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생체공학

3D Microtissues 用于可注射再生疗法和高通量药物筛选

Published: October 04, 2017
doi:
10.3791/55982
, , , , ,
1Department of Biomedical Engineering, School of Medicine,Tsinghua University, 2Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, 3School of Life Sciences,Tsinghua University

Summary

本协议描述了通过将微细加工与 cryogelation 技术相结合的方法制备弹性3D 大孔 microcryogels。在加载与细胞, 3D microtissues 生成, 这可以很容易地注入在体内, 以促进再生治疗或组装成阵列的体外高通量药物筛选。

Abstract

为了将传统的2D 细胞培养技术升级到3D 细胞培养, 我们将微加工与 cryogelation 工艺相结合, 生产出大孔微尺度 cryogels (microcryogels), 可加载多种细胞类型, 形成 3D microtissues。在此, 我们提出了制造多功能 3D microtissues 及其在再生疗法和药物筛选中的应用的协议。尺寸和形状可控 microcryogels 可以在一个阵列芯片上制作, 可以被捕获片作为单独的细胞负载载体的可注射再生疗法或进一步组装在芯片上 3D microtissue 阵列的高通量药物筛选。由于这些微型 cryogels 的高弹性性质, 3D microtissues 在注射过程中保护细胞免受机械剪切力的侵袭, 为微创细胞治疗提供了巨大的可。这就保证了小鼠肢体缺血模型中细胞存活率的提高和治疗效果。同时, 以标准的 384-井格式组装 3D microtissue 阵列, 便于使用通用的实验室设施和设备, 使高通量的药物筛选在这个多才多艺的3D 细胞培养平台。

Introduction

传统的细胞培养在平坦的 two-dimensional (2D) 表面, 如培养皿或井板, 很难引出细胞行为接近他们的本土状态。在三维 (3D) 体系结构中包含各种细胞类型、胞外基质和生物活性可溶性因子的环境的精确重述1,2,3 ,4, 对于组织工程、再生医学、基础生物学研究和药物发现的应用 (5、6、7),在体外构建 biomimicking 组织是必不可少的. ,8,9。

在2D 细胞培养中, 3D 细胞培养被广泛应用于培养细胞的仿生微结构和功能性特征的体外。流行的3D 单元格区域性方法是将单元格聚合为椭球7、8、9、10。细胞椭球可以注射到受伤的组织, 增强细胞的保留和生存相比, 注射的分散细胞。然而, 在注射过程中, 流体剪切力对细胞的非均匀球体大小和不可避免的机械损伤导致细胞治疗效果不佳11,12,13。同样, 在椭球聚合过程中固有的不均匀性使得他们的翻译为3D 细胞高通量药物筛选挑战10。

另一种3D 细胞培养方法是在生物材料的帮助下实现的, 它通常将水凝胶或多孔支架中的细胞封装起来。它允许在构建3D 体系结构时具有更大的灵活性。对于治疗, 细胞包裹在散装支架通常被送到动物身体通过外科植入, 这是侵入性和创伤, 因此限制其广泛的翻译到床边。另一方面, 水凝胶可以通过注射悬浮在水凝胶前体溶液中的细胞进入动物体内进行微创治疗, 使原位凝胶通过热, 化学或酶交联的11。然而, 当细胞被交付, 而水凝胶前体仍然在水状态时, 他们也暴露于机械剪在注射期间。不仅如此, 化学或酶交联过程中, 在原位凝胶的凝胶体也可能造成损害的细胞内。在药物筛选中, 生物材料辅助细胞培养面临着均匀性、可控性和吞吐量等问题。使用水凝胶, 细胞通常涉及在凝胶过程中, 这可能会影响细胞的活力和功能。在细胞播种过程中, 凝胶也会阻碍大多数高通量设备的使用, 因为水凝胶可能需要在冰层上保存, 以防止在细胞播种前凝胶, 而水凝胶可能堵塞配药技巧, 这通常非常薄, 以确保准确性高通量筛选。预成形的支架可能会将生物材料的制造过程从细胞培养中分离出来, 但是大多数 scaffold-based 的产品都可以作为相对较低吞吐量14的散装材料。

