Name: a novel method for the culture and polarized stimulation of human intestinal mucosa explants
Uploaded: 2013-05-01T12:00:00
Description: Watch this Scientific Journal Video about A Novel Method for the Culture and Polarized Stimulation of Human Intestinal Mucosa Explants at JoVE.com

Vi takker Erika Mileti for fremragende teknisk support og Dr. Antonio Di Sabatino for at levere ilt kamre. Finansiering: Dette arbejde blev støttet af tilskud fra det 7. EU-rammeprogram (IBDase, ERC: Dendroworld) og Fondazione Cariplo til MR og støtte af et Marie Curie international uddannelse mobilitet netværk (Cross-Talk, tilskudsaftale nr.: 21.553 til 2) til KT.

1.. Indhentning og Forberedelse af Tissue <ol><li> Væv (sundt eller IBD slimhinde) opnås under operationen. Når prøven udskæres overførsel til laboratoriet snarest muligt at holde det i Ca + + / Mg + +-fri HBSS buffer suppleret med Pen / Strep ved 4 ° C eller på is. </li></ol> * Størrelsen af ​​prøven afhænger af tilgængeligheden af ​​vævet: den del af den opnåede væv, er strengt hvad der ikke er nødvendigt for diagnose og kan variere meget mellem s...

<ol>
	<li>Schuller, S., Lucas, M., Kaper, J. B., Giron, J. A., Phillips, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ex+vivo+response+of+human+intestinal+mucosa+to+enteropathogenic+Escherichia+coli+infection.">The ex vivo response of human intestinal mucosa to enteropathogenic Escherichia coli infection.</a> Cell. Microbiol. 11, 521-530 (2009).</li><li>Tsilingiri, 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=Probiotic+and+postbiotic+activity+in+health+and+disease:+comparison+on+a+novel+polarised+ex-vivo+organ+culture+model.">Probiotic and postbiotic activity in health and disease: comparison on a novel polarised ex-vivo organ culture model.</a> Gut. , (2012).</li><li>Tsilingiri, K., Rescigno, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Should+probiotics+be+tested+on+ex+vivo+organ+culture+models?.">Should probiotics be tested on ex vivo organ culture models?.</a> Gut Microbes. , (2012).</li><li>Mileti, E., Matteoli, G., Iliev, I. D., Rescigno, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+the+immunomodulatory+properties+of+three+probiotic+strains+of+Lactobacilli+using+complex+culture+systems:+prediction+for+in+vivo+efficacy.">Comparison of the immunomodulatory properties of three probiotic strains of Lactobacilli using complex culture systems: prediction for in vivo efficacy.</a> PLoS One. 4, e7056 (2009).</li><li>Cencic, A., Langerholc, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+cell+models+of+the+gut+and+their+applications+in+food+microbiology--a+review.">Functional cell models of the gut and their applications in food microbiology--a review.</a> Int. J. Food Microbiol. 141, S4-S14 (2010).</li><li>te Velde, A. A., Verstege, M. A., Hommes, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Critical+appraisal+of+the+current+practice+in+murine+TNBS-induced+colitis.">Critical appraisal of the current practice in murine TNBS-induced colitis.</a> Inflamm. Bowel Dis. 12, 995-999 (2006).</li><li>Senior, P. V., Pritchett, C. J., Sunter, J. P., Appleton, D. R., Watson, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crypt+regeneration+in+adult+human+colonic+mucosa+during+prolonged+organ+culture.">Crypt regeneration in adult human colonic mucosa during prolonged organ culture.</a> J. Anat. 134, 459-469 (1982).</li><li>Browning, T. H., Trier, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organ+culture+of+mucosal+biopsies+of+human+small+intestine.">Organ culture of mucosal biopsies of human small intestine.</a> J. Clin. Invest. 48, 1423-1432 (1969).</li><li>Besselink, M. 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=Probiotic+prophylaxis+in+predicted+severe+acute+pancreatitis:+a+randomised,+double-blind,+placebo-controlled+trial.">Probiotic prophylaxis in predicted severe acute pancreatitis: a randomised, double-blind, placebo-controlled trial.</a> Lancet. 371, 651-659 (2008).</li><li>Lakhdari, O., 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+NF-kappaB+modulation+capabilities+within+human+intestinal+commensal+bacteria.">Identification of NF-kappaB modulation capabilities within human intestinal commensal bacteria.</a> J. Biomed. Biotechnol. 2011, 282356 (2011).</li></ol>

Behovet for fysiologisk relevante modeller, som behandlinger for human tarmslimhinden gyldigt kan testes har længe været fremhævet. Mange forskere har identificeret mulige artefakter og defekter i både celle 5 og musemodeller 6 hidtil anvendte til dette formål. Selv om lovende prækliniske data opnås ofte, er disse sjældent oversætte til væsentlig klinisk fordel. Fremgangsmåden beskrevet i dette arbejde, præsenterer en forholdsvis enkel, men effektiv og hidti...

