Contributors: M. Marcali, C. L. Ren, M. Aucoin
1 Analysis of formation dynamics of blood droplets in T-junction generators.
Several models have been developed to predict final droplet volume and formation dynamics of Newtonian fluids such as water and glycerol [1],[2]. However, little attention is given to the non-Newtonian fluids. To fill this gap in the literature, the formation of blood droplets in the T-junction generator was analyzed as shown in Figure 1. Furthermore, since many application of droplet-based microfluidic system requires biological and chemical fluids which are mostly non-Newtonian, it is great importance to analyze the formation dynamics of these fluids.
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 Figure 1. Blood droplet formation and stages of the formation cycle. |
2 Quantification of Influenza Virus-Like-Particles via hemagglutination assay in microdroplets.
VLPs are genetically engineered non-infectious particles, which have proteins to mimic the original virus but lack of genetic material to infect host organisms. Therefore, VLPs are good candidates for vaccine production [3]. To determine the final dosage of VLPs in the vaccine, HA is done using 96-well plates. In this assay, diluted particles mixed with the target (RBCs) and the aggregation takes 4 hours to finalize[4]. Although this method is a standard procedure, longer reaction time, cross-contamination, and human errors are the major drawbacks of this system. In this study, it was shown that the reaction time dropped to 4-10 seconds due to small diffusion length in droplets, cross-contamination is prevented due to the compartmentalization nature of droplets, and aggregation is detected by the image analysis to eliminate human error (Figure 2). Besides, with this system binding affinity of the virus to the cells were analyzed by applying shear to the trapped droplets.
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 Figure 2 Schematic of microfluidic channel and hemagglutination reaction. (Top right) formation of blood & virus droplet and hemagglutination reaction in microdroplets. |
3 Analysis of Infection kinetics of influenza Virus-Like Particles in microdroplets.
This study represents the ability of droplet-based microfluidic systems to investigate the infection kinetics influenza virus-like particles (VLP). VLPs are protein complexes that are composed of viral structural proteins. These particles mimic the organization of the original virus, but they do not contain any genetic material. Therefore, they have no ability to infect the host. This property of the particles is utilized in vaccine production to replace low yield standard methods. To increase the yield, the baculovirus-insect cell expression system has been developed [5]. In this system, insect cells are infected with a gene-edited baculovirus. Edited gene is able to express proteins that the influenza virus has such as HA, NA, and M1 and form VLPs in insect cells. Once baculoviruses infect cells, cells start to express influenza virus proteins that are tagged with fluorescent proteins, and the infection cycle becomes observable. This cycle has been investigated using a population of cells in culture for various viruses. Therefore, the behavior of a single cell against the virus infection is masked by the population. Timm et al. [6][7] have shown that virus release was two times faster than the standard infection in culture for a single BHK cell infection. They accomplished the single-cell analysis by applying serial dilution to the cell population in 96-well plates. In this study, to have better control on a single cell, a droplet-based microfluidic system was used. Insect cells and viruses were encapsulated in droplets using flow focusing design, as shown in Figure 3. After encapsulation, droplets flew through the serpentine channel to enhance mixing. They were stored at the chamber for incubation, as shown in Figure 3.a-b, c-d, and cells start to emit fluorescence after 24hrs of infection in the culture. However, by utilizing the shorter diffusion length in droplets, emission time was reduced to 14-18hrs after infection, which reduces the total infection time Figure 3 .e-f, g-h. S.
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 Figure 3. Schematic of microfluidic channel. (Bottom section) Fluorescence imaging of the Infection cycle in microdroplets. |
References
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