Methods of isolating exosomes from a biological sample is provided. In one embodiment, the method may include a series of optional centrifugation steps, and comprises exosome precipitation using a PEG-based solution followed by resuspension in a saccharide-based solution such as trehalose. The metho
Methods of isolating exosomes from a biological sample is provided. In one embodiment, the method may include a series of optional centrifugation steps, and comprises exosome precipitation using a PEG-based solution followed by resuspension in a saccharide-based solution such as trehalose. The method advantageously results in essentially pure exosomes that maintain integrity and stability. The exosomes are useful for the in vivo delivery of cargo, including macromolecules such as protein and nucleic acid.
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1. A method of isolating exosomes from a biological sample comprising the steps of: i) optionally exposing the biological sample to a first centrifugation to remove cellular debris greater than about 7-10 microns in size from the sample and obtaining the supernatant following centrifugation;ii) opti
1. A method of isolating exosomes from a biological sample comprising the steps of: i) optionally exposing the biological sample to a first centrifugation to remove cellular debris greater than about 7-10 microns in size from the sample and obtaining the supernatant following centrifugation;ii) optionally subjecting the supernatant from step i) to centrifugation to remove microvesicles and apoptotic bodies therefrom;iii) optionally microfiltering the supernatant from step ii) and collecting the microfiltered supernatant;iv) combining the microfiltered supernatant from step iii) with a polyethylene glycol (PEG) solution to precipitate the exosomes and subjecting the solution to at least one round of centrifugation to obtain an exosome pellet; andv) re-suspending the exosome pellet from step iv) in a trehalose solution and conducting an optional centrifugation step to remove vesicles having a diameter of greater than 140 nm from the solution. 2. The method of claim 1, wherein the PEG solution comprises PEG chain lengths having an average molecular weight of between about 400 to 20,000 daltons and a concentration of about 10-20% PEG. 3. The method of claim 1, wherein the trehalose solution has a concentration of about 10 mM to 1,000 mM. 4. The method of claim 1, wherein step ii) comprises centrifugation at a speed of between 40,000-60,000×g for 30-90 minutes. 5. The method of claim 4, wherein the centrifugation is conducted at a speed of 50,000 for 1 hour. 6. The method of claim 1, wherein step ii) comprises a second centrifugation of the supernatant at a speed of between 12,000-15,000×g for 30-90 minutes, and the resulting supernatant is subjected to a third centrifugation at a speed of between 40,000-60,000×g for 30-90 minutes. 7. The method of claim 6, wherein the second centrifugation is conducted at a speed of 14,000×g for 1 hour and the third centrifugation is conducted at a speed of 50,000 for 1 hour. 8. The method of claim 1, wherein the first centrifugation is conducted at a speed of between 1000 to 4000×g for 10 to 60 minutes, and wherein the first centrifugation is optionally repeated. 9. The method of claim 1, wherein the supernatant of step iii) is microfiltered at least once using a 0.2-10 micron filter, and preferably a 0.22 micron filter. 10. An exosome pellet or physiological solution comprising exosomes essentially free from particles having a diameter less than 20 nm or greater than 140 nm. 11. The pellet or solution of claim 10, wherein the exosomes are loaded with an exogenous cargo. 12. The pellet or solution of claim 11, wherein the exogenous cargo is selected from the group consisting of DNA, siRNA, mRNA, tRNA, aptamers, miRNA, peptides, proteins, ribozymes, carbohydrates, therapeutic compounds, small molecules, and polymers. 13. The pellet or solution of claim 11, wherein the cargo comprises secondary structure or is greater than 20 base pairs in size. 14. The pellet or solution of claim 11, wherein the cargo is mRNA. 15. The pellet or solution of claim 11, wherein the cargo is a protein. 16. A kit for use in a method of isolating exosomes from a biological sample as defined in claim 1, comprising a polyethylene glycol solution to precipitate the exosomes and a trehalose solution to reduce exosome aggregation. 17. A method of in vivo delivery of exogenous cargo to a mammal comprising the step of administering to the mammal an physiologically acceptable exosome solution which is essentially free from particles having a diameter less than 20 nm or greater than 140 nm, wherein the exosomes are loaded with the exogenous cargo. 18. The method of claim 17, wherein the cargo comprises secondary structure or is greater than 20 base pairs in size. 19. The method of claim 18, wherein the cargo is a protein or mRNA. 20. The method of claim 17, wherein the cargo is delivered to muscle, heart, brain, liver, kidney, lung, inguinal white adipose tissue, brown adipose tissue, pancreas or colon.
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