将3D多细胞乳房球体直接生物打印到内皮网络
Summary
该协议的目标是将生物打印乳房上皮细胞作为多细胞球体直接印在预先形成的内皮网络上，以快速创建可用于药物筛选研究的3D乳腺内皮共生模型。
Abstract
生物打印正在成为制造3D人类癌症模型的有希望的工具，能够更好地回顾体内组织结构的关键特征。在目前的逐层挤压生物打印中，单个细胞与复杂的空间和时间线索一起挤出生物沉淀物，以促进分层组织自我组装。然而，这种生物制药技术依赖于细胞、生物素和生化和生物物理线索之间的复杂相互作用。因此，自组装可能需要数天甚至数周的时间，可能需要特定的生物墨水，并且当涉及多个细胞类型时，可能并不总是发生。因此，我们开发了一种技术，直接生物打印预先形成的3D乳房上皮球体在各种生物墨水。生物打印预形成的3D乳房上皮球体在打印后维持其生存能力和极化结构。此外，我们还将3D球体打印到血管内皮细胞网络上，以创建共培养模型。因此，新的生物打印技术迅速创造了一个与生理相关的3D人体乳房模型，其成本更低，灵活性也比传统生物打印技术高。这种多才多艺的生物打印技术可以推断出来，以创建其他组织的其他生物墨水的3D模型。
Introduction
3D体外血管化肿瘤模型是癌症生长和转移的机械研究的重要工具。特别是对于乳腺癌，在Matrigel培养的乳房上皮细胞组织成极化球形，更类似于体内乳腺癌结构1，2，3，4，5，6，7，8。3D乳房上皮细胞培养也影响细胞功能，3D培养物在表皮生长因子（EGF）受体调节8、9方面表现出差异：电基因功能，包括 ErbB210：生长和凋亡信号11，12：和化疗耐药性13，14。血管内皮细胞在3D与传统2D文化15、16、17、18中对环境刺激的反应相似。然而，对血管内皮和乳房上皮相互作用的理解大部分来自使用条件介质或Transwell插入物的2D培养，或3D模型，其中两种细胞类型物理分离19，20，21，22，23。这些共同培养模型提供了有限的生理洞察力，因为3D培养和细胞接触对血管内皮-乳房上皮细胞相互作用24，25，26至关重要。
3D癌症模型是使用各种技术制造的，包括悬挂下降球形形成，生物打印，磁性组装，培养在水凝胶或工程脚手架5，27，28，29。最近，3D肿瘤模型被创建，在各自的3D结构中排列了多种细胞类型。在片上肿瘤平台的一个例子中，癌症、内皮细胞和频闪细胞被混合到基质中，然后注射到聚二甲基硅氧烷（PDMS）装置中的三个中央组织室中。组织室由代表动脉和静脉的两个外通道包围。经过5-7天的培养，内皮细胞形成微血管网络，癌细胞增殖，在血管附近形成小肿瘤。然后，该平台用于筛选药物和药物组合30。其他肿瘤片上平台已经创建，以研究转移和癌症类型与特定的机械刺激（例如，在肺部的机械应变）31，32。然而，这些平台通常不包括血管和癌症在其各自的3D结构。
生物合成在推进体外血管化肿瘤模型方面显示出巨大的希望，因为它能够对细胞位置进行严格的空间控制。尽管生物印刷在过去十年中增长，但很少有研究特别侧重于肿瘤33，34。在一个例子中，用明胶/藻酸盐/纤维蛋白原水凝胶对HiLa细胞进行3D打印，以创建体外宫颈癌模型。肿瘤细胞被生物打印为单个细胞，然后允许形成球形，这表明更高的增殖率，增加矩阵金属蛋白酶表达，和更高的化学耐附性比细胞在2D培养35。在这些研究中，像许多其他36，37，分离细胞悬浮物被生物打印，然后细胞培养物被提供所需的机械和生化线索，使细胞形成一个3D结构。然而，细胞自组装可能需要数天或数周的时间，可能需要复杂的空间和时间环境提示，或在两种细胞类型共同培养时可能不会发生。例如，乳腺上皮细胞在2D共生中诱发内皮细胞死亡，分离的乳房上皮细胞在生物印在藻酸盐/明胶水凝胶38中时没有形成3D球形。分离的乳腺上皮或癌细胞只有在包裹在圆形 PDMS 模具中时，才会在藻酸盐生物沉积中形成球形。在其他情况下，球体是使用悬浮液滴在超低附件圆形井板中形成的，然后混合成藻酸盐基生物墨水39，40。
我们现在在此协议中描述了一种替代的3D组织生物制造方法。我们描述的不是种子分离细胞和等待这些细胞形成3D结构，而是描述如何在血管管网络上创建和生物打印3D肿瘤球体，以创建一个肿瘤共生模型，几乎可以立即使用。肿瘤球体可以在体外生长或从人体组织（器官）中提取。同样，血管管可以生长，也可以从脂肪组织微血管片段中提取。生物素的范围从生物不活跃的藻酸盐到高度生物活性的马特里格尔41。由于这种3D肿瘤共生模型可以创建与各种细胞结构和生物素，它可以结合多种细胞类型，细胞外矩阵，和化学基质梯度15，42。虽然在其目前的配方中，内皮网络无法注入，但未来的迭代可以将此方法与微流体或片上系统集成。将3D乳房上皮球体打印到内皮网络上，使人类乳房模型能够快速生物合成，用于药物测试和个性化精密医学27。
Protocol
1. 乳房上皮细胞生长和检测介质 MCF10A 乳房上皮细胞注：非肿瘤不朽的乳房上皮细胞系来自纤维囊病患者43。细胞不表达雌激素受体。 要准备 20μg/mL 表皮生长因子 （EGF），在 500 μL 的无菌 dH2O 中溶解 100μg 的亲皮 EGF，使 200 μg/mL EGF。在 dH 2 O 中将 200 微克/mL EGF 的 500 微升添加到 4.5 mL 的无菌0.1%BSA 中，以制作 20μg/mL EGF 库存解决方案。商店准备20μg…
Representative Results
乳房上皮细胞应自我组织成3D球形后，5-8天的培养矩阵溶液和文化介质与2%的矩阵溶液。非肿瘤性MCF10A乳房上皮球体应呈圆形，并具有空心中心，与整数+6偏振到球体的外缘（图1，内嵌显示空心中心）。高度侵入性的MDA-MB-231乳腺癌上皮细胞形成不规则球形。当球形直径约为100-300μm时，应使用球形。当球体变得太大，并接近，球体将连接在一起，形成巨型球体。此外，MDA-MB-231…
Discussion
