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. Inrättande av Gastric organoida Culture Notera: Detta protokoll kan användas för isoleringen av gastriska körtlar från mus eller human vävnad. Det rekommenderas att använda vävnad av ca 1 cm. Mänsklig vävnad kan erhållas från gastriska resektioner eller biopsier. <ol><li> Framställning av material Obs: Den använda källaren matrisen är Matrigel. Håll källaren matrisen på is vid alla tidpunkter. Förvara källaren matrisen vid -20 ° C och tina på is före anvä...

<ol>
	<li>Aiuto, L., 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=Human+Induced+Pluripotent+Stem+Cell-Derived+Models+to+Investigate+Human+Cytomegalovirus+Infection+in+Neural+Cells.">Human Induced Pluripotent Stem Cell-Derived Models to Investigate Human Cytomegalovirus Infection in Neural Cells.</a> PLoS ONE. 7 (11), e49700 (2012).</li><li>Penkert, R. R., Kalejta, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Embryonic+Stem+Cell+Lines+Model+Experimental+Human+Cytomegalovirus+Latency.">Human Embryonic Stem Cell Lines Model Experimental Human Cytomegalovirus Latency.</a> mBio. 4 (3), e00298-13-e00298-13 (2013).</li><li>Roelandt, P., 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=Human+pluripotent+stem+cell-derived+hepatocytes+support+complete+replication+of+hepatitis+C+virus.">Human pluripotent stem cell-derived hepatocytes support complete replication of hepatitis C virus.</a> J Hepatol. 57 (2), 246-251 (2012).</li><li>Schwartz, R. E., Trehan, 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=Modeling+hepatitis+C+virus+infection+using+human+induced+pluripotent+stem+cells.">Modeling hepatitis C virus infection using human induced pluripotent stem cells.</a> Proc Natl Acad Sci USA. 109 (7), 2544-2548 (2012).</li><li>Shlomai, A., 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=Modeling+host+interactions+with+hepatitis+B+virus+using+primary+and+induced+pluripotent+stem+cell-derived+hepatocellular+systems.">Modeling host interactions with hepatitis B virus using primary and induced pluripotent stem cell-derived hepatocellular systems.</a> Proc Natl Acad Sci USA. 111 (33), 12193-12198 (2014).</li><li>Wu, X., 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=Productive+Hepatitis+C+Virus+Infection+of+Stem+Cell-Derived+Hepatocytes+Reveals+a+Critical+Transition+to+Viral+Permissiveness+during+Differentiation.">Productive Hepatitis C Virus Infection of Stem Cell-Derived Hepatocytes Reveals a Critical Transition to Viral Permissiveness during Differentiation.</a> PLoS Pathogens. 8 (4), e1002617 (2012).</li><li>Yoshida, T., 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=Use+of+human+hepatocyte-like+cells+derived+from+induced+pluripotent+stem+cells+as+a+model+for+hepatocytes+in+hepatitis+C+virus+infection.">Use of human hepatocyte-like cells derived from induced pluripotent stem cells as a model for hepatocytes in hepatitis C virus infection.</a> Biochem Biophys Res Commun. 416 (1-2), 119-124 (2011).</li><li>Ng, 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=Human+iPSC-Derived+Hepatocyte-like+Cells+Support+Plasmodium+Liver-Stage+Infection+In+Vitro.">Human iPSC-Derived Hepatocyte-like Cells Support Plasmodium Liver-Stage Infection In Vitro.</a> Stem Cell Report. 4 (2), (2015).</li><li>Klotz, C., Aebischer, T., Seeber, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell-derived+cell+cultures+and+organoids+for+protozoan+parasite+propagation+and+studying+host-parasite+interaction.">Stem cell-derived cell cultures and organoids for protozoan parasite propagation and studying host-parasite interaction.</a> Int J Med Microbiol. 302 (4-5), 203-209 (2012).</li><li>Engevik, M. A., 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=Loss+of+NHE3+alters+gut+microbiota+composition+and+influences+Bacteroides+thetaiotaomicron+growth.">Loss of NHE3 alters gut microbiota composition and influences Bacteroides thetaiotaomicron growth.</a> AJP: GI. 305 (10), G697-G711 (2013).</li><li>Wilson, S. S., Tocchi, A., Holly, M. K., Parks, W. C., Smith, 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=A+small+intestinal+organoid+model+of+non-invasive+enteric+pathogen-epithelial+cell+interactions.">A small intestinal organoid model of non-invasive enteric pathogen-epithelial cell interactions.</a> Mucosal Immunol. 8 (2), 352-361 (2015).