3D 细胞打印的芯片上低氧癌症，用于回顾固体癌症的病理进展
1Department of Mechanical Engineering,Pohang University of Science and Technology (POSTECH), 2Department of Creative IT Engineering,Pohang University of Science and Technology (POSTECH), 3Department of Rural and Biosystems Engineering, College of Agriculture and Life Sciences,Chonnam National University
Summary
缺氧是肿瘤微环境的标志，在癌症进展中起着至关重要的作用。本文描述了基于3D细胞打印技术的芯片上低氧癌症的制造过程，以回顾癌症的缺氧相关病理学。
Abstract
癌症微环境对疾病的进展有重大影响。特别是缺氧是癌症生存、入侵和耐受性的关键驱动因素。虽然已经开发了几个体外模型来研究缺氧相关的癌症病理学，但由于缺乏精确的空间控制，在体内观察到的癌症微环境的复杂相互作用尚未被复制。相反，提出了3D生物制剂方法，以创建微生理系统，以更好地模拟癌症生态学和准确的抗癌治疗评估。在此，我们提出了一种3D细胞打印方法，以制造一个芯片上的低氧癌症。芯片中的缺氧诱导组件是根据计算机对氧气分布的模拟确定的。使用含有胶质母细胞和内皮细胞的生物因子印刷癌症-斯特罗马同心环，以重新概括一种实体癌症。由此产生的芯片实现了癌症的中枢缺氧和加重恶性肿瘤，并形成具有代表性的病理生理标记。总体而言，为癌症研究创造实体癌-致密微生理系统的拟议方法有望弥合体内模型和体外模型之间的差距。
Introduction
癌症微环境是推动癌症进展的关键因素。多种成分，包括生化、生物物理和细胞线索，决定了癌症的病理特征。其中，缺氧与癌症的生存、增殖和入侵密切相关。由于癌细胞的无限生长和分裂，营养物质和氧气不断枯竭，产生缺氧梯度。在低氧条件下，细胞激活缺氧诱导转录因子（HIF）相关分子级联。这个过程诱导坏死核心，触发代谢变化，并启动血管增生和转移2，3。随后，癌细胞缺氧导致邻近正常组织的破坏。此外，缺氧与多因素的实体肿瘤的治疗耐药性密切相关。缺氧可能严重阻碍放射治疗，因为放射性敏感性有限，由于反应性氧物种1，4。此外，它降低了癌症微环境的pH水平，从而减少了药物积累1。因此，复制体外缺氧病理特征是科学和临床前发现有希望的策略。
模拟癌症的特定微环境对于了解癌症发展和探索适当的治疗方法至关重要。虽然动物模型由于其强烈的生理相关性而得到广泛应用，但与物种差异和伦理问题有关的问题依然存在。此外，虽然传统的2D和3D模型允许对癌细胞进行操作和实时成像，以便进行深入分析，但无法完全重新概括其结构和细胞的复杂性。例如，癌症球形模型已被广泛使用，因为球体中的癌细胞聚集自然会产生核心缺氧。此外，大量大小均匀的细胞球体已经使用塑料或硅基多井系统6，7生产。然而，在用传统平台捕捉癌组织的确切异质结构方面灵活性较低，这就要求建立先进的生物制剂技术，以建立一个高度仿生平台，以改善癌症研究。
3D微生理系统（MPSs）是回顾癌细胞9的复杂几何和病理进展的有用工具。当癌细胞感知生长因子和化疗因子的生化梯度以及系统中复制的机械异质性时，可以在体外研究癌症发展的重要特征。例如，使用MPS10、11研究了癌症的生存能力、转移恶性肿瘤和根据氧气浓度变化而形成的耐药性。尽管最近取得了进步，但产生体外模型的缺氧条件依赖于复杂的制造过程，包括与物理气体泵的连接。因此，需要简单和灵活的方法来建立癌症特有的微观环境。
3D细胞打印技术由于精确控制生物材料的空间排列，以回顾原生生物结构12而备受关注。特别是，该技术克服了3D缺氧模型的现有局限性，因为它具有很高的可控性和构建癌症微环境空间特征的可行性。3D 打印还通过一层一层的过程促进计算机辅助制造，从而提供快速、准确和可重复的复杂几何结构，以模拟实际的组织结构。除了现有的3D MPS制造策略的优势外，癌症进展的病理生理特征可以通过对生化、细胞和生物物理成分13、14的模式进行再现。
在此，我们提出了一个3D细胞打印策略，用于在芯片上复述一种缺氧癌（图1）15的异质性。制造参数是通过系统中中央缺氧形成的计算模拟确定的。使用含有胶质母细胞和内皮细胞的胶原蛋白生物因子印刷癌症-频闪同心环，以模拟胶质母细胞瘤的病理生理学，胶质母细胞瘤是一种实体癌症。径向氧梯度的形成加重了癌症恶性肿瘤，表明攻击性增强。此外，我们为芯片应用于患者特定的预科模型指明了未来的观点。提议的创建实体癌症-致密微生理系统的方法有望弥合体内和体外癌症模型之间的差距。
Protocol
1. 氧气梯度形成的计算机模拟 用于芯片上低氧癌症打印的 3D 几何模型的生成 运行3D CAD软件。 勾勒出芯片上低氧癌症的几何模型。单击 草图 并选择所需的平面来绘制几何形状。请参阅图（图2A）了解每个部分的详细比例。 通过单击 功能突出型主/基座设置几何形状的厚度。在空框中输入所需的厚度（参?…
Representative Results
芯片上的缺氧癌是利用计算机辅助的3D细胞打印技术来重新概括缺氧和癌症相关病理学（图1）的。使用 3D 几何模型模拟氧气运输和消耗。该芯片设计为同心环，以模拟径向氧扩散和耗竭，在癌症组织（图2A）。在定义了氧气扩散并被细胞消耗的空间的控制体积后，通过计算有限元素分析（图2B，C）</…
