Name: 3d cell-printed hypoxic cancer-on-a-chip for recapitulating pathologic progression of solid cancer
Uploaded: 2021-01-05T14:30:00
Description: Watch this Scientific Journal Video about 3D Cell-Printed Hypoxic Cancer-on-a-Chip for Recapitulating Pathologic Progression of Solid Cancer at JoVE.com

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）。

1. 氧气梯度形成的计算机模拟
<ol><li>
用于芯片上低氧癌症打印的 3D 几何模型的生成
	<ol>
<li>运行3D CAD软件。</li>
		<li>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。</li>
		<li>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?...

<ol>
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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia-modified+cancer+cell+metabolism.">Hypoxia-modified cancer cell metabolism.</a> Frontiers in Cell and Developmental Biology. 7, 4 (2019).</li><li>Petrova, V., Annicchiarico-Petruzzelli, M., Melino, G., Amelio, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+hypoxic+tumour+microenvironment.">The hypoxic tumour microenvironment.</a> Oncogenesis. 7 (1), 1-13 (2018).</li><li>Hockel, M., Vaupel, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+hypoxia:+definitions+and+current+clinical,+biologic,+and+molecular+aspects.">Tumor hypoxia: definitions and current clinical, biologic, and molecular aspects.</a> Journal of the National Cancer Institute. 93 (4), 266-276 (2001).</li><li>Kim, H., Lin, Q., Glazer, P. 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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+tumor+spheroids+as+in+vitro+models+to+mimic+in+vivo+human+solid+tumors+resistance+to+therapeutic+drugs.">3D tumor spheroids as in vitro models to mimic in vivo human solid tumors resistance to therapeutic drugs.</a> Biotechnology and Bioengineering. 116 (1), 206-226 (2019).</li><li>Wan, L., Neumann, C., LeDuc, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor-on-a-chip+for+integrating+a+3D+tumor+microenvironment:+chemical+and+mechanical+factors.">Tumor-on-a-chip for integrating a 3D tumor microenvironment: chemical and mechanical factors.</a> Lab on a Chip. 20 (5), 873-888 (2020).</li><li>Nam, H., Funamoto, K., Jeon, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+cell+migration+and+cancer+drug+screening+in+oxygen+tension+gradient+chip.">Cancer cell migration and cancer drug screening in oxygen tension gradient chip.</a> Biomicrofluidics. 14 (4), 044107 (2020).</li><li>Palacio-Casta&#241;eda, V., Kooijman, L., Venzac, B., Verdurmen, W. P., Le Gac, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+switching+of+tumor+cells+under+hypoxic+conditions+in+a+tumor-on-a-chip+model.">Metabolic switching of tumor cells under hypoxic conditions in a tumor-on-a-chip model.</a> Micromachines. 11 (4), 382 (2020).</li><li>Ronaldson-Bouchard, K., Vunjak-Novakovic, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organs-on-a-chip:+a+fast+track+for+engineered+human+tissues+in+drug+development.">Organs-on-a-chip: a fast track for engineered human tissues in drug development.</a> Cell Stem Cell. 22 (3), 310-324 (2018).</li><li>Mi, S., Du, Z., Xu, Y., Sun, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+crossing+and+integration+between+microfluidic+technology+and+3D+printing+for+organ-on-chips.">The crossing and integration between microfluidic technology and 3D printing for organ-on-chips.</a> Journal of Materials Chemistry B. 6 (39), 6191-6206 (2018).</li><li>Yi, H. -. G., Lee, H., Cho, D. -. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+printing+of+organs-on-chips.">3D printing of organs-on-chips.</a> Bioengineering. 4 (1), 10 (2017).</li><li>Yi, H. -. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+bioprinted+human-glioblastoma-on-a-chip+for+the+identification+of+patient-specific+responses+to+chemoradiotherapy.">A bioprinted human-glioblastoma-on-a-chip for the identification of patient-specific responses to chemoradiotherapy.</a> Nature Biomedical Engineering. 3 (7), 509-519 (2019).</li><li>Kang, T. -. Y., Hong, J. M., Jung, J. W., Yoo, J. J., Cho, D. -. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+assessment+of+a+microfluidic+network+system+for+oxygen+transport+in+engineered+tissue.">Design and assessment of a microfluidic network system for oxygen transport in engineered tissue.</a> Langmuir. 29 (2), 701-709 (2013).</li><li>Woo Jung, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+the+effective+diffusivity+of+a+freeform+fabricated+scaffold+using+computational+simulation.">Evaluation of the effective diffusivity of a freeform fabricated scaffold using computational simulation.</a> Journal of Biomechanical Engineering. 135 (8), (2013).</li><li>Brown, A. C., De Beer, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+stereolithography+(STL)+slicing+and+G-code+generation+algorithm+for+an+entry+level+3-D+printer.">Development of a stereolithography (STL) slicing and G-code generation algorithm for an entry level 3-D printer.</a> 2013 Africon (IEEE). , 1-5 (2013).</li><li>Shim, J. -. H., Lee, J. -. S., Kim, J. Y., Cho, D. -. 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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?...

