(341a) A Digital Focus Forming Assay Platform for the Quantification of Infectious Influenza Virus

The focus forming assay (FFA) is a common method for the quantification of infectious virus in a liquid sample. Unlike the more commonly used plaque assay, which relies on the lysis of infected cells to form plaques, the FFA uses immunofluorescent staining of infected cells to visualize foci after shorter incubation periods and is especially useful for viruses that do not cause cytotoxicity in the cells. However, the conventional FFA requires laborious counting of foci and may involve some subjectivity in the interpretation of the results. This study reports a microfluidic digital cell culture platform that modifies the FFA to provide a convenient, binary readout that can be directly correlated to the infectious titer of the sample. Owing to the microfluidic design, it additionally addresses a secondary issue with the conventional FFA by significantly reducing the quantity of antibodies and reagents needed for staining cells, thereby reducing assay cost.

The microwell array and enclosing channel were fabricated through polydimethylsiloxane (PDMS) soft lithography and bonded to a glass slide substrate to allow for imaging at various stages of the process as shown in Figure 1A. The well array comprises 36x30 cuboidal wells, each separated by a 50µm spacing and having a width of 200 µm and depth of 110 µm. The enclosing channel is 110µm high with inlet and outlet ports to interface the chip through microfluidics. The channel also includes herringbone mixers and cylindrical pillars to improve the lateral distribution of cells flowing into the microwells. Hydrophilic surface modifications of the PDMS surface facilitate the retention of aqueous media within the wells when discretized by oil.

The device is operated by filling the channel initially with phosphate-buffered saline (PBS) and then placing it in a desiccator under a vacuum to fill the microwells and remove any trapped air bubbles from the channel. A Madin-Darby Canine Kidney (MDCK) cell suspension is perfused into the device, and the cells are allowed to sediment into the wells. After incubation, the cells adhere to the inside of the microwells, and the influenza viral stock solution is added and allowed to rest for two hours. The remnant solution containing any unbound virus particles is flushed out and replaced with 1.2% microcrystalline cellulose (Avicel) suspension containing a standard maintenance medium to support the infection progression. Mineral oil is perfused into the channel, which partitions the wells while retaining the Avicel-containing media within the wells as shown in Figure 1B. This prevents any cell growth in the space between the wells which prevents potential cross-contamination between the wells. The chips are incubated for 24 hours and then fixed, permeabilized, and stained through immunofluorescence by flowing in primary and secondary antibody solutions.

Preliminary experiments using high virus titers demonstrate that the platform is effective at displaying influenza infection across all the wells while also showing excellent isolation between the microwells through the combined usage of Avicel and mineral oil overlays, as shown in Figure 1C. Experiments to quantify infectious viral titers at lower virus concentrations are currently underway. We believe that by overcoming key constraints associated with conventional focus assays, the digital FFA platform offers significant benefits for the rapid, reliable, and efficient analysis of virus infectivity.