小鼠结肠隐窝三维培养离体研究肠道干细胞功能
Summary
本协议描述了建立鼠结肠类器官系统以研究claudin-7敲除模型中结肠干细胞的活性和功能。
Abstract
肠上皮每5-7天再生一次，并由位于隐窝区域底部的肠上皮干细胞（IESC）群控制。IESC包括活性干细胞，其自我更新并分化为各种上皮细胞类型，以及静止干细胞，在受伤的情况下作为储备干细胞。肠上皮的再生由这些活性IESC的自我更新和分化能力控制。此外，隐窝干细胞群的平衡和干细胞生态位的维持对于肠道再生至关重要。类器官培养是研究调节干细胞存活和功能的蛋白质、信号分子和环境线索的一种重要且有吸引力的方法。该模型比动物模型更便宜，耗时更少，并且更易于操作。类器官还模仿组织微环境，提供 体内 相关性。本协议描述了结肠隐窝的分离，将这些分离的隐窝细胞嵌入到三维凝胶基质系统中并培养隐窝细胞以形成能够自组织，增殖，自我更新和分化的结肠类器官。该模型允许人们操纵环境 – 敲除特定的蛋白质，如claudin-7，激活/停用信号通路等 – 研究这些效应如何影响结肠干细胞的功能。具体而言，研究了紧密连接蛋白claudin-7在结肠干细胞功能中的作用。Claudin-7对于维持肠道稳态和屏障功能和完整性至关重要。在小鼠中敲除claudin-7诱导炎症性肠病样表型，表现出肠道炎症，上皮增生，体重减轻，粘膜溃疡，上皮细胞脱落和腺瘤。此前，据报道，claudin-7是小肠肠上皮干细胞功能所必需的。在该协议中，建立了结肠类器官培养系统以研究claudin-7在大肠中的作用。
Introduction
肠道类器官培养是一种三维（3D）离体系统，其中干细胞从原代组织的肠隐窝中分离并接种到凝胶基质中1，2。这些干细胞能够自我更新，自我组织和器官功能2。类器官模拟组织微环境，与二维（2D）体外细胞培养模型更类似于体内模型，尽管可操作性不如细胞3，4。该模型消除了2D模型中遇到的障碍，例如缺乏适当的细胞-细胞粘附，细胞-基质相互作用和同质群体，并且还减少了动物模型的局限性，包括高成本和长时间5。肠道类器官 – 也称为结肠类器官，用于从结肠隐窝来源的干细胞生长的类结肠 – 本质上是包含上皮的微型器官，包括体内存在的所有细胞类型以及腔。该模型允许操纵系统来研究肠道的许多方面，例如干细胞生态位、肠道生理学、病理生理学和肠道形态发生3，5，6。它还为药物发现、研究炎症性肠病 （IBD） 和结直肠癌等人类肠道疾病、患者特异性个性化治疗开发以及研究组织再生4，7，8，9 提供了一个很好的模型。此外，类器官系统还可用于研究细胞通讯、药物代谢、活力、增殖和对刺激的反应7，8。虽然动物模型可用于测试肠道病理状况的潜在治疗方法，但它们非常有限，因为同时研究多种药物是一项挑战。体内混杂变量较多，相关成本和时间分别高而长。另一方面，类器官培养系统允许在较短的时间内一次筛选多种治疗方法，并且还允许通过使用患者来源的类器官培养物进行个性化治疗4，8。结肠类器官模仿组织组织、微环境和功能的能力也使它们成为研究再生和组织修复的优秀模型9。本实验室建立了小肠类器官培养系统，研究claudin-7对小肠干细胞功能的影响10。在这项研究中，建立了一个大型肠道类器官培养系统来研究干细胞在条件claudin-7敲除（cKO）模型中自我更新，分化和增殖的能力或缺乏能力。
Claudin-7是一种非常重要的紧密连接（TJ）蛋白，在肠道中高度表达，对于维持TJ功能和完整性至关重要11。cKO小鼠患有IBD样表型，表现出严重的炎症，溃疡，上皮细胞脱落，腺瘤和细胞因子水平升高11，12。虽然人们普遍认为克劳迪斯对上皮屏障功能至关重要，但克劳迪斯的新角色正在出现;它们参与增殖、迁移、癌症进展和干细胞功能10，12，13，14，15，16，17。目前尚不清楚claudin-7如何影响结肠干细胞的干细胞生态位和功能。由于肠道大约每5-7天快速自我更新一次，因此维持干细胞生态位和活性干细胞的正常功能至关重要18。在这里，建立了一个系统来检查claudin-7对结肠干细胞生态位的潜在调节作用。
Protocol
Representative Results
Discussion
类器官培养是研究干细胞功能、肠道生理学、药物发现、人类肠道疾病以及组织再生和修复的优秀模型7，8，9，10，11，26。虽然它有很多优点，但建立起来可能具有挑战性。在整个协议的所有步骤中都必须小心，但最重要的是在电镀阶段。将分离的…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项研究由NIH DK103166资助。
Materials
| 0.09 cubic feet space-saver vacuum desiccator | United States Plastic Corp | 78564 | anesthesia chamber |
| 0.5 M EDTA pH 8.0 | Invitrogen | AM9261 | |
| 1.5 mL microcentrifuge tubes | ThermoFisher | 69715 | |
