Name: the ex vivo culture and pattern recognition receptor stimulation of mouse intestinal organoids
Uploaded: 2016-05-18T15:00:00
Description: Watch this Scientific Journal Video about The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids at JoVE.com

The authors would like to thank Dr. Sheryl Coutermarsh-Ott, Dylan McDaniel and Bettina Heid for technical discussions. The authors thank Dr. Nanda Nanthakumar for providing the Caco-2 cells. The authors also thank The Multicultural Academic Opportunities Program (MAOP) at Virginia Tech. This work was supported by the National Institute of Diabetes and Digestive and Kidney Diseases Award K01DK092355 (to I.C.A.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

All research was approved and conducted under Virginia Tech IACUC guidelines
1. Prepare R-Spondin1 Conditioned Media From HEK293T-Rspo1 Cell Line
<ol>
	<li>Generation of HEK293T-Rspondin1 cells has been previously described10. Seed HEK293T-Rspondin1 secreting cells at 5-10% confluency, approximately 8 x 105-1.7 x 106 cells, in a T-175 flask with 40 ml of 1x Dulbecco&#39;s Modified Eagle Medium (DMEM) + 10% Fetal Bovine Serum (FBS) as the growth media, and incubate at 37 &#176;C +...

<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>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>Lancaster, 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=Cerebral+organoids+model+human+brain+development+and+microcephaly.">Cerebral organoids model human brain development and microcephaly.</a> Nature. 501 (7467), 373-379 (2013).</li><li>Freshney, R. I., Freshney, M. 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> Culture of epithelial cells. 2nd edn. , (2002).</li><li>Vachon, P. H., 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=Differentiation+state-selective+roles+of+p38+isoforms+in+human+intestinal+epithelial+cell+anoikis.">Differentiation state-selective roles of p38 isoforms in human intestinal epithelial cell anoikis.</a> Gastroenterology. 123 (6), 1980-1991 (2002).</li><li>Hynds, R. E., Giangreco, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concise+review:+the+relevance+of+human+stem+cell-derived+organoid+models+for+epithelial+translational+medicine.">Concise review: the relevance of human stem cell-derived organoid models for epithelial translational medicine.</a> Stem Cells. 31 (3), 417-422 (2013).</li><li>Kaiko, G. E., Stappenbeck, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host-microbe+interactions+shaping+the+gastrointestinal+environment.">Host-microbe interactions shaping the gastrointestinal environment.</a> Trends Immunol. 35 (11), 538-548 (2014).</li><li>Kim, K. 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=Mitogenic+influence+of+human+R-spondin1+on+the+intestinal+epithelium.">Mitogenic influence of human R-spondin1 on the intestinal epithelium.</a> Science. 309 (5738), 1256-1259 (2005).</li><li>Chomczynski, P., Sacchi, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+single-step+method+of+RNA+isolation+by+acid+guanidinium+thiocyanate-phenol-chloroform+extraction:+twenty-something+years+on.">The single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction: twenty-something years on.</a> Nat Protoc. 1 (2), 581-585 (2006).</li><li>Allen, I. C., 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=NLRX1+protein+attenuates+inflammatory+responses+to+infection+by+interfering+with+the+RIG-I-MAVS+and+TRAF6-NF-kappaB+signaling+pathways.">NLRX1 protein attenuates inflammatory responses to infection by interfering with the RIG-I-MAVS and TRAF6-NF-kappaB signaling pathways.</a> Immunity. 34 (6), 854-865 (2011).</li><li>Zhang, Y. G., Wu, S., Xia, Y., Sun, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salmonella-infected+crypt-derived+intestinal+organoid+culture+system+for+host-bacterial+interactions.">Salmonella-infected crypt-derived intestinal organoid culture system for host-bacterial interactions.</a> Physiol Rep. 2 (9), (2014).</li><li>Grabinger, 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=Ex+vivo+culture+of+intestinal+crypt+organoids+as+a+model+system+for+assessing+cell+death+induction+in+intestinal+epithelial+cells+and+enteropathy.">Ex vivo culture of intestinal crypt organoids as a model system for assessing cell death induction in intestinal epithelial cells and enteropathy.</a> Cell Death Dis. 5, 1228 (2014).</li><li>Slater, T. F., Sawyer, B., Straeuli, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+Succinate-Tetrazolium+Reductase+Systems.+Iii.+Points+of+Coupling+of+Four+Different+Tetrazolium+Salts.">Studies on Succinate-Tetrazolium Reductase Systems. Iii. Points of Coupling of Four Different Tetrazolium Salts.</a> Biochim Biophys Acta. 77, 383-393 (1963).</li><li>Parry, M. F., Neu, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+N-acetylcysteine+on+antibiotic+activity+and+bacterial+growth+in+vitro.">Effect of N-acetylcysteine on antibiotic activity and bacterial growth in vitro.</a> J Clin Microbiol. 5 (1), 58-61 (1977).</li></ol>

The culture and maintenance of intestinal organoids is a procedure that can be mastered by any individual with adequate tissue culture technique. There are subtleties in passaging when compared to growing cells in a more conventional monolayer, but these subtleties are not difficult to overcome. The critical steps of this method involve being able to grow the organoids to a high enough density for optimal seeding. Experiments must be scaled down with organoids as large seeding densities that can commonly be achieved with...

