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本文内容

  • 摘要
  • 摘要
  • 引言
  • 研究方案
  • 结果
  • 讨论
  • 披露声明
  • 致谢
  • 材料
  • 参考文献
  • 转载和许可

摘要

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。

摘要

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。

引言

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。

研究方案

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

  1. 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)。对齐方式是通常手动调节，但如果适当大小的必要垫片可以世俗地夹在刀尖和桶双方协助同心对齐的内、 外针。密封环氧胶桶交界处和对齐间隔从删除提示时适用。
  2. 在中央针在相反方向的桶中插入另一针 (23g)。 然后生成一个洞在上述外针的桶并插入进洞里，紧接着用环氧胶密封匹配大小的金属连接器。
  3. 通过两个 PVC 管单独，连接到 bioink 和交联解决方案，注射双通道注射泵打印头的入口。装载到复印机使用由 poly(methyl methacrylate)) 塑料持有人的头上挤出机。
    注意: 复印机选择取决于可用性。在本例中，我们已经成功地测试此安装程序对几个商用 bioprinters。然而，任何生物，具有 x，y，z 的机动化的阶段应，原则上，启用集成此微流控打印头。

2.生物 Microfibrous 血管床

  1. 使 bioink 4 w/v%（低粘度） 的海藻酸钠、 明胶甲基丙烯酰 （GelMA，1-2 w/v%）57，58，和光引发剂 Irgacure 2959 混合使用 (0.2-0.5 wt.%) 溶解在 25 毫米 2-[4-(2-hydroxyethyl) 哌嗪-1-基] 乙烷磺酸 （复缓冲区，pH 7.4） 含 10 vol.%胎牛血清 (FBS)。
  2. 复缓冲区包含 10 vol.%胎牛血清作为交联载体流体中提出的 0.3 M CaCl2的解决方案。
  3. 立即生物，游离人脐静脉内皮细胞 (Huvec) 从烧瓶用 0.05 w/v%胰蛋白酶 5-10 分钟，治疗前后悬在 5-10 × 106细胞/毫升浓度 bioink 中的单元格。移液器暂停慢慢地 5 至 10 倍，确保均匀分布。
  4. 开始使用双通道注射泵在相同的流率的 5ml µ L bioink/交联流体注射。可以允许流动连续运行达 1 分钟，直到他们稳定。随后，通过控制在大约 4 毫米/s (图 1B) 沉积速度复印机启动打印头的运动。这些速度可能需要精细调谐与每个新的安装程序，以确保最佳的生物。生物过程通常进行室温 (21-25 ° C)，但这个温度可能会改变。生物过程应该允许快离子凝胶化海藻成分和沉积 microfibrous 支架 (图 1B)。
  5. 脚手架是罗后，20-30 s (图 1C) 通过进一步交联的 GelMA 组件，在大约 5-10 兆瓦/厘米2的 UV 光 (360-480 nm) 实现化学凝胶。
  6. 生物和交联后，轻轻地洗净用磷酸盐缓冲液 (PBS)，删除多余的 CaCl2脚手架。文化 Huvec 载货 microfibrous 支架在血管内皮细胞的生长介质 (EGM) 在一个孵化器在 37 ° C 和 5 vol.%CO2长达 16 天与介质改变至少每隔 2 天。文化期间监测在显微镜下 Huvec 的形貌。

3.构建血管的组织

  1. 一旦脐已经迁移到的边缘在脚手架上，形成管腔样结构 (图 1D) 超细纤维，检索脚手架，轻轻地将其放在表面的疏水性表面 （例如，板坯硅氧烷 [PDMS]）。使用一块无菌过滤纸小心地从毛细管力与脚手架的间隙空间卸下所有介质。
  2. 立即添加一滴 （大约 20-40 微升） 悬挂系统的二次电池类型 （例如，心肌细胞），密度为 1-10 × 106细胞/毫升上脚手架，应渗透到整个间隙的支架 (图 1E) 介质中。培养这种配置在孵化器 （37 ° C，5 vol.%CO2，相对湿度 95%） 为 0.5-2 h，使细胞附着个人超细纤维。同期，确保没有明显的蒸发观察监测液滴尺寸。
  3. 轻轻地清洗支架的摇晃中，PBS 浴相关介质中删除任何非贴壁细胞和文化建设，直到形成所需的带血管蒂的组织。

结果

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?...

讨论

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?...

披露声明

作者宣称，他们有没有经济利益的竞争。

致谢

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

材料

NameCompanyCatalog NumberComments
Alginic acid sodium salt from brown algaeSigma-AldrichA0682BioReagent, plant cell culture tested, low viscosity, powder
Gelatin type A from porcine skinSigma-AldrichG2500Gel strength 300
Irgacure 2959 (2-Hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone)Sigma-Aldrich41089698%
HEPES bufferSigma-AldrichH08871 M, pH 7.0 - 7.6, sterile-filtered, BioReagent, suitable for cell culture
Fetal bovine serum Thermo Fisher Scientific10438026Qualified, heat-inactivated, USDA-approved regions
Calcium chloride dihydrateSigma-AldrichC5080BioXtra, ≥99.0%
Phosphate buffered salineThermo Fisher Scientific10010023pH 7.4
Human umbilical vein endothelial cellsAngio-ProteomiecAP-0001Human Umbilical Vein Endothelial Cells (HUVECs)
GFP-expressing human umbilical vein endothelial cellsAngio-ProteomiecAP-0001GFPGFP-Expressing Human Umbilical Vein Endothelial Cells (GFPHUVECs)
Endothelial cell growth mediumLonzaCC-3162EGM-2 BulletKit
Dulbecco’s Modified Eagle Medium Thermo Fisher Scientific12430054High glucose, HEPES
Sylgard 184 silicone elastomer kitEllsworth Adhesives184 SIL ELAST KIT 0.5KGClear 0.5 kg Kit
UV curing lamp systemExcelitas TechnologiesOmniCure S2000Spot UV Light Curing System with Intelligent UV Sensor

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