毛细力光刻技术在心脏组织工程
Summary
In this protocol, we demonstrate the fabrication of biomimetic cardiac cell culture substrata made from two distinct polymeric materials using capillary force lithography. The described methods provide a scalable, cost-effective technique to engineer the structure and function of macroscopic cardiac tissues for in vitro and in vivo applications.
Abstract
心血管疾病仍然是全世界死亡1的首要原因。心脏组织工程拥有许多承诺能够提供突破性的医学发现与开发用于心脏再生和体外筛选分析功能组织的目标。但是，要创建心脏组织的高保真模型的能力已经证明，很难。心脏的细胞外基质（ECM）是一种复杂的结构，包括生物化学和生物力学信号从微至纳米尺度2。局部机械负载条件和细胞-细胞外基质相互作用的最近被确认为在心脏组织工程3-5关键部件。
心脏ECM的很大一部分是由排列的胶原纤维与纳米级直径的显著影响组织结构和机电耦合2。不幸的是，很少有甲肝病毒方法Ë能够模仿的ECM纤维的组织下来到纳米尺度。在纳米材料制备技术的最新发展，但是，已经启用了模仿的心脏6-9 ECM的体内结构和基底刚度线索可伸缩支架的设计和制造。
这里，我们提出的发展2再现的，具有成本效益且可扩展的纳米图案的过程，使用生物相容的聚合物的聚（丙交酯-共-乙交酯）（PLGA）8和聚氨酯（PU）基聚合物心肌细胞的功能对应。这些各向异性nanofabricated基质（ANFS）模仿的良好组织，对准组织相关的ECM，并且可以用于研究纳米形貌对细胞的形态和功能10-14中的作用。
使用nanopatterned（NP）硅原型为模板，聚氨酯丙烯酸酯（PUA）的模具制造。这PUA模具，然后用PA通过紫外线辅助或溶剂介导的毛细管力光刻（CFL），分别为15,16 ttern聚氨酯或PLGA水凝胶。简要地，PU或PLGA的预聚物是滴分配到一个玻璃盖玻片和PUA模具被放置在顶部。用于UV辅助CFL，拾音器，然后暴露于UV辐射（λ= 250-400纳米）进行固化。对于溶剂介导的节能灯相比，PLGA是用热（120℃）和压力（100千帕）压花。固化后，将模具中的PUA被剥离，留下一个ANFS用于细胞培养。主细胞，如新生大鼠心室肌细胞，以及人多能干细胞衍生的心肌细胞，可以维持在ANFS 2。
Introduction
心血管疾病是发病率和死亡率在世界上领先的原因，并提出对已经紧张的全球卫生系统1,17一个沉重的社会经济负担。心脏组织工程有两个不同的目标：（1）缺血性疾病或心肌病后的再生受损心肌或（2）创建的心脏进行体外药物筛选或疾病模型的高保真度模型。
心脏是一个复杂的器官，必须不断地努力提供血液到身体。心肌细胞和组织的支持密密麻麻的层状结构在整个心脏壁18,19排列成螺旋状图案。心脏是机电也以高度协调的方式有效地排出血液输送到全身21加上20。几个主要障碍仍有待解决，但是，之前巧夺天工的设计，能够可靠体外概括。首先，尽管强劲的心肌细胞分化的方法继续发展22，HPSC-CMS仍然表现出相当不成熟的表现型。其机电性能和形态最接近胎儿的水平23。其次，当保持在传统的培养条件下，无论是干细胞衍生和初级心肌不能组装成原生，组织样结构。相反，细胞变得随机取向，并没有表现出成人的心肌24的带状杆形外观。
细胞外基质（ECM）的环境中与细胞相互作用中起着许多细胞过程11,13,25显著作用。细胞外基质组成的复杂的，定义良好的分子和地形线索显著影响细胞6,26的结构和功能。内部的心脏，细胞排列紧密遵循的基本纳米尺度的ECM纤维2。这些nanotopograph的影响对细胞和组织的功能iCal的线索，然而，还远远没有完全理解。纳米尺度的细胞的生物材料相互作用的初步研究表明，亚微米，沟槽宽度为细胞信号27，粘附28-30，生长31和分化32,33的潜在重要性和影响。然而，由于在显影重现的和可扩展的nanofabricated基板的难度，这样的研究不能再现复杂的体内细胞外基质环境中的多尺度的细胞效应。在这个协议中，一个简单和具有成本效益的纳米加工技术生产的细胞培养支架模仿原生心脏细胞外基质纤维排列进行了描述，从而为广泛的心肌生物材料相互作用的新调查。了解心肌细胞与细胞外基质的纳米级环境如何相互作用可能允许控制细胞行为更加紧密地模仿天然组织功能的能力化。此外，单层细胞是一种简化的实验系统相比，三维结构，但仍然表现出对有见地的调查和功能性筛选2,34-36复杂的多细胞行为。最后，这样的支架可以用于改善细胞的移植物功能时进行再生的目的37植入到心脏。
Protocol
Representative Results
Discussion
功能上成熟的心脏组织都在体内和心脏组织工程的体外应用缺乏供。这里所描述的紧凑型荧光灯的纳米加工方法是健壮的技术可以实现蜂窝对准与影响宏观组织的功能，由于该系统的可伸缩性。大区域可以很容易地被图案化，并用于细胞培养。宏观蜂窝对准是在心脏组织工程必要的，以便建立仿生的，因为它影响着心肌38的机械和电气性能的功能组织。
