وأيد هذا العمل من قبل برنامج HIT-CF من CF الأساس الهولندية (NCFS)، ZonMW (40-00812-98-14103)، وصندوق أبحاث مستشفى يلهيلمينا الأطفال وتشيكوسلوفاكيا، وZilverenkruis / Achmea. ونود أن نشكر S. Heida ميشيل، M. GEERDINK، KM دي وينتر دي غروت، وG. Berkers (قسم أمراض الأطفال أمراض الرئة، ومستشفى يلهيلمينا الأطفال، UMC اوتريخت)، وRHJ هوفن (قسم أمراض الأطفال أمراض الجهاز الهضمي، ويلهيلمينا مستشفى الأطفال، UMC أوتريخت) لتقترب من المرضى والحصول على خزعات لجيل من CF البنك الحيوي.

<ol>
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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.

هنا، ونحن نقدم بروتوكول كامل للجيل، والتوسع، التجمد، وذوبان organoids القولون البشري. في حين أنشأنا الثقافات عضي الإنسان منذ بعض الوقت 17، فقد ثبت في بعض الأحيان من الصعب تحديد التكنولوجيا في مختبرات أخرى دون التدريب العملي على التدريب. ونحن نتوقع أن هذه الب?...

ويتسبب CF عن طفرات في جين التليف الكيسي الغشاء تصرف المنظم (CFTR) الذي يشفر قناة أنيون الظهارية. CF يؤثر على ما يقرب من 85،000 شخص حول العالم 1. وقد تم تحديد أكثر من 2،000 طفرات CFTR ( <a href="http://www.genet.sickkids.on.ca">www.genet.sickkids.on.ca</a> ). هذا التنوع يفسر جزئيا طائفة واسعة من الظواهر المرض لوحظ ( <a href="http://www.CFTR2.org">www.CFTR2.org</a> ) 2 و 3. يتم تعريف يستند ستة فصول من طفرات CFTR على تأثيرها على التعبير البروتين CFTR وظيفة: (I) لا التوليف، (الثاني) الاتجار ضعاف، (الثالث) المعيبة قناة النابضة، (رابعا) تصرف تتغير (V) مستويات عادة انخفاض الأداء CFTR، و (السادس) خلية ضعف استقرار سطح 4. على الرغم من أن الطفرات CFTR المشتركة ومدروسة، وظيفة CFTR والعلاقة مع الحالة السريرية لا تزال poorlفهم ذ على مستوى الفرد، لا سيما بالنسبة لمجموعة كبيرة من نادرة &quot;اليتيمة&quot; الطفرات ( <a href="http://www.CFTR2.org">www.CFTR2.org</a> ) 1 و 3. مؤخرا، تم تطوير عقاقير تستهدف بروتين CFTR في أزياء طفرة معينة. فئتين من CFTR الأدوية التي تستهدف البروتينات هي حاليا في الاستخدام السريري ولها طرق متميزة للعمل. Potentiators، مثل VX-770، وتعزيز الاحتمال مفتوحا من CFTR الطافرة المحلية apically وتعمل مباشرة على إضافة لخلايا 5. المصححين، مثل VX-809، واستعادة الاتجار اندوبلازمية مترجمة شبكية تتجمع CFTR وتتطلب مرحلة ما قبل الحضانة مع ويلاحظ الخلايا قبل آثار 6. وpotentiator CFTR، VX-770، وقد تم تسجيل للمواد الدراسية مع الطفرة G551D 7، 8، فضلا عن ثمانية أخرىCFTR النابضة الطفرات، بما في ذلك S1251N 9؛ معا، وتتم هذه الطفرات بنسبة 5٪ من جميع المواد CF. وقد أشارت التجارب السريرية الأخرى التي VX-770، جنبا إلى جنب مع مصحح VX-809، ويقتصر آثار بعد هامة على وظيفة الرئة ويؤدي إلى انخفاض في معدلات تفاقم في مواضيع متماثل لطفرة F508del التي يحملها 45-50٪ من المرضى 10، 11. التجارب السريرية التقليدية لتحديد المواضيع التي تستجيب للأدوية داخل 50٪ المتبقية من مرضى التليف الكيسي مكلفة وتستغرق وقتا طويلا وليست ذات جدوى بالنسبة للأفراد مع الأنماط الوراثية CFTR نادرة للغاية. الرواية، وطرق شخصية فعالة من حيث التكلفة ضرورية لتتناسب مع عدد متزايد من جهري CFTR إلى الأفراد تحمل أي نوع من CFTR التحور. حتى الآن، وقد استرشد إدراج محاكمة مجموعات المرضى تحمل طفرات CFTR محددة من الدراسات باستخدام متحولة ترنسفكأيشن CFTR الجينات في ح<html> نظم خلايا eterologous، تليها الدراسات الكهربية في غرفة أوسينج 5 و 6 و 12. ونظرا لعدم وجود نماذج CF الحيوانية الكافية والدراسات فعالية الدواء في الخلايا الظهارية القصبية متباينة الهواء السائل واجهة المستمدة من المواد يزدرع CF الرئة استخدمت لتطوير العقاقير 13 و 14 و 15. ومع ذلك، فإن محدودية توافر أنسجة الرئة يزدرع وإجراءات الغازية للحصول على خلايا الشعب الهوائية من المواد دون المرض في نهاية المرحلة تعرقل تحليل طفرات CFTR أقل شيوعا ومنع اختبار المخدرات بطريقة شخصية. للتغلب على هذه القيود، الأنسجة &quot;سهولة الوصول&quot;، مثل organoids القولون والمستقيم، وخلايا الشعب الهوائية الأنفية، وخلايا الشعب الهوائية المشتقة من الخلايا الجذعية المحفزة التي يسببها، ويجري حاليا التنقيب فيها عن العلاج بالعقاقير شخصية. = &quot;jove_content&quot;&gt; سابقا، أنشأنا بروتوكولات للخلايا الجذعية ثقافة الظهارية من أي جهاز الهضمي في شكل 3D organoids 16 و 17. عوامل النمو