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。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="4">Cells</td>
		</tr>
		<tr>
			<td>Human umbilical vein endothelial cells</td>
			<td>Promocell</td>
			<td>C-12200</td>
			<td></td>
		</tr>
		<tr>
			<td>U-87 MG cells</td>
			<td>ATCC</td>
			<td>ATCC HTB-14</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">Disposable</td>
		</tr>
		<tr>
			<td>0.2 &#956;m syringe filter</td>
			<td>Sartorius</td>
			<td>16534-K</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL disposable syringe</td>
			<td>Jung Rim</td>
			<td>10ml 21G32</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL glass vial</td>
			<td>Hubena</td>
			<td>A0039</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL Serological pipette tip</td>
			<td>SPL lifescience</td>
			<td>91010</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL conical tube</td>
			<td>SPL lifescience</td>
			<td>50015</td>
			<td></td>
		</tr>
		<tr>
			<td>18G plastic needle</td>
			<td>Musashi engineering</td>
			<td>PN-18G-B</td>
			<td></td>
		</tr>
		<tr>
			<td>20G plastic tapered dispense tip</td>
			<td>Musashi engineering</td>
			<td>TPND-20G-U</td>
			<td></td>
		</tr>
		<tr>
			<td>22x50 glass cover</td>
			<td>MARIENFIELD</td>
			<td>0101142</td>
			<td></td>
		</tr>
		<tr>
			<td>25 mL Serological pipette tip</td>
			<td>SPL lifescience</td>
			<td>90125</td>
			<td></td>
		</tr>
		<tr>
			<td>3 mL disposable syringes</td>
			<td>HENKE-JET</td>
			<td>4020-X00V0</td>
			<td></td>
		</tr>
		<tr>
			<td>40 &#181;m cell strainer</td>
			<td>Falcon</td>
			<td>352360</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL Serological pipette tip</td>
			<td>SPL lifescience</td>
			<td>91005</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL conical tube</td>
			<td>SPL lifescience</td>
			<td>50050</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL Serological pipette tip</td>
			<td>SPL lifescience</td>
			<td>90150</td>
			<td></td>
		</tr>
		<tr>
			<td>50N precision nozzle</td>
			<td>Musashi engineering</td>
			<td>HN-0.5ND</td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminum foil</td>
			<td>SINKWANG</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Capillary tips</td>
			<td>Gilson</td>
			<td>CP1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell-scrapper</td>
			<td>SPL lifescience</td>
			<td>90030</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal dish</td>
			<td>SPL lifescience</td>
			<td>200350</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Bemis</td>
			<td>PM996</td>
			<td></td>
		</tr>
		<tr>
			<td>Pre-coated histology slide</td>
			<td>MATSUNAMI</td>
			<td>MAS-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Reservoir</td>
			<td>SPL lifescience</td>
			<td>23050</td>
			<td></td>
		</tr>
		<tr>
			<td>T-75 cell culture flask</td>
			<td>SPL lifescience</td>
			<td>70075</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">Equipment</td>
		</tr>
		<tr>
			<td>3DX printer</td>
			<td>T&#38;R Biofab</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Autoclave</td>
			<td>JEIOTECH</td>
			<td>AC-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuger</td>
			<td>Cyrozen</td>
			<td>1580MGR</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal laser microscopy</td>
			<td>Olympus Life Science</td>
			<td>FV 1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence microscope</td>
			<td>FISHER SCEINTIFIC</td>
			<td>O221S366</td>
			<td></td>
		</tr>
		<tr>
			<td>Forcep</td>
			<td>Korea Ace Scientific</td>
			<td>HC.203-30</td>
			<td></td>
		</tr>
		<tr>
			<td>Hand tally counter</td>
			<td>KTRIO</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>MARIENFIELD</td>
			<td>0650030</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Panasonic</td>
			<td>MCO-170AIC</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminar flow cabinet</td>
			<td>DAECHUNG SCIENCE</td>
			<td>CB-BMMS C-001</td>
			<td></td>
		</tr>
		<tr>
			<td>Metal syringe</td>
			<td>IWASHITA engineering</td>
