Name: forskolin-induced swelling in intestinal organoids: an in vitro assay for assessing drug response in cystic fibrosis patients
Uploaded: 2017-02-11T14:00:00
Description: 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

Dette arbeidet ble støttet av HIT-CF program av den nederlandske CF foundation (NFH), ZonMW (40-00812-98-14103), Wilhelmina Barnas Hospital Research Fund og CZ og Zilverenkruis / Achmea. Vi vil gjerne takke S. Heida-Michel, M. Geerdink, KM de Winter-de Groot, og G. Berkers (Department of Pediatric Lunge, Wilhelmina barnesykehus, UMC Utrecht), og RHJ Houwen (Department of Pediatric Gastroenterology, Wilhelmina barne~~POS=TRUNC, UMC Utrecht) for nærmer pasientene og få biopsier for generering av et CF Biobank.

All eksperimentering ved hjelp av menneskelig vev beskrevet her ble godkjent av etisk komité ved University Medical Center Utrecht (UMCU; TcBio # 14-008). Informert samtykke til vev innsamling, produksjon, lagring og bruk av organoids er innhentet fra pasienter ved Wilhelmina barnesykehus (WKZ) -UMCU. <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> Utstyr </td><td> Konsum </td><td> Verktøy </td></tr><tr><td...

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Her gir vi en komplett protokoll for generering, ekspansjon, frysing og tining av menneskelige kolorektal organoids. Mens vi har etablert menneskelige organoid kulturer for en tid siden 17, har det noen ganger vist seg vanskelig å etablere teknologien i andre laboratorier uten hands-on trening. Vi forventer at disse protokollene vil erstatte slik opplæring. Wnt-3A-kondisjonert medium er en av de mest avgjørende reagenser for å lykkes i å etablere og opprettholde ...

