Abstract
Purpose :
Small extracellular vesicles (sEVs) derived from 2D cultures of human bone marrow mesenchymal stromal cells (BM-MSCs) provide reproducible neuroprotection in different experimental models. Recent data suggest that 3D cultures of BM-MSCs yield more sEVs with enhanced therapeutic properties than 2D cultures. Hence, we established 3D cultures of BM-MSCs to isolate sEVs and compare their retinal ganglion cell (RGC) neuroprotective properties with sEVs from 2D cultures.
Methods :
BM-MSCs were cultured in 2D monolayer and 3D PBS-Mini Vertical-Wheel Bioreactor systems. After reaching 80% confluency, 2D and 3D cultures were grown for 48 hours in exosome-free media. sEVs were isolated from 200 mL of condition media using buoyant density ultracentrifugation and characterized by NanoSight, Western blotting, and electron microscopy. Mixed primary mouse retinal cell cultures (n=3) were treated with different concentrations of sEVs (1.2x108-6x109 per 5x104 cells) for 72 hours. β-III tubulin staining was used to count RGCs and for in-depth morphometric assessment.
Results :
NanoSight analysis revealed significantly higher (14.4-fold) sEV generation in 3D cultures compared to 2D. Western blots showed the presence of exosome (CD63, CD81, & CD9) and microvesicle (Annexin A2) markers in both 3D and 2D sEVs. Treatment of retinal cells with 1.2x109 sEVs from both cultures showed significantly enhanced RGC survival compared to untreated cells (3D: 86% ±14% & 2D: 75% ±12% increase, respectively), while no significant differences were observed between 2D and 3D sEVs. Compared to the untreated RGCs, sEV-treated RGCs demonstrated significant improvement in their morphometry, i.e. total no. of neurons with neurites/axons (3D: 3-fold), total no. of neurites excluding axon (3D: 4.2-fold & 2D 3.2-fold), total number of axons (3D: 2-fold), total no. of axonal tree branches (3D: 4.5-fold & 2D: 2.9-fold), primary (3D: 3.6-fold & 2D: 3.1-fold), secondary (3D: 4.3-fold & 2D: 3.1-fold), and tertiary neurites (3D: 6.4-fold & 2D: 5.3-fold). Interestingly, we observed a significantly increased no. of RGC axonal tree branches in 3D-sEVs compared to 2D-sEVs (1.5-fold, p<0.05).
Conclusions :
Compared with 2D cultures, 3D BM-MSCs generated a significantly higher yield of sEVs with increased complexity in the axonal tree branches besides their similar levels of RGCs protection.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.