The ability to harvest and culture primary organoids have been described for small intestine, colon, pancreas, liver and brain and are exciting advances germane to understanding a more physiologically representative phenomena for tissue biology1-5. The first methods describing the culture and maintenance of small intestinal organoids was reported by Sato et al. out of the lab of Hans Clevers1. Prior to this method, harvesting and culture of primary intestinal epithelial cells proved to be limited and ineffective in sustaining epithelial cell growth. Methods included dissociation of tissue via incubation with enzymes, such as collagenase and dispase, which would ultimately lead to the outgrowth of intermixed primary fibroblast cells6. These conditions would also be time restricted in sustaining the epithelial cell culture. Minimal to no epithelial cell niche would form, as the epithelial cells would enter apoptosis due to the lack of appropriate growth factors or loss of contact integrity, termed anokis7. The advent of the 3D-organoid culture system has provided a method to culture primary intestinal cells containing a spectrum of intestinal cell types in sustained culture1. These epithelial organoids have advantages over cell lines being that they are composed of several differentiated cells, and better mimic the organ they are derived from in vivo8. The process to ultimately &#34;grow a mini gut in a dish&#34; has proven to be a valuable tool for assessing the response of intestinal epithelium under different stimuli. Investigating the interaction of primary intestinal cells with microbial pathogen associated molecular patterns (PAMPs) is relevant to the field of immunology as these molecular patterns can regulate diverse responses from both host and microbe9. Not only can investigators now explore these interactions with mouse organoids, but they can be cultured from humans as well2. This technology has the potential to dramatically alter personalized medicine and it is tempting to speculate about advances that this technique will make possible in the near future.
The overall goal of this method is to provide a protocol for the culture, expansion, and treatment of intestinal organoids with a variety of stimuli. Such stimuli can ultimately range from vaccines, bacterial PAMPs, live pathogens, gastrointestinal (GI) and cancer therapeutics. The isolation and culture of mouse intestinal organoids has been adapted from Sato et al. Though there are slight deviations from the original method, the end product being organoid culture is still achieved when following this protocol. This method is focused on describing an adequate technique for proper normalization when working with non-homogenous cell structures, which must be taken into consideration when conducting an assay based on cell number.

