Automated Patient-Specific Dendritic Cell Generation for Transcriptomics-Driven Vaccinology

 描述（由申请人提供）：树突状细胞（DC）是研究人类反应的不可或缺的部分，对于癌症和传染病的保护性免疫以及预防自身免疫和移植排斥反应非常重要。这些细胞也是个性化疫苗的关键要素，个性化疫苗是癌症和传染病的主要研究重点。尽管树突状细胞在临床和基础研究中发挥着重要作用，但从个体中获得这些细胞的方法仍然是一个相对欠发达和效率低下的过程。因为DC在血液中以非常低的浓度（<1%）存在，所以这些细胞必须从单核细胞产生，并且这种产生的现有技术涉及静态培养和用培养基中含有的细胞因子刺激的费力过程。从患者来源的血液或外周血单核细胞（PBMC）的样品到足够数量的DC需要许多手动步骤，所述DC可用于疫苗开发、T细胞治疗或机制研究。当涉及一种或两种情况或单独抽血的研究规模甚至扩大到数十个样本的水平时，在人员小时和手动步骤数量方面的资源需求变得很大。考虑到这些细胞在更大规模上的现有和计划使用，例如在疫苗和个性化细胞治疗方案的II期或III期临床试验中，DC生成的当前方法造成了异常大的负担，最重要的是在成本方面，而且在进行全面研究和试验所需的时间方面。该提案旨在通过设计全自动微流体系统（microDEN）来解决对有效DC生成技术的未满足的需求，该微流体系统接受血液或PBMC样品，并在用细胞因子灌注一段时间后直接递送DC。该系统将联合收割机从血液中分离单核细胞和灌注培养结合到个体的患者特异性芯片中。与单核细胞纯化和在含有细胞因子的两种不同培养基类型中培养相关的手动步骤的消除本身将代表相对于本领域DC产生的开始的主要进步。我们进一步假设，在我们的微流体方法中采用的灌注技术将允许减少DC生成所需的时间（目前约6天），从而提供成本和资源的显著额外节省。所提出的microDEN系统将使用一系列功能测定建立并严格基准化传统DC生成技术，其中最关键的是用自体BCG感染的DC刺激的CD4+ T细胞的转录组谱。

 DESCRIPTION (provided by applicant): Dendritic cells (DCs) are an indispensable part of studying human responses that are important for protective immunity against cancer and infectious diseases as well as prevention of autoimmunity and transplant rejection. These cells are also key elements of personalized vaccines which are a major research focus in cancer and infectious diseases. Despite the vital role of DCs in both clinical and basic research contexts, methods for obtaining these cells from individuals remains a comparatively under-developed and inefficient process. Because DCs are present in very low concentrations (<1%) in blood, these cells must be generated from monocytes and the state of the art in such generation involves a laborious process of static culture and stimulation with cytokines contained in culture medium. Numerous manual steps are required to go from a sample of patient-derived blood or peripheral blood mononuclear cells (PBMCs) to sufficient numbers of DCs that can be utilized for vaccine development, T cell therapy, or mechanistic studies. When scaled even to the level of tens of samples for a study involving one or two conditions or separate blood draws, the resource requirement in terms of personnel hours and number of manual steps becomes significant. Considering the existing and projected use of these cells at much larger scale, such as in Phase II or III clinical trials of vaccines and personalized cell therapy regimens, the curret approach to DC generation poses an unusually large burden, most significantly in terms of cost, but also in terms of the time required to perform comprehensive studies and trials. This proposal aims to address the unmet need for effective DC generation technologies by designing of a fully-automated microfluidic system (microDEN) that accepts a blood or PBMC sample and directly delivers DCs following a period of perfusion with cytokines. This system will combine monocyte isolation from blood and perfusion culture into individual, patient-specific chips. The elimination of manual steps associated with monocyte purification and culturing in two different media types containing cytokines will, in itself, represent a major advance relative to start of th art DC generation. We further hypothesize that the perfusion technique employed in our microfluidic method will allow reduction in the time required for DC generation (currently ~ 6 days), thereby offering significant additional savings in cost and resources. The proposed microDEN system will be built and rigorously benchmarked against the conventional DC generation technique using a range of functional assays, the most critical of which will be transcriptomal profiling of CD4+ T cells stimulated with autologous, BCG-infected DCs.