Questo studio è stato sostenuto da sovvenzioni RO1 CA 135561, R01 CA151494, R01 CA174432 e P3013330 dal National Institutes of Health. Vorremmo ringraziare Michele Houston, Elena Dhima e Dott.ssa Anna Velcich per i loro preziosi commenti nello sviluppo del protocollo di isolamento cripta. Ringraziamo anche la formazione del diabete e Centro di Ricerca del Albert Einstein College of Medicine sostenuto da NIH P60DK20541, e il dottor Michael Brownlee e il dottor Xue Liang-Du, che dirigono e operano la struttura Seahorse, rispettivamente.

Questo studio è stato eseguito in conformità con le raccomandazioni nella Guida per la cura e l&#39;uso di animali da laboratorio del National Institutes of Health. Il protocollo è stato approvato dalla commissione etica della sperimentazione animale del Albert Einstein College of Medicine. 1. Isolamento Cripta e Cultura <ol><li> Isolamento delle Cripte dal piccolo intestino: <ol><li> Isolare cripte intestinali da qualsiasi modello di topi di interesse. Eutanasia i topi con CO 2 seguita da dislocazione cervicale. </li><li> Aprire l&#39;addome longitudinalmente e riempire il piccolo intestino (SI) con soluzione fisiologica fredda ghiaccio fosfato tamponata, PBS (-Ca 2+; -Mg 2+) con 2x antibiotico-antimicotico (anti-anti). Rapidamente isolare piccolo intestino. </li><li> Sezionare completamente privo di grasso mesenterico con un bisturi. Fare attenzione a non forare il tessuto - in ogni momento mantenere il tessuto umido con ghiaccio PBS freddo. Aprite inteStine longitudinalmente e lavare accuratamente con ghiaccio freddo PBS. </li><li> Tagliare piccolo intestino in due sezioni e appiattire con una punta di cotone bagnato, delicatamente raschiare il villi con una diapositiva pre-raffreddata. Lavare vigorosamente in PBS freddo più volte. </li><li> Incubare 3 minuti in 20 ml di PBS 1x per 3 mM di acido etilendiamminotetraacetico (EDTA) /0.5 mM ditiotreitolo (DTT) per la dissociazione non enzimatica del tessuto. </li><li> Tagliare il tessuto in piccoli pezzi (2-4 mm) usando una lama di rasoio su un vetrino pre-raffreddato. Trasferire i pezzi in un tubo da 50 ml contenente 20 ml di PBS freddo ghiaccio. </li><li> Pipettare su e giù delicatamente 10x con un 10 ml sterile, pipetta monouso. Lasciate che frammenti di tessuto sedimentano per gravità. Rimuovere il surnatante con una pipetta. Ripetere altre tre volte; assicurarsi che il surnatante è chiaro. </li><li> Aggiungere 20 ml di PBS per 2 mM EDTA. Turbinio e incubare a 4 ° C per 30 minuti con dolce dondolio. </li><li> Lasciate sedimenti tessuti ed eliminare il surnatante. Aggiungere 15 ml di PBS freddo ghiaccio e pipettasu e giù 5x con una pipetta 10 ml. Lasciate che i frammenti di tessuto sedimenti e raccogliere il surnatante come Frazione 1 (F1). Ripetere la raccolta di F2-F5, ogni frazione mantenuto separatamente. </li><li> Aggiungere 15 ml di ghiaccio PBS freddo e questa volta agitare vigorosamente a mano per 15 secondi. Raccogliere F6 e ripetere per F7, F8. Se i pezzi di tessuto cominciano a galleggiare, toccare per aiutarli a stabilirsi. </li><li> Controllare un&#39;aliquota di ciascuna frazione al microscopio. In comune le frazioni contenenti cripte. </li><li> Passare le frazioni aggregati attraverso un filtro 70 micron cellule di nylon, raccogliendo cripte in un tubo da 50 ml. </li><li> Centrifugare a 100 xg per 5 minuti a 4 ° C, scartare il surnatante e risospendere il pellet in 10 ml di PBS freddo ghiaccio con 2x cripte anti-anti e trasferirli in una provetta da 15 ml. Ripetere il lavaggio ancora una volta. </li><li> Lavare le cripte una volta con 10 ml ghiacciata ADF, avanzata DMEM / F-12 (Dulbecco &quot;s Modified Eagle Medium / Ham&quot; s F-12) - 2x anti-anti. </li><li> Lavare le cripte una volta con 10 ml ADF - 1x anti-anti. </li><li> Contare le cripte con un emocitometro. Regolare il volume della soluzione cripta e trasferire in una provetta da 1,5 ml in modo che la concentrazione finale quando cripta risospeso sarà 100-500 cripte per 50 ml di miscela proteica gelatinosa (Matrigel). Centrifugare a 100 xg per 5 min a 4 ° C e risospendere cripte nella miscela proteica gelatinosa. </li><li> Tavola 50 ml di sospensione gelatinosa cripta - miscela proteica per pozzetto in una piastra da 24 pozzetti (area di crescita: 2 cm 2), ponendo attenzione la goccia di sospensione al centro di ciascun pozzetto. Mantenere la piastra in una di CO 2, 37 ° C incubatore per ~ 30 minuti fino a quando la sospensione si solidifica. </li><li> Aggiungere 500 supporti freddo ghiaccio cultura completa ml (ottimo DMEM / F12 con 1x contro - contro, 10 mM 4- (2-idrossietil) -1 acid-piperazineethanesulfonic (HEPES), 1x Glutamax, 1x B27 supplemento, 1x N2 Supplement, 1 mM N-acetil-L-cisteina (NAC), 50 ng / ml di fattore di crescita epidermico (EGF), 100 ng / mlZucca, 500 ng / ml R-spondina). <ol><li> Ricostituire fattori di crescita nello 0,1% albumina sierica