The authors would like to thank Ms. A. De Paoli and Ms. Georgie Wray-McCann for technical assistance. The authors acknowledge use of the facilities and technical assistance of Monash Histology Platform, Department of Anatomy and Developmental Biology, Monash University. The laboratory is supported by funding from the National Health and Medical Research Council (NHMRC) to RLF (APP1079930, APP1107930). RLF is supported by a Senior Research Fellowship from the NHMRC (APP1079904). KD and MC are both supported by Monash Graduate Scholarships. KD is also supported by the Centre for Innate Immunity and Infectious Diseases, Hudson Institute of Medical Research, while MC has an International Postgraduate Scholarship from the Faculty of Medicine, Nursing and Health Sciences, Monash University. Research at the Hudson Institute of Medical Research is supported by the Victorian Government&#8217;s Operational Infrastructure Support Program.

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<li>Kong, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+sensitive+and+specific+PCR+method+to+detect+Helicobacter+felis+in+a+conventional+mouse+model%5BTitle%5D)+AND+%22Clinical+and+Vaccine+Immunology%22%5BJournal%5D)">A sensitive and specific PCR method to detect Helicobacter felis in a conventional mouse model.</a> Clinical and Vaccine Immunology. 3 (1), 73-78 (1996).</li>
<li>Ng, G., Every, A., McGuckin, M., Sutton, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Increased+Helicobacter+felis+colonization+in+male+129%2FSv+mice+fails+to+suppress+gastritis%5BTitle%5D)+AND+%22Gut+Microbes%22%5BJournal%5D)">Increased Helicobacter felis colonization in male 129/Sv mice fails to suppress gastritis.</a> Gut Microbes. 2 (6), 358-360 (2011).</li>
<li>Ferrero, R. L., Ave, P., Radcliff, F. J., Labigne, A., Huerre, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Outbred+mice+with+long-term+Helicobacter+felis+infection+develop+both+gastric+lymphoid+tissue+and+glandular+hyperplastic+lesions%5BTitle%5D)+AND+%22The+Journal+of+Pathology%22%5BJournal%5D)">Outbred mice with long-term Helicobacter felis infection develop both gastric lymphoid tissue and glandular hyperplastic lesions.</a> The Journal of Pathology. 191 (3), 333-340 (2000).</li>
</ol>

The authors have nothing to disclose.

This protocol describes the use of an in vivo mouse model for Helicobacter infection. The critical steps of the procedure are the: 1) preparation of Helicobacter inocula containing viable and motile bacteria; 2) delivery of the appropriate numbers of bacteria to the mouse via intragastric gavage; 3) detection of bacterial infection by colony counting and/or PCR; and 4) processing of gastric tissues to enable the assessment of histopathology in infected stomachs. Further suggestions for modifica...

Helicobacter pylori is a spiral-shaped, Gram-negative, human gastric pathogen present in all populations across the world, with infection rates in developing countries estimated to be in the order of 80%1. Although most H. pylori-infected individuals are asymptomatic, some develop more severe diseases, ranging from peptic ulceration to gastric cancer2. H. pylori-associated cancers are broadly characterized either by malignant changes in epithelial cells (GECs) or by the formation of extra-nodal lymphoid tissues in the stomach, resulting in gastric adenocarcinoma or mucosa-associated lymphoid tissue (MALT) lymphoma, respectively. H. pylori is highly adapted to survive in the harsh ecological niche of the stomach due to the presence of various virulence factors and mechanisms facilitating its adherence, growth and metabolism in this niche. In particular, virulent strains of H. pylori possess the 40 kb cag Pathogenicity Island (cagPAI) that encodes approximately 30 genes required for the production of a Type 4 secretion system (T4SS)3,4. cagPAI-positive H. pylori strains are associated with the induction of higher levels of chronic inflammation in the host, which has been implicated as an essential precursor of gastric adenocarcinoma5.
In vivo animal models, particularly mice, have been highly informative by allowing researchers to investigate the relative contributions of host, bacterial and environmental factors on H. pylori infection and disease outcome6. Studies have previously demonstrated that prolonged H. pylori infection of mice on the C57BL/6 genetic background results in the development of chronic gastritis and gland atrophy, both hallmarks of H. pylori infection7. Furthermore, infection with the related feline/canine bacterial species, H. felis, has been shown to induce MALT formation in mice with similar pathology and disease progression as seen in human MALT lymphoma8,9. The most commonly used H. pylori isolate in mouse colonization studies is the &#8220;Sydney Strain 1&#8221; (SS1) strain10, which is cagPAI+ but has a non-functional T4SS (T4SS&#8722;)11. Other widely used strains include H. pylori B128 7.13 (cagPAI+/T4SS+)12 and X47-2AL (cagPAI-/T4SS&#8722;)13. For H. felis infections, the strain CS1 (&#8220;Cat Spiral 1&#8221;, cagPAI-/T4SS&#8722;) is generally used14.
Herein, we provide a protocol describing the preparation of Helicobacter inocula for in vivo infection, the procedure for intragastric gavage of mice, as well as methods for the processing of tissues for the study of histopathological changes in the stomach. In particular, this article will focus on the histological methods used to visualize bacterial colonization and assess histopathological changes, including MALT formation, in the gastric mucosa of infected mice. Some of the methods described here may be adapted to the study of other gut pathogens such as S. Typhimurium or C. rodentium.

