结合人新生儿肠道类生物和菌群失调微生物组的坏死性小肠结肠炎微流控模型
Summary
该方案描述了坏死性小肠结肠炎（NEC）的 体外 模型，可用于疾病发病机制的机制研究。它的特点是微流控芯片，其中植入了源自人类新生儿肠道、内皮细胞和患有严重 NEC 的新生儿肠道微生物组的肠道类生物。
Abstract
坏死性小肠结肠炎 （NEC） 是一种严重且可能致命的肠道疾病，由于其复杂的发病机制而难以研究，目前尚不完全清楚。NEC 的病理生理学包括肠道紧密连接破坏、肠道屏障通透性增加、上皮细胞死亡、微生物菌群失调和炎症失调。研究NEC的传统工具包括动物模型、细胞系以及人类或小鼠肠道类器官。虽然使用这些模型系统的研究提高了该领域对疾病病理生理学的理解，但它们概括人类NEC复杂性的能力是有限的。现在已经开发了一种使用微流控技术的改进的NEC 体外 模型，称为NEC-on-a-chip。NEC-on-a-chip 模型由一个微流控装置组成，该装置接种了源自早产新生儿的肠样蛋白，与人内皮细胞和来自患有严重 NEC 的婴儿的微生物组共培养。该模型是NEC病理生理学机制研究的宝贵工具，也是新生儿肠道疾病药物发现测试的新资源。在本手稿中，将提供 NEC-on-a-chip 模型的详细说明。
Introduction
坏死性小肠结肠炎 （NEC） 影响早产儿，出生体重 < 1500 g 的早产儿发病率高达 10%1。NEC 的病理生理学很复杂，包括肠上皮损伤、肠紧密连接破坏、肠道屏障通透性增加、免疫失调和上皮细胞死亡 2,3。我们对NEC发病机制的理解仍然不完整，尽管进行了数十年的研究，但仍然没有有效的靶向治疗。
推进NEC研究的一个重大障碍是从人类婴儿中分离出的原代肠道组织的可用性有限且尺寸小。从NEC患儿身上切除的肠道组织通常坏死并严重受损，这使得对疾病发病前机制的研究变得复杂。例如，患有NEC的婴儿的小肠被免疫细胞淹没，并且还观察到肠道干细胞数量减少，上皮细胞增殖减少和上皮细胞凋亡增加4,5,6,7。这导致从这些样品中培养肠上皮细胞以及分离RNA和蛋白质的困难，这些RNA和蛋白质可以在这种恶劣的炎症环境中降解。此外，由于手术NEC婴儿的疾病过程已经晚期，因此对诱发疾病的因素进行机制研究是不可行的。这些局限性导致依赖动物模型进行NEC的机理研究。
已经为小鼠、大鼠、仔猪、兔子和狒狒建立了NEC的动物模型5,8,9,11。动物模型的一个优势是，NEC样肠道疾病是由与人类NEC发病相关的因素诱发的，包括菌群失调的微生物组、反复缺氧发作和缺乏母乳喂养5,8,10,11。此外，在实验性NEC期间观察到的炎症反应和病理变化与人类疾病平行5,9,12。虽然这些模型模仿了人类NEC的许多特征，但NEC在动物和人类中的病理生理学之间存在固有的差异。例如，NEC的小鼠模型是在足月出生的小鼠中诱导的，尽管它们的肠道发育不完整，但NEC的病理生理学在这种临床背景下本质上是不同的。出生时小鼠肠道基因表达与存活前人类胎儿相似，直到第 14 天才接近妊娠 22-24 周的早产新生儿 （P14）13。这混淆了小鼠NEC模型，因为在P10之后通常不能在小鼠中诱导肠道损伤。此外，小鼠的近交系缺乏人类新生儿的免疫学14 和微生物多样性15，这是另一个混杂因素。因此，在NEC研究中增加将原发性人类样本纳入可提高该领域研究的临床相关性。
传统上，对 NEC 体外机制的研究利用了源自成人肠道癌细胞的单型细胞系，例如结直肠腺癌 （Caco2） 和人结肠腺癌 （HT-29） 细胞16。这些模型很方便，但由于它们从成体癌细胞生长、非极化结构以及与培养物重复传代相关的表型变化，因此生理相关性有限。肠样蛋白改进了这些模型，因为它们可以从肠组织的隐窝生长，分化为所有肠上皮亚型，并形成三维 （3D） 绒毛状结构17,18,19,20。最近，肠样蛋白与微流控技术相结合，开发了一种小肠芯片模型，并提供了更具生理相关性的体外模型系统21。
最初的器官芯片微流控设备是在 2000 年代初期推出的22,23,24。第一个器官芯片模型是人类呼吸肺芯片25。紧随其后的是许多单器官模型，如肠 21、肝脏 26、肾脏27、骨髓 28、血脑屏障 29 和心脏30。这些器官芯片模型已用于研究急性、慢性和罕见疾病，包括急性放射综合征、31 慢性阻塞性肺病、32 和神经退行性疾病 33。这些芯片上细胞的极化性质以及由多孔膜隔开的两个细胞区室的存在允许对复杂的生理过程进行建模，例如灌注、化学浓度梯度和免疫细胞趋化性34,35。因此，这些微流控系统为研究人类疾病的病理生理学和机制提供了新的工具。
Kasendra 等人于 2018 年描述了小肠芯片模型，他们利用儿科（10-14 岁）小肠活检标本分化成肠样并在微流控设备上培养21。血管内皮细胞、连续介质流动和拉伸/松弛也被纳入该模型。他们观察到肠上皮亚型分化、3D 绒毛状轴的形成、粘液产生和小肠基因表达模式21。随着 NEC-on-a-chip 系统的开发，该微流控模型被应用于新生儿疾病，该系统结合了新生儿肠样蛋白、内皮细胞和来自 NEC36 新生儿的微生物组。NEC-on-a-chip 概括了人类 NEC 的许多关键特征，包括炎症基因表达、特化上皮细胞的丧失和肠道屏障功能降低36。因此，该模型在NEC研究中有许多应用，包括机理研究和药物发现。在这篇手稿中，提供了NEC-on-a-chip模型性能的详细协议。
