CAREER: A new class of microfluidic ex vivo culture models to understand whole organ physiology and disease

职业：一类新型微流体离体培养模型，用于了解整个器官生理学和疾病

• 批准号: 1943686
• 负责人: Jason Gleghorn
• 金额: $ 55万
• 依托单位: University of Delaware
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-02-01 至 2025-01-31
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1943686&HistoricalAwards=false
• 关键词: CAREER; new; class; microfluidic; ex

Lymph nodes are critically important organs of the immune system that train immune cells to fight off infections from bacteria and viruses. Despite this essential role, few techniques exist to study the dynamic interactions between cells within lymph nodes. Additionally, the structure of the lymph node is so unique that the complex architecture prevents drugs from penetrating into the lymph node to target viruses, bacteria, and metastatic cancer cells that collect there causing infection. Current methods do not allow straightforward testing of immune cell interactions and drug transport a lymph node without animal testing. This CAREER project will develop a microfluidics device to culture an entire lymph node to observe and quantify cell behaviors and interactions in real time. In addition to answering fundamental questions as to how the immune system activates, this lymph node model has potential broad applications to study 1) chronic infection and inflammation, 2) cancer metastasis to the lymph node, and 3) drug delivery strategies for chemotherapeutic and antiretroviral therapies for cancer and HIV treatment. It can also serve as a screening platform for immune response to foreign materials and/or transplant rejection. The complementary education plan focuses on cross-training students in microphysiological engineering and immune biology at the undergraduate and graduate level. Through a partnership with Delaware public school teachers, middle and high school curricular materials will be developed and implemented on immune system physiology, vaccine function, and bioengineering for broad dissemination of these important and advanced concepts into the secondary school system.The investigator’s long-term research goal is to develop a new class of microfluidic ex vivo organ culture platforms to serve as a microphysiological system to mechanistically interrogate physiology, development, and remodeling in a multicellular context with native tissue architecture. Toward this goal, the research objective of this CAREER project is to develop an ex vivo lymph node (LN) platform for extended culture and to validate proper adaptive immune system response, cell trafficking, and drug pharmacokinetics, which will enable mechanistic interrogation of LN physiology, in a multicellular context, with native tissue architecture and circulatory flows. The Research Plan is organized under two Aims. The FIRST Aim is to create a framework for non-invasive monitoring LN health and physiology over extended culture (21 days) in an ex vivo LNChip microfluidic platform. Individual LNs will be dissected from pig tissue and the afferent and efferent lymphatic and vascular vessels will be mobilized for cannulation and connection to the microfluidic platform, which enables precise control and measurement of the cellular and fluid input and output of the LN. Rigorous assessment and quantification of organ health will be determined with “minimally invasive” longitudinal measures and end-point assays. These data will be coupled with a computational model of transport in the LN to create a framework for “non-invasive” monitoring organ health from temporal analyte concentrations in the efferent lymphatic and venous flows. Modeling the creation, degradation, and transport of factors that are indicators of cellular stress, apoptosis and necrosis will enable monitoring the health of the LN from measurements made from these input and output flows without disturbing the LN. The SECOND Aim is to assess key cellular interactions, cell trafficking, and drug pharmacokinetics in a LNChip platform with an embedded window for longitudinal real-time imaging, with the goal of confirming that the platform demonstrates immune cell trafficking from both vascular and lymph pathways as documented in the literature and has similar transport characteristics for small molecules and drugs during culture. For trafficking studies, fluorescently labeled cells will be recirculated through either the vascular or lymphatic networks, and the number and locations of cells will be quantified in each fluid network and within the LN itself. Incorporation of a glass "window" into the LN will allow for real-time imaging and timelapse cell tracking. This will enable studies focusing on the dynamics of cell trafficking in a physiologically relevant microenvironment. T cell activation assays will serve as confirmation of normal function following culture and transport studies (passive molecules of varying sizes and drug) in a LN will be performed to assess the transport of species between vascular and lymphatic networks and throughout the lobule. To broaden impact, in addition to the fundamental knowledge of LN physiology directly assessed in this project and the impact that such an ex vivo culture system would have, the investigator’s lab will set up a website to share the numerous tricks and techniques developed that enable cannulation of small vessels, organ preparation, harvest, and culture, and the design of pump systems needed to sustain this class of microfluidic device. The website will serve as a source for how-to videos, device designs, and resource sharing around development of tissue fluidic connectors for the global scientific community.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.

