# US 12,599,711 B2 · Device and method of isolating extracellular vesicles > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 12,599,711 B2 - Document ID: US12599711B2 - Status: Granted - Kind: B2 - Application number: 17/893,846 - Filed: 2022-08-23 - Published: 2023-03-02 - Granted: 2026-04-14 - Earliest claimed: 2021-08-24 - Assignees: Forever Labs, Inc. - Applicants: Forever Labs, Inc. - Inventors: Mark Katakowski; Ann Hozeska-Solgot - Web page: https://patentgrep.com/patent/US12599711B2 ## CPC - A61M 1/029: {Separating blood components present in distinct layers in a container, not otherwise provided for (containers for storing blood or blood components A61J1/05; sampling or analysing blood by separating blood components G01N33/491)} - A61M 1/3693: {using separation based on different densities of components, e.g. centrifuging} - B01D 21/262: {by using a centrifuge} - A61M 2202/0415: Plasma - A61M 2202/0427: Platelets; Thrombocytes - A61M: DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY (introducing media into or onto the bodies of animals A61D7/00; means for inserting tampons A61F13/26; devices for administering food or medicines orally A61J; containers for collecting, storing or administering blood or medical fluids A61J1/05); DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY (surgery A61B; chemical aspects of surgical articles A61L); DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR {(Electrotherapy, e.g. producing anaesthesia by the use of alternating or intermittent currents A61N1/36021)} - B01D: SEPARATION (separating solids from solids by wet methods B03B, B03D; by pneumatic jigs or tables B03B; by other dry methods B07; magnetic or electrostatic separation of solid materials from solid materials or fluids, separation by high-voltage electric fields B03C; centrifuges, vortex apparatus B04; presses per se for squeezing-out liquid from liquid-containing material B30B9/02; treatment of water C02F, e.g. softening by ion-exchange C02F1/42; {arrangements of air intake cleaners in gas turbine plants F02C7/05} ; arrangements or mounting of filters in air-conditioning, air-humidification or ventilation F24F13/28) ## Abstract A centrifugal device includes a container having a body with a first end and a second end disposed opposite to the first end. A cap is coupled to the second end of the container, and the cap includes a top surface having at least one port configured to receive or transmit one or more of air or fluid. So configured, the container is moveable between an upright position, in which a first fluid disposed in the container is centrifuged to precipitate at least one extracellular vesicle separate from the first fluid, and an inverted position in which one or more of the first fluid having at least one extracellular vesicle depleted therefrom is removed from the container and a second fluid mixed with the at least one extracellular vesicle removed is withdrawn from the container for injection. ## Claims ### Claim 1 A method of isolating extracellular vesicles, the method comprising: adding an aqueous two-phase solution and a first fluid to a container of a centrifugal device, the container in an upright position; centrifuging the aqueous two-phase solution and the first fluid in the container to form a fraction of extracellular vesicles disposed near a first end of the container separate from the first fluid; moving the container of the centrifugal device from an upright position to an inverted position and removing the first fluid with the fraction of extracellular vesicles depleted therefrom from the container through at least one port of the container; moving the container back to the upright position and adding a second fluid to the container; mixing the second fluid with the fraction of extracellular vesicles disposed near the first end of the container; and moving the container back to the inverted position and removing the second fluid with the fraction of extracellular vesicles through the at least one port of the container for injection. ### Claim 2 (depends on 1) The method of claim 1, further comprising centrifuging a platelet-rich plasma and aseptically removing a portion of platelet-poor plasma from the platelet-rich plasma before adding the aqueous two-phase solution and the first fluid to a container of the centrifugal device. ### Claim 3 (depends on 1) The method of claim 1, wherein adding an aqueous two-phase solution and a first fluid to a container of a centrifugal device comprises adding a concentrated polyethylene glycol-dextran (PEG-DEX) solution and a platelet-poor plasma as the aqueous two-phase solution; and wherein the aqueous two-phase solution and the first fluid are added to the container as via at least one port disposed at a second end of the container of the centrifugal device. ### Claim 4 (depends on 3) The method of claim 3, wherein adding the aqueous two-phase solution and the first fluid to the container via at least one port disposed at the second end of the container of the centrifugal device comprises adding the aqueous two-phase solution and platelet-poor plasma to the container via a first port of the container and allowing air to simultaneously exit through a second port of the container. ### Claim 5 (depends on 1) The method of claim 1, wherein centrifuging the aqueous two-phase solution and the first fluid in the container to form a fraction of extracellular vesicles comprises removing the fraction of extracellular vesicles from platelet-poor plasma and forming the fraction of extracellular vesicles disposed near the first end of the container separate from the platelet-poor plasma in the container. ### Claim 6 (depends on 1) The method of claim 1, wherein adding a second fluid to the container of moving the container back to the upright position and adding a second fluid to the container further comprises adding platelet-rich plasma through a first port disposed on a second end of the container. ### Claim 7 (depends on 1) The method of claim 1, wherein removing the second fluid with the fraction of extracellular vesicles through the at least one port of the container