Name: culture and imaging of human nasal epithelial organoids
Uploaded: 2021-12-17T14:00:00
Description: Watch this Scientific Journal Video about Culture and Imaging of Human Nasal Epithelial Organoids at JoVE.com

我们非常感谢所有捐赠HNE刷式活检的参与者为开发该协议所做的贡献。我们感谢Latona Kersh和儿童研究部门的工作人员协调研究志愿者招募和样本收集。我们感谢Lily Deng，Johnathan Bailey和Stephen Mackay，他们是我们实验室的前学员，为他们提供了技术援助。我们感谢刘忠和赵锐的技术帮助。UAB CF研究中心主任Steven M. Rowe提供领导和资源，没有这些，这项工作是不可能的。我们还要感谢Biotek的Sarah Guadiana在仪器培训方面的帮助，Robert Grabski在UAB高分辨率成像设施的共聚焦显微镜协助，以及Dezhi Wang在UAB组织学核心的组织学帮助。这项工作得到了美国国立卫生研究院（NIH）的支持。授予K23HL143167（JSG），囊性纤维化基金会（CFF）授予GUIMBE18A0-Q（授予JSG），Gregory Fleming James囊性纤维化中心[NIH赠款R35HL135816和DK072482以及CFF阿拉巴马大学伯明翰分校（UAB）研发计划（Rowe19RO）]和UAB临床和转化科学中心（NIH赠款UL1TR001417）。

HNE样本是在阿拉巴马州儿童医院收集的。此处描述的所有程序和方法均已获得伯明翰阿拉巴马大学IRB（UAB IRB #151030001）的批准。为了促进人鼻上皮细胞（HNEs）的扩增和功能的提高，本培养方法改编自公知的气液界面（ALI）培养方法28，34。如前面描述的12，14所示，最初通过刷子活检收集HNE，唯一的区别是使用...

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本手稿提供了对源自 HNE 刷式活检的气道上皮类器官进行全面活体和固定成像的详细方法。它描述了可以确定个体CFTR活性的功能测定。HNE为各种应用提供微创的初级组织。这里提供的扩展技术可用于模拟气道疾病，包括类器官。类器官可用于精确治疗方法和监测基于基因或mRNA的疗法随时间推移的稳定性，用于精确试验设计，并帮助解决不确定的诊断39。目前的研究是关于CF的，?...