<table>
	<tbody>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Atlanta Biologicals</td>
			<td>S11050</td>
			<td>(Section 1,3,6) Or equivalent brand</td>
		</tr>
		<tr>
			<td>Sorvall Legend XTR Centrifuge</td>
			<td>Thermo</td>
			<td></td>
			<td>(Section 1,3)</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>GE Healthcare</td>
			<td>Sh30243.01</td>
			<td>(Section 1,6) For Caco-2 and HEK293 Rspondin1 cells</td>
		</tr>
		<tr>
			<td>HEK293T-Rspondin1 secreting cell line</td>
			<td></td>
			<td></td>
			<td>(Section 1) Described and modified from Kim, K.A. et al. Lentiviral particles contained RSPO1(NM_138683) ORF cDNA cloned into a pReceiver-Lv105 backbone custom ordered and purchased from GeneCopoeia.&#160;</td>
		</tr>
		<tr>
			<td>50 ml conical tube</td>
			<td>Falcon</td>
			<td>352070</td>
			<td>(Section 1) Or equivalent brand</td>
		</tr>
		<tr>
			<td>T-175 Flask</td>
			<td>Corning</td>
			<td>431079</td>
			<td>(Section 1) Or equivalent brand&#160;</td>
		</tr>
		<tr>
			<td>Protein Matrix</td>
			<td>Corning</td>
			<td>356231</td>
			<td>(Section 2,3,4,5,6) Matrigel Growth Factor Reduced&#160;</td>
		</tr>
		<tr>
			<td>HyClone Dulbecco&#39;s (DPBS)</td>
			<td>GE Healthcare</td>
			<td>SH30264.01</td>
			<td>(Section 2,3)</td>
		</tr>
		<tr>
			<td>DMEM/F12&#160;</td>
			<td>Life Technologies</td>
			<td>12634-010</td>
			<td>(Section 2,3) Advanced DMEM/F12</td>
		</tr>
		<tr>
			<td>Corning 24 Well TC Plates</td>
			<td>Corning</td>
			<td>3524</td>
			<td>(Section 2)</td>
		</tr>
		<tr>
			<td>N2 Supplement 100x&#160;</td>
			<td>Life Technologies</td>
			<td>17502-048</td>
			<td>(Section 2)</td>
		</tr>
		<tr>
			<td>B27 without vitamin A 50x&#160;</td>
			<td>Life Technologies</td>
			<td>12587-010&#160;</td>
			<td>(Section 2)</td>
		</tr>
		<tr>
			<td>Trizol</td>
			<td>Life Technologies</td>
			<td>15596-026</td>
			<td>(Section 2)</td>
		</tr>
		<tr>
			<td>Glutamine Supplement (Glutamax)</td>
			<td>Life Technologies</td>
			<td>35050-061</td>
			<td>(Section 2) Can Combine with Advanced DMEM/ F12</td>
		</tr>
		<tr>
			<td>HEPES (1 M)</td>
			<td>Life Technologies</td>
			<td>15630-080</td>
			<td>(Section 2) Can Combine with Advanced DMEM/ F12&#160;</td>
		</tr>
		<tr>
			<td>10ml Serological Pipet</td>
			<td>Falcon</td>
			<td>357551</td>
			<td>(Section 2) Or equivalent brand</td>
		</tr>
		<tr>
			<td>Murine Noggin</td>
			<td>Peprotech</td>
			<td>250-38</td>
			<td>(Section 2) Stock = 100 mg/ml</td>
		</tr>
		<tr>
			<td>N-Acetyl-L-cysteine</td>
			<td>Sigma-Aldrich</td>
			<td>A9165</td>
			<td>(Section 2) Stock = 1M</td>
		</tr>
		<tr>
			<td>Recombinant Mouse EGF</td>
			<td>Biolegend</td>
			<td>585608</td>
			<td>(Section 2) Stock = 500 mg/ml</td>
		</tr>
		<tr>
			<td>Rocker Variable</td>
			<td>Bioexpres</td>
			<td></td>
			<td>(Section 3)</td>
		</tr>
		<tr>
			<td>dissecting scissors</td>
			<td></td>
			<td></td>
			<td>(Section 3)</td>
		</tr>
		<tr>
			<td>forceps</td>
			<td></td>
			<td></td>
			<td>(Section 3)</td>
		</tr>
		<tr>
			<td>glass slides</td>
			<td></td>
			<td></td>
			<td>(Section 3)</td>
		</tr>
		<tr>
			<td>dissecting tweezers</td>
			<td></td>
			<td></td>
			<td>(Section 3)</td>
		</tr>
		<tr>
			<td>25 ml Serological Pipet</td>
			<td>Falcon</td>
			<td></td>
			<td>(Section 3)</td>
		</tr>
		<tr>
			<td>EDTA&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>SLBB9821</td>
			<td>(Section 3) 0.5M or alternative TC grade EDTA</td>
		</tr>
		<tr>
			<td>Sterile Petri Dish 100mm x 15mm</td>
			<td>Fisher</td>
			<td>FB0875712</td>
			<td>(Section 3) Or equal sized TC dish</td>
		</tr>
		<tr>
			<td>1ml Syringe</td>