bovina (BSA) in PBS. </li></ol></li></ol></li><li> Cripta organoide cultura e del passaggio: <ol><li> Passaggio organoidi cripta 14-21 giorni dopo la semina (o secondo necessità) come segue: <ol><li> Cambia i media ogni venerdì e lunedì. Il mercoledì, sostituire la metà della media con mezzi freschi. </li></ol></li><li> Rimuovere il terreno di coltura. Lavare il campione due volte con 500 microlitri di PBS a intervalli di 5 min. </li><li> Rimuovere PBS e rompere delicatamente la miscela proteica gelatinosa con un P1000 sterile punta micro pipetta. </li><li> Aggiungi terreni di coltura 1 ml completa di un singolo pozzo e risospendere le organoidi in 1 ml di media. </li><li> Interrompere delicatamente i organoidi con un P1000 pipettando su e giù 20-30x (controllare sotto il microscopio per una corretta dissociazione organoide con una buona resa cripta, fino a quando una tecnica consistente si sviluppa). </li><li> Trasferire le cripte in una provetta da microcentrifuga da 1,5 ml;centrifugare a 100 xg a 4 ° C, 5 min. </li><li> Lavare il pellet due volte con 1 ml di terreno di coltura completo. </li><li> Split a un rapporto di 1: 3 o 1: 6 come necessario, cripte risospeso in 50 microlitri Matrigel per pozzetto. Procedere come descritto in precedenza (sezione 1.1.16-18). </li></ol></li><li> Cripta di congelamento organoide: <ol><li> Rimuovere il terreno di coltura. Lavare il campione in 500 l di PBS. </li><li> Disperdere la miscela proteica gelatinosa con una punta P1000 pipetta. </li><li> Aggiungere 1 ml di media per un singolo pozzo e risospendere le organoidi in 1 ml di media. </li><li> Rompere delicatamente le organoidi con un P1000 pipettando su e giù 20-30x. </li><li> Trasferire i organoidi in una provetta da 1,5 ml. Centrifugare a 100 xga 4 ° C, 5 min. </li><li> Lavare il pellet due volte con 1 ml di terreno di coltura completo. </li><li> Cripte risospendere in 500 microlitri di congelamento media. </li><li> Trasferimento cripte ad un cryotube, posizionare il tubo in un contenitore di congelamento e conservare in freezer -80 ° C overnight. </li><li> Trasferimento cripte di liquido N 2 serbatoio. <ol><li> Recuperare organoidi cripta congelati rapidamente scongelamento in un bagno a 37 ° C Acque. Lavare con 500 microlitri mezzi di coltura completo una volta, centrifugare a 100 xg, 5 min e risospendere in miscela proteica gelatinosa e cultura come descritto sopra. Per una migliore ripresa, aggiungere inibitore ROCK (Y-27632) per il supporto di congelamento. </li></ol></li></ol></li></ol> 2. Cripta organoide Metabolismo Assay <ol><li> 24-ben Preparazione del piatto: <ol><li> Isolare e contare cripte secondo il protocollo (vedere la sezione 1.1 per l&#39;isolamento cripta e la sezione 1.2 per il passaggio cripta). </li><li> Cripte Risospendere in miscela proteica gelatinosa (100-200 cripte per 20 ml). </li><li> Piastra cripta-gelatinosa sospensione miscela proteica in una piastra a 24 pozzetti test (assicurarsi che non vi siano almeno triplicato per ogni campione) e permettono la sospensione a solidificare a 37 ° C in CO 2incubatore; quindi aggiungere 500 microlitri Terreni di coltura completo. </li><li> Cripte cultura (come descritto nella sezione 1.1) e osservare al microscopio fino cripte crescere in organoidi completamente sviluppati. </li></ol></li><li> Extracellulare Flux saggio: <ol><li> Cartuccia Hydrate durante la notte in un non-CO 2 (0% CO 2), 37 ° C incubatore. </li><li> Rimuovere il terreno di coltura e lavare organoidi due volte con 500 microlitri DMEM (senza: glucosio, L-glutamina, piruvato di sodio e bicarbonato di sodio, e con: rosso fenolo). Attendere 5 min. </li><li> Aggiungere 675 ml per medie dimensioni, ben dosaggio (DMEM con 2 mM L-glutammina e 5 mm D-glucosio) in ciascun pozzetto. </li><li> Controllare cripta organoidi morfologia microscopica per garantire che organoidi e miscela proteica gelatinosa sono intatti dopo i lavaggi. Incubare 1 ora in un non-CO 2, 37 ° C incubatore. </li><li> Preparare 10 mM composti iniettabili (oligomicina, cianuro di carbonile-p-trifluoro-metossi-fenil-idrazone (FCCP), rotenone) nel terreno di coltura. </li><li> Set-up della cartuccia caricando 75 ml di 10 micron composti iniettabili nei porti della sequenza della cartuccia: Port A - oligomicina; Port B - FCCP; e Port C - rotenone (Le concentrazioni finali durante il test sarà di 1 micron). </li><li> Incubare cartuccia 30 min - 1 ora a 37 ° C in un non-CO 2 incubatore. </li><li> Allo stesso tempo, attivare la XF Analyzer e creare il modello di protocollo del test. </li><li> Posizionare la cartuccia e la piastra di utilità in XF Analyzer ed eseguire &quot;calibrare&quot; cartuccia. </li><li> Controllare organoidi cripta al microscopio per assicurarsi che essi sono attaccati alla miscela proteica gelatinosa. </li><li> Cella di carico piastra di coltura nel protocollo dello strumento ed eseguire test. </li></ol></li></ol>

