Name: forskolin-induced swelling in intestinal organoids: an in vitro assay for assessing drug response in cystic fibrosis patients
Uploaded: 2017-02-11T14:00:00
Description: Watch this Scientific Journal Video about Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients at JoVE.com

这项工作是由荷兰CF基金会（NCFS），ZonMW（40-00812-98-14103）时，威廉敏娜儿童医院研究基金和CZ和Zilverenkruis / Achmea的HIT-CF项目的支持。我们要感谢S.哈大 - 米歇尔，M. Geerdink，KM去冬德格鲁特和G Berkers（小儿呼吸科，威廉敏娜儿童医院，UMC乌得勒支系）和RHJ厚文（小儿消化内科，威廉敏娜的部儿童医院，UMC乌德勒支）对接近患者获得活检为CF生物资料库的产生。

使用本文中描述人类组织的所有试验批准在乌德勒支大学医学中心的伦理委员会（UMCU; TcBio＃14-008）。从病人威廉敏娜儿童医院（WKZ）-UMCU获得用于组织收集，生成，存储和使用的组织体的知情同意。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 设备 </td><td> 耗材 </td><td> 工具 </td></tr><tr><td>层流罩</td><td> 15和50毫升锥形管?...

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这里，我们提供了用于产生，膨胀，凝固，和人大肠组织体的解冻一完整的协议。虽然我们已经建立了人类器官样的文化前段时间17，它有时被证明很难建立在其他实验室的技术，而无需实际操作培训。我们预计，这些协议将取代这种培训。 的Wnt-3A条件培养基是为了在建立和维护长期化培养成功最关键的试剂之一。事实上，器官样的文化可能“撞车”暴露在...

CF是通过在编码上皮阴离子通道的囊性纤维化跨膜传导调节蛋白（CFTR）基因的突变引起的。 CF影响全世界约1 85000人。超过2000 CFTR突变已经确定（ <a href="http://www.genet.sickkids.on.ca">www.genet.sickkids.on.ca</a> ）。这种多样性也部分解释观察到的疾病表型（的广泛<a href="http://www.CFTR2.org">www.CFTR2.org</a> ）2，3。六类CFTR突变是基于其对CFTR蛋白表达和功能作用定义的：（I）中没有的合成，（II）中受损的贩卖，（Ⅲ）有缺陷的通道门控，（IV）中改变的电导，（V）的降低的正常水平功能CFTR，和（VI）受损细胞表面稳定性4。虽然常见CFTR突变很好的研究，CFTR功能和临床状态的关系保持poorlŸ了解在个人的水平，特别是对于大集团的罕见的“孤儿”突变（ <a href="http://www.CFTR2.org">www.CFTR2.org</a> ）1,3。 最近，药物已经开发了靶向特定突变的方式CFTR蛋白。两个班的CFTR蛋白，靶向药物，目前在临床上使用，并有行动不同的模式。增效剂，例如VX-770，增强顶部本地化突变体CFTR的开放概率和在他们除了细胞5直接作用。校正，如VX-809，恢复内质网本地化错误折叠的CFTR的贩运和要求预孵育的影响细胞前观察到6。该CFTR增强剂，VX-770，已注册的G551D突变的7，8个学科，以及为其他八位CFTR突变门控，包括S1251N 9;一起，这些突变通过所有CF患者的5％进行。其它的临床试验已经表明VX-770，与校正的VX-809相结合，限制对肺功能尚未显著影响，并导致恶化率在受试者纯合的患者10 45-50％承载的F508del突变降低， 11。 常规的临床试验，以CF患者的剩余的50％的范围内确定药物响应受试者是昂贵和费时的，并且不与非常罕见的CFTR基因型的个体是可行的。新颖，具有成本效益的，个性化的方法是，以匹配于携带任何类型的CFTR突变的个体数量的增加CFTR调节剂的关键。到现在为止，承载特定的CFTR突变的患者群体的审判列入遵循了使用H CFTR突变基因转染的研究eterologous细胞系统，随后电生理学研究中尤斯灌流室5，6，12。由于缺乏足够的CF动物模型中，在从CF肺外植体材料来源的空气-液体界面分化支气管上皮细胞的药物的功效研究已用于药物开发13，14，15。然而，肺组织外植体和侵入性操作的使用限制，以获得无疾病终末期患者支气管细胞阻碍不太常见CFTR突变的分析和预防药物测试的个性时尚。为了克服这些限制，“绿色通道”的组织，例如结肠组织体，鼻气道细胞，和从诱导的多能干细胞衍生的气道细胞，目前正在探索的个性化的药物治疗。 16，17的形式建立的协议，以培养上皮干细胞从任何胃肠器官。为人类结肠/直肠中，培养条件包括限定生长因子（表皮生长因子（EGF），胃泌素，的Wnt-3A，R脊椎蛋白3（Rspo3），和头蛋白）结合的小分子（烟酰胺，A83-01，和SB202190）在基底膜基质。在这些条件下，单个干细胞或小组织片段生长出到由高度偏振光上皮朝向外侧面向其基部侧形成封闭，囊状，三维结构。所有类型的细胞通常出现在他们正常的比例和位置。组织体可以在由每周机械破碎和再镀长时间周期进行扩展。它们是遗传和表型稳定，可以进行储存，从而允许长期扩增和生物银行17。他们是服从所有标准的细胞生物学/遗传操作和分析技术的二维细胞系18开发的。 我们最近证明CFTR功能可以在大肠癌组织体可容易地测得在毛喉素诱导肿胀（FIS）测定19,20。当暴露于毛喉素（FSK）或者，霍乱毒素，类器官迅速增加其环磷酸腺苷（cAMP）的水平，这在CFTR通道19的开口又导致。类器官来自健康个体，或从与剩余功能相关CFTR突变，受试者将随后溶胀离子和水输送到组织体腔，分泌性腹泻的体外等效的结果。大肠组织体的FIS响应以前证明是完全的CFTR相关的，通过从CFTR空个人取得组织体所指示的一个通过使用特定的药理CFTR抑制剂19次。大型主题特定数据集可以在几个星期内采取活检后得到。 在这里详细描述的FIS测定中，类器官是从可以在任何年龄和只有有限的不适21得到直肠活检中培养。类器官是由机械破碎每周传代成单隐窝，可以轻松地重新密封，并形成新的类器官。用于运行FIS测定中，〜这些扰乱小组织体30-80铺板在96孔板19的每个孔中。在测定的当天，将组织体染色用钙黄绿素绿色，它被保持活细胞内，便利实时成像的荧光细胞渗透性染料。然后，FSK，这引起了细胞内cAMP，从而激活CFTR，是为了刺激类器官肿胀加入。在心尖CFTR充当增效剂同时W¯¯添加第i个毛喉素，而认为CFTR恢复贩卖校正器加FSK之前所加的24小时。的器官样肿胀是由计算用于在福斯克林除了各时间点的所有荧光物体的总面积的相对增加的自动图像分析进行定量。 三维类器官溶胀提供了超过在2D培养的气道细胞现有电CFTR读数在尤斯灌流室的优点和缺点。一个主要优点是溶胀测定的吞吐量。细胞进行培养，并使用单一类型的培养基的测定，和一个有经验的技术人员可以培养多达25类器官样本每周，而在12患者样品定量每周约1,200个数据点。我们通常通过键入每板重复或三次测量单个实验条件，并在三个独立的孵化时间点重复这种测量。在总计约300-500小号英格尔类器官结构，则每个实验条件下，从而导致有限的技术可变性CFTR功能的非常精确的测量进行测定。这种精度使我们清楚地界定残留的功能和应对CFTR调节差异，并允许我们随手拿起携带相同的CFTR突变19，22，23，24，25例患者的遗传背景的影响。数据质量可以从显微镜图像容易地评估。而FIS是充分CFTR依赖性，它是CFTR功能的间接的测量结果，所造成的离子输送的流体传输的耦合其读出。与此相反，在尤斯灌流室直接CFTR功能测量，测量跨膜离子流26。尤斯灌流室心尖允许基底或C的选择刺激ompartments（其类器官测定不允许）;由透基底膜，心尖CFTR依赖性阴离子分泌可以有选择地测量27。