Protocol
肠样蛋白来源于早产儿（妊娠 22 至 36 周出生）的小肠样本，这些样本是在 NEC 或其他具有非炎症性病因的肠道疾病手术时获得的。所有样本采集和处理均在圣路易斯华盛顿大学机构审查委员会（IRB 协议编号 201706182 和 201804040）和北卡罗来纳大学教堂山分校（IRB 协议编号 21-3134）的知情同意和批准后进行。 1.从人新生儿小肠中分离并铺板隐窝以建立肠样体 …
Representative Results
将肠样体接种到微流控装置上（图1）并如上所述培养。通过明场显微镜监测接种前细胞培养基质水凝胶中肠样蛋白的生长，然后在接种装置后随后的肠上皮细胞单层扩增（图2）。形成汇合的肠上皮细胞单层，随后发育成成熟的 3D 绒毛状结构（图 2）。这种接种有肠源性新生儿肠上皮和 HIMEC 的微流控装置被称为新生儿肠芯片模型<…
Discussion
这种 NEC-on-a-chip 系统是一种强大的新工具，可用于模拟 NEC 的病理生理学。该平台提供了一个复杂的微环境，通过结合具有连续管腔流动和拉伸的共培养系统，比以前的模型更接近 体内 肠道环境。这些条件促进了 3D 绒毛状结构的发展，该结构由高度极化的上皮组成，由成熟的上皮亚型和紧密连接组成（图 2）36。此外，顶端上皮表面很容易接触到实?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这份手稿得到了美国国立卫生研究院的 R01DK118568 （MG）、R01DK124614 （MG） 和 R01HD105301 （MG）、Chan Zuckerberg Initiative Grant 2022-316749 （MG）、Thrasher 研究基金早期职业奖 （LCF）、UNC 儿童发展早期职业调查员资助 （LCF） 的支持，通过捐赠者对北卡罗来纳大学教堂山分校的慷慨支持， 以及北卡罗来纳大学教堂山分校的儿科系。
Materials
| [Leu15]-Gastrin I human | Sigma-Aldrich | G9145 | |
| A 83-01 | Sigma-Aldrich | SML0788 | |
| Advanced Dulbecco's Modified Eagle Medium/Ham's F-12 | Gibco | 12634010 | |
| B-27 Supplement, serum free (50x) | Gibco | 17504044 | |
| Basic Bio-kit | Emulate | N/A | |
| BioTek Synergy 2 Multi-Mode Microplate Reader | Agilent | 7131000 | |
| BRAND Methacrylate (PMMA) Cuvettes, Semi-Micro | BrandTech | 759085D | |
| Cell Recovery Solution | Corning | 354270 | |
| CFX Opus Real-Time PCR Systems | Bio-Rad | 12011319 | |
| Chip Cradle | Emulate | N/A | |
| Chip-S1 Stretchable Chip | Emulate | N/A | |
| CHIR99021 | Sigma-Aldrich | SML1046 | |
| Clear TC-treated Multiple Well Plates, 48 well | Corning | 3548 | |
| Collagen from human placenta | Sigma-Aldrich | C5533 | |
| Collagenase, Type I, powder | Gibco | 17018029 | |
| Complete Human Endothelial Cell Medium with Kit | Cell Biologics | H-1168 | |
| Conical Polypropylene Centrifuge Tubes, 15 mL | Fisher Scientific | 05-539-12 | |
| Conical Polypropylene Centrifuge Tubes, 50mL | Fisher Scientific | 05-539-8 | |
