Name: organoids as model for infectious diseases: culture of human and murine stomach organoids and microinjection of helicobacter pylori
Uploaded: 2015-11-12T15:00:00
Description: Watch this Scientific Journal Video about Organoids as Model for Infectious Diseases: Culture of Human and Murine Stomach Organoids and Microinjection of Helicobacter Pylori at JoVE.com

This work was supported by EU Marie Curie Fellowship (EU/300686-InfO) to S.B. and a Research Prize from the United European Gastroenterology Foundation to H.C. We would like to thank Harry Begthel, Jeroen Korving and the Hubrecht Imaging Center for technical assistance, Meritxell Huch for help with initial organoid culture and Yana Zavros for discussion.

1. إنشاء المعدة عضي الثقافة ملاحظة: هذا البروتوكول يمكن استخدامها لعزل الغدد المعدة من الماوس أو الأنسجة البشرية. ينصح لاستخدام الأنسجة من حوالي 1 سم مربع. ويمكن الحصول على الأنسجة البشرية من استئصال المعدة أو الخزعات. <ol style=";text-al...

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This protocol describes the use of ever-expanding, untransformed primary organoids from adult stem cells for infection biology. Critical steps are i) the isolation of viable glands, ii) expansion of organoids and iii) the microinjection. Below are some suggestions for modifications, troubleshooting and technical considerations. 
Compared to other isolation methods, which use vigorous shaking or pipetting to release glands and can be equally successful, the technique presented here has the adva...

تعتمد دراسة مسببات الأمراض على أنظمة نموذج كافية لمحاكاة العدوى في الجسم الحي. وبالنسبة لبعض العوامل المعدية، ونظم نموذج كافية وتفتقر بينما بعض الأنظمة المستخدمة هي بعيدة كل البعد عن المثالية. ومن الأمثلة على ذلك بكتيريا المعدة هيليكوباكتر بيلوري (H. بيلوري)، والذي يرتبط سببيا لتطوير سرطان المعدة. ولكن في غياب نظام زراعة الخلايا أكثر ملاءمة، العديد من الدراسات التي تهدف إلى تحليل الآليات الجزيئية الكامنة وراء تطور مرض السرطان السرطان استخدام خطوط الخلايا، التي تمثل نقطة النهاية لسلسلة السرطانية. أن الخلايا الأولية، غير حولت يكون نموذجا أفضل لهذه الدراسات. ومع ذلك، هي الخلايا الأولية متوفرة فقط من عدد قليل من المانحين ولا يمكن أن تكون مثقف لفترات أطول من الوقت. في السنوات الأخيرة، جعلت أبحاث الخلايا الجذعية تقدما كبيرا لتوفير مصادر جديدة للثقافات الخلية الأولية لدراسة البيولوجيا العدوى. ثقافات منوقد استخدمت ثلاثة مصادر الخلايا الجذعية: الخلايا الجذعية الجنينية (ESC)، والخلايا التي يسببها المحفزة الجذعية (التوجيهية) أو الخلايا الجذعية البالغة. تم استخدامها في تصميم نموذج العدوى بفيروسات، مثل الفيروس المضخم للخلايا 1،2 أو الالتهاب الكبدي الفيروسي ج 3-7، والطفيليات، مثل المنجلية 8 أو 9 التوكسوبلازما، والبكتيريا، مثل باكتيريويدز thetaiotaomicron 10 أو السالمونيلا الملهبة لل11. وفي الآونة الأخيرة، تم نشر العديد من المناهج لنموذج عدوى H. بيلوري مع organoids المستمدة من ESC أو الجذع خلايا 12، الخلايا الجذعية الماوس الكبار 21،22 أو الإنسان الخلايا الجذعية البالغة 13-15. تطوير الثقافات عضي من الخلايا الجذعية البالغة نشأت من الدراسة، والتي كانت المصنفة الخلايا الجذعية واحدة معزولة عن ظهارة الأمعاء الفئران في مصفوفة 3 الأبعاد وجزءا لا يتجزأ من المتوسطة التي تحاكي البيئة من الخلايا الجذعية المعوية تحتوي EGF كما mitogen، R-spondin لتعزيز إشارات Wnt ورأس لمنع البروتين morphogenic العظام (BMP) مما يشير إلى 16. ولا سيما هذه الثقافات لا تتطلب ثقافة مشتركة مع خلايا اللحمة المتوسطة. في هذه الظروف، فإن الخلايا الجذعية تتكاثر وتشكل هياكل صغيرة مع المجالات التي تؤوي خلايا الخبايا المعوية، والمجالات التي تحتوي على خلايا زغابة المعوية. وبالتالي فإن organoids تنظيم الذاتي لتقليد الوضع في الجسم الحي. اليوم، والخلايا الجذعية البالغة من العديد من الفئران والأنسجة إنسانية يمكن زراعتها في المختبر وتقرير المصير، تنتظم organoids التي تشبه في الجسم الحي نظيرهما، مثل الأمعاء الدقيقة والقولون 17، 13،18 المعدة والكبد 19،20، 21 و البنكرياس البروستاتا 22. نحن هنا نقدم بروتوكول الفيديو إلى الماوس الثقافة أو organoids المعدة البشري من سل الجذعية للبالغينليرة سورية وmicroinject لهم H. بيلوري. ويستند هذا البروتوكول على تقارير سابقة 13،18. هذه الطريقة يمكن تكييفها للزراعة واصابة الثقافات عضي أخرى مثل organoids الأمعاء.

