在开顶器官芯片上重建人上皮组织的细胞结构和功能
Summary
本协议描述了开顶器官芯片在成功建立和成熟原代组织（皮肤、肺泡、气道和肠）的全层器官芯片培养物方面的能力和基本培养方式，提供了研究人上皮/间充质和血管生态 位界面的不同功能方面的机会体外。
Abstract
几乎所有人体器官都衬有上皮组织，包括一层或多层紧密连接的细胞，这些细胞组织成三维（3D）结构。上皮的主要功能之一是形成屏障，保护下层组织免受物理和化学侮辱和感染因子的侵害。此外，上皮介导营养物质、激素和其他信号分子的转运，通常产生指导细胞在器官内定位和区室化的生化梯度。由于其在决定器官结构和功能方面的核心作用，上皮是许多人类疾病的重要治疗靶点，这些疾病并不总是被动物模型捕获。除了明显的物种间差异外，对动物上皮的屏障功能和运输特性进行研究由于难以在生命系统中访问这些组织而进一步复杂化。虽然二维（2D）人类细胞培养物可用于回答基本的科学问题，但它们通常产生较差 的体内 预测。为了克服这些限制，在过去十年中，大量被称为器官芯片的微工程仿生平台已成为传统 体外 和动物试验的有前途的替代方案。在这里，我们描述了一个开顶器官芯片（或开顶芯片），一个设计用于模拟器官特异性上皮组织的平台，包括皮肤、肺和肠道。该芯片为重建上皮组织的多细胞结构和功能提供了新的机会，包括通过将组织特异性成纤维细胞和内皮细胞整合到机械活性系统中来重建3D基质成分的能力。这种开顶芯片为从单细胞到多层组织构建体的多种分辨率尺度研究上皮/间充质和血管相互作用提供了一种前所未有的工具，从而允许分子解剖健康和疾病中上皮化器官的细胞间串扰。
Introduction
从历史上看，科学家们一直依靠临床前动物试验来发现药物，但由于与人类结果的相关性差，越来越多的这些方法受到质疑1。实施“3R”原则来替代、减少和改进动物实验，促使科学家寻找新的体外替代方法，以支持临床前药物和化学毒理学风险评估2。然而，迄今为止开发的许多体外模型缺乏概括人类活体器官动态性质所必需的生物结构，细胞复杂性和机械环境3，4。
传统的 体外 临床前系统通常采用在刚性塑料表面上生长的人类细胞的2D单培养物。这些方法为进行简单的机理研究提供了一种工具，并能够快速筛选候选药物。由于其相对较低的成本和高稳健性，2D模型通常与自动高通量系统配对，并在药物开发过程的早期阶段用于快速识别潜在的候选药物5，6。然而，这种2D模型不能提供一种转化方法来模拟对候选治疗药物的组织水平、器官水平或全身反应，这是在其开发的临床前阶段准确预测药物安全性和有效性所必需的。扁平细胞培养不能概括天然组织微环境，包括复杂的多细胞相互作用、生物力学特性和人体组织的三维（3D）结构7。在平坦表面上生长的细胞通常不会获得成熟的表型，因此不能像在天然组织中那样对药理学刺激做出反应。例如， 体外 生长的原代人肺泡上皮细胞表现出鳞状表型并失去关键表型标记，包括表面活性蛋白 C 和 B（SP-C 和 SP-B）8。除了分化不足外，原代细胞经常在 体外对生物应激源不敏感，因为与组织炎症相关的某些生化途径变得无功能9。这种细胞功能的丧失似乎主要与使用坚硬的底物以及缺乏组织特异性基质细胞（如肺成纤维细胞和平滑肌细胞）自然释放的可溶性因子有关10，11。
认识到缺乏化学物理和生物学复杂性限制了体外细胞的生理行为，促进了更复杂的多细胞模型的发展，这些模型已被证明可以更好地捕获体 外 人体组织的复杂性12，13。自 1970 年代初创建第一个共培养模型14 以来，合成和天然水凝胶的引入显着提高了模拟天然组织微环境的能力，并已成为驱动细胞分化、引导细胞自组织成组织样结构和恢复天然组织功能的宝贵工具15，16.例如，当在适当的3D支架中生长时，人类细胞可以自我排列成功能结构，如球状体或类器官，表达干细胞标志物，并且能够自我更新17。相比之下，人类细胞（包括干细胞）在传统的2D基质上生长时，在几次传代后会迅速老化并衰老18。此外，水凝胶可以“定制”以匹配特定的组织特性，如孔隙率、孔径、纤维厚度、粘弹性、形貌和刚度，或者进一步设计组织来源的细胞成分和/或生物活性分子，从而模拟生理或病理条件19，20.尽管它们在药物测试方面具有巨大的潜力，但用于药物研究的基于3D水凝胶的模型并不能完全概括 体内 组织的复杂细胞结构，并且缺乏通常存在于人体中的重要血液动力学和机械刺激，包括静水压力，循环拉伸和流体剪切21。
微生理系统（MPS），如器官芯片（OOCs）最近成为能够在体外捕获复杂生理反应的工具22，23。这些模型通常使用微流体平台，可以对活器官的动态微环境进行建模。
我们将3D组织生物工程和机械生物学的原理相结合，创建了复杂人类上皮组织的开顶芯片模型。这使我们能够密切概括上皮组织的多细胞和动态微环境。这包括天然存在于活体器官中的组织特异性生化和生物力学线索，但经常被传统的体外模型所忽视24。开顶芯片包含两个隔室：一个血管隔室（图1A）和一个基质隔室（图1B），由多孔膜隔开，允许营养物质在两个腔室之间扩散（图1C）。血管室暴露于连续的流体流动以概括生理剪切应力，而基质室的可拉伸设计允许模拟与呼吸运动或肠道蠕动相关的机械应变。基质室容纳可调谐的3D水凝胶支架，旨在支持组织特异性成纤维细胞的生理生长。它有一个可拆卸的盖子，便于建立气液界面，这种情况允许更好地模拟粘膜组织的人体生理学，以及直接进入组织以将药物直接施用到上皮层。补充图1捕获了开顶芯片设计的一些关键组件，包括尺寸和生物隔室（补充图1A-D）以及该协议中描述的主要技术步骤（补充图1E）。
