人鼻上皮类器官的培养和成像
Summary
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。
Abstract
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）。
Introduction
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。
Protocol
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…
Representative Results
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?…
Discussion
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，?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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）。
Materials
| Nasal brush | Medical Packaging CYB1 | CYB-1 | Length: 8 inches, width approximately 7 mm |
| Large-Orifice Pipette Tips | ThermoFisher Scientific | 02-707-141 | Large bore pipette tips |
| Accutase | ThermoFisher Scientific | A1110501 | Cell detachment solution |
| 0.05% trypsin -EDTA | Gibco | 25300-054 | |
| Trypsin inhibitor from soybean | Sigma | T6522 | Working solution: 1mg/mL in 1XDPBS |
| Matrigel matrix | Corning | 356255 | Extracellular matrix (EM) |
| µ-Slide Angiogenesis | Ibidi | 81506 | 15-well slide |
| 24-Well Transwell | Corning | 7200154 | Culture insert |
| Chambered Coverglass | ThermoFisher Scientific | 155409 | 8-well glass-bottom chamber slides |
| Cell-Tak Cell and Tissue Adhesive | ThermoFisher Scientific | 354240 | Cell adhesive |
| Paraformaldehyde | Electron Microscopy Sciences | 50980487 | |
| Triton X-100 | Alfa Aesar | A16046 | |
| BSA | ThermoFisher Scientific | BP1600-100 | |
| NucBlue | ThermoFisher Scientific | R37605 | DAPI |
| Eclipse Ts2-FL (Inverted Routine Microscope) | Nikon | Inverted epi-fluorescence microscope or bright-field microscope | |
| Nikon A1R-HD25 | Nikon | Confocal microscope | |
| NIS Elements- Basic Research | Nikon | manual imaging analysis software | |
| Histogel | ThermoFisher Scientific | HG-4000-012 | |
| Disposable Base Molds | ThermoFisher Scientific | 41-740 | |
| Lionheart FX | BioTek | BTLFX | Automated image system |
| Lionheart Cover | BioTek | BT1450009 | Environmental Control Lid |
| Humidity Chamber | BioTek | BT1450006 | Stage insert (environmental chamber) |
| Gas Controller for CO2 and O2 | BioTek | BT1210013 | Gas controller |
| Microplate/Slide Stage Insert | BioTek | BT1450527 | Slide holder |
| Gen5 Imaging Prime Software | BioTek | BTGEN5IPRIM | Automated imaging analysis software |
| 4x Phase Contrast Objective | BioTek | BT1320515 | |
| 10x Phase Contrast Objective | BioTek | BT1320516 | |
| LED Cube | BioTek | BT1225007 | |
| Filter Cube (DAPI) | BioTek | BT1225100 | DAPI |
| CFTRinh-172 | Selleck Chemicals | S7139 | |
| Forskolin | Sigma | F6886 | |
| IBMX | Sigma | I5879 | |
| Expansion Media | |||
| DMEM | ThermoFisher Scientific | 11965 | |
| F12 Nutrient mix | ThermoFisher Scientific | 11765 | |
| Fetal Bovine Serum | ThermoFisher Scientific | 16140-071 | |
| Penicillin/Streptomycin | ThermoFisher Scientific | 15-140-122 | |
| Cholera Toxin | Sigma | C8052 | |
| Epidermal Growth Factor (EGF) | ThermoFisher Scientific | PHG0314 | |
| Hydrocortisone (HC) | Sigma | H0888 | |
| Insulin | Sigma | I9278 | |
| Adenine | Sigma | A2786 | |
| Y-27632 | Stemgent | 04-0012-02 | |
| Antibiotic Media | |||
| Ceftazidime | Alfa Aesar | J66460-03 | |
| Tobramycin | Alfa Aesar | J67340 | |
| Vancomycin | Alfa Aesar | J67251 | |
| Amphotericin B | Sigma | A2942 | |
| Differentiation Media | |||
| DMEM/F-12 (1:1) | ThermoFisher Scientific | 11330-32 | |
| Ultroser-G | Pall | 15950-017 | |
| Fetal Clone II | Hyclone | SH30066.03 | |
| Bovine Brain Extract | Lonza | CC-4098 | |
| Insulin | Sigma | I-9278 | |
| Hydrocortisone | Sigma | H-0888 | |
| Triiodothyronine | Sigma | T-6397 | |
| Transferrin | Sigma | T-0665 | |
| Ethanolamine | Sigma | E-0135 | |
| Epinephrine | Sigma | E-4250 | |
| O-Phosphorylethanolamine | Sigma | P-0503 | |
| Retinoic Acid | Sigma | R-2625 | |
| Primary antibodies | |||
| Human CFTR antibody | R&D Systems | MAB1660 | Dilution: 100x |
| ZO-1 antibody | Thermo Fisher | MA3-39100-A647 | Dilution: 1000x |
| Anti-MUC5B antibody | Sigma | HPA008246 | Dilution: 100x |
| Anti-acetylated tubulin | Sigma | T7451 | Dilution: 100x |
| Anti-beta IV Tubulin antibody | Abcam | Ab11315 | Dilution: 100x |
| Secondary antibodies | |||
| Donkey anti-Mouse IgG (H+L), Alexa Fluor 488 | Invitrogen | A21202 | Dilution: 2000x |
| Donkey anti-Rabbit IgG (H+L), Alexa Fluor 594 | Invitrogen | A21207 | Dilution: 2000x |
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Cite This Article
Liu, Z., Anderson, J. D., Natt, J., Guimbellot, J. S. Culture and Imaging of Human Nasal Epithelial Organoids. J. Vis. Exp. (178), e63064, doi:10.3791/63064 (2021).