小鼠小肠上皮类器官与先天性淋巴细胞的共培养
Summary
该协议为建立小鼠小肠类器官，从小鼠小肠固有层中分离1型先天淋巴细胞，以及在两种细胞类型之间建立3维（3D）共培养物以研究肠上皮细胞和1型先天淋巴细胞之间的双向相互作用提供了详细的说明。
Abstract
类器官与免疫细胞的复杂共培养物为询问支持粘膜稳态微妙平衡的双向相互作用提供了一种多功能工具。这些3D，多细胞系统提供了一种还原论模型，用于解决多因素疾病并解决在研究组织驻留先天淋巴细胞（ILC）等稀有细胞类型时出现的技术难题。本文介绍了一种小鼠系统，该系统结合了小肠类器官和小肠固有层衍生的辅助性1型ILCs（ILC1s），可以很容易地扩展到其他ILC或免疫人群。ILCs是一种组织居民群体，在粘膜中特别丰富，在那里它们促进体内平衡并对损伤或感染迅速反应。与ILC的类器官共培养已经开始揭示肠道中新的上皮免疫信号传导模块，揭示了不同的ILC亚群如何影响肠道上皮屏障的完整性和再生。该协议将能够进一步研究上皮细胞和免疫细胞之间的相互相互作用，这有可能为粘膜稳态和炎症的机制提供新的见解。
Introduction
肠上皮和肠道驻留免疫系统之间的沟通是维持肠道稳态的核心 1。这些相互作用的破坏与局部和全身性疾病有关，包括炎症性肠病（IBD）和胃肠道癌2。最近描述的稳态关键调节因子的一个显着例子来自对先天性淋巴细胞（ILCs）的研究，这些细胞已成为肠道免疫景观中的关键参与者3。ILC是一组异质性先天免疫细胞，主要通过细胞因子介导的信号传导来调节肠道稳态并协调炎症4。
小鼠 ILC 根据转录因子、受体和细胞因子表达谱广泛分为亚型5。1 型 ILC，包括细胞毒性自然杀伤 （NK） 细胞和辅助性 1 型 ILCs （ILC1s），分别由 T 细胞中表达的转录因子 （eomesodermin） Eomes 和 T-box 蛋白 （T-bet）6 以及与 T 辅助性 1 型 （TH1） 免疫相关的分泌细胞因子来定义：干扰素-γ （IFNγ） 和肿瘤坏死因子 （TNF），以响应白细胞介素 （IL）-12， IL-15和IL-187。在体内平衡期间，组织常驻ILC1s分泌转化生长因子β（TGF-β）以驱动上皮增殖和基质重塑8。2 型 ILC （ILC2s） 主要 通过 分泌 2 型 T 辅助性 T 辅助细胞 （TH2） 相关细胞因子 IL-4、IL-5 和 IL-13 对蠕虫感染作出反应，其特征在于表达视黄酸相关孤儿受体 （ROR） α （ROR-α）9 和 GATA 结合蛋白 3 （GATA-3）10，11，12.在小鼠中，肠道“炎症性”ILC2s进一步表征为表达杀伤细胞凝集素样受体（亚家族G成员1，KLRG）13 ，其中它们响应上皮簇状细胞衍生的IL-2514，15。最后，3型ILCs，包括淋巴组织诱导细胞和辅助性3型ILCs（ILC3s），依赖于转录因子ROR-γt16，并聚集在分泌粒细胞巨噬细胞集落刺激因子（GM-CSF），IL-17或IL-22以响应局部IL-1β和IL-23信号17的组。淋巴组织诱导细胞聚集在Peyer贴片中，对于发育过程中这些次级淋巴器官的发育至关重要18，而ILC3s是成年鼠小肠固有层中最丰富的ILC亚型。利用最早的鼠肠类器官共培养系统之一与ILC3s来梳理细胞因子IL-22对信号传感器和转录激活剂3（STAT-3）介导的富含亮氨酸重复序列含有G蛋白偶联受体5（Lgr5）+ 肠干细胞增殖的影响19，这是再生ILC上皮相互作用的有力例子。ILC表现出器官20，21 之间的印记异质性，并且响应于极化细胞因子22，在亚群之间表现出可塑性。是什么推动了这些组织特异性印记和可塑性差异，以及它们在IBD23等慢性疾病中的作用，仍然是令人兴奋的话题，可以使用类器官共培养来解决。
肠道类器官已成为研究肠道上皮24，25的成功和可靠的模型。这些是由培养肠上皮Lgr5 + 干细胞或整个分离的隐窝产生的，其中包括作为Wnt家族成员3A（Wnt3a）的内源性来源的Paneth细胞。这些3D结构在合成水凝胶26 或模仿基底层固有体的生物材料中维持，例如，热交联基底细胞外基质（TBEM），并进一步补充模仿周围生态位的生长因子，最着名的是上皮生长因子（EGF），骨形态发生蛋白（BMP）抑制剂Noggin，以及Lgr5配体和Wnt激动剂R-Spondin127.在这些条件下，类器官维持上皮蜂基极性，并用萌芽的干细胞隐窝来概括肠上皮的隐窝绒毛结构，这些隐窝在类器官的中心最终分化为吸收细胞和分泌细胞，然后通过anoikis28脱落到内部假羽化物中。虽然单独使用肠道类器官作为上皮发育和动力学的还原论模型具有巨大的优势29，30，但它们在理解这些行为如何被免疫区室调节，影响甚至破坏方面具有巨大的未来潜力。
在下面的方案中，描述了小鼠小肠类器官和固有层衍生的ILC1s之间的共培养方法，该方法最近用于鉴定该人群如何意外地减少炎症的肠道特征，而不是 通过 该系统中的TGF-β促进上皮增殖的增加8。
Protocol
Representative Results
Discussion
该协议描述了建立小鼠小肠类器官的方法，通过在肠道解离方案期间最小化淋巴细胞的丢失来分离罕见的ILC1，并在这两个区室之间建立共培养。该方案有许多步骤，虽然有些步骤特定于ILC1s，但这种方法可以应用于其他肠道免疫细胞类型，并且共培养设置可以模块化地适应个别研究问题。这里重点介绍了几个关键步骤（建议不要偏离），以及该协议中技术上更具挑战性的元素的故障排除指南。