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

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

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

图1A显示嵌入BMM隐窝代表新鲜隔离。隐窝是从CF受试者的结肠活检。通常情况下，从每个地穴（ - C 图1A）生成的类器官。由于CFTR的功能障碍，大部分结肠的CF类器官的不囊状，而是结构紧凑，有突起和buddings（ 图2A - 2B）。然而，一些CF类器官培养物，尤其是具有高的残留功能，具有与囊性形状（ 图2C）?...

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.

毛喉素诱导在肠组织体溶胀：一个<em&gt;体外</em&gt;检测方法的评估囊性纤维化患者对药物的反应

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

Research

JoVE Journal

Medicine

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

Assessing Teratogenic Changes in a Zebrafish Model of Fetal Alcohol Exposure

在斑马鱼模型的胎儿酒精暴露评估致畸的变化

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

科研

医学

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

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

评估传染性海绵状脑病物种界限与<em&gt;在体外</em&gt;朊蛋白转化测定法

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

Assessing Specificity of Anticancer Drugs In Vitro

Assessing Specificity of Anticancer Drugs In Vitro

评估抗癌药物的特异性<em&gt;体外</em

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

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

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

新生儿鼠耳蜗外科手术技术<em&gt;体外</em&gt;听力研究筛选工具

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

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

经优化的 LC/ms 方法对囊性纤维化患者 Ivacaftor 及其主要代谢物和 Lumacaftor 的高通量分析

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

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

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

一种基于猪脑内皮细胞的体外血脑屏障模型的改进方法

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

人类肠道器官脂滴形成特征化和定量的荧光测定

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

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

大鼠胡萝卜动脉压力控制段气球损伤与腹腔ventit 治疗应用

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

Investigating Intestinal Barrier Breakdown in Living Organoids

研究活体器官的肠道屏障分解

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

使用化学活检进行移植质量评估

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