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。

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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.

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"暴露在低?...

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><tbody><tr><td>Advanced Dulbecco&amp;rsquo;s Modified Eagles Medium with Nutrient Mixture F-12 Hams (Ad-DF) 500 ml&amp;nbsp;</td><td>Thermo Fisher Scientific:&amp;nbsp; Invitrogen</td><td>#12634</td><td>stored at 4 &amp;deg;C</td></tr><tr><td>GlutaMax</td><td>Thermo Fisher Scientific:&amp;nbsp; Invitrogen</td><td>#35050</td><td>stored at 4 &amp;deg;C</td></tr><tr><td>Hepes</td><td>Thermo Fisher Scientific:&amp;nbsp; Invitrogen</td><td># 15630-056</td><td>stored at 4 &amp;deg;C</td></tr><tr><td>Penicillin/Streptomycin</td><td>Thermo Fisher Scientific:&amp;nbsp; Invitrogen</td><td>#15140-122</td><td>stored at -20 &amp;deg;C</td></tr><tr><td>96 well culture plate</td><td>Cellstar</td><td>#655180</td><td></td></tr><tr><td>24 well culture plate</td><td>Cellstar</td><td>#662160</td><td></td></tr><tr><td>6 well culture plate</td><td>Cellstar</td><td>#657160</td><td></td></tr><tr><td>Dulbecco&#039;s Phosphate Buffered Saline (-) CaCl&lt;sub&gt;2 &lt;/sub&gt;(-) MgCl&lt;sub&gt;2&lt;/sub&gt;) (DPBS)</td><td>Life Technologies: Gibco</td><td>#14190-094</td><td>stored at 4 &amp;deg;C</td></tr><tr><td>Dulbecco&amp;rsquo;s Modified Eagles Medium&amp;nbsp; (DMEM) 500 ml&amp;nbsp;</td><td>Thermo Fisher Scientific:&amp;nbsp; Invitrogen</td><td>#31966-021</td><td>For Wnt-3A Conditioned Medium Production. Stored at 4 &amp;deg;C</td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Bovogen</td><td>#SFBS LOT#11113</td><td>For Wnt-3A Conditioned Medium Production. Stored at -20 &amp;deg;C</td></tr><tr><td>L Wnt3A cell line</td><td>ATCC</td><td>#CRL-2647</td><td>For Wnt-3A Conditioend Medium Production.</td></tr><tr><td>TOP/FOP plasmids</td><td>Millipore&amp;nbsp;</td><td>#17-285</td><td>For measuring Wnt activity</td></tr><tr><td>pTK-Renilla</td><td>Promega&amp;nbsp;</td><td>#E2241</td><td>For measuring Wnt activity</td></tr><tr><td>HEK-293</td><td>ATCC</td><td>#CRL-1573</td><td>For measuring Wnt activity</td></tr><tr><td>Dual-Luciferase Reporter Assay System</td><td>Promega&amp;nbsp;</td><td>#E1910</td><td>For measuring Wnt activity</td></tr><tr><td>Zeocin&amp;nbsp;</td><td>Thermo Fisher Scientific:&amp;nbsp; Invitrogen</td><td>#R250-01</td><td>For Wnt-3A Cell line selection</td></tr><tr><td>B27 supplement&amp;nbsp;</td><td>Thermo Fisher Scientific:&amp;nbsp; Invitrogen</td><td>#17504-044</td><td>stored at -20 &amp;deg;C</td></tr><tr><td>N-Acetylcysteine</td><td>Sigma Aldrich</td><td>#A9165-5G</td><td>stored at -20 &amp;deg;C</td></tr><tr><td>Nicotinamide</td><td>Sigma Aldrich</td><td>#N0636</td><td>stored at -20 &amp;deg;C</td></tr><tr><td>Human Epithelial Growth Factor (hEGF)</td><td>PrepoTech</td><td>#AF-100-15</td><td>stored at -20 &amp;deg;C</td></tr><tr><td>Gastrin</td><td>Sigma Aldrich</td><td>#G9145</td><td>stored at -20 &amp;deg;C</td></tr><tr><td>TGFb type I Receptor inhibitor (A83-01)&amp;nbsp;</td><td>Tocris</td><td>#2939</td><td>stored at -20 &amp;deg;C</td></tr><tr><td>Y-27632 dihydrochloride (RhoKi)</td><td>Selleckchem</td><td>#S1049</td><td>stored at -20 &amp;deg;C</td></tr><tr><td>p38 MAPK inhibitor (p38i) (SB202190)</td><td>Sigma Aldrich</td><td>#S7067</td><td>stored at -20 &amp;deg;C</td></tr><tr><td>Primocin</td><td>InvivoGen</td><td>#ant-pm-1</td><td>stored at -20 &amp;deg;C</td></tr><tr><td>Human Noggin (hNoggin)</td><td>PrepoTech</td><td>#120-10C</td><td>stored at -20 &amp;deg;C</td></tr><tr><td>Human R-spondin 3 (hRspo-3)</td><td>R&amp;amp;D Systems</td><td>#3500-RS/CF</td><td>stored at -20 &amp;deg;C</td></tr><tr><td>Vancomycin</td><td>Sigma Aldrich</td><td>#861987- 250mg</td><td>stored at -20 &amp;deg;C</td></tr><tr><td>Gentamycin</td><td>Life Technologies: Gibco</td><td>#15710-049</td><td>stored at -20 &amp;deg;C</td></tr><tr><td>Ethylenediamine tetraacetic acid (EDTA)</td><td>Sigma Aldrich</td><td>#431788</td><td>Stored at 4 &amp;deg;C</td></tr><tr><td>Matrigel</td><td>Corning</td><td>#354230</td><td>stored at -80 &amp;deg;C</td></tr><tr><td>TryplE Express&amp;nbsp;</td><td>Life Technologies: Gibco</td><td>#12605-010</td><td>for trypsinizing organoids for freezing</td></tr><tr><td>Recovery Cell Culture Freezing Medium</td><td>Life Technologies: Gibco</td><td>#12648010</td><td>for freezing</td></tr><tr><td>Calcein</td><td>Life Technologies: Gibco</td><td>#C3100MP</td><td>stored at -20 &amp;deg;C</td></tr><tr><td>Forskolin</td><td>R&amp;amp;D Systems</td><td>#1099-50 mg</td><td>stored at -80 &amp;deg;C</td></tr><tr><td>Lumacaftor (VX-809)</td><td>Selleckchem</td><td>#s1565</td><td>stored at -80 &amp;deg;C</td></tr><tr><td>Ivacaftor (VX-770)</td><td>Selleckchem</td><td>#s1144</td><td>stored at -80 &amp;deg;C</td></tr><tr><td>&lt;strong&gt;Name of Reagents/Material&lt;/strong&gt;</td><td>&lt;strong&gt;Solvent&lt;/strong&gt;</td><td>&lt;strong&gt;Stock Concentration&lt;/strong&gt;</td><td>&lt;strong&gt;Final Concentration&lt;/strong&gt;</td></tr><tr><td>GlutaMax</td><td></td><td>200 mM</td><td>2 mM</td></tr><tr><td>Hepes</td><td></td><td>1 M</td><td>10 mM</td></tr><tr><td>Penicillin/Streptomycin</td><td></td><td>10K U/ml 10K &amp;micro;g/ml</td><td>100 U/ml 100 &amp;micro;g/ml</td></tr><tr><td>Zeocin&amp;nbsp;</td><td></td><td>100 mg/ml&amp;nbsp;</td><td>125 &amp;micro;g/ml</td></tr><tr><td>B27 supplement&amp;nbsp;</td><td></td><td>100x</td><td>1x</td></tr><tr><td>N-Acetylcysteine</td><td>MiliQ H&lt;sub&gt;2&lt;/sub&gt;O</td><td>500 mM</td><td></td></tr><tr><td>Nicotinamide</td><td>DPBS</td><td>1 M</td><td>10 mM</td></tr><tr><td>Human Epithelial Growth Factor (hEGF)</td><td>DPBS 0.1% BSA</td><td>0.5 mg/ml</td><td>50 ng/ml</td></tr><tr><td>Gastrin</td><td>DPBS</td><td>100 &amp;micro;M</td><td>10 nM</td></tr><tr><td>TGFb type I Receptor inhibitor (A83-01)&amp;nbsp;</td><td>DMSO</td><td>5 mM</td><td>500 nM</td></tr><tr><td>Y-27632 dihydrochloride (RhoKi)</td><td>DMSO</td><td>10 mM</td><td>10 &amp;micro;M</td></tr><tr><td>p38 MAPK inhibitor (p38i) (SB202190)</td><td>DMSO</td><td>30 mM</td><td>10 &amp;micro;M</td></tr><tr><td>Primocin</td><td></td><td>50 mg/ml&amp;nbsp;</td><td>100 &amp;micro;g/ml</td></tr><tr><td>Human Noggin (hNoggin)</td><td>DPBS 0.1%BSA</td><td>100 &amp;micro;g/ml</td><td>100 ng/ml</td></tr><tr><td>Human R-spondin 3 (hRspo-3)</td><td></td><td>varies per lot</td><td>300 ng/ml</td></tr><tr><td>Vancomycin</td><td></td><td>10 mg/ml</td><td>50 &amp;micro;g/ml</td></tr><tr><td>Gentamycin</td><td></td><td>10 mg/ml</td><td>50 &amp;micro;g/ml</td></tr><tr><td>Ethylenediamine tetraacetic acid (EDTA)</td><td>MiliQ H&lt;sub&gt;2&lt;/sub&gt;O</td><td>0.5 M</td><td>2 mM</td></tr><tr><td>Calcein</td><td>DMSO</td><td>10 &amp;micro;g/ml</td><td>3.3 ng/ml</td></tr><tr><td>Forskolin</td><td>DMSO</td><td>10 mM</td><td>variable</td></tr><tr><td>Lumacaftor (VX-809)</td><td>DMSO</td><td>20 mM</td><td>variable</td></tr><tr><td>Ivacaftor (VX-770)</td><td>DMSO</td><td>20 mM</td><td>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 ...

