Exosomes are nanoscale extracellular vesicles, ranging from 40 to 160 nm, secreted by nearly all cell types. They contain a variety of molecular components, including nucleic acids (notably RNAs), proteins, lipids, and metabolites, which can reflect the characteristics of their originating cells. However, the utilization of undifferentiated embryonic or fetal cells poses a tumorigenic risk. In therapeutic programs, exosome-based approaches seek to take advantage of their regeneration capabilities, while minimizing associated risks of cell therapy. To this aim, we established a new method to isolate exosomes from fetal tissues with potential application in regenerative medicine. A 2-week pregnant WISTAR rat was euthanized under the approved animal application protocol of Ferdowsi University of Mashhad, and related fetuses were extracted from the uterine tubes. The combined fetal tissue was first mechanically homogenized and then underwent sequential centrifugations to remove cells and debris. The precleared supernatant was subsequently combined with a 16% polyethylene glycol (PEG 4000) solution and incubated overnight at 4 °C to precipitate exosomes. The isolated particles were analyzed by dynamic light scattering (DLS), zeta potential assessment, and field-emission scanning electron microscopy (FE-SEM). DLS data revealed a vesicle population concentration peak at approximately 50-150 nm, whereas zeta-potential analysis demonstrated a negative surface charge (peak at -25mV), compatible with lipid bilayer vesicles. FE-SEM imaging confirmed a uniform population of small, approximately spherical nanovesicles. These results align with established exosome features, including a size of around 100 nm and a cup-shaped morphology.

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