类器官来源的上皮单层：临床相关的肠道屏障功能体外模型
Summary
在这里，我们描述了用于研究肠屏障功能，通透性和运输的人类器官衍生的肠上皮单层的制备。由于类器官代表原始的上皮组织对外部刺激的反应，这些模型结合了细胞系可扩增性的优势以及原代组织的相关性和复杂性。
Abstract
过去，肠道上皮模型系统仅限于转化的细胞系和原代组织。这些模型系统具有固有的局限性，因为前者不能忠实地代表原始组织生理学，并且后者的可用性有限。因此，它们的应用阻碍了基础和药物开发研究。基于成体干细胞的类器官（以下简称类器官）是正常或患病上皮组织的缩影，它们来自这些组织。它们可以从不同的胃肠道（GI）区域非常有效地建立，具有长期可扩展性，并模拟组织和患者对体外治疗的特定反应。在这里，已经证明了肠类器官来源的上皮单层的建立以及测量上皮屏障完整性，通透性和运输，抗菌蛋白分泌以及组织学的方法。此外，肠道类器官来源的单层可以富含增殖的干细胞和运输扩增细胞以及关键的分化上皮细胞。因此，它们代表了一个模型系统，可以定制以研究化合物对靶细胞的影响及其作用方式。虽然类器官培养在技术上比细胞系要求更高，但一旦建立，它们可以减少药物开发后期阶段的失败，因为它们真正代表了体内上皮的复杂性和患者间异质性。
Introduction
肠上皮充当肠腔含量与下层组织之间的物理屏障。该屏障包括主要由吸收性肠细胞组成的单个上皮层，这些肠细胞通过紧密的连接连接，在相邻细胞之间建立强大的细胞间连接。这些细胞形成极化上皮衬里，分离肠的顶端（腔）和基底外侧，同时调节消化的营养物质和代谢物的副细胞运输。除肠细胞外，其他重要的上皮细胞（如高脚杯、Paneth和肠内分泌细胞）也分别通过产生粘液、抗菌肽和激素来促进肠道稳态。通过在肠隐窝底部分裂富含亮氨酸的含有G蛋白偶联受体5阳性（LGR5 +）干细胞来不断补充肠上皮，产生向上迁移并分化成其他细胞类型的1的富含亮氨酸的重复物。遗传和环境因素（例如暴露于食物过敏原、药物化合物和微生物病原体）破坏肠道上皮稳态，导致肠道屏障功能破坏。这些疾病可引起多种肠道疾病，包括炎症性肠病 （IBD）、乳糜泻和药物诱发的胃肠道毒性2。
使用几种体外平台系统（例如膜插入物，芯片上器官系统，Ussing室和肠环）对肠上皮进行研究。这些平台适用于建立极化上皮单层，具有进入膜的顶端和基外侧，使用转化的细胞系或原代组织作为模型。虽然转化的细胞系，如结直肠（腺）癌细胞系Caco-2，T84和HT-29，能够在一定程度上分化成极化肠肠细胞或产生粘液的细胞，但它们并不代表体内上皮细胞，因为缺少几种细胞类型，并且各种受体和转运蛋白异常表达3.此外，由于细胞系来源于单个供体，因此它们不代表患者间异质性，并且复杂性和生理相关性降低。虽然用于Ussing室和作为肠环的原代组织更能代表体内情况，但它们的可用性有限，短期生存能力和缺乏可膨胀性使它们不适合作为高通量（HT）研究的介质。
类器官是从不同器官（如肠、肾、肝、胰腺和肺）建立的体外上皮培养物。它们被证明具有长期，稳定的可扩展性以及遗传和表型稳定性，因此是原始器官上皮的代表性生物学微缩模型，对外部刺激具有忠实的反应4，5，6，7，8，9。从切除或活检的正常、患病、发炎或癌组织中有效地建立类器官，代表异质性患者特异性反应10，11，12，13，14，15，16。本文展示了如何建立来自类器官培养物的肠上皮单层。单层已经成功地从小肠以及结肠和直肠类器官培养物中建立。该模型为研究上皮细胞对药物的运输和通透性及其对上皮的毒理学影响创造了机会。此外，该模型允许与免疫细胞和细菌共培养以研究它们与肠上皮的相互作用17，18，19。此外，该模型可用于以患者特异性方式研究对治疗的反应，并开始筛查工作以寻找下一波上皮屏障聚焦的治疗方法。这种方法可以扩展到诊所，并为个性化治疗铺平道路。
尽管该方案中的上皮单层是由人类正常肠道类器官制备的，但该方案可以应用于其他类器官模型并进行优化。上皮类器官单层在含有Wnt的肠道类器官扩张培养基中培养，以支持干细胞增殖并代表肠隐窝细胞组成。通过调节 Wnt、Notch 和表皮生长因子 （EGF） 途径，可以富集肠道类器官以具有不同的肠上皮命运，例如肠细胞、Paneth、高脚杯和肠内分泌细胞。在这里，在扩增培养基中建立单层后，它们被驱动向更分化的肠上皮细胞，如前面描述的20，21，22，23，24，25。出于筛选目的，根据目标化合物的作用模式，其靶细胞和实验条件，单层可以被驱动到所选的细胞组成，以测量具有相关功能读数的化合物的效果。
Protocol
1. 制备培养试剂 注意：在生物安全柜内执行所有步骤，并遵循使用细胞培养物的标准指南。在启动生物安全柜之前使用紫外线10分钟。使用前后，用浸有70%乙醇的薄纸清洁生物安全柜的表面。为了促进细胞外基质（ECM）的三维液滴的形成，请在37°C的培养箱中保持96孔，24孔和6孔板的预热储备。 基础培养基制备 通过添加5 mL 200 mM谷氨酰胺，5 mL 1 M4-（2-羟基乙硫基?…
Representative Results
图1A 显示了从冷冻管中解冻后肠道类器官的代表性明场图像。重要的是要以高密度解冻类器官，以确保最佳恢复。将类器官电镀在约10μL的ECM圆顶中的24孔或6孔板中（图1B）。大多数正常肠道类器官具有囊性形态。从解冻过程中恢复后，类器官生长到更大的尺寸，并准备在3-7天后传代，具体取决于类器官培养物（图1C）。在收获类?…
Discussion
该协议描述了肠道类器官的一般操作和维持，以及来自这些类器官的上皮单层的制备和可能的应用。迄今为止，已经成功地从十二指肠，回肠和结肠类器官的不同区域制备了单层，这些区域来自正常以及先前和主动发炎的肠组织（未发表的数据）。患者衍生的类器官单层的应用有助于以疾病和患者特异性方式研究屏障功能，以及研究患者对各种药物治疗的特异性反应。虽然细胞系可以形成含有肠?…
Declarações
The authors have nothing to disclose.
Acknowledgements
这项工作得到了 荷兰LSH部门的Topsector Life Sciences & Health – Topconsortium voor Kennis en Innovatie Health~Holland （LSH-TKI）公私合作伙伴关系（PPP）津贴的支持，项目编号LSHM16021 Organoids作为Hubrecht Organoid Technology（HUB）的毒理学建模新工具，以及HUB内部资助疾病建模和毒理学部门。我们感谢Sabine Middendorp（威廉明娜儿童医院儿科胃肠病学科，UMC，乌得勒支）和Hugo R. de Jonge和Marcel J.C. Bijvelds（鹿特丹伊拉斯谟MC胃肠病学和肝病学系）的实验室为在膜插入物上设置单层提供了初步技术支持。