</li><li>McCracken, K. W., 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=Modelling+human+development+and+disease+in+pluripotent+stem-cell-derived+gastric+organoids.">Modelling human development and disease in pluripotent stem-cell-derived gastric organoids.</a> Nature. 516 (7531), 400-404 (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=In+Vitro+Expansion+of+Human+Gastric+Epithelial+Stem+Cells+and+Their+Responses+to+Bacterial+Infection.">In Vitro Expansion of Human Gastric Epithelial Stem Cells and Their Responses to Bacterial Infection.</a> Gastroenterology. 148 (1), (2014).</li><li>Schlaermann, P., Toelle, B., 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=A+novel+human+gastric+primary+cell+culture+system+for+modelling+Helicobacter+pylori+infection+in+vitro.">A novel human gastric primary cell culture system for modelling Helicobacter pylori infection in vitro.</a> Gut. , (2014).</li><li>Bertaux-Skeirik, N., 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=CD44+Plays+a+Functional+Role+in+Helicobacter+pylori-induced+Epithelial+Cell+Proliferation.">CD44 Plays a Functional Role in Helicobacter pylori-induced Epithelial Cell Proliferation.</a> PLOS Pathogens. 11 (2), e1004663 (2015).</li><li>Sato, T., 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=Single+Lgr5+stem+cells+build+crypt+villus+structures+in+vitro+without+a+mesenchymal+niche.">Single Lgr5 stem cells build crypt villus structures in vitro without a mesenchymal niche.</a> Nature. 459 (7244), 262-265 (2009).</li><li>Sato, T., 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=Long-term+Expansion+of+Epithelial+Organoids+From+Human+Colon,+Adenoma,+Adenocarcinoma,+and+Barrett's+Epithelium.">Long-term Expansion of Epithelial Organoids From Human Colon, Adenoma, Adenocarcinoma, and Barrett's Epithelium.</a> Gastroenterology. 141 (5), 1762-1772 (2011).</li><li>Barker, N., 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=Lgr5+ve+Stem+Cells+Drive+Self-Renewal+in+the+Stomach+and+Build+Long-Lived+Gastric+Units+In+Vitro.">Lgr5+ve Stem Cells Drive Self-Renewal in the Stomach and Build Long-Lived Gastric Units In Vitro.</a> Cell Stem Cell. 6 (1), 25-36 (2010).</li><li>Huch, M., 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=In+vitro+expansion+of+single+Lgr5++liver+stem+cells+induced+by+Wnt-driven+regeneration.">In vitro expansion of single Lgr5+ liver stem cells induced by Wnt-driven regeneration.</a> Nature. 494 (7436), 247-250 (2013).</li><li>Huch, M., 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=Long-Term+Culture+of+Genome-Stable+Bipotent+Stem+Cells+from+Adult+Human+Liver.">Long-Term Culture of Genome-Stable Bipotent Stem Cells from Adult Human Liver.</a> Cell. 160 (1-2), 299-312 (2015).</li><li>Boj, S. 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. A., Aihara, 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+use+of+murine-derived+fundic+organoids+in+studies+of+gastric+physiology.">The use of murine-derived fundic organoids in studies of gastric physiology.</a> Journal Physiol. 593 (8), 1809-1827 (2015).</li><li>Schwank, G., Andersson-Rolf, A., Koo, B. -. K., Sasaki, N., Clevers, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+BAC+Transgenic+Epithelial+Organoids.">Generation of BAC Transgenic Epithelial Organoids.</a> PLoS ONE. 8 (10), e76871 (2013).</li><li>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=Controlled+gene+expression+in+primary+Lgr5+organoid+cultures.">Controlled gene expression in primary Lgr5 organoid cultures.</a> Nat Meth. 9 (1), 81-83 (2012).</li><li>Schwank, G., 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=Functional+repair+of+CFTR+by+CRISPR/Cas9+in+intestinal+stem+cell+organoids+of+cystic+fibrosis+patients.">Functional repair of CFTR by CRISPR/Cas9 in intestinal stem cell organoids of cystic fibrosis patients.</a> Cell Stem Cell. 13 (6), 653-658 (2013).</li><li>Li, V. S. W., Ng, S. 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=Wnt+Signaling+through+Inhibition+of+&#946;-Catenin+Degradation+in+an+Intact+Axin1+Complex.">Wnt Signaling through Inhibition of &#946;-Catenin Degradation in an Intact Axin1 Complex.</a> Cell. 149 (6), 1245-1256 (2012).</li><li>Van de Wetering, M., 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=Prospective+derivation+of+a+'Living+Organoid+Biobank'+of+colorectal+cancer+patients.">Prospective derivation of a 'Living Organoid Biobank' of colorectal cancer patients.</a> Cell. 161 (4), 933-945 (2015).</li></ol>

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...

