Fig. 3

The impact of exosome production protocols on both cAD-MSCs characteristics and cAD-MSC-derived exosome qualitative specifications. Schematic illustration of (A) 2D-based exosome production protocol and (B) 3D-based exosome production protocol. (C) Morphology representation of cAD-MSCs undergoing exosome production was obtained at the beginning (day 0) and the end (day 2) of the conditioning period. (D) Live/dead staining of cAD-MSCs was conducted at the beginning and final days of the conditioning period during exosome production. Live cells were labeled with calcein-AM dye, emitting green fluorescence, while dead cells were stained with propidium iodide dye, emitting red fluorescence. The scale bar indicates 250 μm. (E) The morphology of cAD-MSC-derived exosomes isolated from different production protocols was visualized using TEM. The scale bar marked 100 nm. (F) Particle size and distribution and (G) mode size of cAD-MSC-derived exosomes produced from either 2D platforms or 3D platform were analyzed using NTA. The value indicating mean was plotted (n = 4). (H) Pseudo-contour diagram generated from Nano-scale flow cytometry analysis illustrating gates of CD9+ exosome produced from different protocols. The mean ± SD are plotted (n = 3). (I) CD9 positivity of exosome was generated from nano-scale flow cytometry findings. The results are presented as fold change normalized to the exosome from SF-DMEM-2D as control. The mean and p-value are denoted (n = 4). (J) Zeta potential value of cAD-MSC-derived exosome obtained from various production protocols. The mean and p-value are plotted (n = 3). The asterisks illustrate the significance levels (*p < 0.05, **p < 0.01, ***p < 0.001).