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

淋巴结是免疫系统中至关重要的器官，它训练免疫细胞抵抗细菌和病毒的感染。尽管这一重要作用，很少有技术存在，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穿透淋巴结，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实验的情况下直接测试免疫细胞相互作用和药物在淋巴结的转运。这个CAREER项目将开发一种微流体设备来培养整个淋巴结，实时观察和量化细胞行为和相互作用。除了回答免疫系统如何激活的基本问题外，这种淋巴结模型在研究1)慢性感染和炎症，2)癌症转移到淋巴结，以及3)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方面具有潜在的广泛应用。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在本科和研究生阶段交叉培养微生理工程和免疫生物学的学生。通过与特拉华州公立学校教师的合作，将开发和实施有关免疫系统生理学、疫苗功能和生物工程的初中和高中课程材料，以便在中学系统中广泛传播这些重要和先进的概念。研究者的长期研究目标是开发一种新型的微流体离体器官培养平台，作为一种微生理系统，在具有天然组织结构的多细胞环境中机械地询问生理、发育和重塑。为了实现这一目标，本CAREER项目的研究目标是开发一个体外淋巴结（LN）平台，用于扩展培养，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，这将使在多细胞环境下，具有天然组织结构和循环流动的LN生理学的机制询问成为可能。研究计划有两个目标。第一个目标是在离体LNChip微流控平台上创建一个非侵入性监测LN健康和生理的框架，并延长培养时间（21天）。将从猪组织中解剖单个LN，并动员传入和传出淋巴管和血管进行插管并连接到微流体平台，从而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，以创建一个“非侵入性”的框架，从传出淋巴和静脉流动中的时间分析物浓度监测器官健康。对作为细胞应激、细胞凋亡和坏死指标的因子的产生、降解和运输进行建模，可以在不干扰LN的情况下，通过测量这些输入和输出流来监测LN的健康状况。第二个目标是评估具有纵向实时成像嵌入式窗口的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，目的是确认该平台显示了文献中记录的血管和淋巴途径的免疫细胞运输，并且在培养过程中对小分子和药物具有相似的运输特性。对于贩运研究，荧光标记的细胞将通过血管或淋巴网络再循环，细胞的数量和位置将在每个流体网络和LN本身进行量化。在LN中加入一个玻璃“窗口”将允许实时成像和延时细胞跟踪。这将使研究重点放在生理相关微环境中细胞运输的动力学上。在LN中进行培养和运输研究（不同大小的被动分子和药物）后，T细胞活化试验将作为正常功能的确认，以评估物种在血管和淋巴网络之间以及整个小叶之间的运输。为了扩大影响，除了本项目直接评估的LN生理学基础知识和这种离体培养系统的影响外，研究者的实验室还将建立一个网站，分享开发的许多技巧和技术，这些技巧和技术可以实现小血管的套管，器官制备，收获和培养，以及维持这类微流体装置所需的泵系统设计。该网站将为全球科学界提供有关组织流体连接器开发的入门视频、设备设计和资源共享的来源。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。

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.