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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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19.2K 次观看.  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.

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

The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids

Primary intestinal organoids are a valuable model system that has the potential to significantly impact the field of mucosal immunology. However, the complexities of the organoid growth characteristics carry significant caveats for the investigator. Specifically, the growth patterns of each individual organoid are highly variable and create a heterogeneous population of epithelial cells in culture. With such caveats, common tissue culture practices cannot be simply applied to the organoid system due to the complexity of the cellular structure. Counting and plating based solely on cell number, which is common for individually separated cells, such as cell lines, is not a reliable method for organoids unless some normalization technique is applied. Normalizing to total protein content is made complex due to the resident protein matrix. These characteristics in terms of cell number, shape and cell type should be taken into consideration when evaluating secreted contents from the organoid mass. This protocol has been generated to outline a simple procedure to culture and treat small intestinal organoids with microbial pathogens and pathogen associated molecular patterns (PAMPs). It also emphasizes the normalization techniques that should be applied when protein analysis are conducted after such a challenge.

The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids

Here, a protocol to harvest, maintain, and treat mouse small intestinal organoids with pathogen associated molecular patterns (PAMPs) and Listeria monocytogenes is described, as well as emphasis on gene expression and proper normalization techniques for protein.

该<em&gt;离体</em&gt;文化与模式识别小鼠肠道组织体受体刺激

Primary intestinal organoids are a valuable model system that has the potential to significantly impact the field of mucosal immunology. However, the ...