Materials
| 100% ethanol | Fisher Emergo | 10644795 | |
| 1250, 300, and 20 µL low-retention filter-tips | Greiner bio-one | 732-1432 / 732-1434 / 732-2383 | |
| 15 mL conical tubes | Greiner bio-one | 188271 | |
| 24-well cell culture plates | Greiner bio-one | 662160 | |
| 24-well HTS Fluoroblok Transwell plate (light-tight) | Corning | 351156 | Plates require REMS AutoSampler for TEER measurements |
| 24-well HTS Transwell plates (Table 1) | Corning | 3378 | |
| 24-well plate with Transwell inserts | Corning | 3470 | membrane inserts |
| 40 µm cell strainer | PluriSelect | 43-50040-01 | |
| 50 mL conical tubes | Greiner bio-one | 227261 | |
| 6-well cell culture plates | Greiner bio-one | 657160 | |
| 96-well black plate transparent bottom | Greiner bio-one | 655090 | |
| 96-well fast thermal cycling plates | Life Technologies Europe BV | 4346907 | |
| 96-well HTS Fluoroblok Transwell plate | Corning | 351162 | |
| 96-well HTS Transwell plates (Table 1) | Corning | 7369 | |
| 96-well transparent culture plate | Greiner bio-one | 655180 | |
| A83-01 | Bio-Techne Ltd | 2939 | |
| Accutase Cell Dissociation Reagent | Life Technologies Europe BV | A11105-01 | |
| Advanced DMEM/F-12 | Life Technologies Europe BV | 12634028 | |
| B27 supplement | Life Technologies Europe BV | 17504001 | |
| Cell culture microscope (light / optical microscope) | Leica | ||
| CellTiter-Glo | Promega | G9683 | |
| Centrifuge | Eppendorf | ||
| CO2 incubator | PHCBI | ||
| DAPT | Sigma-Aldrich | D5942 | |
| DEPC treated H2O | Life Technologies Europe BV | 750024 | |
| Dulbecco's phosphate-buffered saline (DPBS) with Ca2+ and Mg2+ | Life Technologies Europe BV | 14040091 | |
| DPBS, powder, no calcium, no magnesium | Life Technologies Europe BV | 21600069 | |
| EnzChek Lysozyme Assay Kit | Life Technologies Europe BV | E22013 | |
| EVOM2 meter with STX electrode | WTI | ||
| Gastrin | Bio-Techne Ltd | 3006 | |
| Glass pipettes | Volac | ||
| GlutaMAX | Life Technologies Europe BV | 35050038 | |
| hEGF | Peprotech | AF-100-15 | |
| HEPES | Life Technologies Europe BV | 15630056 | |
| Human Noggin | Peprotech | 120-10C | |
| Human Rspo3 | Bio-Techne Ltd | 3500-RS/CF | |
| IWP-2 | Miltenyi Biotec | 130-105-335 | |
| Ki67 primary antibody | Sanbio | BSH-7302-100 | |
| Ki67 secondary antibody | Agilent | K400111-2 | |
| Kova International Glasstic Slide with Counting grids | Fisher Emergo | 10298483 | |
| Laminar flow hood | Thermo scientific | ||
| Lucifer Yellow CH dilithium salt | Sigma-Aldrich | L0259 | |
| Matrigel, Growth Factor Reduced (GFR) | Corning | 356231 | extracellular matrix (ECM) |
| MicroAmp Fast 8-Tube Strip, 0.1 mL | Life Technologies Europe BV | 4358293 | |