<table><tbody><tr><td>&lt;strong&gt;Bacteriological reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Oxoid Blood Agar Base No.2</td><td>Thermo Fischer Scientific</td><td>CM0271B</td><td>Dissolve in deinonized water prior to sterilization</td></tr><tr><td>Premium Defibrinated Horse blood</td><td>Australian Ethical Biologicals</td><td>PDHB100</td><td></td></tr><tr><td>Bacto Brain Heart Infusion Broth</td><td>BD Bioscience</td><td>237500</td><td>Dissolve in deinonized water prior to sterilization</td></tr><tr><td>CampyGen gas packs</td><td>Thermo Fischer Scientific</td><td>CN0035A/CN0025A</td><td></td></tr><tr><td>&lt;strong&gt;Histological reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Formalin, neutral buffered, 10%</td><td>Sigma Aldrich</td><td>HT501128</td><td></td></tr><tr><td>Absolute alcohol, 100% Denatured</td><td>ChemSupply</td><td>AL048-20L-P</td><td></td></tr><tr><td>Isopropanol (2-propanol)</td><td>Merck</td><td>100995</td><td></td></tr><tr><td>Xylene (sulphur free)</td><td>ChemSupply</td><td>XT003-20L</td><td></td></tr><tr><td>Mayer&#039;s Haematoxylin</td><td>Amber Scientific</td><td>MH-1L</td><td>Filter before use</td></tr><tr><td>Eosin, Aqueous Stain</td><td>Amber Scientific</td><td>EOCA-1L</td><td>Filter before use</td></tr><tr><td>Wright-Giemsa Stain, modified</td><td>Sigma Aldrich</td><td>WG80-2.5L</td><td>Dilute before use (20% Giemsa, 80% deionized water)</td></tr><tr><td>Histolene</td><td>Grale Scientific</td><td>11031/5</td><td></td></tr><tr><td>DPX mounting medium</td><td>VWR</td><td>1.00579.0500</td><td></td></tr><tr><td>&lt;strong&gt;Molecular biology reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Qubit dsDNA BR Assay Kit</td><td>Thermo Fischer Scientific</td><td>Q32850</td><td></td></tr><tr><td>Oligonucleotides</td><td>Sigma Aldrich</td><td></td><td>The annealing temperature of&lt;em&gt; ureB&lt;/em&gt; primers used in this study is 61 &amp;deg;C</td></tr><tr><td>GoTaq Flexi DNA Polymerase</td><td>Promega</td><td>M8291</td><td>Kit includes 10x PCR buffer and Magnesium Chloride</td></tr><tr><td>dNTPs</td><td>Bioline</td><td>BIO-39028</td><td>Dilute to 10 mM in sterile nuclease free water before use</td></tr><tr><td>Molecular Grade Agarose</td><td>Bioline</td><td>BIO-41025</td><td></td></tr><tr><td>Sodium Hydrogen Carbonate</td><td>Univar (Ajax Fine Chemicals)</td><td>A475-500G</td><td></td></tr><tr><td>Magnesium Sulphate Heptahydrate</td><td>Chem-Supply</td><td>MA048-500G</td><td></td></tr><tr><td>&lt;strong&gt;Antibiotics&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Vancomycin</td><td>Sigma Aldrich</td><td>V2002-1G</td><td>Dissolve in deionized water</td></tr><tr><td>Polymyxin B</td><td>Sigma Aldrich</td><td>P4932-5MU</td><td>Dissolve in deionized water</td></tr><tr><td>Trimethoprim (&amp;ge;98% HPLC)</td><td>Sigma