Extracellular vesicles are membrane bounded sacs containing crucial biomolecules which are released into extracellular matrix. Exosomes, with peculiar properties, are nano-scaled extracellular vesicles ranging 30_150 nm in size and are found in body fluids. Amniotic fluid is a temporary organ, which is essential for embryonic development and is rich in peptides, carbohydrates, and signaling molecules and the amniotic fluid exosomes have shown a great potential in regenerative medicine. Therefore, establishing a practical method to extract amniotic fluid from laboratory animals is important for research or even clinical applications. In current study, we established a protocol for isolating rat amniotic fluid and extract its exosomes by Poly-ethylene glycol (PEG-4000) precipitation method. To this aim, a pregnant Wistar rat was anesthetized, the abdominal region was cut and the yellow jelly-like liquid between fetal sacs was extracted using a normal syringe. The liquid was sequentially centrifuged (300g, 2000g, 10,000g) to remove intact cells, organelles, and cell debris. The resulting supernatant was filtered (0.22 µm), then mixed with PEG (1:4 v/v) and NaCl (1:30 v/v). After 16 h incubation at 4°C, exosomes were precipitated by centrifugation at 5000g for 50 min. Finally, the pellet was resuspended in NaCl and applied for further characterization assays. BCA assay showed a protein concentration of 1852 µg/ml. DLS analysis revealed a Z-average of 210.28 nm, mean number of 73.19 nm, and PDI of 0.28, confirming uniform nanoparticle distribution. A Zeta potential of -15.48 mV for the exosomes indicated their colloidal stability, and AFM imaging confirmed their size range. In conclusion amniotic fluid exosomes can be extracted using PEG and with potential of this approach in clinical applications.

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