Ex Vivo Generation of Dendritic Cells Within a Vascular Bioreactor
Abstract
Dendritic cells (DCs) play a prime role in the activation and control of the immune system and have promising potential in the treatment of cancer, viral infection, autoimmune diseases and transplantation rejection. DCs can be generated ex vivo from human monocytes by the use of growth factors or during transendothelial migration. Many studies have generated DCs under static, “no flow” conditions. However, such studies do not mimic the “flow” occurring in the capillaries. The objective of this study was to see if we could generate functional DCs within a novel bioreactor system under flow conditions. A HUVEC monolayer was grown on a porous membrane and added it to a bioreactor at 0.4 mL/min. Inflammation was mimicked by adding TNF-α to the system for 48 h. Cell morphology, viability, CAM expression and MCP-1 were measured. CFSE-labeled monocytes were added above the flow path. Monocytes were analyzed for location based on fluorescent intensity and migration markers. Finally, we looked at the differentiation of monocytes into DCs by looking at common DC markers. To test for functionality, we performed a mixed leukocyte reaction (MLR) for seven days and measured proliferation and activation of T-cells. HUVECs maintained a compact network after 72 h. Cell viability with and without TNF-α was 78% and 82%, respectively. Increased VCAM-1 expression (37%) and MCP-1 (6x) act as inflammatory responses to send signals for recruitment of leukocytes. Increased monocyte migration through the stimulated endothelium could be due loose HUVEC junctions. All migration marker expression was downregulated in a stimulated system, possibly due to monocyte shifting away from lineage or differentiation into DCs. DCs from the bioreactor had no morphological differences when compared to static culture. DCs attached to the endothelium expressed 80% CD206, possibly playing a role in their attachment to the endothelium. DCs after culture expressed CD86 and after adding maturation cytokines, bioreactor and static DCs matured as indicated by CD83 expression (72% and 84.9%, respectively). DCs from the bioreactor proliferated activated T-cells more than static DCs as indicated by MFI. Overall, the system was able to generate functional DCs that promoted better T-cell proliferation than traditional methods.
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- OSU Theses [15752]