Este trabajo fue apoyado por el programa HIT-CF de los holandeses CF fundación (CNSF), ZonMW (40-00812-98-14103), el Fondo de Investigación del Hospital Infantil Wilhelmina y CZ, y Zilverenkruis / Achmea. Nos gustaría dar las gracias a S. Heida-Michel, M. Geerdink, KM de Winter-de Groot, y G. Berkers (Departamento de Neumología Pediátrica, el Hospital de Niños Wilhelmina, UMC Utrecht), y RHJ Houwen (Departamento de Gastroenterología Pediátrica, Wilhelmina hospital de niños, UMC Utrecht) para acercarse a los pacientes y obtener las biopsias para la generación de un CF Biobanco.

<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. 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=Defining+the+disease+liability+of+variants+in+the+cystic+fibrosis+transmembrane+conductance+regulator+gene.">Defining the disease liability of variants in the cystic fibrosis transmembrane conductance regulator gene.</a> Nature Genet. 45 (10), 1160-1167 (2013).</li><li>Zielenski, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genotype+and+phenotype+in+cystic+fibrosis.">Genotype and phenotype in cystic fibrosis.</a> Respiration. 67 (2), 117-133 (2000).</li><li>Van Goor, F., Hadida, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rescue+of+CF+airway+epithelial+cell+function+in+vitro+by+a+CFTR+potentiator,+VX-770.">Rescue of CF airway epithelial cell function in vitro by a CFTR potentiator, VX-770.</a> Proc Natl Acad Sci U.S.A. 106 (44), 18825-18830 (2009).</li><li>Van Goor, F., Hadida, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correction+of+the+F508del-CFTR+protein+processing+defect+in+vitro+by+the+investigational+drug+VX-809.">Correction of the F508del-CFTR protein processing defect in vitro by the investigational drug VX-809.</a> Proc Natl Acad Sci U.S.A. 108 (46), 18843-18848 (2011).</li><li>Accurso, F. J., Rowe, S. 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=Effect+of+VX-770+in+persons+with+cystic+fibrosis+and+the+G551D-CFTR+mutation.">Effect of VX-770 in persons with cystic fibrosis and the G551D-CFTR mutation.</a> N Engl J Med. 363 (21), 1991-2003 (2010).</li><li>Ramsey, B. 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. 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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.

A continuación, ofrecemos un protocolo completo para la generación, la expansión, la congelación, descongelación y de organoides colorrectal humano. Si bien hemos establecido culturas organoides humanos alguna vez hace 17 años, que a veces ha sido difícil establecer la tecnología en otros laboratorios sin la formación práctica. Anticipamos que estos protocolos se sustituya dicha formación. Wnt-3A-medio condicionado es uno de los reactivos más importantes p...

