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Bioengineering

多路人工细胞微环境阵列的制备

Published: September 07, 2018
doi:
10.3791/57377
, , , , , , , , , , , , , , , ,
1Institute for Integrated Cell-Material Sciences (WPI-iCeMS),Kyoto University, 2Department of Life Science and Technology, School of Life Science and Technology,Tokyo Institute of Technology, 3Biomaterials Center for Regenerative Medical Engineering,Foundation for Advancement of International Science, 4Faculty of Science and Natural Resources,Universiti Malaysia Sabah, 5Institute for Chemical Research,Kyoto University, 6Ecole Normale Supérieure

Summary

本文介绍了用于制备多路人工细胞微环境 (MACME) 阵列的详细方法, 用于对模拟体内细胞微环境的物理和化学线索进行高通量操作, 并用单细胞分析方法确定人类多潜能干细胞 (hPSCs) 的最佳细胞环境。

Abstract

细胞微环境由多种线索组成, 如生长因子、细胞外基质和细胞间相互作用。这些线索是精心编排的, 是调节细胞功能在一个生活系统中的关键。虽然许多研究人员试图研究环境因素与所期望的细胞功能之间的相关性, 但仍有许多未知之处在。这主要是由于缺乏一个适当的方法来模仿这种环境线索的体外, 并同时测试不同的环境提示细胞。在这里, 我们报告一个综合平台的微流控通道和碳纤维阵列, 其次是高含量单细胞分析, 以检查干细胞表型改变的不同环境因素。为了证明该平台的应用, 本研究重点研究了自我更新的人多潜能干细胞 (hPSCs) 的表型。在这里, 我们提出了一个碳纤维阵列的准备程序和微流控结构制造的多复用人工细胞微环境 (MACME) 阵列。此外, 还介绍了单细胞分析、多荧光标记细胞染色、多荧光成像和统计分析的总体步骤。

Introduction

人类多潜能干细胞 (hPSCs)1,2自我更新无限和分化为各种组织血统, 这可能会革命性的药物开发, 细胞治疗, 组织工程和再生医学3,4,5,6. 然而, 一般文化菜肴和微量滴定板的设计并不是为了在细胞层面上精确的物理和化学细胞操作, 而纳米到微表的范围是细胞扩张的关键因素,自我更新和分化。为了解决这个缺点, 研究已经研究了细胞微环境在调节细胞命运决定和细胞功能方面的作用4。近年来, 越来越多的研究已经进行了重建细胞微环境的体外7,8。纳米和微加工工艺通过化学9、10、11、12、13的操作建立了这些微环境, 14、15、16、17和物理18、19、20环境提示。到目前为止, 还没有报告系统地调查在单个平台内对细胞命运决定和功能的化学和物理环境线索的基本机制。

在这里, 我们引入了一个基于简单设计原则的策略来建立一个健壮的筛选平台 (图 1)。首先, 我们描述了使用碳纤维阵列和微流控结构创建多功能、人工细胞微环境的集成平台的开发过程: 多路人工细胞微环境 (MACME) 阵列(图 1A和2A)。碳纤维阵列在碳纤维材料和密度的不同组合中有12种不同的微环境。静电纺丝是用来制造纳米纤维的。碳纤维材料, 如聚苯乙烯 (PS)21, polymethylglutarimide (PMGI)22, 和明胶 (GT)23, 旨在测试其化学性质, 这可能影响细胞黏附和维护干细胞 (图 2B)。碳纤维密度随静电纺丝时间的改变而变化, 生成的纳米纤维根据其密度 (DNF, D = XLow/低/中/高) 来定义。微流控结构由烷48细胞培养室组成, 可沿96井微板块的标准尺寸定位。是一种生物相容和气体可交换聚合物, 通常用于制造微流控器件24。每个微流控通道设计为700µm 宽和8.4 毫米长, 其边缘有两个入口 (表 1)。分庭有不同的高度 (250, 500 和1000µm) 来操纵初始细胞播种密度 (0.3, 0.6, 1.2 x 10 细胞/厘米 5), 这可能与生存, 增殖和分化的 hPSCs2(图 2C)。进入腔室的细胞数量与室底板上的柱密度成正比, 从而控制了初始细胞播种密度, 并将同一细胞悬浮液引入不同高度的培养室。所有通道的设计是≥ 250-µm-高26 , 以尽量减少低氧张力27和剪切应力28对细胞的影响。通道高度为250、500和1000µm, 这里缩写为 XCD, 分别为 X = 低、中、高。不同碳纤维密度和初始细胞播种密度的环境被缩短为 “Material_NF density_Cell 密度” (如 GT_HighNF_HighCD: 一种以高密度 GT 纳米纤维和高初始细胞播种为特征的环境;密度)。

