METHODS AND SYSTEMS FOR POINT OF CARE EXOSOME ISOLATION
Described herein are methods and systems for isolating exosomes or extracellular vesicles from a blood sample. In one embodiment, the method comprises isolating and fractionating whole blood and using beads to shear platelets and leukocytes causing the release of exosomes or extracellular vesicles. The resulting exosome-rich platelet rich plasma (ER-PRP) can be further refined by microfiltration.
1 . A method for isolating exosomes or extracellular vesicles from a blood sample, the method comprising:
(a) adding a blood sample to an isolation container comprising:
a physical barrier configured to separate non-red blood cell blood components from red blood cells;
a plurality of particles having a particle density that is less than a density of the physical barrier, the plurality of particles being configured to generate shear stress on non-red blood cell blood components; and
an anticoagulant;
(b) centrifuging the isolation container to separate non-red blood cell blood components from red blood cells, thereby generating a platelet-rich plasma (PRP); and
(c) agitating the isolation container to generate shear stress on one or more non-red blood cell blood components and release exosomes or extracellular vesicles from the PRP, thereby generating an exosome-rich PRP (ER-PRP).
2 . The method of claim 1 , further comprising filtering the ER-PRP to remove platelets and generate an exosome-rich plasma (ERP).
3 . The method of claim 2 , wherein filtering comprises the use of a filter having pores ranging from about 0.1 μm to about 5 μm in size.
4 . The method of claim 1 , further comprising filtering the ER-PRP to remove white blood cells.
5 . The method of claim 4 , wherein filtering comprises the use of a filter having pores ranging from about 0.1 μm to about 20 μm in size.
6 . The method of claim 1 , wherein the isolation container comprises a glass tube or a plastic tube.
7 . The method of claim 1 , wherein the physical barrier comprises a valve, a polymer disc or buoy, or a thixotropic gel.
8 . The method of claim 1 , wherein the physical barrier comprises a density ranging from about 1.05 g/cm 3 to about 1.10 g/cm 3 .
9 . The method of claim 1 , wherein the plurality of particles comprises a plurality of beads comprised of polypropylene, zirconium, or a combination thereof.
10 . The method of claim 1 , wherein the plurality of particles comprises a density ranging from about 0.95 g/cm 3 to about 1.05 g/cm 3 .
11 . The method of claim 1 , wherein the anticoagulant comprises anticoagulant citrate dextrose solution A (ACDA), calcium citrate, or a combination thereof.
12 . The method of claim 1 , wherein centrifuging the isolation container comprises centrifugation at about 100-7000×g for about 5-60 minutes to separate non-red blood cell blood components from red blood cells.
13 . The method of claim 12 , wherein centrifuging the isolation container comprises centrifugation at about 1000-1500×g for about 10-20 minutes to separate non-red blood cell blood components from red blood cells.
14 . The method of claim 1 , wherein agitating the isolation container comprises agitation at an angle of about 30° to about 60° to generate shear stress on the one or more non-red blood cell blood components.
15 . The method of claim 14 , wherein agitating the isolation container comprises agitation at an angle of about 45° to generate shear stress on the one or more non-red blood cell blood components.
16 . (canceled)
17 . A method for isolating exosomes or extracellular vesicles from a platelet-rich plasma (PRP) sample, the method comprising:
(a) adding a PRP sample to an isolation container comprising:
a plurality of particles configured to generate shear stress on non-red blood cell blood components; and
an anticoagulant; and
(b) agitating the isolation container to generate shear stress on one or more non-red blood cell blood components and release exosomes or extracellular vesicles from the PRP sample, thereby generating an exosome-rich PRP (ER-PRP).
18 . The method of claim 17 , further comprising filtering the ER-PRP to remove platelets and generate an exosome-rich plasma (ERP).
19 . The method of claim 18 , wherein filtering comprises the use of a filter having pores ranging from about 0.1 μm to about 5 μm in size.
20 - 22 . (canceled)
23 . A system for isolating exosomes or extracellular vesicles from a blood sample, the system comprising:
an isolation container comprising a physical barrier configured to separate non-red blood cell blood components from red blood cells in a blood sample;
a plurality of particles having a particle density that is less than a density of the physical barrier, the plurality of particles being configured to generate shear stress on non-red blood cell blood components; and
an anticoagulant.
24 . (canceled)
25 . The system of claim 23 , further comprising an agitator to generate shear stress on non-red blood cell blood components.
26 - 28 . (canceled)