技术介绍 基于离体培养的检测是精准医学和疾病病理生理学研究中日益常用的工具。原发性人鼻上皮（HNE）细胞培养物已被用于囊性纤维化1，2，3，4，5，6，7，8，9，10，11，12，13的众多研究中，一种影响多个器官上皮细胞功能的常染色体隐性遗传病。HNE 培养提供可再生的气道上皮来源，可前瞻性地获得，并概括电生理和生化特性，以测试囊性纤维化跨膜传导调节剂 （CFTR） 活性。HNE细胞可以以最小的副作用14进行采样，类似于普通的病毒呼吸道拭子。描述源自HNE刷式活检的囊性纤维化研究模型的研究工作最近发表了11，13。虽然与使用原代HNE2，3和肠组织15，16，17，18，19的其他模型相似，但此处描述了该模型的分化和成像的详细表征，以用于CF研究和辅助其他气道疾病的研究13.类器官模型不像永生化细胞系那样是无限的，但可以通过条件重编程（使用辐照和灭活的饲养层成纤维细胞和Rho激酶抑制剂）扩展到更像干细胞的状态20，21，22，23。使用这种方法处理HNE刷活检可产生大量上皮细胞，用于多种应用，具有更高的通量，同时仍保留完全分化的能力。虽然该方案是使用饲养层细胞开发的，但希望避免使用饲养层细胞技术的研究者可以使用其他方法14，24。
该技术对肺生物学的重要性 一项重要的研究致力于了解上皮细胞细胞膜中缺乏规则的，功能正常的CFTR如何导致肺，胰腺，肝脏，肠道或其他组织的功能障碍。功能失调的上皮离子转运，特别是氯化物和碳酸氢盐的上皮离子转运，导致上皮衬里液体积减少和粘液分泌物改变，导致粘液淤滞和梗阻。在其他气道疾病中，例如原发性纤毛运动障碍，睫状体运动改变会损害粘膜纤毛清除并导致粘液淤滞和梗阻25。因此，目前的HNE类器官模型已经针对各种应用而开发，这取决于研究者的实验设计和资源。这包括使用活细胞染色剂的活细胞成像;固定和切片以表征形态;用抗体和全贴片共聚焦成像进行免疫荧光染色，以避免破坏腔内结构;和明场成像和微光学相干断层扫描，用于定量测量纤毛搏动频率和粘膜纤毛转运13.为了便于扩展到其他研究者，市售试剂和用品被用于培养。开发了一种功能测定法，使用常见的显微镜技术和更专业的设备。总体而言，虽然本模型被设计为在基线或响应于治疗时评估CFTR活性，但该方案中描述的技术可以应用于涉及上皮细胞功能的其他疾病，特别是上皮细胞液运输。
与其他方法的比较 最近，通过将患者类器官的体外CFTR调节剂反应与其临床反应相关联，开发了这种类器官模型的实用性11。值得注意的是，本模型还证明了在相同患者中并行的短路电流响应，这是评估CFTR功能的当前金标准。短路电流不同于溶胀测定，因为前者通过离子输运26测量CFTR功能。相比之下，该测定测量流体运输的下游效应，提供有关CFTR27，28，29，30，31，32的整体功能的额外信息。短路电流测量仍然是确定CFTR氯化物通道活性1，33的常用可靠方法。这些电生理测定需要专门的昂贵设备，每个实验重复需要比类器官测定多许多倍的细胞，不容易自动化，并且不适合扩大规模以适应更高的通量应用。另一种衍生自肠上皮细胞的类器官模型具有15、16、17、18的附加优点，例如更优异的复制能力，但既不是从气道组织衍生的，也不是普遍可用的。HNE刷牙是用廉价的细胞学刷获得的，无需镇静，风险最小。刷牙不需要临床医生，可以由训练有素的研究协调员和其他研究人员进行14.HNE类器官模型可以由任何具有原代细胞培养能力的实验室进行培养，并且某些应用可以使用标准显微镜技术进行。总而言之，这些优势为评估气道上皮功能提供了额外的技术，否则某些实验室可能无法获得这些技术。此外，HNE类器官可用于研究影响气道的其他疾病状态，例如原发性睫状体运动障碍25或病毒感染，而肠道类器官则不能。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Nasal brush</td>
			<td>Medical Packaging CYB1</td>
			<td>CYB-1</td>
			<td>Length: 8 inches, width approximately 7 mm</td>
		</tr>
		<tr>
			<td>Large-Orifice Pipette Tips</td>
			<td>ThermoFisher Scientific</td>
			<td>02-707-141</td>
			<td>Large bore pipette tips</td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>ThermoFisher Scientific</td>
			<td>A1110501</td>
			<td>Cell detachment solution</td>
		</tr>
		<tr>
			<td>0.05% trypsin -EDTA</td>
			<td>Gibco</td>
			<td>25300-054</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin inhibitor from soybean</td>
			<td>Sigma</td>
			<td>T6522</td>
			<td>Working solution: 1mg/mL in 1XDPBS</td>
		</tr>
		<tr>
			<td>Matrigel matrix</td>
			<td>Corning</td>
			<td>356255</td>
			<td>Extracellular matrix (EM)</td>
		</tr>
		<tr>
			<td>&#181;-Slide Angiogenesis</td>
			<td>Ibidi</td>
			<td>81506</td>
			<td>15-well slide</td>
		</tr>
		<tr>
			<td>24-Well Transwell</td>
			<td>Corning</td>
			<td>7200154</td>
			<td>Culture insert</td>
		</tr>
		<tr>
			<td>Chambered Coverglass</td>
			<td>ThermoFisher Scientific</td>
			<td>155409</td>
			<td>8-well glass-bottom chamber slides</td>
		</tr>
		<tr>
			<td>Cell-Tak Cell and Tissue Adhesive</td>
			<td>ThermoFisher Scientific</td>
			<td>354240</td>
			<td>Cell adhesive</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Electron Microscopy Sciences</td>
			<td>50980487</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Alfa Aesar</td>
