微流控装置中组织模型的 Perfusable 血管网
Summary
该协议描述了如何在球体中设计一个 perfusable 的血管网。球体周围的微环境被设计来诱导血管生成, 并将球体连接到微流控装置中的微。该方法允许球状的灌注, 这是一个期待已久的技术在三维文化。
Abstract
球体 (多细胞聚合体) 被认为是人体内活体组织的良好模型。尽管在球体培养方面有了显著的进步, 球体的 perfusable 血管网仍然是维持和发展其功能所需的长期文化的关键挑战, 如蛋白质表达和形态发生。该协议提出了一种新的方法来集成 perfusable 血管网络在球体内的微流控装置。为了诱导球体中的 perfusable 血管网, 利用球形培养的人肺成纤维细胞的血管生成因子, 引导与微相连的血管生成苗。血管生成苗达到球形, 与球体中的内皮细胞合并, 形成连续的血管网络。血管网可以灌注球体内部, 而不会有任何渗漏。构建的血管网络可进一步用作营养物质的供应和废弃物的去除, 模拟血液循环在体内。该方法为球形培养提供了一个新的平台, 可以更好地对活体组织进行重述。
Introduction
从单层 (二维) 文化转移到三维文化, 是因为需要使用模仿活体组织的细胞功能的文化模型1,2,3。细胞培养中常用的扁平和硬质塑料基底不像人体内大部分的胞外环境。事实上, 许多研究表明, 三维的文化重现了组织特异性的结构, 机械和生物化学的线索, 和细胞通讯, 没有在常规的二维文化中观察到4, 5,6,7,8。
多细胞聚合或球体, 是实现此三维区域性9、10的最有希望的技术之一。细胞分泌细胞外基质 (ECM), 可以与球体中的其他人相互作用。虽然一些其他生物工程方法11,12,13,14, 如单元格堆叠, 成功地复制人体的空间复杂性, 这些方法只有两个或三个种类的细胞, 便于分析, 只专注于一个功能的靶器官。相比之下, 球体中的细胞由于在球体中的养分、氧、分泌和分泌信号分子的异构供应而暴露在不同的文化环境中, 这取决于它们在球体中的位置。球体的此功能部分模仿体内区域性条件, 并使球体中的单元格在体外创建比堆叠组织9中培养的更复杂、组织化的组织结构.15,16. 请注意, 如果球体由单个细胞组成, 则球体中的细胞的功能由于球体中的异构环境而不均匀。在过去的几年中, 球体培养允许胚胎干细胞 (ESCs), 诱导多能干细胞 (iPSCs) 或组织驻留干细胞模仿体内发育序列, 并重新创建微型器官, 如大脑17,肝脏18和肾脏19,20。
尽管球形培养技术取得了重大进展, 但长期培养大型球体仍然存在问题。在一个三维的组织中, 细胞需要位于血管的150-200 µm 内, 因为氧气和营养素的供应有限21。在球体内的血管网络是必要的, 以重述血液和组织之间的交换物质在体内。为达到这一目的, 其他组与靶细胞共培养内皮细胞22,23,24或诱导多潜能细胞分化为 CD31-positive 细胞20。然而, 报告的船状结构没有腔的开放端向球体中心提供氧气和养分。为了模拟血管的作用, 滋养细胞在三维的文化, 开放和 perfusable 血管网络必须发展在球体。
在过去几年中, microengineering 领域的一些研究小组报告了通过利用共培养成纤维细胞的血管生成因子在微流控装置中自发形成 perfusable 血管网络的方法 25 ,26。这些血管网络与它们的体内对应物具有相似的形态学, 可以被环境因素重塑, 使它们适合于在球状培养中模仿血管功能。本协议的目的是使用微流控平台27在球体中构造一个 perfusable 的血管网络。微流控设备从先前报告的设备25中进行了修改, 以便可以合并球体。通过将球状成纤维细胞的血管生成分泌物定向到微的内皮细胞, 微与球体吻合, 形成 perfusable 血管网络。这种方法允许直接交付广泛的物质, 如荧光分子和微米级的珠子进入球体的内部, 这为长期组织培养与血管网络提供了框架。
Protocol
1. 微流控装置模具的制作 使用商用软件 (Clewin5 或 AutoCAD 2016,等) 设计微流控设备的模式。有关 Clewin5 的功能, 请参阅用户手册 (http://manualzz.com/doc/7159150/clewin-user-s-manual)。注意: 设计文件在辅助文件 1中可用。 将设计文件传送到微阵列生成器, 并将该工具加载到涂有正胶的铬罩上。 使用微阵列发生器在图案区域中暴露正光刻胶。 使用开发人员 (<str…
Representative Results
图 1显示了微流控设备的设计和照片。它有三个平行的渠道, 其中渠道2包含球形井。通道1和3用于 HUVEC 文化, 通道2为球体。每个通道由梯形 microposts 分离, 设计为模式。microposts 防止2通道中的水凝胶通过表面张力泄漏到通道1和 3, 允许在微28中的球体和血管内皮细胞之间交换物质。 <strong c…
Discussion
以前的报告表明, hLFs 分泌多种血管生成因子的鸡尾酒, 如 angiopoietin-1, 染, 肝细胞生长因子, 转化生长因子α, 肿瘤坏死因子和一些细胞外基质蛋白29, 30。这种检测依赖于共培养球体中 hLFs 的血管生成物分泌, 这是该技术的局限性。因此, 在井底设置一个共培养球体是稳定的血管形成的关键, 因此共培养球体与血管内皮细胞通道1和3之间的距离可以缩短。成?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了 JST (JPMJCR14W4)、促进科学协会 (jsp) KAKENHI (赠款编号25600060、16K16386)、下个和 JST 创新项目中心的支持, 项目重点是开发关键的评估技术, 从日本医药研发机构, 阿曼德, 瑞穗科技促进会。《京都大学纳米技术中心》支持微细加工。
Materials
| AutoCAD 2017 | Autodesk | AutoCAD 2017 | |
| A chromium mask coated with AZP 1350. | CLEAN SURFACE TECHNOLOGY | CBL2506Bu-AZP | |
| Micro pattern generator | Heidelberg | uPG101 | |
| MF CD-26 developer | Rohm and haas electronic materials | – | Developer in protocol 1.4 |