<table border="0">
	<tbody>
		<tr>
			<td>Medium</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Invitrogen</td>
			<td>15630-056</td>
			<td></td>
		</tr>
		<tr>
			<td>Advanced DMEM/F12</td>
			<td>Invitrogen</td>
			<td>12634-028</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel, GFR, phenol free</td>
			<td>BD</td>
			<td>356231</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX</td>
			<td>Invitrogen</td>
			<td>35050-079</td>
			<td>Stock concentration 200 mM, final concentration 2 mM</td>
		</tr>
		<tr>
			<td>B27</td>
			<td>Invitrogen</td>
			<td>17504-044</td>
			<td>Stock concentration 50 x, final concentration 1x</td>
		</tr>
		<tr>
			<td>N-Acetylcysteine</td>
			<td>Sigma-Aldrich</td>
			<td>A9165-5G</td>
			<td>Stock concentration 500 mM, final concentration 1 mM</td>
		</tr>
		<tr>
			<td>Murine recombinant EGF</td>
			<td>Invitrogen</td>
			<td>PMG8043</td>
			<td>Stock concentration 500 &#181;g/mL, final concentration 50 ng/mL</td>
		</tr>
		<tr>
			<td>Human recombinant FGF10</td>
			<td>Peprotech</td>
			<td>100-26</td>
			<td>Stock concentration 100 &#181;g/mL, final concentration 200 ng/mL</td>
		</tr>
		<tr>
			<td>TGF&#946;i A-83-01</td>
			<td>Tocris</td>
			<td>2939</td>
			<td>Stock concentration 500 &#181;M, final concentration 2 &#181;M&#160;</td>
		</tr>
		<tr>
			<td>Nicotinamide</td>
			<td>Sigma-Aldrich</td>
			<td>N0636</td>
			<td>Stock concentration 1 M, final concentration 10 mM&#160;</td>
		</tr>
		<tr>
			<td>[Leu15]-Gastrin</td>
			<td>Sigma-Aldrich</td>
			<td>G9145</td>
			<td>Stock concentration 100 &#181;M, final concentration 1 nM</td>
		</tr>
		<tr>
			<td>RHOKi Y-27632</td>
			<td>Sigma-Aldrich</td>
			<td>Y0503</td>
			<td>Stock concentration 10 mM, final concentration 10 &#181;M</td>
		</tr>
		<tr>
			<td>Wnt3A conditioned medium</td>
			<td></td>
			<td></td>
			<td>Stable cell line generated in the Clevers Lab. Final concentration 50%. Cells can be obtained from Hans Clevers.</td>
		</tr>
		<tr>
			<td>R-spondin1 conditioned medium</td>
			<td></td>
			<td></td>
			<td>Stable cell line generated in the Kuo Lab. Final concentration 10%. Cell line can be obtained from Calvin Kuo, Stanford.</td>
		</tr>
		<tr>
			<td>Noggin conditioned medium</td>
			<td></td>
			<td></td>
			<td>Stable cell line generated in the Clevers Lab. Final concentration 10%. Cells can be obtained from Hans Clevers.</td>
		</tr>
		<tr>
			<td>R-spondin3</td>
			<td>R&#38;D</td>
			<td>3500-RS/CF</td>
			<td>Alternative source for R-spondin. This has been tested on human intestine organoids (1 &#181;g/mL), but not yet on gastric organoids.</td>