开顶芯片的灌注是通过可编程蠕动泵实现的（图1D）。蠕动泵设置允许同时灌注 12 个开顶芯片。大多数培养箱可以容纳两种设置，每个培养箱最多可培养 24 个芯片。机械拉伸是使用定制的可编程真空压力调节器实现的（图1E）。它由一个由数模转换器电子控制的电动气动真空调节器组成。换句话说，电-气真空调节器产生正弦真空曲线，其幅度和频率由用户决定。通过以 0 至 -90 kPa 的振幅和 0.2 Hz 的频率向开顶芯片的真空通道施加负压，产生 0% 至 15% 的循环应变。它是一个定制的系统，相当于之前在其他论文25中采用和描述的市售Flexcell应变单元。为了模拟与例如肺的呼吸运动或肠道蠕动相关的机械组织变形，气动执行器应用正弦真空/应变波，其幅度和振幅可以调节以匹配人体细胞在其天然组织中经历的生理应变水平和频率。
在这里，我们描述了一种在原型开顶芯片平台上工程和培养器官型上皮等效物的高效且可重复的方法。它允许生成复杂的器官模型，如皮肤、肺泡、气道和结肠，同时整合血管液流和机械拉伸。我们将概述在实施组织工程原理以生成复杂的上皮模型时必须考虑的关键技术方面。我们将讨论当前设计的优点和可能的局限性。
用于实现组织和器官成熟的主要步骤的概述，包括流动和拉伸参数，报告如下：图2为皮肤，图3为肺泡，图4为气道，图5为肠道。有关培养基组成和用于培养不同器官模型的试剂的其他信息包含在补充表中（皮肤补充表1;肺泡补充表2;补充表3为气道，补充表4为肠道）。
Protocol
根据辛辛那提儿童医院机构生物安全委员会（IBC 2017-2011）的指南，从肠切除术中获得人类结肠样体。 1. 表面活化 活化缓冲液的制备将交联剂和溶剂缓冲试剂置于生物安全柜（BSC）下，并在使用前在室温（RT）下平衡10分钟。 使用无菌不透光容器或用铝箔包裹的透明 15 mL 锥形管在 5 mL 溶剂缓冲液中复溶 5 mg 交联剂，以保护交联剂溶液免受直?…
Representative Results
表面微图案化细胞外基质（ECM）的微图案可用于复制肠隐窝界面的空间配置。可以修改开顶芯片配置，以集成专门设计的微图案邮票，以模仿结肠上皮-基质界面（图6A，B）和微米级的肠隐窝的自然形貌（图6C-E）。请注意，我们为皮肤、气道和肺泡模型使用了平坦（无图案）的表面。在这种情况下?…
Discussion
Open-Top Chip代表了一个使能平台，用于实时研究受控微环境中内皮、基质和上皮之间发生的复杂细胞相互作用。与传统的器官型和类器官培养相比，该技术具有关键优势，例如整合与重建人体组织微环境相关的物理和生化线索，包括流体剪切（流动）、循环拉伸和通过微图案 实现 的上皮表面形貌重建。在该平台内生长的人类细胞协同作用，以概括可以通过常规技术 分析 的组织特异?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
没有
Materials
| 10x EMEM | Lonza | 12-684F | Medium; Stroma |
| 18 Gauge needle | MicroGroup | 316H18RW | Tube stainless steel 316 welded, 18RW Full Hard |
| 19 Gauge needle | MicroGroup | 316H19RW | Tube stainless steel 316 welded, 19RW Full Hard |
| 2-Stop PharMed BPT | Cole-Palmer | EW-95723-12 | Tube, 0.25 mm, 12/pack |
| 70% ethanol and wipes | - | - | For surface sterilization |
| 8-Bromoadenosine 3′,5′-cyclic monophosphate sodium salt (8-Br-cAMP) | Sigma | B7880 | Medium supplement |
| A-83-01 | Tocris | 2939 | |
| Adenine | Sigma | A9795 | |
| Advanced DMEM/F12 | Thermo | 12634010 | |
| Airway Epithelial Cells | Lifeline Cell Technology | FC-0016 | |
| Aluminum foil | - | - | - |
| Alveolar cells | Cell Biologics | H6621 | |
| Anti-ABCA3 | ABCAM | ab24751 | Mouse monoclonal antibody [3C9] |
| Anti-Aquaporin5 Alexa Fluor 647 | ABCAM | ab215225 | Rabbit monoclonal antibody [EPR3747] |
| Anti-Aquaporin5 | ABCAM | ab92320 | Rabbit monoclonal antibody [EPR3747] |
| Anti-beta IV Tubulin | ABCAM | ab11315 | Mouse monoclonal antibody [ONS.1A6] |
| Anti-CD31 (PECAM-1) | ABCAM | ab9498 | Mouse monoclonal [JC/70A] antibody |