Aldrich</td><td>T7883</td><td>Dissolve in 100% (absolute) Ethanol</td></tr><tr><td>Amphotericin</td><td>Amresco (Astral Scientific)</td><td>E437-100MG</td><td>Dissolve in deionized water</td></tr><tr><td>Bacitracin from &lt;em&gt;Bacillus licheniformis&lt;/em&gt;</td><td>Sigma Aldrich</td><td>B0125</td><td>Dissolve in deionized water</td></tr><tr><td>Naladixic acid</td><td>Sigma Aldrich</td><td>N8878</td><td>Dissolve in deionized water</td></tr><tr><td>&lt;strong&gt;Other reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Methoxyflurane (Pentrhox)</td><td>Medical Developments International</td><td>Not applicable</td><td></td></tr><tr><td>Paraffin Wax</td><td>Paraplast Plus, Leica Biosystems</td><td>39601006</td><td></td></tr><tr><td>&lt;strong&gt;Equipment and plasticware&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Oxoid Anaerobic Jars</td><td>Thermo Fischer Scientific</td><td>HP0011/HP0031</td><td></td></tr><tr><td>COPAN Pasteur Pipettes</td><td>Interpath Services</td><td>200CS01</td><td></td></tr><tr><td>Eppendorf 5810R centrifuge</td><td></td><td></td><td>Collect bacterial pellets by centrifugation at 2,200 rpm for 10 mins at 4 &amp;deg;C</td></tr><tr><td>23 g precision glide needle</td><td>BD Bioscience</td><td>301805</td><td></td></tr><tr><td>Parafilm M</td><td>Bemis, VWR</td><td>PM996</td><td></td></tr><tr><td>Portex fine bore polythene tubing</td><td>Smiths Medical</td><td>800/100/200</td><td></td></tr><tr><td>Plastic feeding catheters</td><td>Instech&amp;nbsp; Laboratories</td><td>FTP20-30</td><td></td></tr><tr><td>1 mL tuberculin luer slip disposable syringes</td><td>BD Bioscience</td><td>302100</td><td></td></tr><tr><td>Eppendorf micropestle for 1.2 - 2 mL tubes</td><td>Sigma Aldrich</td><td>Z317314</td><td>Autoclavable polypropylene pestles used for stomach homogenization</td></tr><tr><td>GentleMACs Dissociator</td><td>Miltenyi Biotec</td><td>130-093-235</td><td>Use a pre-set gentleMACS Programs for mouse stomach tissue</td></tr><tr><td>M Tubes (orange cap)</td><td>Miltenyi Biotec</td><td>30-093-236</td><td></td></tr><tr><td>Qubit Fluorometer</td><td>Thermo Fischer Scientific</td><td>Q33216</td><td></td></tr><tr><td>Sterile plastic loop</td><td>LabServ</td><td>LBSLP7202</td><td></td></tr><tr><td>Cold Plate, Leica EG1160 Embedding System</td><td>Leica Biosystems</td><td>Not applicable</td><td></td></tr><tr><td>Tissue-Tek Base Mould System, Base Mold 38 x 25 x 6</td><td>Sakura, Alphen aan den Rijn</td><td>4124</td><td></td></tr><tr><td>Tissue-Tek III Uni-Casette System</td><td>Sakura, Alphen aan den Rijn</td><td>4170</td><td></td></tr><tr><td>Microtome, Leica RM2235</td><td>Leica Biosystems</td><td></td><td></td></tr><tr><td>Charged SuperFrost Plus glass slides</td><td>Menzel Glaser, Thermo Fischer Scientific</td><td>4951PLUS4</td><td></td></tr></tbody></table>