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Combining Human Organoids and Organ-on-a-Chip Technology to Model Intestinal Region-Specific Functionality

The intestinal mucosa is a complex physical and biochemical barrier that fulfills a myriad of important functions. It enables the transport, absorption, and metabolism of nutrients and xenobiotics while facilitating a symbiotic relationship with microbiota and restricting the invasion of microorganisms. Functional interaction between various cell types and their physical and biochemical environment is vital to establish and maintain intestinal tissue homeostasis. Modeling these complex interactions and integrated intestinal physiology in vitro is a formidable goal with the potential to transform the way new therapeutic targets and drug candidates are discovered and developed.
Organoids and Organ-on-a-Chip technologies have recently been combined to generate human-relevant intestine chips suitable for studying the functional aspects of intestinal physiology and pathophysiology in vitro. Organoids derived from the biopsies of the small (duodenum) and large intestine are seeded into the top compartment of an organ chip and then successfully expand&#160;as monolayers while preserving the distinct cellular, molecular, and functional features of each intestinal region. Human intestine tissue-specific microvascular endothelial cells are incorporated in the bottom compartment of the organ chip to recreate the epithelial-endothelial interface. This novel platform facilitates luminal exposure to nutrients, drugs, and microorganisms, enabling studies of intestinal transport, permeability, and host-microbe interactions.
Here, a detailed protocol is provided for the establishment of intestine chips representing the human duodenum (duodenum chip) and colon (colon chip), and their subsequent culture under continuous flow and peristalsis-like deformations. We demonstrate methods for assessing drug metabolism and CYP3A4 induction in duodenum chip using prototypical inducers and substrates. Lastly, we provide a step-by-step procedure&#160;for the in vitro modeling of interferon gamma (IFN&#947;)-mediated barrier disruption (leaky gut syndrome) in a colon chip, including methods for evaluating the alteration of paracellular permeability, changes in cytokine secretion, and transcriptomic profiling of the cells within the chip.

Biopsy-derived intestinal organoids and organ-on-a-chips technologies are combined into a microphysiological platform to recapitulate region-specific intestinal functionality.

结合人体类器官和芯片器官技术，对肠道区域特异性功能进行建模

The intestinal mucosa is a complex physical and biochemical barrier that fulfills a myriad of important functions. It enables the transport, absorption, ...

生物工程

Generation of Human Nasal Epithelial Cell Spheroids for Individualized Cystic Fibrosis Transmembrane Conductance Regulator Study

While the introduction of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) modulator drugs has revolutionized care in Cystic Fibrosis (CF), the genotype-directed therapy model currently in use has several limitations. First, rare or understudied mutation groups are excluded from definitive clinical trials. Moreover, as additional modulator drugs enter the market, it will become difficult to optimize the modulator choices for an individual subject. Both of these issues are addressed with the use of patient-derived, individualized preclinical model systems of CFTR function and modulation. Human nasal epithelial cells (HNEs) are an easily accessible source of respiratory tissue for such a model. Herein, we describe the generation of a three-dimensional spheroid model of CFTR function and modulation using primary HNEs. HNEs are isolated from subjects in a minimally invasive fashion, expanded in conditional reprogramming conditions, and seeded into the spheroid culture. Within 2 weeks of seeding, spheroid cultures generate HNE spheroids that can be stimulated with 3',5'-cyclic adenosine monophosphate (cAMP)-generating agonists to activate CFTR function. Spheroid swelling is then quantified as a proxy of CFTR activity. HNE spheroids capitalize on the minimally invasive, yet respiratory origin of nasal cells to generate an accessible, personalized model relevant to an epithelium reflecting disease morbidity and mortality. Compared to the air-liquid interface HNE cultures, spheroids are relatively quick to mature, which reduces the overall contamination rate. In its current form, the model is limited by low throughput, though this is offset by the relative ease of tissue acquisition. HNE spheroids can be used to reliably quantify and characterize CFTR activity at the individual level. An ongoing study to tie this quantification to in vivo drug response will determine if HNE spheroids are a true preclinical predictor of patient response to CFTR modulation.