<ol>
	<li>Jemal, 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=.">.</a> CA Cancer J Clin. 58, 71-96 (2008).</li><li>Slattery, M. L., Boucher, K. M., Caan, B. J., Potter, J. D., Ma, K. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eating+patterns+and+risk+of+colon+cancer.">Eating patterns and risk of colon cancer.</a> Am J Epidemiol. 148, 4-16 (1998).</li><li>Warburg, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+origin+of+cancer+cells.">On the origin of cancer cells.</a> Science. 123, 309-314 (1956).</li><li>Coles, N. W., Johnstone, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutamine+metabolism+in+Ehrlich+ascites-carcinoma+cells.">Glutamine metabolism in Ehrlich ascites-carcinoma cells.</a> Biochem J. 83, 284-291 (1962).</li><li>Medina, M. A., Castro I, N. u. n. e. z. d. e. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutaminolysis+and+glycolysis+interactions+in+proliferant+cells.">Glutaminolysis and glycolysis interactions in proliferant cells.</a> Int J Biochem. 22, 681-683 (1990).</li><li>Anso, 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=Metabolic+changes+in+cancer+cells+upon+suppression+of+MYC.">Metabolic changes in cancer cells upon suppression of MYC.</a> Cancer Metab. 1, 7 (2013).</li><li>Malmgren, 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=Coordinate+changes+in+histone+modifications,+mRNA+levels,+and+metabolite+profiles+in+clonal+INS-1+832/13+&#946;-cells+accompany+functional+adaptations+to+lipotoxicity.">Coordinate changes in histone modifications, mRNA levels, and metabolite profiles in clonal INS-1 832/13 &#946;-cells accompany functional adaptations to lipotoxicity.</a> J Biol Chem. 288 (17), 11973-11987 (2013).</li><li>Whitehead, R. 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=Establishment+of+conditionally+immortalized+epithelial+cell+lines+from+both+colon+and+small+intestine+of+adult+H-2Kb-tsA58+transgenic+mice.">Establishment of conditionally immortalized epithelial cell lines from both colon and small intestine of adult H-2Kb-tsA58 transgenic mice.</a> Proc Natl Acad Sci U S A. 90 (2), 587-591 (1993).</li><li>Roig, A. I., 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=Immortalized+epithelial+cells+derived+from+human+colon+biopsies+express+stem+cell+markers+and+differentiate+in+vitro.">Immortalized epithelial cells derived from human colon biopsies express stem cell markers and differentiate in vitro.</a> Gastroenterology. 138 (3), 1012-1021 (2010).</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, 262-265 (2009).</li><li>Sato, T., 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=Growing+self-organizing+mini-guts+from+a+single+intestinal+stem+cell:+mechanism+and+applications.">Growing self-organizing mini-guts from a single intestinal stem cell: mechanism and applications.</a> Science. 340, 1190-1194 (2013).</li><li>Yilmaz, O. 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=mTORC1+in+the+Paneth+cell+niche+couples+intestinal+stem-cell+function+to+calorie+intake.">mTORC1 in the Paneth cell niche couples intestinal stem-cell function to calorie intake.</a> Nature. 486, 490-495 (2012).</li></ol>

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Abbiamo testato il tasso di consumo di ossigeno (OCR) e il tasso di acidificazione extracellulare (ECAR) delle cripte isolate da 8 mesi topi e cresciuti in organoidi ex vivo. Dopo la misurazione del tasso basale, il metabolismo cripta è stata valutata con l&#39;aggiunta di oligomicina, il cianuro di carbonile -p-trifluoromethoxyphenylhydrazone (FCCP) e rotenone, in sequenza. Basale OCR e ECAR basale sono stati registrati 0-29 min (Figura 2A e 2B). ...