			<td>A16046</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>ThermoFisher Scientific</td>
			<td>BP1600-100</td>
			<td></td>
		</tr>
		<tr>
			<td>NucBlue</td>
			<td>ThermoFisher Scientific</td>
			<td>R37605</td>
			<td>DAPI</td>
		</tr>
		<tr>
			<td>Eclipse Ts2-FL (Inverted Routine Microscope)</td>
			<td>Nikon</td>
			<td></td>
			<td>Inverted epi-fluorescence microscope or bright-field microscope</td>
		</tr>
		<tr>
			<td>Nikon A1R-HD25</td>
			<td>Nikon</td>
			<td></td>
			<td>Confocal microscope</td>
		</tr>
		<tr>
			<td>NIS Elements- Basic Research</td>
			<td>Nikon</td>
			<td></td>
			<td>manual imaging analysis software</td>
		</tr>
		<tr>
			<td>Histogel</td>
			<td>ThermoFisher Scientific</td>
			<td>HG-4000-012</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Base Molds</td>
			<td>ThermoFisher Scientific</td>
			<td>41-740</td>
			<td></td>
		</tr>
		<tr>
			<td>Lionheart FX</td>
			<td>BioTek</td>
			<td>BTLFX</td>
			<td>Automated image system</td>
		</tr>
		<tr>
			<td>Lionheart Cover</td>
			<td>BioTek</td>
			<td>BT1450009</td>
			<td>Environmental Control Lid</td>
		</tr>
		<tr>
			<td>Humidity Chamber</td>
			<td>BioTek</td>
			<td>BT1450006</td>
			<td>Stage insert (environmental chamber)</td>
		</tr>
		<tr>
			<td>Gas Controller for CO2 and O2</td>
			<td>BioTek</td>
			<td>BT1210013</td>
			<td>Gas controller</td>
		</tr>
		<tr>
			<td>Microplate/Slide Stage Insert</td>
			<td>BioTek</td>
			<td>BT1450527</td>
			<td>Slide holder</td>
		</tr>
		<tr>
			<td>Gen5 Imaging Prime Software</td>
			<td>BioTek</td>
			<td>BTGEN5IPRIM</td>
			<td>Automated imaging analysis software</td>
		</tr>
		<tr>
			<td>4x Phase Contrast Objective</td>
			<td>BioTek</td>
			<td>BT1320515</td>
			<td></td>
		</tr>
		<tr>
			<td>10x Phase Contrast Objective</td>
			<td>BioTek</td>
			<td>BT1320516</td>
			<td></td>
		</tr>
		<tr>
			<td>LED Cube</td>
			<td>BioTek</td>
			<td>BT1225007</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter Cube (DAPI)</td>
			<td>BioTek</td>
			<td>BT1225100</td>
			<td>DAPI</td>
		</tr>
		<tr>
			<td>CFTRinh-172</td>
			<td>Selleck Chemicals</td>
			<td>S7139</td>
			<td></td>
		</tr>
		<tr>
			<td>Forskolin</td>
			<td>Sigma</td>
			<td>F6886</td>
			<td></td>
		</tr>
		<tr>
			<td>IBMX</td>
			<td>Sigma</td>
			<td>I5879</td>
			<td></td>
		</tr>
		<tr>
			<td>Expansion Media</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>ThermoFisher Scientific</td>
			<td>11965</td>
			<td></td>
		</tr>
		<tr>
			<td>F12 Nutrient mix</td>
			<td>ThermoFisher Scientific</td>
			<td>11765</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>ThermoFisher Scientific</td>
			<td>&#160;16140-071</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>ThermoFisher Scientific</td>
			<td>&#160;15-140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Cholera Toxin</td>
			<td>Sigma</td>
			<td>&#160;C8052</td>
			<td></td>
		</tr>
		<tr>
			<td>Epidermal Growth Factor (EGF)</td>
			<td>ThermoFisher Scientific</td>
			<td>&#160;PHG0314</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrocortisone (HC)</td>
			<td>Sigma</td>