		</tr>
		<tr>
			<td>Noggin</td>
			<td>Peprotech</td>
			<td>120-10</td>
			<td>Alternative source for noggin. This has been tested on human intestine organoids (100 ng/mL), but not yet on gastric organoids.</td>
		</tr>
		<tr>
			<td>TrypLE express</td>
			<td>Life Technologies</td>
			<td>12605036</td>
			<td>Enzymatic dissociation solution&#160;</td>
		</tr>
		<tr>
			<td>CoolCell&#174; Alcohol-Free Cell Freezing Containers</td>
			<td>biocision</td>
			<td>BCS-405</td>
			<td></td>
		</tr>
		<tr>
			<td>Recovery Cell Culture Freezing Medium</td>
			<td>Invitrogen</td>
			<td>12648-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotics</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Primocin</td>
			<td>Invivogen</td>
			<td>ant-pm-1</td>
			<td>An antibiotics composition agains bacteria and fungi. It is helpful after initiation of a culture. For long term culture you can switch to other antibiotics or none.</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Invitrogen</td>
			<td>15140-122</td>
			<td>Stock concentration 10000/10000 U/mL, final concentration 100/100 U/mL. Can be used alternatively to Primocin in long term culture.</td>
		</tr>
		<tr>
			<td>Other</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezers</td>
			<td>Neolabs</td>
			<td>2-1033</td>
			<td>Tweezers with fine tips are helpful for the removal of muscle layer from the tissue.</td>
		</tr>
		<tr>
			<td>4 Well Multidishes</td>
			<td>Thermo Scientific</td>
			<td>144444</td>
			<td>You can use other Multidishes. These were particularly helpful for microinjections because they have a low outer rim and allow more mobility for the manipulator.</td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>Narishige</td>
			<td>M-152</td>
			<td></td>
		</tr>
		<tr>
			<td>Microinjector</td>
			<td>Narishige</td>
			<td>IM-5B</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Leica</td>
			<td>MZ75</td>
			<td></td>
		</tr>
		<tr>
			<td>Workbench</td>
			<td>Clean Air</td>
			<td></td>
			<td>Custom made to fit the stereomicroscope in ML2 condition</td>
		</tr>
		<tr>
			<td>Cappillaries</td>
			<td>Harvard Apparatus</td>
			<td>GC100T-10</td>
			<td>1 mm outer diameter, 0,78 mm inner diameter.</td>
		</tr>
		<tr>
			<td>Micropipette Puller</td>
			<td>Sutter Instruments</td>
			<td>Flaming Brown Micropipette Puller</td>
			<td></td>
		</tr>
		<tr>
			<td>anti Cag A antibody</td>
			<td>Santa Cruz</td>
			<td>sc-25766</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