			<td>Becton Dickinson</td>
			<td>309659</td>
			<td>(Section 4)</td>
		</tr>
		<tr>
			<td>Precision Glide Needle</td>
			<td>Becton Dickinson</td>
			<td>305120</td>
			<td>(Section 4) 23G x 1 1/4 (0.6mm x 30mm)</td>
		</tr>
		<tr>
			<td>Flagellin from Bacillus subtilis</td>
			<td>Invivogen</td>
			<td>tlrl-bsfla&#160;</td>
			<td>(Section 5,6)</td>
		</tr>
		<tr>
			<td>Listeria monocytogenes</td>
			<td>ATCC</td>
			<td>19115</td>
			<td>(Section 5,6)&#160;(Murray&#160;et&#160;al.)&#160;</td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>Sigma-Aldrich</td>
			<td>Z359629-1EA</td>
			<td>(Section 5,6)Or equivalent brand</td>
		</tr>
		<tr>
			<td>BBL Brain Heart Infusion Agar</td>
			<td>Becton Dickinson</td>
			<td>211065</td>
			<td>(Section 5)</td>
		</tr>
		<tr>
			<td>Bacto Brain Heart Infusion</td>
			<td>Becton Dickinson</td>
			<td>237500</td>
			<td>(Section 5)</td>
		</tr>
		<tr>
			<td>Caco-2</td>
			<td>ATCC</td>
			<td>HTB-37</td>
			<td>(Section 6)</td>
		</tr>
		<tr>
			<td>Trypsin&#160;</td>
			<td>gibco</td>
			<td>25200056</td>
			<td>(section 6)</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Fisher</td>
			<td>A412-4</td>
			<td>(Section 6)</td>
		</tr>
		<tr>
			<td>SpectraMax M5</td>
			<td>Molecuar Devices</td>
			<td></td>
			<td>(Section 6)</td>
		</tr>
		<tr>
			<td>96 Well Assay Plate</td>
			<td>Corning</td>
			<td>3603</td>
			<td>(Section 6) Black Plate, Clear Bottom TC treated</td>
		</tr>
		<tr>
			<td>Nuclear Staining Dye</td>
			<td>Life Technologies</td>
			<td>H1399</td>
			<td>(section 6) Hoechst 33342</td>
		</tr>
		<tr>
			<td>T-75 Flask</td>
			<td>Corning</td>
			<td>430641</td>
			<td>(Section 6) Or equivalent brand</td>
		</tr>
		<tr>
			<td>15 ml conical tube</td>
			<td>Falcon</td>
			<td>352096</td>
			<td>(Section1,3) Or equivalent brand</td>
		</tr>
		<tr>
			<td>1.7 ml polypropylene tube</td>
			<td>Bioexpress</td>
			<td>C-3262-1</td>
			<td>Or equivalent brand</td>
		</tr>
		<tr>
			<td>Quick-RNA MiniPrep</td>
			<td>Zymo Research</td>
			<td>R1054</td>
			<td>Or equivalent brand</td>
		</tr>
		<tr>
			<td>TNF-alpha&#160;</td>
			<td>Applied Biosystems</td>
			<td>Mm 00443260_g1</td>
			<td>Taqman gene expression assay kit</td>
		</tr>
		<tr>
			<td>IL-6&#160;</td>
			<td>Applied Biosystems</td>
			<td>(Mm 00446190_m1</td>
			<td>Taqman gene expression assay kit</td>
		</tr>
		<tr>
			<td>IL-1beta</td>
			<td>Applied Biosystems</td>
			<td>Mm 00434228_m1</td>
			<td>Taqman gene expression assay kit</td>
		</tr>
		<tr>
			<td>IL-18</td>
			<td>Applied Biosystems</td>
			<td>Mm 00434225_m1</td>
			<td>Taqman gene expression assay kit</td>
		</tr>
		<tr>
			<td>18s</td>
			<td>Applied Biosystems</td>
			<td>Hs 99999901_s1</td>
			<td>Taqman gene expression assay kit</td>
		</tr>
		<tr>
			<td>7500 Fast Real Time PCR System</td>
			<td>Applied Biosystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nexus gradient Mastercycler</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TaqMan Fast Universal PCR Master Mix</td>
			<td>Life Technologies</td>
			<td>4352042</td>
			<td></td>
		</tr>
		<tr>
			<td>High Capacity cDNA Reverse Transcription Kit</td>
			<td>Life Technologies/Applied Biosystems</td>
			<td>4368814</td>
			<td></td>
		</tr>
		<tr>
			<td>Fast Optical 96-Well Reaction Plate, 0.1 mL</td>
			<td>Life Technologies/Applied Biosystems</td>
			<td>4346907</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Mouse R-Spondin 1 Protein</td>
			<td>R&#38;D Systems</td>
			<td>3474-RS-050</td>
			<td>500 ng/ml</td>
		</tr>
		<tr>
			<td>chloroform</td>
			<td>Sigma-Aldrich</td>
			<td>C7559</td>
			<td></td>
		</tr>
	</tbody>
</table>