此协议是首次在其 3D 架构中与内皮细胞在其 3D 架构中共同培养的生物打印球体。关键协议步骤包括乳房上皮球体和HUVEC网络的初始形成。在喂养乳房上皮球体时必须格外小心，因为它们很容易被矩阵溶液打乱。同样，乳房上皮球体在从矩阵溶液上移开并混合到网络中时，必须小心对待。HUVEC 网络不应以过高的密度进行镀层，也不应离开超过 16 小时，因为它们将分别形成单层或死亡。最后，所有?…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
这项研究由NIH 1R01HL140239-01向AMC资助。我们要感谢德雷塞尔大学的细胞成像中心。
Materials
| 37°C incubator, 5% CO2 and 95% humidity | Sanyo | MCO-20AIC | Cell incubation |
| 3D Bio printer | custom-made | None | Used for bioprinting |
| 8-well chamber slides | VWR, Radnor, PA | 53106-306 | for seeding spheroids |
| 25-gauge needle | Sigma, St. Louis, MO | Z192406-100EA | bioprinting syringe needle |
| Absolute ethanol (200 proof ) | Sigma, St.Louis, MO | E7023-500ML | reconsitution of media components |
| Affinipure F(ab′)2 fragment goat anti-mouse IgG | Jackson ImmunoResearch, West Grove, PA | 115006020 | secondary block – Immunofluorescence |
| Alexa Fluor 488 (1:200) | Thermo Fisher, Waltham, MA | A-11006 | Seconday antibody-Immunofluorescence |
| Bovine insulin | Sigma, St.Louis, MO | I-035-0.5ML | MCF10A Media additive |
| Bovine serum albumin (BSA) | Sigma, St.Louis, MO | A2153-500G | Blocking agent -Immunofluorescence |
| Falcon 70 µm Cell Strainer | Corning, Corning, NY | 352350 | Remove large or clustered spheroids |
| CellTracker™ Red CMTPX Dye | Thermo Fisher, Waltham, MA | C34552 | pre-stain for HUVEC tubes |
| Compact Centrifuge | Hermle- Labnet, Edison ,NJ | Z206A | For cell centrifugations |
| Cholera Toxin | Sigma, St.Louis, MO | C8052-.5MG | MCF10A Media additive |
| Conical tubes 15 mL | VWR, Radnor, PA | 62406-200 | Collecting and resuspending cells |
| Countess II-FL Cell counter | Thermo Fisher, Waltham, MA | AMQAF1000 | counting cells |
| Glass pipettes (10 mL) | VWR, Radnor, PA | 76184-746 | cell resuspension |
| DMEM F:12 | Thermo Fisher, Waltham, MA | 11320033 | MCF10A basal media |
| DMEM 1X | VWR, Radnor, PA | 10-014-CV | MDA-MB-231 basal media |
| Endothelial Basal Medium-2 (EBM-2) | Lonza, Durham, NC | CC-3156 | HUVEC basal media |
| Endothelial Growth Medium-2 (EGM-2) | Lonza, Durham, NC | CC-3162 | Accompanied with a Bulletkit (containing growth factors) |
| Alexa Fluor™ 488 Phalloidin | Thermo Fisher, Waltham, MA | Labelling MDA-MB-231 spheroids | |
| Fetal Bovine serum | Cytiva, Logan, UT | SH30071.03 | HUVEC/MDA-MB-231 media additive |
| Goat serum | Thermo Fisher, Waltham, MA | 16210064 | Live and dead cell stain assay for cell viability |
| Glycine | Sigma, St.Louis, MO | G8898-500G | immunofluorescence buffer component |