为了克服目前3D 文化方法的一些缺点, 我们开发了一种微加工-cryogelation 集成技术来制造现成的和 user-friendly 的 microcryogel 阵列芯片15。在本协议中, 选择明胶作为 microcryogel 制造技术的例证, 因为它具有生物相容性、可降解、cost-effective, 并且不需要对细胞附着进行进一步的修改。其他天然或合成来源的聚合物也可用于制造, 这取决于应用。通过这一技术, 我们可以制造小型化和高弹性 microcryogels 的大小, 形状和布局可控。当加载各种细胞类型, 3D microtissues 可以形成各种应用。这些独特的功能使理想的可, 细胞保护和现场定向保留后, 注入在体内, 以加强治疗效果。不仅如此, microcryogels 可以进一步处理, 形成 3D microtissue 阵列, 这是兼容的通用实验室设备和仪器, 以实现高通量细胞培养多功能药物筛选和其他细胞化验。在这里, 我们将详细介绍 microcryogels 及其后处理作为单独的 3D microtissues 或 3D microtissue 阵列用于两个重要的应用, 细胞治疗和药物筛选, 分别为10,15.

Protocol

动物实验遵循了清华大学生物医学分析中心动物伦理委员会批准的严格的协议。在伦理委员会的批准下, 从北京协和医院整形外科部门获得了患者的知情同意, 得到了人体脂肪组织. 1. 制作 3D Microcryogels microstencil 阵列芯片的设计和制作 使用商用软件设计特定几何图形的数组, 如圆、椭圆、三角形或草 14 , 具体取决于后续应用?…

Representative Results

3D microtissue 形成的 microcryogels 的制备和表征。 根据该协议, microcryogels 是捏造的, 形成 3D microtissues 和个人 microcryogels 或 microcryogel 阵列, 并分别用于再生疗法和药物筛选 (图 1)。用 PMMA 制备的 Microstencil 阵列芯片作为 microcryogel 阵列芯片的 micromolds。microstencil 阵列芯片可进行可变几何设计。我们…

Discussion

再生医学和体外药物筛选模型是组织工程的两个重要应用:5,6,7,8,9。虽然这两种应用有着截然不同的需求, 但它们之间的共同点在于需要一个更具仿生的培养条件来增强细胞功能19。只有改进的细胞功能研究才能更好的治疗疾病20,<…

Disclosures

The authors have nothing to disclose.

