Abstract
此详细的协议概述图像制导，激光为基础的水凝胶降解嵌入在PEGDA水凝胶血管源性微流体网络的制造执行。在这里，我们描述的虚拟面具，使图像引导的激光控制的建立;一个微成形PEGDA水凝胶，适用于微流体网络制造和压头驱动流的光聚合;设置和使用飞秒脉冲钛配对市售的激光扫描共聚焦显微镜：秒激光诱导水凝胶退化;并使用制造的微流体网络的成像荧光物质和共焦显微镜。多协议的集中在显微镜软件和显微镜宏的适当的设置和实施方式中，因为这些是在使用市售的显微镜对包含许多复杂的微流体制造目的的关键步骤。此techniqu的图像引导部件e可以为3D图像堆栈或用户生成3D模型的实现，从而使创意微设计和几乎所有的配置复杂的微流体系统的制造。在组织工程中的预期效果，在该协议中概述的方法可以在先进的仿生microtissue构建用于器官和人体上的单芯片器件的制造提供帮助。通过模仿复杂结构，扭曲，尺寸和体内脉管系统的密度，基本生物运输过程可以在这些构造中复制，导致药代动力学和疾病的更准确的体外模型。
Introduction
血管系统，包括两个淋巴和cardiovasculature的，形式高度致密的网络，是用于营养和氧气的传输和用于去除代谢废物是必不可少的。因此，居住在血管化组织细胞从来没有超过50-100微米远离容器1。重现体内血管结构体外的能力，关键是要精确地模拟使用工程结构在体内的运输过程。随着最近的驱动开发用于高通量药物器官上的单芯片器件2筛选3,4和疾病建模5,6，方法创建一个概括的合成或天然水凝胶在体内般的交通正在绘制微流控网络显著的兴趣。以提高在这些设备中使用microtissues的仿生学，我们开发了利用三dimensiona一个图像引导，基于激光的水凝胶降解法作为模板天然脉管l的（3D）图像栈以生成嵌入PEGDA水凝胶7血管源性的微流体网络中。这个协议概括了使用配备有飞秒脉冲激光经由图像引导，基于激光的降解来制造在PEGDA凝胶血管源性，仿生微流体网络的市售激光扫描共焦显微镜的。
来流化水凝胶当前的方法包括的血管8-10或血管生成10-12内皮细胞自组装和微制造技术中的诱导对内皮13-16创建预定义的信道。而自组装网络概括密度和微血管的复杂结构，它们往往比体内网络11,17,18，这可能是在造型运输用于药物筛选应用问题更加可渗透的。自组装网络由physiolog的ically相关，毛细管尺寸的容器，但它们可能难以与散装流体流动整合由于在产生较大的动脉大小船只限制。因为是在这些网络的装配没有直接控制，最终的结构可以从样本变化来样，使得难以重复地制备具有相同的流体流动和输运性质网络。
为了产生3D，水凝胶的嵌有重复的几何形状和良好定义的体系结构的微流体网络中，若干微细加工技术得到了发展，其中包括模块式组件13，牺牲材料16，直接写入组件14，和全向印刷15的三维印刷。应用这些方法，微流体结构，因此，流体流动和输运性质，可以在许多结构被重复制造。这些方法的主要限制，然而，这是无法创造微法luidic网络与毛细管大小的功能，4〜10微米19。多数的微细加工技术往往局限于特征在直径13,16从150至650微米。一些现有技术能够在很宽的直径范围内具有通道产生分层网络，10至300微米为直接写入组件14，和18至600微米为全向印刷15，但它们在他们的产生密集网络或能力有限到单个构建体7内产生极为接近的多个微流体网络。
为了克服这些限制，我们开发的图像制导，激光为基础的水凝胶降解技术，使仿生的重复制造，即概括体内微血管的结构层次微流体网络。要做到这一点，一个790纳米，140飞秒脉冲激光在80 MHz工作是光栅扫描我Ñ所需的水凝胶内的三维位置， 通过体内脉管系统的图像作为限定。我们推测，降解过程通过水的激光诱导光学击穿，所得等离子体形成时，水的随后的快速热弹扩张，和水凝胶的局部降解操作作为水膨胀20。这种机制从基于蛋白的水凝胶21-24的基于激光的降解略有不同。不像PEGDA，其具有低多光子截面，蛋白质常常具有大的多光子截面，因此通过多光子吸收引起的化学断裂23降解。以生成图像引导的微流体网络中，激光快门被图像衍生虚拟口罩，其由限定微流体结构的兴趣9区域的镶嵌的控制。使用这种方法，我们已经证明，制作三维血管源性仿生微流体的能力集成电路网络，概括在体内脉管系统的密集和曲折结构，以便通过改变能量的降解期间输送的量局部地控制水凝胶的孔隙率。我们也已经能够产生交织在接近（15微米），但从未直接连接7两个独立的微流控网络。我们还证明，通过交降解官能与整结扎肽序列endothelialize激光恶化微通道的能力，精氨酸-甘氨酸-天冬氨酸-丝氨酸（RGDS），以促进内皮细胞的粘附和管腔形成7。
有了这个协议，复杂的微流控网络在PEGDA水凝胶的生成是使用在许多大学校园访问市售显微镜通过影像引导下，基于激光的下降成为可能。作为降解过程是由数字，虚拟掩模导向，这法布里卡化技术是适合于微流体网络的创意设计，允许其在各种应用中使用。我们预计，在这里所描述的方法将是在设计能够复制生物运输过程建模药物转运2重要的仿生器官和人体上的单芯片器件最有利的。这种制造技术也可用于在体外疾病模型，包括癌症转移5,6-和血脑屏障模型25的产生兴趣。如基于激光的水凝胶降解以前已用于创建神经元向外生长21-23的引导轨道，该技术的图像引导的延伸可在高级组织工程策略证明是有用的定位在用户定义三维空间排列的细胞。
Materials
| Name | Company | Catalog Number | Comments |
| MATLAB | Mathworks | R2015a | named "programming software" in protocol; refer to source 9 for details on algorithm |
| FIJI (Fiji is Just Image J) | NIH | version 1.51a | named "image processing software" in protocol |
| LSM 780 Confocal Microscope | Zeiss | named "laser-scanning confocal microscope" in protocol; for laser-based hydrogel degradation | |
