Questo lavoro è stato sostenuto dal programma HIT-CF della olandese CF fondazione (NCFS), ZonMw (40-00812-98-14103), Fondo di ricerca dell&#39;ospedale dei bambini di Wilhelmina e CZ, e Zilverenkruis / Achmea. Vorremmo ringraziare S. Heida-Michel, M. Geerdink, KM de Winter-de Groot, e G. Berkers (Dipartimento di Pneumologia Pediatrica, Ospedale Wilhelmina bambini, UMC Utrecht), e RHJ Houwen (Dipartimento di Gastroenterologia Pediatrica, Wilhelmina Ospedale pediatrico, UMC Utrecht) per avvicinare i pazienti e ottenere le biopsie per la generazione di un CF Biobank.

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	<li>De Boeck, K., Amaral, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+in+therapies+for+cystic+fibrosis.">Progress in therapies for cystic fibrosis.</a> Lancet Respir Med. 4 (8), 662-674 (2016).</li><li>Cutting, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cystic+fibrosis+genetics:+from+molecular+understanding+to+clinical+application.">Cystic fibrosis genetics: from molecular understanding to clinical application.</a> Nature Rev Genet. 16 (1), 45-56 (2015).</li><li>Sosnay, P. R., Siklosi, K. 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W., Davies, J., 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+CFTR+potentiator+in+patients+with+cystic+fibrosis+and+the+G551D+mutation.">A CFTR potentiator in patients with cystic fibrosis and the G551D mutation.</a> N Engl J Med. 365 (18), 1663-1672 (2011).</li><li>De Boeck, K., Munck, 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=Efficacy+and+safety+of+ivacaftor+in+patients+with+cystic+fibrosis+and+a+non-G551D+gating+mutation.">Efficacy and safety of ivacaftor in patients with cystic fibrosis and a non-G551D gating mutation.</a> J Cyst Fibros. 13 (6), 674-680 (2014).</li><li>Boyle, M. P., Bell, S. 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=A+CFTR+corrector+(lumacaftor)+and+a+CFTR+potentiator+(ivacaftor)+for+treatment+of+patients+with+cystic+fibrosis+who+have+a+phe508del+CFTR+mutation:+a+phase+2+randomised+controlled+trial.">A CFTR corrector (lumacaftor) and a CFTR potentiator (ivacaftor) for treatment of patients with cystic fibrosis who have a phe508del CFTR mutation: a phase 2 randomised controlled trial.</a> Lancet Respir Med. 2 (7), 527-538 (2014).</li><li>Wainwright, C. E., Elborn, J. 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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=Single+Lgr5+stem+cells+build+crypt-villus+structures+in+vitro+without+a+mesenchymal+niche.">Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche.</a> Nature. 459 (7244), 262-265 (2009).</li><li>Sato, T., Stange, D. 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=Long-term+expansion+of+epithelial+organoids+from+human+colon,+adenoma,+adenocarcinoma,+and+Barrett's+epithelium.">Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett's epithelium.</a> Gastroenterology. 141 (5), 1762-1772 (2011).</li><li>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=Modeling+development+and+disease+with+Organoids.">Modeling development and disease with Organoids.</a> Cell. 165 (7), 1586-1597 (2016).</li><li>Dekkers, J. F., Wiegerinck, C. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+functional+CFTR+assay+using+primary+cystic+fibrosis+intestinal+organoids.">A functional CFTR assay using primary cystic fibrosis intestinal organoids.</a> Nature Med. 19 (7), 939-945 (2013).</li><li>Dekkers, J. F., van der Ent, C. K., Beekman, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+opportunities+for+CFTR-targeting+drug+development+using+organoids.">Novel opportunities for CFTR-targeting drug development using organoids.</a> Rare Diseases. 1 (1), e27112 (2014).</li><li>Servidoni, M. F., Sousa, 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=Rectal+forceps+biopsy+procedure+in+cystic+fibrosis:+technical+aspects+and+patients+perspective+for+clinical+trials+feasibility.">Rectal forceps biopsy procedure in cystic fibrosis: technical aspects and patients perspective for clinical trials feasibility.