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 DMEM/F12 supplemented with HEPES; an appropriate glutamine source such as Glutamax and appropriate antibiotics such as 1x Primocin. The incubator used is a standard cell culture incubator (37 &#176;C, humidified atmosphere, 5% CO2)
	<ol>
		<li>Sterilize preparation utensils by autoclaving or using 100% isopropyl alcohol: sharp forceps, scissors, a scalpel and a glass microscopy slide.</li>
		<li>The day before isolation, place a 24 well cell culture plate in the incubator.</li>
		<li>Prepare the basement matrix according to manufacturers&#8217; recommendation. Prepare aliquots of 1 ml to avoid repeated freeze thaw cycles.</li>
		<li>Prepare 500 ml chelating buffer and cool on ice. Prepare 1x buffer from sterile distilled water with 5.6 mM Na2HPO4, 8.0 mM KH2PO4, 96.2 mM NaCl, 1.6 mM KCl, 43.4 mM sucrose, 54.9 mM D-sorbitol, 0.5 mM DL-dithiothreitol (CAUTION), pH 7. If planning several isolations, prepare a 5x concentrated solution without the DL-dithiothreitol. Sterile filter the buffer. Before isolation, dilute the 5x buffer with sterile water to 1x and add DL-dithiothreitol just before isolation.</li>
		<li>Prepare and dilute all growth factors according to manufacturers&#8217; recommendations. Use small-sized aliquots and avoid freeze-thaw cycles. Functional growth factors are crucial for the results.</li>
		<li>Cool basal medium on ice and cool the centrifuge to 4 &#176;C.</li>
		<li>Warm another aliquot of basal medium to 37 &#176;C.</li>
	</ol>
	</li>
	<li>Isolation of Glands
	<ol>
		<li>Collect approximately 1 cm&#178; of tissue in ice cold basal medium. Keep on ice until preparation. While it is preferable to process tissue as soon as possible, store tissue in medium on ice if necessary.</li>
		<li>Place the tissue in a 10 cm Petri dish and cover it with cold 1x chelation buffer. Wash by gently moving back and forth.</li>
		<li>Place the tissue in a dry 10 cm Petri dish. Using forceps, carefully remove the mucus as well as the muscle layer under a stereomicroscope. Wash the tissue in cold 1x chelating buffer by gently moving back and forth.</li>
		<li>Place 1 cm2 tissue on a new, dry 10 cm Petri dish. Using scissors cut it into 20-50 small pieces of approximately 2-5 mm&#178; size.</li>
		<li>Using forceps, place all pieces into a 50 ml tube.</li>
		<li>Take a sterile 10 ml plastic pipette. Wet the pipette in 1x chelating buffer. Keep using this same pipette throughout the procedure.</li>
		<li>Add 10 ml cold 1x chelating buffer to the 50 ml tube. Wash the pieces by vigorously pipetting up and down 10 times. Let the pieces settle. Remove the supernatant.</li>
		<li>Repeat washing (step 1.2.7) until the supernatant is clear. This takes approximately 50-100 ml of 1x chelating buffer for 1 cm&#178; of human tissue.</li>
		<li>Incubate the tissue pieces in 20 ml 1x chelating buffer supplemented with 10 mM EDTA at RT for 10 min with gentle inverting every 2 min. 
		Note: Time and temperature of this incubation can be optimized for either speed and/or preservation of glandular architecture. They can range from several hours incubation at 4 &#176;C on a rolling platform to 5 min at 37 &#176;C in a shaker with 200 rpm. Good starting points for mouse stomach are 30 min at 4 &#176;C on a rolling platform and for human stomach, 10 min at RT.</li>
		<li>Pipette once gently up and down using the 10 ml pipette. Allow the pieces to settle. Discard the supernatant.</li>
		<li>Using the pipette, transfer the pieces to a clean 10 cm Petri dish. Remove the liquid.</li>
		<li>Place a glass microscopy slide on top of the tissue pieces. Observe the intact tissue under an inverted microscope for cell culture with 10X magnification.</li>
		<li>Apply pressure to the glass slide and Petri dish. Hold both in hand (with sterile gloves) and pressing the glass with the thumb. The rim of the tissue will appear cloudy indicating that glands are released out of the tissue.</li>
		<li>Observe the released glands under an inverted microscope for cell culture with 10X magnification.</li>
		<li>Collect glands and tissue in 30 ml cold basal medium. Let large tissue fragments settle.</li>
		<li>Collect the supernatant containing glands in two 15 ml tubes.</li>
		<li>Centrifuge 5 min at 200 x g and 4 &#176;C. Discard supernatant. The pellet contains isolated glands. Keep them on ice.</li>
	</ol>
	</li>
	<li>Seeding of Glands
	<ol>
		<li>For the seeding, keep basement matrix ice-cold at all times, so it will stay liquid.</li>
		<li>Alternative 1: Seed 6 wells of a dilution row.