			<td>&#160;H0888</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Sigma</td>
			<td>&#160;I9278</td>
			<td></td>
		</tr>
		<tr>
			<td>Adenine</td>
			<td>Sigma</td>
			<td>&#160;A2786</td>
			<td></td>
		</tr>
		<tr>
			<td>Y-27632</td>
			<td>Stemgent</td>
			<td>&#160;04-0012-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic Media</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ceftazidime</td>
			<td>Alfa Aesar</td>
			<td>&#160;J66460-03</td>
			<td></td>
		</tr>
		<tr>
			<td>Tobramycin</td>
			<td>Alfa Aesar</td>
			<td>&#160;J67340</td>
			<td></td>
		</tr>
		<tr>
			<td>Vancomycin</td>
			<td>Alfa Aesar</td>
			<td>&#160;J67251</td>
			<td></td>
		</tr>
		<tr>
			<td>Amphotericin B</td>
			<td>Sigma</td>
			<td>&#160;A2942</td>
			<td></td>
		</tr>
		<tr>
			<td>Differentiation Media</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F-12 (1:1)</td>
			<td>ThermoFisher Scientific</td>
			<td>&#160;11330-32</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultroser-G</td>
			<td>Pall</td>
			<td>&#160;15950-017</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Clone II</td>
			<td>Hyclone</td>
			<td>&#160;SH30066.03</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Brain Extract</td>
			<td>Lonza</td>
			<td>&#160;CC-4098</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Sigma</td>
			<td>&#160;I-9278</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrocortisone</td>
			<td>Sigma</td>
			<td>&#160;H-0888</td>
			<td></td>
		</tr>
		<tr>
			<td>Triiodothyronine</td>
			<td>Sigma</td>
			<td>&#160;T-6397</td>
			<td></td>
		</tr>
		<tr>
			<td>Transferrin</td>
			<td>Sigma</td>
			<td>&#160;T-0665</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanolamine</td>
			<td>Sigma</td>
			<td>&#160;E-0135</td>
			<td></td>
		</tr>
		<tr>
			<td>Epinephrine</td>
			<td>Sigma</td>
			<td>E-4250</td>
			<td></td>
		</tr>
		<tr>
			<td>O-Phosphorylethanolamine</td>
			<td>Sigma</td>
			<td>P-0503</td>
			<td></td>
		</tr>
		<tr>
			<td>Retinoic Acid</td>
			<td>Sigma</td>
			<td>R-2625</td>
			<td></td>
		</tr>
		<tr>
			<td>Primary antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Human CFTR antibody</td>
			<td>R&#38;D Systems</td>
			<td>MAB1660</td>
			<td>Dilution: 100x</td>
		</tr>
		<tr>
			<td>ZO-1 antibody</td>
			<td>Thermo Fisher</td>
			<td>MA3-39100-A647</td>
			<td>Dilution: 1000x</td>
		</tr>
		<tr>
			<td>Anti-MUC5B antibody</td>
			<td>Sigma</td>
			<td>HPA008246</td>
			<td>Dilution: 100x</td>
		</tr>
		<tr>
			<td>Anti-acetylated tubulin</td>
			<td>Sigma</td>
			<td>T7451</td>
			<td>Dilution: 100x</td>
		</tr>
		<tr>
			<td>Anti-beta IV Tubulin antibody</td>
			<td>Abcam</td>
			<td>Ab11315</td>
			<td>Dilution: 100x</td>
		</tr>
		<tr>
			<td>Secondary antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey anti-Mouse IgG (H+L), Alexa Fluor 488</td>
			<td>Invitrogen</td>
			<td>A21202</td>
			<td>Dilution: 2000x</td>
		</tr>
		<tr>
			<td>Donkey anti-Rabbit IgG (H+L), Alexa Fluor 594</td>
			<td>Invitrogen</td>
			<td>A21207</td>
			<td>Dilution: 2000x</td>
		</tr>
	</tbody>
</table>