</a> BMC gastroenterology. 13, 91 (2013).</li><li>Ommen, D. D. Z. -. V., Vijftigschild, L. A. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Limited+premature+termination+codon+suppression+by+read-through+agents+in+cystic+fibrosis+intestinal+organoids.">Limited premature termination codon suppression by read-through agents in cystic fibrosis intestinal organoids.</a> J Cyst Fibros. 15 (2), 158-162 (2016).</li><li>Dekkers, J. F., Gogorza Gondra, R. 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=Optimal+correction+of+distinct+CFTR+folding+mutants+in+rectal+cystic+fibrosis+organoids.">Optimal correction of distinct CFTR folding mutants in rectal cystic fibrosis organoids.</a> Eur Respir J. , (2016).</li><li>Dekkers, J. F., Van Mourik, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potentiator+synergy+in+rectal+organoids+carrying+S1251N,+G551D,+or+F508del+CFTR+mutations.">Potentiator synergy in rectal organoids carrying S1251N, G551D, or F508del CFTR mutations.</a> J Cyst Fibros. , (2016).</li><li>Dekkers, J. F., Berkers, 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=Characterizing+responses+to+CFTR-modulating+drugs+using+rectal+organoids+derived+from+subjects+with+cystic+fibrosis.">Characterizing responses to CFTR-modulating drugs using rectal organoids derived from subjects with cystic fibrosis.</a> Sci Transl Med. 8 (344), 344ra84 (2016).</li><li>Li, H., Sheppard, D. N., Hug, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transepithelial+electrical+measurements+with+the+Ussing+chamber.">Transepithelial electrical measurements with the Ussing chamber.</a> J Cyst Fibros. 3, 123-126 (2004).</li><li>Sheppard, D. N., Carson, M. R., Ostedgaard, L. S., Denning, G. M., Welsh, M. 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H., 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=Redundant+sources+of+Wnt+regulate+intestinal+stem+cells+and+promote+formation+of+Paneth+cells.">Redundant sources of Wnt regulate intestinal stem cells and promote formation of Paneth cells.</a> Gastroenterology. 143 (6), 1518-1529 (2012).</li><li>Drost, J., 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+culture+systems+for+prostate+epithelial+and+cancer+tissue.">Organoid culture systems for prostate epithelial and cancer tissue.</a> Nat Protoc. 11 (2), 347-358 (2016).</li><li>Vijftigschild, L. A. W., Berkers, 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=&#946;2-adrenergic+receptor+agonists+activate+CFTR+in+organoids+and+subjects+with+cystic+fibrosis.">&#946;2-adrenergic receptor agonists activate CFTR in organoids and subjects with cystic fibrosis.</a> Eur Respir J. 48 (3), 768-779 (2016).</li><li>Dekkers, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+bioassay+using+intestinal+organoids+to+measure+CFTR+in+human+plasma.">A bioassay using intestinal organoids to measure CFTR in human plasma.</a> J Cyst Fibros. 14 (2), 178-181 (2015).</li><li>Graeber, S. Y., 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=Intestinal+Current+Measurements+Detect+Activation+of+Mutant+CFTR+in+Patients+with+Cystic+Fibrosis+with+the+G551D+Mutation+Treated+with+Ivacaftor.">Intestinal Current Measurements Detect Activation of Mutant CFTR in Patients with Cystic Fibrosis with the G551D Mutation Treated with Ivacaftor.</a> Am J Respir Crit Care Med. 192 (10), 1252-1255 (2015).</li><li>Van Goor, 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=Ivacaftor+potentiation+of+multiple+CFTR+channels+with+gating+mutations.">Ivacaftor potentiation of multiple CFTR channels with gating mutations.</a> Journal of Cystic Fibrosis: Official Journal of the Eur Cyst Fibrosis. 11 (3), 237-245 (2012).</li></ol>

Hans Clevers is an inventor on patents for organoid culture and the Forskolin-induced swelling assay. Jeffrey M Beekman is an inventor on a patent for the Forskolin-induced swelling assay. Sylvia F Boj and Robert Vries are employed by the Foundation Hubrecht Organoid Technology (HUB), which holds the exclusive license to the Organoid Technology. Otherwise, the authors have nothing to disclose.