CF er forårsaket av mutasjoner i cystisk fibrose transmembrane konduktans regulator (CFTR) genet som koder for en epitelial anion kanal. CF påvirker om lag 85 000 mennesker over hele verden en. Over 2000 CFTR mutasjoner er blitt identifisert ( <a href="http://www.genet.sickkids.on.ca">www.genet.sickkids.on.ca</a> ). Dette mangfoldet delvis forklarer et bredt spekter av observerte sykdoms fenotyper ( <a href="http://www.CFTR2.org">www.CFTR2.org</a> ) 2, 3. Seks klasser av CFTR mutasjoner er definert på grunnlag av deres virkning på CFTR protein ekspresjon og funksjon: (I) ingen syntese, (II) svekket handel, (III) defekt kanal gating, (IV) endret konduktans, (V) reduserte nivåer av vanlig fungerende CFTR, og (VI) svekket celleoverflaten stabilitet fire. Selv om felles CFTR mutasjoner er godt studert, CFTR funksjon og relasjon med klinisk status forbli poorly forstått på nivå med den enkelte, spesielt for den store gruppen av sjeldne &quot;orphan&quot; mutasjoner ( <a href="http://www.CFTR2.org">www.CFTR2.org</a> ) 1, 3. Nylig, narkotika har blitt utviklet som målrette CFTR protein i en mutasjonsspesifikk måte. To klasser av CFTR protein-måls narkotika er nå i klinisk bruk, og har forskjellige moduser av handlingen. Potensiatorer, så som VX-770, forbedre den åpne sannsynlighet for apikalt-lokaliserte mutant CFTR, og virker direkte på deres tilsetning til celler 5. Korrektur, slik som VX-809, gjenopprette smugling av endoplasmatiske retikulum-lokaliserte misfolded CFTR og krever pre-inkubasjon med celler før effekten er observert seks. CFTR-potensiator, VX-770, er blitt registrert for fag med G551D mutasjon 7, 8, så vel som for andre åtteCFTR gating mutasjoner, inkludert S1251N 9; sammen, er disse mutasjonene båret av 5% av CF-individer. Andre kliniske forsøk har indikert at VX-770, kombinert med korrigeringsanordningen VX-809, har begrenset ennå betydelige virkninger på lungefunksjonen og fører til en reduksjon i forverring priser fag homozygote for mutasjonen F508del båret av 45-50% av pasientene 10, 11. Vanlige kliniske forsøk for å identifisere medikament responsive fag innenfor de resterende 50% av CF-pasienter er kostbare og tidkrevende, og er ikke gjennomførbare for personer med ekstremt sjeldne CFTR genotyper. Novel, kostnadseffektive, tilpassede metoder er avgjørende for å matche det økende antall CFTR modulatorer til enkeltpersoner som frakter alle typer CFTR mutasjon. Til nå har rettssaken inkludering av pasientgrupper som bærer spesifikke CFTR mutasjoner vært ledet av studier med mutant CFTR genet transfeksjon i heterologous cellesystemer, etterfulgt av elektrofysiologiske studier i Ussing-kammere 5, 6, 12. På grunn av mangel på tilstrekkelige CF dyremodeller, narkotika effektstudier i luft-væske-grensesnittet differensiert bronkiale epitelceller avledet fra CF lunge eksplantering materialer har blitt brukt for legemiddelutvikling 13, 14, 15. Men den begrensede tilgjengeligheten av lunge eksplantering vev og invasiv undersøkelse innhente bronkial celler fra individer uten sluttstadiet sykdom hemme analysen av mindre vanlige CFTR mutasjoner og forebygge narkotikatesting i en personlig måte. For å overvinne disse begrensningene, &quot;lett tilgang&quot; vev, for eksempel tykktarms organoids, nese Airway celler, og luftveis celler avledet fra induserte pluripotente stamceller, blir nå undersøkt for personlige medikamentelle behandlinger. 16, 17. For den humane tykktarm / endetarm, involverer dyrkningsforholdene definerte vekstfaktorer (epithelial vekstfaktor (EGF), Gastrin, Wnt-3A, R-spondin 3 (Rspo3), og Noggin) kombinert med små molekyler (nikotinamid, A83-01, og SB202190) i en basalmembran matrise. Under disse betingelser, enkelt stamceller eller små vevsfragmenter vokse ut i lukket, cystisk, 3D-strukturer dannet av høyt-polarisert epitel med sin basal side er rettet mot utsiden. Alle celletyper vanligvis vises i sine normale forhold og posisjoner. Organoids kan utvides over lange tidsperioder etter ukentlig mekanisk ødeleggelse og re-plating. De er genetisk og fenotypisk stabil og kan lagres, slik at langvarig utvidelse og bio-bank 17. De er mottagelig foralle standard celle-biologiske / genetiske manipulasjoner og analytiske teknikker utviklet for 2D cellelinjer 18. Vi har nylig vist at CFTR funksjon lett kan måles i kolorektale organoids i en forskolin-indusert svelling (FIS) assay 19, 20. Når de utsettes for forskolin (Fsk) eller, alternativt, til koleratoksin, organoids raskt øke sin cyklisk adenosin monofosfat (cAMP) nivåer, noe som igjen resulterer i åpningen av kanalen 19 CFTR. Organoids fra friske individer, eller fra individer med CFTR mutasjoner assosiert med restfunksjon, vil deretter hovne som en konsekvens av ion og vann transport til organoid lumen, in vitro tilsvarer sekretorisk diaré. FIS responsen av kolorektale organoids ble tidligere vist å være fullt ut CFTR-avhengige, som angitt ved organoids avledet fra CFTR-null individer ennd ved bruk av spesifikke farmakologiske CFTR-hemmere 19. Store fagspesifikke datasett kan utledes innenfor flere uker etter å ha tatt en biopsi. For FIS analysen beskrevet i detalj her, organoids dyrkes fra endetarms biopsier som kan fås i alle aldre og med bare begrenset ubehag 21. Organoids passeres ukentlig av mekanisk avbrudd i enkelt krypter som lett forsegle og danner nye organoids. For å kjøre FIS-analysen, ~ 30-80 av disse forstyrret små organoids er belagt i hver brønn av en 96-brønns plate 19. På dagen for analysen, blir organoids farget med calcein grønn, en fluorescerende celle-gjennomtrengelige fargestoff som det kan beholdes i levende celler, legge til rette for levende avbildning. Deretter Fsk, noe som øker intracellulært cAMP og derved aktiverer CFTR, tilsettes for å stimulere organoid hevelse. Potensiatorer som virker på apikal CFTR tilsettes samtidig wed forskolin, mens korrektur som gjenoppretter CFTR menneskehandel legges 24 timer før tilsetting av Fsk. Den organoid svelling er kvantifisert ved en automatisert bildeanalyse som beregner den relative økning i det totale areal av alle fluorescerende gjenstander for hvert tidspunkt ved tilsetning forskolin. 3D organoid hevelse gir fordeler og ulemper i forhold til eksisterende elektro CFTR avlesninger i 2D kultivert Airway celler i Ussing-kammere. En stor fordel er at gjennomstrømningen av den svellende analysen. Celler dyrkes og analyseres ved hjelp av en enkelt type dyrkingsmedium, og en erfaren tekniker kan kultur opptil 25 organoid prøver på en ukentlig basis, mens kvantifisering av ca 1200 datapunkter per uke i 12 pasientprøver. Vi konvensjonelt skriver en enkelt eksperimentell tilstand ved like eller tredoble målinger per plate og gjenta slike målinger på tre uavhengige inkubasjon tidspunkter. Totalt, ca 300-500 single organoid strukturer er deretter målt per eksperimentell tilstand, noe som fører til svært nøyaktige målinger av CFTR-funksjon med begrenset teknisk variasjon. Dette presisjons tillater oss å klart definere forskjeller i rest funksjon og respons til CFTR modulatorer og tillater oss å lett plukke opp genetiske bakgrunn effekter mellom pasienter bærer identiske CFTR mutasjoner 19, 22, 23, 24, 25. Datakvalitet lett kan vurderes fra mikroskop bilder. Mens FIS er fullt CFTR-avhengig, er det en indirekte effektmål for CFTR-funksjon, dens utlesning forårsaket av kobling av ione-transporten til fluid-transport. Dette i motsetning til direkte målinger CFTR funksjon i Ussing kamre, som måler transepitelial ion strømninger 26. Ussing kamre tillater velger stimulering av apikale eller basolateral compartments (som organoid analysen ikke tillater); av permeabilizing basolaterale membraner, kan den apikale CFTR-avhengig anion sekresjon selektivt målt 27.

