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

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 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.