?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
D. H. Kim thanks the Department of Bioengineering at the University of Washington for the new faculty startup fund. D. H. Kim is also supported by the Perkins Coie Award for Discovery, the Wallace H. Coulter Foundation Translational Research Partnership Award, the Washington State Life Science Discovery Fund, and the American Heart Association Scientist Development Grant (13SDG14560076). J. Macadangdang and A. Jiao thank the support from the NIH Bioengineering Cardiovascular Training Grant Fellowship. Additional support for this work comes from the National Institutes of Health (NIH) grant R01HL111197 to M. Regnier.
Materials
| Name | Company | Catalogue number | Comments (optional) |
| Fibronectin | BD Biosciences | 354008 | |
| NOA 76 | Norland Products, Inc. | 7606B | |
| Surface Adhesion Promotor (Glass Primer) | Minuta Tech | ||
| PUA | Minuta Tech | MINS-311RM | |
| Soft Rubber Roller | Speedball | ||
| Silicon Wafers | NOVA Electronic Materials | FA01-9900 | |
| Photoresist | Shipley | SPRT510 | |
| Photoresist Developer | Shipley | MF320 | |
| Electron-Beam Lithography System | JEOL | JBX-9300FS | |
| Etching System | Surface Technology Systems | NP10 8UJ | |
| Plasma Asher System | BMR Technology Co. | DSF-200 | |
| Ozone Cure System | Minuta Tech | MT-UV-O- 08 | |
| Fusion Cure System | Minuta Tech | MT-UV-A 11 | |
| NOA 83H | Norland Products, Inc. | 8301 | |
| Spin Coater | Laurel Technology | WS-400-6NPP | |
| Skyrol PET Film | SKC Co., Ltd. | 23038-59-9 | |
| 25mm Glass Slides | Corning | 2948 | |
| Sylgard 184 Silicone Elastomer Kit | Dow Corning | 6/5/2553 | |
| Poly(D,L-lactide-co-glycolide) | Sigma-Aldrich | P2191-1G | |
| Chloroform | Sigma-Aldrich | 372978-1L | |
| 500g Weights | Global Insustrial | T9FB503120 | |
| Isopropyl Alcohol | EMD Millipore | PX1835-2 | |
| Hot Plate | Corning | PC-420D | |
| Sonicator | Branson | B2510MTH |
References
- Lozano, R., et al. Global and regional mortality from 235 causes of death for 20 age groups in 1990 and 2010: a systematic analysis for the Global Burden of Disease Study 2010. The Lancet. 380, 2095-2128 (2012).