Qui, forniamo un protocollo completo per la generazione, l&#39;espansione, il congelamento, lo scongelamento e di organoidi colorettali umani. Mentre abbiamo stabilito le culture umane organoide qualche tempo fa 17, è a volte dimostrato difficile stabilire la tecnologia in altri laboratori, senza una formazione pratica. Prevediamo che questi protocolli sostituiranno tale formazione. Wnt-3A-condizionata mezzo è uno dei reagenti più importanti al fine di riuscire a s...

CF è causata da mutazioni nel gene della fibrosi cistica regolatore della conduttanza transmembrana (CFTR), che codifica per un canale anionico epiteliale. CF colpisce circa 85.000 persone in tutto il mondo 1. Oltre 2.000 mutazioni CFTR sono state identificate ( <a href="http://www.genet.sickkids.on.ca">www.genet.sickkids.on.ca</a> ). Questa diversità spiega in parte un ampio spettro di fenotipi della malattia osservati ( <a href="http://www.CFTR2.org">www.CFTR2.org</a> ) 2, 3. Sei classi di mutazioni CFTR sono definiti in base alla loro effetto sulla espressione della proteina CFTR e la funzione: (I) N. sintesi, (II) il traffico compromessa, (III) del canale difettoso gating, (IV) la conduttanza alterato, (V) i livelli di norma ridotta funzionamento CFTR, e (VI) cellula compromessa la stabilità della superficie 4. Anche se le mutazioni CFTR comuni sono ben studiati, la funzione CFTR e relazione con lo stato clinico rimangono poorly inteso a livello del singolo, in particolare per il grande gruppo di rare &quot;orfani&quot; mutazioni ( <a href="http://www.CFTR2.org">www.CFTR2.org</a> ) 1, 3. Recentemente, i farmaci sono stati sviluppati che hanno come bersaglio la proteina CFTR in maniera specifica per la mutazione. Due classi di farmaci proteici CFTR-targeting sono attualmente in uso clinico e hanno distinte modalità di azione. Potenziatori, come il VX-770, migliorano l&#39;open-probabilità di CFTR mutata direzione apicale-localizzata e agiscono direttamente sulla loro aggiunta a cellule 5. Correttori, come il VX-809, ripristinare il traffico di reticolo endoplasmatico-localizzato misfolded CFTR e richiedono pre-incubazione con si osservano cellule prima che gli effetti 6. Il potenziatore CFTR, VX-770, è stato registrato per i soggetti con la mutazione G551D 7, 8, così come per altri ottoCFTR gating mutazioni, tra cui S1251N 9; insieme, queste mutazioni sono trasportati da 5% di tutti i soggetti CF. Altri studi clinici hanno indicato che VX-770, in combinazione con il correttore VX-809, ha effetti limitati ma significativi sulla funzione polmonare e provoca una diminuzione della frequenza di esacerbazioni nei soggetti omozigoti per la mutazione F508del portato da 45-50% dei pazienti 10, 11. studi clinici convenzionali per identificare i soggetti farmaco-sensibile all&#39;interno del restante 50% dei pazienti affetti da FC sono costosi e che richiede tempo e non sono realizzabili per gli individui con genotipo CFTR estremamente rari. Novel, metodi personalizzati di costo-efficacia sono cruciali per soddisfare il crescente numero di modulatori CFTR a individui portatori di qualsiasi tipo di CFTR mutazione. Fino ad ora, l&#39;inclusione di prova di gruppi di pazienti che trasportano specifiche mutazioni CFTR è stata guidata da studi che utilizzano mutante trasfezione del gene CFTR in hsistemi cellulari eterologous, seguiti da studi elettrofisiologici in Camera di Ussing 5, 6, 12. A causa della mancanza di modelli animali CF adeguate, studi di efficacia del farmaco nelle cellule epiteliali bronchiali aria-liquido-interfaccia dissociati derivati da CF polmonari materiali espianti sono stati utilizzati per lo sviluppo di farmaci 13, 14, 15. Tuttavia, la limitata disponibilità di tessuti espianto polmonari e le procedure invasive per ottenere cellule bronchiali da soggetti senza malattia allo stadio terminale ostacolare l&#39;analisi delle mutazioni CFTR meno comuni e prevenire test anti-droga in un modo personalizzato. Per superare queste limitazioni, i tessuti &quot;easy access&quot;, come organoidi del colon-retto, cellule delle vie aeree nasali, e le cellule delle vie aeree derivate da cellule staminali pluripotenti indotte, sono attualmente oggetto di studio per trattamenti farmacologici personalizzati. <p class= &quot;jove_content&quot;&gt; In precedenza, abbiamo stabilito i protocolli per le cellule staminali epiteliali cultura da qualsiasi organo gastrointestinale in forma di 3D organoidi 16, 17. fattori di crescita per il colon / retto umana, le condizioni di coltura comportano definiti (fattore di crescita epiteliale (EGF), gastrina, Wnt-3A, R-spondina 3 (Rspo3), e Noggin) in combinazione con piccole molecole (nicotinamide, A83-01, e SB202190) in una matrice