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Bioengenharia

微流控生物工程血管化组织和细胞器解体

Published: August 11, 2017
doi:
10.3791/55957
, ,
1Division of Engineering in Medicine, Department of Medicine, Brigham and Women’s Hospital,Harvard Medical School, 2Department of Plastic and Reconstructive Surgery, Renji Hospital,Shanghai Jiao Tong University School of Medicine, 3Division of Pharmacology, Utrecht Institute for Pharmaceutical Sciences,Utrecht University

Summary

我们提供一种基于微流控生物战略工程 microfibrous 血管床，在二次电池类型可以进一步播种进入这 microfibrous 结构来生成血管的组织和细胞器解体的间隙空间的广义的协议。

Abstract

工程血管化的组织构造和细胞器解体以来历史上具有挑战性。在这里，我们描述一种基于微流控生物生成多层交错水凝胶超细纤维支架的新颖方法。要实现平滑生物，包含从核心流和交联解决方案由鞘流，挤压复合 bioink 制定的核心鞘微流控打印头设计和装在复印机。通过混合与海藻酸钠明胶丙烯 (GelMA)，经历了在场的瞬时离子交联多糖选择二价离子，其次是要实现长期的稳定的 GelMA 组件二次交联，microfibrous 支架可使用这种生物策略。重要的是，封装在罗超细纤维内的内皮细胞可以形成 16 天，当然文化类似血管管腔样结构。置 microfibrous 支架可进一步用于血管床作为构建血管化的组织，通过后续播的超细纤维间质太空的二次电池类型。微流控生物提供了方便的高保真在血管组织工程中的广义的策略。

Introduction

组织工程的目标，以生成可用于取代、 还原或增加那些受伤或患病在人体1，2，3，4，往往是通过所需单元格类型、 生物活性分子5，6和生物材料7，8，，910的组合的功能性组织代用品。最近，组织工程技术也越来越多地采取了生成体外组织和器官模型模拟体内的同行，药物开发，在常规过度简化平面单元格文化11，12，13，14，15，16，17，，1819更换等应用程序的重要功能。在这两种情况下，能够概括复杂的微体系结构和人体组织层次结构是关键在启用功能的工程化的组织10，和特别是，如何融入工程化组织的血管网络的需求因为血管化提出了场20，21，，2223的最大挑战之一。

到目前为止，各种方法已在这方面，试图构建血管结构组织工程的构建和不同程度的成功8成。例如，自组装的内皮细胞允许生成的微血管网络24;交付的新生血管生长因子诱导新生血管持续的25，26;利用血管祖细胞和周细胞促进内皮细胞生长和大会24，27;设计脚手架属性使精确调制的血管28，29;和电池板技术允许的血管分层30操作控制方便。然而，这些策略不赋予控制血管，往往导致的血管组织工程的构建和因而有限的重现内随机分布的空间模式的能力。在过去几年期间生物已成为一类有利的技术来实现的这种挑战，因为他们无与伦比的多功能性，在自动或半自动方式31，，3233沉积复合组织模式在高保真和重现性的解决方案。祭祀的生物34，35，36，，3738、 嵌入式的生物39，，4041和空心结构生物/待客42，43，44，45，46，47，48，49，50，51，52，53有所有论证了生成血管或带血管蒂的组织的可行性。

另外，微流控生物战略 microfibrous 支架制造最近已发展了、 哪里混合 bioink 组成的海藻和甲基丙烯酰明胶 (GelMA) 通过同心打印头和氯化钙 (CaCl2) 解决方案的核心贯彻执行打印头54，55外鞘流。这两种流允许立即物理交联的海藻组件启用超细纤维的形成，而随后的光交联确保长期稳定的多层 microfibrous 支架共挤。值得注意的是，发现封装在罗超细纤维内的内皮细胞增殖和迁移向超细纤维假设模仿血管床54，55的管腔样结构的边缘。这些生物打印，置血管床可以随后使用填充所需二次电池类型进一步构建血管的组织55。本议定书从而提供这种微流控生物战略由同心喷嘴设计，确保在组织工程和组织化建模中的潜在应用带血管蒂组织制作方便启用的详细的过程。