mouse models of helicobacter infection and gastric pathologies

Helicobacter pylori is a gastric pathogen that is present in half of the global population and is a significant cause of morbidity and mortality in humans. Several mouse models of gastric Helicobacter infection have been developed to study the molecular and cellular mechanisms whereby H. pylori bacteria colonize the stomach of human hosts and cause disease. Herein, we describe protocols to: 1) prepare bacterial suspensions for the in vivo infection of mice via intragastric gavage; 2) determine bacterial colonization levels in mouse gastric tissues, by polymerase chain reaction (PCR) and viable counting; and 3) assess pathological changes, by histology. To establish Helicobacter infection in mice, specific pathogen-free (SPF) animals are first inoculated with suspensions (containing &#8805;105 colony-forming units, CFUs) of mouse-colonizing strains of either Helicobacter pylori or other gastric Helicobacter spp. from animals, such as Helicobacter felis. At the appropriate time-points post-infection, stomachs are excised and dissected sagittally into two equal tissue fragments, each comprising the antrum and body regions. One of these fragments is then used for either viable counting or DNA extraction, while the other is subjected to histological processing. Bacterial colonization and histopathological changes in the stomach may be assessed routinely in gastric tissue sections stained with Warthin-Starry, Giemsa or Haematoxylin and Eosin (H&#38;E) stains, as appropriate. Additional immunological analyses may also be undertaken by immunohistochemistry or immunofluorescence on mouse gastric tissue sections. The protocols described below are specifically designed to enable the assessment in mice of gastric pathologies resembling those in human-related H. pylori diseases, including inflammation, gland atrophy and lymphoid follicle formation. The inoculum preparation and intragastric gavage protocols may also be adapted to study the pathogenesis of other enteric human pathogens that colonize mice, such as Salmonella Typhimurium or Citrobacter rodentium.

This protocol describes an oral gavage technique to achieve intragastric infection with H. pylori or H. felis in murine mouse models (Figure 1). Following euthanasia, stomachs are removed, weighed and divided into 2 equal halves comprising the antrum, body and non-glandular regions of gastric tissues (Figure 2). The non-glandular region is removed prior to performing any analyses.
Successful colonization of animals i...

Watch this Scientific Journal Video about Mouse Models Of Helicobacter Infection And Gastric Pathologies at JoVE.com

Mouse Models Of Helicobacter Infection And Gastric Pathologies

Mice represent an invaluable in vivo model to study infection and diseases caused by gastrointestinal microorganisms. Here, we describe the methods used to study bacterial colonization and histopathological changes in mouse models of Helicobacter pylori-related disease.

Mouse Models Of Helicobacter Infection And Gastric Pathologies

Helicobacter pylori is a gastric pathogen that is present in half of the global population and is a significant cause of morbidity and mortality in ...

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19.9K Views.  Hudson Institute of Medical Research. Mice represent an invaluable in vivo model to study infection and diseases caused by gastrointestinal microorganisms. Here, we describe the methods used to study bacterial colonization and histopathological changes in mouse models of Helicobacter pylori-related disease.

Video: Mouse Models Of Helicobacter Infection And Gastric Pathologies

Transurethral Induction of Mouse Urinary Tract Infection

Uropathogenic bacterial strains of interest are grown on agar. Generally, uropathogenic E. coli (UPEC) and other strains can be grown overnight on Luria-Bertani (LB) agar at 37&deg;C in ambient air. UPEC strains grow as yellowish-white translucent colonies on LB agar. Following confirmation of appropriate colony morphology, single colonies are then picked to be cultured in broth. LB broth can be used for most uropathogenic bacterial strains. Two serial, overnight LB broth cultures can be employed to enhance expression of type I pili, a well-defined virulence factor for uropathogenic bacteria. Broth cultures are diluted to the desired concentration in phosphate buffered saline (PBS). Eight to 12 week old female mice are placed under isoflurane anesthesia and transurethrally inoculated with bacteria using polyethylene tubing-covered 30 gauge syringes. Typical inocula, which must be empirically determined for each bacterial/mouse strain combination, are 106 to 108 cfu per mouse in 10 to 50 microliters of PBS. After the desired infection period (one day to several weeks), urine samples and the bladder and both kidneys are harvested. Each organ is minced, placed in PBS, and homogenized in a Blue Bullet homogenizer. Urine and tissue homogenates are serially diluted in PBS and cultured on appropriate agar. The following day, colony forming units are counted.

This video will demonstrate methods to transurethrally induce mouse urinary tract infections and quantify the extent of resulting infections.

Uropathogenic bacterial strains of interest are grown on agar. Generally, uropathogenic E. coli (UPEC) and other strains can be grown overnight on ...

