Table 1.
Three types of culture media for UC-MSC-derived EX manufacturing.
Fig 1.
Characteristic of UCMSCs cultured in five different media.
(A) A representative morphology of UCMSCs cultured in DM-PLT medium at P5. Magnification 10X. Scale bar 50 μm. (B) A comparative population doubling time of three UCMSC lines cultured in 10% FBS supplemented (DM-FBS), a 5% PLT supplemented (DM-PLT), and three serum-free media (STEMin1, NutriStem, and StemMACS) (n = 3). (C) Expression of MSC surface markers (n = 3) and (D) colony-forming potential of UCMSCs cultured in five different culture media (n = 3). A representative image from DM-PLT culture medium independent experiments is shown. Magnification 4X. Scale bar 200 μm. (E) A representative trilineage differentiation of UCMSCs cultured in DM-PLT medium. Magnification 10X. Scale bar 50 μm. Sin1, NutriS, DM-PLT, SMACS, DM-FBS are UCMSCs cultured in STEMin1, NutriStem, DMEM/F12 medium supplemented with 5% PLT, StemMACS and DMEM/F12 medium supplemented with 10% FBS, respectively. P: Passage, CFU: Colonies forming unit.
Fig 2.
Exosomes derived from UCMSCs displayed typical exosome morphology and surface marker.
(A) Representative morphology of EXs derived from UCMSCs cultured in DM-PLT medium observed under transmission electron microscopy. (B) Internal control GAPDH and CD9 were detected in all EX samples with 15 μg total exosomal protein loaded into each lane. DM-FBS_EXs, DM-PLT_EXs, SMACS_EXs, NutriS_EXs, Sin1_EXs are EXs derived from UCMSCs cultured in DMEM/12 + 10% FBS, DMEM/F12 + 5% PLT, StemMACS, NutriStem, and STEMin1, respectively.
Fig 3.
Quantity of growth factors and cytokines in exosomes purified from 108 UCMSCs cultured in different media (n = 3).
DM-FBS_EXs, DM-PLT_EXs, SMACS_EXs, NutriS_EXs, Sin1_EXs are EXs from UCMSCs cultured in DMEM/12 + 10% FBS, DMEM/F12 + 5% PLT, StemMACS, NutriStem, and STEMin1, respectively. Statistical significance was determined by ANOVA and post-hoc Tukey HSD tests and is indicated by: * where p < 0.05; ** where p < 0.01; *** where p < 0.001; **** where p < 0.0001.
Fig 4.
Effects of EXs on fibroblast proliferation and migration (n = 3).
(A) Confocal images of fibroblasts incubated for two hours with ExoGlow (green) labeled UCMSC-derived EXs. DAPI was used to stain nuclei (B) Fibroblast proliferation rates induced by EXs from different media. The percentage of cell proliferation was normalized to medium supplement with DM-FBS_EXs. (C) EXs-mediated fibroblast migration as determined by a scratch assay. DM-FBS_EXs, DM-PLT_EXs, SMACS_EXs, NutriS_EXs, Sin1_EXs are EXs from UCMSCs cultured in DMEM/12 + 10% FBS, DMEM/F12 + 5% PLT, StemMACS, NutriStem, and STEMin1, respectively. Statistical significance was determined by ANOVA and post-hoc Tukey HSD tests, two-way ANOVA, and is indicated by: * where p < 0.05; ** where p < 0.01; *** where p < 0.001; **** where p < 0.0001.
Fig 5.
Influence of EX treatment on the tube formation (n = 3).
(A) Uptake of ExoGlow-labeled EXs (green) in endothelial cells after two hours. The nucleus was stained with DAPI. (B) The representative tube structures of hUVECs under an inverted microscope. (C) Quantitative analysis of hUVEC tube formation normalized to the control group. DM-FBS_EXs, DM-PLT_EXs, SMACS_EXs, NutriS_EXs, Sin1_EXs are EXs from UCMSCs cultured in DMEM/12 + 10% FBS, DMEM/F12 + 5% PLT, StemMACS, NutriStem, and STEMin1, respectively. Statistical significance was determined by ANOVA and post-hoc Tukey HSD tests and is indicated by: * where p < 0.05; ** where p < 0.01; *** where p < 0.001; **** where p < 0.0001.