Discussion
在这项研究中，我们描述了基于3D细胞打印技术的芯片上低氧癌症的制造过程。通过计算机模拟预测了设计芯片中低氧梯度的形成。通过结合 3D 打印气体透气屏障和玻璃盖的简单策略，再现了可诱导异质缺氧梯度的环境。胶质母细胞瘤的缺氧相关病理特征，包括伪帕利塞德和少量癌症干细胞，在芯片的低氧梯度条件下被重新概括。
为了提高生产率和可重复性，与之前公布的?…
Declarações
The authors have nothing to disclose.
Acknowledgements
这项研究得到了韩国国家研究基金会（NRF）的支持，该基金会由教育部（第2020R1A6A1A03047902号和NRF-2018H1A2A1062091）和韩国政府（MSIT）资助（ NO.NRF-2019R1C1C1009606和NRF-2019R1A3A3005437）。
Materials
| Cells | |||
| Human umbilical vein endothelial cells | Promocell | C-12200 | |
| U-87 MG cells | ATCC | ATCC HTB-14 | |
| Disposable | |||
| 0.2 μm syringe filter | Sartorius | 16534-K | |
| 10 mL disposable syringe | Jung Rim | 10ml 21G32 | |
| 10 mL glass vial | Hubena | A0039 | |
| 10 mL Serological pipette tip | SPL lifescience | 91010 | |
| 15 mL conical tube | SPL lifescience | 50015 | |
| 18G plastic needle | Musashi engineering | PN-18G-B | |
| 20G plastic tapered dispense tip | Musashi engineering | TPND-20G-U | |
| 22×50 glass cover | MARIENFIELD | 0101142 | |
| 25 mL Serological pipette tip | SPL lifescience | 90125 | |
| 3 mL disposable syringes | HENKE-JET | 4020-X00V0 | |
| 40 µm cell strainer | Falcon | 352360 | |
| 5 mL Serological pipette tip | SPL lifescience | 91005 | |
| 50 mL conical tube | SPL lifescience | 50050 | |
| 50 mL Serological pipette tip | SPL lifescience | 90150 | |
| 50N precision nozzle | Musashi engineering | HN-0.5ND | |
| Aluminum foil | SINKWANG | ||
| Capillary tips | Gilson | CP1000 | |
| Cell-scrapper | SPL lifescience | 90030 | |
| Confocal dish | SPL lifescience | 200350 | |
| Parafilm | Bemis | PM996 | |
| Pre-coated histology slide | MATSUNAMI | MAS-11 | |
| Reservoir | SPL lifescience | 23050 | |
| T-75 cell culture flask | SPL lifescience | 70075 | |
| Equipment | |||
| 3DX printer | T&R Biofab | ||
| Autoclave | JEIOTECH | AC-12 | |
| Centrifuger | Cyrozen | 1580MGR | |
| Confocal laser microscopy | Olympus Life Science | FV 1000 | |
| Fluorescence microscope | FISHER SCEINTIFIC | O221S366 | |
| Forcep | Korea Ace Scientific | HC.203-30 | |
| Hand tally counter | KTRIO | ||
| Hemocytometer | MARIENFIELD | 0650030 | |
| Incubator | Panasonic | MCO-170AIC | |
| Laminar flow cabinet | DAECHUNG SCIENCE | CB-BMMS C-001 | |
| Metal syringe | IWASHITA engineering | SUS BARREL 10CC | |
