Ce travail a été soutenu par le programme des FC fondation néerlandaise (NCFS), ZonMW (40-00812-98-14103), le Fonds de recherche de l&#39;Hôpital de Wilhelmina enfants et CZ et Zilverenkruis / Achmea HIT-CF. Nous tenons à remercier S. Heida-Michel, M. Geerdink, KM de Winter-de Groot, et G. Berkers (Département de pneumologie pédiatrique, Hôpital de Wilhelmina enfants, UMC Utrecht), et RHJ Houwen (Département de gastro-entérologie pédiatrique, Wilhelmina Hôpital pour enfants, UMC Utrecht) pour approcher les patients et d&#39;obtenir les biopsies pour la génération d&#39;un Biobanque CF.

<ol>
	<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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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.

Ici, nous fournissons un protocole complet pour la génération, l&#39;expansion, la congélation et la décongélation des organites colorectales humaines. Alors que nous avons établi des cultures de organoïdes humains il y a quelque temps 17, il a parfois avéré difficile d&#39;établir la technologie dans d&#39; autres laboratoires sans formation pratique. Nous prévoyons que ces protocoles remplaceront une telle formation. Wnt-3A-milieu conditionné est l&#39;u...

CF est causée par des mutations dans le gène régulateur de la conductance transmembranaire de la fibrose kystique (CFTR) qui code pour un canal anionique épithéliale. CF touche environ 85.000 personnes dans le monde 1. Plus de 2000 mutations du gène CFTR ont été identifiées ( <a href="http://www.genet.sickkids.on.ca">www.genet.sickkids.on.ca</a> ). Cette diversité explique en partie un large spectre de phénotypes de la maladie observés ( <a href="http://www.CFTR2.org">www.CFTR2.org</a> ) 2, 3. Six classes de mutations du gène CFTR sont définies en fonction de leur effet sur l&#39;expression et la fonction de la protéine CFTR: (I) pas de synthèse, (II) avec facultés affaiblies le trafic, (III) canal défectueux gating, (IV) conductance modifié, (V) les niveaux de normalement réduite fonctionnant CFTR et (VI) ayant une déficience cellulaire stabilité de la surface 4. Bien que les mutations du gène CFTR communes sont bien étudiés, la fonction CFTR et relation avec l&#39;état clinique restent poorly compris au niveau de l&#39;individu, en particulier pour le grand groupe de rares &quot;orphelines&quot; mutations ( <a href="http://www.CFTR2.org">www.CFTR2.org</a> ) 1, 3. Récemment, les médicaments ont été développés qui ciblent la protéine CFTR de manière spécifique à la mutation. Deux classes de CFTR médicaments protéiques de ciblage sont actuellement en usage clinique et ont des modes d&#39;action distincts. Potentialisateurs, tels que le VX-770, d&#39; améliorer l&#39;open-probabilité de CFTR mutant apicale-localisé et agissent directement sur leur addition aux cellules 5. Correcteurs, tels que le VX-809, restaurer le trafic de endoplasmique misfolded CFTR réticulum localisée et nécessitent une pré-incubation avec des cellules avant que les effets sont observés 6. Le potentialisateur de CFTR, le VX-770, a été enregistrée pour les sujets atteints de la mutation G551D 7, 8, ainsi que pour les huit autresCFTR gating mutations, y compris S1251N 9; ensemble, ces mutations sont portés par 5% de tous les sujets des FC. D&#39; autres essais cliniques ont indiqué que le VX-770, combiné avec le correcteur VX-809, a des effets limités mais significatifs sur la fonction pulmonaire et provoque une diminution des taux d&#39;exacerbation chez les sujets homozygotes pour la mutation F508del porté par 45-50% des patients 10, 11. essais cliniques classiques pour identifier les sujets de drogue sensible dans les 50% restants des patients atteints de mucoviscidose sont coûteux et prend du temps et ne sont pas réalisables pour les personnes ayant de très rares génotypes de CFTR. Novel, méthodes personnalisées rentables sont cruciales pour correspondre au nombre croissant de modulateurs de CFTR à des individus porteurs de tout type de mutation CFTR. Jusqu&#39;à présent, l&#39;inclusion d&#39;essai de groupes de patients porteurs de mutations du gène CFTR spécifique a été guidée par des études utilisant mutant transfection du gène CFTR dans hdes systèmes de cellules eterologous, suivie par des études électrophysiologiques dans des chambres d&#39; Ussing 5, 6, 12. En raison d&#39;un manque de modèles animaux de CF adéquates, des études d&#39;efficacité des médicaments dans les cellules épithéliales bronchiques air-liquide-interfaces différenciées dérivées de matériaux explants pulmonaires CF ont été utilisées pour le développement de médicaments 13, 14, 15. Cependant, la disponibilité limitée des tissus d&#39;explants pulmonaires et les procédures invasives pour obtenir des cellules bronchiques de sujets sans maladie en phase terminale d&#39;entraver l&#39;analyse des mutations du gène CFTR moins fréquentes et prévenir le dépistage des drogues d&#39;une manière personnalisée. Pour surmonter ces limitations, les tissus «accès facile», comme organites colorectaux, les cellules des voies respiratoires nasales, et les cellules des voies respiratoires dérivées de cellules souches pluripotentes induites, sont actuellement à l&#39;étude pour les traitements médicamenteux personnalisés. 