culture and imaging of human nasal epithelial organoids

囊性纤维化（CF）患者的个体化治疗可以通过 体外 疾病模型来实现，以了解基线囊性纤维化跨膜电导调节剂（CFTR）活性和小分子化合物的恢复。我们小组最近专注于建立一种直接来自原代人类鼻上皮细胞（HNE）的良好分化类器官模型。切片类器官的组织学、全装载免疫荧光染色和成像（使用共聚焦显微镜、免疫荧光显微镜和明场）对于表征类器官和确认上皮分化以准备功能测定至关重要。此外，HNE类器官产生与CFTR活性相关的不同大小的管腔，区分CF和非CF类器官。在本手稿中，详细描述了培养HNE类器官的方法，重点是使用成像方式评估分化，包括基线管腔面积的测量（任何具有显微镜的实验室都可以使用的类器官CFTR活性测量方法）以及开发的功能测定的自动化方法（需要更专业的设备）。

高净值物质的扩张对于有机物文化的蓬勃发展至关重要。成功采集样本的HNE应在10天左右扩大到超过70%的汇合度。成功和不成功样本的示例分别如图1A和图1B所示。如果细胞在与辐照的3T3细胞共培养14天后不能达到70%汇合，则必须丢弃这些细胞。如果无法用其他抗菌剂快速抢救，任何受污染的细胞都应立即丢弃。
在15孔?...

Watch this Scientific Journal Video about Culture and Imaging of Human Nasal Epithelial Organoids at JoVE.com

Culture and Imaging of Human Nasal Epithelial Organoids

这里提出了一个详细的方案来描述来自人类鼻上皮细胞的 体外 类器官模型。该协议具有需要标准实验室设备的测量选项，以及用于专用设备和软件的其他可能性。

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 ...