A Double Humanized BLT-mice Model Featuring a Stable Human-Like Gut Microbiome and Human Immune System

Humanized mice (hu-mice) that feature a functional human immune system have fundamentally changed the study of human pathogens and disease. They can be used to model diseases that are otherwise difficult or impossible to study in humans or other animal models. The gut microbiome can have a profound impact on human health and disease. However, the murine gut microbiome is very different than the one found in humans. &#160;There is a need for improved pre-clinical hu-mice models that have an engrafted human gut microbiome. Therefore, we created double hu-mice that feature both a human immune system and stable human-like gut microbiome. NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice are one of the best animals for humanization due to their high level of immunodeficiency. However, germ-free NSG mice, and various other important germ-free mice models are not currently commercially available. Further, many research settings do not have access to gnotobiotic facilities, and working under gnotobiotic conditions can often be expensive and time consuming. Importantly, germ-free mice have several immune deficiencies that exist even after the engraftment of microbes. Therefore, we developed a protocol that does not require germ-free animals or gnotobiotic facilities. To generate double hu-mice, NSG mice were treated with radiation prior to surgery to create bone-marrow, liver, thymus-humanized (hu-BLT) mice. The mice were then treated with broad spectrum antibiotics to deplete the pre-existing murine gut microbiome. After antibiotic treatment, the mice were given fecal transplants with healthy human donor samples via oral gavage. Double hu-BLT mice had unique 16S rRNA gene profiles based on the individual human donor sample that was transplanted. Importantly, the transplanted human-like microbiome was stable in the double hu-BLT mice for the duration of the study up to 14.5 weeks post-transplant.

We describe a novel method for generating double humanized BLT-mice that feature a functional human immune system and a stable engrafted human-like gut microbiome. This protocol can be followed without the need for germ-free mice or gnotobiotic facilities.

Humanized mice (hu-mice) that feature a functional human immune system have fundamentally changed the study of human pathogens and disease. They can be ...

Biochemistry

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. ...

Risk Prediction Model of Gastric Low-Grade Intraepithelial Neoplasia

Establishment and Evaluation of a Risk Prediction Model for Pathological Escalation of Gastric Low-Grade Intraepithelial Neoplasia

The study aims to explore the risk factors for pathological escalation after endoscopic surgery for gastric low-grade intraepithelial neoplasia (LGIN) and to establish and evaluate a risk prediction model for LGIN. A total of 120 patients diagnosed with gastric LGIN by biopsy and endoscopic submucosal dissection (ESD) between November 2020 and June 2022 were retrospectively analyzed. Gender, age, Helicobacter pylori (HP) infection, lesion size, lesion location, morphology, gastric mucosal congestion, nodules status, surface ulceration and erosion, and ME-observation of all patients were collected and divided into upgraded and non-upgraded groups according to the biopsy and ESD postoperative pathological diagnosis results. Independent risk factors for pathological escalation after ESD surgical treatment were screened by logistic regression analysis, and a risk prediction model was established. Among the 120 patients with gastric LGIN, 49 patients developed postoperative pathological upgrading; the rate of pathological upgrading was 40.83%. Among them, 42 patients were upgraded to high-grade intraepithelial neoplasia (HGIN), 1 case was upgraded to advanced gastric cancer, and 6 cases were upgraded to early gastric carcinoma (EGC). Univariate analysis showed that age, lesion size, gastric mucosal congestion, surface ulcers, and erosion were significantly different between the groups (p &lt; 0.05). Multivariate Logistic regression analysis revealed that age &#8805;60 years, focal length &#8805;2 cm, gastric mucosal congestion, and surface ulceration and erosion were independent risk factors for postoperative pathological escalation in patients with gastric LGIN. Final joint probability forecasting model for P = 1/[1 + e(26.515-0.161 x &#946;1-0.357 x &#946;2+0.039 x &#946;3-0.269 x &#946;4)]. Age, lesion size &#8805;2 cm, gastric mucosal congestion, and lesion surface ulceration and erosion are risk factors for postoperative pathological upgrading in patients with gastric LGIN. The risk prediction model established in this study based on risk factors has predictive value and can provide a scientific reference for the clinical treatment of patients with gastric LGIN.

Author Spotlight: Advancing Early Detection and Treatment of Gastrointestinal Tumors

Here, we performed a systematic evaluation of patients diagnosed with gastric low-grade intraepithelial neoplasia by previous endoscopic biopsy and obtained a pathological diagnosis by complete resection of the lesion by endoscopic submucosal dissection (ESD), analyzing factors that potentially increase the risk of pathological escalation.