16, 17. facteurs de croissance pour le côlon / rectum humain, les conditions de culture impliquent définis (épithéliale Growth Factor (EGF), Gastrin, Wnt-3A, R-spondine 3 (Rspo3), et Noggin) combiné avec de petites molécules (nicotinamide, A83-01, et SB202190) dans une matrice de membrane basale. Dans ces conditions, les cellules souches individuelles ou de petits fragments de tissu développent dans fermées, kystique, des structures 3D formées par un épithélium hautement polarisé avec son côté de base orientée vers l&#39;extérieur. Tous les types de cellules apparaissent généralement dans leurs rapports et positions normales. Organites peuvent être étendues sur de longues périodes par rupture mécanique hebdomadaire et re-placage. Ils sont génétiquement et phénotypiquement stables et peuvent être stockés, ce qui permet l&#39; expansion à long terme et de la bio-banking 17. Ils se prêtent àtoutes les manipulations de cellules biologiques standards / génétiques et des techniques analytiques développées pour les lignées cellulaires 2D 18. Nous avons récemment démontré que la fonction CFTR peut être facilement mesurée en organites colorectaux dans une enflure induite par la forskoline (FIS) dosage 19, 20. Lorsqu&#39;il est exposé à la forskoline (FSK) ou, en variante, à la toxine du choléra, organoïdes augmentent rapidement leur adénosine monophosphate cyclique (AMPc) niveaux, ce qui entraîne à son tour l&#39;ouverture du canal CFTR 19. Organites d&#39;individus sains ou de sujets avec des mutations du gène CFTR associées à la fonction résiduelle, seront ensuite gonfler comme une conséquence de l&#39; ion et le transport de l&#39; eau à la lumière organoïdes, l&#39;équivalent in vitro de la diarrhée sécrétoire. La réponse des organites FIS colorectales a été montré précédemment pour être entièrement dépendant de la CFTR, comme indiqué par les organites provenant d&#39;individus CFTR-null, unnd par l&#39;utilisation d&#39;inhibiteurs de CFTR pharmacologiques spécifiques 19. Les grands ensembles de données spécifiques au sujet peuvent être obtenus en quelques semaines après la prise d&#39;une biopsie. Pour le dosage de la FIS décrit en détail ici, organites sont cultivées à partir de biopsies rectales qui peuvent être obtenus à tout âge et avec seulement une gêne limitée 21. Organites sont repiquées chaque semaine par une rupture mécanique dans cryptes simples qui RESEAL facilement et forment de nouveaux organites. Pour exécuter le test FIS, ~ 30-80 de ces petits organites dissociées sont étalées dans chaque puits d&#39;une plaque à 96 puits 19. Le jour de l&#39;essai, les organites sont colorées avec du vert de la calcéine, un colorant fluorescent perméable aux cellules qui est retenu à l&#39;intérieur des cellules vivantes, ce qui facilite l&#39;imagerie en temps réel. Ensuite, FSK, ce qui augmente l&#39;AMPc intracellulaire et active CFTR ainsi, est ajouté afin de stimuler le gonflement organoïde. Potentialisateurs qui agissent sur le CFTR apicale sont ajoutés simultanément wvec la forskoline, alors que les correcteurs qui rétablissent le trafic de CFTR sont ajoutés 24 heures avant l&#39;addition de Fsk. Le gonflement organoide est quantifiée par une analyse d&#39;image automatisé qui calcule l&#39;augmentation relative de la superficie totale de tous les objets fluorescents pour chaque point de temps après l&#39;addition de forskoline. 3D organoide gonflement fournit des avantages et des inconvénients sur les lectures CFTR électrophysiologiques existantes en 2D cellules des voies respiratoires cultivées dans des chambres de Ussing. Un avantage majeur est le débit de l&#39;essai de gonflement. Les cellules sont cultivées et testées en utilisant un seul type de milieu de culture, et un technicien expérimenté peut culture jusqu&#39;à 25 échantillons organoïdes sur une base hebdomadaire tout en quantifiant environ 1200 points de données par semaine dans 12 échantillons de patients. Nous tapons classiquement une seule condition expérimentale par des mesures double ou en triple par plaque et répéter ces mesures en trois points de temps d&#39;incubation indépendants. Au total, environ 300-500 sIngle organoide structures sont ensuite mesurées par condition expérimentale, ce qui conduit à des mesures très précises de la fonction CFTR avec la variabilité technique limitée. Cette précision nous permet de définir clairement les différences de fonction résiduelle et la réponse à des modulateurs de CFTR et nous permet de prendre facilement des effets de fond génétiques entre les patients porteurs de mutations du gène CFTR identiques 19, 22, 23, 24, 25. La qualité des données peut être facilement évaluée à partir d&#39;images de microscope. Alors que la FIS est entièrement dépendant de la CFTR, il est une mesure de résultat indirect pour la fonction CFTR, son hors lecture causé par le couplage du transport d&#39;ions pour le transport des fluides. Cela contraste avec les mesures de la fonction CFTR directe dans des chambres de Ussing, qui mesurent les courants ioniques transépithéliaux 26. chambres Ussing permettent la stimulation de sélection de apical ou basolatéral compartments (dont le test organoide ne permet pas); par perméabilisation membranes basolatérale, la apical anion sécrétion dépendant de la CFTR peut être mesurée de manière sélective 27.

