Stacked, paper-based culture models of heart valve layers: effects of hypoxia gradients and heterogeneous extracellular matrix

项目负责人：Grande-Allen， Kathryn J.提案编号：1404008机构：William Marsh Rice University标题：心脏瓣膜层的堆叠、纸质培养模型：缺氧梯度和异质细胞外基质的影响心脏瓣膜疾病影响全世界所有年龄和社会经济阶层的数十万人。鉴于钙化性主动脉瓣疾病与衰老、糖尿病和代谢综合征的关联越来越大，仅治疗这种疾病就意味着现在和未来的重大医疗保健费用。目前，瓣膜疾病只能通过手术或介入治疗；没有一种药丸可以预防或逆转瓣膜疾病。目前尚不清楚瓣膜疾病是如何开始或如何恶化的。由于细胞和细胞周围物质（如胶原蛋白和复合碳水化合物）之间的相互作用似乎在瓣膜的健康和疾病中起着重要作用，PI开发了一种独特的方法来研究这些相互作用。使用先进的创新方法在模拟正常、健康或患病瓣膜的环境中培养瓣膜细胞，将为心脏瓣膜的基础生物学提供重要的新信息，并有助于了解疾病过程和测试新的非手术治疗方法。此外，本研究的目标和活动将分发给广泛的学生（从高中到研究生院）以及高中教师。心脏瓣膜的功能是由组织内细胞外基质（ECM）成分的独特微观结构安排实现的。包括PI在内的许多小组已经开始研究ECM如何从根本上调节瓣膜细胞和组织行为，通常使用2D细胞培养和免疫组织化学，但这些方法可能很耗时，并且对活性组织重塑的了解有限。细胞培养环境，如由ECM或合成聚合物构建的水凝胶或纤维支架，为细胞提供了一个高度仿生的3D环境，但在整个3D支架厚度上分析细胞行为是具有挑战性的。因此，本研究将利用纸质细胞培养技术的新领域来评估瓣膜ECM和瓣膜间质细胞（VICs）的正常和病理行为。特别是，用凝胶支架浸渍的纸堆具有高通量、自动分析整个堆中的细胞行为的吸引力，并可用于评估缺氧的影响。事实上，这项技术非常适合研究VIC-ECM关系。更具体地说，这项技术将被用来组装成堆的纸——每一堆纸都浸渍着不同的基于ecm的凝胶支架——形成一个分层结构，模仿心脏瓣膜的异质分层结构。这种方法将允许进行一些深入的研究，以检查某些类型的细胞外基质的影响，细胞接受的氧气量，以及心脏瓣膜的异质性层状性质，对瓣膜细胞的表型和信号传导行为。这些反应将被评估和比较两个原始心脏瓣膜源（主动脉瓣和二尖瓣）的vic。这项工作的总体目标是评估vic对ECM和缺氧的正常和病理反应。预计这个独特的实验系统将产生关于瓣膜细胞的基本行为以及如何调节这种行为的见解，这将对开发新的疾病治疗方法和组织工程瓣膜的设计产生影响。

PI: Grande-Allen, Kathryn J. Proposal Number: 1404008Institution: William Marsh Rice University Title: Stacked, paper-based culture models of heart valve layers: effects of hypoxia gradients and heterogeneous extracellular matrixHeart valve disease affects hundreds of thousands of people of all ages and socioeconomic classes worldwide. Given the growing association of calcific aortic valve disease with aging, diabetes, and metabolic syndrome, the treatment of just this disease alone represents significant health care costs now and in the future. Currently, valve diseases can only be treated through surgical or interventional means; there is no pill one can take to prevent or reverse valve disease. It is unclear exactly how valve diseases get started or how they worsen. Since the interactions between the cells and the material surrounding the cells - like collagen and complex carbohydrates - appear to play an important role in the health of the valve, as well as in the diseases, the PI has developed a unique way to study these interactions. The use of advanced, innovative approaches to grow the valve cells in an environment mimicking a normal, healthy or diseased valve will provide significant new information on the fundamental biology of heart valves and will help understand the disease process and test new non-surgical therapies. In addition, the goals and activities of this research will be distributed to a broad audience of students (ranging from high school to graduate school) as well as high school teachers.The function of heart valves is made possible by the unique microstructural arrangement of extracellular matrix (ECM) components within the tissue. Many groups including the PI's have begun to investigate how ECM fundamentally regulates valve cell and tissue behavior, often using 2D cell cultures and immunohistochemistry, but these methods can be time-consuming and provide limited insight into active tissue remodeling. Cell culture environments such as hydrogels or fibrous scaffolds constructed from ECM or synthetic polymers offer cells a 3D environment that can be highly biomimetic, but analysis of the cell behavior throughout the entire thickness of the 3D scaffold is challenging. Thus, the proposed studies will evaluate the normal and pathological behavior of valvular ECM and valvular interstitial cells (VICs) using the new field of paper-based cell culture technology. In particular, stacks of paper impregnated with gel scaffolds are appealing for high throughput, automated analysis of cell behavior throughout the stack, and can be used to evaluate the effects of hypoxia. Indeed, this technology is exceptionally well suited to investigate VIC-ECM relationships. More specifically, this technology will be used to assemble stacks of papers - each impregnated with a different ECM-based gel scaffold - into a layered structure that mimics the heterogeneous layered structure of the heart valve. This method will allow several in-depth studies to examine the effects of certain types of extracellular matrix, the amount of oxygen the cells are receiving, and the heterogeneous layered nature of heart valves, on the phenotypic and signaling behavior of the valve cells. These responses will be evaluated and compared for two original heart valve sources of the VICs (aortic vs. mitral valve). The overall goal of the work is to evaluate the normal and pathological responses of VICs to ECM and hypoxia. It is expected that this unique experimental system will generate insight regarding the fundamental behavior of valve cells and how this behavior can be regulated, which will be impactful on developing new therapies for disease and the design of tissue engineered valves.