Generation of a stable Müller cell line spontaneously releasing fluorescently labeled exosomes; An in vitro model to study trafficking of retinal extracellular vesicles.

Abstract

Purpose : Extracellular vesicles including exosomes are means of intercellular communication. It is interesting to develop both in vitro and in vivo models to study the trafficking of these biological nanovesicles across the retina. We aimed to generate a stable retinal Müller cell line that spontaneously releases fluorescently labeled exosomes. The same approach can be used for other retina cells.

Methods : Exosome Cyto-Tracer, pCT-CD63-GFP was purchased (SBI, CA, USA). A lentiviral expression system was used to package the construct into pseudoviral particles. 293TN cell line was used as producer cells for the packaging procedure. Both the CD63-GFP lentivector construct and the plasmids were cotransfected into 293TN cells. 18-24 hours before the transfection, 8 x 10⁶ 293TN cells were seeded in 150 cm² culture plate. After the cells reached 80% confluency on the next day, the lentivector construct, the plasmids, and the packaging mix were added to the media. The media was collected 72 hours after the transfection. The media was centrifuged to get rid of any cell debris and then the supernatant was transferred to a new tube. Polyethylene glycol precipitation solution was used to concentrate the viral particles. The viral particles were then aliquoted and stored at -80^oC. Ultra rapid Lentivirl titer kit was used to determine the pseudoviral titer by real-time PCR. 50,000 rat Müller cells per well were seeded into a 24-well plate. Different concentrations of the CD63-GFP pseudovirus particles were used to treat different wells. 5 days after the transfection, FACs sorting was used to isolate GFP-positive Müller cells.

Results : CD63-GFP pseudoviral particles were generated and were able to transfect rat Müller cells. The transfection and the transduction were successful in allowing the CD63-GFP construct to be included in the genome of the Müller cells. The released CD63-GFP-positive exosomes can be visualized under a fluorescence microscope.

Conclusions : The generation of Müller cell lines that spontaneously release fluorescent exosomes can be of great help in studying the process of trafficking of the retinal extracellular vesicles. Coculture of these cell lines with other retinal cell lines can help visualize the dynamics of the release and the uptake of the extracellular vesicles including exosomes.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.