the ex vivo culture and pattern recognition receptor stimulation of 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 complexities of the organoid growth characteristics carry significant caveats for the investigator. Specifically, the growth patterns of each individual organoid are highly variable and create a heterogeneous population of epithelial cells in culture. With such caveats, common tissue culture practices cannot be simply applied to the organoid system due to the complexity of the cellular structure. Counting and plating based solely on cell number, which is common for individually separated cells, such as cell lines, is not a reliable method for organoids unless some normalization technique is applied. Normalizing to total protein content is made complex due to the resident protein matrix. These characteristics in terms of cell number, shape and cell type should be taken into consideration when evaluating secreted contents from the organoid mass. This protocol has been generated to outline a simple procedure to culture and treat small intestinal organoids with microbial pathogens and pathogen associated molecular patterns (PAMPs). It also emphasizes the normalization techniques that should be applied when protein analysis are conducted after such a challenge.

When following this protocol to cultivate intestinal organoids, characteristic sphere shaped organoids will be present after harvesting. The addition of R-spondin1 conditioned media daily will initiate the growth and budding of the organoids. The growth of organoids is shown in Figure 1A-F, and is representative of intestinal organoids on days 1, 2, 4, 5, 6 and day 14. Figure 1F represents the non-homogeneous growth chara...

Watch this Scientific Journal Video about The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids at JoVE.com

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

Here, a protocol to harvest, maintain, and treat mouse small intestinal organoids with pathogen associated molecular patterns (PAMPs) and Listeria monocytogenes is described, as well as emphasis on gene expression and proper normalization techniques for protein.

The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of 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 ...

The overall goal of this procedure is to measure the effects of pathogenic challenge on small intestine organoids with an emphasis on normalization techniques against the total cell number.This method could help answer key questions in the innate as well as mucosal immunology fields by providing a means to investigate host pathogen interactions of bacteria with intestinal epithelial cells in vitro.Essential to this procedure is the method for normalization against the total cell number, a necessity when measuring organoid secreted proteins via techniques such as ELISA.To harvest mouse small intestinal crypts for organoid culture, begin by using a sterile glass slide to gently scrape away the villi from the luminal surface of the small intestine.Next, use dissecting scissors to cut the tissue into 1-2 centimeter length strips.And place the strips into a 50 milliliter conical tube containing 10 milliliters of ice cold PBS.Mix the contents by gentle inversion and allow the tissue contents to settle to the bottom of the tube.Then, aspirate the PBS and wash the strips three more times in 10 milliliters of fresh PBS, each time in the same manner.At the end of the final wash, place the tube on ice for ten minutes.Then, replace the PBS with 25 milliliters of PBS supplemented with 2 millimolar EDTA, in an ice bucket on a rocking platform for 45 minutes.At the end of the incubation, allow the tissue strips to settle to the bottom of the tube and replace the PBS-EDTA with 10 milliliters of PBS supplemented with 10%FBS.Shake the tube vigorously by hand ten times.Then, allow the tissue to settle to the bottom of the tube and transfer the supernatant to a 15 milliliter conical tube labeled, Fraction 1.Agitate the tissues in PBS FBS five more times.Transferring the supernatant into a new fraction tube each time.After the last agitation, centrifuge all of the samples and resuspend the pellets in five milliliters of prewarmed DMEM/F12 with no added growth factors.Now, spin down the samples again and aspirate four milliliters of the supernatant from each tube.Resuspend the pellets in the last milliliter of remaining medium and use 20 microliter aliquots from each fraction to visualize the crypts and the debris on glass slides.After pooling the appropriate fractions to achieve the greatest percentage of crypt to debris ratio, centrifuge the pooled fractions and aspirate all but the last 50-100 microliters of the supernatants.Keeping the tubes on ice, add 1 milliliter of protein matrix to the pooled fractions, pipetting up and down slowly to prevent the addition of air bubbles.Then, add 50 microliters of the protein matrix/crypt suspension into the middle of each well of 37

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Research

JoVE Journal

Immunology and Infection

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the protocols described here use freshly isolated human peripheral blood mononuclear cells (PBMCs) or plasmacytoid dendritic cells (pDCs) to sense infections in either T cell line (MT4) or heterologous primary CD4+ T cells. In order to ensure proper sensing, it is essential that PBMC are isolated immediately after blood collection and that optimal percentage of infected T cells are used. Furthermore, multi-parametric flow cytometric staining can be used to confirm that PBMC samples contain the different cell lineages at physiological ratios. A number of controls can also be included to evaluate viability and functionality of pDCs. These include, the presence of specific surface markers, assessing cellular responses to known agonist of Toll-Like Receptors (TLR) pathways, and confirming a lack of spontaneous type-I interferon (IFN) production. In this system, freshly isolated PBMCs or pDCs are co-cultured with HIV-1 infected cells in 96 well plates for 18-22 hr. Supernatants from these co-cultures are then used to determine the levels of bioactive type-I IFNs by monitoring the activation of the ISGF3 pathway in HEK-Blue IFN-&#945;/&#946; cells. Prior and during co-culture conditions, target cells can be subjected to flow cytometric analysis to determine a number of parameters, including the percentage of infected cells, levels of specific surface markers, and differential killing of infected cells. Although, these protocols were initially developed to follow type-I IFN production, they could potentially be used to study other imuno-modulatory molecules released from pDCs and to gain further insight into the molecular mechanisms governing HIV-1 innate sensing.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Unlike cell-free HIV-1 particles, infected CD4+ T cells are effectively sensed by plasmacytoid dendritic cells (pDCs). This manuscript describes a method where peripheral blood mononuclear cells (PBMCs) or isolated pDCs are co-cultured with HIV-1 infected T cells to evaluate innate sensing by pDCs as assessed by release of type-I IFN.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