		<ol>
			<li>Suspend the pellet in 60 &#181;l of basement matrix.</li>
			<li>Prepare 5 sterile 1.5 ml tubes on ice with each containing 50 &#181;l of basement matrix. Transfer 10 &#181;l of glands/basement matrix suspension into the first 1.5 ml tube. Suspend the glands and transfer 10 &#181;l of this solution to the next 1.5 ml tube. Repeat for the next tubes.</li>
		</ol>
		</li>
		<li>Alternative 2: Seed a calculated amount of glands per well. Start with 100 glands per 50 &#181;l basement matrix in one well of a 24 well plate.
		<ol>
			<li>Carefully suspend the pelleted glands in 10 ml basal medium. Place 50 &#181;l on a clean 10 ml Petri dish. Count number of glands under an inverted microscope for cell culture with 10X magnification.</li>
			<li>Calculate the concentration of glands in the solution. Transfer the volume that contains 400 glands to a new 15 ml tube.</li>
			<li>Centrifuge 5 min at 200 x g and 4 &#176;C. Discard supernatant and keep the tube on ice. Add 200 &#181;l basement matrix and carefully resuspend. Keep on ice.</li>
		</ol>
		</li>
		<li>Take the 37 &#176;C warm 24-well plate (see step 1.1.2) from the incubator. Place one drop of 50 &#181;l glands in basement matrix in the center of the well. The drop will form a dome.</li>
		<li>Carefully transfer the plate back to the cell culture incubator. Let the basement matrix solidify 10 min.</li>
		<li>Prepare warm medium with all growth factors.
		<ol>
			<li>For human glands: Supplement the basal medium with the following growth factors for human gastric organoids: 50 ng/ml EGF, 10% noggin-conditioned medium, 10% R-spondin1&#8211;conditioned medium, 50% Wnt-conditioned medium, 200 ng/ml fibroblast growth factor (FGF)10, 1 nM gastrin, and 2 &#181;M TGFbeta inhibitor and 10 &#181;M rho-associated coiled coil forming protein serine/threonine kinase inhibitor (RHOKi).</li>
			<li>For murine glands: Supplement the basal medium with the following growth factors for mouse gastric organoids: 50 ng/ml EGF, 10% noggin-conditioned medium, 10% R-spondin1&#8211;conditioned medium, 50% Wnt-conditioned medium, 100 ng/ml FGF10, 10 nM gastrin, 0.5 mM TGFbeta inhibitor and 10 &#181;M RHOKi.</li>
		</ol>
		</li>
		<li>Take the plate from the incubator. Carefully add 500 &#181;l medium with all growth factors (1.3.6) to each well without disturbing the basement matrix. Place it back to the incubator.</li>
		<li>Refresh the medium 3 times per week (every 2-3 days). For refreshing medium, remove the old medium with leaving the drop of basement matrix intact. Carefully add fresh, warm medium with all growth factors. Do not add RHOKi. Only add RHOKi directly after seeding of glands or after splitting of the organoids (step 2).</li>
	</ol>
	</li>
</ol>
2. Passage of Gastric Organoid Culture Before Microinjection.
Note: Every type of organoid culture has its own doubling time. Mouse intestinal as well as gastric cultures are usually split 1:5 every 5-7 days. Human intestinal cultures are split 1:5 every 10-12 days. Human gastric cultures are split 1:5 every 14 days. If initiated from single cells, human gastric organoids may also take 20 days to form properly. It is a good sign if budding structures surround the central lumen. In this protocol, organoids are split in 4 well plates for microinjection. Maintenance of organoids follows the same protocol, but can use any other cell culture plate, such as standard 24 well cell culture plates.