			<td>SUS BARREL 10CC</td>
			<td></td>
		</tr>
		<tr>
			<td>Operating Scissors</td>
			<td>Hirose</td>
			<td>HC.13-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Oven</td>
			<td>JEIOTECH</td>
			<td>OF-12, H070023</td>
			<td></td>
		</tr>
		<tr>
			<td>Positive displacement pipette</td>
			<td>GILSON</td>
			<td>NJ05652</td>
			<td></td>
		</tr>
		<tr>
			<td>Refrigerator</td>
			<td>SAMSUNG</td>
			<td>CRFD-1141</td>
			<td></td>
		</tr>
		<tr>
			<td>Voltex Mixer</td>
			<td>DAIHAN scientific</td>
			<td>VM-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td>DAIHAN SCIENTIFIC</td>
			<td>WB-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Water purifier</td>
			<td>WASSER LAB</td>
			<td>DI-GR</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">Materials</td>
		</tr>
		<tr>
			<td>0.25 % Trypsin-EDTA</td>
			<td>Gibco</td>
			<td>25200-072</td>
			<td></td>
		</tr>
		<tr>
			<td>10x PBS</td>
			<td>Intron</td>
			<td>IBS-BP007a</td>
			<td></td>
		</tr>
		<tr>
			<td>4% Paraformaldehyde</td>
			<td>Biosesang</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>70% Ethanol</td>
			<td>Daejung</td>
			<td>4018-4410</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-CD31 antibody</td>
			<td>Abcam</td>
			<td>ab28364</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-HIF-1 alpha antibody</td>
			<td>Abcam</td>
			<td>ab16066</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-SHMT2/SHMT antibody</td>
			<td>Abcam</td>
			<td>ab88664</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-SOX2 antibody</td>
			<td>Abcam</td>
			<td>ab75485</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Thermo scientific</td>
			<td>J10857-22</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen from porcine skin</td>
			<td>Dalim tissen</td>
			<td>PC-001-1g</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI (4&#39;,6-Diamidino-2-Phenylindole, Dihydrochloride)</td>
			<td>Thermofisher</td>
			<td>D1306</td>
			<td></td>
		</tr>
		<tr>
			<td>Endothelial Cell Growth Medium-2</td>
			<td>Promocell</td>
			<td>C22011</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Gibco</td>
			<td>12483-020</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488</td>
			<td>Theromofisher</td>
			<td>A-11001</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 594</td>
			<td>Theromofisher</td>
			<td>A-11012</td>
			<td></td>
		</tr>
		<tr>
			<td>High-glucose Dulbecco&#8217;s Modified Eagle Medium(DMEM)</td>
			<td>Hyclone</td>
			<td>SH30243-0</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>Sigma-Aldrich</td>
			<td>311413-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Live/dead assay kit</td>
			<td>Invitrogen</td>
			<td>L3224</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse IgG1, kappa monoclonal [15-6E10A7] - Isotype Control</td>
			<td>Abcam</td>
			<td>ab170190</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenol red solution</td>
			<td>Sigma-Aldrich</td>
			<td>P0290-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly(ethylene-vinyl acetate)&#160;</td>
			<td>Poly science</td>
			<td>06108-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Polydimethylsiloxane</td>
			<td>Dowhitech</td>
			<td>sylgard 184</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit IgG, polyclonal - Isotype Control</td>
			<td>Abcam</td>
			<td>ab37415</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide solution</td>
			<td>Samchun</td>
			<td>S0610</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Biosesang</td>
			<td>TRI020-500-50</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue</td>
			<td>Sigma-Aldrich</td>
			<td>T8154</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">Software</td>
		</tr>
		<tr>
			<td>COMSOL Multiphysics 3.5a</td>
			<td>COMSOL AB</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>IMS beamer</td>
			<td></td>
			<td></td>
			<td>in-house software</td>
		</tr>
		<tr>
			<td>SolidWorks Package</td>
			<td colspan="2">Dassault Systems SolidWorks Corporation</td>
			<td></td>
		</tr>
	</tbody>
</table>

