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.
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