用于分析肠道粘膜免疫反应的免疫活性肠芯片模型
概要
我们的详细方案概述了高级肠芯片模型的创建和使用，该模型模拟具有 3D 结构和各种细胞类型的人类肠粘膜，能够深入分析响应微生物定植的免疫反应和细胞功能。
Abstract
已经开发了一种先进的肠芯片模型，可重建上皮 3D 器官型绒毛样和隐窝样结构。免疫活性模型包括人脐静脉内皮细胞 （HUVEC）、Caco-2 肠上皮细胞、组织驻留巨噬细胞和树突状细胞，它们在组织内自组织，反映了人肠粘膜的特征。该平台的一个独特之处在于它能够整合循环的人类原代免疫细胞，增强生理相关性。该模型旨在研究肠道免疫系统对细菌和真菌定植和感染的反应。由于其扩大的腔体尺寸，该模型提供了多种功能读数，例如渗透分析、细胞因子释放和免疫细胞浸润，并且与上皮细胞层形成的 3D 结构的免疫荧光测量兼容。因此，它提供了对细胞分化和功能的全面见解。在微生理灌注生物芯片平台中，肠道芯片平台已证明其在阐明活微生物群的替代物与人类宿主组织之间的复杂相互作用方面的潜力。
Introduction
器官芯片 （OoC） 系统代表了一种新兴的 3D 细胞培养技术，能够弥合传统 2D 细胞培养和动物模型之间的差距。OoC 平台通常由一个或多个隔室组成，这些隔室包含在各种支架（如膜或水凝胶）上生长的组织特异性细胞1。这些模型能够模拟一个或多个定义的器官型功能。泵能够对细胞培养基进行连续微流控灌流，以去除细胞废物，提供营养和生长因子以改善细胞分化，并重建必要的体内条件。随着免疫细胞的整合，OoC 系统可以在体外模拟人类免疫反应 2。迄今为止，已经提出了广泛的器官和功能单位1。这些系统包括脉管系统3（vaschorue）、肺4（lung）、肝脏2（h）2,5和肠道6（intestine 6）的模型，可用于药物检测5,7和感染研究6,8。
我们在这里提出了一个人肠芯片模型，该模型整合了人上皮细胞，形成了绒毛样和隐窝样结构的器官型 3D 地形，并结合了内皮衬里和组织驻留的巨噬细胞。该模型以显微载玻片的形式在微流体灌注的生物芯片中培养。每个生物芯片由两个独立的微流体腔组成。每个腔体由多孔聚对苯二甲酸乙二醇酯 （PET） 膜分为上腔室和下腔室。膜本身也充当细胞在每一侧生长的支架。膜的孔隙使细胞串扰和细胞层之间的细胞迁移成为可能。每个腔室都可以通过两个母鲁尔锁大小的端口进入。或者，一个额外的微型鲁尔锁大小的端口可以提供对上腔室或下腔室的访问（图 1）。
OoC 平台提供了许多可以从单个实验中获得的读数。肠芯片专为结合灌注 3D 细胞培养、流出物分析和荧光显微镜检查而设计，以评估细胞标志物表达、代谢率、免疫反应、微生物定植和感染以及屏障功能 3,6,8。该模型包括组织驻留的免疫细胞和活微生物与宿主组织的直接接触，与其他已发表的模型相比，这是一个优势9。此外，上皮细胞自组织成三维结构，为活微生物群的定植提供生理相关的界面6。
Protocol
该方案要求每个生物芯片从健康供体那里获得 ~20 mL 的新鲜血液，以分离原代人单核细胞。所有捐献者都书面知情同意参与这项研究，该研究得到了耶拿大学医院伦理委员会的批准（许可号 2018-1052-BO）。有关材料的详细信息，请参阅 材料表。有关所有解决方案和介质的组成的详细信息，请参阅 表 1。 1. 一般生物芯片处理备注 小?…
Representative Results
这些代表性结果显示了 intestine-on-chip 模型的不同组织层。它们按照方案第 11 节中的说明进行免疫荧光染色。用落射荧光或共聚焦荧光显微镜作为 z 堆栈拍摄图像，并处理为正交投影。有关显微镜设置和软件的详细信息，请参阅 材料表 。 图 5 显示了血管层，一种形成屏障的内皮单层，由 HUVEC 和巨噬细胞组成。对代表性细胞标志物如内皮细胞的 VE-钙粘蛋白和血?…
Discussion
所提出的协议详细介绍了生成免疫活性肠芯片模型的必要步骤。我们描述了特定的技术和可能的读出方法，例如免疫荧光显微镜、细胞因子和代谢物分析、流式细胞术、蛋白质和遗传分析以及通透性测量。
所描述的模型由原代 HUVEC、单核细胞衍生的巨噬细胞和单核细胞衍生的树突状细胞组成，与代表粘液分泌、吸收、肠内分泌和 Paneth 细胞样群体的各个方面的肠上皮细胞 3D ?…
開示
The authors have nothing to disclose.
