研究体 外 肠道炎症对已建立的小鼠结肠样的上皮影响
Summary
我们描述了一个协议，详细说明了小鼠结肠隐窝的分离以开发三维结肠。然后，可以在接受炎症激发或被引导建立上皮单层之前对已建立的结肠样蛋白进行终末分化以反映宿主上皮的细胞组成。
Abstract
肠上皮在人体健康中起着至关重要的作用，在宿主和外部环境之间提供了屏障。这种高度动态的细胞层提供了微生物和免疫群体之间的第一道防线，并有助于调节肠道免疫反应。上皮屏障的破坏是炎症性肠病（IBD）的标志，对于治疗靶向具有重要意义。三维结肠培养系统是研究IBD发病机制中肠道干细胞动力学和上皮细胞生理学的非常有用的 体外 模型。理想情况下，从动物发炎的上皮组织中建立结肠样体对于评估遗传和分子对疾病的影响最有益。然而，我们已经表明， 体内 上皮变化不一定保留在由急性炎症小鼠建立的结肠样中。为了解决这一限制，我们开发了一种方案，用通常在IBD期间升高的炎症介质混合物来治疗结肠样。虽然该系统可以普遍应用于各种培养条件，但该方案强调对分化结肠和源自已建立的结肠的二维单层的治疗。在传统培养环境中，结肠富含肠道干细胞，为研究干细胞生态位提供了理想的环境。然而，该系统不允许分析肠道生理学的特征，例如屏障功能。此外，传统的结肠样不能提供研究终末分化上皮细胞对促炎刺激的细胞反应的机会。这里介绍的方法提供了一个替代的实验框架来解决这些限制。二维单层培养系统也为 离体治疗药物筛选提供了机会。这种极化细胞层可以用细胞基底侧的炎症介质处理，并伴有假定的治疗方法，以确定它们在IBD治疗中的效用。
Introduction
炎症性肠病 （IBD） 是一种慢性、缓解和复发性疾病，其特征是炎症发作和临床静止。IBD 的病因是多因素的，但该疾病的关键特征包括屏障功能缺陷和肠上皮通透性增加，此外还有上皮区间内激活的促炎信号级联反应 1,2。几种体外和体内模型已被用于概括IBD期间的上皮反应，包括炎症的细胞培养和小鼠模型3。然而，所有这些系统都有重要的缺点，限制了它们在IBD4期间概括上皮变化的能力。大多数用于研究IBD的细胞系都是转化的，具有形成单层的能力，并且可以分化3种，但本质上的繁殖方式与宿主中未转化的肠上皮细胞不同。几种不同的小鼠炎症模型用于研究IBD，其中一些包括敲除模型，感染模型，化学炎症模型和T细胞转移模型5,6,7,8。虽然每个人都可以研究IBD的某些病因方面，例如遗传易感性，屏障功能障碍，免疫失调和微生物组，但他们研究疾病的多因素性质的能力有限。
肠道类器官，包括类肠类和结肠类，在过去十年中已被建立为一种有用的体外模型，不仅可以研究肠道干细胞的动力学，还可以研究它们在肠道稳态和疾病中的屏障完整性和功能的作用。这些实体为我们理解IBD9的发病机制做出了重大贡献，并为个性化医疗开辟了新的机会。结肠样或干细胞衍生的自组织组织培养物已经从小鼠和人体组织发展而来，其过程允许位于肠隐窝内的干细胞繁殖并无限期维持10。体内干细胞生态位依赖于细胞外因子来支持其生长，特别是典型的Wnt信号传导和骨形态发生蛋白信号通路11。这些因素的添加促进了结肠样细胞的健康和寿命，但也推动培养物走向干细胞样状态，这种状态不反映体内上皮细胞结构，该结构由自我更新和终末分化的细胞组成12,13。虽然肠上皮的功能取决于干细胞区室和分化细胞之间的持续串扰，但在结肠样培养系统中同时具有两者的能力相当有限。尽管存在这些限制，类器官培养系统仍然是研究体外上皮内在特性的金标准。尽管如此，可能需要考虑替代文化策略来回答手头的科学问题。
已经表明，连续7天葡聚糖硫酸钠（DSS）方案的小鼠发生上皮炎症和屏障功能障碍14。此外，线粒体生物发生失败和肠上皮内的代谢重编程，已被证明在人类IBD中很明显，也已捕获在结肠炎15的DSS模型中。然而，我们的初步数据表明，线粒体生物发生失败的特征并未保留在源自DSS处理动物隐窝的结肠中（补充图1）。因此，在检查小鼠肠道炎症期间炎症如何驱动上皮变化时，必须使用替代培养方法。在这里，我们概述了我们开发的一个协议，该协议描述了1）如何从整个结肠组织中分离隐窝以建立鼠结肠样，2）如何最终分化该细胞群以反映 体内的细胞群，以及3）如何在此 体外 模型中诱导炎症。为了研究发炎上皮内的药物相互作用，我们开发了一种方案，从鼠结肠样体中建立2-二脉（2D）单层，可以用炎症介质进行基础治疗，并用药物治疗进行顶端治疗。
Protocol
本文描述的所有使用小鼠组织的实验均已获得匹兹堡大学机构审查委员会的批准，并根据匹兹堡大学动物研究和护理委员会和 UPMC 制定的指导方针进行。 1. 培养准备 注意：所有试剂都列在 材料 表部分，所有溶液组成都可以在溶液成分表中找到（表1）。 如 表1所述制备小鼠洗涤培养基。在 4°C 下…
Representative Results
3D肠结肠样培养系统是研究上皮对肠粘膜稳态的内在贡献的宝贵工具。所描述的协议提供了有关如何在8周龄时从C57BL / 6J（WT）小鼠中分离隐窝并建立可用于多个下游应用的长期结肠样培养系统的详细说明。在基底膜基质中分离和电镀隐窝后，当通过明场显微镜观察时，隐窝在结构上显得致密和多细胞。它们可以是球形的，也可以是细长的圆柱形，类似于通常在 体内 肠道结构中观察到的单?…