membrana basale. In queste condizioni, le cellule staminali singoli o piccoli frammenti di tessuto crescono in chiusi, cistica, strutture 3D formate da epitelio altamente polarizzata con il lato basale orientato verso l&#39;esterno. Tutti i tipi di cellule compaiono tipicamente nei loro rapporti normali e posizioni. Organoidi possono essere espanse per periodi di tempo lunghi per rottura meccanica settimanale e ri-placcatura. Sono geneticamente e fenotipicamente stabili e possono essere memorizzati, permettendo l&#39;espansione a lungo termine e bio-banking 17. Esse sono suscettibili ditutte le manipolazioni standard di cellulari-biologici / genetici e tecniche analitiche sviluppate per linee cellulari 2D 18. Abbiamo recentemente dimostrato che la funzione CFTR può essere facilmente misurato in organoidi del colon-retto in un saggio forskolin indotta gonfiore (FIS) 19, 20. Se esposto a forskolin (FSK) o, in alternativa, tossina del colera, organoidi rapidamente aumentano la loro adenosina monofosfato ciclico livelli (cAMP), che a sua volta provoca l&#39;apertura del canale CFTR 19. Organoidi da individui sani o da pazienti con CFTR mutazioni associate alla funzione residua, saranno successivamente gonfiarsi come conseguenza di trasporto di ioni e acqua al lume organoide, l&#39;equivalente in vitro di diarrea secretoria. La risposta FIS di organoidi del colon-retto è stato precedentemente dimostrato di essere pienamente CFTR-dipendente, come indicato dalla organoidi derivati ​​da individui CFTR-udenti and mediante l&#39;uso di specifici inibitori farmacologici CFTR 19. Grandi insiemi di dati specifici per soggetti possono essere derivate entro alcune settimane dopo l&#39;assunzione di una biopsia. Per il saggio FIS descritto in dettaglio qui, organoidi sono coltivati da biopsie rettali ottenibili a qualsiasi età e con solo disagio limitata 21. Organoidi sono diversi passaggi ogni settimana dalla rottura meccanica in singoli cripte che facilmente richiudere e formano nuove organoidi. Per l&#39;esecuzione del test FIS, ~ 30-80 di queste piccole organoidi perturbato sono placcati in ciascun pozzetto di una piastra a 96 pozzetti 19. Al giorno del test, i organoidi sono colorate con calceina verde, un colorante cellula-permeabile fluorescente che viene trattenuto all&#39;interno delle cellule viventi, facilitando l&#39;imaging dal vivo. Poi, FSK, che solleva cAMP intracellulare e quindi attiva CFTR, viene aggiunto per stimolare gonfiore organoide. Potenziatori che agiscono su CFTR apicale sono aggiunti contemporaneamente won la forskolina, mentre correttori che ripristinano il traffico di CFTR si aggiungono 24 ore prima dell&#39;aggiunta di Fsk. Il gonfiore organoide viene quantificato un&#39;analisi automatica delle immagini che calcola il relativo aumento della superficie totale di tutti gli oggetti fluorescenti per ogni punto di tempo dopo l&#39;aggiunta forskolin. 3D organoide gonfiore offre vantaggi e svantaggi sopra letture CFTR elettrofisiologici esistenti in cellule in coltura delle vie aeree 2D in Camera di Ussing. Un importante vantaggio è la velocità del test gonfiore. Le cellule sono coltivate e analizzati utilizzando un unico tipo di terreno di coltura, e un tecnico esperto può cultura fino a 25 campioni organoide su base settimanale, mentre la quantificazione circa 1.200 punti dati a settimana in 12 campioni di pazienti. Abbiamo convenzionalmente digitare una singola condizione sperimentale da misure doppio o triplo, per piastra e ripetere tali misurazioni in tre punti di tempo di incubazione indipendenti. In totale, circa 300-500 single organoide strutture sono poi misurati per ogni condizione sperimentale, che porta a misure molto precise della funzione CFTR con variabilità tecnico limitato. Questa precisione ci permette di definire in modo chiaro le differenze nella funzione residua e la risposta a modulatori CFTR e ci permette di raccogliere facilmente fino effetti di sfondo genetiche tra i pazienti portatori di mutazioni CFTR identici 19, 22, 23, 24, 25. La qualità dei dati può essere facilmente valutata da immagini al microscopio. Mentre FIS è completamente CFTR-dipendente, è una misura di esito indiretto per la funzione CFTR, la sua out lettura causata dall&#39;accoppiamento di trasporto ionico per il trasporto di fluidi. Questo contrasta con le misurazioni della funzione CFTR dirette in camere ussing, che misurano correnti ioniche transepiteliali 26. camere ussing permettono di selezione stimolazione apicale del o basolaterale compartments (che il test organoide non permette); da permeabilizzante membrane basolaterale, il apicale CFTR-dipendente la secrezione di anione può essere selettivamente misurato 27.