| Anti-CK5 | ABCAM | ab75869 | Rabbit recombinant monoclonal [AY1E6] |
| Anti-Cytokeratin 10 | ThermoFisher | MA5-13705 | Mouse monoclonal antibody (DE-K10) |
| Anti-Cytokeratin 14 | ABCAM | ab7800 | Mouse monoclonal antibody |
| Anti-E-Cadherin | ABCAM | ab1416 | Mouse monoclonal antibody |
| Anti-Filaggrin | ThermoFisher | PA5-79267 | Rabbit polyclonal antibody |
| Anti-HTI-56 | Terrace Biotech | TB-29AHT1-56 | Mouse monoclonal antibody (IgG1) |
| Anti-HTII-280 | Terrace Biotech | TB-27AHT2-280 | Mouse monoclonal antibody (IgM) |
| Anti-Involucrin | ThermoFisher | MA5-11803 | Mouse monoclonal antibody (SY5) |
| Anti-Isoforms TA p63-α, -β, -γ | Biolengend | 618902 | Rabbit polyclonal antibody |
| Anti-Ki67 | ABCAM | ab8191 | Mouse monoclonal antibody [B126.1] |
| Anti-LAMP3 | ABCAM | ab111090 | Rabbit polyclonal antibody |
| Anti-Mature SP-B | Seven Hill | WRAB-48604 | Rabbit polyclonal antibody |
| Anti-MUC5AC | ThermoFisher | PA5-34612 | Rabbit polyclonal antibody |
| Anti-Mucin-2 | SantaCruz Biotechnology | sc-7314 | Mouse monoclonal antibody (IgG1) |
| Anti-p63 | Dako | GA662 | Mouse monoclonal antibody p63 Protein (Dako Omnis) Clone DAK-p63 |
| Anti-PCNA | ThermoFisher | PA5-32541 | Rabbit polyclonal antibody |
| Anti-Podoplanin (AT-1α) | ABCAM | ab128994 | Rabbit polyclonal antibody |
| Anti-Pro + Mature Surfactant Protein B | ABCAM | ab40876 | Rabbit polyclonal antibody |
| Anti-Surfactant C | Seven Hill | WRAB-9337 | Rabbit polyclonal antibody |
| Anti-Uteroglobin/SCGB1A1 | Hycult Biotech | HM2178 | Mouse monoclonal antibody [AY1E6] |
| Anti-VE-cadherin | ABCAM | ab33168 | Rabbit polyclonal antibody |
| Anti-ZO-1 | ThermoFisher | 33-9100 | Mouse monoclonal antibody [1A12] |
| Ascorbic acid | Sigma | A4544 | |
| Aspirating pipettes | Corning / Falcon | 357558 | 2 mL, polystyrene, individually wrapped |
| Aspirating tips | - | - | Sterile (autoclaved) |
| B27 | Thermo | 17504044 | |
| Blocker BSA (10X) in PBS solution | ThermoFisher | 37525 | Blocker agent |
| Calcium Chloride | Sigma | C7902 | |
| CHIR 99021 | Tocris | 4423 | |
| Collagen I | Advanced Biomatrix | 5133 | 10 mg/mL (Stroma) |
| Collagen I | Advanced BioMatrix | 5005 | 3 mg/mL (Vascular ECM) |
| Collagen IV | Sigma | C5533 | |
| Collagen-IV | Sigma | C5533-5MG | Collagen from human placenta, 5 mg powder, reconstitute to 1 mg/mL |