Discussion
类器官的发展彻底改变了科学界在 体外 研究器官系统的方式，能够在培养皿中部分概括动物或人类的细胞结构和功能。此外，源自患有疾病的人类的类器官系统为个性化医疗提供了一种有前途的工具，可以指导治疗决策。在这里，我们描述了一种运行良好的加密隔离协议，并介绍了允许在电镀前清理隔离中多余碎屑的关键步骤。此外，我们还解释了一个在这个过程中经常被忽视的关键细?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了美国国立卫生研究院拨款R01DK120986（KPM）的支持。
Materials
| 0.4-μM transparent transwell, 24-well | Greiner Bio-one | 662-641 | |
| 15-mL conical tubes | Thermo Fisher | 12-565-269 | |
| 50-mL conical tubes | Thermo Fisher | 12-565-271 | |
| 70-μM cell strainer | VWR | 76327-100 | |
| Advanced DMEM/F12 | Invitrogen | 12634-010 | Stock Concentration (1x); Final Concentration (1x) |
| B-27 supplement | Invitrogen | 12587-010 | Stock Concentration (50x); Final Concentration (1x) |
| Chopsticks Electrode Set for EVO | World Precision Instruments | STX2 | |
| Corning Matrigel GFR Membrane Mix | Corning | 354-230 | Stock Concentration (100%); Final Concentration (100%) |
| Dithiothreitol (DTT) | Sigma-Aldrich | D0632-5G | Stock Concentration (1 M); Final Concentration (1.5 mM); Solvent (ultrapure water) |
| DMEM high glucose | Thermo Fisher | 11960-069 | Stock Concentration (1x); Final Concentration (1x) |
| Dulbecco's phosphate-buffered saline without Calcium and Magnesium | Gibco | 14190-144 | Stock Concentration (1x); Final Concentration (1x) |
| Ethylenediaminetetraacetic acid (ETDA) | Sigma-Aldrich | E7889 | Stock Concentration (0.5 M); Final Concentration (30 mM) |
| Fetal Bovine Serum | Bio-Techne | S11150H | Stock Concentration (100%); Final Concentration (1%) |
| Fisherbrand Superfrost Plus Microscope Slides, White, 25 x 75 mm | Thermo Fisher | 12-550-15 | |
| G418 | InvivoGen | ant-ga-1 | Final Concentration (400 µg/µL) |
| Gentamicin Reagent | Gibco/Fisher | 15750-060 | Stock Concentration (50 mg/mL); Final Concentration (250 μg/mL) |
| GlutaMAX-1 | Fisher Scientific | 35050-061 | Stock Concentration (100x); Final Concentration (1x) |
| HEPES 1 M | Gibco | 15630-080 | Stock Concentration (1 M); Final Concentration (10 mM) |
| hIFNγ | R&D Systems | 285-IF | Stock Concentration (1000 ng/µL); Final Concentration (10 ng/mL); Solvent (ultrapure water) |