CF está causada por mutaciones en el gen de la fibrosis quística regulador de la conductancia transmembrana (CFTR) que codifica un canal de aniones epitelial. CF afecta a aproximadamente 85.000 personas en todo el mundo 1. Más de 2.000 mutaciones CFTR se han identificado ( <a href="http://www.genet.sickkids.on.ca">www.genet.sickkids.on.ca</a> ). Esta diversidad explica parcialmente un amplio espectro de fenotipos de la enfermedad observados ( <a href="http://www.CFTR2.org">www.CFTR2.org</a> ) 2, 3. Seis clases de mutaciones CFTR se definen en función de su efecto sobre la expresión de la proteína CFTR y la función: (I) la síntesis de NO, (II) el tráfico deteriorado, (III) canal gating defectuoso, (IV) conductancia alterado, (V) niveles de reduce normalmente funcionamiento CFTR, y (VI) la estabilidad de la superficie celular alterada 4. Aunque las mutaciones CFTR comunes están bien estudiados, la función de CFTR y su relación con el estado clínico permanecen poorlY entiende a nivel del individuo, en particular para el gran grupo de mutaciones raras &quot;huérfanos&quot; ( <a href="http://www.CFTR2.org">www.CFTR2.org</a> ) 1, 3. Recientemente, se han desarrollado medicamentos que se dirigen a la proteína CFTR en una manera-mutación específica. Dos clases de fármacos de proteínas CFTR de metas se encuentran actualmente en uso clínico y tienen modos de acción diferentes. Potenciadores, como el VX-770, aumentan la probabilidad de apertura de-CFTR mutante localizada apicalmente y actúan directamente sobre su adición a las células 5. Correctores, tales como VX-809, restauran el tráfico de endoplásmico CFTR misfolded retículo-localizados y requieren pre-incubación con las células antes de que los efectos se observan 6. El potenciador CFTR, VX-770, ha sido registrada para los sujetos con la mutación G551D 7, 8, así como para otros ochoCFTR gating mutaciones, incluyendo S1251N 9; En conjunto, estas mutaciones se realizan en un 5% de todos los sujetos con FQ. Otros ensayos clínicos han indicado que VX-770, combinado con el corrector VX-809, ha limitado efectos todavía significativos sobre la función pulmonar y causa una disminución en las tasas de exacerbación en los sujetos homocigotos para la mutación F508del llevado por 45 a 50% de los pacientes 10, 11. ensayos clínicos convencionales para identificar sujetos sensibles al fármaco dentro del 50% restante de los pacientes con FQ son costosos y consumen mucho tiempo y no son factibles para los individuos con genotipos CFTR extremadamente raros. Novela, personalizados, métodos rentables son cruciales para que coincida con el creciente número de moduladores de CFTR a los individuos portadores de cualquier tipo de mutación CFTR. Hasta ahora, la inclusión de los ensayos de grupos de pacientes portadores de mutaciones CFTR específicos se ha guiado por los estudios que utilizan la transfección mutante del gen CFTR en hsistemas de células de eterologous, seguido de estudios electrofisiológicos en cámaras Ussing 5, 6, 12. Debido a la falta de modelos animales adecuados CF, estudios de eficacia de los medicamentos en las células epiteliales bronquiales de aire-líquido-interfaz-diferenciadas derivadas a partir de materiales de explantes de pulmón CF se han utilizado para el desarrollo de fármacos 13, 14, 15. Sin embargo, la limitada disponibilidad de tejidos de explantes de pulmón y los procedimientos invasivos para obtener células bronquiales de los sujetos sin enfermedad en fase terminal obstaculizar el análisis de mutaciones CFTR menos comunes y evitar las pruebas de drogas de una manera personalizada. Para superar estas limitaciones, los tejidos &quot;fácil acceso&quot;, como organoides colorrectal, células de las vías respiratorias nasales y las vías respiratorias células derivadas de células madre pluripotentes inducidas, están actualmente siendo explorado por los tratamientos farmacológicos personalizados. 16, 17. factores de crecimiento para el colon / recto humano, las condiciones de cultivo implican definido (factor de crecimiento epitelial (EGF), gastrina, Wnt-3A, R-espondina 3 (Rspo3), y Noggin) en combinación con moléculas pequeñas (nicotinamida, A83-01, y SB202190) en una matriz de membrana basal. En estas condiciones, las células madre individuales o fragmentos de tejido pequeños crecen hacia fuera en, quística, estructuras 3D cerrado formado por epitelio altamente polarizada con su lado basal orientada hacia el exterior. Todos los tipos de células suelen aparecer en sus proporciones y posiciones normales. Organoides pueden ampliarse durante períodos de tiempo largos por rotura mecánica semanal y re-recubrimiento. Ellos son genéticamente y fenotípicamente estables y pueden ser almacenados, lo que permite la expansión a largo plazo y bio-banca 17. Son susceptibles detodas las manipulaciones de células / biológica estándar genéticos y técnicas de análisis desarrolladas para líneas celulares 2D 18. Recientemente hemos demostrado que la función de CFTR se puede medir fácilmente en organoides colorrectal en un ensayo de inflamación inducida por la forskolina (FIS) 19, 20. Cuando se expone a la forskolina (FSK) o, alternativamente, a la toxina del cólera, organoides aumentan rápidamente su monofosfato de adenosina cíclico niveles (AMPc), que a su vez se traduce en la apertura del canal CFTR 19. Organoides de individuos sanos o de sujetos con mutaciones CFTR asociados con la función residual, posteriormente se hincharán como consecuencia de transporte de iones y agua al lumen de organoides, el equivalente in vitro de la diarrea secretora. La respuesta FIS de organoides colorrectal fue previamente demostrado ser totalmente dependiente de CFTR, como se indica por organoides derivados de individuos CFTR nulo unand por el uso de inhibidores de CFTR farmacológicas específicas 19. Grandes conjuntos de datos de temas específicos se pueden derivar dentro de varias semanas después de tomar una biopsia. Para el ensayo de la FIS se describe en detalle aquí, organoides se cultivan a partir de biopsias rectales que se pueden obtener a cualquier edad y con la única incomodidad limitada 21. Organoides se pasan semanalmente por la ruptura mecánica en criptas individuales que se vuelva a sellar y formar nuevos organoides fácilmente. Para ejecutar el ensayo de FIS, ~ 30-80 de estos pequeños organoides alterado son sembraron en cada pocillo de una placa de 96 pocillos 19. En el día del ensayo, los organoides se tiñeron con calceína verde, un colorante fluorescente de células permeable que está retenido dentro de las células vivas, lo que facilita imágenes en vivo. Entonces, FSK, lo que eleva el AMPc intracelular y por lo tanto activa CFTR, se añade con el fin de estimular la hinchazón organoide. Potenciadores que actúan sobre CFTR apical se añaden simultáneamente won la forskolina, mientras que los correctores que restauran el tráfico de CFTR se añaden 24 h antes de la adición de FSK. La hinchazón organoide se cuantifica mediante un análisis de imagen automatizado que calcula el aumento relativo de la superficie total de todos los objetos fluorescentes para cada punto de tiempo después de la adición de forskolina. 3D organoid hinchazón ofrece ventajas y desventajas sobre las lecturas de CFTR electrofisiológicos existentes en las vías respiratorias células cultivadas 2D en cámaras Ussing. Una ventaja importante es el rendimiento del ensayo de hinchamiento. Las células se cultivan y se ensayaron utilizando un único tipo de medio de cultivo, y un técnico con experiencia pueden cultivo hasta 25 muestras de organoides sobre una base semanal, mientras que la cuantificación de aproximadamente 1200 puntos de datos por semana en 12 muestras de pacientes. Que convencionalmente escribir una sola condición experimental por medio de mediciones por duplicado o triplicado por placa y repetir dichas mediciones en tres momentos de incubación independientes. En total, aproximadamente 300-500 single organoide estructuras se miden por condición experimental, lo que lleva a mediciones muy precisas de la función de CFTR con variabilidad técnica limitada. Esta precisión nos permite definir con claridad las diferencias en la función y la respuesta residual a moduladores de CFTR y nos permite recoger fácilmente a efectos de fondo genéticas entre los pacientes portadores de mutaciones CFTR idénticos 19, 22, 23, 24, 25. calidad de los datos se puede evaluar fácilmente a partir de imágenes de microscopio. Mientras FIS es totalmente dependiente de CFTR, que es una medida de resultado indirecto de la función de CFTR, su lectura fuera causado por el acoplamiento del transporte de iones para el transporte de fluidos. Esto contrasta con las mediciones de la función de CFTR directos en cámaras Ussing, que miden las corrientes de iones transepiteliales 26. cámaras Ussing permiten la estimulación de selección de c apical o basolateralompartments (que el ensayo no permite organoid); por permeabilización de la membrana basolateral, la secreción de aniones CFTR dependiente apical se puede medir de forma selectiva 27.

