and can be subdivided into the buccal mucosa, ...

Co-Culture of Murine Small Intestine Epithelial Organoids with Innate Lymphoid Cells

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