Figure 3.

Characterization of extracellular vesicles secreted by encapsulated W8B2⁺ CSCs. (A) Transduced W8B2+ cells expressing both the GlucB-IRES-GFP and sshBirA-IRES-mCherry vectors (W8B2⁺ CSC^{GlucB+sshBirA}), resulting in extracellular vesicles with GlucB and sshBirA labelled and biotinylated on the surface of the plasma membrane. (B) W8B2⁺ CSC^{GlucB+sshBirA} encapsulated within a TheraCyte device and expressing mCherry and GFP fluorescence proteins in vitro. (C, D) Gluc activity (C) and concentration of microvesicles (D) in conditioned media (50 µL and centrifuged at 500 g) harvested at 1, 2, and 3 days from W8B2⁺ CSC^{GlucB+sshBirA} cultured as monolayer (2D) or encapsulated within a TheraCyte device (3D). n = 3 independent experiments. Data are shown as mean ± SEM. *P < 0.05, ****P < 0.0001 by one-way ANOVA with Bonferroni post hoc test. (E, F) Nanoparticle tracking analysis showing the size distribution of EVs in conditioned media harvested at 1, 2, and 3 days from W8B2⁺ CSC^{GlucB+sshBirA} cultured as monolayer (E) or encapsulated within a TheraCyte device (F). (G) Size distribution of EVs in conditioned media harvested on Day 3 (∼1.5 mL and centrifuged at 2000 g followed by 110 000 g twice) from untransduced W8B2⁺ CSCs and W8B2⁺ CSC^{GlucB+sshBirA} encapsulated within a TheraCyte device. n = 3 independent experiments.