Exosomes from 3D cultures of Bone Marrow Mesenchymal Stem Cell (BM-MSCs) have higher neuro-regenerative potential than those generated from 2D conditions

Abstract

Purpose : Corneal nerves and innervation are susceptible to several diseases including diabetes. Despite the clinical need to promote corneal nerve regeneration, few specific therapeutic interventions are available. MSCs and their paracrine effect mediated by exosomes presents an attractive treatment for enhancing nerve regeneration. In this study, we assessed the neuro regenerative potential of exosomes from BM-MSCs cultured in 2D and 3D systems.

Methods : Human BM-MSCs were cultured in 2D monolayer and 3D bioreactor systems. Exosomes were isolated using ultracentrifugation followed by size and concentration measurements utilizing NanoSight and Exoview. Mouse trigeminal ganglia (TG) neurons were isolated from Thy1-YFP mice possessing fluorescent corneal nerves. The neurons were plated in the presence and absence of exosomes derived from 2D or 3D culture systems. Neuronal growth and morphology were monitored over 5 days. Thereafter, the neurons were fixed and immune-stained with β3 tubulin followed by confocal microscopy. Images were analyzed by Neurolucida software to obtain the density and length of the neurites.

Results : The Nanosight tracking analysis revealed significant increase in concentration of exosomes obtained from 3D vs. 2D culture condition (24X fold-change). Exoview analysis showed significantly higher concentration of CD63, CD81 and CD91 exosomal markers in 3D vs. 2D condition. Furthermore, a notable shift towards a more heterogeneous exosome phenotype was observed in the 3D compared to 2D culture systems. Exosomes derived from both 2D and 3D condition significantly induced neurite growth after 5 days in culture compared to untreated control. Neurolucida analysis of the Immunostaining images (β3 tubulin) showed a significant increase in density and complexity of the neuronal growth of TG neurons in 3D vs. 2D condition. Additionally, a significant increase in neuron branching was observed in 3D versus 2D.

Conclusions : Our results demonstrate the distinct effect of BM-MSCs-derived exosomes in neurites growth and elongation. Exosomes obtained from 3D culture system enhanced the neurite complexity and branching compared to the effect of vesicles from 2D culture. This specific property could potentially enhance the therapeutic effect of EVs in vivo and provide new therapeutic interventions for the restoration of damaged corneal nerves.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.