في القولون البشري / المستقيم، والظروف ثقافة تنطوي على تعريف (طلائي عامل النمو (EGF)، الغاسترين، WNT-3A، R-spondin 3 (Rspo3)، ورأس) جنبا إلى جنب مع جزيئات صغيرة (نيكوتيناميد، A83-01، وSB202190) في مصفوفة الغشاء القاعدي. في ظل هذه الظروف، والخلايا الجذعية واحدة أو أجزاء الأنسجة الصغيرة على النمو للخروج الى مغلقة، الكيسي، هياكل 3D التي شكلتها ظهارة درجة عالية من الاستقطاب مع الجانب القاعدي لها موجهة نحو الخارج. تظهر جميع أنواع الخلايا عادة في النسب والمواقف المعتادة. Organoids يمكن توسيعها على مدى فترات زمنية طويلة بسبب عرقلة الميكانيكية الأسبوعية وإعادة الطلاء. هم جينيا ومستقر ظاهريا، ويمكن تخزينها، مما يتيح التوسع على المدى الطويل والحيوي المصرفي 17. وهي قابلة للجميع التلاعب القياسية الخلايا البيولوجية / الجينية والتقنيات التحليلية وضعت لخطوط الخلايا 2D 18. نحن في الآونة الأخيرة أظهرت أن وظيفة CFTR يمكن قياسها بسهولة في organoids القولون والمستقيم في يسببها forskolin تورم (FIS) فحص 19 و 20. عندما تتعرض لforskolin (FSK) أو، بدلا من ذلك، لبكتيريا الكوليرا، organoids زيادة أعدادها بسرعة الأدينوزين الأدينوزين الحلقي (المخيم) المستويات، والتي تؤدي بدورها في افتتاح قناة CFTR 19. سوف Organoids من الأفراد الأصحاء، أو من الموضوعات مع طفرات CFTR المرتبطة مع وظيفة المتبقية، تضخم في وقت لاحق نتيجة لايون ونقل المياه إلى التجويف عضي، أي ما يعادل في المختبر من الإسهال إفرازية. وقد أظهرت استجابة الجبهة الإسلامية للإنقاذ من organoids القولون والمستقيم في السابق أن تكون تماما CFTR التي تعتمد، كما يتضح من organoids المستمدة من الأفراد CFTR باطل لالثانية عن طريق استخدام مثبطات CFTR محدد بالعقاقير 19. مجموعات البيانات موضوعا محددا كبيرة يمكن استخلاصها في غضون عدة أسابيع بعد أخذ الخزعة. للمقايسة الجبهة الإسلامية للإنقاذ وصفها في التفاصيل هنا، organoids ومثقف من الخزعات المستقيم التي يمكن الحصول عليها في أي سن وفقط مع عدم الراحة محدود 21. و passaged Organoids أسبوعيا بسبب عرقلة الميكانيكية في الأقبية واحدة أن ختم بسهولة وتشكيل organoids الجديد. لتشغيل الفحص الجبهة الإسلامية للإنقاذ، ~ ومطلي 30-80 من هذه organoids القليل تعطلت في كل بئر من لوحة 96-جيدا 19. في يوم الفحص، ملطخة organoids مع الأخضر calcein، وهي خلايا قابلة للاختراق صبغة الفلورسنت التي يتم الاحتفاظ بها داخل الخلايا الحية، وتسهيل التصوير الحي. ثم، يضاف FSK، الأمر الذي يثير مخيم داخل الخلايا، وبالتالي ينشط CFTR من أجل تحفيز تورم عضوي الشكل. تضاف Potentiators التي تعمل على CFTR قمي في وقت واحد ثإيث forskolin، في حين أن المصححين أن استعادة الاتجار CFTR تضاف 24 ساعة قبل إضافة FSK. وكميا تورم عضوي الشكل عن طريق تحليل الصور الآلي التي تحسب الزيادة النسبية في مساحة إجمالية قدرها كافة الكائنات الفلورسنت لكل نقطة زمنية على إضافة forskolin. عضي 3D تورم يوفر مزايا وعيوب على قراءات CFTR الكهربية الموجودة في 2D خلايا الشعب الهوائية مثقف في غرفة أوسينج. والميزة الرئيسية هي مرت من فحص التورم. الخلايا مثقف ويعاير باستخدام نوع واحد من مستنبت، ويمكن للفني من ذوي الخبرة ثقافة ما يصل الى 25 عينة عضي على أساس أسبوعي بينما قياس ما يقرب من 1200 نقطة البيانات أسبوعيا في 12 عينات من المرضى. نحن تقليديا اكتب حالة تجريبية واحدة من القياسات مكررة أو ثلاث نسخ لكل لوحة، وتكرار مثل هذه القياسات في ثلاث نقاط فترة حضانة مستقلة. في المجموع، ما يقرب من 300-500 قثم يتم قياس إنجل عضي الهياكل في حالة تجريبية، الأمر الذي يؤدي إلى قياسات دقيقة للغاية من وظيفة CFTR مع تقلب فنيا محدودا. هذه الدقة تتيح لنا أن تحدد بوضوح الاختلافات في وظيفة المتبقية وردا على جهري CFTR وتتيح لنا اختيار ما يصل بسهولة آثار خلفية وراثية بين المرضى الذين يحملون طفرات CFTR متطابقة 19، 22، 23، 24، 25. جودة البيانات التي يمكن تقييمها بسهولة من الصور المجهر. بينما الجبهة الإسلامية للإنقاذ هو تماما CFTR التي تعتمد، وهو قياس النتيجة غير المباشرة لوظيفة CFTR، للخروج قراءة الناجم عن اقتران النقل أيون لنقل السوائل. وهذا يتناقض مع القياسات وظيفة CFTR مباشرة في غرف Ussing، التي تقيس بطريق الظهارة التيارات أيون 26. غرف Ussing تسمح حدد تحفيز ج قمي أو basolateralompartments (الذي فحص عضوي الشكل لا يسمح)؛ بواسطة permeabilizing الأغشية basolateral، والقمي تعتمد على CFTR إفراز أنيون يمكن قياسها بشكل انتقائي (27).