…Declarações
The authors have nothing to disclose.
Acknowledgements
E.R.承认惠康信托基金会的博士学位奖学金（215027/Z/18/Z）。G.M.J.承认惠康信托基金会的博士学位（203757/Z/16/A）。华盛顿特区承认NIHR GSTT BRC的博士学位。J.F.N.授予Marie Skłodowska-Curie奖学金，国王奖奖学金，RCUK / UKRI卢瑟福基金奖学金（MR / R024812 / 1）以及Wellcome Trust的科学种子奖（204394 / Z / 16 / Z）。我们还感谢盖伊医院的BRC流式细胞术核心团队。Rorc（γt）-GfpTG C57BL/6报告小鼠是G. Eberl（法国巴黎巴斯德研究所）的慷慨礼物。CD45.1 C57BL / 6小鼠由T. Lawrence（伦敦国王学院，伦敦）和P. Barral（伦敦国王学院，伦敦）善意地给予。
Materials
| Reagents | |||
| 2-Mercaptoethanol | Gibco | 21985023 | |
| Anti-mouse CD45 (BV510) | BioLegend | 103137 | |
| Anti-mouse NK1.1 (PE) | Thermo Fisher Scientific | 12-5941-83 | |
| B-27 Supplement (50X), serum free | Gibco | 17504044 | |
| CD127 Monoclonal Antibody (APC) | Thermo Fisher Scientific | 17-1271-82 | |
| CD19 Monoclonal Antibody (eFluor 450) | Thermo Fisher Scientific | 48-0193-82 | |
| CD3e Monoclonal Antibody (eFluor 450) | Thermo Fisher Scientific | 48-0051-82 | |
| CD5 Monoclonal Antibody (eFluor 450) | Thermo Fisher Scientific | 48-0031-82 | |
| CHIR99021 | Tocris | 4423/10 | |
| COLLAGENASE D, 500MG | Merck | 11088866001 | |
| Cultrex HA- RSpondin1-Fc HEK293T Cells | Cell line was used to harvest conditioned RSpondin1 supernatant, the cell line and Materials Transfer Agreement was provided by the Board of Trustees of the Lelands Stanford Junior University (Calvin Kuo, MD,PhD, Stanford University) | ||
| DISPASE II (NEUTRAL PROTEASE, GRADE II) | Merck | 4942078001 | |
| DMEM/F12 (1:1) (1X) Dulbecco's Modified Eagle Medium Nutrient Mixture F-12 (Advanced DMEM/F12) | Gibco | 11320033 | |
| DNASE I, GRADE II | Merck | 10104159001 | |
| Dulbecco's Modified Eagle Medium (1X) | Gibco | 21969-035 | |
| Ethilenediamine Tetraacetate Acid | Thermo Fisher Scientific | BP2482-100 | |
| FC block | 2B Scientific | BE0307 | |
| Fetal Bovine Serum, qualified, hear inactivated | Gibco | 10500064 | |
| GlutaMAX (100X) | Gibco | 3050-038 | |
| Hanks' Balanced Salt Solution (10X) | Gibco | 14065056 | |
| HBSS (1X) | Gibco | 12549069 | |
| HEK-293T- mNoggin-Fc Cells | Cell line was used to harvest conditioned Noggin supernatant, cell line acquired through Materials Transfer Agreement with the Hubrecth Institute, Uppsalalaan8, 3584 CT Utrecht, The Netherlands, and is based on the publication by Farin, Van Es, and Clevers Gastroenterology (2012). | ||
