Figure 5.
Comparison of the Immunomodulatory Effect and Protein Profiles between EVs Derived from MSCs Expanded under 2D versus 3D Culture System
(A) Representative photograph of fluorescence (Dil)-labeled MSCs on microcarriers (Corning Life Sciences) at 4 h after seeding. (B) IFN-γ, IL-6, TGF-β1, and IL-10 ELISA with conditioned medium of anti-CD3/28-stimulated splenocytes for 72 h with or without 2D or 3D MSC-EVs (1.5 × 109 particles/mL; n = 4). (C) ELISA for serum cytokines (TNF-α, IL-6, and IL-17) at 5 h after EV injection (1 × 1010 particles/mouse) in LPS-challenged mice (30 μg/mouse; i.v.; n = 5/group). EVs were isolated from PD15 MSCs expanded in 2D culture (CellStack cell culture chambers; Corning Life Sciences) or 3D culture (microcarriers in a spinner flask; Corning Life Sciences). All data are presented as means ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 by one-way ANOVA with Tukey’s test. (D) Venn diagram of protein profiling of 2D and 3D MSC-EVs. (E) Classification of proteins found in both, but enriched (1.5-fold) in 3D MSC-EVs compared to 2D according to molecular function. (F) Classification of binding proteins in (E). (G) Classification of proteins found in both, but enriched (1.5-fold) in 3D MSC-EVs compared to 2D according to biological process. (H) Classification of proteins found only in 3D MSC-EVs according to biological process. (I) RT-PCR assays for TGF-β1, TGF-β2, TGFβI, and TSG-6 mRNA in 2D and 3D MSCs and ELISAs for TGF-β1, TGF-β2, TGFβI, and TSG-6 protein in 2D and 3D MSC-EVs (n = 3; three different preparations). (J) ELISAs of TGF-β1 and TGF-β2 proteins in PD15 and PD40 MSC-EVs with or without lysis buffer treatment (n = 3; three different preparations). All data are presented as means ± SD. ∗p < 0.05 by one-way ANOVA with Tukey’s test.