Acknowledgements
这项工作得到了合作研究中心 PolyTarget 1278（项目编号 316213987）对 V.D.W. 和 A.S.M. A.F. 和 A.S.M. 的财政支持，进一步感谢德国卓越战略 – EXC 2051 – 项目 ID 690 390713860 下卓越集群“微宇宙平衡”的财政支持。我们要感谢 Astrid Tannert 和 Jena 生物光子学和成像实验室 （JBIL） 为我们提供他们的共聚焦激光扫描显微镜蔡司 LSM980。 图 1C 和 图 2 是使用 Biorender.com 创建的。
Materials
| 96-well plate black, clear bottom | Thermo Fisher | 10000631 | Consumables |
| Acetic acid | Roth | 3738.4 | Chemicals |
| Alexa Fluor 488 AffiniPure, donkey, anti-mouse IgG (H+L) | Jackson Immuno Research | 715-545-150 | Secondary Antibody Vascular Staining and Epithelial Staining |
| Alexa Fluor 647 AffiniPure, donkey, anti-rabbit IgG (H+L) | Jackson Immuno Research | 711-605-152 | Secondary Antibody Epithelial Staining |
| Alexa Fluor 647, donkey, anti-rabbit IgG (H+L) | Thermo Fisher Scientific, Invitrogen | A31573 | Secondary Antibody Vascular Staining |
| Axiocam ERc5s camera | Zeiss | 426540-9901-000 | Technical equipment |
| Basal Medium MV, phenol red-free | Promocell | C-22225 | Cell culture consumables |
| Biochip | Dynamic 42 | BC002 | Microfluidic consumables |
| BSA fraction V | Gibco | 15260-037 | Cell culture consumables |
| C2BBe1 (clone of Caco-2) | ATCC | CRL-2102 | Epithelial Cell Source |
| Chloroform | Sigma | C2432 | Chemicals |
| CO2 Incubator | Heracell | 150i | Technical equipment |
| Collagen IV from human placenta | Sigma-Aldrich | C5533 | Cell culture consumables |
| Coverslips (24 x 40 mm; #1.5) | Menzel-Gläser | 15747592 | Consumables |
| Cy3 AffiniPure, donkey, anti-goat IgG (H+L) | Jackson Immuno Research | 705-165-147 | Secondary Antibody Vascular Staining |
| Cy3 AffiniPure, donkey, anti-rat IgG (H+L) | Jackson Immuno Research | 712-165-150 | Secondary Antibody Epithelial Staining |
| DAPI (4',6-Diamidin-2-phenylindol, Dilactate) | Thermo Fisher Scientific, Invitrogen | D3571 | Vascular and Epithelial Staining |
| Descosept PUR | Dr.Schuhmacher | 00-323-100 | Cell culture consumables |
| DMEM high glucose | Gibco | 41965-062 | Cell culture consumables |
| DMEM high glucose w/o phenol red | Gibco | 31053028 | Cell culture consumables |
| DPBS (-/-) | Gibco | 14190-169 | Cell culture consumables |
| DPBS (+/+) | Gibco | 14040-133 | Cell culture consumables |
| EDTA solution | Invitrogen | 15575-038 | Cell culture consumables |
| Endothelial Cell Growth Medium | Promocell | C-22020 | Cell culture consumables |
| Endothelial Cell Growth Medium supplement mix | Promocell | C-39225 | Cell culture consumables |
| Ethanol 96%, undenatured | Nordbrand-Nordhausen | 410 | Chemicals |
| Fetal bovine Serum | invitrogen | 10270106 | Cell culture consumables |
| Fluorescein isothiocyanate (FITC)-dextran (3-5 kDa) | Sigma Aldrich | FD4-100MG | Chemicals |
| Fluorescent Mounting Medium | Dako | S3023 | Chemicals |
| Gentamycin (10mg/mL) | Sigma Aldrich | G1272 | Cell culture consumables |
| GlutaMAX Supplement (100x) | Gibco | 35050061 | Cell culture consumables |
| Histopaque | Sigma-Aldrich | 10771 | Cell culture consumables |
| Hoechst (bisBenzimid) H33342 | Sigma-Aldrich | 14533 | Epithelial Staining |
| Holotransferrin (5mg/mL) Transferrin, Holo, Human Plasma | Millipore | 616397 | Cell culture consumables |
| Human recombinant GM-CSF | Peprotech | 300-30 | Cell culture consumables |
| Human recombinant M-CSF | Peprotech | 300-25 | Cell culture consumables |