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	<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 figure 1A montre un isolement frais représentatif de cryptes embarqués sur BMM. Les cryptes sont d&#39;une biopsie du côlon d&#39;un sujet CF. Habituellement, un organoïde est généré à partir de chaque crypte (figure 1A - C). En raison du dysfonctionnement du CFTR, la plupart des organites colon des FC ne sont pas kystique, mais sont compacts et avec des projections et des bourgeonnements (Figure 2A - ...

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.

Forskoline induite par Gonflement dans Intestinal organites: Un<em&gt; In vitro</em&gt; Essai pour évaluer la réponse des médicaments dans les patients atteints de fibrose kystique

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

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Unit 1

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

Other Respiratory Disorders

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19.0K vues.  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.

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

Assessing Teratogenic Changes in a Zebrafish Model of Fetal Alcohol Exposure

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, including mental retardation due to disordered and damaged brain development. Fetal alcohol spectrum disorder (FASD) is a term used to cover a continuum of birth defects that occur due to maternal alcohol consumption, and occurs in approximately 4% of children born in the United States. With 50% of child-bearing age women reporting consumption of alcohol, and half of all pregnancies being unplanned, unintentional exposure is a continuing issue2. In order to best understand the damage produced by ethanol, plus produce a model with which to test potential interventions, we developed a model of developmental ethanol exposure using the zebrafish embryo. Zebrafish are ideal for this kind of teratogen study3-8. Each pair lays hundreds of eggs, which can then be collected without harming the adult fish. The zebrafish embryo is transparent and can be readily imaged with any number of stains. Analysis of these embryos after exposure to ethanol at different doses and times of duration and application shows that the gross developmental defects produced by ethanol are consistent with the human birth defect. Described here are the basic techniques used to study and manipulate the zebrafish FAS model.

In order to understand the molecular mechanisms of the ethanol-induced developmental damage, we have developed a zebrafish model of ethanol exposure and are exploring the physical, cellular, and genetic alterations that occur after ethanol exposure1. We then seek to find potential interventions and rapidly test them in this animal model.