| 15 mL conical centrifuge tubes | Fisher Scientific | 14-959-53A | |
| 1x Dulbecco’s Phosphate buffered saline | Gibco | 14190-144 | |
| 2-methylbutane | Sigma | 277258 | |
| 4% paraformaldehyde | ThermoFisher | J61899.AK | |
| 4-hydroxytamoxifen (4OH-TAM) | Sigma | 579002 | |
| 50 mL conical centrifuge tubes | Fisher Scientific | 14-432-22 | |
| 70 µm nylon cell strainer | Corning | 352350 | |
| 96 well culture plate | Greiner Bio-One | 655180 | |
| B-27 Supplement (50x) | Gibco | 12587-010 | |
| Bovine serum albumin | Fisher Scientific | BP1605-100 | |
| Claudin-7 anti-murine rabbit antibody | Immuno-Biological Laboratories | 18875 | |
| Cover glass (24 x 50-1.5) | Fisher Scientific | 12544E | |
| Cryomolds | vwr | 25608-916 | |
| Cultrex RCF BME, Type 2 | R&D Systems | 3533-005-02 | gel matrix |
| Cy3 anti-rabbit antibody | Jackson Immunoresearch | 111-165-003 | |
| Dewar Flask | Thomas Scientific | 1173F61 | |
| DMEM High Glucose with L-Glutamine | ATCC | 30-2002 | |
| EVOS FLoid Imaging System | ThermoFisher | 4477136 | |
| Fluoro-Gel II with DAPI | Electron Microscopy Sciences | 17985-50 | |
| GlutaMAX (100x) | Gibco | 35050-061 | |
| Glycine | JT Baker | 4059-02 | |
| HEPES (1 M) Buffer Solution | Gibco | 15630-080 | |
| Hoechst | ThermoFisher | 62249 | |
| In situ cell death detection kit, TMR Red | Roche | 12156792910 | |
| Isoflurane | Pivetal | 07-893-8440 | |
| L-WRN Media | Harvard Medical School Gastrointestinal Organoid Derivation and Culture Core | N/A | |
| Mouse surgical kit | Kent Scientific Corporation | INSMOUSEKIT | |
| Murine EGF | PeproTech | 315-09-500UG | |
| N2 Supplement (100x) | Gibco | 17502-048 | |
| Optimum cutting temperature (OCT) compound | Agar Scientific | AGR1180 | |
| Penicillin-Streptomycin | Gibco | 15140-122 | |
| Sequenza Rack | vwr | 10129-584 | |
| Sodium Citrate | Fisher Scientific | S-279 | |
| Sucrose | Sigma | S9378 | |
| Triton X-100 | Sigma | X100 | |
| Vacuum filter (0.22 µm; cellulose acetate) | Corning | 430769 | |
| Y-27632 dihydrochloride | Tocris Bioscience | 1254 |
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