| Zen 2010B SP1 | Zeiss | release version 6.0 | named "microscope software" in protocol; for use on Zeiss LSM-780 |
| Multitime, 2010 | Zeiss | v16.0 | named "microscope macro" in protocol; for use on Zeiss LSM-780 (in conjunction with Zen) |
| Objective W Plan-Apochromat 20X/1.0 DIC D=0.17 M27 75 mm | Zeiss | 421452-9880-000 | for use on Zeiss LSM-780 |
| Chameleon Vision II Modelocked Ti:S Laser | Coherent | named "high-powered pulsed laser" in protocol | |
| Sodium chloride | Sigma-Aldrich | S5886-1KG | |
| HEPES | Sigma-Aldrich | H3375-250G | |
| Triethanolamine (TEOA) | Sigma-Aldrich | 90279-100ML | flammable; skin and eye irritant; work with in a fume hood |
| Sodium hydroxide | Sigma-Aldrich | S5881-500G | |
| 150 mL Vacuum Filtration Cups with 0.2 µm PES Membrane | VWR | 10040-460 | |
| PEGDA | synthesized in house | refer to source 33 for synthesis methods; store under argon | |
| Eosin Y disodium salt | Sigma-Aldrich | E6003-25G | |
| 1-vinyl-2-pyrrolidinone (NVP) | Sigma-Aldrich | V3409-5G | store under argon; carcinogenic; work with in a fume hood |
| 3M Double Coated Tape, 9500PC, 6.0 mil | Thomas Goldkamp | 37728 | |
| Flexmark 90 PFW Liner | FLEXcon | FLX000620 | backing for handling of double coated tape |
| Model SC Plotter (adhesive cutter) | USCutter | SC631E | used to cut adhesive in ring shapes to connect coverslips to petri dishes |
| 60 mm Petri Dish with 20 mm Hole | MatTek Corporation | P60-20-F-NON | |
| High Intensity Illuminator (white light source) | Fiber-Lite | 4715MS-12WB10 | |
| Power Meter | Newport | 1916-R | detect power at 524 nm when using white light source |
| Slim Profile Wand Detector | Newport | 918D-ST | for use with power meter |
| Sylgard 184 Silicone Elastomer Kit | Dow Corning | 3097358-1004 | used to make PDMS molds; refer to source 7 for methods |
| TMPSA-Functionalized #1.5 Coverslips, 40 mm Round | synthesized in house | refer to source 7 for methods | |
| Dextran, Fluorescein, 2,000,000 MW, Anionic, Lysine Fixable | Life Technologies | D7137 | can use alternative tagged dextrans; 2,000 kDa does not diffuse readily into a 5% 3.4 kDa PEGDA hydrogel |
| 1 mL Syringe, Luer-Lok | BD | 309628 | |
| Acrodisc Syring Filter, 0.2 µm Supor Membrane, Low Protein Binding | Pall | PN 4602 | |
| sticky-Slide VI0.4 | Ibidi | 80601 | microfluidic devices that can be used to house hydrogels |
References
- Baish, J. W., Stylianopoulos, T., et al. Scaling rules for diffusive drug delivery in tumor and normal tissues. Proc. Natl. Acad. Sci. USA. 108 (5), 1799-1803 (2011).