| Colonic Fibroblasts | Cell Biologics | H6231 | |
| Colonic microvascular endothelial cells | Cell Biologics | H6203 | |
| Conical tubes | - | - | 15 mL and 50 mL polypropylene, sterile |
| Crosslinker (ER-1) | Emulate | 10461 | 5 mg powder |
| DAPI (4',6-Diamidino-2-Phenylindole, Dilactate) | ThermoFisher | D3571 | DNA probe |
| Dermal fibroblasts | ATCC | PCS-201-010 | |
| Dermal microvascular endothelial cells | ATCC | CRL-3243 | |
| Dexamethasone | Sigma | D4902 | |
| DMEM | ThermoFisher | 11054020 | |
| DMEM/F-12 | GIBCO | 11320082 | |
| DMEM/F-12, GlutaMAX | GIBCO | 10565-018 | Basal medium for ALI medium |
| Donkey Anti-Mouse IgG H&L (Alexa Fluor 488) | ABCAM | ab150105 | Donkey Anti-Mouse secondary antibody |
| Donkey Anti-Mouse IgG H&L (Alexa Fluor 568) | ABCAM | ab175472 | Donkey Anti-Mouse secondary antibody |
| Donkey Anti-Mouse IgG H&L (Alexa Fluor 647) | ABCAM | ab150107 | Donkey Anti-Mouse secondary antibody |
| Donkey Anti-Rabbit IgG H&L (Alexa Fluor 488) | ABCAM | ab150073 | Donkey Anti-Mouse secondary antibody |
| Donkey Anti-Rabbit IgG H&L (Alexa Fluor 568) | ABCAM | ab175470 | Donkey Anti-Mouse secondary antibody |
| Donkey Anti-Rabbit IgG H&L (Alexa Fluor 647) | ABCAM | ab150075 | Donkey Anti-Mouse secondary antibody |
| Dulbecco’s PBS (DPBS-/-) (without Ca2+, Mg2+) | Corning | 21-031-CV | 1x |
| Epidermal Growth Factor (EGF) human, recombinant in E. coli | PromoCell | C-60170 | Medium supplement |
| F-12 Ham’s | Invitrogen | 21700-108 | For vascular ECM |
| FibriCol | Advanced BioMatrix | 5133-20ML | Collagen-I solution (10 mg/mL) |
| Fibronectin | Corning | 356008 | |
| Fibronectin, Human, Natural, | Corning | 47743-654 | human plasma fibronectin |
| Fine-tip precision tweezers | Aven | 18056USA | Technik Style 5B-SA Precision Stainless Steel Tweezers |
| Glutamax | Invitrogen | 21700-108 | |
| Glutamax | Invitrogen | 35050061 | |
| Goat Anti-Mouse IgG H&L (Alexa Fluor 594) | ABCAM | ab150080 | Goat Anti-Mouse secondary antibody |
| Goat Anti-Mouse IgG H&L (Alexa Fluor 647) | ABCAM | ab150115 | Goat Anti-Mouse secondary antibody |
| Goat Anti-Mouse IgG H&L (FITC) | ABCAM | ab6785 | Goat Anti-Mouse secondary antibody |
| Goat Anti-Mouse IgG1 Alexa Fluor 568 | ThermoFisher | A-21124 | Goat Anti-Mouse IgG1 secondary antibody |
