Collaborative Research: Engineering Architecture for Tissue Models

合作研究：组织模型的工程架构

• 批准号: 1805043
• 负责人: Rebecca Carrier
• 金额: $ 16.22万
• 依托单位: Northeastern University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-08-01 至 2022-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1805043&HistoricalAwards=false
• 关键词: Collaborative; Research; Engineering; Architecture; Tissue

This project focuses on developing a screen printing process to build models of both neural networks and the innervated colon (i.e., a colon with imbedded nerves to mimic the natural structure). The screen printing process has been around for thousands of years and is used to make art, to mass produce t-shirts, and to make microelectronics. It is simple, low cost, reproducible, and scalable, making it ideal for printing highly accessible tissue models. While there have been many exciting approaches to print tissue models, most involve expensive equipment and subject the cells to either ultraviolet rays or shearing through fine needles, both of which can impact cell survival and behavior. The objective of this project is to determine the resolution and reproducibility of the screen printing approach and then use the process developed to make the two new models. The neural network model (based on human derived stem cells) will lay the groundwork to study diseases and investigate therapies relevant to nerve tissue in a more physiology-based/meaningful way that is still suitable for high throughput systems. The colon model (consisting of epithelial cells, goblet cells producing mucous, immune cells, and neurons) will provide one of the first innervated gut models that is suitable for high throughput techniques. High throughput screening opens the possibilities of investigating the interplay between these cells in the development of food allergies and conditions such as colitis. The research will provide new tools for researchers interested in understanding the complex cross-talk between cells in these tissues and developing new therapies for insults to these systems. The screen printing technique will also be used to engage young researchers. Screen printing demonstrations will be built into outreach programs in schools and utilized to educate researchers in classrooms and labs. Screen printing is exceptionally familiar and "user-friendly," thus a great platform for engaging and keeping people engaged in science. For example, Jello will be screen printed on edible paper to explain methods to make tissues as part of outreach program demonstrations in the Baltimore and Boston public schools. Educational and outreach efforts will be complemented by a website that highlights scientists from diverse backgrounds and the accessible, low tech parts of their work, like screen printing.The objective of this project is to determine the resolution and reproducibility of a screen printing approach to making highly scalable hydrogel-cell tissue models and to then use the system developed to make new models of neural networks and the innervated colon. The investigators hypothesize that screen-printing will provide a cost-effective approach to making tissue models that surpass those made via bioprinting approaches in terms of ease, cost and cell survival and thus enable more relevant models of tissues from cells that are highly sensitive to shear. The Research Plan is organized under four aims. THE FIRST AIM is to characterize the screen-printed materials (PLL(poly(L-lysine))/protein and hydroxy appetite-based gels functionalized with PEG-thiol groups printed on glass slides) with regards to the resolution, repeatability and lamination of layers. Expected aim outcomes include: determining the gelation time and mechanics of the different materials, confirming the concentration and distribution of proteins on the gels, determining the strength of lamination between similar and different layers, and knowing the limits on how thin gels can be reproducibly screen printed. THE SECOND AIM is to characterize the multilamellar, patterned gels with regards to architecture including resolution and reproducibility of features in the printing plane. Expected aim outcomes include: determining the finest resolution that can be reproducibly achieved for a range of gels, confirming the concentration and distribution of proteins on and in the gels and determining if there are any types of features that are more or less easily printed at high resolution. THE THIRD AIM is to determine the relationship between printing resolution, cell survival, and cellular behavior using human induced pluripotent stem (iPS) cells and human iPS-derived neurons to build neural circuits including excitatory and inhibitory neurons. Expected aim outcomes include: knowing which hydrogel augments iPS cell survival the most, knowing the impact of the matrix on the genetic and phenotypic cellular behavior of iPS and their progeny, knowing the impact screen printing has on the genetic and phenotypic cellular behavior of iPS cells and their progeny and developing a reproducible, scalable system for high throughput screening of 3D neural interactions that lead to neural circuits. THE FOURTH AIM is to build a model of the innervated colon to investigate the application of screen printing in a multicellular, physiologically relevant, high throughput model. Cell lines include enterocytes and goblet cells derived from Caco-2 cells, primary human intestinal epithelial cells, immune cells (dendritic cells) and neural cells (from Aim 3). Matrix materials include PLL-PEG gel systems printed with laminin and collagen I. The matrix optimized in Aim 3 will be used for the neural layer. Expected aim outcomes include: the development of a novel gut/colon model 1) that lays the foundation for study and enhanced understanding of the role of the enteric nervous system, 2) that, even without the neural component, will be one of the first colon models that can be prepared in a high throughput manner and interface with real time electrical recording and imaging modalities without a substantial investment in materials or equipment and 3) that lays for foundation for adding the next critical component--the microbiome.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

