Human serum-derived extracellular vesicles support corneal epithelial wound healing in vitro

Abstract

Purpose : In ocular surface disease there are limited non-surgical therapies to restore normal corneal epithelial function. We propose that extracellular vesicles derived from human serum can act as a biostable and readily available therapeutic alternative. We assessed their effect on a human corneal epithelial cell line.

Methods : Human serum (n=3 donors) diluted 50% in PBS (phosphate buffered saline) was subjected to serial ultracentrifugation, up to 110,000xg. The resulting pellet was resuspended in PBS. Analysis included NTA (nanoparticle tracking analysis), BCA assay, and transmission electron microscopy (TEM).
To assess proliferation, cells were seeded at 50000 cells/well and the number of cells were counted after 72h of culture in serum-free medium, and with the addition of 10% human serum, or ECVs (1x10¹¹ particles/mL). Additionally, confluent cells were scratched with a pipette tip and the rate of wound closure was monitored.
For the scratch assay multiple unpaired t-tests analysis was performed with a false discovery rate correction at 0.1. For Proliferation, a one -way ANOVA with Dunnett’s multiple comparisons test was performed.

Results : NTA demonstrated a mean concentration of 4.51x10¹² particles/mL, and the modal particle size range was 97.4-113.4nm. Mean protein concentration was 4685mg/mL. TEM revealed membrane bound particles 90-110nm in diameter.
After 72h culture in serum-free medium, mean cell doubling time was 36.8h±7.5h (mean, SD). With serum-supplemented medium it was 18.8h±0.8h (p<0.005), and in ECV-supplemented medium 27.0h±1.5h (p<0.05), n=3.
The addition of both human serum and serum-derived ECVs significantly accelerated wound closure as compared with the serum-free control medium at the 9h, 12h, and 24h timepoints (p<0.005 for human serum, p<0.05 for ECVs), n=3.

Conclusions : ECVs derived from human serum causes a significant increase in the proliferation and wound healing rate of corneal epithelial cells in serum-free medium. This result supports the further study of the therapeutic potential of serum-derived ECVs.

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

 

Vesicle isolated from human serum after ultracentrifugation

 

(a) Wound closure of epithelial cells treated with serum-free and ECV-supplemented media. (b) The rate of wound closure in serum-free and ECV-supplemented media. *p<0.05.