| S-Clean | Sasaki Chemical | S-24 | Chromium etchant in protocol 1.5 |
| Aceton | Wako | 012-00343 | |
| Silicon Wafer | Canosis | SiJ-4 | |
| Spin Coater | MIKASA | 1H-D7 | |
| Hexamethyldisilazane (HMDS) | Tokyo Ohka Kogyo | H0089 | |
| SU-8 3050 | MicroChem | – | Negative photoresist in protocol 1.9 |
| UV Exposure | Nanometric Technology Inc | LA310s | |
| SU-8 Developer | MicroChem | Y020100 | Developer for the negative photoresist in protocol 1.13 |
| 2-propanol | Wako | 163-04841 | |
| Surhace profiler | Vecco | Veeco Dektak XT-S | |
| (Trichloro (1H, 1H, 2H, 2H-perfluorooctyl) silane | Sigma | 448931 | |
| Polydimethylsiloxane (PDMS) | Dow Corning Toray | 184W/C | |
| Biopsy Punch (1.0mm) | Kai Industries | BP-10F | |
| Biopsy Punch (2.0mm) | Kai Industries | BP-20F | |
| Plasma System | Femto Science | COVANCE | |
| Cover glass | MATSUNAMI GLASS | C024241 | |
| Culture Dishes | Iwaki | 1000-035 | |
| RFP Expressing Human Umbilical Vein Endothelial Cell | Angio Proteomie | cAP-0001RFP | |
| Normal Human Lung Fibroblasts | Lonza | CC-2512 | |
| Endothelial Cell Growth Medium | Lonza | CC-3162 | |
| Fibroblast Growth Media Kits | Lonza | CC-3132 | |
| DMEM | Thermo Fisher Scientific | 11965092 | |
| Fetal Bovine Serum | Thermo Fisher Scientific | 26140079 | |
| Penicillin-Streptomycin Solution | Wako | 168-23191 | |
| 0.05w/v% Trypsin-0.53mmol/l EDTA• 4Na Solution with Phenol Red | Wako | 204-16935 | |
| PBS (Phosphate Buffered Salts) | Takara bio | T900 | |
| 96-well plate | Sumitomo bakelite | 631-21031 | |
| 1000ul Chip | NIPPON Genetics | FG-402 | |
| 200ul Chip | NIPPON Genetics | FG-301 | |
| 10ul Chip | NIPPON Genetics | 37650 | |
| CO2 incubator | Thermo Fisher Scientific | Model 370 | |
| GFP Expressing Human Umbilical Vein Endothelial Cell | Angio Proteomie | cAP-0001GFP | |
| Fibrinogen from bovine plasma | Sigma | F8630 | |
| Aprotinin from bovine lung | Sigma | A6279 | |
| Collagen I | Corning | 354236 | |
| Thrombin from bovine plasma | Sigma | T4648 | |
| Hoechst 33342 | Invitrogen | H21492 | Fluorescent dye to stain nuclei in protocol 5.5 |
| Paraformaldehyde Solution | Wako | 163-25983 | |
| Inverted Fluorescence Microscope | OLYMPUS | IX71 | |
| Degital CCD Camera | OLYMPUS | ORCA-R2 | |
| Confocal Laser Scanning Biological Microscope | OLYMPUS | FV1000 | |
| Inverted Fluorescence Microscope | OLYMPUS | IX-83 | |
| Fluorescein isothiocyanate-dextran | Sigma | FD70S |
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Cite This Article
Nashimoto, Y., Teraoka, Y., Banan Sadeghian, R., Nakamasu, A., Arima, Y., Hanada, S., Kotera, H., Nishiyama, K., Miura, T., Yokokawa, R. Perfusable Vascular Network with a Tissue Model in a Microfluidic Device. J. Vis. Exp. (134), e57242, doi:10.3791/57242 (2018).