Method and system for point-of-care exosome isolation

Abstract

This specification describes methods and systems for isolating exosomes or extracellular vesicles from blood samples. In one embodiment, the method includes the steps of isolating and fractionating whole blood and shearing platelets and leukocytes using beads, thereby causing the release of exosomes or extracellular vesicles. The resulting exosome-rich platelet-rich plasma (ER-PRP) can be further refined by microfiltration.

Claims

1. A method for isolating exosomes or extracellular vesicles from a blood sample, wherein: (a) A physical barrier configured to separate non-red blood cell components from red blood cells; Multiple particles having a particle density lower than the density of a physical barrier and configured to generate shear stress in non-erythrocyte blood components; and Anticoagulants The steps include: adding a blood sample to an isolation container containing; (b) The isolation vessel is centrifuged to separate the non-erythrocyte blood components from the red blood cells, thereby producing platelet-rich plasma (PRP); (c) The step of stirring the isolation vessel to generate shear stress in one or more non-erythrocyte blood components, causing exosomes or extracellular vesicles to be released from the PRP, thereby generating exosome-rich PRP (ER-PRP). The above method, including.

2. The method according to claim 1, further comprising the steps of filtering ER-PRP to remove platelets and generating exosome-rich plasma (ERP).

3. The method according to claim 2, wherein the filtering step includes using a filter having pores ranging in size from about 0.1 μm to about 5 μm.

4. The method according to claim 1, further comprising the step of filtering ER-PRP to remove leukocytes.

5. The method according to claim 4, wherein the filtering step includes using a filter having pores ranging in size from about 0.1 μm to about 20 μm.

6. The method according to claim 1, wherein the isolation container includes a glass tube or a plastic tube.

7. The method according to claim 1, wherein the physical barrier comprises a valve, a polymer disc or buoy, or a thixotropy gel.

8. The physical barrier is approximately 1.05 g / cm³. ３ Approximately 1.10 g / cm³ ３ The method according to claim 1, comprising densities in the range up to [a certain value].

9. The method according to claim 1, wherein the plurality of particles comprises a plurality of beads composed of polypropylene, zirconium, or a combination thereof.

10. Multiple particles, approximately 0.95 g / cm³ ３ Approximately 1.05 g / cm³ ３ The method according to claim 1, comprising densities in the range up to [a certain value].

11. The method according to claim 1, wherein the anticoagulant comprises the anticoagulant dextrose citrate solution A (ACDA), calcium citrate, or a combination thereof.

12. The method according to claim 1, wherein the step of centrifuging the isolation vessel includes centrifugation at approximately 100 to 7000 × g for approximately 5 to 60 minutes to separate non-erythrocyte blood components from erythrocytes.

13. The method according to claim 12, wherein the step of centrifuging the isolation vessel includes centrifugation at about 1000 to 1500 × g for about 10 to 20 minutes to separate non-erythrocyte blood components from erythrocytes.

14. The method according to claim 1, wherein the step of stirring the isolation vessel includes stirring at an angle of about 30° to about 60°, which generates shear stress in one or more non-erythrocyte blood components.

15. The method according to claim 14, wherein the step of stirring the isolation vessel includes stirring at an angle of about 45°, which generates shear stress in one or more non-erythrocyte blood components.

16. An exosome or extracellular vesicle isolated using the method described in claim 1.

17. A method for isolating exosomes or extracellular vesicles from platelet-rich plasma (PRP) samples, comprising: (a) Multiple particles configured to generate shear stress in non-red blood cell components; and Anticoagulants The steps include: adding the PRP sample to an isolation container containing; (b) The step of stirring the isolation vessel to generate shear stress in one or more non-erythrocyte blood components, causing exosomes or extracellular vesicles to be released from the PRP sample, thereby generating exosome-rich PRP (ER-PRP). The above method, including.

18. The method according to claim 17, further comprising the steps of filtering ER-PRP to remove platelets and generating exosome-rich plasma (ERP).

19. The method according to claim 18, wherein the filtration step includes using a filter having pores ranging in size from about 0.1 μm to about 5 μm.

20. The method according to claim 17, further comprising the step of filtering ER-PRP to remove leukocytes.

21. The method according to claim 20, wherein the filtering step includes using a filter having pores ranging in size from about 0.1 μm to about 20 μm.

22. An exosome or extracellular vesicle isolated using the method described in claim 17.

23. A system for isolating exosomes or extracellular vesicles from blood samples: A physical barrier configured to separate non-red blood cell components from red blood cells in a blood sample; Multiple particles having a particle density lower than the density of a physical barrier and configured to generate shear stress in non-erythrocyte blood components; and Isolation container containing anticoagulant The above system, including.

24. The system according to claim 23, further comprising a centrifuge for separating non-red blood cell components from red blood cells.

25. The system according to claim 23, further comprising an agitator that generates shear stress in non-red blood cell components.

26. An exosome or extracellular vesicle isolated using the system described in claim 23.

27. Use of the system according to claim 23 for the isolation of exosomes or extracellular vesicles.

28. A kit for isolating exosomes or extracellular vesicles from blood samples: A physical barrier configured to separate non-red blood cell components from red blood cells in a blood sample; Multiple particles having a particle density lower than the density of a physical barrier and configured to generate shear stress in non-erythrocyte blood components; and Anticoagulants An isolation container containing; If necessary, one or more filters, a subcutaneous needle, or a syringe; Buffers and containers as needed; and If necessary, one or more packages or instruction manuals Includes the above kit.

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