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. Istituzione di gastrico organoide Cultura Nota: Questo protocollo può essere utilizzato per l&#39;isolamento delle ghiandole gastriche da mouse o tessuti umani. Si consiglia di usare il tessuto di circa 1 cm². Tessuto umano può essere ottenuto da resezioni gastriche o biopsie. <ol><li> Preparazione del materiale Nota: La matrice seminterrato utilizzato è Matrigel. Mantenere la matrice seminterrato su ghiaccio in ogni momento. Conservare la matrice seminterrato a -20 ° C e sc...

<ol>
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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...

Lo studio di patogeni si basa su adeguati sistemi modello per imitare l&#39;infezione in vivo. Per alcuni agenti infettivi, adeguati sistemi modello sono carenti, mentre alcuni dei sistemi utilizzati sono ben lungi dall&#39;essere ottimale. Un esempio è il batterio gastrico Helicobacter pylori (H. pylori), che è causalmente legata alla sviluppo del cancro gastrico. Tuttavia, in assenza di un sistema di coltura cellulare più adatto, molti studi che mirano ad analizzare i meccanismi molecolari alla base dello sviluppo del cancro linee cellulari tumorali uso, che rappresentano il punto finale della cascata cancerose. , Cellule non trasformate primarie sarebbe un modello migliore per questi studi. Tuttavia, le cellule primarie sono disponibili solo da un piccolo numero di donatori e non possono essere coltivate su periodi di tempo più lunghi. Negli ultimi anni, la ricerca sulle cellule staminali ha compiuto progressi significativi per fornire nuove fonti per colture cellulari primarie per lo studio della biologia infezione. Culture disono state utilizzate tre fonti di cellule staminali: le cellule staminali embrionali (ESC), le cellule indotte pluripotenti staminali (IPSC) o le cellule staminali adulte. Essi sono stati utilizzati per modellare infezioni da virus, come il citomegalovirus 1,2 o virus dell&#39;epatite C 3 - 7, parassiti, come Plasmodium falciparum 8 o 9 Toxoplasma gondii, e batteri, quali Bacterioides thetaiotaomicron 10 o Salmonella enterica 11. Più di recente, diversi approcci sono stati pubblicati per modellare l&#39;infezione da H. pylori con organoidi derivati ​​da ESC o iPS cellule 12, le cellule staminali adulte del mouse 21,22 o umani cellule staminali adulte di 13 - 15. Lo sviluppo delle colture organoide da cellule staminali adulte origine da uno studio, in cui le cellule staminali singole isolate da murino epitelio intestinale sono state seminate in una matrice 3-dimensionale eincorporato in mezzo che imitava l&#39;ambiente delle cellule staminali intestinali che contengono FEG mitogeno, R-spondina per migliorare la segnalazione Wnt e Noggin di inibire la proteina morfogenetica dell&#39;osso (BMP) di segnalazione 16. In particolare queste colture non richiedono co-coltura con cellule mesenchimali. In queste condizioni, le cellule staminali proliferano e formano piccole strutture con domini ospitare cellule delle cripte intestinali, e domini che contengono le cellule del villi intestinali. I organoidi quindi auto-organizzano per simulare la situazione in vivo. Oggi, le cellule staminali adulte da molte murino e tessuti umani possono essere coltivate in vitro e auto-organizzarsi in organoidi che ricordano la loro controparte in vivo, come l&#39;intestino tenue e colon 17, stomaco 13,18, fegato 19,20, pancreas e 21 22 della prostata. Qui forniamo un protocollo video al topo cultura o organoidi gastriche umane da cel staminali adultels e li microinject con H. pylori. Questo protocollo si basa sulle precedenti relazioni 13,18. Questo metodo può essere adattato per la coltura e infettare altre culture organoide come organoidi intestinali.

<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.

Questo protocollo permette isolamento delle ghiandole gastriche (Figura 2). Ghiandole vengono seminate in matrice seminterrato, che solidifica come goccia all&#39;interno di un pozzo, fornendo una struttura tridimensionale 3 ricco di laminina e collagene per consentire le ghiandole crescono in organoidi (Figura 3). Organoidi tipicamente iniziano come piccole cisti e entro 12-16 giorni, si espandono in sfere con un diametro di 50-300 micron (Figura 4). Alcuni organoidi r...

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.

Organoidi come modello per le malattie infettive: cultura dei diritti dell&#39;uomo e organoidi stomaco murini e microiniezione dell&#39;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. ...

Research

JoVE Journal

Immunology and Infection

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

Il rilevamento del virus infettivo da Campo-zanzare raccolte mediante test coltura cellulare Vero

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

Ricerca

Immunologia e infezioni

Rapid Identification of Gram Negative Bacteria from Blood Culture Broth Using MALDI-TOF Mass Spectrometry

Rapida identificazione dei batteri Gram-negativi dal sangue brodo di coltura Uso MALDI-TOF

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional ...

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

Ad alta risoluzione microscopia elettronica del<em&gt; Helicobacter pylori</em&gt; Cag secrezione di tipo IV Sistema Pili Prodotto in diverse condizioni di Ferro Disponibilità

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 ...

Isolation and Flow Cytometric Characterization of Murine Small Intestinal Lymphocytes

Isolamento e caratterizzazione citometria a flusso di Murine Piccolo intestinale Linfociti

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

Il Madagascar sibilo scarafaggio come modello alternativo Non mammiferi animali per studiare la virulenza, patogenesi e l'efficacia dei farmaci

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

Modelli murini di patologie gastriche e infezione da Helicobacter

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

Preparazione della ghiandola salivare sottomandibolare murino per microscopia Intravital verticale

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

Identificazione dei Mouse e dei repertori di anticorpo umano mediante sequenziamento di nuova generazione

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

Isolamento di singole comunità batteriche intracellulari generati da un modello murino di infezione delle vie urinarie per analisi unicellulare a valle

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

Infusione retrograda della ghiandola parotide attraverso il dotto di Stensen in un primate non umano per la consegna di geni vettoriali

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