| HEPES Buffer Solution (1M) | Gibco | 15630-056 | |
| KLRG1 Monoclonal Antibody (PerCP eFluor-710) | Thermo Fisher Scientific | 46-5893-82 | |
| Live/Dead Fixable Blue Dead Cell Stain Kit, for UV excitation | Thermo Fisher Scientific | L23105 | |
| Ly-6G/Ly-6C Monoclonal Antibody (eFluor 450) | Thermo Fisher Scientific | 48-5931-82 | |
| Matrigel Growth Factor Reduced Basement Membrane Matrix, Phenol Red-free, LDEV-free | Corning | 356231 | |
| N-2 Supplement (100X) | Gibco | 17502048 | |
| N-acetylcysteine (500mM) | Merck | A9165 | |
| NKp46 Monoclonal Antibody (PE Cyanine7) | Thermo Fisher | 25-3351-82 | |
| PBS (1 X) 7.2 pH | Thermo Fisher Scientific | 12549079 | |
| PBS (10X) | Gibco | 70013032 | |
| Percoll | Cytiva | 17089101 | |
| Recombinant Human EGF, Animal-Free Protein | R&D Systems | AFL236 | |
| Recombinant Human IL-15 GMP Protein, CF | R&D Systems | 247-GMP | |
| Recombinant Human IL-2 (carrier free) | BioLegend | 589106 | |
| Recombinant Mouse IL-7 (carrier free) | R&D Systems | 407-ML-005/CF | |
| UltraComp eBeads | Thermo Fisher Scientific | 01-2222-42 | |
| Y-27632 dihydrochloride (ROCK inhibitor) | Bio-techne | 1254 | |
| Plastics | |||
| 50 mL tube | Falcon | 10788561 | |
| 1.5 mL tube | Eppendorf | 30121023 | |
| 10 mL pippette | StarLab | E4860-0010 | |
| 15 mL tube | Falcon | 11507411 | |
| 25 mL pippette | StarLab | E4860-0025 | |
| p10 pippette tips | StarLab | S1121-3810-C | |
| p1000 pippette tips | StarLab | I1026-7810 | |
| p200 pippette tips | StarLab | E1011-0921 | |
| Standard tissue culture treated 24-well plate | Falcon | 353047 | |
| Equipment | |||
| Centrifuge | Eppendorf | 5810 R | |
| CO2 and temperature controled incubator | Eppendorf | Galaxy 170 R/S | |
| Flow Assisted Cellular Sorter | BD equipment | FACS Aria II | |
| Heated shaker | Stuart Equipment | SI500 | |
| Ice box | – | – | |
| Inverted light microscope | Thermo Fisher Scientific | EVOS XL Core Imaging System (AMEX1000) | |
| p10 pippette | Eppendorf | 3124000016 | |
| p1000 pippette | Eppendorf | 3124000063 | |
| p200 pippette | Eppendorf | 3124000032 | |
| Pippette gun | Eppendorf | 4430000018 | |
| Wet ice | – | – |
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