(23b) Light-Induced Extracellular Vesicle Adsorption

The role of extracellular vesicles (EVs) in human health and disease has garnered considerable attention over the past two decades. However, while several types of EVs are known to interact dynamically with the extracellular matrix and there is great potential value in producing high-fidelity EV micropatterns, there are currently no label-free, high-resolution, and tunable platform technologies with this capability. We introduce Light-induced Extracellular Vesicle Adsorption (LEVA) as a powerful solution to rapidly advance the study of matrix- and surface-bound EVs and other particles. The versatility of LEVA is demonstrated using commercial GFP-EV standards, EVs from glioblastoma bioreactors, and E. coli outer membrane vesicles (OMVs), with the resulting patterns used for glioblastoma cell migration on migrasome-mimetic trails and OMV-mediated neutrophil swarming.

Methods

Bioreactors with defined, EV-free media are utilized for the generation of glioblastoma EVs, and E. coli OMVs are isolated from bacterial-conditioned media. For both the OMVs and glioblastoma EVs the media is purified through size exclusion chromatography and characterized using nanoparticle tracking analysis. The migrasome-mimetic EVs are isolated from the bioreactor conditioned media through sequential ultracentrifugation. LEVA’s optimized micropatterning technique provides control of the desired microdomain by shining ultraviolet (UV) light through a digital micromirror device (DMD). The DMD converts grayscale images to relevant UV intensities and shines the UV light onto a benzophenone comprised photoactivator. The photoactivator, once exposed to UV light, will then reveal the substrate below it, poly-L-lysine (PLL), which has the capability to electrostatically bind EVs and OMVs. Human blood is drawn from a healthy donor control and human peripheral blood neutrophils are isolated through negative immunoselection.

Results / Discussion

LEVA’s high-resolution capabilities are shown as it converts the grayscale image (Figure 1A) to the desired UV intensities, exposing the PLL substrate and allowing for the binding of EVs (Figure 1B). For the migrasome-mimetic studies, the grayscale mask (Figure 1C) is utilized to pattern migrasome-mimetic EVs (Figure 1D) and when U87 glioblastoma cell line is added to the migrasome-mimetic trails, they adhere to the pattern and begin to migrate and oscillate along the patterns (Figure 1E). An advantage of LEVA is the ability to have multiple different patterns within the same microdomain, as a circle and star of the same area are patterned next to each other and filled with OMVs. The OMVs contain the same surface components as the whole bacteria, this allows for neutrophil recognition and induces the immune signaling cascade (Figure 1F). Interestingly, the increased perimeter of the star pattern caused the neutrophils to have differential migration towards it instead of the circle (Figure 1G). Where during the initial swarming phase neutrophils migrate towards both patterns equally, but then cells towards the circle and have increased migration towards the star pattern. This is quantified through a chemotactic index, which is a measure of directionality either towards or away from the defined pattern.

Conclusions

LEVA will enable rapid advancements in the study of matrix- and surface-bound EVs and other particles and should encourage researchers from many disciplines to create novel diagnostic, biomimetic, immunoengineering, and therapeutic screening assays.