| Illumination device | Zeiss | HXP 120 C | Fluorescence Microscope Setup |
| Laser Scanning Microscope | Zeiss | CLSM980 | Fluorescence Microscope Setup |
| Lidocain hydrochloride | Sigma-Aldrich | L5647 | Cell culture consumables |
| Lipopolysaccharide (LPS) | Sigma | L2630 | Cell culture consumables |
| Loftex Wipes | Loftex | 1250115 | Consumables |
| Low attachment tubes (PS, 5 mL) | Falcon | 352052 | Consumables |
| Luer adapter for the top cap (M) | Mo Bi Tec | M3003 | Microfluidic consumables |
| Male mini luer plugs, row of four,PP, opaque | Microfluidic chipshop | 09-0556-0336-09 | Microfluidic consumables |
| MEM Non-Essential Amino Acids Solution | Gibco | 11140 | Cell culture consumables |
| Methanol | Roth | 8388.2 | Chemicals |
| Microscope | Zeiss | Axio Observer 5 | Fluorescence Microscope Setup |
| Microscope slides | Menzel | MZ-0002 | Consumables |
| Monoclonal, mouse, anti-human CD68 Antibody (KP1) | Thermo Fisher Scientific, Invitrogen | 14-0688-82 | Primary Antibody Vascular Staining |
| Monoclonal, rat, anti-human E-Cadherin antibody (DECMA-1) | Sigma-Aldrich, Millipore | MABT26 | Primary Antibody Epithelial Staining |
| Multiskan Go plate reader | Thermo Fisher | 51119300 | Technical equipment |
| Normal donkey serum | Biozol | LIN-END9010-10 | Chemicals |
| Optical Sectioning | Zeiss | ApoTome | Fluorescence Microscope Setup |
| Penicillin-Streptomycin (10,000 U/mL) | Gibco | 15140-122 | Cell culture consumables |
| Plugs | Cole Parmer | GZ-45555-56 | Microfluidic consumables |
| Polyclonal, goat, anti-human VE-Cadherin Antibody | R&D Systems | AF938 | Primary Antibody Vascular Staining |
| Polyclonal, rabbit, anti-human Von Willebrand Factor Antibody | Dako | A0082 | Primary Antibody Vascular Staining |
| Polyclonal, rabbit, anti-human ZO-1 antibody | Thermo Fisher Scientific, Invitrogen | 61-7300 | Primary Antibody Epithelial Staining |
| Power Supply Microscope | Zeiss | Eplax Vp232 | Fluorescence Microscope Setup |
| Primovert microscope | Zeiss | 415510-1101-000 | Technical equipment |
| Reglo ICC peristaltic pump | Ismatec | ISM4412 | Technical equipment |
| SAHA (Vorinostat) | Sigma Aldrich | SML0061-25MG | Chemicals |
| Saponin | Fluka | 47036 | Chemicals |
| S-Monovette, 7.5 mL Z-Gel | Sarstedt | 01.1602 | Consumables |
| S-Monovette, 9.0 mL K3E | Sarstedt | 02.1066.001 | Consumables |
| Sodium Pyruvate | Gibco | 11360-088 | Cell culture consumables |
| Tank 4.5 mL | ChipShop | 10000079 | Microfluidic consumables |
| Trypane blue stain 0.4% | Invitrogen | T10282 | Cell culture consumables |
| Trypsin | Gibco | 11538876 | Cell culture consumables |
| Tubing | Dynamic 42 | ST001 | Microfluidic consumables |
| Tweezers (Präzisionspinzette DUMONT abgewinkelt Inox08, 5/45, 0,06 mm) | Roth | K343.1 | Consumables |
| Wheat Germ Agglutinin (WGA) | Thermo Fisher Scientific, Invitrogen | W32464 | Epithelial Staining |
| X-VIVO 15 | Lonza | BE02-060F | Cell culture consumables, Hematopoietic cell medium |
| Zellkultur Multiwell Platten, 24 Well, sterile | Greiner Bio-One | 662 160 | Consumables |
| Zellkultur Multiwell Platten, 6 Well, sterile | Greiner Bio-One | 657 160 | Consumables |
| Zen Blue Software | Zeiss | Version 3.7 | Microscopy Software |
参考文献
- Alonso-Roman, R., et al. Organ-on-chip models for infectious disease research. Nat Microbiol. 9 (4), 891-904 (2024).