随后, 我们描述了如何进行单细胞分析, 系统地调查细胞的行为, 以应对环境因素 (图 1B)。作为概念证明, 我们确定了 hPSC 自我更新的最佳细胞环境, 这是 hPSC 维护的关键功能 (图 1B)29。基于图像的细胞术, 其次是统计分析, 允许对细胞环境的个体细胞表型反应进行定量解释。在多种细胞功能中, 本文提供了一个详细的程序来确定保持 hPSC 自我更新的最佳条件。

Protocol

1. MACME 阵列的制作 注: 所有材料和设备都列在材料表中。 碳纤维阵列和微流控结构模具的掩模制备 使用3维计算机图形软件包 (表 1) 创建用于微流控结构的碳纤维阵列和模具的三维 (3D) 掩码图像。注:3D 图像由3D 打印机读取和打印。打印的掩码和模具具有与在步骤1.1.1 中使用图形软件定义的3D 图像相同的尺寸。 ?…

Representative Results

MACME 阵列: 设计和制造:结合碳纤维技术, 我们利用微流控细胞培养和筛选技术, 以前使用的确定最佳条件 hPSC 自我更新或分化35,36 (图 1)。这非常适合于建立强健的高通量细胞检测方法, 因为细胞培养室和条件是精确可控和可扩展的42,43,<sup class…

Discussion

该协议的第一个筛选方法, 建立一个健全的文化系统, 以维护合格的 hPSCs。首先, 我们描述了如何使用与碳纤维阵列 (MACME 阵列) 集成的微流控设备来准备一个具有不同人工 ECMs 和细胞播种密度的平台。第二, 以定量图像为基础的单细胞分型进行50 , 以评估个别细胞结局和行为改变的独特的生物化学和生物物理特征。在该协议中, 在高初始细胞播种密度条件下, 由 GT 碳纤维和正控 ECM ?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

我们感谢京都大学 iCeMS 的 Nakatsuji 教授提供人类 ES 细胞。我们还感谢东京理工大学的圆山教授在使用原子力显微镜方面的支持。日本促进科学学会慷慨提供资金 (jsp; 22350104、23681028、25886006和 24656502);新能源和工业技术发展组织 (尼德) 和泰尔茂生命科学基金会也提供了资金。iCeMS 由日本教育、文化、体育、科学和技术部 (下个) 世界总理国际研究中心倡议支持。这项工作的一部分是由京都大学纳米技术中心和 AIST 纳米处理设施的支持, 在 “纳米技术平台项目” 赞助的日本下个。