Acknowledgements

这项工作得到了中国国家自然科学基金的资助 (赠款: 81522022, 51461165302)。作者想感谢所有杜实验室的成员提供一般的帮助。

Materials

Gelatin sigma G7041 All other reagents were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise indicated.
Glutaraldehyde  J&K 902042 Used as crosslinker in preparation of material.
Glass cover slip (24X50mm) CITOGLASS, China 10212450C To scrape prcursor solution onto microstencils array chips.
Sodium borohydride, NaBH4 Beijing Chemical Works 116-8 To wash remaining glutaraldehyde away after gelation.
Vacuum jar asperts, China VC8130 To preserve microgels under vacuum.
Polymethylmethacrylate (PMMA) sheets  Sunjin Electronics Co., Ltd, China Ordinary PMMA sheets.
Rayjet laser system Rayjet, Australia Rayjet 50 C30 To engrave PMMA sheets to form wells.
Plasma Cleaner Mycro Technologies, USA PDC-32G To make PMMA hyphophilic.
Lyophilizer Boyikang, China SC21CL To lyophilize materials.
Trypan Blue solution (0.4%) Zhongkekeao, China DA0065 To dye microgels.
Doxorubicin hydrochloride ENERGY CHEMICAL, China A01E0801360010 To test drug resistance of cells in 2D or 3D microgel.
Live/dead assay Dojindo Molecular Technologies (Kumamoto, Japan) CS01-10 To distinguish alive and dead cells.
Cell Titer-Blue Promega (Wisconsin, USA). G8080 To test cell viability.
Cell strainer BD Biosciences, USA 352360 To collect microgels.
D-Luciferin SYNCHEM (Germany) s039 To tack cells.
Scanning electron microscope FEI, USA Quanta 200 To characterize microgel morphology.
 Mechanical testing machine Bose, USA 3230 To measure mechanical features.
Programmable syringe pump  World Precision Instruments, USA ALADINI 1000 To test injactabiliy.
Digital force gauge HBO, Yueqing Haibao Instrument Co., Ltd., China H-50  To test injactabiliy.
Ethylene oxide sterilization system Anprolene, Anderson Sterilization, Inc., Haw River, NC AN74i To sterilize microgels with ethylene oxide gas.
Microplate reader Molecular Devices,USA M5 To measure fluorescence intensity in micro-array.
Confocal microscope Nikon, Japan A1Rsi To observe cell distribution in 3D.
Xenogen  Lumina II imaging system Caliper Life Sciences, USA IVIS To track cell in animals.
Liquid work stataion Apricot design,USA S-pipette To load medium or cell suspension high-throuputly.
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References