3d cell-printed hypoxic cancer-on-a-chip for recapitulating pathologic progression of solid cancer

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。

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）</...

Watch this Scientific Journal Video about 3D Cell-Printed Hypoxic Cancer-on-a-Chip for Recapitulating Pathologic Progression of Solid Cancer at JoVE.com

3D Cell-Printed Hypoxic Cancer-on-a-Chip for Recapitulating Pathologic Progression of Solid Cancer

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。

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

Cancer microenvironment has a significant impact on the progression of the disease. In particular, hypoxia is the key driver of cancer survival, invasion, ...

缺氧是癌症发展的关键驱动因素，诱发基因组不稳定和肿瘤形成。我们演示了基于 3D 生物打印技术的体外固体癌症中产生中央缺氧的方法。使用这种方法，可以使用简单的策略复制无线电低氧梯度，该策略结合了 3D 打印气体透气屏障和玻璃盖。这种缺氧癌症片上技术可用于预测药物疗效，以方便患者特定的抗癌药物处方。而且，它也有望使侵略性癌症的快速诊断。为了帮助制备三毫升中和胶原蛋白预凝胶溶液，将 30 毫克胶原蛋白海绵切成 5 x 5 毫米平方块。将碎片放入无菌的 10 毫升玻璃瓶中，在小瓶中加入 2.4 毫升 0.2 微米注射器过滤器 0.1 普通盐酸，在摄氏 4 度和每分钟 15 圈的三天孵化中。消化后，通过40微米细胞过滤器应变任何未消化的胶原蛋白颗粒，并将酸性胶原蛋白溶液储存在4摄氏度，持续7天。要调整 1%中和胶原蛋白预凝胶溶液的 pH， 在混合和离心后，将 30 微升酚红色溶液添加到最终浓度为 1%和 300 微升的 10X PBS 中，最终浓度为 10%，使用一种普通氢氧化钠将 pH 值中和到 7，验证颜色变化并加入蒸馏水，获得总容量为 3 毫升。然后将 pH 调整后的 1% 中和胶原蛋白预凝胶溶液存放在 4 摄氏度，在三天内使用。要预先检查中和胶原蛋白预凝胶溶液的凝胶，请使用正位移移液化剂将 50 微升胶原蛋白液滴添加到小菜中，并在 37 摄氏度的孵化器中孵化液滴一小时。在孵化结束时，检查胶原蛋白是否从透明颜色变为不透明的白色。倾斜容器以确认胶原蛋白粘附在容器底部。并将 PBS 倒入液滴，以确认胶原蛋白构造不会破坏溶液。有关牺牲的 PEVA 模具的 3D 打印，请单击"文件"并保存文件类型为 STL，将 3D CAD 文件转换为 STL 文件，然后单击选项和输出表单作为 ASCII 生成 G 代码。要导入生成的 STL 文件，请单击"文件"并打开 STL 文件并选择已保存的 STL 文件。要自动生成牺牲的 PEVA 模具的 G 代码，请选择 STL-CAD 交换器的切片模型。然后，要生成芯片制造的打印路径，请在 110 摄氏度的 500 千帕气压下使用 50 度精确喷嘴，将牺牲的 PEVA 模具打印到无菌粘合亲水组织学幻灯片上。为了铸就 PDMS 屏障，在塑料储液池中混合 6 毫升 PDMS 碱基弹性体和 600 微升固化剂 5 分钟，并将均匀混合溶液加载到 10 毫升一次性注射器中。为注射器配备 20 仪表塑料锥形分配尖端，并使用混合 PDMS 解决方案填充牺牲的 PEVA 模具。混合的 PDMS 将用凸面填充此牺牲的 PEVA 模具，屏障将高于模具。在 40 摄氏度烤箱中固化 PDMS 屏障超过 36 小时后，为避免 PEVA 熔化，使用一对精密钳子分离牺牲的 PEVA 模具，并在高压灭菌器中在 120 摄氏度处对气体透气屏障进行消毒。要将溶液与癌细胞混合，在 20 微升培养介质中再吸收每种类型的收集细胞颗粒，并在湿冰上每个恢复的细胞悬浮液中加入 1%中和胶原蛋白预凝胶溶液，然后轻轻混合。使用正位移移移器混合每个单元格悬架。获得同质解决方案后，使用正一次性移液器将细胞封装胶原蛋白生物因子转移到单个三毫升一次性注射器中，并将注射器储存在摄氏四度，直到 3D 细胞打印。对于癌症-频闪同心环的 3D 打印，将适当的 3D CAD 文件转换为 STL 文件格式，并使用 STL-CAD 交换器生成癌症频闪同心环的 G 代码。将细胞封装胶原蛋白放入 3D 打印机头，并将头部和板材的温度设置为 15 摄氏度。在 3D 打印机的控制软件上加载生成的打印路径，然后单击"开始"，根据加载的 G 码将胶原蛋白生物因子打印到气体渗透屏障上，该 G 码带有 18 个仪表塑料针，气压约为 20 千帕，温度约为 15 摄氏度。每次打印操作结束时，手动在气体透气屏障顶部放置消毒的 22 x 50 毫米玻璃盖，以产生低氧梯度。在产生三个缺氧癌后，芯片转移到37摄氏度的孵化器一个小时，将胶原蛋白生物素交叉连接。当所有低氧癌片都打印出来后，用细胞刮刀轻轻擦过气体透气屏障顶部的盖眼镜，以紧密结合。为了在不分离癌症构造的情况下将细胞培养介质刷新到芯片上，将芯片倾斜，并使用移液器将 1.5 毫升内皮细胞生长介质引入每个芯片的侧面。芯片上的缺氧癌以同心环的形式设计，以模拟癌症组织中观察到的径向氧扩散和耗竭。在确定供氧和细胞消耗的空间的控制体积后，可以通过计算有限元素分析确定中央缺氧生成的适当细胞密度。在3D细胞打印后，可以创建一个分隔的癌症-频闪同心环结构，以再现实体癌症的解剖特征。从数量上讲，打印后细胞的生存能力通常大于 96%，这证实了制造条件适合癌症和频闪细胞。在这个具有代表性的分析中，根据氧气梯度的存在和缺失对两组进行了比较，以验证缺氧梯度对癌症进展的影响。在这两种条件下，外围区域都存在成熟的CD31正内皮细胞，表明空间模式的活体结构是利用3D生物打印技术产生的。与氧梯度负条件相比，梯度阳性状况表现出低氧梯度，表示HIF1α的逐渐表达。还观察到SHMT2阳性伪帕利塞德细胞和SIX2阳性多能细胞，表明固体癌症具有侵略性病理生理特征。芯片上的缺氧癌是研究实体癌症病理生理特征和癌细胞与肿瘤形成促进微环境的动态相声的有用工具。由于该方法可在合理的时间范围内适用于患者特定药物设计，因此该方法有望弥合体内和体外癌症模型之间的差距。