- Bhatia, S. N., Ingber, D. E. Microfluidic organs-on-chips. Nature Biotechnol. 32 (8), 760-772 (2014).
- Esch, E. W., Bahinski, A., Huh, D. Organs-on-chips at the frontiers of drug discovery. Nat. Rev. Drug Discov. 14 (4), 248-260 (2015).
- Ghaemmaghami, A. M., Hancock, M. J., Harrington, H., Kaji, H., Khademhosseini, A. Biomimetic tissues on a chip for drug discovery. Drug Discov. Today. 17 (3-4), 173-181 (2012).
- Jeon, J. S., Bersini, S., et al. Human 3D vascularized organotypic microfluidic assays to study breast cancer cell extravasation. Proc. Natl. Acad. Sci. USA. 112 (1), 214-219 (2015).
- Pisano, M., Triacca, V., Barbee, K. A., Swartz, M. A. An in vitro model of the tumor-lymphatic microenvironment with simultaneous transendothelial and luminal flows reveals mechanisms of flow enhanced invasion. Integr. Biol. 7 (5), 525-533 (2015).
- Heintz, K. A., Bregenzer, M. E., Mantle, J. L., Lee, K. H., West, J. L., Slater, J. H. Fabrication of 3D Biomimetic Microfluidic Networks in Hydrogels. Adv. Healthc. Mater. , (2016).
- Cuchiara, M. P., Gould, D. J., McHale, M. K., Dickinson, M. E., West, J. L. Integration of Self-Assembled Microvascular Networks with Microfabricated PEG-Based Hydrogels. Adv. Funct. Mater. 22 (21), 4511-4518 (2012).
- Culver, J. C., Hoffmann, J. C., Poché, R. A., Slater, J. H., West, J. L., Dickinson, M. E. Three-Dimensional Biomimetic Patterning in Hydrogels to Guide Cellular Organization. Adv. Mater. 24 (17), 2344-2348 (2012).
- Kim, S., Lee, H., Chung, M., Jeon, N. L. Engineering of functional, perfusable 3D microvascular networks on a chip. Lab Chip. 13 (8), 1489 (2013).
- Saik, J. E., Gould, D. J., Keswani, A. H., Dickinson, M. E., West, J. L. Biomimetic Hydrogels with Immobilized EphrinA1 for Therapeutic Angiogenesis. Biomacromolecules. 12 (7), 2715-2722 (2011).
- Zisch, A. H., Lutolf, M. P., et al. Cell-demanded release of VEGF from synthetic, biointeractive cell-ingrowth matrices for vascularized tissue growth. FASEB J. , (2003).
- He, J., Zhu, L., Liu, Y., Li, D., Jin, Z. Sequential assembly of 3D perfusable microfluidic hydrogels. J. Mater. Sci. Mater. Med. 25 (11), 2491-2500 (2014).
- Therriault, D., White, S. R., Lewis, J. A. Chaotic mixing in three-dimensional microvascular networks fabricated by direct-write assembly. Nat. Mater. 2 (4), 265-271 (2003).
- Wu, W., DeConinck, A., Lewis, J. A. Omnidirectional Printing of 3D Microvascular Networks. Adv. Mater. 23 (24), H178-H183 (2011).
- Kolesky, D. B., Truby, R. L., Gladman, A. S., Busbee, T. A., Homan, K. A., Lewis, J. A. 3D Bioprinting of Vascularized, Heterogeneous Cell-Laden Tissue Constructs. Adv. Mater. 26 (19), 3124-3130 (2014).
- Zervantonakis, I. K., Hughes-Alford, S. K., Charest, J. L., Condeelis, J. S., Gertler, F. B., Kamm, R. D. Three-dimensional microfluidic model for tumor cell intravasation and endothelial barrier function. Proc. Natl. Acad. Sci. USA. 109 (34), 13515-13520 (2012).