| Goat Anti-Mouse IgM Alexa Fluor 488 | ThermoFisher | A-21042 | Goat Anti-Mouse IgM secondary antibody |
| Handheld vacuum aspirator | Corning | 4930 | - |
| Heat Inactivated HyClone FetalClone II Serum (FCS) | GE Healthcare Life Sciences | SH30066.03 | |
| Hemocytometer | - | - | - |
| Heparin sodium salt from porcine intestinal mucosa | Sigma | H3149 | |
| HEPES | Thermo | 15630080 | |
| Human [Leu15] – Gastrin | Sigma | G9145 | |
| Human colonoids | Obtained from clinical resections | Obtained from clinical resections | |
| Human EGF Recombinant Protein | Thermo | PHG0311L | |
| human epithelial growth factor | Thermo | PHG0311 | |
| HyClone FetalClone II Serum (U.S.) | GE Healthcare | SH30066.02HI | Sterile FBS heat-inactivated |
| Hydrocortisone 21-hemisuccinate sodium salt | Sigma | H4881 | |
| Hydrocortisone | PromoCell | C-64420 | Medium supplement |
| Ice bucket | - | - | - |
| Ismatec IPC-N | Cole-Palmer | EW-78000-41 | Low-Speed Digital Peristaltic Pump; q24-Channel (1 per 12 Chips) |
| ITES | BioWhittaker | 17-839Z | |
| Keratinocyte Growth Factor (KGF), also known as Basic Fibroblast Growth Factor 7 (FGF-7), human, recombinant in HEK | PromoCell | C-63821 | |
| Keratinocytes | ATCC | PCS-200-010 | |
| Laminin | Biolamina | CT521-0501 | |
| Laminin, 521 CTG (CT521) | Biolamina | CT521-0501 | human recombinant laminin 521 |
| Lung Fibroblast | Cell Biologics | H6013 | |
| Lung Fibroblast | Lifeline Cell Technology | FC-0049 | |
| Lung microvascular endothelial cells | Lonza | CC-2527 | |
| Lung smooth muscle cells | Lifeline Cell Technology | FC-0046 | |
| Manual counter | - | - | - |
| Masterflex (TPE) Transfer Tubing | Cole-Palmer | FV-96880-02 | PharMed BPT, 1/32" ID x 5/32" OD |
| Medium 199, no phenol red | Thermo | 11043023 | |
| Microcentrifuge tube | - | - | 1.5 mL, sterile |
| Microscope (with camera) | - | - | For bright-field imaging |
| N2 | Sigma | 17502001 | |
| N-acetyl cysteine | Sigma | A5099 | |
| Noggin (HEK293T conditioned medium) | Sigma | N17001 | |
| Normal Goat Serum | ThermoFisher | 50062Z | Blocking solution |
| O-phosphosrylethanolamine | Sigma | P0503 | |
| Paraformaldehyde (4% wt/vol) | EMS | 15710 | Fixing agent |
| Penicillin Streptomycin | GIBCO | 15140122 | |
| Penicillin-streptomycin | Sigma | P4333 | 10,000 U/mL; 10 mg/mL |