Studiet av patogener bygger på lämpliga modellsystem för att efterlikna in vivo infektion. För vissa smittämnen, är lämpliga modellsystem saknas medan vissa av de använda systemen är långt ifrån optimal. Ett exempel är den gastriska bakterien Helicobacter pylori (H. pylori), som kausalt relaterad till utveckling av magcancer. Men i avsaknad av en mer lämplig cellodlingssystem, många studier som syftar analysera de molekylära mekanismerna bakom cancerutveckling användning cancercellinjer, som representerar slutpunkten för cancer kaskaden. Primära, icke-transformerade celler skulle vara en bättre modell för dessa studier. Men primära celler är endast tillgängliga från ett litet antal givare och kan inte odlas över längre tidsperioder. Under de senaste åren har stamcellsforskning gjort betydande framsteg för att ge nya källor för primära cellkulturer för studiet av infektionsbiologi. Kulturer fråntre stamcells källor har använts: Embryonala stamceller (ESC), inducerade pluripotenta stamceller (IPSC) eller adulta stamceller. De har använts för att modellera infektioner med virus, såsom cytomegalovirus 1,2 eller hepatit C-virus 3-7, parasiter, såsom Plasmodium falciparum 8 eller Toxoplasma gondii 9, och bakterier, såsom Bacterioides thetaiotaomicron 10 eller Salmonella enterica 11. Senast har flera metoder publicerats att modellera infektion med H. pylori med organoids härrör från ESC eller iPS-celler 12, mus adulta stamceller 21,22 eller mänskliga adulta stamceller 13 - 15. Utvecklingen av organoid kulturer från adulta stamceller härstammar från en studie, i vilken enstaka stamceller som isolerats från murina tarmepitel ympades i en 3-dimensionell matris ochinbäddad i medium som härmade miljön i tarm stamceller innehållande EGF som mitogen, R-spondin att förbättra Wnt-signalering och Noggin att inhibera benmorfogent protein (BMP) signalering 16. Notably dessa kulturer inte kräver samodling med mesenkymala celler. Under dessa förhållanden, stamcellerna proliferera och bilda små strukturer med domäner som härbärgerar celler av tarmkryptorna och domäner som innehåller cellerna i villi. De organoids sålunda själv organisera sig för att efterlikna in vivo situationen. Idag kan adulta stamceller från många murina och humana vävnader odlas in vitro och själv organisera sig i organoids som liknar deras in vivo motsvarighet, såsom tunntarmen och tjocktarmen 17, mage 13,18, lever 19,20, bukspottkörtel 21 och prostata 22. Här ger vi en video protokoll till kultur mus eller människa gastric organoids från adulta stamceller cells och mikroinjicera dem med H. pylori. Detta protokoll är baserad på tidigare rapporter 13,18. Denna metod kan anpassas för odling och infektera andra organoid kulturer såsom intestinala 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.

Detta protokoll möjliggör isolering av magkörtlar (Figur 2). Körtlar sås i källaren matris, som stelnar som droppe inuti en brunn, vilket ger en 3 dimensionell ram rik på laminin och kollagen för att tillåta körtlar växa till organoids (Figur 3). Organoids börjar vanligtvis som små cystor och inom 12-16 dagar, de expanderar till sfärer med en diameter av 50-300 pm (Figur 4). Vissa organoids kommer att stanna cystisk, vissa kommer att utveckla små buddings...

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 som modell för Infektionssjukdomar: Culture of Human och Murina mage Organoids och mikroinjektion av 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

Detektion av infektiösa virus från Field-in Myggor av vero-cellodling analys

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

Högupplöst elektronmikroskopi av<em&gt; Helicobacter pylori</em&gt; Cag Typ IV sekretionssystemet Pili Producerad i varierande förhållanden of Iron Tillgänglighet

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

De<em&gt; Ex vivo</em&gt; Kultur och mönsterigenkänning receptorstimulering av 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 ...

Isolation and Flow Cytometric Characterization of Murine Small Intestinal Lymphocytes

Isolering och Flödescytometriska karakterisering av murina tunntarms lymfocyter

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

Madagaskar väsande kackerlacka som en alternativ icke däggdjur djurmodell att undersöka virulens, patogenes och drogen effekt

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

Musmodeller av Helicobacter -infektion och Gastric patologier

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

Beredning av murina Submandibular spottkörteln för upprätt Intravital mikroskopi

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

Identifiering av mus och Human antikropp repertoarer av nästa generations sekvensering

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

Isolering av enda intracellulära bakteriella samhällen som genereras från en murin modell av urinvägsinfektion för nedströms Single-cell analys

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

Retrograd parotidkörtelinfusion genom Stensens kanal i en icke-mänsklig primat för vektor genleverans

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