三维细胞培养模型研究嗜酸性粒细胞性食管炎中上皮屏障
Summary
在这里，提供了人食管类器官培养和气液界面培养的方案。食管类器官的气液界面培养可用于研究细胞因子对食管上皮屏障的影响。
Abstract
食管鳞状上皮直接暴露在环境中，不断面对外来抗原，包括食物抗原和微生物。保持上皮屏障的完整性对于预防感染和避免由无害的食物源性抗原引起的炎症至关重要。本文提供了从患者活检生成人食管类器官和气液界面培养物的简化方案，以在组织稳态和疾病的背景下研究食管上皮室。这些方案是过去十年中重要的科学里程碑，描述了来自患者来源的原代细胞、类器官和气液界面培养物的三维器官样结构。它们提供了在三维框架内研究食管上皮中特定细胞因子、生长因子和信号通路功能的可能性，同时保持供体的表型和遗传特性。类器官通过评估细胞因子刺激后的转录组和蛋白质组来提供有关组织微结构的信息。相比之下，气液界面培养允许通过跨上皮阻力 （TEER） 或大分子通量测量来评估上皮屏障的完整性。将这些类器官和气液界面培养物相结合是推进食管上皮屏障受损条件研究的有力工具。
Introduction
食管炎症会损害上皮屏障的完整性 1,2,3,4,5，如嗜酸性粒细胞性食管炎 （EoE） 所观察到的那样，这是一种以 Th2 为主的食管慢性炎症性疾病 6。EoE 于 1990 年代首次描述 7,8，主要由食物抗原 9,10,11,12,13 诱导。成人中最常见的 EoE 症状是吞咽困难和食物嵌塞14。在儿童中，EoE 通常表现为生长迟缓、食物拒绝、呕吐和腹痛15。全基因组关联研究 （GWAS） 已确定与上皮屏障完整性相关的 EoE 风险基因，使上皮成为 EoE 研究的重点 16,17,18。EoE 转录组学进一步揭示，分化过程受损和反应性基底区增生导致食管上皮屏障功能受损 3,5,19,20,21,22。对 EoE 是 Th2 介导的疾病6 的早期理解导致发现 IL-13 通过干扰上皮完整性作为驱动介质 3,4,21,23。实验系统允许解剖细胞因子介导的对上皮完整性的影响，从内在屏障损伤到遗传易感性，为研究免疫细胞与EoE中上皮细胞之间的复杂相互作用提供了可能性。人食管类器官和气液界面 （ALI） 培养物已被提议作为分析细胞因子刺激对上皮完整性后果的宝贵工具5。
在2009年首次发表的肠道类器官报告使用肠道Lgr5 + ASCs概括小肠上皮室24之后，建立了第一个产生成体组织特异性干细胞（ASC）衍生食管类器官的方案。DeWard等人率先从小鼠食管上皮细胞中产生类器官25。2018 年，Kasagi 等人从永生化的人食管鳞状上皮细胞系 EPC2-hTERT 和原代患者来源的细胞中生成了人食管类器官26。同年，Zhang等人成功制备了诱导多能干细胞（iPSC）衍生的食管类器官。他们阐述了 TGFβ 和骨形态发生蛋白 （BMP） 抑制对食管祖细胞 （EPC） 发育的意义，以及 Notch 信号转导在分层鳞状上皮分化中的关键作用26,27。Trisno及其同事通过将Sox2确定为Wnt抑制剂来补充这些发现，该抑制剂将发育命运指向食管分化28。随后对方案、培养基组成和培养条件的改进提高了类器官形成率，并使得冷冻保存后传代培养和回收类器官成为可能26,29,30,31,32。尽管这些类器官是研究细胞因子刺激后组织结构和潜在靶基因表达的强大工具，但食管类器官无法提供测量跨上皮抵抗 （TEER） 或大分子通量作为屏障完整性的直接测量方法。正如 Sherrill 及其同事22 之前所描述的，模拟上皮分化的 ALI 培养物4 允许直接评估上皮完整性。结合患者来源的类器官和 ALI 培养物是研究 EoE 中组织结构和上皮屏障完整性的有力工具。
以下是从食管活检中分离活细胞和建立食管类器官和 ALI 培养物的程序，这些程序可用于进一步研究细胞因子对屏障完整性的影响。
Protocol
这些程序得到了瑞士西北部和中部伦理委员会（EKNZ;项目ID 2019-00273）。所有患者在内窥镜检查前都提供了实验性使用活检的书面知情同意书。研究中使用的试剂和设备列在 材料表中。 1. 患者来源的食管类器官的细胞分离 注： 表1中提供了用于培养人食管类器官的培养基成分列表。 进行活检。注：在本研究…
Representative Results
食管类器官将根据所提供的方案的指示从患者活检中提取的原代细胞生长，如倒置明场显微镜所记录的那样（图1）。上皮 ASC 在将分离的细胞接种到基底膜提取物中后，在培养的前两天开始以自组织方式形成细胞簇，充当支架。细胞簇的大小和数量在第一周内持续增加，用倒置明场显微镜观察（图2）。然而，在这一点上，细胞簇缺乏 ASC 衍生的食管类?…
Discussion
所提供的程序允许培养患者来源的类器官和 ALI 培养物，成功的可能性很高。类器官方案改编自第一个发表的报告人类食管类器官产生的方案26 和最近发表的方案32。Sherill 及其同事描述了 ALI 模型22。类器官和 ALI 培养模型在研究食管疾病（如 EoE 5,26）中细胞因子和其他介质对食管上皮屏障的影响方?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