- Kim, D. -. H., et al. Nanoscale cues regulate the structure and function of macroscopic cardiac tissue constructs. Proceedings of the National Academy of Sciences. 107, 565-570 (2010).
- Tulloch, N. L., et al. Growth of Engineered Human Myocardium With Mechanical Loading and Vascular Coculture. Circulation Research. 109, 47-59 (2011).
- Bursac, N., Parker, K., Irvanian, S., Tung, L. Cardiomyocyte Cultures With Controlled Macroscopic Anisotropy: A Model for Functional Electrophysiological Studies of Cardiac Muscle. Circulation Research. 91, (2002).
- Fink, C., et al. Chronic stretch of engineered heart tissue induces hypertrophy and functional improvement. The FASEB Journal. 14, 669-679 (2000).
- Stevens, M. M. Exploring and Engineering the Cell Surface Interface. Science. 310, (2005).
- Mark, K., Park, J., Bauer, S., Schmuki, P. Nanoscale engineering of biomimetic surfaces: cues from the extracellular matrix. Cell Tissue Res. 339, 131-153 (2009).
- Lü, J. -. M., Wang, X., Marin-Muller, C., Wang, H., Lin, P. H., Yao, Q., Chen, C. Current advances in research and clinical applications of PLGA-based nanotechnology. Expert. Rev. Mol. Diagn. 9, 325-341 (2009).
- Kim, H. N., et al. Patterning Methods for Polymers in Cell and Tissue Engineering. Ann Biomed Eng. 40, 1339-1355 (2012).
- Kim, D. -. H., Provenzano, P. P., Smith, C. L., Levchenko, A. Matrix nanotopography as a regulator of cell function. The Journal of Cell Biology. 197, 351-360 (2012).
- Park, J., Kim, H. -. N., Kim, D. -. H., Levchenko, A., Kahp-Yang, S. Quantitative Analysis of the Combined Effect of Substrate Rigidity and Topographic Guidance on Cell Morphology. IEEE Trans.on Nanobioscience. 11, 28-36 (2012).
- Kim, D. -. H., Lee, H., Lee, Y. K., Nam, J. -. M., Levchenko, A. Biomimetic Nanopatterns as Enabling Tools for Analysis and Control of Live Cells. Adv. Mater. 22, 4551-4566 (2010).
- Kim, D. -. H., Wong, P. K., Park, J., Levchenko, A., Sun, Y. Microengineered Platforms for Cell Mechanobiology. Annu. Rev. Biomed. Eng. 11, 203-233 (2009).
- Kim, D. -. H., et al. Nanopatterned cardiac cell patches promote stem cell niche formation and myocardial regeneration. Integr Biol. 4, 1019 (2012).
- Kim, P., et al. Fabrication of nanostructures of polyethylene glycol for applications to protein adsorption and cell adhesion. Nanotechnology. 16, 2420-2426 (2005).
- Hwang, S. Y., et al. Adhesion Assays of Endothelial Cells on Nanopatterned Surfaces within a Microfluidic Channel. Anal. Chem. 82, 3016-3022 (2010).
- Heidenreich, P. A., et al. Forecasting the Future of Cardiovascular Disease in the United States: A Policy Statement From the American Heart Association. Circulation. 123, 933-944 (2011).
- Legrice, I. J., et al. Laminar structure of the heart: ventricular myocyte arrangement and connective tissue architecture in the dog. Am J Physiol Heart Circ Physiol. 269, 1-12 (2002).
- Sosnovik, D. E., Wang, R., Dai, G., Reese, T. G., Wedeen, V. J. Diffusion MR tractography of the heart. J Cardiovasc Magn Reson. 11, 47 (2009).
- Bers, D. M. Calcium Fluxes Involved in Control of Cardiac Myocyte Contraction. Circulation Research. 87, 275-281 (2000).
- Mohrman, D. E., Heller, L. J. . Cardiovascular Physiology. , (2010).