Materials

Polystyrene (PS) Sigma #182435 Average Mw: 290,000, average Mn: 130,000
Polymethylglutarimide (PMGI) MicroChem G113113
Gelatin (GT) Sigma G2625 From porcine skin, type A
Sylgard 184 silicone elastomer kit Doe Corning Toray #1064291 PDMS curing agent and silicone elastomer base are components of this kit.
OpenSCAD This is a free 3D computer graphics software (http://www.openscad.org/) used for designing the mold of the microfluidic device.
AutoCAD 2014 Autodesk This is a 3D computer graphics software (https://www.autodesk.com/products/autocad/overview) used for design of the mask used on nanofiber-array preparation.
3D printer, AGILISTA-3000 Keyence
UV-curable resin, AR-M2 Keyence This is used for 3D printing by Agilista.
Acetic acid Sigma #338826 ≥99.99%
Ethyl acetate Sigma #270989 Anhydrous, 99.8%
Tetrahydrofuran (THF) Sigma #401757
MSP-30T Vacuum Device Magnetron sputtering machine
Nunc OmniTray Thermo Fisher Scientific #242811 This is a polystyrene baseplate on which the nanofiber array is created. This plate size is typically 127.7 x 85.5 mm. 
Gun-type corona discharge machine Shinko Electric & Instrumentation CFG-500 This handy device is used to generate corona for activation of the bottom surface of the PDMS layer at step 1.5 "Assembly of the MACME arrays" in the protocol.
5 mL syringe Terumo SS-05SZ
Stainless-steel blunt needle (23-gauge) Nipro #2166 Outside diameter and length are 0.6 and 32 mm, respectively.
High-voltage power supply TechDempaz
1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide, hydrochloride Dojindo W001
N-Hydroxysuccinimide Sigma #56480
Matrigel hESC-Qualified Matrix Corning #354277 This protein is refered as basement membrane gel matrix in the protocol.
CellAdhere Vitronectin, Human, Solution STEMCELL Technologies #07004
TeSR-E8 STEMCELL Technologies #05940 Feeder-free, xeno-free culture medium for maintenance of human ES and iPS cells
Y-27632 Wako Pure Chemical Industries #253-00513
TrypLE Express Enzyme (1X), phenol red Thermo Fisher Scientific #12605028 This ia a recombinant trypsin-like protease for dissociation of adherant mammalian cells.
Click-iT EdU Imaging Kit with Alexa Fluor 647 Azides Thermo Fisher Scientific C10086 The fluorescent labeling of proliferating cells in on-plate fluorescent staining was performed along the product manual of this kit.
Annexin V, Alexa Fluor 594 conjugate Thermo Fisher Scientific A13203
4',6-diamidino-2-phenylindole (DAPI) Thermo Fisher Scientific D1306
Oct-3/4 Antibody (C-10) Santa Cruz Biotechnology sc-5279
Donkey Anti-Mouse IgG H&L (DyLight 488) abcam ab96875 This is a secondary antibody used in on-plate fluorescent cell staining.
ECLIPSE Ti-E Nikon This is an inverted fluorescence microscope equipped with a CFI Plan Fluor 4×/0.13 N.A. objective lens (Nikon), CCD camera (ORCA-R2, Hamamatsu), mercury lamp (Intensilight, Nikon), XYZ automated stage (Ti-S-ER motorized stage with encoders, Nikon), and filter cubes for four fluorescence channels (DAPI, GFP HYQ, TRITC, Cy5; Nikon)
NIS-Elements Advanced Research Nikon This is a microscope imaging software used for automatic image acquisition.
CellProfiler, Version 2.1.0 This is a free open software for cell image analysis (http://cellprofiler.org/).
R SOM analysis is performed by kohonen package of this software. This is freely available (https://www.r-project.org/).
Cluster 3.0 This is the open source clustering software (http://bonsai.hgc.jp/~mdehoon/software/cluster/software.htm). Unsupervised hierarchical clustering is performed with this software.
Java TreeView This open source software (http://jtreeview.sourceforge.net/) is used to visualize clustering data as a heatmap and a dendrogram.
H9 human embryonic stem cell WiCell Stem Cell Bank WA09
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References