Protocol

在本协议中使用的乳鼠心肌细胞株从 2 日龄大鼠后批准的机构动物护理和使用委员会布里格姆妇女医院的既定程序56 。 1.仪器仪表的复印机 插入小钝针 （例如，27 G，1 英寸） 为核心的更大的钝针 （例如，18 G，半英寸） 中心作为鞘构建双层、 同心微流控打印头;请确保核心针微微伸出 （~ 1 毫米） 长于外壳(图 1</…

Representative Results

微流控生物策略，可以直接挤出使用低粘度 bioinks54，55microfibrous 支架材料的生物。如图 2A，脚手架，其大小为 6 × 6 × 6 毫米3包含 > 30 层的超细纤维可能会在 10 分钟内的罗。同时确保长期稳定的罗 microfibrous 支架，所述顶视图和侧视图图 2B 和 2c所示的 GelMA 组件随后物?…

Discussion

建设的同轴打印头表示成功的微流控生物，以便这两个 bioink 从核心和交联剂用量从鞘的同时交付的关键步骤。虽然在本议定书中使用 27 G 针为核心，18g 针为壳创建了示例打印头，它可能很容易扩展到各种组合使用不同大小的针。然而，改变在针大小，流量变动的结果发表在每个阶段，将需要进一步优化的流率的 bioink 和交联剂 （可分别调节两个注射器泵使用一种双通道），和可能也招致复印机?…

Declarações

The authors have nothing to disclose.

Acknowledgements

作者承认国家癌症研究所的独立奖 (K99CA201603) 通往国家机构健康。

Materials

Alginic acid sodium salt from brown algae Sigma-Aldrich A0682 BioReagent, plant cell culture tested, low viscosity, powder
Gelatin type A from porcine skin Sigma-Aldrich G2500 Gel strength 300
Irgacure 2959 (2-Hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone) Sigma-Aldrich 410896 98%
HEPES buffer Sigma-Aldrich H0887 1 M, pH 7.0-7.6, sterile-filtered, BioReagent, suitable for cell culture
Fetal bovine serum  Thermo Fisher Scientific 10438026 Qualified, heat-inactivated, USDA-approved regions
Calcium chloride dihydrate Sigma-Aldrich C5080 BioXtra, ≥99.0%
Phosphate buffered saline Thermo Fisher Scientific 10010023 pH 7.4
Human umbilical vein endothelial cells Angio-Proteomie cAP-0001 Human Umbilical Vein Endothelial Cells (HUVECs)
GFP-expressing human umbilical vein endothelial cells Angio-Proteomie cAP-0001GFP GFP-Expressing Human Umbilical Vein Endothelial Cells (GFPHUVECs)
Endothelial cell growth medium Lonza CC-3162 EGM-2 BulletKit
Dulbecco’s Modified Eagle Medium  Thermo Fisher Scientific 12430054 High glucose, HEPES
Sylgard 184 silicone elastomer kit Ellsworth Adhesives 184 SIL ELAST KIT 0.5KG Clear 0.5 kg Kit
UV curing lamp system Excelitas Technologies OmniCure S2000 Spot UV Light Curing System with Intelligent UV Sensor
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Tags

Microfluidic BioprintingOrganoidsVascular VatLiverSkinCancerBioprinterAlginateGelMAPhotoinitiatorHEPES BufferFBSCalcium ChlorideHUVECsDual Channel Syringe PumpCrosslinking

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Zhang, Y. S., Pi, Q., van Genderen, A. M. Microfluidic Bioprinting for Engineering Vascularized Tissues and Organoids. J. Vis. Exp. (126), e55957, doi:10.3791/55957 (2017).
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