| MicroAmp Optical 8-Cap Strips | Life Technologies Europe BV | 4323032 | |
| Microcentrifuge tubes | Eppendorf | 0030 120 086 | |
| Micropipettes (1000, 200, and 20 µL) | Gilson | ||
| Microtome | Leica | ||
| MUC2 primary antibody | Santa Cruz Biotechnology | sc-15334 | |
| MUC2 secondary antibody | VWR | VWRKS/DPVR-HRP | |
| Multichannel pipette (200 µL) | Gilson | ||
| N-acetylcysteine | Sigma-Aldrich | A9165 | |
| NGS Wnt | U-Protein Express | N001-0.5mg | |
| Nicotinamide | Sigma-Aldrich | N0636 | |
| Oligonucleotide ALPI1/Forward | Custom-made | GGAGTTATCCTGCTCCCCAC | |
| Oligonucleotide ALPI1/Reverse | Custom-made | CTAGGAGGTGAAGGTCCAACG | |
| Oligonucleotide LGR5/Forward | Custom-made | ACACGTACCCACAGAAGCTC | |
| Oligonucleotide LGR5/Reverse | Custom-made | GGAATGCAGGCCACTGAAAC | |
| Oligonucleotide MUC2/Forward | Custom-made | AGGATCTGAAGAAGTGTGTCACTG | |
| Oligonucleotide MUC2/Reverse | Custom-made | TAATGGAACAGATGTTGAAGTGCT | |
| Oligonucleotide TBP/Forward | Custom-made | ACGCCGAATATAATCCCAAGCG | |
| Oligonucleotide TBP/Reverse | Custom-made | AAATCAGTGCCGTGGTTCGTG | |
| Optical adhesive covers | Life Technologies Europe BV | 4311971 | |
| PD0325901 | Stemcell Technologies | 72184 | |
| Penicillin/streptomycin | Life Technologies Europe BV | 15140122 | |
| Plate shaker | Panasonic | ||
| PowerUp SYBR Green Master Mix | Fisher Emergo | A25776 | |
| Primocin | InvivoGen | ANT-PM-2 | antimicrobial formulation for primary cells |
| Qubit RNA HS Assay Kit | Life Technologies Europe BV | Q32852 | |
| Reagent reservoir for multichannel pipet | Sigma-Aldrich | CLS4870 | |
| REMS AutoSampler with 24-probe or 96C-probe | WTI | ||
| Richard-Allan Scientific Alcian Blue/PAS Special Stain Kit | Thermo scientific | 87023 | |
| RNase-Free DNase Set | Qiagen | 79254 | |
| RNeasy Mini Kit | Qiagen | 74106 | |
| SB202190 | Sigma-Aldrich | S7076 | |
| Serological pipettes | Greiner bio-one | 606180 / 607180 / 760180 | |
| Serological pipettor (Pipet-Aid) | Drummond | ||
| Single edge razor blade | GEM Scientific | ||
| Superscript 1st strand system for RT-PCR | Life Technologies Europe BV | 11904018 | |
| Tecan Spark 10M plate reader | Tecan | ||
| Trypan Blue Solution, 0.4% | Life Technologies Europe BV | 15250-061 | |
| TrypLE Express Enzyme (1x) | Life Technologies Europe BV | 12605-010 | Cell dissociation reagent |
| Water bath | Grant | ||
| Y27632 (ROCK inhibitor) | AbMole | M1817 |
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Citar este artigo
van Dooremalen, W. T. M., Derksen, M., Roos, J. L., Higuera Barón, C., Verissimo, C. S., Vries, R. G. J., Boj, S. F., Pourfarzad, F. Organoid-Derived Epithelial Monolayer: A Clinically Relevant In Vitro Model for Intestinal Barrier Function. J. Vis. Exp. (173), e62074, doi:10.3791/62074 (2021).