Évaluer les changements tératogènes dans un modèle de poisson zèbre de l&#39;exposition d&#39;alcoolisation fœtale

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, ...

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Médecine

Assessing Transmissible Spongiform Encephalopathy Species Barriers with an In Vitro Prion Protein Conversion Assay

Studies to understanding interspecies transmission of transmissible spongiform encephalopathies (TSEs, prion diseases) are challenging in that they typically rely upon lengthy and costly in vivo animal challenge studies. A number of in vitro assays have been developed to aid in measuring prion species barriers, thereby reducing animal use and providing quicker results than animal bioassays. Here, we present the protocol for a rapid in vitro prion conversion assay called the conversion efficiency ratio (CER) assay. In this assay cellular prion protein (PrPC) from an uninfected host brain is denatured at both pH 7.4 and 3.5 to produce two substrates. When the pH 7.4 substrate is incubated with TSE agent, the amount of PrPC that converts to a proteinase K (PK)-resistant state is modulated by the original host&#8217;s species barrier to the TSE agent. In contrast, PrPC in the pH 3.5 substrate is misfolded by any TSE agent. By comparing the amount of PK-resistant prion protein in the two substrates, an assessment of the host&#8217;s species barrier can be made. We show that the CER assay correctly predicts known prion species barriers of laboratory mice and, as an example, show some preliminary results suggesting that bobcats (Lynx rufus) may be susceptible to white-tailed deer (Odocoileus virginianus) chronic wasting disease agent.

Assessing Transmissible Spongiform Encephalopathy Species Barriers with an In Vitro Prion Protein Conversion Assay

Measuring the barrier to the interspecies transmission of prion diseases is challenging and typically involves animal challenges or biochemical assays. Here, we present an in vitro prion protein conversion assay with the ability to predict species barriers.

Évaluation de l&#39;encéphalopathie spongiforme transmissible barrières d&#39;espèces avec une<em&gt; In Vitro</em&gt; Test Prion de conversion des protéines

Studies to understanding interspecies transmission of transmissible spongiform encephalopathies (TSEs, prion diseases) are challenging in that they ...

Assessing Specificity of Anticancer Drugs In Vitro

A procedure for assessing specificity of anticancer drugs in vitro using cultures containing both tumor and non-tumor cells is demonstrated. The key element is the quantitative determination of a tumor-specific genetic alteration in relation to a universal sequence using a dual-probe digital PCR assay and the subsequent calculation of the proportion of tumor cells. The assay is carried out on a culture containing tumor cells of an established line and spiked-in non-tumor cells. The mixed culture is treated with a test drug at various concentrations. After the treatment, DNA is prepared directly from the survived adhesive cells in wells of 96-well plates using a simple and inexpensive method, and subjected to a dual-probe digital PCR assay for measuring a tumor-specific genetic alteration and a reference universal sequence. In the present demonstration, a heterozygous deletion of the NF1 gene is used as the tumor-specific genetic alteration and a RPP30 gene as the reference gene. Using the ratio NF1/RPP30, the proportion of tumor cells was calculated. Since the dose-dependent change of the proportion of tumor cells provides an in vitro indication for specificity of the drug, this genetic and cell-based in vitro assay will likely have application potential in drug discovery. Furthermore, for personalized cancer-care, this genetic- and cell-based tool may contribute to optimizing adjuvant chemotherapy by means of testing efficacy and specificity of candidate drugs using primary cultures of individual tumors.

Assessing Specificity of Anticancer Drugs In Vitro

The goal of this protocol is to assess specificity of anticancer drugs in vitro using mixed cultures containing both tumor and non-tumor cells.

Évaluation Spécificité de Anticancer Drugs<em&gt; In vitro</em

A procedure for assessing specificity of anticancer drugs in vitro using cultures containing both tumor and non-tumor cells is demonstrated. The key ...