SNSF 向 J.H.N. 提供的 310030_219210 拨款支持了此手稿的出版，没有限制。 图 1 是在 BioRender.com 的帮助下创建的。
Materials
| 1250 µL Griptip – Filter | Integra | 4445 | |
| 300 µL Griptip – Filter | Integra | 4435 | |
| 70 µM cell strainer | Sarstedt | 83.3945.070 | |
| Ascorbic Acid | Sigma-Aldrich (Merck) | A4544 | |
| Bovine pituitary extract | Gibco (Thermo Fischer Scientific) | 3700015 | |
| Calcium chloride | Sigma-Aldrich (Merck) | 21115 | |
| Cell Culture Multiwell Plates CELLSTAR for suspension cultures | Greiner Bio-One | 7.657 185 | |
| Cultrex Basement Membrane Extract (BME), Type 2, Pathclear | R&D Systems (Bio-Techne) | 3532-010-02 | |
| Dimethyl sulfoxide (DMSO), >99,5% BioScience Grade | Carl Roth | A994 | |
| Dispase I | Corning | 354235 | |
| Dispase II | Sigma-Aldrich (Merck) | D4693 | |
| Dulbeccos Phosphate Buffered Saline (DPBS) | Sigma-Aldrich (Merck) | D8537 | |
| EVE Automated Cell Counter | NanoEntek | EVE-MC | |
| EVE Cell counting slide | NanoEntek | EVS-050 | |
| Falcon 5 mL Round Bottom Polystyrene Test Tube, with Cell Strainer Snap Cap | Falcon | 352235 | |
| Fluorescin isothiocyanate (FITC)-dextran | Sigma-Aldrich (Merck) | FD4 | average mol wt 3000-5000 |
| Heraeus – Megafuge 40R | Thermo Fisher Scientific | 75004518 | |
| Human recombinant epidermal growth factor | Gibco (Thermo Fischer Scientific) | 3700015 | |
| Keratinocyte-SFM | Gibco (Thermo Fischer Scientific) | 17005042 | |
| Penicillin-Streptomycin | Gibco (Thermo Fischer Scientific) | 15140122 | |
| Recombinant Human KGF/FGF-7 Protein | R&D Systems (Bio-Techne) | 251-KG-010/CF | |
| Screw cap tube, 15 mL | Sarstedt | 62.554.502 | |
| Single Channel EVOLVE 100-1000 µL | Integra | 3018 | |
| Single Channel EVOLVE 20-200 µL | Integra | 3016 | |
| Syringe 1 mL | 1134950 | ||
| ThermoMixer C | Eppendorf | 5382000015 | |
| Trypsin inhibitor from Glycine max (soybean) | Sigma-Aldrich (Merck) | T9128 | |
| Trypsin-EDTA | SAFC Biosciences (Merck) | 59418C | |
| Y27632 dihydrochloride | Tocris (Bio-Techne) | 1254 |
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Cite This Article
Kaymak, T., Niess, J. H. Three-Dimensional Cell Culture Models to Investigate the Epithelial Barrier in Eosinophilic Esophagitis . J. Vis. Exp. (207), e66503, doi:10.3791/66503 (2024).