Research

JoVE Journal

Bioengineering

Tracking Hypoxic Signaling within Encapsulated Cell Aggregates

在封装的细胞聚集跟踪缺氧信号

In Diabetes mellitus type 1, autoimmune destruction of the pancreatic &beta;-cells results in loss of insulin production and potentially lethal ...

科研

生物工程

Monitoring Protein Adsorption with Solid-state Nanopores

监测与固态纳米孔蛋白质吸附

Solid-state nanopores have been used to perform measurements at the single-molecule level to examine the local structure and flexibility of nucleic acids ...

Sample Preparation Strategies for Mass Spectrometry Imaging of 3D Cell Culture Models

样品制备策略的3D细胞培养模型质谱成像

Three dimensional cell cultures are attractive models for biological research. They combine the flexibility and cost-effectiveness of cell culture with ...

Solid Lipid Nanoparticles (SLNs) for Intracellular Targeting Applications

Solid Lipid Nanoparticles SLNs for Intracellular Targeting Applications

固体脂质纳米粒（前哨淋巴结）细胞内定位的应用

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter ...

Selective Cell Elimination from Mixed 3D Culture Using a Near Infrared Photoimmunotherapy Technique

从混合三维培养使用近红外Photoimmunotherapy技术选择性细胞消除

Recent developments in tissue engineering offer innovative solutions for many diseases. For example, tissue engineering using induced pluripotent stem ...

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

3D胶原凝胶和微通道3D相互作用的研究准备<em&gt;在体内</em

Historically, most cellular processes have been studied in only 2 dimensions.  While these studies have been informative about general cell signaling ...

Micropatterning and Assembly of 3D Microvessels

缩微和3D微血管大会

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass ...

Modeling Ovarian Cancer Multicellular Spheroid Behavior in a Dynamic 3D Peritoneal Microdevice

模拟卵巢癌多细胞球体行为动态三维微型腹膜

Ovarian cancer is characterized by extensive peritoneal metastasis, with tumor spheres commonly found in the malignant ascites. This is associated with ...

Bioprinting of Cartilage and Skin Tissue Analogs Utilizing a Novel Passive Mixing Unit Technique for Bioink Precellularization

Bioink Precellularization 无源混合装置打印软骨及皮肤组织类似物的应用

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both ...

Rigid Embedding of Fixed and Stained, Whole, Millimeter-Scale Specimens for Section-free 3D Histology by Micro-Computed Tomography

用微计算机断层扫描法对无节段3D 组织学进行固定染色、全尺寸、毫米度试样的刚性嵌入

For over a hundred years, the histological study of tissues has been the gold standard for medical diagnosis because histology allows all cell types in ...