Circulatory system and integrated muscle tissue for drug and tissue toxicity

描述（由申请人提供）：鉴于肌肉质量和通过肌床的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、其在介导外周动脉疾病和心力衰竭严重程度中的作用以及治疗需求，骨骼肌对于药物和毒性试验非常重要 用于肌肉疾病如肌肉萎缩症和肌肉减少症。为了开发在生理条件下进行功能和药物测试的系统，我们将在循环系统中加入三维骨骼肌培养物，该循环系统由高压动脉系统组成，高压动脉系统将培养基运送到各种组织微循环器官床，并通过低压静脉系统返回。动脉血管将由内皮内层和分化的血管平滑肌细胞或间充质干细胞层组成。计算机控制的泵和阀系统将泵送少量流体以模拟动脉流动。O2、CO2和压力的测量将用于控制流向各个床的流量。微循环器官床的模块化设计有助于与广泛的其他器官和组织模拟物整合，作为合作协议UH3阶段的一部分。所有实验将使用原代人类细胞。为了推广测试床的适用性，我们将从iPS细胞中培养成熟的平滑肌细胞和骨骼肌。在目标1中，我们将制造和测试由几层可收缩的人平滑肌细胞或间充质干细胞和汇合的内皮层组成的小口径血管的分支网络。流速、血管扩张和收缩以及内衬有内皮的微流体微循环床的阻力将控制到不同微循环床的流量分布。在目标2中，我们将开发张力下骨骼肌和成纤维细胞的三维结构。动脉和静脉流入管路中不同水平的氧分压将用于产生一系列氧梯度。肌肉将连接到包含铁凝胶的柱子上，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，作为灌注介质和骨骼肌纤维之间的界面。穿过肌肉层的氧梯度将由细胞密度和细胞层的厚度控制。我们将制作一个电极系统来电刺激纤维并测量力的产生。在目标3中，我们将联合收割机结合血管和肌肉单元，并运行该单元四周。O2、CO2和压力的测量将用于控制总流量并调节流向不同床的流量。我们将评估血管扩张和肌肉功能。在目标4中，完整的系统将用于测试血管扩张剂和血管收缩剂的局部释放对血流分布、葡萄糖代谢和肌肉摄氧量的影响。我们将检查血管和肌肉对炎症刺激的反应。将进行代谢分析，以模拟不同的生理条件以及对药物和毒素的反应。 公共卫生相关性：本项目将为在实验室中检测人体骨骼肌的功能以及测试药物的有效性和毒性提供新的方法。我们期望本研究的结果将有助于筛选治疗骨骼肌和血管疾病的候选药物。

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. PUBLIC HEALTH RELEVANCE: This project will provide new methodology to examine the function of human skeletal muscle in the laboratory and to test the effectiveness and toxicity of drugs. We expect that the results of this project will facilitate the screening of candidate drugs o treat disorders of skeletal muscle and blood vessels.