- Burridge, P. W., Keller, G., Gold, J. D., Wu, J. C. Production of De Novo Cardiomyocytes: Human Pluripotent Stem Cell Differentiation and Direct Reprogramming. Stem Cell. 10, 16-28 (2012).
- Zhang, J., et al. Functional Cardiomyocytes Derived From Human Induced Pluripotent Stem Cells. Circulation Research. 104, (2009).
- Qian, J. -. Y., Guo, L. Altered cytosolic Ca2+ dynamics in cultured Guinea pig cardiomyocytes as an in vitro model to identify potential cardiotoxicants. Toxicology in Vitro. 24, 960-972 (2010).
- You, M. -. H., et al. Synergistically Enhanced Osteogenic Differentiation of Human Mesenchymal Stem Cells by Culture on Nanostructured Surfaces with Induction Media. Biomacromolecules. 11, 1856-1862 (2010).
- Kim, H. N., et al. Nanotopography-guided tissue engineering and regenerative medicine. Advanced Drug Delivery Reviews. 65, 536-558 (2013).
- Mannix, R. J., et al. Nanomagnetic actuation of receptor-mediated signal transduction. Nature Nanotech. 3, 36-40 (2007).
- Karuri, N. W., et al. Biological length scale topography enhances cell-substratum adhesion of human corneal epithelial cells. J Cell Sci. 117, 3153-3164 (2007).
- Cavalcanti-Adam, E. A., et al. Cell Spreading and Focal Adhesion Dynamics Are Regulated by Spacing of Integrin Ligands. Biophysical Journal. 92, 2964-2974 (2007).
- Koo, L. Y., Irvine, D. J., Mayes, A. M., Lauffenburger, D. A., Griffith, L. G. Co-regulation of cell adhesion by nanoscale RGD organization and mechanical stimulus. J Cell Sci. 115, 1-11 (2002).
- Yim, E. K. F., et al. Nanopattern-induced changes in morphology and motility of smooth muscle cells. Biomaterials. 26, 5405-5413 (2008).
- Dalby, M. J., et al. The control of human mesenchymal cell differentiation using nanoscale symmetry and disorder. Nat Mater. 6, 997-1003 (2007).
- Park, J., Bauer, S., Mark, v. o. n. . d. e. r. . K., Schmuki, P. Nanosize and Vitality: TiO 2Nanotube Diameter Directs Cell Fate. Nano Lett. 7, 1686-1691 (2007).
- Entcheva, E., Bien, H. Macroscopic optical mapping of excitation in cardiac cell networks with ultra-high spatiotemporal resolution. Progress in Biophysics and Molecular Biology. 92, 232-257 (2006).
- Tung, L., Zhang, Y. Optical imaging of arrhythmias in tissue culture. Journal of Electrocardiology. 39, (2006).
- Himel, H. D., Bub, G., Lakireddy, P., El-Sherif, N. Optical imaging of arrhythmias in the cardiomyocyte monolayer. Heart Rhythm. 9, 2077-2082 (2012).
- Kim, J., Hayward, R. C. Mimicking dynamic in vivo environments with stimuli-responsive materials for cell culture. Trends in Biotechnology. 30, 426-439 (2012).
- Henderson, D. J., Anderson, R. H. The Development and Structure of the Ventricles in the Human Heart. Pediatr Cardiol. 30, 588-596 (2009).
- Badie, N., Bursac, N. Novel Micropatterned Cardiac Cell Cultures with Realistic Ventricular Microstructure. Biophysj. 96, 3873-3885 (2009).
- Badrossamay, M. R., McIlwee, H. A., Goss, J. A., Parker, K. K. Nanofiber Assembly by Rotary Jet-Spinning. Nano Lett. 10, 2257-2261 (2010).
- Rao, C., et al. The effect of microgrooved culture substrates on calcium cycling of cardiac myocytes derived from human induced pluripotent stem cells. Biomaterials. 34, 2399-2411 (2013).