<table border="1">
	<tbody>
		<tr>
			<td>Regeant</td>
			<td>Company</td>
			<td>Catalog No.</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Reagents</td>
		</tr>
		<tr>
			<td>10X HBSS</td>
			<td>Eurocalne</td>
			<td>ECM4006XL</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Peni/Strepto</td>
			<td>Lonza</td>
			<td>DE17602E</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Gentamycin</td>
			<td>Gibco</td>
			<td>15750-037</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Lonza</td>
			<td>BE12614</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>FBS-Na</td>
			<td>Gibco</td>
			<td>16000-044</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>100X ITS-X</td>
			<td>Gibco</td>
			<td>51500-056</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>Tebu-bio</td>
			<td>100-15</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Lonza</td>
			<td>BE17605E</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Non essential aminoacids 100X</td>
			<td>Gibco</td>
			<td>11140-035</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NaPyr</td>
			<td>Gibco</td>
			<td>11360-039</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Materials</td>
		</tr>
		<tr>
			<td>Cloning cylinders 6x8 mm</td>
			<td>BellCo</td>
			<td>2090-00608</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cloning cylinders 8x8 mm</td>
			<td>BellCo</td>
			<td>2090-0080</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cloning cylinders 10x10 mm</td>
			<td>BellCo</td>
			<td>2090-01010</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Metal grids</td>
			<td>Home made</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Centre well organ culture plate</td>
			<td>BD Falcon</td>
			<td>353037</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Surgical glue (Vetbond)</td>
			<td>3M</td>
			<td>1469SB</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Oxygen Jars</td>
			<td>Home made</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Oxygen tanks</td>
			<td>Air Liquid Sanit&agrave;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

a novel method for the culture and polarized stimulation of human intestinal mucosa explants

Enkelte modeller i øjeblikket eksisterer for at realistisk simulere den komplekse menneskelige tarmens mikro-miljø, hvor en bred vifte af interaktioner finder sted. Korrekt homeostase direkte afhængig af disse interaktioner, da de forme en hel immunologisk respons inducing tolerance overfor fødevarer antigener, mens på samme tid iværksætte effektive immunreaktioner mod patogene mikrober uheld indtager med fødevarer. Intestinal homeostase bevares også gennem forskellige komplekse interaktioner mellem mikrobiota (herunder mad-associerede gavnlige bakteriestammer) og værten, der regulerer den vedhæftede fil / nedbrydning af slim, produktion af antimikrobielle peptider ved epitelbarrieren, og &quot;undervisning&quot; af epitelceller &#39;, der styrer tolerogent eller immunogene fænotype af unikke, gut-hjemmehørende lymfoide celler befolkninger. Disse interaktioner har været så langt meget vanskelige at reproducere med in vitro assaysanvendelse af enten dyrkede cellelinier eller perifere mononukleære blodceller. Endvidere afviger musemodeller væsentligt i komponenterne i tarmslimhinden (slimlag organisation, kommensale bakterier samfund) med hensyn til den menneskelige tarm. Således har undersøgelser af en række behandlinger, der skal bringes i klinikkerne for vigtige stress-relaterede eller patologiske tilstande såsom irritabel tyktarm, inflammatorisk tarmsygdom eller kolorektal cancer været vanskeligt at udføre. For at løse disse problemer, har vi udviklet et nyt system, der gør det muligt for os at stimulere eksplantater af human tarmslimhinden, der bevarer deres in situ konditionering af værten mikrobiota og immunrespons i et polariseret måde. Polariseret apikale stimulering er af stor betydning for resultatet af den fremkaldte immunrespons. Det har gentagne gange vist, at de samme stimuli kan producere helt forskellige reaktioner, når de omgå den apikale overflade af tarmen epithelium, stimulere epitelceller basolateralt eller kommer i direkte kontakt med lamina propria komponenter skifte fænotype fra tolerogent til immunogene og forårsager unødvendig og overdreven betændelse i området. Vi opnåede polariseret stimulation ved limning en hule cylinder, som afgrænser området stimulering på den apikale overflade af slimhinden som det vil blive beskrevet i protokollen. Vi brugte denne model til at undersøge, blandt andre, differential effekter af tre forskellige Lactobacilli stammer. Vi viser, at denne model system er meget stærke til at vurdere immunmodulerende egenskaber af probiotika i sunde og sygdomstilstande.

Det lykkedes os at opretholde en sund og IBD menneske tarmslimhinden i kultur for mindst 24 timer bevare trivsel af vævet. I overensstemmelse med tidligere observationer 1, var dette kun muligt, hvis størstedelen af kulturen tid (mindst 85% af totalen) fandt sted i O 2, da vævet ikke overlevede i 24 timer i konventionelle inkubatorer (figur 2). Vi har også vist, at forskellige reaktioner af eksplantaterne kan iagttages på denne model efter polariteten af stimuleringen (apikale...

Watch this Scientific Journal Video about A Novel Method for the Culture and Polarized Stimulation of Human Intestinal Mucosa Explants at JoVE.com

A Novel Method for the Culture and Polarized Stimulation of Human Intestinal Mucosa Explants

Vi introducerer en hidtil ukendt fremgangsmåde til opretholdelse af menneskers tarmslimhinden i kultur og overvågning af reaktion på forskellige typer af stimuli over mindst 24 timer. Med vores metode er polariteten af ​​vævet bibeholdes, giver mulighed for en fysiologisk stimulering via apikale rute.

En ny metode til Kultur-og polariseret Stimulation for Human tarmslimhinden Eksplantater

Few models currently exist to realistically simulate the complex human intestine's micro-environment, where a variety of interactions take place. Proper ...

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