  1. Thomson, J. A., et al. Embryonic stem cell lines derived from human blastocysts. Science. 282 (5391), 1145-1147 (1998).
  2. Takahashi, K., et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell. 131 (5), 861-872 (2007).
  3. Ameen, C., et al. Human embryonic stem cells: Current technologies and emerging industrial applications. Crit Rev Oncol Hematol. 65 (1), 54-80 (2008).
  4. Nishikawa, S., Goldstein, R. A., Nierras, C. R. The promise of human induced pluripotent stem cells for research and therapy. Nat Rev Mol Cell Biol. 9 (9), 725-729 (2008).
  5. Robinton, D. A., Daley, G. Q. The promise of induced pluripotent stem cells in research and therapy. Nature. 481 (7381), 295-305 (2012).
  6. Sartipy, P., Bjorquist, P., Strehl, R., Hyllner, J. The application of human embryonic stem cell technologies to drug discovery. Drug Discovery Today. 12 (17-18), 688-699 (2007).
  7. Discher, D. E., Mooney, D. J., Zandstra, P. W. Growth factors, matrices, and forces combine and control stem cells. Science. 324 (5935), 1673-1677 (2009).
  8. Joyce, J. A., Pollard, J. W. Microenvironmental regulation of metastasis. Nat Rev Cancer. 9 (4), 239-252 (2009).
  9. Danhier, F., Feron, O., Preat, V. To exploit the tumor microenvironment: Passive and active tumor targeting of nanocarriers for anti-cancer drug delivery. J Control Release. 148 (2), 135-146 (2010).
  10. Murphy, W. L., McDevitt, T. C., Engler, A. J. Materials as stem cell regulators. Nat Mater. 13 (6), 547-557 (2014).
  11. Patel, A. K., et al. A defined synthetic substrate for serum-free culture of human stem cell derived cardiomyocytes with improved functional maturity identified using combinatorial materials microarrays. Biomaterials. 61, 257-265 (2015).
  12. Zhang, R., et al. A thermoresponsive and chemically defined hydrogel for long-term culture of human embryonic stem cells. Nat Commun. 4, (2013).
  13. Anderson, D. G., Putnam, D., Lavik, E. B., Mahmood, T. A., Langer, R. Biomaterial microarrays: rapid, microscale screening of polymer-cell interaction. Biomaterials. 26 (23), 4892-4897 (2005).
  14. Celiz, A. D., et al. Discovery of a Novel Polymer for Human Pluripotent Stem Cell Expansion and Multilineage Differentiation. Adv Mater. 27 (27), 4006-4012 (2015).
  15. Hansen, A., et al. High-Density Polymer Microarrays: Identifying Synthetic Polymers that Control Human Embryonic Stem Cell Growth. Adv Healthcare Mater. 3 (6), 848-853 (2014).
  16. Mei, Y., et al. A high throughput micro-array system of polymer surfaces for the manipulation of primary pancreatic islet cells. Biomaterials. 31 (34), 8989-8995 (2010).
  17. Saha, K., et al. Surface-engineered substrates for improved human pluripotent stem cell culture under fully defined conditions. Proc Natl Acad Sci U S A. 108 (46), 18714-18719 (2011).
  18. Bettinger, C. J., Langer, R., Borenstein, J. T. Engineering substrate topography at the micro- and nanoscale to control cell function. Angew Chem Int Ed Engl. 48 (30), 5406-5415 (2009).
  19. McMurray, R. J., et al. Nanoscale surfaces for the long-term maintenance of mesenchymal stem cell phenotype and multipotency. Nat Mater. 10 (8), 637-644 (2011).
  20. Sun, Y., Jallerat, Q., Szymanski, J. M., Feinberg, A. W. Conformal nanopatterning of extracellular matrix proteins onto topographically complex surfaces. Nat Methods. 12 (2), 134-136 (2015).
  21. Nitanan, T., et al. Effects of processing parameters on morphology of electrospun polystyrene nanofibers. Korean J Chem Eng. 29 (2), 173-181 (2012).
  22. Liu, L., et al. Chemically-defined scaffolds created with electrospun synthetic nanofibers to maintain mouse embryonic stem cell culture under feeder-free conditions. Biotechnol Lett. 34 (10), 1951-1957 (2012).
  23. Liu, L., et al. Nanofibrous gelatin substrates for long-term expansion of human pluripotent stem cells. Biomaterials. 35 (24), 6259-6267 (2014).
  24. Kamei, K., et al. Phenotypic and transcriptional modulation of human pluripotent stem cells induced by nano/microfabrication materials. Adv Healthcare Mater. 2 (2), 287-291 (2013).
  25. Peerani, R., et al. Niche-mediated control of human embryonic stem cell self-renewal and differentiation. EMBO J. 26 (22), 4744-4755 (2007).
  26. Yamamoto, K., et al. Fluid shear stress induces differentiation of Flk-1-positive embryonic stem cells into vascular endothelial cells in vitro. Am J Physiol Heart Circ Physiol. 288 (4), 1915-1924 (2005).
  27. Charati, S. G., Stern, S. A. Diffusion of gases in silicone polymers: Molecular dynamics simulations. Macromolecules. 31 (16), 5529-5535 (1998).
  28. Prado-Lopez, S., et al. Hypoxia promotes efficient differentiation of human embryonic stem cells to functional endothelium. Stem Cells. 28 (3), 407-418 (2010).