Here we describe a method to generate three-dimensional spheroid cultures of human nasal epithelial cells. Spheroids are then stimulated to drive Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)-dependent ion and fluid secretion, quantifying the change in the spheroid luminal size as a proxy for CFTR function.

人鼻上皮细胞球体的生成与个体化囊性纤维膜电导调节的研究

While the introduction of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) modulator drugs has revolutionized care in Cystic Fibrosis (CF), the ...

医学

Canine Intestinal Organoids in a Dual-Chamber Permeable Support System

The permeable support system is typically used in conjunction with traditional two-dimensional (2D) cell lines as an in vitro tool for evaluating the oral permeability of new therapeutic drug candidates. However, the use of these conventional cell lines has limitations, such as altered expression of tight junctions, partial cell differentiation, and the absence of key nuclear receptors. Despite these shortcomings, the Caco-2 and MDCK models are widely accepted and validated for the prediction of human in vivo oral permeability.
Dogs are a relevant translational model for biomedical research due to their similarities in gastrointestinal anatomy and intestinal microflora with humans. Accordingly, and in support of parallel drug development, the elaboration of an efficient and accurate in vitro tool for predicting in vivo drug permeability characteristics both in dogs and humans is highly desirable. Such a tool could be the canine intestinal organoid system, characterized by three-dimensional (3D), self-assembled epithelial structures derived from adult stem cells.
The (1) Permeable Support Seeding Protocol describes the experimental methods for dissociating and seeding canine organoids in the inserts. Canine organoid isolation, culture, and harvest have been previously described in a separate set of protocols in this special issue. Methods for general upkeep of the canine intestinal organoid monolayer are discussed thoroughly in the (2) Monolayer Maintenance Protocol. Additionally, this protocol describes methods to assess the structural integrity of the monolayer via transepithelial electrical resistance (TEER) measurements and light microscopy. Finally, the (3) Permeability Experimental Protocol&#160;describes the tasks directly preceding an experiment, including in vitro validation of experimental results.
Overall, the canine organoid model, combined with a dual-chamber cell culture technology, overcomes limitations associated with 2D experimental models, thereby improving the reliability of predictions of the apparent oral permeability of therapeutic drug candidates both in the canine and human patient.

Here, we present a protocol describing the culture of canine intestinal organoids in a dual-chamber, permeable support system. Organoid seeding in the permeable supports, the monolayer maintenance, and subsequent drug permeability experiments are described.

双室渗透支持系统中的犬肠类器官

The permeable support system is typically used in conjunction with traditional two-dimensional (2D) cell lines as an in vitro tool for evaluating the oral ...

评估生物功能自组装肽的细胞粘附和形态学

Fabrication and Characterization of Colorectal Cancer Organoids from SW1222 Cell Line in Ultrashort Self&#45;Assembling Peptide Matrix

Ultrashort self-assembling peptides (SAPs) can spontaneously form nanofibers that resemble the extracellular matrix. These fibers allow the formation of hydrogels that are biocompatible, biodegradable, and non-immunogenic. We have previously proven that SAPs, when biofunctionalized with protein-derived motifs, can mimic the extracellular matrix characteristics that support colorectal organoid formation. These biofunctional peptide hydrogels retain the original parent peptide's mechanical properties, tunability, and printability while incorporating cues that allow cell-matrix interactions to increase cell adhesion. This paper presents the protocols needed to evaluate and characterize the effects of various biofunctional peptide hydrogels on cell adhesion and lumen formation using an adenocarcinoma cancer cell line able to form colorectal cancer organoids cost-effectively. These protocols will help evaluate biofunctional peptide hydrogel effects on cell adhesion and luminal formation using immunostaining and fluorescence image analysis. The cell line used in this study has been previously utilized for generating organoids in animal-derived matrices.