for injection of moving the container back to the inverted position and removing the second fluid with the fraction of extracellular vesicles through the at least one port of the container for injection further comprises one or more of: (1) removing platelet-rich plasma with the fraction of extracellular vesicles through a first port of the container via a syringe for injection; or (2) removing platelet-rich plasma with the fraction of extracellular vesicles through a port of a Y-connector coupled to the at least one port of the container of the centrifugal device via a syringe for injection. ### Claim 8 A method of isolating extracellular vesicles from a fluid, the method comprising: transferring a first volume of plasma from a transfer device to a container of a centrifugal device, maintaining a second volume of plasma in the transfer device; adding an aqueous two-phase solution to the container of the centrifugal device; centrifuging the aqueous two-phase solution and the first volume of plasma in the container to form a fraction of extracellular vesicles and platelet-rich plasma; inverting the container of the centrifugal device and removing remaining aqueous two-phase solution and depleted platelet-poor plasma from the container; moving the container from an inverted position back to an upright position and adding the second volume of plasma from the transfer device into the container of the centrifugal device; resuspending the fraction of extracellular vesicles and platelet-rich plasma with the second volume of plasma in the container by one or more of shaking, inverting, vortexing, and/or centrifuging the container; and inverting the container and removing platelet-rich plasma with extracellular vesicles for placement in an injection device, the volume of platelet-rich plasma and extracellular vesicles equal to the second volume of plasma in the transfer device. ### Claim 9 (depends on 8) The method of claim 8, wherein the centrifugal device is a first centrifugal device, and, before transferring the first volume of plasma from the transfer device to the container of the first centrifugal device, the method further comprises adding whole blood to a container of a second centrifugal device, centrifuging the whole blood, and removing red blood cells from the container of the second centrifugal device. ### Claim 10 (depends on 9) The method of claim 9, further comprising transferring the second volume of plasma from the container of the second centrifugal device to the transfer device after removing the red blood cells from the container of the second centrifugal device. ### Claim 11 (depends on 8) The method of claim 8, wherein adding an aqueous two-phase solution to the container of the centrifugal device comprises adding a concentrated polyethylene glycol-dextran (PEG-DEX) solution as the aqueous two-phase solution; and wherein the aqueous two-phase solution is added to the container via at least one port of the container. ### Claim 12 (depends on 11) The method of claim 11, wherein the aqueous two-phase solution is added to the container via at least one port of the container comprises wherein the aqueous two-phase solution is added to the container via a first port of the container and air is allowed to simultaneously exit through a second port of the container. ### Claim 13 (depends on 8) The method of claim 8, wherein centrifuging the aqueous two-phase solution and the plasma in the container comprises centrifuging the aqueous two-phase solution and the plasma in the container to form the fraction of extracellular vesicles and platelet-rich plasma disposed near a first end of the container. ### Claim 14 (depends on 8) The method of claim 8, wherein removing remaining aqueous two-phase solution and depleted platelet-poor plasma from the container of inverting the container of the centrifugal device and removing remaining aqueous two-phase solution and depleted platelet-poor plasma from the container comprises removing the remaining aqueous two-phase solution and depleted platelet-poor plasma from the container through a first port of the container via a. ### Claim 15 (depends on 8) The method of claim 8, wherein adding the second volume of plasma from the transfer device into the container of the centrifugal device of moving the container from an inverted position back to an upright position and adding the second volume of plasma from the transfer device into the container of the centrifugal device comprises adding the second volume of plasma from the transfer device and through a first port disposed on a second end of the container. ### Claim 16 (depends on 8) The method of claim 8, wherein removing platelet-rich plasma with extracellular vesicles for injection of inverting the container and removing platelet-rich plasma with extracellular vesicles for injection comprises one or more of: (1) removing platelet-rich plasma with extracellular vesicles through a first port of the container via a syringe for injection; or (2) removing platelet-rich plasma with extracellular vesicles through a port of a Y-connector coupled to the at least one port of the container of the centrifugal device via a syringe for injection. ### Claim 17 A method of isolating extracellular vesicles from a fluid, the method comprising: transferring a first volume of plasma from a transfer device to a container of a centrifugal device; adding an aqueous two-phase solution to the container; centrifuging the aqueous two-phase solution and the first volume of plasma in the container to form a fraction of extracellular vesicles and platelet-rich plasma; inverting the container of the centrifugal device and removing remaining aqueous two-phase solution and depleted platelet-poor plasma from the container; moving the container to an upright position and adding a second volume of plasma from the transfer device into the container; resuspending the fraction of extracellular vesicles and platelet-rich plasma with the second volume of plasma in the container; and inverting the container and removing platelet-rich plasma with extracellular vesicles for placement in an injection device.