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. Establishment of Gastric Organoid Culture
Note: This protocol can be used for the isolation of gastric glands from mouse or human tissue. It is advised to use tissue of approximately 1 cm&#178;. Human tissue can be obtained from gastric resections or biopsies.
<ol>
	<li>Preparation of Material 
	Note: The used basement matrix is Matrigel. Keep the basement matrix on ice at all times. Store the basement matrix at -20 &#176;C and thaw on ice before use. Basal medium refers to Advanced ...

<ol>
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F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organoid+Models+of+Human+and+Mouse+Ductal+Pancreatic+Cancer.">Organoid Models of Human and Mouse Ductal Pancreatic Cancer.</a> Cell. 160 (1-2), 324-338 (2015).</li><li>Karthaus, W. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+multipotent+luminal+progenitor+cells+in+human+prostate+organoid+cultures.">Identification of multipotent luminal progenitor cells in human prostate organoid cultures.</a> Cell. 159 (1), 163-175 (2014).</li><li>Bartfeld, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+and+single-cell+imaging+of+NF-kappaB+oscillations+using+monoclonal+cell+lines.">High-throughput and single-cell imaging of NF-kappaB oscillations using monoclonal cell lines.</a> BMC cell. 11, 21 (2010).</li><li>Blanchard, T. G., Nedrud, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laboratory+Maintenance+of+Helicobacter+Species.">Laboratory Maintenance of Helicobacter Species.</a> Curr Protoc Microbiol. , (2006).</li><li>Van Es, J. H., de Geest, N., van de Born, M., Clevers, H., Hassan, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intestinal+stem+cells+lacking+the+Math1+tumour+suppressor+are+refractory+to+Notch+inhibitors.">Intestinal stem cells lacking the Math1 tumour suppressor are refractory to Notch inhibitors.</a> Nat Commun. 1 (2), 1-5 (2010).</li><li>Andersson-Rolf, A., Fink, J., Mustata, R. C., Koo, B. -. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Video+Protocol+of+Retroviral+Infection+in+Primary+Intestinal+Organoid+Culture.">A Video Protocol of Retroviral Infection in Primary Intestinal Organoid Culture.</a> J Vis Exp. (90), (2014).</li><li>Stange, D. E., Koo, B. -. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiated+Troy++Chief+Cells+Act+as+Reserve+Stem+Cells+to+Generate+All+Lineages+of+the+Stomach+Epithelium.">Differentiated Troy+ Chief Cells Act as Reserve Stem Cells to Generate All Lineages of the Stomach Epithelium.</a> Cell. 155 (2), 357-368 (2013).</li><li>Van de Wetering, M., Sancho, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+beta-catenin/TCF-4+complex+imposes+a+crypt+progenitor+phenotype+on+colorectal+cancer+cells.">The beta-catenin/TCF-4 complex imposes a crypt progenitor phenotype on colorectal cancer cells.</a> Cell. 111 (2), 241-250 (2002).</li><li>Schumacher, M. 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Hans Clevers is an inventor on several patents for organoid culture. Otherwise the authors have nothing to disclose.

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...

The study of pathogens relies on adequate model systems to mimic the in vivo infection. For some infective agents, adequate model systems are lacking while some of the used systems are far from optimal. One example is the gastric bacterium&#160;Helicobacter pylori (H. pylori), which is causally related to development of gastric cancer. Yet in absence of a more suitable cell culture system, many studies that aim to analyze the molecular mechanisms underlying cancer development use cancer cell lines, which represent the endpoint of the cancerous cascade. Primary, non-transformed cells would be a better model for these studies. However, primary cells are only available from a small number of donors and cannot be cultured over longer periods of time. In recent years, stem cell research has made significant progress to provide new sources for primary cell cultures for the study of infection biology.
Cultures from three stem cell sources have been used: Embryonic stem cells (ESC), induced pluripotent stem cells (iPSC) or adult stem cells. They have been used to model infections with viruses, such as Cytomegalovirus1,2 or Hepatitis C Virus3&#8211;7, parasites, such as Plasmodium falciparum8 or Toxoplasma gondii9, and bacteria, such as Bacterioides thetaiotaomicron10 or Salmonella enterica11. Most recently, several approaches have been published to model infection with H. pylori with organoids derived from ESC or iPS cells12, mouse adult stem cells21,22 or human adult stem cells13&#8211;15.
The development of organoid cultures from adult stem cells originated from a study, in which single stem cells isolated from murine intestinal epithelium were seeded into a 3-dimensional matrix and embedded in medium that mimicked the environment of the intestinal stem cells containing EGF as mitogen, R-spondin to enhance Wnt signalling and Noggin to inhibit bone morphogenic protein (BMP) signalling16. Notably these cultures do not require co-culture with mesenchymal cells. In these conditions, the stem cells proliferate and form small structures with domains harboring cells of the intestinal crypts, and domains that contain the cells of the intestinal villus. The organoids thus self-organize to mimic the in vivo situation. Today, adult stem cells from many murine and humane tissues can be grown in vitro and self-organize into organoids that resemble their in vivo counterpart, such as small intestine and colon17, stomach13,18, liver19,20, pancreas21 and prostate22.
Here we provide a video protocol to culture mouse or human gastric organoids from adult stem cells and microinject them with H. pylori. This protocol is based on previous reports13,18. This method can be adapted for culturing and infecting other organoid cultures such as intestinal organoids.

<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.

This protocol allows isolation of gastric glands (Figure 2). Glands are seeded into basement matrix, which solidifies as drop within a well, providing a 3 dimensional framework rich in laminin and collagen to allow the glands grow into organoids (Figure 3). Organoids typically start as small cysts and within 12-16 days, they expand into spheres with a diameter of 50-300 &#181;m (Figure 4). Some organoids will stay cystic, some will develop small buddings. The latter is u...

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.

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

organoids-as-model-for-infectious-diseases-culture-human-murine

Research

JoVE Journal

Immunology and Infection

34.8K ビュー.  University Medical Centre Utrecht. 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.