- Fahrner, R., Groger, M., Settmacher, U., Mosig, A. S. Functional integration of natural killer cells in a microfluidically perfused liver on-a-chip model. BMC Res Notes. 16 (1), 285 (2023).
- Raasch, M., et al. Microfluidically supported biochip design for culture of endothelial cell layers with improved perfusion conditions. Biofabrication. 7 (1), 015013 (2015).
- Deinhardt-Emmer, S., et al. Co-infection with Staphylococcus aureus after primary influenza virus infection leads to damage of the endothelium in a human alveolus-on-a-chip model. Biofabrication. 12 (2), 025012 (2020).
- Kaden, T., et al. Generation & characterization of expandable human liver sinusoidal endothelial cells and their application to assess hepatotoxicity in an advanced in vitro liver model. Toxicology. 483, 153374 (2023).
- Maurer, M., et al. A three-dimensional immunocompetent intestine-on-chip model as in vitro platform for functional and microbial interaction studies. Biomaterials. 220, 119396 (2019).
- Hoang, T. N. M., et al. Invasive aspergillosis-on-chip: A quantitative treatment study of human aspergillus fumigatus infection. Biomaterials. 283, 121420 (2022).
- Kaden, T., et al. Modeling of intravenous caspofungin administration using an intestine-on-chip reveals altered Candida albicans microcolonies and pathogenicity. Biomaterials. 307, 122525 (2024).
- Shah, P., et al. A microfluidics-based in vitro model of the gastrointestinal human-microbe interface. Nat Commun. 7, 11535 (2016).
- Jaffe, E. A., Nachman, R. L., Becker, C. G., Minick, C. R. Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria. J Clin Invest. 52 (11), 2745-2756 (1973).
- Mosig, S., et al. Different functions of monocyte subsets in familial hypercholesterolemia: Potential function of cd14+ cd16+ monocytes in detoxification of oxidized ldl. FASEB J. 23 (3), 866-874 (2009).
- Peterson, M., Mooseker, M. Characterization of the enterocyte-like brush border cytoskeieton of the c2bbe clones of the human intestinal cell line, caco-2. J Cell Sci. 102, 581-600 (1992).
- Shin, W., Hinojosa, C. D., Ingber, D. E., Kim, H. J. Human intestinal morphogenesis controlled by transepithelial morphogen gradient and flow-dependent physical cues in a microengineered gut-on-a-chip. iScience. 15, 391-406 (2019).
- Kim, H. J., Ingber, D. E. Gut-on-a-chip microenvironment induces human intestinal cells to undergo villus differentiation. Integr Biol (Camb). 5 (9), 1130-1140 (2013).
- Kim, H. J., Huh, D., Hamilton, G., Ingber, D. E. Human gut-on-a-chip inhabited by microbial flora that experiences intestinal peristalsis-like motions and flow. Lab Chip. 12 (12), 2165-2174 (2012).
- Karra, N., Fernandes, J., James, J., Swindle, E. J., Morgan, H. The effect of membrane properties on cell growth in an ‘airway barrier on a chip’. Organs-on-a-Chip. 5, 10025 (2023).
