Automated dendritic cell generation in a closed system

Abstract

Background & Aim Dendritic cells (DC) are an attractive vehicle for therapeutic manipulation and are used in antigen-pulsed autologous DC therapies, DC-stimulated autologous T cell therapies, and a variety of in vitro cellular immunology assays. Given the low abundance of DC in blood, they are typically generated ex vivo from monocytes or stem cell precursors in a manual culture process. The standard multiwell plate or flask culture used for DC generation requires several solution exchanges including cytokine refresh and wash steps. When DCs must be generated in a patient-specific manner, the multistep nature of the protocol presents significant logistical challenges for consistent results. To this end, the Corning MicroDEN® system relies upon medium perfusion to supply fresh cytokines throughout the duration of monocyte-derived DC generation. This study demonstrates performance of the MicroDEN system for immature DC (iDC) generation from a source of enriched monocytes. Methods, Results & Conclusion Monocytes were isolated from whole blood via Ficoll separation and CD14 magnetic bead purification. Monocyte differentiation to iDCs was achieved by a 6 day culture with CellGenix DC medium containing IL4 (350 U/mL;CellGenix), GM-CSF (350 U/mL;CellGenix), and 1% penicillin-streptomycin in both 6-well tissue culture-treated (TCT) multiwell plates (Corning) and the MicroDEN system. Flow cytometry data indicated that the iDCs from MicroDEN and 6-well plates were phenotypically comparable with regard to expression of CD209, CD80, CD83, CD86 and CD14. Allogeneic T cell proliferation assays and an antigen-specific assay demonstrated that iDCs generated via MicroDEN are functionally competent and can successfully generate a T cell response. A DOE-based approach was employed to characterize the relationship between perfusion flow rate, monocyte seeding density, and yield of dendritic cells. DCs generated by MicroDEN are functionally equivalent to DCs generated by the standard well plate culture. The larger surface area of MicroDEN cartridge allows generation of a higher number of dendritic cells in single run when compared to the standard 6-well plate, thereby reducing time and labor involved in harvesting cells from multiple plates. Higher perfusion flow rates enable the processing of larger numbers of seeded monocytes, upto a seeding density level of 1.3 million monocytes/cm2. MicroDEN is an effective tool to generate dendritic cells for applications ranging from cell-based assays to cell-based immunotherapies.