| Hoescht 33342 | Thermo Fisher, Waltham, MA | 62249 | Nuclei stain immunofluorescence |
| Horse Serum | Thermo Fisher, Waltham, MA | 16050130 | MCF10A Media additive |
| Hydrocortisone | Sigma, St.Louis, MO | H0888-5G | MCF10A Media additive |
| Human Umblical Vein Endothelial cells (HUVECs) | Cell applications, San Diego , CA | 200-05f | Endothelial cell lines |
| Integrin α6 | Millipore, Billerica, MA | MAB1378 | Immunofluorescence spheroid labelling component |
| Live Dead assay | Thermo Fisher, Waltham, MA | L3224 | Live and dead cell stain assay for cell viability |
| LSM 700 Confocal microscope | Zeiss, Thornwood, NY | Used to visualize cells | |
| Matrigel – growth factor reduced 10 mg/ml | VWR, Radnor, PA | 354230 | Spheroid formation |
| MCF10A cells | ATCC | CRL-10317 | Breast cell line |
| MDA-MB-231 cells | ATCC | HTB-26 | Breast cell line |
| Paraformaldehyde | Sigma, St.Louis, MO | 158127-500G | cell fixative |
| Penicillin and streptomycin | Thermo Fisher, Waltham, MA | 15140122 | MCF10A / MDA-MB-231/HUVEC Media additive |
| Phosphate Buffered Saline 1X (PBS) | Thermo Fisher, Waltham, MA | 7001106 | Wash buffer for cells before trypsinization |
| Phosphate buffer saline 10X | Thermo Fisher, Waltham, MA | AM9625 | immunofluorescence buffer component |
| Prolong gold antifade | Thermo Fisher, Waltham, MA | P36934 | immunofluorescence mountant medium |
| Recombinant Human Epidermal Growth Factor, EGF | Peprotech, Rocky Hill, NJ | AF-100-15 | MCF10A/ assay media component |
| Sodium Azide | Sigma, St.Louis, MO | S2002-25G | immunofluorescence buffer component |
| Sterile syringe (10 mL) | VWR, Radnor, PA | 75846-757 | bioprinting process |
| Tissue culture dish (10cm) | VWR, Radnor, PA | 25382-166 | monolayer cell culture |
| Triton X-100 | Sigma, St.Louis, MO | T8787-250ML | immunofluorescence buffer component |
| Trypan blue 0.4% | Thermo Fisher, Waltham, MA | 15250061 | cell counter additive |
| Trypsin-EDTA 0.05% | Thermo Fisher, Waltham, MA | 25300054 | cell detachment |
| Tween -20 | Thermo Fisher, Waltham, MA | 85113 | immunofluorescence buffer component |
| >Vascular Endothelial Growth factor (VEGF165) | Peprotech, Rocky Hill, NJ | 100-20 | HUVEC tube additive |
| Volocity 6.3 cell imaging software | PerkinElmer, Hopkinton, MA | Z stack compresser |
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Citar este artículo
Swaminathan, S., Clyne, A. M. Direct Bioprinting of 3D Multicellular Breast Spheroids onto Endothelial Networks. J. Vis. Exp. (165), e61791, doi:10.3791/61791 (2020).