Exosome Uptake into Hey Ovarian Cancer Cells and its Potential to Serve as a Vessel for Gene Therapy
Shin, Esther Jeeyoung
MetadataShow full item record
Exosomes are microvesicles that are released from several different types of cells. Exosomes are thought to play an important role in functions such as immune regulation and coagulation; however their full functionality is not completely understood. Current research has started to explore their potential utilization in gene therapy and drug delivery. Their derivation from different proteins and RNA make them a versatile transport target in microbiology research. Although exosomes are being increasingly used in current research for gene therapy applications, the actual mechanism is unknown once the exosomes are taken into the cells. Using microfluidic channels, the entire process of exosome uptake can be imaged and monitored. The design of the microfluidic device allows for the manipulation of cellular flow and imitates the real flow of cells during exosome uptake and interaction. The microfluidic device is made from a mold using polydimethylsiloxane (PDMS) and the channels are coated with fibronectin for the cells to adhere to. The device is plasma bonded to a thin sheet of PDMS, incubated, and then left to cure. Because of its ability to grow quickly and efficiently in less-than-ideal conditions, hey ovarian cancer cells are used to seed the device. The hey cells are seeded at a density between five million and 10 million cells in the device, and fresh media is pumped through the device. The cells are left to adhere and proliferate for between 24 hours while fresh media is passed through the device in the 37 degrees C incubator. The hey cells are dyed using a DAPI fluorescent stain which causes the hey cells to illuminate blue fluorescence. Exosomes that are stained with PKH to illuminate green fluorescence are then seeded into the channels, and images are taken using confocal microscopy at several time points. The images showing blue fluorescent hey cells and green fluorescent exosomes are overlayed to show the exosomes uptake into the hey cells. Several images are taken across approximately a 20-minute time period to show the interaction between the exosomes and the cells in the channels.