  29. Yanagihara, K., et al. Prediction of Differentiation Tendency Toward Hepatocytes from Gene Expression in Undifferentiated Human Pluripotent Stem Cells. Stem Cells and Development. 25 (24), 1884-1897 (2016).
  30. Kamei, K., et al. 3D printing of soft lithography mold for rapid production of polydimethylsiloxane-based microfluidic devices for cell stimulation with concentration gradients. Biomed Microdevices. 17 (2), (2015).
  31. Song, J. H., Kim, H. E., Kim, H. W. Production of electrospun gelatin nanofiber by water-based co-solvent approach. J Mater Sci Mater Med. 19 (1), 95-102 (2008).
  32. Owen, M. J., Smith, P. J. Plasma Treatment of Polydimethylsiloxane. J Adhes Sci Technol. 8 (10), 1063-1075 (1994).
  33. Chen, G. K., et al. Chemically defined conditions for human iPSC derivation and culture. Nat Methods. 8 (5), 424-476 (2011).
  34. Watanabe, K., et al. A ROCK inhibitor permits survival of dissociated human embryonic stem cells. Nat Biotechnol. 25 (6), 681-686 (2007).
  35. Kamei, K., et al. An integrated microfluidic culture device for quantitative analysis of human embryonic stem cells. Lab Chip. 9 (4), 555-563 (2009).
  36. Kamei, K., et al. Microfluidic image cytometry for quantitative single-cell profiling of human pluripotent stem cells in chemically defined conditions. Lab Chip. 10 (9), 1113-1119 (2010).
  37. Carpenter, A. E., et al. CellProfiler: image analysis software for identifying and quantifying cell phenotypes. Genome Biol. 7 (10), 100 (2006).
  38. Kohonen, T. Self-Organized Formation of Topologically Correct Feature Maps. Biol Cybern. 43 (1), 59-69 (1982).
  39. Wehrens, R., Buydens, L. M. C. Self- and super-organizing maps in R: The kohonen package. J Stat Softw. 21 (5), 1-19 (2007).
  40. Eisen, M. B., Spellman, P. T., Brown, P. O., Botstein, D. Cluster analysis and display of genome-wide expression patterns. Proc Natl Acad Sci U S A. 95 (25), 14863-14868 (1998).
  41. Saldanha, A. J. Java Treeview–extensible visualization of microarray data. Bioinformatics. 20 (17), 3246-3248 (2004).
  42. Du, G. S., Fang, Q., den Toonder, J. M. J. Microfluidics for cell-based high throughput screening platformsd-A review. Anal Chim Acta. 903, 36-50 (2016).
  43. Huang, S. B., et al. An integrated microfluidic cell culture system for high-throughput perfusion three-dimensional cell culture-based assays: effect of cell culture model on the results of chemosensitivity assays dagger. Lab Chip. 13 (6), 1133-1143 (2013).
  44. Wang, B. L., et al. Microfluidic high-throughput culturing of single cells for selection based on extracellular metabolite production or consumption. Nat Biotechnol. 32 (5), 473 (2014).
  45. Becker, K. A., et al. Self-renewal of human embryonic stem cells is supported by a shortened G1 cell cycle phase. J Cell Physiol. 209 (3), 883-893 (2006).
  46. Neganova, I., Lako, M. G1 to S phase cell cycle transition in somatic and embryonic stem cells. J Anat. 213 (1), 30-44 (2008).
  47. Fox, V., et al. Cell-cell signaling through NOTCH regulates human embryonic stem cell proliferation. Stem Cells. 26 (3), 715-723 (2008).
  48. Xu, C., et al. Feeder-free growth of undifferentiated human embryonic stem cells. Nat Biotechnol. 19 (10), 971-974 (2001).
  49. Kamei, K., et al. Microfluidic-Nanofiber Hybrid Array for Screening of Cellular Microenvironments. Small. 13 (18), (2017).
  50. Sun, J., et al. A Microfluidic Platform for Systems Pathology: Multiparameter Single-Cell Signaling Measurements of Clinical Brain Tumor Specimens. Cancer Res. 70 (15), 6128-6138 (2010).
  51. Theunissen, T. W., Jaenisch, R. Molecular Control of Induced Pluripotency. Cell Stem Cell. 14 (6), 720-734 (2014).
  52. Yoo, H. S., Kim, T. G., Park, T. G. Surface-functionalized electrospun nanofibers for tissue engineering and drug delivery. Adv Drug Del Rev. 61 (12), 1033-1042 (2009).
  53. Dixon, J. E., et al. Combined hydrogels that switch human pluripotent stem cells from self-renewal to differentiation. Proc Natl Acad Sci U S A. 111 (15), 5580-5585 (2014).

Tags

Keywords: 3D PrintingElectrospinningPolymer SolutionsNanofiber ArraysMicrofluidic StructuresCrosslinkingCellular MicroenvironmentStem Cell Engineering
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Mashimo, Y., Yoshioka, M., Tokunaga, Y., Fockenberg, C., Terada, S., Koyama, Y., Shibata-Seki, T., Yoshimoto, K., Sakai, R., Hakariya, H., Liu, L., Akaike, T., Kobatake, E., How, S., Uesugi, M., Chen, Y., Kamei, K. Fabrication of a Multiplexed Artificial Cellular MicroEnvironment Array. J. Vis. Exp. (139), e57377, doi:10.3791/57377 (2018).
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