Microfluidic on-demand engineering of exosomes towards cancer immunotherapy

Extracellular vesicles (EVs), particularly exosomes (30-150 nm), are an emerging delivery system in mediating cellular communications, which have been observed for priming immune responses by presenting parent cell signaling proteins or tumor antigens to immune cells. Therefore, preparation of antigenic exosomes that can play therapeutic roles, particularly in cancer immunotherapy, is emerging. However, standard benchtop methods (e.g., ultracentrifugation and filtration) lack the ability to purify antigenic exosomes specifically among other microvesicle subtypes, due to the non-selective and time-consuming (>10 h) isolation protocols. Exosome engineering approaches, such as the transfection of parent cells, also suffer from poor yields, low purity, and time-consuming operations. In this paper, we introduce a streamlined microfluidic cell culture platform for integration of harvesting, antigenic modification, and photo-release of surface engineered exosomes in one workflow, which enables the production of intact, MHC peptide surface engineered exosomes for cytolysis activation. A PDMS microfluidic cell culture chip is simply cast from a 3D-printed mold. This proof-of-concept study demonstrated the enhanced ability of harvested exosomes in antigen presentation and T cell activation, by decorating melanoma tumor peptides on the exosome surface (e.g., gp-100, MART-1, and MAGE-A3). Such surface engineered antigenic exosomes were harvested in real-time from the on-chip culture of leukocytes isolated from human blood, leading to much faster cellular uptake. The activation of gp100-specific CD8 T cells which were purified from the spleen of 2 Pmel1 transgenic mice was evaluated using surface engineered exosomes prepared from murine antigen presenting cells. Antigen-specific CD8 T cell proliferation was significantly induced by the engineered exosomes compared to that by native, non-engineered exosomes. This microfluidic platform serves as an automated and highly integrated cell culture device for rapid and real-time production of therapeutic exosomes that could advance cancer immunotherapy.