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 yield, low purity, and time-consuming operations.

Researchers from the University of Kansas 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. The PDMS microfluidic cell culture chip is simply cast from a 3D-printed mold. The 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, 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 muring antigen presenting cells. Antigen-specific CD8 T cell proliferation was significantly induced by the engineered exosomes compared to 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.

3D-printing Molded Microfluidic Cell Culture Device for
Real-time Engineering and Harvesting Antigenic Exosomes

a)  Illustration  of  3D -printing  molded  microfluidic   culture  device  for   engineering  immunogenic  exosomes  directly  from  on -chip  cultured  cells  in   real    time.    b)    Fluorescence    dye    solution    for    showing    the    flow    of     immunomagnetic  beads  (A -Inlet)  mixing  with  cell  culture  media  (B -Inlet)  eluted from cell  culture chamber. c)  Bright -field microscopic image showing  the  immunomagnetic  microbeads  mixing  process  for  isolating  exosomes  in   the serpentine microchannel. d) Bright -field microscopic image showing the  morphology  of  on -chip  cultured  leukocytes.  e)  SEM  image  showing  the   engineered exosomes released out of the chip.