هذا البروتوكول يسمح عزل الغدد المعدة (الشكل 2). هي المصنفة الغدد في مصفوفة الطابق السفلي، والذي يتصلب قطرة داخل بئر، وتوفير إطار الأبعاد 3 غني laminin والكولاجين للسماح للغدد تنمو لتصبح organoids (الشكل 3). بدء Organoids عادة الخراجات الصغيرة، وداخل 12-16 يوما، والت...

Watch this Scientific Journal Video about Organoids as Model for Infectious Diseases: Culture of Human and Murine Stomach Organoids and Microinjection of Helicobacter Pylori at JoVE.com

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

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.

Organoids كنموذج للأمراض المعدية: ثقافة الإنسان وOrganoids المعدة الفئران وهيليكوباكتر بيلوري Microinjection من

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

Research

JoVE Journal

Immunology and Infection

Detection of Infectious Virus from Field-collected Mosquitoes by Vero Cell Culture Assay

الكشف عن الفيروسات المعدية التي جمعت من الحقل عن طريق البعوض فيرو خلية الفحص الثقافة

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many ...

High Resolution Electron Microscopy of the Helicobacter pylori Cag Type IV Secretion System Pili Produced in Varying Conditions of Iron Availability

High Resolution Electron Microscopy of the Helicobacter pylori Cag Type IV Secretion System Pili Produced in Varying Conditions of Iron Availability

عالية الدقة المجهر الإلكتروني ل<em&gt; هيليكوباكتر بيلوري</em&gt; CAG النوع الرابع إفراز نظام بيلي المنتجة في ظروف مختلفة من توافر الحديد

Helicobacter pylori is a helical-shaped, gram negative bacterium that colonizes the human gastric niche of half of the human population1,2.  H. pylori is ...

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

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

ال<em&gt; فيفو السابقين</em&gt; الثقافة والتعرف على الأنماط مستقبلات تحفيز ماوس المعوية Organoids

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

Isolation and Flow Cytometric Characterization of Murine Small Intestinal Lymphocytes

العزلة وتدفق Cytometric توصيف الفئران الصغيرة المعوية اللمفاويات

The intestines &#8212; which contain the largest number of immune cells of any organ in the body &#8212; are constantly exposed to foreign antigens, both ...

The Madagascar Hissing Cockroach as an Alternative Non-mammalian Animal Model to Investigate Virulence, Pathogenesis, and Drug Efficacy

أن مدغشقر الهسهسة الصرصور كنموذج بديل غير الثدييات حيوانية للتحقيق في الفوعة، والمرضية، وفعالية المخدرات

Many aspects of innate immunity are conserved between mammals and insects. An insect, the Madagascar hissing cockroach from the genus Gromphadorhina, can ...

Mouse Models Of Helicobacter Infection And Gastric Pathologies

Mouse Models Of Helicobacter Infection And Gastric Pathologies

نماذج الماوس الملوية العدوى وأمراض المعدة

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 ...

Preparation of Murine Submandibular Salivary Gland for Upright Intravital Microscopy

إعداد موريني الغدة اللعابية اللهاة تستقيم مجهرية إينترافيتال

The submandibular salivary gland (SMG) is one of the three major salivary glands, and is of interest for many different fields of biological research, ...

Identification of Mouse and Human Antibody Repertoires by Next-Generation Sequencing

تحديد الماوس وجسم الإنسان مبدع بتسلسل الجيل المقبل

The immense adaptability of antigen recognition by antibodies is the basis of the acquired immune system. Despite our understanding of the molecular ...

Isolation of Single Intracellular Bacterial Communities Generated from a Murine Model of Urinary Tract Infection for Downstream Single-cell Analysis

عزل واحد المجتمعات البكتيرية داخل الخلايا التي تم إنشاؤها من نموذج موريني لعدوى المسالك البولية لتحليل خلية واحدة أسفل المجرى

In this article, we outline a procedure used to isolate individual intracellular bacterial communities from a mouse that has been experimentally infected ...

Retrograde Parotid Gland Infusion through Stensen's Duct in a Non-Human Primate for Vectored Gene Delivery

ضخ الغدة الباروتية الرجعية من خلال قناة ستينسن في الرئيسيات غير البشرية لتسليم الجينات المتجهة

Salivary glands are an attractive tissue target for gene therapy with promising results already leading to human trials. They are inherently capable of ...