Cancro del colon-retto (CRC) è la terza causa di decessi per cancro correlati negli Stati Uniti. Cancro del colon sporadica - vale a dire che derivanti nel corso della vita (&gt; 50 anni di età) e senza fattori predisponenti genetici chiare - conti per ~ 80% di tutti i casi, con un&#39;incidenza fortemente influenzata da abitudini alimentari a lungo termine 1,2. Questi tumori presentano uno spostamento metabolico verso dipendenza glicolisi ossidativa, noto come effetto Warburg, che può in parte rendere concentrazioni elevate di particelle elementari cellulari e di energia disponibili (attraverso glutaminolysis) permettere e forse guidare alti tassi di proliferazione delle cellule tumorali 3-5 . Studi di tumore del colon e altri tumori gastrointestinali, inclusi piccoli tumori intestinali fornire informazioni importanti sulle cause della formazione di tumori. Indagare le differenze metaboliche tra gli stati normali, pro-tumorali e tumorali di sistemi di organi gastrointestinali possono aiutare detine di rischio relativo per lo sviluppo del tumore e la diagnosi precoce della neoplasia. Inoltre, la comprensione del metabolismo bioenergetico che coinvolge la respirazione mitocondriale e la glicolisi si forniscono informazioni fondamentali sul modo in cui la fisiologia delle cellule, l&#39;invecchiamento e la malattia lo stato perturba l&#39;omeostasi intestinale. L&#39;utilizzo della tecnologia bioenergetica test per l&#39;analisi del flusso extracellulare in grado di valutare i tassi di respirazione mitocondriale e glicolisi contemporaneamente in cellule in crescita in coltura in tempo reale 6,7. Fino a poco tempo, studi in vitro di tenue sono stati limitati a linee cellulari derivate da tumori benigni o maligni 8,9 e non rappresentano la fisiologia dei normali epiteli intestinali e l&#39;influenza del microambiente in cui risiedono. Nel 2009, Sato et al. 10 ha introdotto un sistema ex vivo della cultura a crescere tridimensionale (3D) del mouse intestinali organoidi epiteliali, o epithelial &quot;mini-coraggio&quot;, adatto a fini sperimentali, diagnostiche e terapeutiche indagini 10,11. Inoltre, cripte isolate da topi calorico limitati mantengono le loro proprietà di crescita alterati come organoidi in tali colture 12. Rispetto a linee cellulari trasformate, culture organoide cripta possono essere utilizzati per generare dati fisiologicamente rilevanti che presentano un modello molto meglio comprendere lo stato in vivo. Abbiamo adattato la tecnologia di analisi bioenergetica per saggiare il metabolismo energetico di organoidi cripta intestinale. Mouse organoidi cripta intestinale sono state coltivate ex vivo per lo sviluppo degli studi cripta metabolismo energetico organoide presentati. Il tasso di consumo di ossigeno (OCR) e il tasso di acidificazione extracellulare (ECAR) di organoidi cripta sono stati misurati in assenza e in presenza di due diversi inibitori metabolici (oligomicina, rotenone) e un vettore di ioni (Carbonil cianuro-p-trifluorometossifenil idrazone). L&#39;orga criptarisposta metabolica noid a questi composti chimici sono riflessi con successo attraverso la modifica ECAR e valori OCR. Studi bioenergetici cellulari potranno chiarire le reciproche interazioni tra stato metabolico e rischio di malattia e fenotipo nel cancro, obesità, diabete, malattie metaboliche e le malattie mitocondriali e contribuire a metodi di screening anticipo aventi implicazioni dirette per la medicina traslazionale. Qui, descriviamo un protocollo dettagliato per isolare piccole cripte intestinali e alla cultura organoidi cripta. Inoltre, si introduce un nuovo metodo per usare le culture organoide cripta per le analisi metaboliche.

<table border="0" cellpadding="0" cellspacing="0" width="1633">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">BD Matrigel Basement Membrane Matrix, GFR, Phenol Red-free, LDEV-free</td>
			<td style="width:229px;">BD Biosciences</td>
			<td style="width:135px;">356231</td>
			<td style="width:456px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">PBS (phosphate buffered saline), no magnesium, no calcium, pH 7.2</td>
			<td style="width:229px;">Life Technologies</td>
			<td style="width:135px;">20012-027</td>
			<td style="width:456px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">Advanced DMEM/F-12 (1X)</td>
			<td style="width:229px;">Life Technologies</td>
			<td style="width:135px;">12634-028</td>
			<td style="width:456px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">Dulbecco&#8242;s Modified Eagle&#8242;s Medium without glucose, L-glutamine, Phenol Red, sodium pyruvate and sodium bicarbonate</td>
			<td style="width:229px;">Sigma-Aldrich</td>
			<td style="width:135px;">D5030</td>
			<td style="width:456px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">Phenol Red sodium salt&#160;</td>
			<td style="width:229px;">Sigma-Aldrich</td>
			<td style="width:135px;">P4758</td>
			<td style="width:456px;">Final Concentration 15 mg / L in DMEM (D5030) - step 2.2.2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">Antibiotic-Antimycotic, 100X, 100ml</td>
			<td style="width:229px;">Life Technologies</td>
			<td style="width:135px;">15240-062&#160;</td>
			<td style="width:456px;">Final Concentration 1 X or 2 X</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">Penicilin-Streptomycin, liquid</td>
			<td style="width:229px;">Life Technologies</td>
			<td style="width:135px;">15140-122</td>
			<td style="width:456px;">Final Concentration 1 X</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">Gibco&#174; GlutaMAX&#8482; supplement</td>
			<td style="width:229px;">Life Technologies</td>
			<td style="width:135px;">35050061</td>
			<td style="width:456px;">Final Concentration 1 X</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">Gibco&#174; HEPES (N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid), 1 M</td>
			<td style="width:229px;">Life Technologies</td>
			<td style="width:135px;">15630-080</td>
			<td style="width:456px;">Final Concentration 10 mM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">N-Acetyl-L-Cysteine, 25g</td>
			<td style="width:229px;">Sigma-Aldrich</td>
			<td style="width:135px;">A9165-25G</td>
			<td style="width:456px;">Final Concentration 1 mM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">100X N-2 supplement, liquid</td>
			<td style="width:229px;">Invitrogen</td>
			<td style="width:135px;">17502-048</td>
			<td style="width:456px;">Final Concentration 1 X</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">50X B-27&#174; supplement minus Vitamin A, liquid</td>
			<td style="width:229px;">Invitrogen</td>
			<td style="width:135px;">12587-010</td>
			<td style="width:456px;">Final Concentration 1 X</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">Recombinant Mouse R-Spondin 1, CF, 50ug</td>
			<td style="width:229px;">R&#38;D Systems</td>
			<td style="width:135px;">3474-RS-050&#160;</td>
			<td style="width:456px;">Final Concentration 500 ng / mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">Recombinant Murine EGF, 100ug&#160;</td>
			<td style="width:229px;">Peprotech</td>
			<td style="width:135px;">315-09&#160;</td>
			<td style="width:456px;">Final Concentration 50 ng / mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">Recombinant Murine Noggin, 20ug&#160;</td>
			<td style="width:229px;">Peprotech</td>
			<td style="width:135px;">250-38</td>
			<td style="width:456px;">Final Concentration 100 ng / mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">Gibco&#174; L-glutamine, 200 mM</td>
			<td style="width:229px;">Life Technologies</td>
			<td style="width:135px;">25030-081</td>
			<td style="width:456px;">Final Concentration 2 mM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">Gibco&#174; Glucose powder</td>
			<td style="width:229px;">Life Technologies</td>
			<td style="width:135px;">15023-021</td>
			<td style="width:456px;">Final Concentration 5 mM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">Ambion&#174; 0.5 M EDTA (Ethylenediaminetetraacetic acid), pH 8.0</td>
			<td style="width:229px;">Life Technologies</td>
			<td style="width:135px;">AM9260G</td>
			<td style="width:456px;">Final Concentration 3 mM for step 1.1.5; 2 mM for step 1.1.8&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">DTT (Dithiothreitol), 1M</td>
			<td style="width:229px;">Life Technologies</td>
			<td style="width:135px;">P2325</td>
			<td style="width:456px;">Final Concentration 3 mM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">Albumin from bovine serum (BSA)</td>
			<td style="width:229px;">Sigma-Aldrich</td>
			<td style="width:135px;">A2058</td>
			<td style="width:456px;">0.1 % in PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">Fetal Bovine Serum (FBS)</td>
			<td style="width:229px;">Life Technologies</td>
			<td style="width:135px;">16000-044</td>
			<td style="width:456px;">1 % in PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">Recovery&#8482; Cell Culture Freezing Medium</td>
			<td style="width:229px;">Life Technologies</td>
			<td style="width:135px;">12648-010</td>
			<td style="width:456px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">ROCK inhibitor (Y-27632)&#160;</td>
			<td style="width:229px;">Sigma-Aldrich</td>
			<td style="width:135px;">Y0503</td>
			<td style="width:456px;">Final Concentration 10 &#181;M</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">Oligomycin</td>
			<td style="width:229px;">Sigma-Aldrich</td>
			<td style="width:135px;">O4876</td>
			<td style="width:456px;">Final Concentration 1 &#181;M&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">Carbonyl cyanide-p-trifluoro-methoxy-phenyl-hydrazone (FCCP)</td>
			<td style="width:229px;">Sigma-Aldrich</td>
			<td style="width:135px;">C2920</td>
			<td style="width:456px;">Final Concentration 1 &#181;M&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">Rotenone</td>
			<td style="width:229px;">Sigma-Aldrich</td>
			<td style="width:135px;">R8875</td>
			<td style="width:456px;">Final Concentration 1 &#181;M&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">Sodium hydroxide</td>
			<td style="width:229px;">Sigma-Aldrich</td>
			<td style="width:135px;">221465</td>
			<td style="width:456px;">Final Concentration 0.1 N in PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">XF24 Extracellular Flux Analyzer (XF Analyzer)</td>
			<td style="width:229px;">Seahorse Bioscience</td>
			<td style="width:135px;"></td>
			<td style="width:456px;"></td>
		</tr>
	</tbody>
</table>