| Countess Cell Counting Chamber Slides | Invitrogen | C10283 | |
| Countess II automated cell counter | Invitrogen | AMQAX1000 | |
| DAPI (4',6-Diamidino-2-Phenylindole, Dilactate) | Invitrogen | D3571 | |
| DAPT | Sigma-Aldrich | D5942 | |
| Dextran, Cascade Blue, 3000 MW, Anionic, Lysine Fixable | Invitrogen | D7132 | Permeability dye |
| Dimethyl sulfoxide (DMSO) | Sigma-Aldrich | D8418 | |
| Disposable PES Filter Units, 0.2um aPES membrane | Fisher Scientific | FB12566504 | |
| DMEM/F-12 | Gibco | 11320033 | |
| Donkey serum | Sigma-Aldrich | D9663 | |
| Dulbecco′s Modified Eagle′s Medium – high glucose | Sigma-Aldrich | D5796 | |
| Dulbecco′s Phosphate Buffered Saline (DPBS) | Gibco | 14190-136 | |
| EDTA, 0.5 M, pH 8.0 | Corning | 46-034-CI | |
| ER-1 surface activation reagent | Emulate | ER-1 | Chip Activation Reagent 1 |
| ER-2 surface activation reagent | Emulate | ER-2 | Chip Activation Reagent 2 |
| Fibronectin Human Protein, Plasma | Gibco | 33016015 | |
| Fisherbrand Petri Dishes with Clear Lid, 100mm | Fisher Scientific | FB0875713 | |
| Gelatin-Based Coating Solution | Cell Biologics | 6950 | |
| Genie Temp-Shaker 300 | Scientific Industries, Inc. | SI-G300 | |
| Gentamicin | Gibco | 15750060 | |
| HEPES, Liquid 1M Solution (238.3 mg/ mL) | Corning | 25-060-CI | |
| Hoechst 33342, Trihydrochloride, Trihydrate | Invitrogen | H3570 | |
| Human Collagen Type I | Sigma-Aldrich | CC050 | |
| Human Primary Small Intestinal Microvascular Endothelial Cells | Cell Biologics | H-6054 | |
| Inverted Microscope | Fisher Scientific | 03-000-013 | |
| Isotemp General Purpose Deluxe Water Baths | Fisher Scientific | FSGPD10 | |
| L-Glutamine | Gibco | 25030-081 | |
| Luria Broth (LB) agar, Miller | Supelco | L3027 | |
| L-WRN Cells | American Type Culture Collection | CRL-3276 | |
| Matrigel Growth Factor Reduced Basement Membrane Matrix, LDEV-free | Corning | 356231 | Cell Culture Matrix |
| N-2 Supplement (100x) | Gibco | 17502048 | |
| N-acetyl-L-cysteine | Sigma-Aldrich | 1009005 | |
| NAILSTAR UV LAMP | NailStar | NS-01-US | |