  1. Cukierman, E., Pankov, R., Stevens, D. R., Yamada, K. M. Taking cell-matrix adhesions to the third dimension. Science. 294 (5547), 1708-1712 (2001).
  2. Abbott, A. Cell culture: biology’s new dimension. Nature. 424 (6951), 870-872 (2003).
  3. Loessner, D., et al. Bioengineered 3D platform to explore cell-ECM interactions and drug resistance of epithelial ovarian cancer cells. Biomaterials. 31 (32), 8494-8506 (2010).
  4. Fischbach, M. A., Bluestone, J. A., Lim, W. A. Cell-based therapeutics: the next pillar of medicine. Sci Transl Med. 5 (179), 179 (2013).
  5. Kuraitis, D., Giordano, C., Ruel, M., Musaro, A., Suuronen, E. J. Exploiting extracellular matrix-stem cell interactions: a review of natural materials for therapeutic muscle regeneration. Biomaterials. 33 (2), 428-443 (2012).
  6. Breslin, S., O’Driscoll, L. Three-dimensional cell culture: the missing link in drug discovery. Drug Discov Today. 18 (5-6), 240-249 (2013).
  7. Lovitt, C. J., Shelper, T. B., Avery, V. M. Miniaturized three-dimensional cancer model for drug evaluation. Assay Drug Dev Technol. 11 (7), 435-448 (2013).
  8. Yoshii, Y., et al. High-throughput screening with nanoimprinting 3D culture for efficient drug development by mimicking the tumor environment. Biomaterials. 51, 278-289 (2015).
  9. Li, X., et al. Micro-scaffold array chip for upgrading cell-based high-throughput drug testing to 3D using benchtop equipment. Lab Chip. 14 (3), 471-481 (2014).
  10. Qi, C., Yan, X., Huang, C., Melerzanov, A., Du, Y. Biomaterials as carrier, barrier and reactor for cell-based regenerative medicine. Protein Cell. 6 (9), 638-653 (2015).
  11. Li, Y., et al. Primed 3D injectable microniches enabling low-dosage cell therapy for critical limb ischemia. Proc Natl Acad Sci U S A. 111 (37), 13511-13516 (2014).
  12. Liu, W., et al. Magnetically controllable 3D microtissues based on magnetic microcryogels. Lab Chip. 14 (15), 2614-2625 (2014).
  13. Zhao, S., Zhao, H., Zhang, X., Li, Y., Du, Y. Off-the-shelf microsponge arrays for facile and efficient construction of miniaturized 3D cellular microenvironments for versatile cell-based assays. Lab Chip. 13 (12), 2350-2358 (2013).
  14. Liu, W., et al. Microcryogels as injectable 3-D cellular microniches for site-directed and augmented cell delivery. Acta Biomater. 10 (5), 1864-1875 (2014).
  15. Hakanson, M., et al. Controlled breast cancer microarrays for the deconvolution of cellular multilayering and density effects upon drug responses. PLoS One. 7 (6), e40141 (2012).
  16. Du, Y., et al. Rapid generation of spatially and temporally controllable long-range concentration gradients in a microfluidic device. Lab Chip. 9 (6), 761-767 (2009).
  17. He, J., et al. Microfluidic synthesis of composite cross-gradient materials for investigating cell-biomaterial interactions. Biotechnol Bioeng. 108 (1), 175-185 (2011).
  18. Zeng, Y., et al. Preformed gelatin microcryogels as injectable cell carriers for enhanced skin wound healing. Acta Biomater. 25, 291-303 (2015).
  19. Yang, F., et al. Genetic engineering of human stem cells for enhanced angiogenesis using biodegradable polymeric nanoparticles. Proc Natl Acad Sci U S A. 107 (8), 3317-3322 (2010).
  20. Zhang, L., et al. Delayed administration of human umbilical tissue-derived cells improved neurological functional recovery in a rodent model of focal ischemia. Stroke. 42 (5), 1437-1444 (2011).
  21. Kinnaird, T., et al. Local delivery of marrow-derived stromal cells augments collateral perfusion through paracrine mechanisms. Circulation. 109 (12), 1543-1549 (2004).
  22. Fischbach, C., et al. Engineering tumors with 3D scaffolds. Nat Methods. 4 (10), 855-860 (2007).
  23. Dhiman, H. K., Ray, A. R., Panda, A. K. Three-dimensional chitosan scaffold-based MCF-7 cell culture for the determination of the cytotoxicity of tamoxifen. Biomaterials. 26 (9), 979-986 (2005).
  24. Gimble, J. M., Guilak, F., Bunnell, B. A. Clinical and preclinical translation of cell-based therapies using adipose tissue-derived cells. Stem Cell Res Ther. 1 (2), (2010).
  25. Thai, H. M., et al. Implantation of a three-dimensional fibroblast matrix improves left ventricular function and blood flow after acute myocardial infarction. Cell Transplant. 18 (3), 283-295 (2009).
  26. Moreira Teixeira, L. S., et al. High throughput generated micro-aggregates of chondrocytes stimulate cartilage formation in vitro and in vivo. Eur Cell Mater. 23, 387-399 (2012).
