Paper-based cultures supporting tissue-like structures for biochemical studies of oxygen gradients and screening applications

项目摘要：Lockett，Matthew Ryen 尽管梯度在调节组织形成、结构和体内平衡中起重要作用， 少数分析工具能够产生具有实验定义的氧气梯度的组织样环境， ents。可用的工具尚未被组织培养实验室广泛采用，因为它们 需要专门的设备和工程专业知识来设置、维护和分析。为了使研究能够进行 氧气在细胞、组织和器官水平上指导反应的作用，需要新的培养平台- ed.在过去的四年里，我的实验室一直在开发一个基于纸张的文化平台。这个平台是 与众不同采用简单的技术解决方案，我们可以很容易地生成3D文化与定义 细胞外环境，无论细胞类型或组织结构。实验控制水平提供- 艾德的纸培养平台，使他们成为一个强大的使能技术，探测细胞反应， 明确的细胞外环境 这个MIRA应用程序建立在我们之前的成功之上，并继续创新纸质平台。在同等条件下- 特别是，我们将利用我们的能力，以产生定义的细胞外梯度，研究氧气的作用， 调节细胞表型、调节蛋白质表达和活性以及促进定向运动。 在我们的平台中形成的扩散主导梯度与健康组织中形成的梯度相似， 我们能够产生健康的组织环境以及由局部缺血或不良组织引起的组织环境， 血管化特别是，我们将：1）开发工具，以表征在纸丘形成的梯度- tures，重点是氧气，pH值，葡萄糖和乳酸。通过使用发光的细胞外，细胞内， 和基于转录的传感器，我们将确定设计规则以按需生成梯度。等 设计规则将帮助其他对研究组织微环境的特定方面感兴趣的人。2)评估- 不动细胞和高侵袭性细胞之间的差异。这些数据集将揭示微环境- ronment的作用，特别是氧气梯度，在促进不同的细胞反应，导致移动， 3）产生具有生理相关氧梯度的组织样共培养物。 通过生成肝脏和乳腺腔模型，我们将能够通过实验确定氧气梯度是如何变化的。 雌激素导致分区（肝脏）和调节激素受体活性（乳房）。4)开发一个筛选平台 多个3D组织结构并行。能够并行生成和评估许多组织结构 将大大改善评估肝脏毒性和识别潜在内分泌干扰物的筛选过程。

Project Abstract: Lockett, Matthew Ryen Despite the importance gradients play in regulating tissue formation, structure, and homeostasis there are few analytical tools capable of generating tissue-like environments with experimentally defined oxygen gradi- ents. The tools that are available have not been widely adopted by tissue culture laboratories because they require specialized equipment and engineering expertise to setup, maintain, and analyze. To enable the study of oxygen’s role in directing responses at the cellular, tissue, and organ level new culture platforms are need- ed. My laboratory has been developing a paper-based culture platform for the last four years. This platform is unlike any other. Employing simple technological solutions, we can readily generate 3D cultures with defined extracellular environments, regardless of cell type or tissue structure. The level of experimental control afford- ed by the paper culture platform, makes them a powerful enabling technology to probe cellular responses in well-defined extracellular environments This MIRA application builds upon our prior successes and continues to innovate the paper platform. In par- ticular, we will leverage our ability to generate defined extracellular gradients to study the role of oxygen in regulating cellular phenotype, modulating protein expression and activity, and promoting directed movement. The diffusion-dominated gradients formed in our platform are similar to those that form in healthy tissue, and we are able to generate both healthy tissue environments as well as those resulting from ischemia or poor vascularization. In particular we will: 1) Develop tools to characterize the gradients that form in the paper cul- tures, focusing on oxygen, pH, glucose, and lactate. Through the use of luminescent extracellular, intracellular, and transcription-based sensors we will determine the design rules to generate gradients on demand. Such design rules will help others interested in studying particular aspects of the tissue microenvironment. 2) Evalu- ate differences between immotile and highly invasive cells. These datasets will shed light on the microenvi- ronment’s role—in particular oxygen gradients—in promoting varied cellular responses that result in move- ment, drug resistance, etc. 3) Generate tissue-like co-cultures with physiologically relevant oxygen gradients. By generating liver and breast lumen models, we will be able to experimentally determine how oxygen gradi- ents lead to zonation (liver) and regulate hormone receptor activity (breast). 4) Develop a platform to screen multiple 3D tissue structures in parallel. The ability to generate and evaluate many tissue structures in parallel will greatly improve screening processes to assess liver toxicity and identify potential endocrine disruptors.