囊性纤维化患者的个体化治疗可以通过体外疾病模型来实现，以了解基线时的CFTR活性。人鼻上皮类器官或HNE类器官产生与CFTR活性相关的不同大小的管腔，区分CF和非CF类器官。在这里，我们详细描述了一种培养和成像这些HNE类器官的方法。功能失调的上皮离子转运，特别是氯离子和碳酸氢盐的上皮离子转运，导致上皮衬里液体积减少。这也会影响导致粘液淤积和梗阻的粘液分泌物。因此，我们的HNE模型已经针对各种应用进行了开发，具体取决于研究者的实验设计和资源。除了在基线或响应治疗时评估CFTR活性外，该技术还可以应用于涉及上皮细胞功能的其他疾病，特别是上皮细胞液运输。这些方法主要用于囊性纤维化研究。其他评估气道上皮的研究，如原发性睫状体运动障碍，也可能发现这些方法有帮助。这些技术需要注意细节和精度。它们还需要仔细观察，以确保细胞的初始扩增是适当的。每天监控所有文化，成功的可能性将更大。首先，通过将细胞学刷通过1毫升大口径移液器尖端几次，在15毫升锥形管中将鼻刷活检解离到8毫升RPMI培养基中。将细胞以500倍g在四摄氏度下离心五分钟。弃去上清液，然后将细胞沉淀重新悬浮在三毫升的细胞分离溶液中。在室温下孵育16分钟以消化。使用五毫升的扩增培养基将细胞洗涤两次。然后，将它们加入T75烧瓶中，预先播种有辐照，灭活和50%至60%融合的3T3成纤维细胞。让细胞在扩增培养基中共培养和扩增7至14天。如果对囊性纤维化患者的细胞引入抗生素，应在培养的最初三天后用无抗生素扩增培养基替换培养基，并可继续每两天更换培养基，直至 80% 汇合。用1x DPBS洗涤细胞。然后，向T75烧瓶中加入1.5毫升0.05%胰蛋白酶-EDTA，并在37摄氏度下孵育4分钟，以从培养物中除去照射和灭活的3T3成纤维细胞。用1x DPBS冲洗T75烧瓶两次，以彻底洗掉任何剩余的3T3成纤维细胞。将1.5毫升0.05%胰蛋白酶-EDTA加入T75烧瓶中，并在37摄氏度下孵育10分钟以分离HNE。用大豆胰蛋白酶抑制剂以一比一的比例中和胰蛋白酶。将细胞以500倍gi离心五分钟。然后，弃去上清液并用五毫升扩增培养基洗涤细胞一次。这些细胞现在已经准备好接种以生长类器官。将类器官培养物细胞外基质或ECM在四摄氏度下解冻过夜。在相同温度下冷却15孔血管生成载玻片过夜。接下来，将移液器吸头冷却至四摄氏度。在冰上涂上五微升冷的100%ECM。将它们放入37摄氏度的细胞培养箱中至少30分钟以进行巩固。使用血细胞计数器计算从共培养中收获的HNE。然后，将细胞稀释至每微升500个细胞，用20%的ECM在冰上用分化培养基稀释。将 ECM 包衣载玻片从培养箱中取出，将五微升冷池 ECM 混合物接种到每个孔中。立即将载玻片转移到37摄氏度的培养箱中一小时，以巩固细胞ECM混合物。之后，将载玻片从培养箱中取出，并用50微升分化培养基将细胞送入15孔血管生成载玻片的每个孔中。将载玻片放回37摄氏度的培养箱中，每隔一天更换培养基，直到类器官准备好进行进一步的实验。用细胞粘合剂预处理8孔玻璃底腔载玻片30分钟。丢弃溶液，将孔再风干30分钟。接下来，从ECM顶部丢弃培养基，然后将50微升的冷1x PBS加入冰上15孔载玻片的每个孔中。使用200微升的大口径移液器吸头上下移液三到五次。将溶液分配到8孔载玻片孔的中心。通过细吸管立即从孔中除去多余的液体。然后，将腔室载玻片放入37摄氏度的培养箱中40分钟，以增强粘附在玻璃底部的类器官。40分钟后，用1x PBS轻轻洗涤类器官两次，并用300微升4%多聚甲醛在每个孔中固定30分钟，设定室温。用1x PBS洗涤两次，并将类器官储存在1x PBS设定的4摄氏度中，以进行免疫染色长达两周。为了收获用于组织学研究的类器官，从培养物中取出培养基，并在冰上的每个载玻片孔中加入50微升的1x PBS。使用200微升大口径移液器吸头上下移液三到五次。将15孔载玻片或培养插件中的所有溶液合并到冰上的15毫升锥形管中。通过加入冷的1x PBS将管中的总溶液体积调节至10毫升。将管子在四摄氏度下离心，300倍g，持续五分钟。然后，吸出上清液并加入60微升温组织凝胶，使用200微升大口径移液器吸头与类器官沉淀混合。立即将悬浮液转移到组织学模具中。在室温下固结组织凝胶后，将模具块放入4%多聚甲醛中，在4摄氏度下固定过夜。打开软件，然后右键单击屏幕底部。然后，选择“分析控件”，然后选择“自动测量结果”。打开类器官图像，选择5至10个具有可见管腔的类器官。右键单击图像以打开菜单，然后选择多边形ROI以勾勒出完整的类器官，以获得类器官的总表面积。然后，勾勒出流明以获得流明面积。对孔中剩余的类器官以及测定中的所有孔重复此操作。最后，将数据导出到 Excel。将管腔面积除以总表面积，并平均样品中的所有类器官，以获得基线流明比。明场图像代表成功和不成功样本收集的HNE。HNEs在一个大集群中增长良好。相比之下，HNE在照射的3T3细胞周围的两个小簇中生长不良。15孔载玻片中的类器官比培养插入物中的类器官具有更精确，更清晰的图像。除此之外，在这两种培养方法中没有观察到显着的形态学差异。非CF类器官通常比CF类器官具有更大的管腔和更多的液体。这些图像显示了来自非CF，F508del / F508del受试者的类器官中的HNE染色以及类器官中纤毛的免疫荧光染色。这里显示了用乙酰化微管蛋白染色的纤毛，FITC标记的二抗和用DAPI标记的细胞核。通过全贴片免疫荧光染色对两种代表性类器官的最大投影图像及其相应的三维重建图像如下所示。显示类器官腔内的粘液和纤毛。图形图像代表毛喉素诱导的肿胀测定，以测试原发性鼻上皮细胞上的CFTR功能。这里显示了非CF志愿者的具有代表性的毛喉素剂量反应实验。将 FSK 剂量反应与 1 小时时的平均分数变化进行比较，而 8 小时时，这表明 8 小时测定在不同 FSK 剂量之间产生的肿胀差异比 1 小时时的肿胀差异更显著。与八小时时的平均分数变化相比，曲线下方的区域可以显示肿胀的微小差异。在尝试此程序时，请记住，在收集，固定和染色过程中会丢失类器官。因此，在每个步骤中都必须小心谨慎，并且必须获得足够的起始数字以确保成功。随着这些技术的发展，在培养物插入物中生长类器官可以在这方面有所帮助。在这里，我们使用市售的试剂和耗材，以促进扩展到其他研究人员。还开发了一种功能测定法，使用常见的显微镜技术和更专业的设备。