real time analysis of metabolic profile in ex vivo mouse intestinal crypt organoid cultures

La mucosa dell&#39;intestino tenue presenta un&#39;architettura ripetitiva organizzata in due strutture fondamentali: villi, sporgenti nel lume intestinale e composti da enterociti maturi, cellule caliciformi e cellule enteroendocrine; e cripte, che risiedono in prossimità del sottomucosa e muscolare, l&#39;ospitare staminali adulte e cellule progenitrici e cellule mature Paneth, così come stromali e cellule del sistema immunitario del microambiente cripta. Fino agli ultimi anni, studi in vitro di piccolo intestino era limitato a linee cellulari derivate da tumori benigni o maligni, e non rappresentano la fisiologia dei normali epiteli intestinali e l&#39;influenza del microambiente in cui risiedono. Qui, dimostriamo un metodo adattato da Sato et al. (2009) per la coltura di organoidi cripta del mouse intestinali primarie derivate da topi C57BL / 6. Inoltre, vi presentiamo l&#39;uso di colture organoide cripta di saggiare il profilo metabolico cripta in tempo reale per misurazione del consumo basale di ossigeno, glicolisi, la produzione di ATP e la capacità respiratoria. Organoidi mantengono proprietà definite per la loro origine e conservano gli aspetti del loro adattamento metabolico riflessa dal consumo di ossigeno e tassi di acidificazione extracellulare. In tempo reale studi metabolici in questo organoide sistema di coltura cripta sono un potente strumento per studiare il metabolismo energetico organoide cripta, e come possono essere modulati da fattori nutrizionali e farmacologici.

Organoidi cripta sono state stabilite da 8 mesi C57BL / 6 topi nutriti purificato roditore dieta American Institute of Nutrition 76A (AIN76A). Organoidi cripta intestinali possono essere coltivate in coltura per lunghi periodi da un unico crypt (Figura 1A, singola freccia rossa). Organoidi crescono strutture cripta-come in 18-20 giorni in coltura (Figura 1B, frecce rosse). Cripte sono stati diversi passaggi ogni 3 settimane e organoidi recuperati in modo efficiente dopo ogni passaggio. ...