Optimized LC-MS/MS Method for the High-throughput Analysis of Clinical Samples of Ivacaftor, Its Major Metabolites, and Lumacaftor in Biological Fluids of Cystic Fibrosis Patients

Defects in the cystic fibrosis trans-membrane conductance regulator (CFTR) are the cause of cystic fibrosis (CF), a disease with life-threatening pulmonary manifestations. Ivacaftor (IVA) and ivacaftor-lumacaftor (LUMA) combination are two new breakthrough CF drugs that directly modulate the activity and trafficking of the defective CFTR-protein. However, there is still a dearth of understanding on pharmacokinetic/pharmacodynamic parameters and the pharmacology of ivacaftor and lumacaftor. The HPLC-MS technique for the simultaneous analysis of the concentrations of ivacaftor, hydroxymethyl-ivacaftor, ivacaftor-carboxylate, and lumacaftor in biological fluids in patients receiving standard ivacaftor or ivacaftor-lumacaftor combination therapy has previously been developed by our group and partially validated to FDA standards. However, to allow the high-throughput analysis of a larger number of patient samples, our group has optimized the reported method through the use of a smaller pore size reverse-phase chromatography column (2.6 &#181;m, C8 100 &#197;; 50 x 2.1 mm) and a gradient solvent system (0-1 min: 40% B; 1-2 min: 40-70% B; 2-2.7 min: held at 70% B; 2.7-2.8 min: 70-90% B; 2.8-4.0 min: 90% B washing; 4.0-4.1 min: 90-40% B; 4.1-6.0 min: held at 40% B) instead of an isocratic elution. The goal of this study was to reduce the HPLC-MS analysis time per sample dramatically from ~15 min to only 6 min per sample, which is essential for the analysis of a large amount of patient samples. This expedient method will be of considerable utility for studies into the exposure-response relationships of these breakthrough CF drugs.

Ivacaftor and ivacaftor-lumacaftor combination are two new CF drugs. However, there is still a dearth of understanding on their PK/PD and pharmacology. We present an optimized HPLC-MS technique for the simultaneous analysis of ivacaftor and its major metabolites, and lumacaftor.

Optimisé méthode LC-MS/MS pour l’analyse de haut débit d’échantillons cliniques de Ivacaftor, ses principaux métabolites et Lumacaftor dans les fluides biologiques des patients atteints de fibrose kystique

Defects in the cystic fibrosis trans-membrane conductance regulator (CFTR) are the cause of cystic fibrosis (CF), a disease with life-threatening ...

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) models based on pBECs in mono-culture (MC), MC with astrocyte-conditioned medium (ACM), and non-contact co-culture (NCC) with astrocytes of porcine or rat origin. pBECs were isolated and cultured from fragments of capillaries from the brain cortices of domestic pigs 5-6 months old. These fragments were purified by careful removal of meninges, isolation and homogenization of grey matter, filtration, enzymatic digestion, and centrifugation. To further eliminate contaminating cells, the capillary fragments were cultured with puromycin-containing medium. When 60-95% confluent, pBECs growing from the capillary fragments were passaged to permeable membrane filter inserts and established in the models. To increase barrier tightness and BBB characteristic phenotype of pBECs, the cells were treated with the following differentiation factors: membrane permeant 8-CPT-cAMP (here abbreviated cAMP), hydrocortisone, and a phosphodiesterase inhibitor, RO-20-1724 (RO). The procedure was carried out over a period of 9-11 days, and when establishing the NCC model, the astrocytes were cultured 2-8 weeks in advance. Adherence to the described procedures in the protocol has allowed the establishment of endothelial layers with highly restricted paracellular permeability, with the NCC model showing an average transendothelial electrical resistance (TEER) of 1249 &#177; 80 &#937; cm2, and paracellular permeability (Papp) for Lucifer Yellow of 0.90 10-6 &#177; 0.13 10-6 cm sec-1 (mean &#177; SEM, n=55). Further evaluation of this pBEC phenotype showed good expression of the tight junctional proteins claudin 5, ZO-1, occludin and adherens junction protein p120 catenin. The model presented can be used for a range of studies of the BBB in health and disease and, with the highly restrictive paracellular permeability, this model is suitable for studies of transport and intracellular trafficking.

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of the protocol is to present an optimized procedure for the establishment of an in vitro blood-brain barrier (BBB) model based on primary porcine brain endothelial cells (pBECs). The model shows high reproducibility, high tightness, and is suitable for studies of transport and intracellular trafficking in drug discovery.

Procédé amélioré pour la création d’un In Vitro barrière hémato - encéphalique modèle basé sur les cellules endothéliales du cerveau porcin

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) ...