The study aims to explore the risk factors for pathological escalation after endoscopic surgery for gastric low-grade intraepithelial neoplasia (LGIN) and ...

Cancer Research

Surgical Models of Gastroesophageal Reflux with Mice

Multiple surgical procedures have been reported to induce gastroesophageal reflux in animals. Herein, we report three surgical models with mice aiming to induce reflux of gastric contents, duodenal contents or mixed contents. Surgical procedures and general principles have been described in detail. A researcher with surgical experience should be able to grasp the technique after a short period of practice. After surgery, most mice can survive and develop reflux esophagitis similar to that in humans. However, it should be noted that histological differences between mouse and human esophagus are the inherent limitations of these surgical models. If used for research on Barrett&#8217;s esophagus and adenocarcinoma, these procedures may need to be combined with genetic modifications.

This article demonstrates surgical procedures of gastroesophageal reflux with mice. These models are useful tools for research on mechanisms and treatment of gastroesophageal reflux disease and potentially Barrett&#8217;s esophagus and esophageal adenocarcinoma.

Multiple surgical procedures have been reported to induce gastroesophageal reflux in animals. Herein, we report three surgical models with mice aiming to ...

Medicine

Mouse- and Human-derived Primary Gastric Epithelial Monolayer Culture for the Study of Regeneration

In vitro studies of gastric wound repair typically involves the use of gastric cancer cell lines in a scratch-wound assay of cellular proliferation and migration. One critical limitation of such assays, however, is their homogenous assortment of cellular types. Repair is a complex process which demands the interaction of several cell types. Therefore, to study a culture devoid of cellular subtypes, is a concern that must be overcome if we are to understand the repair process. The gastric organoid model may alleviate this issue whereby the heterogeneous collection of cell types closely reflects that of the gastric epithelium or other native tissues in vivo. Demonstrated here is a novel, in vitro scratch-wound assay derived from human or mouse 3-dimensional organoids which can then be transferred to a gastric epithelial monolayer as either intact organoids or as a single cell suspension of dissociated organoids. The goal of the protocol is to establish organoid-derived gastric epithelial monolayers that can be used in a novel scratch-wound assay system to study gastric regeneration.

Here we describe a protocol for establishing and culturing human- and mouse-derived 3-dimensional (3D) gastric organoids, and the method for the transfer of 3D organoids to a 2-dimensional monolayer. The use of the gastric epithelial monolayer as a novel scratch-wound assay for regeneration studies is also described.

In vitro studies of gastric wound repair typically involves the use of gastric cancer cell lines in a scratch-wound assay of cellular proliferation and ...

Adapting Gastrointestinal Organoids for Pathogen Infection and Single Cell Sequencing under Biosafety Level 3 (BSL-3) Conditions

Human intestinal organoids constitute the best cellular model to study pathogen infections of the gastrointestinal tract. These organoids can be derived from all sections of the GI tract (gastric, jejunum, duodenum, ileum, colon, rectum) and, upon differentiation, contain most of the cell types that are naturally found in each individual section. For example, intestinal organoids contain nutrient-absorbing enterocytes, secretory cells (Goblet, Paneth, and enteroendocrine), stem cells, as well as all lineage-specific differentiation intermediates (e.g.,&#160;early or immature cell types). The greatest advantage in using gastrointestinal tract-derived organoids to study infectious diseases is the possibility of precisely identifying which cell type is targeted by the enteric pathogen and to address whether the different sections of the gastrointestinal tract and their specific cell types similarly respond to pathogen challenges. Over the past years, gastrointestinal models, as well as organoids from other tissues, have been employed to study viral tropism and the mechanisms of pathogenesis. However, utilizing all the advantages of using organoids when employing highly pathogenic viruses represents a technical challenge and requires strict biosafety considerations. Additionally, as organoids are often grown in three dimensions, the basolateral side of the cells is facing the outside of the organoid while their apical side is facing the inside (lumen) of the organoids. This organization poses a challenge for enteric pathogens as many enteric infections initiate from the apical/luminal side of the cells following ingestion. The following manuscript will provide a comprehensive protocol to prepare human intestinal organoids for infection with enteric pathogens by considering the infection side (apical vs. basolateral) to perform single-cell RNA sequencing to characterize cell-type-specific host/pathogen interactions. This method details the preparation of the organoids as well as the considerations needed to perform this work under biosafety level 3 (BSL-3) containment conditions.