Watch this Scientific Journal Video about Real Time Analysis of Metabolic Profile in Ex Vivo Mouse Intestinal Crypt Organoid Cultures at JoVE.com

Real Time Analysis of Metabolic Profile in Ex Vivo Mouse Intestinal Crypt Organoid Cultures

Piccoli organoidi cripta intestinali in coltura ex vivo fornire un sistema di coltura tessuto che racchiude in sintesi la crescita delle cripte dipendenti sulle cellule staminali e la loro nicchia. Abbiamo stabilito un metodo per dosare il profilo metabolico in tempo reale in organoidi cripta del mouse primario. Abbiamo trovato organoidi mantengono le proprietà fisiologiche definite dalla loro fonte.

Real Time Analisi di profilo metabolico in<em&gt; Ex Vivo</em&gt; Culture del mouse intestinale Cripta organoide

The small intestinal mucosa exhibits a repetitive architecture organized into two fundamental structures: villi, projecting into the intestinal lumen and ...

real-time-analysis-metabolic-profile-ex-vivo-mouse-intestinal-crypt

Research

JoVE Journal

Biology

Real Time Analysis of Metabolic Profile in Ex Vivo Mouse Intestinal Crypt Organoid Cultures

16.1K Views.  Albert Einstein College of Medicine.  Piccoli organoidi cripta intestinali in coltura ex vivo fornire un sistema di coltura tessuto che racchiude in sintesi la crescita delle cripte dipendenti sulle cellule staminali e la loro nicchia. Abbiamo stabilito un metodo per dosare il profilo metabolico in tempo reale in organoidi cripta del mouse primario. Abbiamo trovato organoidi mantengono le proprietà fisiologiche definite dalla loro fonte. 

Video: Real Time Analysis of Metabolic Profile in Ex Vivo Mouse Intestinal Crypt Organoid Cultures

Eukaryotic Polyribosome Profile Analysis

Protein synthesis is a complex cellular process that is regulated at many levels. For example, global translation can be inhibited at the initiation phase or the elongation phase by a variety of cellular stresses such as amino acid starvation or growth factor withdrawal. Alternatively, translation of individual mRNAs can be regulated by mRNA localization or the presence of cognate microRNAs. Studies of protein synthesis frequently utilize polyribosome analysis to shed light on the mechanisms of translation regulation or defects in protein synthesis. In this assay, mRNA/ribosome complexes are isolated from eukaryotic cells.  A sucrose density gradient separates mRNAs bound to multiple ribosomes known as polyribosomes from mRNAs bound to a single ribosome or monosome. Fractionation of the gradients allows isolation and quantification of the different ribosomal populations and their associated mRNAs or proteins. Differences in the ratio of polyribosomes to monosomes under defined conditions can be indicative of defects in either translation initiation or elongation/termination. Examination of the mRNAs present in the polyribosome fractions can reveal whether the cohort of individual mRNAs being translated changes with experimental conditions. In addition, ribosome assembly can be monitored by analysis of the small and large ribosomal subunit peaks which are also separated by the gradient. In this video, we present a method for the preparation of crude ribosomal extracts from yeast cells, separation of the extract by sucrose gradient and interpretation of the results.  This procedure is readily adaptable to mammalian cells.

This article describes a protocol for the extraction of translating ribosomes from eukaryotic cells. Once extracted, ribosomes are separated into monosomes and polyribosomes by sucrose gradient fractionation to allow different ribosomal populations to be analyzed. As such, this method is the gold standard for examining the regulation of translation.

Eucarioti Analisi Profilo Polyribosome

Protein synthesis is a complex cellular process that is regulated at many levels. For example, global translation can be inhibited at the initiation phase ...

Ricerca

Biologia

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

Imaging in vivo e trattamenti terapeutici in un modello di mouse ortotopico di cancro ovarico

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Metabolic Profile Analysis of Zebrafish Embryos

A growing goal in the field of metabolism is to determine the impact of genetics on different aspects of mitochondrial function. Understanding these relationships will help to understand the underlying etiology for a range of diseases linked with mitochondrial dysfunction, such as diabetes and obesity. Recent advances in instrumentation, has enabled the monitoring of distinct parameters of mitochondrial function in cell lines or tissue explants. Here we present a method for a rapid and sensitive analysis of mitochondrial function parameters in vivo during zebrafish embryonic development using the Seahorse bioscience XF 24 extracellular flux analyser. This protocol utilizes the Islet Capture microplates where a single embryo is placed in each well, allowing measurement of bioenergetics, including: (i) basal respiration; (ii) basal mitochondrial respiration (iii) mitochondrial respiration due to ATP turnover; (iv) mitochondrial uncoupled respiration or proton leak and (iv) maximum respiration. Using this approach embryonic zebrafish respiration parameters can be compared between wild type and genetically altered embryos (mutant, gene over-expression or gene knockdown) or those manipulated pharmacologically. It is anticipated that dissemination of this protocol will provide researchers with new tools to analyse the genetic basis of metabolic disorders in vivo in this relevant vertebrate animal model.

Zebrafish represent a powerful vertebrate model that has been under-utilised for metabolic studies. Here we describe a rapid way to measure the in vivo metabolic profile of developing zebrafish that allows the comparison of different mitochondrial function parameters between genetically or pharmacologically manipulated embryos, thereby increasing the applicability of this organism.

Analisi Profilo metabolico di embrioni di zebrafish

A growing goal in the field of metabolism is to determine the impact of genetics on different aspects of mitochondrial function. Understanding these ...

