3-D MICROTISSUES WITH PERFUSED HUMAN CAPILLARIES

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 该应用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，以减少研究中动物的使用：创建微型多细胞器官，用于高通量筛选，用于化学毒性测试。人体组织是三维的，需要通过毛细血管网络对营养物质和废物进行对流运输，以满足代谢需求。化学毒素主要通过皮肤、肺部和胃肠道的微循环吸收。然而，目前还没有包含灌流的人体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人体微组织平台(~1mm3)，通过灌流的人体毛细血管接收营养并清除废物。该平台将由平行的内皮细胞排列的微流体通道组成，模仿小动脉和小静脉，由第三个中央平行通道分隔，其中包含嵌入纤维蛋白的基质细胞。这些通道充满了富含氧气和其他营养物质的流动介质，并以固定的间隔多孔化，这定义了微组织的长度。这些毛孔允许内皮细胞通过萌发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。我们的策略使用微制造技术来创建流体通道和毛孔，但通过模仿体内血管生成的步骤而受到生物学的启发。由此产生的平台将在不超过500平方厘米的单个设备上包含1000个微组织，非常适合于高通量化学毒性筛选，在这种筛选中，可以同时研究&gt；50种不同的化学物质或化学浓度。我们提出了两个具体的目标：1)制造能够高通量地产生注入人体毛细血管的三维微组织的微流控装置；2)制造注入人体毛细血管的三维微组织，并表征毛细血管网络的通透性。该方案的创新之处在于将微制造、微流体、光学成像和内皮/基质细胞生物学相结合的设计策略，首次实现了体外灌流人体毛细血管床。该项目的完成将提供一个高通量的受控平台来研究人体微循环，直接应用于高通量的化学毒性测试，而且还包括广泛的附加领域，包括药物发现、正常和缺血伤口愈合、运动适应、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.