<ol>
	<li>Preparation of Material
	<ol>
		<li>Autoclave Pasteur pipettes.</li>
		<li>The day before passage, place a 4 well cell culture plate in the incubator at 37 &#176;C. The 4 well plates have low edges and allow micromanipulation.</li>
		<li>Prepare the basement matrix according to manufacturers&#8217; recommendation. On the day of passaging, thaw basement matrix on ice.</li>
		<li>Prepare and dilute all growth factors according to manufacturers&#8217; recommendations.</li>
		<li>Cool basal medium on ice and cool the centrifuge to 4 &#176;C.</li>
	</ol>
	</li>
	<li>Passage of Organoid Cultures
	<ol>
		<li>Take the plate with the organoids from the incubator. Remove medium from one well.</li>
		<li>Add 1 ml of cold basal medium to the basement matrix in the well. Using a 1 ml micropipette, vigorously pipette up and down until the basement matrix is broken up. Transfer to a 15 ml tube. Place on ice.</li>
		<li>Take a glass Pasteur pipette and a fire source such as a Bunsen burner.
		<ol>
			<li>Hold the tip of the pipette into the fire until the opening of the tip has narrowed from 1.5 mm to about 0.5 mm (Figure 1). Let the pipette cool to RT.</li>
			<li>Wet the pipette in basal medium. An optimally narrowed pipette will take up the medium visibly slower than an un-narrowed pipette, but it will still allow pipetting well.</li>
		</ol>
		</li>
		<li>Take up organoids in the pipette and vigorously pipette 10x up and down. The breaking of the organoids can be observed by eye.</li>
		<li>Add 9 ml of cold basal medium and centrifuge for 5 min at 200 x g and 4 &#176;C.</li>
		<li>Carefully discard all supernatant. Suspend the pellet in 250 &#181;l basement matrix. Keep on ice.</li>
		<li>Take the 37 &#176;C warm 4 well culture plate and place a drop of 50 &#181;l organoids/basement matrix in each well.</li>
		<li>Transfer the plate carefully back to the incubator. Let the basement matrix solidify at 37 &#176;C for 10 min.</li>
		<li>Prepare appropriate medium for either mouse or human organoids as listed in step 1.3.6.</li>
		<li>Take the plate from the incubator and overlay each basement matrix drop with 500 &#181;l medium.</li>
		<li>Transfer the plate to the incubator.</li>
		<li>Refresh the medium 3 times per week every 2-3 days (see 1.3.8.).</li>
		<li>For freezing of organoids, mechanically disrupt organoids as described for splitting in this protocol (Steps 2.2.1-2.2.5.). Suspend the organoid fragments in 500 ml&#160;freezing medium and freeze to -80 &#176;C using cryotubes and a freezing container. For long-term storage, transfer the tubes to liquid nitrogen.</li>
		<li>For thawing of organoids, prepare the culture plate, basement matrix, growth factors, cold and warm medium as outlined in steps 1.1.2, 1.1.3., 1.1.5., 1.1.6., 1.1.7. Warm the frozen vial using a water bath, and transfer cells immediately after thawing to a 15 ml tube with 10 ml cold basal medium. Centrifuge and plate the cells as outlined in 2.2.5 to 2.2.12. 
		Note: Microinjection of organoids is laborious, but allows the unique possibility to study interaction in 3D.</li>
		<li>If 3D studies are not needed, seed organoids in two dimensions. For this, disrupt the organoids as laid out in steps 2.2.1 to 2.2.6. Add 2,250 &#181;l cold medium with all growth factors to 250 &#181;l of basement matrix with organoid fragments.
		<ol>
			<li>Seed in 5 wells of a 24 well plate with 500 &#181;l per well. Change medium 3 times a week, with carefully replacing the upper 400 &#181;l only, to keep the basement matrix at the bottom of the plate undisturbed. 
			Note: Organoids will attach to the cell culture plate and expand in 2D. Electron microscopy has shown that the apical side of the cells faces the medium in the well (data not shown).</li>
			<li>Before infection, exchange all medium including basement matrix, to allow bacteria to attach to the cells. A similar protocol has also been described for infection with H. pylori14. 2D cell layers may contain not all cell types that are present in 3D organoids.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
3. Microinjection of Organoid Culture.
Note: This protocol can be used to microinject bacteria into organoids. It may be helpful to start the injection with organoids that are more permissive to microinjection. For example, mouse gastric organoids can grow into very large cystic organoids that are easy to target.