| NanoDrop OneC Microvolume UV-Vis Spectrophotometer | Thermo Scientific | 840-274200 | |
| Nicotinamide | Sigma-Aldrich | 72340 | |
| Orb-HM1 Hub Module | Emulate | N/A | |
| Paraformaldehyde | ThermoFisher | 047392.9L | |
| Penicillin-Streptomycin | Gibco | 15140122 | |
| Phosphate buffered saline (PBS) | Gibco | 10010023 | |
| Pipet-Lite Multi Pipette L8-200XLS+ | Rainin | 17013805 | |
| Pipette Tips TR LTS 1000µL S 768A/8 | Rainin | 17014966 | |
| Pod Portable Module | Emulate | N/A | |
| Premium Grade Fetal Bovine Serum (FBS)(Heat Inactivated) | Avantor Seradigm | 1500-500 | |
| QuantiTect Reverse Transcription Kit | QIAGEN | 205313 | |
| Recombinant Murine Epidermal Growth Factor (EGF) | PeproTech | 315-09 | |
| SB 431542 | Tocris | 1614 | |
| Square BioAssay Dish with Handles, not TC-treated | Corning | 431111 | |
| SsoAdvanced Universal SYBR Green Supermix | Bio-Rad | 1725271 | |
| Steriflip-GV Sterile Centrifuge Tube Top Filter Unit | Millipore | SE1M179M6 | |
| Sterile Cell Strainers, 70um | Fisher Scientific | 22-363-548 | |
| Sterile Syringes, 10mL | Fisher Scientific | 14-955-453 | |
| Straight, fine, sharp point scissors | Miltex Instruments | MH5-300 | |
| Thermo Scientific Sorvall X4R Pro-MD Centrifuge | Thermo Scientific | 75016052 | |
| Triton X-100 | Sigma-Aldrich | T8787 | Detergent |
| TRIzol Reagent | Invitrogen | 15596026 | RNA extraction reagent |
| Trypan Blue Solution, 0.4% (w/v) in PBS, pH 7.5 ± 0.5 | Corning | 25-900-CI | |
| TrypLE Express Enzyme (1X), no phenol red | Gibco | 12604013 | Enzymatic Dissociation Reagent |
| Trypsin-EDTA solution | Sigma-Aldrich | T4174 | |
| VIOS 160i CO2 Incubator, 165 L | Thermo Scientific | 13-998-252 | |
| Y-27632 | Tocris | 1254 | |
| Zoë-CM1 Culture Module | Emulate | N/A |
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Cite This Article
Frazer, L. C., Yamaguchi, Y., Jania, C. M., Lanik, W. E., Gong, Q., Singh, D. K., Mackay, S., Akopyants, N. S., Good, M. Microfluidic Model of Necrotizing Enterocolitis Incorporating Human Neonatal Intestinal Enteroids and a Dysbiotic Microbiome. J. Vis. Exp. (197), e65605, doi:10.3791/65605 (2023).