该项目的重点是开发一种丝网印刷工艺，以建立神经网络和受神经支配的结肠（即嵌入神经以模仿自然结构的结肠）的模型。丝网印刷工艺已经存在了数千年，用于制作艺术品，批量生产t恤和制造微电子产品。它简单，低成本，可复制和可扩展，使其成为打印高度可访问的组织模型的理想选择。虽然有许多令人兴奋的方法来打印组织模型，但大多数都需要昂贵的设备，并将细胞置于紫外线下或通过细针剪切，这两种方法都会影响细胞的存活和行为。该项目的目标是确定丝网印刷方法的分辨率和再现性，然后使用开发的过程来制作两种新模型。神经网络模型（基于人类来源的干细胞）将为研究疾病和研究与神经组织相关的治疗奠定基础，以更基于生理学/有意义的方式，仍然适用于高通量系统。结肠模型（由上皮细胞、产生粘液的杯状细胞、免疫细胞和神经元组成）将提供适合高通量技术的首批神经支配肠道模型之一。高通量筛选开启了研究这些细胞在食物过敏和结肠炎等疾病发展过程中相互作用的可能性。这项研究将为研究人员提供新的工具，帮助他们了解这些组织中细胞之间复杂的串扰，并开发出针对这些系统损伤的新疗法。丝网印刷技术也将用于吸引年轻的研究人员。丝网印刷示范将被纳入学校的推广项目，并用于教育教室和实验室的研究人员。丝网印刷是非常熟悉和“用户友好的”，因此是一个很好的平台，吸引和保持人们从事科学。例如，果冻将被丝网印刷在可食用的纸上，解释制作纸巾的方法，作为巴尔的摩和波士顿公立学校推广项目示范的一部分。教育和推广工作将由一个网站来补充，该网站突出了来自不同背景的科学家以及他们工作中容易获得的、低技术含量的部分，如丝网印刷。该项目的目标是确定丝网印刷方法的分辨率和可重复性，以制作高度可扩展的水凝胶细胞组织模型，然后使用开发的系统制作神经网络和神经支配结肠的新模型。研究人员假设，丝网印刷将提供一种具有成本效益的方法来制作组织模型，在易用性、成本和细胞存活率方面超过生物打印方法，从而能够从对剪切高度敏感的细胞中获得更相关的组织模型。研究计划有四个目标。第一个目的是表征丝网印刷材料（PLL（聚赖氨酸））/蛋白质和羟基食欲基凝胶与聚乙二醇巯基印刷在玻片上)的分辨率，可重复性和层压。预期的目标结果包括：确定不同材料的凝胶时间和力学，确认凝胶上蛋白质的浓度和分布，确定相似层和不同层之间的层压强度，并了解薄凝胶可重复丝网印刷的限制。第二个目标是表征多层，图案凝胶的结构，包括打印平面上特征的分辨率和再现性。预期的目标结果包括：确定一系列凝胶可重复实现的最佳分辨率，确认凝胶上和凝胶中的蛋白质浓度和分布，并确定是否存在任何类型的特征或多或少容易在高分辨率下打印。第三个目标是确定打印分辨率、细胞存活和细胞行为之间的关系，使用人类诱导多能干细胞和人类诱导多能干细胞衍生的神经元来构建包括兴奋性和抑制性神经元在内的神经回路。预期的目标结果包括：了解哪种水凝胶最能提高iPS细胞的存活率，了解基质对iPS细胞及其后代的遗传和表型细胞行为的影响，了解丝网印刷对iPS细胞及其后代的遗传和表型细胞行为的影响，以及开发可重复的、可扩展的系统，用于高通量筛选导致神经回路的3D神经相互作用。第四个目标是建立一个受神经支配的结肠模型，以研究丝网印刷在多细胞、生理相关、高通量模型中的应用。细胞系包括从Caco-2细胞衍生的肠细胞和杯状细胞、原代人肠上皮细胞、免疫细胞（树突状细胞）和神经细胞（来自Aim 3）。基质材料包括用层粘胶蛋白和胶原i打印的PLL-PEG凝胶系统。Aim 3中优化的基质将用于神经层。预期目标成果包括：一种新型肠道/结肠模型的发展1)为研究奠定了基础，并增强了对肠神经系统作用的理解，2)即使没有神经成分，将成为第一个可以以高通量方式制备并与实时电记录和成像模式接口的结肠模型之一，而无需在材料或设备上进行大量投资；3)为添加下一个关键组件-微生物组奠定基础。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。