Combining Microfluidics and Fluorescence to Quantify the Timing of Viral Release

Abstract

Release of viral particles from a host cell is an important step in the viral life cycle. This has been well studied in a variety of viral systems, but quantitative data on the exact timing of viral release and potential cell-to-cell variations is still needed. This data could shed light on the underlying mechanisms that affect the timing and rate of viral production. In this poster, we explain the experimental setup that will allow us to gather this data using viral like particles produced in the HIV-1 test system.HIV-1 is an important and extensively studied retrovirus. Transfecting cells with only the structural protein Gag produces viral like particles (VLPs). The budding and release of these VLPs is similar to viruses produced from cells transfected with the full viral genome. There is a temporal distribution in the release of viral like particles from transfected cells, but the exact distribution and its cause are not fully known. We are developing an experimental setup that combines microfluidics and fluorescence to determine these.Here, we summarize previous microfluidic designs and elaborate on the design fabrication and testing process specific to the adherent mammalian cells needed to produce VLPs. These adherent cells are grown inside a PDMS/glass based microfluidic device. They are transiently transfected with HIV-1 Gag-YFP. A very slow flow of medium is established and a narrow channel section is observed downstream from the cell colony. A CCD camera is used to detect each VLP and a histogram of observed VLP vs. post transfection time is made. We explain the testing of our detection system limits using fluorescent beads as a model. Finally, we discuss the design and initial testing of microfluidic devices for single cell VLP production studies.