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		<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.

Figur 1A viser en representativ frisk isolering av krypter innebygd på BMM. Krypter er fra en kolorektal biopsi av et CF emne. Vanligvis er en organoid generert fra hver krypten (figur 1A - C). På grunn av dysfunksjon i CFTR, de fleste av colon CF organoids er ikke cystisk, men snarere er kompakte og med fremspring og buddings (Figur 2A - 2B). Men noen CF organoid kulturer, spesielt med høy restfunksj...

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-indusert Hevelse i tarmen Organoids: An<em&gt; In Vitro</em&gt; Analyse for å vurdere Drug Response i pasienter med cystisk fibrose

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

Research

JoVE Journal

Medicine

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

Vurdere Teratogene Endringer i en sebrafisk 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, ...

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

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

Vurdere Overfør spongiform encefalopati Arter Barrierer med en<em&gt; In Vitro</em&gt; Prionproteinet Conversion Assay

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

Assessing Specificity of Anticancer Drugs In Vitro

Assessing Specificity of Anticancer Drugs In Vitro

Vurdere Spesifisitet av kreftmedisiner<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 ...

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

Neonatal Murine Cochlear Explanting Technique som en<em&gt; I Vitro</em&gt; Screening Tool in Hearing Research

While there have been remarkable advances in hearing research over the past few decades, there is still no cure for Sensorineural Hearing Loss (SNHL), a ...

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

Optimalisert LC--MS-/ MS metoden for høy gjennomstrømming analyse av klinisk prøver av Ivacaftor, store metabolitter og Lumacaftor i biologiske væsker Cystic Fibrosis pasienter

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

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

Forbedret metoden for etablering av en In Vitro blod - hjerne barrieren modell basert på svin hjernen endotelceller

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

En fluorescens-basert analyse for karakterisering og kvantifisering av lipid dråpe dannelse i 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) ...

A Rat Carotid Artery Pressure-Controlled Segmental Balloon Injury with Periadventitial Therapeutic Application

En rotte carotis arterie trykkkontrollert segmental ballong skade med periadventitial terapeutisk applikasjon

Cardiovascular disease remains the leading cause of death and disability worldwide, in part due to atherosclerosis. Atherosclerotic plaque narrows the ...

Investigating Intestinal Barrier Breakdown in Living Organoids

Undersøke intestinal barriere sammenbrudd i levende organoider

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

Bruke en kjemisk biopsi for graftkvalitetsvurdering

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