Adapting Gastrointestinal Organoids for Pathogen Infection and Single Cell Sequencing under Biosafety Level 3 BSL-3 Conditions

This protocol describes how to infect human intestinal organoids from either their apical or basolateral side to characterize host/pathogen interactions at the single-cell level using single-cell RNA sequencing (scRNAseq) technology.

Human intestinal organoids constitute the best cellular model to study pathogen infections of the gastrointestinal tract. These organoids can be derived ...

Establishing an Infection Model Through Microinjection of Gastric Organoids with Pathogenic Bacteria

Source: Bartfeld, S. et al. <a href="https://app.jove.com/v/53359">Organoids as Model for Infectious Diseases: Culture of Human and Murine Stomach Organoids and Microinjection of Helicobacter Pylori.</a> J. Vis. Exp.(2015).
This video demonstrates a method to establish a gastric organoid infection model by microinjecting gastric pathogen Helicobacter pylori into the lumen of the organoids. This model helps in studying infection-induced changes in gastric cells.

Ustalenie modelu infekcji poprzez mikroiniekcję organoidów żołądkowych z bakteriami chorobotwórczymi

1. Passage of Organoid Cultures

	Add 1 ml of cold basal medium to the basement matrix in the well. Using a 1 ml micropipette, vigorously pipette up and ...

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

In Vitro and In Vivo Models

Quantification of Helicobacter pylori Load in an Infected Mouse Stomach

Source: D&#39;Costa, K. et al., <a href="https://app.jove.com/v/56985">Mouse Models Of Helicobacter Infection And Gastric Pathologies.</a> J. Vis. Exp. (2018)
In this video, we describe a protocol to quantify Helicobacter pylori load in mouse stomach tissues post-infection to confirm its colonization.

Kwantyfikacja obciążenia Helicobacter pylori w zakażonym żołądku myszy

Source: D&#39;Costa, K. et al., Mouse Models Of Helicobacter Infection And Gastric Pathologies. J. Vis. Exp. (2018)
In this video, we describe a protocol ...

Immunopathology

Assay and Analysis

An Intragastric Gavage Technique for Controlled Helicobacter Infection in Mice

Source: D&#39;Costa, K. et al., <a href="https://app.jove.com/v/56985">Mouse Models Of Helicobacter Infection And Gastric Pathologies.</a>&#160;J. Vis. Exp.&#160; (2018)
This video demonstrates the establishment of Helicobacter pylori infection through the intragastric gavage technique in a mouse model. The bacterial suspension is administered via a catheter inserted through the mouth into the stomach. Inoculated bacteria colonize and establish an infection in the stomach, which can be used to study the host-pathogen interaction.

An Intragastric Gavage Technique for Controlled Helicobacter Infection in Mice

Technika zgłębnika dożołądkowego w kontrolowanym zakażeniu Helicobacter u myszy

Source: D&#39;Costa, K. et al., Mouse Models Of Helicobacter Infection And Gastric Pathologies.&#160;J. Vis. Exp.&#160; (2018)
This video demonstrates the ...

Mouse Models Of Helicobacter Infection And Gastric Pathologies

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Przeglądaj więcej filmów

Transurethral Induction of Mouse Urinary Tract Infection

A Double Humanized BLT-mice Model Featuring a Stable Human-Like Gut Microbiome and Human Immune System

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

Establishment and Evaluation of a Risk Prediction Model for Pathological Escalation of Gastric Low-Grade Intraepithelial Neoplasia

Surgical Models of Gastroesophageal Reflux with Mice

Mouse- and Human-derived Primary Gastric Epithelial Monolayer Culture for the Study of Regeneration

Adapting Gastrointestinal Organoids for Pathogen Infection and Single Cell Sequencing under Biosafety Level 3 (BSL-3) Conditions

Ustalenie modelu infekcji poprzez mikroiniekcję organoidów żołądkowych z bakteriami chorobotwórczymi

Kwantyfikacja obciążenia Helicobacter pylori w zakażonym żołądku myszy

Technika zgłębnika dożołądkowego w kontrolowanym zakażeniu Helicobacter u myszy