| Operating Scissors | Hirose | HC.13-122 | |
| Oven | JEIOTECH | OF-12, H070023 | |
| Positive displacement pipette | GILSON | NJ05652 | |
| Refrigerator | SAMSUNG | CRFD-1141 | |
| Voltex Mixer | DAIHAN scientific | VM-10 | |
| Water bath | DAIHAN SCIENTIFIC | WB-11 | |
| Water purifier | WASSER LAB | DI-GR | |
| Materials | |||
| 0.25 % Trypsin-EDTA | Gibco | 25200-072 | |
| 10x PBS | Intron | IBS-BP007a | |
| 4% Paraformaldehyde | Biosesang | ||
| 70% Ethanol | Daejung | 4018-4410 | |
| Anti-CD31 antibody | Abcam | ab28364 | |
| Anti-HIF-1 alpha antibody | Abcam | ab16066 | |
| Anti-SHMT2/SHMT antibody | Abcam | ab88664 | |
| Anti-SOX2 antibody | Abcam | ab75485 | |
| Bovine Serum Albumin | Thermo scientific | J10857-22 | |
| Collagen from porcine skin | Dalim tissen | PC-001-1g | |
| DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride) | Thermofisher | D1306 | |
| Endothelial Cell Growth Medium-2 | Promocell | C22011 | |
| Fetal bovine serum | Gibco | 12483-020 | |
| Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 | Theromofisher | A-11001 | |
| Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 594 | Theromofisher | A-11012 | |
| High-glucose Dulbecco’s Modified Eagle Medium(DMEM) | Hyclone | SH30243-0 | |
| Hydrochloric acid | Sigma-Aldrich | 311413-100ML | |
| Live/dead assay kit | Invitrogen | L3224 | |
| Mouse IgG1, kappa monoclonal [15-6E10A7] – Isotype Control | Abcam | ab170190 | |
| Penicillin/streptomycin | Gibco | 15140-122 | |
| Phenol red solution | Sigma-Aldrich | P0290-100ML | |
| Poly(ethylene-vinyl acetate) | Poly science | 06108-500 | |
| Polydimethylsiloxane | Dowhitech | sylgard 184 | |
| Rabbit IgG, polyclonal – Isotype Control | Abcam | ab37415 | |
| Sodium hydroxide solution | Samchun | S0610 | |
| Triton X-100 | Biosesang | TRI020-500-50 | |
| Trypan Blue | Sigma-Aldrich | T8154 | |
| Software | |||
| COMSOL Multiphysics 3.5a | COMSOL AB | ||
| IMS beamer | in-house software | ||
| SolidWorks Package | Dassault Systems SolidWorks Corporation | ||
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Park, W., Bae, M., Hwang, M., Jang, J., Cho, D., Yi, H. 3D Cell-Printed Hypoxic Cancer-on-a-Chip for Recapitulating Pathologic Progression of Solid Cancer. J. Vis. Exp. (167), e61945, doi:10.3791/61945 (2021).