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling processes. Extracting sterol-enriched membrane microdomains from plant cells for proteomic analysis is a difficult task mainly due to multiple preparation steps and sources for contaminations from other cellular compartments. The plasma membrane constitutes only about 5-20% of all the membranes in a plant cell, and therefore isolation of highly purified plasma membrane fraction is challenging. A frequently used method involves aqueous two-phase partitioning in polyethylene glycol and dextran, which yields plasma membrane vesicles with a purity of 95% 1. Sterol-rich membrane microdomains within the plasma membrane are insoluble upon treatment with cold nonionic detergents at alkaline pH. This detergent-resistant membrane fraction can be separated from the bulk plasma membrane by ultracentrifugation in a sucrose gradient 2. Subsequently, proteins can be extracted from the low density band of the sucrose gradient by methanol/chloroform precipitation. Extracted protein will then be trypsin digested, desalted and finally analyzed by LC-MS/MS. Our extraction protocol for sterol-rich microdomains is optimized for the preparation of clean detergent-resistant membrane fractions from Arabidopsis thaliana cell cultures.
We use full metabolic labeling of Arabidopsis thaliana suspension cell cultures with K15NO3 as the only nitrogen source for quantitative comparative proteomic studies following biological treatment of interest 3. By mixing equal ratios of labeled and unlabeled cell cultures for joint protein extraction the influence of preparation steps on final quantitative result is kept at a minimum. Also loss of material during extraction will affect both control and treatment samples in the same way, and therefore the ratio of light and heave peptide will remain constant. In the proposed method either labeled or unlabeled cell culture undergoes a biological treatment, while the other serves as control 4.

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Here we describe a robust method for the fractionation of plant plasma membranes into detergent resistant and detergent soluble membranes based on a mixture of unlabeled and in vivo fully 15N labeled Arabidopsis thaliana cell cultures. The procedure is applied for comparative proteomic studies to understand signaling processes.

Metabolic Etichettatura e con membrana frazionamento per proteomica comparativa Analisi del<em&gt; Arabidopsis thaliana</em&gt; Culture sospensione cellulare

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling ...

A Video Protocol of Retroviral Infection in Primary Intestinal Organoid Culture

Lgr5-positive stem cells can be supplemented with the essential growth factors Egf, Noggin, and R-Spondin, which allows us to culture ever-expanding primary 3D epithelial structures in vitro. Both the architecture and physiological properties of these &#39;mini-guts&#39;, also called organoids, closely resemble their in vivo counterparts. This makes them an attractive model system for the small intestinal epithelium. Using retroviral transduction, functional genetics can now be performed by conditional gene overexpression or knockdown. This video demonstrates the procedure of organoid culture, the generation of retroviruses, and the retroviral transduction of organoids to assist phenotypic analysis of the small intestinal epithelium in vitro. This novel organotypic model system in combination with retroviral mediated gene expression provides a valuable tool for rapid analysis of gene function in vitro without the need of costly and time-consuming generation for transgenic animals.

This protocol explains primary Lgr5-positve organoid culture and the subsequent performance of retroviral transduction. This enables Cre-inducible overexpression or knockdown of the delivered transgene and allows functional studies to be carried out in the novel in vitro organotypic model system.

Un protocollo video di infezione retrovirale in Primaria intestinale organoide Cultura

Lgr5-positive stem cells can be supplemented with the essential growth factors Egf, Noggin, and R-Spondin, which allows us to culture ever-expanding ...

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a versatile analytical tool for characterizing viscoelastic mechanical properties for a variety of materials ranging from hard (plastic, glass, metal, etc.) surfaces to cells on any substrate. It has been widely used to measure the stiffness of cells, but less frequently used to measure the stiffness of aortas. In this paper, we will describe the procedures for using AFM in contact mode to measure the ex vivo elastic modulus of unloaded mouse arteries. We describe our procedure for isolation of mouse aortas, and then provide detailed information for the AFM analysis. This includes step-by-step instructions for alignment of the laser beam, calibration of the spring constant and deflection sensitivity of the AFM probe, and acquisition of force curves. We also provide a detailed protocol for data analysis of the force curves.

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

We present detailed protocols for isolation of aortas from mouse and measurement of their elastic modulus using atomic force microscopy.

Misurare la rigidità<em&gt; Ex Vivo</em&gt; Mouse aorte Utilizzando microscopia a forza atomica

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a ...

Mixed Primary Cultures of Murine Small Intestine Intended for the Study of Gut Hormone Secretion and Live Cell Imaging of Enteroendocrine Cells

The gut is the largest endocrine organ of the body, with hormone-secreting enteroendocrine cells located along the length of the gastrointestinal epithelium. Despite their physiological importance, enteroendocrine cells represent only a small fraction of the epithelial cell population and in the past, their characterization has presented a considerable challenge resulting in a reliance on cell line models. Here, we provide a detailed protocol for the isolation and culture of mixed murine small intestinal cells. These primary cultures have been used to identify the signaling pathways underlying the stimulation and inhibition of gut peptide secretion in response to a number of nutrients and neuropeptides as well as pharmacological agents. Furthermore, in combination with the use of transgenic fluorescent reporter mice, we have demonstrated that these primary cultures become a powerful tool for the examination of fluorescently-tagged enteroendocrine cells at the intracellular level, using methods such as patch clamping and single-cell calcium and cAMP-FRET imaging.

This protocol describes the isolation and culture of mixed murine small intestinal cells. These primary intestinal cultures enable the investigation of signaling pathways underlying gut peptide secretion using a number of techniques.

Mixed colture primarie di murino tenue finalizzate allo studio di Gut secrezione dell&#39;ormone e imaging cellulare dal vivo di celle enteroendocrine

The gut is the largest endocrine organ of the body, with hormone-secreting enteroendocrine cells located along the length of the gastrointestinal ...