<ol>
	<li>Preparation of Material
	<ol>
		<li>Pull a glass capillary into two injection needles using a micropipette puller, according to manufacturers&#8217; recommendation. The micropipette puller will heat the glass capillary in the middle and will pull the capillary into two parts, thereby generating two glass needles. It is practical to pull several needles and store them in a clean petri dish.</li>
		<li>To allow bacterial infection, remove the antibiotics from all media for at least 3 medium changes (=one week). This will dilute antibiotics from basement matrix. If using a bacterial strain that is resistant to a specific antibiotic, add this specific antibiotic to minimize chances for contamination.</li>
		<li>Prepare the work bench for microinjections. For work at different biosafety levels it may be necessary to place a stereomicroscope inside a sterile bench. In this protocol, inject H. pylori under biosafety level 2 conditions under a stereomicroscope inside a sterile bench.</li>
		<li>Culture the bacteria according to standard protocols13,23.</li>
		<li>To estimate the multiplicity of infection (MOI), estimate two parameters: The number of bacteria and the number of cells per organoid as outlined below. The MOI can correlate with results, for example with hosts IL-8 mRNA expression13.
		<ol>
			<li>To calculate the bacterial density in the infection solution, first establish the standard curve of optical density and colony forming units (CFU) according to standard protocols24. An optical density of 0.1 at 550 nm should yield approximately 107&#160;CFU/ml.</li>
			<li>To estimate the number of cells per organoid, count number of organoids in 1 well. Disrupt organoids as outlined in steps 2.2.1-2.2.4. After centrifugation 5 min at 200 x g and 4 &#176;C, add 1 ml of enzymatic dissociation solution and resuspend.
			<ol>
				<li>Incubate at 37 &#176;C with repeated shaking for 5-10 min. Determine whether the cells have dissociated into single cells under the microscope (inverted microscope such as Leica DMIL at 10X magnification). If necessary prolong the incubation.</li>
				<li>Count cells per organoid using a hemocytometer and calculate the number of cells per organoid. Human gastric organoids with a diameter of about 200 &#181;m have approximately 4,000 cells. Injection of 0.2 &#181;l of a bacterial solution with 1 x 109 bacteria per ml results in an approximate MOI of 50.</li>
			</ol>
			</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Injection of Organoids. 
	Note: For microinjection, the glass needle is stabilized in a holder, which can be maneuvered in 3 dimensions by a micromanipulator. Organoids remain within the basement matrix within the well. Due to size and positioning in the well, not all organoids are equally amenable to injection. Usually, the 30 largest organoids in one well can be targeted in 5 min.
	<ol>
		<li>Harvest bacteria and wash them in basal medium according to standard protocols24.</li>
		<li>Calculate the number of bacteria per ml according to the optical density (see step 3.1.5.1). Dilute the bacteria to 1 x 109 bacteria per ml&#160;in basal medium.</li>
		<li>Take a glass needle. Using forceps break the tip so that the opening will be approximately 10 &#181;m wide.</li>
		<li>Insert the needle into the injection holder and fix it to the micromanipulator.</li>
		<li>Take up approximately 10 &#181;l bacterial solution into the needle.</li>
		<li>Place a 4 well plate with low edges containing organoid cultures under the stereomicroscope.</li>
		<li>Navigating with the micromanipulator, target a single organoids with the needle. Position the needle close to the organoid and then insert the needle into the organoid with one swift movement. Inject approximately 0.2 &#181;l bacterial solution into each organoid using the microinjector.</li>
	</ol>
	</li>
	<li>Harvest Organoids for Any Analysis.
	<ol>
		<li>As example, fix organoids after 4 hr. Remove supernatant and add 1 ml 2% formaldehyde and incubate 20 min at RT or O/N at 4 &#176;C. Organoids can be processed for immunohistochemistry according to standard procedures25.</li>
	</ol>
	</li>
</ol>

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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>

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. ...

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Immunology and Infection

34.9K Views.  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.