<table border="0" cellpadding="0" cellspacing="0" width="1053">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:355px;">Advanced Dulbecco&#8217;s Modified Eagles Medium with Nutrient Mixture F-12 Hams (Ad-DF) 500ml&#160;</td>
			<td style="width:180px;">Thermo Fisher Scientific:&#160; Invitrogen</td>
			<td style="width:119px;">#12634</td>
			<td style="width:399px;">stored at 4 &#176;C</td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:355px;">GlutaMax</td>
			<td style="width:180px;">Thermo Fisher Scientific:&#160; Invitrogen</td>
			<td style="width:119px;">#35050</td>
			<td style="width:399px;">stored at 4 &#176;C</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:355px;">Hepes</td>
			<td style="width:180px;">Thermo Fisher Scientific:&#160; Invitrogen</td>
			<td style="width:119px;"># 15630-056</td>
			<td style="width:399px;">stored at 4 &#176;C</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:355px;">Penicillin/Streptomycin</td>
			<td style="width:180px;">Thermo Fisher Scientific:&#160; Invitrogen</td>
			<td style="width:119px;">#15140-122</td>
			<td style="width:399px;">stored at -20 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">96 well culture plate</td>
			<td style="width:180px;">Cellstar</td>
			<td style="width:119px;">#655180</td>
			<td style="width:399px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">24 well culture plate</td>
			<td style="width:180px;">Cellstar</td>
			<td style="width:119px;">#662160</td>
			<td style="width:399px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">6 well culture plate</td>
			<td style="width:180px;">Cellstar</td>
			<td style="width:119px;">#657160</td>
			<td style="width:399px;"></td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:355px;">Dulbecco&#39;s Phosphate Buffered Saline (-) CaCl2 (-) MgCl2) (DPBS)</td>
			<td style="width:180px;">Life Technologies: Gibco</td>
			<td style="width:119px;">#14190-094</td>
			<td style="width:399px;">stored at 4 &#176;C</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:355px;">Dulbecco&#8217;s Modified Eagles Medium&#160; (DMEM) 500ml&#160;</td>
			<td style="width:180px;">Thermo Fisher Scientific:&#160; Invitrogen</td>
			<td style="width:119px;">#31966-021</td>
			<td style="width:399px;">For Wnt-3A Conditioned Medium Production. Stored at 4 &#176;C</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:355px;">Fetal Bovine Serum (FBS)</td>
			<td style="width:180px;">Bovogen</td>
			<td style="width:119px;">#SFBS LOT#11113</td>
			<td style="width:399px;">For Wnt-3A Conditioned Medium Production. Stored at -20 &#176;C</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:355px;">L Wnt3A cell line</td>
			<td style="width:180px;">ATCC</td>
			<td style="width:119px;">#CRL-2647</td>
			<td style="width:399px;">For Wnt-3A Conditioend Medium Production.</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:355px;">TOP/FOP plasmids</td>
			<td style="width:180px;">Millipore&#160;</td>
			<td style="width:119px;">#17-285</td>
			<td style="width:399px;">For measuring Wnt activity</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">pTK-Renilla</td>
			<td style="width:180px;">Promega&#160;</td>
			<td style="width:119px;">#E2241</td>
			<td style="width:399px;">For measuring Wnt activity</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">HEK-293</td>
			<td style="width:180px;">ATCC</td>
			<td style="width:119px;">#CRL-1573</td>
			<td style="width:399px;">For measuring Wnt activity</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Dual-Luciferase Reporter Assay System</td>
			<td style="width:180px;">Promega&#160;</td>
			<td style="width:119px;">#E1910</td>
			<td style="width:399px;">For measuring Wnt activity</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:355px;">Zeocin&#160;</td>
			<td style="width:180px;">Thermo Fisher Scientific:&#160; Invitrogen</td>
			<td style="width:119px;">#R250-01</td>
			<td style="width:399px;">For Wnt-3A Cell line selection</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:355px;">B27 supplement&#160;</td>
			<td style="width:180px;">Thermo Fisher Scientific:&#160; Invitrogen</td>
			<td style="width:119px;">#17504-044</td>
			<td style="width:399px;">stored at -20 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">N-Acetylcysteine</td>
			<td style="width:180px;">Sigma Aldrich</td>
