3-D MICROTISSUES WITH PERFUSED HUMAN CAPILLARIES

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 该应用程序解决了广泛的挑战领域（06）使能技术和特定的挑战主题，06-ES-102* 3-D或虚拟模型，以减少在研究中使用动物：创建微型多细胞器官，用于化学毒性测试的高通量筛选。 人体组织是三维的，并且需要通过毛细血管网络对流运输营养物和废物以满足代谢需求。 化学毒素主要通过皮肤、肺和胃肠道的微循环吸收。 然而，还没有包含灌注的人毛细血管的人体组织的三维体外模型。 我们的项目将创建一个高通量的3-D人体微组织（~ 1 mm 3）平台，通过灌注人体毛细血管接收营养物质并消除废物。 该平台将由平行的内皮细胞内衬微流体通道组成，模拟小动脉和小静脉，由第三个中心平行通道隔开，该通道含有包埋在纤维蛋白中的基质细胞。这些通道充满了富含氧气和其他营养物质的流动介质，并且在限定微组织长度的固定间隔处是多孔的。 这些孔允许内皮细胞通过发芽和形成毛细血管网络来响应来自基质细胞的血管生成信号，以满足代谢需要。 我们的策略采用微加工技术来创建流体通道和孔，但通过模仿体内血管生成的步骤而受到生物启发。由此产生的平台将在不大于500 cm 2的单个装置上包含> 1，000个微组织，并且非常适合于高通量化学毒性筛选，其中可以同时研究> 50种不同的化学品或化学浓度。 我们提出两个具体目标：1）制造具有以高通量方式产生用人毛细血管灌注的3-D微组织的能力的微流体装置;以及2）产生用人毛细血管灌注的3-D微组织，并表征毛细血管网络渗透性。 该提案的创新之处在于结合了微加工，微流体，光学成像和内皮/基质细胞生物学的设计策略，首次实现了体外灌注的人体毛细血管床。 该项目的完成将提供一个高通量的控制平台，以研究人类微循环，直接应用于高通量化学毒性测试，而且还广泛的其他领域，包括药物发现，正常和缺血性伤口愈合，适应运动，胚胎发生，肿瘤发生，细胞迁移和组织工程。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. This application addresses broad Challenge Area (06) Enabling Technologies and specific Challenge Topic, 06-ES-102* 3-D or virtual models to reduce use of animals in research: Creation of miniature multi-cellular organs for high throughput screening for chemical toxicity testing. Human tissue is three-dimensional, and requires convective transport of nutrients and waste through capillary networks to meet metabolic demands. Chemical toxins are primarily absorbed through the microcirculation of the skin, lungs, and gastrointestinal tract. However, there are no three-dimensional in vitro models of human tissue which contain perfused human capillaries. Our project will create a high throughput platform of 3-D human microtissues (~ 1 mm3) that receive nutrients and eliminate waste products by perfused human capillaries. The platform will be comprised of parallel endothelial cell-lined microfluidic channels, mimicking an arteriole and venule, separated by a third central parallel channel that contains stromal cells embedded in fibrin. The channels are filled with flowing media enriched with oxygen and other nutrients, and are porous at fixed intervals which define the length of the microtissue. The pores allow the endothelial cells to respond to angiogenic signals from the stromal cells by sprouting and forming a capillary network to meet the metabolic needs. Our strategy employs microfabrication technology to create the fluidic channels and pores, but is biology-inspired by mimicking the steps of in-vivo angiogenesis. The resulting platform will contain > 1,000 microtissues on a single device no larger than 500 cm2, and is ideally suited for high throughput chemical toxicity screening in which > 50 different chemicals or chemical concentrations can be studied simultaneously. We propose two specific aims: 1) fabricate the microfluidic device with the capacity to create 3-D microtissues perfused with human capillaries in a high throughput fashion; and 2) create the 3-D microtissues perfused with human capillaries, and characterize the capillary network permeability. The innovation of the proposal lies in the design strategy which combines microfabrication, microfluidics, optical imaging, and endothelial/stromal cell biology to achieve, for the first time, an in-vitro perfused human capillary bed. Completion of the project will provide a high-throughput controlled platform to study the human microcirculation with direct application to high throughput chemical toxicity testing, but also a broad range of additional fields including drug discovery, normal and ischemic wound healing, adaptation to exercise, embryogenesis, oncogenesis, cell migration, and tissue engineering.