A Fluorescence-based Assay for Characterization and Quantification of Lipid Droplet Formation in Human Intestinal Organoids

Dietary lipids are taken up as free fatty acids (FAs) by the intestinal epithelium. These FAs are intracellularly converted into triglyceride (TG) molecules, before they are packaged into chylomicrons for transport to the lymph or into cytosolic lipid droplets (LDs) for intracellular storage. A crucial step for the formation of LDs is the catalytic activity of diacylglycerol acyltransferases (DGAT) in the final step of TG synthesis. LDs are important to buffer toxic lipid species and regulate cellular metabolism in different cell types. Since the human intestinal epithelium is regularly confronted with high concentrations of lipids, LD formation is of great importance to regulate homeostasis. Here we describe a simple assay for the characterization and quantification of LD formation (LDF) upon stimulation with the most common unsaturated fatty acid, oleic acid, in human intestinal organoids. The LDF assay is based on the LD-specific fluorescent dye LD540, which allows for quantification of LDs by confocal microscopy, fluorescent plate reader, or flow cytometry. The LDF assay can be used to characterize LD formation in human intestinal epithelial cells, or to study human (genetic) disorders that affect LD metabolism, such as DGAT1 deficiency. Furthermore, this assay can also be used in a high-throughput pipeline to test novel therapeutic compounds, which restore defects in LD formation in intestinal or other types of organoids.

This protocol describes an assay for the characterization of lipid droplet (LD) formation in human intestinal organoids upon stimulation with fatty acids. We discuss how this assay is used for quantification of LD formation, and how it can be used for high throughput screening for drugs that affect LD formation.

Un test basé sur la fluorescence pour la caractérisation et la quantification de la formation de gouttelettes lipidiques chez les organoïdes intestinaux humains

Dietary lipids are taken up as free fatty acids (FAs) by the intestinal epithelium. These FAs are intracellularly converted into triglyceride (TG) ...

Investigating Intestinal Barrier Breakdown in Living Organoids

Organoids and three-dimensional (3D) cell cultures allow the investigation of complex biological mechanisms and regulations in vitro, which previously was not possible in classical cell culture monolayers. Moreover, monolayer cell cultures are good in vitro model systems but do not represent the complex cellular differentiation processes and functions that rely on 3D structure. This has so far only been possible in animal experiments, which are laborious, time consuming, and hard to assess by optical techniques. Here we describe an assay to quantitatively determine the barrier integrity over time in living small intestinal mouse organoids. To validate our model, we applied interferon gamma (IFN-&#947;) as a positive control for barrier destruction and organoids derived from IFN-&#947; receptor 2 knock out mice as a negative control. The assay allowed us to determine the impact of IFN-&#947; on the intestinal barrier integrity and the IFN-&#947; induced degradation of the tight junction proteins claudin-2, -7, and -15. This assay could also be used to investigate the impact of chemical compounds, proteins, toxins, bacteria, or patient-derived probes on the intestinal barrier integrity.

Here we describe a technique to quantify the barrier integrity of small intestinal organoids. The fact that the method is based on living organoids enables the sequential investigation of different barrier integrity modulating substances or combinations thereof in a time-resolved manner.

Enquêter sur la dégradation des barrières intestinales chez les organoïdes vivants

Organoids and three-dimensional (3D) cell cultures allow the investigation of complex biological mechanisms and regulations in vitro, which previously was ...

Using a Chemical Biopsy for Graft Quality Assessment

Kidney transplantation is a life-saving treatment for a large number of people with end-stage renal dysfunction worldwide. The procedure is associated with an increased survival rate and greater quality of patient&#8217;s life when compared to conventional dialysis. Regrettably, transplantology suffers from a lack of reliable methods for organ quality assessment. Standard diagnostic techniques are limited to macroscopic appearance inspection or invasive tissue biopsy, which do not provide comprehensive information about the graft. The proposed protocol aims to introduce solid phase microextraction (SPME) as an ideal analytical method for comprehensive metabolomics and lipidomic analysis of all low molecular compounds present in kidneys allocated for transplantation. The small size of the SPME probe enables performance of a chemical biopsy, which enables extraction of metabolites directly from the organ without any tissue collection. The minimum invasiveness of the method permits execution of multiple analyses over time: directly after organ harvesting, during its preservation, and immediately after revascularization at the recipient&#8217;s body. It is hypothesized that the combination of this novel sampling method with a high-resolution mass spectrometer will allow for discrimination of a set of characteristic compounds that could serve as biological markers of graft quality and indicators of possible development of organ dysfunction.