			<td style="width:119px;">#A9165-5G</td>
			<td style="width:399px;">stored at -20 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Nicotinamide</td>
			<td style="width:180px;">Sigma Aldrich</td>
			<td style="width:119px;">#N0636</td>
			<td style="width:399px;">stored at -20 &#176;C</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:355px;">Human Epithelial Growth Factor (hEGF)</td>
			<td style="width:180px;">PrepoTech</td>
			<td style="width:119px;">#AF-100-15</td>
			<td style="width:399px;">stored at -20 &#176;C</td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:355px;">Gastrin</td>
			<td style="width:180px;">Sigma Aldrich</td>
			<td style="width:119px;">#G9145</td>
			<td style="width:399px;">stored at -20 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">TGFb type I Receptor inhibitor (A83-01)&#160;</td>
			<td style="width:180px;">Tocris</td>
			<td style="width:119px;">#2939</td>
			<td style="width:399px;">stored at -20 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Y-27632 dihydrochloride (RhoKi)</td>
			<td style="width:180px;">Selleckchem</td>
			<td style="width:119px;">#S1049</td>
			<td style="width:399px;">stored at -20 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">p38 MAPK inhibitor (p38i) (SB202190)</td>
			<td style="width:180px;">Sigma Aldrich</td>
			<td style="width:119px;">#S7067</td>
			<td style="width:399px;">stored at -20 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Primocin</td>
			<td style="width:180px;">InvivoGen</td>
			<td style="width:119px;">#ant-pm-1</td>
			<td style="width:399px;">stored at -20 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Human Noggin (hNoggin)</td>
			<td style="width:180px;">PrepoTech</td>
			<td style="width:119px;">#120-10C</td>
			<td style="width:399px;">stored at -20 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Human R-spondin 3 (hRspo-3)</td>
			<td style="width:180px;">R&#38;D Systems</td>
			<td style="width:119px;">#3500-RS/CF</td>
			<td style="width:399px;">stored at -20 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Vancomycin</td>
			<td style="width:180px;">Sigma Aldrich</td>
			<td style="width:119px;">#861987- 250mg</td>
			<td style="width:399px;">stored at -20 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Gentamycin</td>
			<td style="width:180px;">Life Technologies: Gibco</td>
			<td style="width:119px;">#15710-049</td>
			<td style="width:399px;">stored at -20 &#176;C</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:355px;">Ethylenediamine tetraacetic acid (EDTA)</td>
			<td style="width:180px;">Sigma Aldrich</td>
			<td style="width:119px;">#431788</td>
			<td style="width:399px;">Stored at 4 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Matrigel</td>
			<td style="width:180px;">Corning</td>
			<td style="width:119px;">#354230</td>
			<td style="width:399px;">stored at -80 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">TryplE Express&#160;</td>
			<td style="width:180px;">Life Technologies: Gibco</td>
			<td style="width:119px;">#12605-010</td>
			<td style="width:399px;">for trypsinizing organoids for freezing</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Recovery Cell Culture Freezing Medium</td>
			<td style="width:180px;">Life Technologies: Gibco</td>
			<td style="width:119px;">#12648010</td>
			<td style="width:399px;">for freezing</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Calcein</td>
			<td style="width:180px;">Life Technologies: Gibco</td>
			<td style="width:119px;">#C3100MP</td>
			<td style="width:399px;">stored at -20 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Forskolin</td>
			<td style="width:180px;">R&#38;D Systems</td>
			<td style="width:119px;">#1099-50 mg</td>
			<td style="width:399px;">stored at -80 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Lumacaftor (VX-809)</td>
			<td style="width:180px;">Selleckchem</td>
			<td style="width:119px;">#s1565</td>
			<td style="width:399px;">stored at -80 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Ivacaftor (VX-770)</td>
			<td style="width:180px;">Selleckchem</td>
			<td style="width:119px;">#s1144</td>
			<td style="width:399px;">stored at -80 &#176;C</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:355px;">Name of Reagents/Material</td>
			<td style="width:180px;">Solvent</td>
			<td style="width:119px;">Stock Concentration</td>