Monitoring Activation of the Antiviral Pattern Recognition Receptors RIG-I And PKR By Limited Protease Digestion and Native PAGE

Host defenses to virus infection are dependent on a rapid detection by pattern recognition receptors (PRRs) of the innate immune system. In the cytoplasm, the PRRs RIG-I and PKR bind to specific viral RNA ligands. This first mediates conformational switching and oligomerization, and then enables activation of an antiviral interferon response. While methods to measure antiviral host gene expression are well established, methods to directly monitor the activation states of RIG-I and PKR are only partially and less well established.
Here, we describe two methods to monitor RIG-I and PKR stimulation upon infection with an established interferon inducer, the Rift Valley fever virus mutant clone 13 (Cl 13). Limited trypsin digestion allows to analyze alterations in protease sensitivity, indicating conformational changes of the PRRs. Trypsin digestion of lysates from mock infected cells results in a rapid degradation of RIG-I and PKR, whereas Cl 13 infection leads to the emergence of a protease-resistant RIG-I fragment. Also PKR shows a virus-induced partial resistance to trypsin digestion, which coincides with its hallmark phosphorylation at Thr 446. The formation of RIG-I and PKR oligomers was validated by native polyacrylamide gel electrophoresis (PAGE). Upon infection, there is a strong accumulation of RIG-I and PKR oligomeric complexes, whereas these proteins remained as monomers in mock infected samples.
Limited protease digestion and native PAGE, both coupled to western blot analysis, allow a sensitive and direct measurement of two diverse steps of RIG-I and PKR activation. These techniques are relatively easy and quick to perform and do not require expensive equipment.

Innate defenses to virus infections are triggered by pattern recognition receptors (PRRs). The two cytoplasmic PRRs RIG-I and PKR bind to viral signature RNAs, change conformation, oligomerize, and activate antiviral signaling. Methods are described which allow to conveniently monitor the conformational switching and the oligomerization of these cytoplasmic PRRs.

Host defenses to virus infection are dependent on a rapid detection by pattern recognition receptors (PRRs) of the innate immune system. In the cytoplasm, ...

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the skin is relatively easy to access, it provides an ideal platform to study peripheral immune regulatory mechanisms. Immune resident cells in healthy skin conduct immunosurveillance, but also play an important role in the development of inflammatory skin disorders, such as psoriasis. Despite emerging insights, our understanding of the biology underlying various inflammatory skin diseases is still limited. There is a need for good quality (single) cell populations isolated from biopsied skin samples. So far, isolation procedures have been seriously hampered by a lack of obtaining a sufficient number of viable cells. Isolation and subsequent analysis have also been affected by the loss of immune cell lineage markers, due to the mechanical and chemical stress caused by the current dissociation procedures to obtain single cell suspension. Here, we describe a modified method to isolate T cells from both healthy and involved psoriatic human skin by combining mechanical skin dissociation using an automated tissue dissociator and collagenase treatment. This methodology preserves expression of most immune lineage markers such as CD4, CD8, Foxp3 and CD11c upon the preparation of single cell suspensions. Examples of successful CD4+ T cell isolation and subsequent phenotypic and functional analysis are shown.

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

We describe a protocol to efficiently isolate skin resident T cells from human skin biopsies. This protocol yields sufficient numbers of viable human skin resident lymphocytes for flow cytometric analysis and ex vivo culture.

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the ...

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.

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. ...

Isolation and Ex Vivo Culture of V&#948;1+CD4+&#947;&#948; T Cells, an Extrathymic &#945;&#946;T-cell Progenitor