Research

JoVE Journal

Medicine

Isolation, Culture, and Imaging of Human Fetal Pancreatic Cell Clusters

分离，培养和成像胎儿胰腺细胞集群

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning ...

科研

医学

Primary Culture of Human Vestibular Schwannomas

人体前庭神经鞘瘤的原代培养

Vestibular schwannomas (VSs) represent Schwann cell (SC) tumors of the vestibular nerve, compromising 10% of all intracranial neoplasms. VSs occur in ...

Isolation, Cryopreservation and Culture of Human Amnion Epithelial Cells for Clinical Applications

分离，冻存和文化的人羊膜上皮细胞的临床应用

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a ...

Establishment of Human Epithelial Enteroids and Colonoids from Whole Tissue and Biopsy

从整体组织和活检建立人类上皮Enteroids和Colonoids的

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem ...

Absorption of Nasal and Bronchial Fluids: Precision Sampling of the Human Respiratory Mucosa and Laboratory Processing of Samples

鼻腔和支气管液的吸收: 人体呼吸道黏膜的精确取样和样品的实验室处理

The methods of nasal absorption (NA) and bronchial absorption (BA) use synthetic absorptive matrices (SAM) to absorb the mucosal lining fluid (MLF) of the ...

Standardized Measurement of Nasal Membrane Transepithelial Potential Difference (NPD)

Standardized Measurement of Nasal Membrane Transepithelial Potential Difference NPD

鼻膜 Transepithelial 电位差的标准化测量 (NPD)

We describe a standardized measurement of nasal potential difference (NPD). In this technique, cystic fibrosis transmembrane conductance regulator (CFTR) ...

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

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 ...

Muscle Imbalances: Testing and Training Functional Eccentric Hamstring Strength in Athletic Populations

肌肉失衡: 运动人群功能性偏心肌筋强度的检测与训练

Many hamstring injuries that occur during physical activity occur while the muscles are lengthening, during eccentric hamstring muscle actions. Opposite ...

Quantitative Mapping of Specific Ventilation in the Human Lung using Proton Magnetic Resonance Imaging and Oxygen as a Contrast Agent

以质子磁共振成像和氧为造影剂对人肺特异性通气进行定量定位

Specific ventilation imaging (SVI) is a functional magnetic resonance imaging technique capable of quantifying specific ventilation &#8213; the ratio of ...