			<td style="width:399px;">Final Concentration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">GlutaMax</td>
			<td style="width:180px;"></td>
			<td style="width:119px;">200 mM</td>
			<td style="width:399px;">2m M</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Hepes</td>
			<td style="width:180px;"></td>
			<td style="width:119px;">1 M</td>
			<td style="width:399px;">10 mM</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:355px;">Penicillin/Streptomycin</td>
			<td style="width:180px;"></td>
			<td style="width:119px;">10K U/ml 10K &#181;g/ml</td>
			<td style="width:399px;">100 U/ml 100 &#181;g/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Zeocin&#160;</td>
			<td style="width:180px;"></td>
			<td style="width:119px;">100 mg/ml&#160;</td>
			<td style="width:399px;">125 &#181;g/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">B27 supplement&#160;</td>
			<td style="width:180px;"></td>
			<td style="width:119px;">100 x</td>
			<td style="width:399px;">1 x</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:355px;">N-Acetylcysteine</td>
			<td style="width:180px;">MiliQ H20</td>
			<td style="width:119px;">500 mM</td>
			<td style="width:399px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Nicotinamide</td>
			<td style="width:180px;">DPBS</td>
			<td style="width:119px;">1 M</td>
			<td style="width:399px;">10 mM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Human Epithelial Growth Factor (hEGF)</td>
			<td style="width:180px;">DPBS 0.1%BSA</td>
			<td style="width:119px;">0.5 mg/ml</td>
			<td style="width:399px;">50 ng/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Gastrin</td>
			<td style="width:180px;">DPBS</td>
			<td style="width:119px;">100 &#181;M</td>
			<td style="width:399px;">10 nM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">TGFb type I Receptor inhibitor (A83-01)&#160;</td>
			<td style="width:180px;">DMSO</td>
			<td style="width:119px;">5 mM</td>
			<td style="width:399px;">500 nM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Y-27632 dihydrochloride (RhoKi)</td>
			<td style="width:180px;">DMSO</td>
			<td style="width:119px;">10 mM</td>
			<td style="width:399px;">10 &#181;M</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">p38 MAPK inhibitor (p38i) (SB202190)</td>
			<td style="width:180px;">DMSO</td>
			<td style="width:119px;">30 mM</td>
			<td style="width:399px;">10 &#181;M</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Primocin</td>
			<td style="width:180px;"></td>
			<td style="width:119px;">50 mg/ml&#160;</td>
			<td style="width:399px;">100 &#181;g/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Human Noggin (hNoggin)</td>
			<td style="width:180px;">DPBS 0.1%BSA</td>
			<td style="width:119px;">100 &#181;g/ml</td>
			<td style="width:399px;">100 ng/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Human R-spondin 3 (hRspo-3)</td>
			<td style="width:180px;"></td>
			<td style="width:119px;">varies per lot</td>
			<td style="width:399px;">300 ng/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Vancomycin</td>
			<td style="width:180px;"></td>
			<td style="width:119px;">10 mg/ml</td>
			<td style="width:399px;">50 &#181;g/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Gentamycin</td>
			<td style="width:180px;"></td>
			<td style="width:119px;">10 mg/ml</td>
			<td style="width:399px;">50 &#181;g/ml</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:355px;">Ethylenediamine tetraacetic acid (EDTA)</td>
			<td style="width:180px;">MiliQ H20</td>
			<td style="width:119px;">0.5 M</td>
			<td style="width:399px;">2 mM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Calcein</td>
			<td style="width:180px;">DMSO</td>
			<td style="width:119px;">10 &#181;g/ml</td>
			<td style="width:399px;">3.3 ng/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Forskolin</td>
			<td style="width:180px;">DMSO</td>
			<td style="width:119px;">10 mM</td>
			<td style="width:399px;">variable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Lumacaftor (VX-809)</td>
			<td style="width:180px;">DMSO</td>
			<td style="width:119px;">20 mM</td>
			<td style="width:399px;">variable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Ivacaftor (VX-770)</td>
			<td style="width:180px;">DMSO</td>
			<td style="width:119px;">20 mM</td>
			<td style="width:399px;">variable</td>
		</tr>
	</tbody>
</table>

