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

 描述(申请人提供)：树突状细胞(DC)是研究人类反应不可或缺的一部分，对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，而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，但从个体获取这些细胞的方法仍然是一个相对不发达和低效的过程。由于DC在血液中的浓度很低(1%)，这些细胞必须从单核细胞产生，而这种产生的技术状态涉及到一个繁琐的静态培养和用培养基中包含的细胞因子刺激的过程。从患者的血液或外周血单个核细胞(PBMC)样本到足够数量的DC，可用于疫苗开发、T细胞治疗或机制研究，需要大量的手动步骤。对于一项涉及一种或两种情况或单独抽血的研究，当规模扩大到数十个样本的水平时，人力小时和手动步骤数量方面的资源需求变得非常重要。考虑到这些细胞的现有和计划在更大范围内的使用，如在疫苗和个性化细胞治疗方案的第二阶段或第三阶段临床试验中，DC生成的快速方法构成了异常巨大的负担，最显著的是在成本方面，但也在进行全面研究和试验所需的时间方面。这项建议旨在通过设计一种全自动微流控系统(MicroDEN)来满足对有效DC生成技术的未得到满足的需求，该系统可以接受血液或PBMC样本，并在细胞因子灌流一段时间后直接输送DC。该系统将把血液中的单核细胞分离和灌流培养结合到单独的、针对患者的芯片中。与单核细胞纯化和在含有细胞因子的两种不同类型的培养液中培养相关的手动步骤的消除，本身将代表着相对于开始产生ART DC的重大进步。我们进一步假设，在我们的微流控方法中采用的灌流技术将允许减少DC产生所需的时间(目前为~6天)，从而在成本和资源方面提供显著的额外节省。建议的microDEN系统将使用一系列功能分析建立并严格参照传统的DC生成技术，其中最关键的将是由自体、卡介苗感染的DC刺激的CD4+T细胞的转录图谱。

 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.