The protocol presents utilization of the chemical biopsy approach followed by comprehensive metabolomic and lipidomic analysis for quality assessment of kidney grafts allocated for transplantation.

Utilisation d’une biopsie chimique pour l’évaluation de la qualité de la greffe

Kidney transplantation is a life-saving treatment for a large number of people with end-stage renal dysfunction worldwide. The procedure is associated ...

Culture and Imaging of Human Nasal Epithelial Organoids

Individualized therapy for cystic fibrosis (CF) patients can be achieved with an in vitro disease model to understand baseline Cystic Fibrosis Transmembrane conductance Regulator (CFTR) activity and restoration from small molecule compounds. Our group recently focused on establishing a well-differentiated organoid model directly derived from primary human nasal epithelial cells (HNE). Histology of sectioned organoids, whole-mount immunofluorescent staining, and imaging (using confocal microscopy, immunofluorescent microscopy, and bright field) are essential to characterize organoids and confirm epithelial differentiation in preparation for functional assays. Furthermore, HNE organoids produce lumens of varying sizes that correlate with CFTR activity, distinguishing between CF and non-CF organoids. In this manuscript, the methodology for culturing HNE organoids are described in detail, focusing on the assessment of differentiation using the imaging modalities, including the measurement of baseline lumen area (a method of CFTR activity measurement in organoids that any laboratory with a microscope can employ) as well as the developed automated approach to a functional assay (which requires more specialized equipment).

A detailed protocol is presented here to describe an in vitro organoid model from human nasal epithelial cells. The protocol has options for measurements requiring standard laboratory equipment, with additional possibilities for specialized equipment and software.

Culture et imagerie des organoïdes épithéliaux nasaux humains

Individualized therapy for cystic fibrosis (CF) patients can be achieved with an in vitro disease model to understand baseline Cystic Fibrosis ...

Rectal Organoid Morphology Analysis (ROMA): A Diagnostic Assay in Cystic Fibrosis

Diagnosis of cystic fibrosis (CF) is not always straightforward, especially when sweat chloride concentration is intermediate and/or less than two disease-causing CFTR mutations can be identified. Physiological CFTR assays (nasal potential difference, intestinal current measurement) have been included in the diagnostic algorithm but are not always readily available or feasible (e.g., in infants). Rectal organoids are 3D structures that grow from stem cells isolated from crypts of a rectal biopsy when cultured under specific conditions. Organoids from non-CF subjects have a round shape and a fluid-filled lumen, as CFTR-mediated chloride transport drives water into the lumen. Organoids with defective CFTR function do not swell, retaining an irregular shape and having no visible lumen. Differences in morphology between CF and non-CF organoids are quantified in the &#39;Rectal Organoid Morphology Analysis&#39; (ROMA) as a novel CFTR physiological assay. For the ROMA assay, organoids are plated in 96-well plates, stained with calcein, and imaged in a confocal microscope. Morphological differences are quantified using two indexes: The circularity index (CI) quantifies the roundness of organoids, and the intensity ratio (IR) is a measure of the presence of a central lumen. Non-CF organoids have a high CI and low IR compared to CF organoids. ROMA indexes perfectly discriminated 167 subjects with CF from 22 subjects without CF, making ROMA an appealing physiological CFTR assay to aid in CF diagnosis. Rectal biopsies can be routinely performed at all ages in most hospitals and tissue can be sent to a central lab for organoid culture and ROMA. In the future, ROMA might also be applied to test the efficacy of CFTR modulators in vitro. The aim of the present report is to fully explain the methods used for ROMA, to allow replication in other labs.

Rectal Organoid Morphology Analysis ROMA: A Diagnostic Assay in Cystic Fibrosis

This protocol describes rectal organoid morphology analysis (ROMA), a novel diagnostic assay for cystic fibrosis (CF). Morphological characteristics, namely the roundness (circularity index, CI) and the presence of a lumen (intensity ratio, IR), are a measure of CFTR function. Analysis of 189 subjects showed perfect discrimination between CF and non-CF.