The thymus, the primary organ for the generation of &#945;&#946; T cells and backbone of the adaptive immune system in vertebrates, has long been considered as the only source of &#945;&#946;T cells. Yet, thymic involution begins early in life leading to a drastically reduced output of na&#239;ve &#945;&#946;T cells into the periphery. Nevertheless, even centenarians can build immunity against newly acquired pathogens. Recent research suggests extrathymic &#945;&#946;T cell development, however our understanding of pathways that may compensate for thymic loss of function are still rudimental. &#947;&#948; T cells are innate lymphocytes that constitute the main T-cell subset in the tissues. We recently ascribed a so far unappreciated outstanding function to a &#947;&#948; T cell subset by showing that the scarce entity of CD4+ V&#948;1+&#947;&#948; T cells can transdifferentiate into &#945;&#946;T cells in inflammatory conditions. Here, we provide the protocol for the isolation of this progenitor from peripheral blood and its subsequent cultivation. V&#948;1 cells are positively enriched from PBMCs of healthy human donors using magnetic beads, followed by a second step wherein we target the scarce fraction of CD4+ cells with a further magnetic labeling technique. The magnetic force of the second labeling exceeds the one of the first magnetic label, and thus allows the efficient, quantitative and specific positive isolation of the population of interest. We then introduce the technique and culture condition required for cloning and efficiently expanding the cells and for identification of the generated clones by FACS analysis. Thus, we provide a detailed protocol for the purification, culture and ex vivo expansion of CD4+ V&#948;1+&#947;&#948; T cells. This knowledge is prerequisite for studies that relate to this &#945;&#946;T cell progenitor`s biology and for those who aim to identify the molecular triggers that are involved in its transdifferentiation.

Isolation and Ex Vivo Culture of V&amp;#948;1+CD4+&amp;#947;&amp;#948; T Cells, an Extrathymic &amp;#945;&amp;#946;T-cell Progenitor

Here, we provide an optimized protocol for the isolation and cloning of the scarce T-cell entity of peripheral V&#948;1+CD4+ T cells that is, as we showed recently, an extrathymic &#945;&#946; T-cell progenitor. This technique allows to quantitatively isolate, clone and efficiently expand these cells in ex vivo culture.

The thymus, the primary organ for the generation of &#945;&#946; T cells and backbone of the adaptive immune system in vertebrates, has long been ...

Using X-ray Crystallography, Biophysics, and Functional Assays to Determine the Mechanisms Governing T-cell Receptor Recognition of Cancer Antigens

Human CD8+ cytotoxic T lymphocytes (CTLs) are known to play an important role in tumor control. In order to carry out this function, the cell surface-expressed T-cell receptor (TCR) must functionally recognize human leukocyte antigen (HLA)-restricted tumor-derived peptides (pHLA). However, we and others have shown that most TCRs bind sub-optimally to tumor antigens. Uncovering the molecular mechanisms that define this poor recognition could aid in the development of new targeted therapies that circumnavigate these shortcomings. Indeed, present therapies that lack this molecular understanding have not been universally effective. Here, we describe methods that we commonly employ in the laboratory to determine how the nature of the interaction between TCRs and pHLA governs T-cell functionality. These methods include the generation of soluble TCRs and pHLA and the use of these reagents for X-ray crystallography, biophysical analysis, and antigen-specific T-cell staining with pHLA multimers. Using these approaches and guided by structural analysis, it is possible to modify the interaction between TCRs and pHLA and to then test how these modifications impact T-cell antigen recognition. These findings have already helped to clarify the mechanism of T-cell recognition of a number of cancer antigens and could direct the development of altered peptides and modified TCRs for new cancer therapies.

Here, we describe methods that we commonly employ in the laboratory to determine how the nature of the interaction between the T-cell receptor and tumor antigens, presented by human leukocyte antigens, governs T-cell functionality; these methods include protein production, X-ray crystallography, biophysics, and functional T-cell experiments.

Human CD8+ cytotoxic T lymphocytes (CTLs) are known to play an important role in tumor control. In order to carry out this function, the cell ...

The Ex Vivo Colon Organ Culture and Its Use in Antimicrobial Host Defense Studies

The intestine displays an architecture of repetitive crypt structures consisting of different types of epithelial cells, lamina propia containing immune cells, and stroma. All of these heterogeneous cells contribute to intestinal homeostasis and participate in antimicrobial host defense. Therefore, identifying a surrogate model for studying immune response and antimicrobial activity of the intestine in an in vitro setting is extremely challenging. In vitro studies using immortalized intestinal epithelial cell lines or even primary crypt organoid culture do not represent the exact physiology of normal intestine and its microenvironment. Here, we discuss a method of culturing mouse colon tissue in a culture dish and how this ex vivo organ culture system can be implemented in studies related to antimicrobial host defense responses. In representative experiments, we showed that colons in organ culture express antimicrobial peptides in response to exogenous IL-1&#946; and IL-18. Further, the antimicrobial effector molecules produced by the colon tissues in the organ culture efficiently kill Escherichia coli in vitro. This approach, therefore, can be utilized to dissect the role of pathogen- and danger-associated molecular patterns and their cellular receptors in regulating intestinal innate immune responses and antimicrobial host defense responses.

The Ex Vivo Colon Organ Culture and Its Use in Antimicrobial Host Defense Studies

The ex vivo organ culture allows investigation of biological processes in the context of the intact tissue architecture. Here, we introduce a method of ex vivo culture of the mouse colon, which can be used to study innate immunity and antimicrobial host defense in the intestine.

