Circulatory system and integrated muscle tissue for drug and tissue toxicity

说明(申请人提供)：骨骼肌对于药物和毒性测试是重要的，因为通过肌床的肌肉质量和心输出量的相对大小，肌肉在能量底物代谢和糖尿病中的关键作用，它在调节外周动脉疾病和心力衰竭的严重程度中的作用，以及治疗的需要。 用于肌肉疾病，如肌肉营养不良和骨质疏松症。为了开发一种在生理条件下进行功能和药物测试的系统，我们将把三维骨骼肌培养物整合到循环系统中，该循环系统由一个高压动脉系统组成，该系统将介质输送到各种组织微循环器官床，并通过低压静脉系统返回。动脉血管将由内层内皮和分化的血管平滑肌细胞或间充质干细胞组成。计算机控制的泵和阀系统将泵出少量的液体，以模拟动脉的流动。氧气、二氧化碳和压力的测量将被用来控制流向各个床层的流量。作为《合作协议》UH3阶段的一部分，微循环器官床的模块化设计促进了与广泛的其他器官和组织仿制品的整合。所有实验都将使用原代人类细胞。为了推广试验台的适用性，我们将从iPS细胞培养成熟的平滑肌细胞和骨骼肌。在目标1中，我们将构建和测试由多层可收缩的人类平滑肌细胞或间充质干细胞和一层融合层的内皮细胞组成的小口径血管分支网络。内衬内皮的微流控微循环床的流速、血管的扩张和收缩以及阻力将控制流向不同微循环床的流量分配。在目标2中，我们将开发骨骼肌和张力下的成纤维细胞的三维结构。动脉和静脉流入管道中不同水平的氧分压将被用来产生一系列的氧气梯度。肌肉将连接到含有铁凝胶的柱子上，铁凝胶在电磁刺激下收缩，从而加载肌肉纤维。内皮细胞将覆盖三维肌肉培养物的外部，作为灌流介质和骨骼肌纤维之间的接口。肌肉层的氧气梯度将由细胞密度和细胞层厚度控制。我们将制作一个电极系统来对纤维进行电刺激并测量作用力。在目标3中，我们将结合血管和肌肉单元，并运行四周的单元。氧气、二氧化碳和压力的测量将用于控制总体流量和调节流向不同床层的流量。我们将评估血管扩张和肌肉功能。在目标4中，完成的系统将用于测试局部释放血管扩张剂和血管收缩药对肌肉的流量分配、葡萄糖代谢和摄氧量的影响。我们将研究血管和肌肉对炎症刺激的反应。将进行新陈代谢分析，以模拟不同的生理条件以及对药物和毒素的反应。

DESCRIPTION (provided by applicant): Skeletal muscle is important for drug and toxicity testing given the relative size of the muscle mass and cardiac output that passes through muscle beds, the key role of muscle in energy substrate metabolism and diabetes, its role in mediating the severity of peripheral arterial disease and heart failure, and the need for therapies for muscle diseases such as muscular dystrophy and sarcopenia. To develop a system for functional and drug testing under physiological conditions, we will incorporate three-dimensional skeletal muscle cultures in a circulatory system that consists of a high-pressure arterial system that carries media to various tissue microcirculatory organ beds and returned via a low-pressure venous system. Arterial vessels will consist of an inner layer of endothelium and layers of differentiated vascular smooth muscle cells or mesenchymal stem cells. A computer controlled pump and valve system will pump small volumes of fluid to mimic arterial flow. Measurement of O2, CO2 and pressure will be used to control flow to the various beds. The modular design of the microcirculatory organ beds facilitates integration with a broad array of other organ and tissue mimics as part of the UH3 phase of the Cooperative Agreement. All experiments will use primary human cells. To generalize the applicability of the test bed, we will develop mature smooth muscle cells and skeletal muscle from iPS cells. In Aim 1, we will fabricate and test a branching network of small caliber blood vessels consisting of several layers of contractile human smooth muscle cells or mesenchymal stem cells and a confluent layer of endothelium. Flow rates, vessel distension and contraction, and the resistance of the microfluidic microcirculatory beds lined with endothelium will control the flow distribution to the different microcirculatory beds. In Aim 2, we will develop three-dimensional constructs of skeletal muscle and fibroblasts under tension. Different levels of oxygen partial pressure in the arterial and venous inflow lines will be used to produce a range of oxygen gradients. The muscle will be connected to posts containing a ferrogel that contracts under electromagnetic stimulation and thereby loads the muscle fibers. Endothelium will cover the outside of the three-dimensional muscle cultures, serving as an interface between the perfusion medium and skeletal muscle fibers. Oxygen gradients across the muscle layer will be controlled by cell density and thickness of the cell layer. We will fabricate an electrode system to electrically stimulate the fibers and measure force production. In Aim 3, we will combine the vascular and muscle units and run the unit for four weeks. Measurement of O2, CO2 and pressure will be used to control overall flow and regulate flow to the different beds. We will assess vessel dilation and muscle function. In Aim 4, the completed system will be used to test the effect of local release of vasodilators and vasoconstrictors on flow distribution, glucose metabolism and oxygen uptake by muscle. We will examine the response of blood vessels and muscle to an inflammatory stimulus. Metabolic profiling will be performed to simulate different physiological conditions and response to drugs and toxins.