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			<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 muestra un aislamiento fresca representante de criptas incrustados en BMM. Las criptas son de una biopsia de colon de un sujeto CF. Por lo general, un organoide se genera a partir de cada cripta (Figura 1A - C). Debido a la disfunción de la CFTR, la mayoría de los organoides de colon CF no son quística, sino más bien son compactos y con proyecciones y injertos (Figura 2A - 2B). Sin embar...

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.

Inducida por la forskolina Hinchazón en organoides intestinal: Una<em&gt; In Vitro</em&gt; Ensayo para evaluar la respuesta de drogas en pacientes con fibrosis quística

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

forskolin-induced-swelling-intestinal-organoids-an-vitro-assay-for

Research

Education

JoVE Core

JoVE Journal

Pharmacology

Medicine

Unit 1

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

Other Respiratory Disorders

SCIENTISTS IN ACTION

19.0K vistas.  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.

Video: 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.

Evaluación de los cambios teratogénicos en un modelo de pez cebra de la exposición fetal al alcohol

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

Investigación

Medicina

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.

La evaluación de la encefalopatía espongiforme transmisible Especies Barreras con un<em&gt; In Vitro</em&gt; Prion Análisis de Conversión de proteínas

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.

La especificidad de la evaluación de Medicamentos contra el cáncer<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.

Método optimizado de LC-MS/MS para el análisis de alto rendimiento de las muestras clínicas de Ivacaftor, sus principales metabolitos y Lumacaftor en fluidos biológicos de pacientes con Fibrosis quística

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.

Mejor método para el establecimiento de un In Vitro Barrera Blood - Brain modelo basado en las células endoteliales de cerebro porcino

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 ensayo basado en fluorescencia para la caracterización y cuantificación de la formación de gotas lipídicas en organoides intestinales humanos

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.

Investigación de la ruptura de la barrera intestinal en los organoides vivos

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.

Uso de una biopsia química para la evaluación de la calidad del injerto

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.

Cultivo e imágenes de organoides epiteliales nasales humanos

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.