<table border="0" cellpadding="0" cellspacing="0" width="1053">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:355px;">Advanced Dulbecco&#8217;s Modified Eagles Medium with Nutrient Mixture F-12 Hams (Ad-DF) 500ml&#160;</td>
			<td style="width:180px;">Thermo Fisher Scientific:&#160; Invitrogen</td>
			<td style="width:119px;">#12634</td>
			<td style="width:399px;">stored at 4 &#176;C</td>
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		<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>
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			<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>
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		<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>
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		<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>
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		<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>
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		<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>
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		<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>
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		<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>
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		<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>
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		<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>
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		<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>
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		<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>
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		<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>
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		<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>
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		<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>
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		<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>
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		<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>
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		<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>
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		<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>
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		<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>
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		<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.

ويبين الشكل 1A عزلة جديدة تمثيلية من أقبية جزءا لا يتجزأ من على BMM. الخبايا هي من خزعة القولون والمستقيم من موضوع CF. عادة، يتم إنشاء عضي من كل سرداب (الشكل 1A - C). ويرجع ذلك إلى خلل في CFTR، أكثر من organoids القولون CF لا الكيسي، وإنما ...

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-الناجم عن تورم في الأمعاء Organoids: ل<em&gt; في المختبر</em&gt; الفحص لتقييم الاستجابة للدواء في التليف الكيسي المرضى

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

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

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

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.

تقييم التغييرات المشوهة في نموذج الزرد من التعرض الكحول الجنينية

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

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.

تقييم المنقولة جنون الأنواع دماغي الحواجز مع<em&gt; في المختبر</em&gt; بريون تحويل البروتين الفحص

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.

تقييم خصوصية المضاد للسرطان المخدرات<em&gt; في المختبر</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.

أسلوب LC-MS/MS الأمثل لتحليل العينات السريرية إيفاكافتور، والايضات الرئيسية، ولوماكافتور في السوائل البيولوجية لمرضى التليف الكيسي الفائق

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.

تحسين أسلوب لإنشاء في المختبر حاجز الدم – المخ نموذج استناداً إلى خلايا المخ الخنزيري بطانية

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.

مقايسة علي أساس الفلورية لتوصيف وتحديد كميات الدهون التجميعية في تشكيل الأمعاء البشرية

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.

التحقيق في انهيار الحاجز المعوي في الأعضاء الحية

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.

استخدام خزعة كيميائية لتقييم جودة الكسب غير المشروع

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.

زراعة وتصوير المواد العضوية الظهارية الأنفية البشرية

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.