| Pipette tips | - | - | P20, P200, and P1000 sterile, low adhesion |
| Pipette | Gilson | F167380 | P20, P200, and P1000 |
| PluriQ Serum Replacement (or alternatively KO Serum replacement) | AMSBIO (or Thermo) | N/A (or C1910828010) | |
| Poly-L-Lysine coated microscope glass slides | Sigma | P0425 | Glass slides |
| Primocin | InvivoGen | ant-pm-1 | |
| Progesterone | Sigma | P8783 | |
| ProLong Gold | ThermoFisher | P36931 | Antifade Mountant with DAPI |
| Retinoic Acid | Sigma | R2625 | |
| ROCK inhibitor (Y27632) | Tocris | TB1254-GMP/10 | |
| R-spondin (HEK293T conditioned medium) | Sigma | SCC111 | |
| SAGM SingleQuots supplements | Lonza | CC-4124 | |
| SAGMTM Small Airway Epithelial Cell Growth medium BulletKitTM | Lonza | CC-4124 | Medium supplements |
| SB2001190 | Tocris | 1264/10 | |
| Serological pipettes | - | - | 2 mL, 5 mL, 10 mL, and 25 mL low endotoxin, sterile |
| Small Airway Epithelial Cell Growth medium (SAGM) | Lonza | CC-4124 | |
| Solvent Buffer (ER-2) | Emulate | 10462 | 25 mL bottle |
| Steriflip-HV | Millipore | SE1M003M00 | Sterile filtering conical tube |
| Sterilin 100 mm Square Petri Dishes | Thermo | 103 | Sterile, 1 per 6 chips |
| T25 flasks | - | - | - |
| T75 flasks | - | - | - |
| Tri-iodothyronine | Sigma | T5516 | |
| Triton X-100 (0.3% (vol/vol) | Sigma | T8787 | Permeabilization agent |
| Trypan blue | Sigma | 93595 | 0.4% solution |
| TrypEE solution | Sigma | 12604013 | Cell detaching solution |
| TWEEN-20 | Sigma | P2287 | Permeabilization agent |
| UV Light Oven (peak frequency 365nm, intensity of 100 µJ/cm2) | VWR | 21474-598 | UVP, Long Range UV, 365 nm 60Hz Model CL-1000L |
| Vacuum set-up | - | - | Minimum pressure: -70 kPa |
| Vascular Endothelial Growth Factor 165 (VEGF-165) human, recombinant in E. coli | PromoCell | C-64420 | |
| VEGF-165 | PromoCell | C-64420 | Medium supplement |
| Von Willebrand Factor conjugated FITC | ABCAM | ab8822 | Sheep polyclonal antibody |
| Water bath (or beads) | - | - | Set to 37 °C |
| Wnt3A (L-Wnt3A conditioned medium) | ATCC | CRL-2647 |
Riferimenti
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Citazione di questo articolo
Antonio, V., Panchal, A., Kasendra, M., Riccardo, B. Reconstituting Cytoarchitecture and Function of Human Epithelial Tissues on an Open-Top Organ-Chip. J. Vis. Exp. (192), e64633, doi:10.3791/64633 (2023).