| hIL-1β | R&D Systems | 201-LB | Stock Concentration (10 ng/µL); Final Concentration (20 ng/mL); Solvent (ultrapure water) |
| hTNFα | R&D Systems | 210-TA | Stock Concentration (10 ng/µL); Final Concentration (40 ng/mL); Solvent (ultrapure water) |
| Hydrogen Peroxide | Sigma | H1009 | Stock Concentration (30%); Final Concentration (0.003%); Solvent (Mouse wash media) |
| Hygromycin B Gold | InvivoGen | ant-hg-1 | Final Concentration (400 µg/µL) |
| L-WRN Cell Line | ATCC | CRL-3276 | |
| mEGF | Novus | NBP2-35176 | Stock Concentration (0.5 µg/µL); Final Concentration (50 ng/mL); Solvent (D-PBS + 1% BSA) |
| N-2 supplement | Invitrogen | 17502-048 | Stock Concentration (100x); Final Concentration (1x) |
| N-Acetyl-L-cysteine | Sigma | A9165-5G | Stock Concentration (500 mM); Final Concentration (1 mM); Solvent (ultrapure water) |
| Noggin | Peprotech | 250-38 | Stock Concentration (0.1 ng/µL); Final Concentration (100 ng/mL); Solvent (UltraPure water + 0.1% BSA) |
| Penicillin-Streptomycin (10,000 U/mL) | Thermo Fisher | 15140-122 | Stock Concentration (100x); Final Concentration (1x) |
| Petri dishes (sterilized; 100 mm x 15 mm) Polystrene disposable | VWR | 25384-342 | |
| Polystyrene Microplates, 24 well tissue culture treated, sterile | Greiner Bio-one | 5666-2160 | |
| R-Spondin | R&D Systems | 3474-RS-050 | Stock Concentration (0.25 µg/µL); Final Concentration (500 ng/mL); Solvent (D-PBS + 1% BSA) |
| Tryp LE Express | Thermo Fisher | 12604-013 | Stock Concentration (10x); Final Concentration (1x); Solvent (1 mM EDTA) |
| UltraPure Water | Invitrogen | 10977-023 | Stock Concentration (1x); Final Concentration (1x) |
| Y-27632 dihyddrochloride | Abcam | ab120129 | Stock Concentration (10 mM); Final Concentration (10 µM); Solvent (UltraPure Water) |
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Cite This Article
Crawford, E., Mentrup, H. L., Novak, E. A., Mollen, K. P. Studying the Epithelial Effects of Intestinal Inflammation In Vitro on Established Murine Colonoids. J. Vis. Exp. (196), e64804, doi:10.3791/64804 (2023).