forskolin-induced swelling in intestinal organoids: an in vitro assay for assessing drug response in cystic fibrosis patients

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant CFTR channel in subjects with cystic fibrosis (CF). Identification of subjects that may benefit from these drugs is challenging because of the extensive heterogeneity of CFTR mutations, as well as other unknown factors that contribute to individual drug efficacy. Here, we describe a simple and relatively rapid assay for measuring individual CFTR function and response to CFTR modulators in vitro. Three dimensional (3D) epithelial organoids are grown from rectal biopsies in standard organoid medium. Once established, the organoids can be bio-banked for future analysis. For the assay, 30-80 organoids are seeded in 96-well plates in basement membrane matrix and are then exposed to drugs. One day later, the organoids are stained with calcein green, and forskolin-induced swelling is monitored by confocal live cell microscopy at 37 &#176;C. Forskolin-induced swelling is fully CFTR-dependent and is sufficiently sensitive and precise to allow for discrimination between the drug responses of individuals with different and even identical CFTR mutations. In vitro swell responses correlate with the clinical response to therapy. This assay provides a cost-effective approach for the identification of drug-responsive individuals, independent of their CFTR mutations. It may also be instrumental in the development of future CFTR modulators.

La figura 1A mostra un isolamento rappresentante fresco di cripte incorporati su BMM. Le cripte sono da una biopsia del colon-retto di un soggetto CF. Di solito, un organoide è generato da ogni cripta (Figura 1A - C). A causa della disfunzione del CFTR, la maggior parte dei organoidi colon CF non sono cistica, ma piuttosto sono compatti e con proiezioni e buddings (Figura 2A - 2B). Tuttavia, alcuni CF o...

Watch this Scientific Journal Video about Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients at JoVE.com

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

This protocol describes an assay for measuring CFTR function and CFTR modulator responses in cultured tissue from subjects with cystic fibrosis (CF). Biopsy-derived intestinal organoids swell in a cAMP-driven fashion, a response that is defective (or strongly reduced) in CF organoids and can be restored by exposure to CFTR modulators.

Forskolin indotta Gonfiore intestinale organoidi: An<em&gt; In Vitro</em&gt; Test per la valutazione della risposta farmacologica nella fibrosi cistica pazienti

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant ...

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Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

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19.0K Views.  Foundation Hubrecht Organoid Technology. This protocol describes an assay for measuring CFTR function and CFTR modulator responses in cultured tissue from subjects with cystic fibrosis (CF). Biopsy-derived intestinal organoids swell in a cAMP-driven fashion, a response that is defective (or strongly reduced) in CF organoids and can be restored by exposure to CFTR modulators.