- Roudsari, L. C., West, J. L. Studying the influence of angiogenesis in in vitro cancer model systems. Adv. Drug Deliv. Rev. , (2015).
- Dawson, T. H. Scaling laws for capillary vessels of mammals at rest and in exercise. Proc. R. Soc. Long. [Biol.]. 270 (1516), 755-763 (2003).
- Vogel, A., Noack, J., Hüttman, G., Paltauf, G. Mechanisms of femtosecond laser nanosurgery of cells and tissues. Appl. Phys. B. 81 (8), 1015-1047 (2005).
- Sarig-Nadir, O., Livnat, N., Zajdman, R., Shoham, S., Seliktar, D. Laser Photoablation of Guidance Microchannels into Hydrogels Directs Cell Growth in Three Dimensions. Biophys. J. 96 (11), 4743-4752 (2009).
- Three-dimensional guidance of DRG neurite outgrowth using multi-photon photo-ablation. Livnat, N., Sarig-Nadir, O., Seliktar, D., Shoham, S. 2009 4th International IEEE/EMBS Conference on Neural Engineering, , 116-119 (2009).
- Applegate, M. B., Coburn, J., et al. Laser-based three-dimensional multiscale micropatterning of biocompatible hydrogels for customized tissue engineering scaffolds. Proc. Natl. Acad. Sci. USA. 112 (39), 12052-12057 (2015).
- Ilina, O., Bakker, G. J., Vasaturo, A., Hoffman, R. M., Friedl, P. Two-photon laser-generated microtracks in 3D collagen lattices: principles of MMP-dependent and -independent collective cancer cell invasion. PB. 8 (2), (2011).
- Naik, P., Cucullo, L. In Vitro Blood-Brain Barrier Models: Current and Perspective Technologies. J. Pharm. Sci. 101 (4), 1337-1354 (2012).
- Choe, Y., Mayerich, D., et al. Specimen Preparation, Imaging, and Analysis Protocols for Knife-edge Scanning Microscopy. JoVE. (58), (2011).
- Mayerich, D., Kwon, J., Sung, C., Abbott, L., Keyser, J., Choe, Y. Fast macro-scale transmission imaging of microvascular networks using KESM. Biomed. Opt. Express. 2 (10), 2888-2896 (2011).
- Chung, J. R., Sung, C., et al. Multiscale Exploration of Mouse Brain Microstructures Using the Knife-Edge Scanning Microscope Brain Atlas. Front. Neuroinform. 5, (2011).
- Schindelin, J., Arganda-Carreras, I., et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods. 9 (7), 676-682 (2012).
- Shukla, A., Slater, J. H., Culver, J. C., Dickinson, M. E., West, J. L. Biomimetic Surface Patterning Promotes Mesenchymal Stem Cell Differentiation. ACS Appl. Mater. Interfaces. , (2015).
- Slater, J. H., Culver, J. C., et al. Recapitulation and Modulation of the Cellular Architecture of a User-Chosen Cell of Interest Using Cell-Derived, Biomimetic Patterning. ACS Nano. 9 (6), 6128-6138 (2015).
- Slater, J. H., West, J. L. Fabrication of Multifaceted, Micropatterned Surfaces and Image-Guided Patterning Using Laser Scanning Lithography. Methods Cell Biol. 119, 193-217 (2014).
- DeLong, S. A., Gobin, A. S., West, J. L. Covalent immobilization of RGDS on hydrogel surfaces to direct cell alignment and migration. J. Control. Release. 109 (1-3), 139-148 (2005).
- DeForest, C. A., Anseth, K. S. Cytocompatible click-based hydrogels with dynamically-tunable properties through orthogonal photoconjugation and photocleavage reactions. Nat. Chem. 3 (12), 925-931 (2011).
- Kloxin, A. M., Kasko, A. M., Salinas, C. N., Anseth, K. S. Photodegradable Hydrogels for Dynamic Tuning of Physical and Chemical Properties. Science. 324 (5923), 59-63 (2009).
- Tibbitt, M. W., Kloxin, A. M., Dyamenahalli, K. U., Anseth, K. S. Controlled two-photon photodegradation of PEG hydrogels to study and manipulate subcellular interactions on soft materials. Soft Matter. 6 (20), 5100-5108 (2010).