淋巴结是免疫系统中至关重要的器官，可以训练免疫细胞抵抗细菌和病毒的感染。尽管有这种重要作用，但很少有技术可以研究淋巴结内细胞之间的动态相互作用。此外，淋巴结的结构是如此独特，以至于复杂的结构阻止药物渗透到淋巴结中以靶向聚集在那里引起感染的病毒、细菌和转移性癌细胞。目前的方法不允许在没有动物试验的情况下直接测试免疫细胞相互作用和药物转运淋巴结。这个CAREER项目将开发一种微流体设备来培养整个淋巴结，以观察和量化细胞行为和真实的时间相互作用。除了回答关于免疫系统如何激活的基本问题外，该淋巴结模型还具有潜在的广泛应用，以研究1）慢性感染和炎症，2）癌症转移到淋巴结，以及3）用于癌症和HIV治疗的化疗和抗逆转录病毒疗法的药物递送策略。它还可以作为对外来物质和/或移植排斥的免疫反应的筛选平台。补充教育计划的重点是交叉培训学生在微生理工程和免疫生物学在本科和研究生水平。 通过与特拉华州公立学校教师的合作，将开发和实施关于免疫系统生理学、疫苗功能、和生物工程，以便在中学系统中广泛传播这些重要和先进的概念。长期研究目标是开发一类新的微流体离体器官培养平台，发育和在具有天然组织结构的多细胞环境中重塑。 为了实现这一目标，该CAREER项目的研究目标是开发一种用于扩展培养的离体淋巴结（LN）平台，并验证适当的适应性免疫系统反应，细胞运输和药物药代动力学，这将使LN生理学的机械询问，在多细胞背景下，具有天然组织结构和循环流动。 研究计划是根据两个目标组织的。 第一个目的是建立一个框架，用于在体外LNChip微流体平台中在延长培养（21天）期间非侵入性监测LN健康和生理学。 将从猪组织中解剖出单个LN，并且将动员传入和传出淋巴管和血管用于插管并连接到微流体平台，这使得能够精确控制和测量LN的细胞和流体输入和输出。 器官健康的严格评估和量化将通过“微创”纵向测量和终点分析来确定。这些数据将与LN中运输的计算模型相结合，以创建用于从传出淋巴和静脉流中的时间分析物浓度监测器官健康的“非侵入性”框架。 对作为细胞应激、细胞凋亡和坏死的指标的因子的产生、降解和运输进行建模将使得能够在不干扰LN的情况下根据从这些输入和输出流进行的测量来监测LN的健康。第二个目的是评估LNChip平台中的关键细胞相互作用、细胞运输和药物药代动力学，该平台具有用于纵向实时成像的嵌入式窗口，目的是确认该平台证明了文献中记载的来自血管和淋巴途径的免疫细胞运输，并且在培养期间具有小分子和药物的相似运输特征。对于运输研究，荧光标记的细胞将通过血管或淋巴网络再循环，并且细胞的数量和位置将在每个流体网络和LN本身内定量。将玻璃“窗口”并入LN将允许实时成像和时移细胞跟踪。这将使研究重点放在生理相关的微环境中的细胞贩运的动态。在LN中进行培养和转运研究（不同大小的被动分子和药物）后，T细胞活化试验将用于确认正常功能，以评估血管和淋巴网络之间以及整个小叶中的物质转运。 为了扩大影响，除了在该项目中直接评估的LN生理学的基本知识以及这种离体培养系统的影响外，研究者实验室还将建立一个网站来分享开发的许多技巧和技术，这些技巧和技术能够实现小血管插管，器官准备，收获和培养，以及维持这类微流体装置所需的泵系统的设计。该网站将成为全球科学界关于组织流体连接器开发的操作视频、设备设计和资源共享的来源。该奖项反映了NSF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。

项目成果

Targeted Gq-GPCR activation drives ER-dependent calcium oscillations in chondrocytes

• DOI: 10.1016/j.ceca.2021.102363
• 发表时间: 2021-02-05
• 期刊: CELL CALCIUM
• 影响因子: 4
• 作者: McDonough, Ryan C.;Gilbert, Rachel M.;Price, Christopher
• 通讯作者: Price, Christopher

Emergent Behavior at the Calcite–Water Interface during Reactive Transport in a Simple Microfluidic Channel

简单微流体通道中反应传输过程中方解石与水界面的突现行为

• DOI: 10.1021/acsearthspacechem.1c00424
• 发表时间: 2022
• 期刊: ACS Earth and Space Chemistry
• 影响因子: 3.4
• 作者: Abdilla, Bektur;Minahan, Daniel J.;Gleghorn, Jason P.;Kim, YoungJae;Lee, Sang Soo;Fenter, Paul;Sturchio, Neil C.
• 通讯作者: Sturchio, Neil C.

Significant Unresolved Questions and Opportunities for Bioengineering in Understanding and Treating COVID-19 Disease Progression

• DOI: 10.1007/s12195-020-00637-w
• 发表时间: 2020-07-27
• 期刊: CELLULAR AND MOLECULAR BIOENGINEERING
• 影响因子: 2.8
• 作者: Shirazi, Jasmine;Donzanti, Michael J.;Gleghorn, Jason P.
• 通讯作者: Gleghorn, Jason P.