The intestine displays an architecture of repetitive crypt structures consisting of different types of epithelial cells, lamina propia containing immune ...

Isolating Lymphocytes from the Mouse Small Intestinal Immune System

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to dietary antigens and commensal bacteria while mounting effective immune responses to enteropathogenic microbes. In addition, it has become clear that local intestinal immunity has a profound impact on distant and systemic immunity. Therefore, it is important to study how an intestinal immune response is induced and what the immunologic outcome of the response is. Here, a detailed protocol is described for the isolation of lymphocytes from small intestine inductive sites like the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes and effector sites like the lamina propria and the intestinal epithelium. This technique ensures isolation of a large numbers of lymphocytes from small intestinal tissues with optimal purity and viability and minimal cross compartmental contamination within acceptable time constraints. The technical capability to isolate lymphocytes and other immune cells from intestinal tissues enables the understanding of immune responses to gastrointestinal infections, cancers, and inflammatory diseases.

Here we describe a detailed protocol for the isolation of lymphocytes from the inductive sites including the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes, and the effector sites including the lamina propria and the intestinal epithelium of the small intestinal immune system.

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to ...

An Ex Vivo Chicken Primary Bursal-cell Culture Model to Study Infectious Bursal Disease Virus Pathogenesis

Infectious bursal disease virus (IBDV) is a birnavirus of economic importance to the poultry industry. The virus infects B cells, causing morbidity, mortality, and immunosuppression in infected birds. In this study, we describe the isolation of chicken primary bursal cells from the bursa of Fabricius, the culture and infection of the cells with IBDV, and the quantification of viral replication. The addition of chicken CD40 ligand significantly increased cell proliferation fourfold over six days of culture and significantly enhanced cell viability. Two strains of IBDV, a cell-culture adapted strain, D78, and a very virulent strain, UK661, replicated well in the ex vivo cell cultures. This model will be of use in determining how cells respond to IBDV infection and will permit a reduction in the number of infected birds used in IBDV pathogenesis studies. The model can also be expanded to include other viruses and could be applied to different species of birds.

An Ex Vivo Chicken Primary Bursal-cell Culture Model to Study Infectious Bursal Disease Virus Pathogenesis

Here we describe the isolation of chicken primary bursal cells from the bursa of Fabricius, the culture and infection of the cells with infectious bursal disease virus, and the quantification of viral replication.

Infectious bursal disease virus (IBDV) is a birnavirus of economic importance to the poultry industry. The virus infects B cells, causing morbidity, ...

Confocal Imaging of Double-Stranded RNA and Pattern Recognition Receptors in Negative-Sense RNA Virus Infection

Double-stranded (ds) RNA is produced as a replicative intermediate during RNA virus infection. Recognition of dsRNA by host pattern recognition receptors (PRRs) such as the retinoic acid (RIG-I) like receptors (RLRs) RIG-I and melanoma differentiation-associated protein 5 (MDA-5) leads to the induction of the innate immune response. The formation and intracellular distribution of dsRNA in positive-sense RNA virus infection has been well characterized by microscopy. Many negative-sense RNA viruses, including some arenaviruses, trigger the innate immune response during infection. However, negative-sense RNA viruses were thought to produce low levels of dsRNA, which hinders the imaging study of PRR recognition of viral dsRNA. Additionally, infection experiments with highly pathogenic arenaviruses must be performed in high containment biosafety level facilities (BSL-4). The interaction between viral RNA and PRRs for highly pathogenic RNA virus is largely unknown due to the additional technical challenges that researchers need to face in the BSL-4 facilities. Recently, a monoclonal antibody (Mab) (clone 9D5) originally used for pan-enterovirus detection has been found to specifically detect dsRNA with a higher sensitivity than the traditional J2 or K1 anti-dsRNA antibodies. Herein, by utilizing the 9D5 antibody, we describe a confocal microscopy protocol that has been used successfully to visualize dsRNA, viral protein and PRR simultaneously in individual cells infected by arenavirus. The protocol is also suitable for imaging studies of dsRNA and PRR distribution in pathogenic arenavirus infected cells in BSL4 facilities.

Double-stranded RNA produced during RNA virus replication can be recognized by pattern recognition receptors to induce an innate immune response. For negative-sense RNA viruses, the interaction between the low-level dsRNA and PRRs remains unclear. We have developed a confocal microscopy method to visualize arenavirus dsRNA and PRR in individual cells.

Double-stranded (ds) RNA is produced as a replicative intermediate during RNA virus infection. Recognition of dsRNA by host pattern recognition receptors ...