作者聚焦：结直肠癌类器官的超短肽基质优化

This protocol aims to evaluate biofunctional self-assembling peptides for cell adhesion, organoid morphology, and gene expression by immunostaining. We will use a colorectal cancer cell line to provide a cost-effective way of obtaining organoids for intensive testing.

在超短自组装肽基质中制备和表征来自 SW1222 细胞系的结直肠癌类器官

Ultrashort self-assembling peptides (SAPs) can spontaneously form nanofibers that resemble the extracellular matrix. These fibers allow the formation of ...

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.

人鼻上皮类器官的培养和成像

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.

直肠类器官形态学分析（ROMA）:囊性纤维化的诊断测定

Diagnosis of cystic fibrosis (CF) is not always straightforward, especially when sweat chloride concentration is intermediate and/or less than two ...

Methods to Study Epithelial Transport Protein Function and Expression in Native Intestine and Caco-2 Cells Grown in 3D

The intestinal epithelium has important transport and barrier functions that play key roles in normal physiological functions of the body while providing a barrier to foreign particles. Impaired epithelial transport (ion, nutrient, or drugs) has been associated with many diseases and can have consequences that extend beyond the normal physiological functions of the transporters, such as by influencing epithelial integrity and the gut microbiome. Understanding the function and regulation of transport proteins is critical for the development of improved therapeutic interventions. The biggest challenge in the study of epithelial transport is developing a suitable model system that recapitulates important features of the native intestinal epithelial cells. Several in vitro cell culture models, such as Caco-2, T-84, and HT-29-Cl.19A cells are typically used in epithelial transport research. These cell lines represent a reductionist approach to modeling the epithelium and have been used in many mechanistic studies, including their examination of epithelial-microbial interactions. However, cell monolayers do not accurately reflect cell-cell interactions and the in vivo microenvironment. Cells grown in 3D have shown to be promising models for drug permeability studies. We show that Caco-2 cells in 3D can be used to study epithelial transporters. It is also important that studies in Caco-2 cells are complemented with other models to rule out cell specific effects and to take into account the complexity of the native intestine. Several methods have been previously used to assess the functionality of transporters, such as everted sac and uptake in isolated epithelial cells or in isolated plasma membrane vesicles. Taking into consideration the challenges in the field with respect to models and the measurement of transport function, we demonstrate here a protocol to grow Caco-2 cells in 3D and describe the use of an Ussing chamber as an effective approach to measure serotonin transport, such as in intact polarized intestinal epithelia.

We describe simple methods to study the regulation of intestinal serotonin transporter (SERT) function and expression using an in vitro cell culture model of Caco-2 cells grown in 3D&#160;and an ex vivo model of mouse intestines. These methods are applicable to the study of other epithelial transporters.

方法来研究上皮转运蛋白功能和表达在纯肠和Caco-2细胞生长在3D

The intestinal epithelium has important transport and barrier functions that play key roles in normal physiological functions of the body while providing ...

毛喉素诱导在肠组织体溶胀:一个

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结合人体类器官和芯片器官技术，对肠道区域特异性功能进行建模

人鼻上皮细胞球体的生成与个体化囊性纤维膜电导调节的研究

双室渗透支持系统中的犬肠类器官

在超短自组装肽基质中制备和表征来自 SW1222 细胞系的结直肠癌类器官

在超短自组装肽基质中制备和表征来自 SW1222 细胞系的结直肠癌类器官

在超短自组装肽基质中制备和表征来自 SW1222 细胞系的结直肠癌类器官

人鼻上皮类器官的培养和成像

直肠类器官形态学分析（ROMA）:囊性纤维化的诊断测定

方法来研究上皮转运蛋白功能和表达在纯肠和Caco-2细胞生长在3D