  27. Ifkovits, J. L., et al. Injectable hydrogel properties influence infarct expansion and extent of postinfarction left ventricular remodeling in an ovine model. Proc Natl Acad Sci U S A. 107 (25), 11507-11512 (2010).
  28. Murphy, A. R., Laslett, A., O’Brien, C. M., Cameron, N. R. Scaffolds for 3D in vitro culture of neural lineage cells. Acta Biomater. , (2017).
  29. Cheng, V., et al. High-content analysis of tumour cell invasion in three-dimensional spheroid assays. Oncoscience. 2 (6), 596-606 (2015).
  30. Huber, J. M., et al. Evaluation of assays for drug efficacy in a three-dimensional model of the lung. J Cancer Res Clin Oncol. 142 (9), 1955-1966 (2016).
  31. Lamichhane, S. P., et al. Recapitulating epithelial tumor microenvironment in vitro using three dimensional tri-culture of human epithelial, endothelial, and mesenchymal cells. BMC Cancer. 16, 581 (2016).
  32. Ware, M. J., et al. Generation of an in vitro 3D PDAC stroma rich spheroid model. Biomaterials. 108, 129-142 (2016).
  33. Monjaret, F., et al. Fully Automated One-Step Production of Functional 3D Tumor Spheroids for High-Content Screening. J Lab Autom. 21 (2), 268-280 (2016).
  34. Shologu, N., et al. Recreating complex pathophysiologies in vitro with extracellular matrix surrogates for anticancer therapeutics screening. Drug Discov Today. 21 (9), 1521-1531 (2016).
  35. Ho, W. J., et al. Incorporation of multicellular spheroids into 3-D polymeric scaffolds provides an improved tumor model for screening anticancer drugs. Cancer Sci. 101 (12), 2637-2643 (2010).
  36. Pathak, A., Kumar, S. Independent regulation of tumor cell migration by matrix stiffness and confinement. Proc Natl Acad Sci U S A. 109 (26), 10334-10339 (2012).
  37. Wei, S. C., et al. Matrix stiffness drives epithelial-mesenchymal transition and tumour metastasis through a TWIST1-G3BP2 mechanotransduction pathway. Nat Cell Biol. 17 (5), 678-688 (2015).
  38. Romero-Lopez, M., et al. Recapitulating the human tumor microenvironment: Colon tumor-derived extracellular matrix promotes angiogenesis and tumor cell growth. Biomaterials. 116, 118-129 (2017).
  39. Xu, X., Sabanayagam, C. R., Harrington, D. A., Farach-Carson, M. C., Jia, X. A hydrogel-based tumor model for the evaluation of nanoparticle-based cancer therapeutics. Biomaterials. 35 (10), 3319-3330 (2014).
  40. Xu, X., et al. Recreating the tumor microenvironment in a bilayer, hyaluronic acid hydrogel construct for the growth of prostate cancer spheroids. Biomaterials. 33 (35), 9049-9060 (2012).
  41. Nyga, A., Loizidou, M., Emberton, M., Cheema, U. A novel tissue engineered three-dimensional in vitro colorectal cancer model. Acta Biomater. 9 (8), 7917-7926 (2013).
  42. Yip, D., Cho, C. H. A multicellular 3D heterospheroid model of liver tumor and stromal cells in collagen gel for anti-cancer drug testing. Biochem Biophys Res Commun. 433 (3), 327-332 (2013).
  43. Hoare, T. R., Kohane, D. S. Hydrogels in drug delivery: Progress and challenges. Polymer. 49 (8), 1993-2007 (2008).
  44. Delgado, L. M., Bayon, Y., Pandit, A., Zeugolis, D. I. To cross-link or not to cross-link? Cross-linking associated foreign body response of collagen-based devices. Tissue Eng Part B Rev. 21 (3), 298-313 (2015).
  45. Florczyk, S. J., et al. Porous chitosan-hyaluronic acid scaffolds as a mimic of glioblastoma microenvironment ECM. Biomaterials. 34 (38), 10143-10150 (2013).
  46. Kimlin, L. C., Casagrande, G., Virador, V. M. In vitro three-dimensional (3D) models in cancer research: an update. Mol Carcinog. 52 (3), 167-182 (2013).
  47. Zhang, M., Boughton, P., Rose, B., Lee, C. S., Hong, A. M. The use of porous scaffold as a tumor model. Int J Biomater. 2013, 396056 (2013).
  48. Wang, J., et al. Engineering EMT using 3D micro-scaffold to promote hepatic functions for drug hepatotoxicity evaluation. Biomaterials. 91, 11-22 (2016).

Tags

Regenerative TherapyDrug ScreeningCryogelationGelatinMicrocryogelsPMMA MicrostencilPDMS Ejector PinLyophilizationHigh-throughput

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Li, Y., Yan, X., Liu, W., Zhou, L., You, Z., Du, Y. 3D Microtissues for Injectable Regenerative Therapy and High-throughput Drug Screening. J. Vis. Exp. (128), e55982, doi:10.3791/55982 (2017).
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