Real-time In Vivo Recording of Arabidopsis Calcium Signals During Insect Feeding Using a Fluorescent Biosensor

Calcium ions are predicted to be key signaling entities during biotic interactions, with calcium signaling forming an established part of the plant defense response to microbial elicitors and to wounding caused by chewing insects, eliciting systemic calcium signals in plants. However, the role of calcium in vivo during biotic stress is still unclear. This protocol describes the use of a genetically-encoded calcium sensor to detect calcium signals in plants during feeding by a hemipteran pest. Hemipterans such as aphids pierce a small number of cells with specialized, elongated sucking mouthparts, making them the ideal tool to study calcium dynamics when a plant is faced with a biotic stress, which is distinct from a wounding response. In addition, fluorescent biosensors are revolutionizing the measurement of signaling molecules in vivo in both animals and plants. Expressing a GFP-based calcium biosensor, GCaMP3, in the model plant Arabidopsis thaliana allows for the real-time imaging of plant calcium dynamics during insect feeding, with a high spatial and temporal resolution. A repeatable and robust assay has been developed using the fluorescence microscopy of detached GCaMP3 leaves, allowing for the continuous measurement of cytosolic calcium dynamics before, during, and after insect feeding. This reveals a highly-localized rapid calcium elevation around the aphid feeding site that occurs within a few minutes. The protocol can be adapted to other biotic stresses, such as additional insect species, while the use of Arabidopsis thaliana allows for the rapid generation of mutants to facilitate the molecular analysis of the phenomenon.

Real-time In Vivo Recording of Arabidopsis Calcium Signals During Insect Feeding Using a Fluorescent Biosensor

This protocol outlines a simple method for analyzing calcium signals in plants generated by feeding hemipteran insects, such as aphids. Arabidopsis thaliana transformed with the GFP calcium biosensor GCaMP3 allow for the real-time in vivo imaging of calcium dynamics with a high temporal and spatial resolution.

Registrazione in tempo reale In Vivo di Arabidopsis segnali di calcio durante l'alimentazione dell'insetto utilizzando un biosensore fluorescente

Calcium ions are predicted to be key signaling entities during biotic interactions, with calcium signaling forming an established part of the plant ...

An Ex Vivo Choroid Sprouting Assay of Ocular Microvascular Angiogenesis

Pathological choroidal angiogenesis, a salient feature of age-related macular degeneration, leads to vision impairment and blindness. Endothelial cell (EC) proliferation assays using human retinal microvascular endothelial cells (HRMECs) or isolated primary retinal ECs are widely used in vitro models to study retinal angiogenesis. However, isolating pure murine retinal endothelial cells is technically challenging and retinal ECs may have different proliferation responses than choroidal endothelial cells and different cell/cell interactions. A highly reproducible ex vivo choroidal sprouting assay as a model of choroidal microvascular proliferation was developed. This model includes the interaction between choroid vasculature (EC, macrophages, pericytes) and retinal pigment epithelium (RPE). Mouse RPE/choroid/scleral explants are isolated and incubated in growth-factor-reduced basal membrane extract (BME) (day 0). Medium is changed every other day and choroid sprouting is quantified at day 6. The images of individual choroid explant are taken with an inverted phase microscope and the sprouting area is quantified using a semi-automated macro plug-in to the ImageJ software developed in this lab. This reproducible ex vivo choroidal sprouting assay can be used to assess compounds for potential treatment and for microvascular disease research to assess pathways involved in choroidal micro vessel proliferation using wild type and genetically modified mouse tissue.

This protocol presents choroid sprouting assay, an ex vivo model of microvascular proliferation. This assay can be used to assess pathways involved in proliferating choroidal micro vessels and assess drug treatments using wild type and genetically modified mouse tissue.

Un test di germinazione coroide ex vivo di angiogenesi microvascolare oculare

Pathological choroidal angiogenesis, a salient feature of age-related macular degeneration, leads to vision impairment and blindness. Endothelial cell ...

Organoid-Derived Epithelial Monolayer: A Clinically Relevant In Vitro Model for Intestinal Barrier Function

In the past, intestinal epithelial model systems were limited to transformed cell lines and primary tissue. These model systems have inherent limitations as the former do not faithfully represent original tissue physiology, and the availability of the latter is limited. Hence, their application hampers fundamental and drug development research. Adult stem-cell-based organoids (henceforth referred to as organoids) are miniatures of normal or diseased epithelial tissue from which they are derived. They can be established very efficiently from different gastrointestinal (GI) tract regions, have long-term expandability, and simulate tissue- and patient-specific responses to treatments in vitro. Here, the establishment of intestinal organoid-derived epithelial monolayers has been demonstrated along with methods to measure epithelial barrier integrity, permeability and transport,&#160;antimicrobial protein secretion, as well as histology. Moreover, intestinal organoid-derived monolayers can be enriched with proliferating stem and transit-amplifying cells as well as with key differentiated epithelial cells. Therefore, they represent a model system that can be tailored to study the effects of compounds on target cells and their mode of action. Although organoid cultures are technically more demanding than cell lines, once established, they can reduce failures in the later stages of drug development as they truly represent in vivo epithelium complexity and interpatient heterogeneity.

Here, we describe the preparation of human organoid-derived intestinal epithelial monolayers for studying intestinal barrier function, permeability, and transport. As organoids represent original epithelial tissue response to external stimuli, these models combine the advantages of expandability of cell lines and the relevance and complexity of primary tissue.

Monostrato epiteliale derivato da organoidi: un modello in vitro clinicamente rilevante per la funzione di barriera intestinale

In the past, intestinal epithelial model systems were limited to transformed cell lines and primary tissue. These model systems have inherent limitations ...