Devices and methods for isolating extracellular vesicles

Abstract

The centrifugation device includes a container having a body with a first end and a second end disposed opposite the first end. A cap is coupled to the second end of the container, the cap including a top surface having at least one port configured to receive or deliver one or more of air or a fluid. As so configured, the container is movable between an upright position in which a first fluid disposed within the container is centrifuged to separate and sediment at least one extracellular vesicle from the first fluid, and an inverted position in which one or more of the first fluids depleted of at least one extracellular vesicle are removed from the container and a second fluid mixed with the removed at least one extracellular vesicle is withdrawn from the container for injection.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is a PCT patent application that claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 236,643, filed August 24, 2021, the entire disclosure of which is incorporated herein by reference.

[0002] The present disclosure relates to cell isolation methods and devices, in particular devices and methods for rapidly isolating extracellular vesicles. [Background technology]

[0003] Extracellular vesicles (such as exosomes) are released by cells to efficiently transfer molecular cargo to other cells. The therapeutic effect of extracellular vesicles arises from their cargo (such as miRNA, other non-coding RNA, and proteins) and surface molecules. In addition, extracellular vesicles can be functional elements of the extracellular matrix, which is involved in the organization, cell regulation, and determining the physical properties of connective tissue and bone.

[0004] Injections of platelet-rich plasma (PRP) and bone marrow concentrate (BMC) have been used in clinical applications to promote healing, stimulate tissue regrowth, angiogenesis, ameliorate inflammation, and regenerate intact endogenous tissues. Extracellular vesicles are found in all biological fluids, including blood and bone marrow, and have been demonstrated to confer many of the effects of the cells in which they are produced. For example, extracellular vesicles from umbilical cord or bone marrow MSCs have been demonstrated to stimulate human skin regeneration or improve engraftment of transplanted fat grafts. It has been demonstrated that extracellular vesicles from bone mesenchymal stem cells exert similar chondroprotective and anti-inflammatory functions and protect mice from the development of osteoarthritis, suggesting that extracellular vesicles recapitulate the primary therapeutic effects of MSCs. Indeed, recent scientific and clinical evidence suggests that MSCs may not exert their therapeutic functions primarily intracellularly, but rather in a paracrine manner, and extracellular vesicles (such as exosomes and microvesicles) have been identified as the primary mediators of these paracrine effects. Extracellular vesicles isolated from fluids, such as biological fluids, have also been used for diagnostic purposes, as their contents may reflect injury, infection, cancer, immune dysfunction, or other pathologies.

[0005] Due to their low density and small size, extracellular vesicles are generally isolated by filtration, ultracentrifugation, immunoaffinity, microfluidics, or polymer precipitation. Current devices used to partition blood or bone marrow (into fractions such as red blood cells (RBC), platelet poor plasma (PPP), and bone marrow concentrate (BMC) or PRP) use low-speed centrifugation, which does not effectively isolate extracellular vesicles or concentrate them into one fraction. Therefore, devices that concentrate whole blood or bone marrow do not concentrate biological agents such as extracellular vesicles, which may produce a significant portion of the therapeutic effect.

[0006] Furthermore, platelet-rich plasma (PRP) is a preparation for therapeutic purposes that is increasingly being accepted for various musculoskeletal disorders, wound healing, cosmetics, and regenerative medicine, at least for its theoretical potential to repair tissue. PRP therapy uses injections of the patient's own platelets. As described, platelets are concentrated by centrifugation from a peripheral blood draw, resulting in three fractions: RBC, PPP, and PRP.

[0007] In a standard PRP procedure, a PRP fraction is injected, leaving behind biomolecules such as extracellular vesicles that are discarded along with the PRP. Extracellular vesicles mediate a range of cellular functions, including transport of materials and intercellular communication. Therefore, increasing the concentration of extracellular vesicles in PRP can improve the therapeutic effect of PRP. Summary of the Invention

[0008] According to one exemplary aspect of the present disclosure, the centrifugation device includes a container having a body with a first end and a second end disposed opposite the first end. A cap is coupled to the second end of the container, the cap including a top surface having at least one port configured to receive or deliver one or more of air or a fluid. As so configured, the container is movable between an upright position in which a first fluid disposed in the container is centrifuged to separate and precipitate at least one extracellular vesicle from the first fluid, and an inverted position in which one or more of the first fluids depleted of at least one extracellular vesicle are removed from the container, and a second fluid mixed with the removed at least one extracellular vesicle is withdrawn from the container for injection.

[0009] According to another aspect of the present disclosure, a method of isolating extracellular vesicles from a fluid includes adding an aqueous two-phase solution and a first fluid to a container of a centrifugal device, the container being in an upright position. The method also includes centrifuging the aqueous two-phase solution and the first fluid in the container to separate and form a fraction of extracellular vesicles located near a first end of the container from the first fluid. The method still further includes moving the container of the centrifugal device from the upright position to an inverted position and removing the first fluid depleted of extracellular vesicles from the container through at least one port of the container. The method still further includes returning the container to the upright position and adding a second fluid to the container. The method still further includes mixing the second fluid with the fraction of extracellular vesicles located near the first end of the container. The method also includes returning the container to an inverted position and removing the second fluid including the fraction of extracellular vesicles through at least one port of the container for injection.

[0010] According to another aspect of the present disclosure, another centrifugal device includes a container having a body with a first end and a second end disposed opposite the first end. A cap is coupled to the second end of the container, the cap including a top surface having a port configured to receive or deliver one or more of air or a fluid. In addition, a Y-shaped connector is coupled to the port of the cap, the first port for receiving or delivering air and the second port for receiving or delivering a fluid. As so configured, the container is movable between an upright position, in which a first fluid and an aqueous two-phase solution disposed in the container are centrifuged to precipitate at least one extracellular vesicle, and an inverted position. The inverted position is a position in which one or more of the first fluids depleted of at least one extracellular vesicle are removed from the container through the second port, and a second fluid mixed with at least one extracellular vesicle is removed from the container through the second port for injection.

[0011] According to another aspect of the present disclosure, a method of isolating extracellular vesicles from a fluid includes 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, and adding an aqueous two-phase solution to the container of the centrifugal device. The method also includes centrifuging the aqueous two-phase solution and plasma in the container to form a fraction of extracellular vesicles and platelet-rich plasma, inverting the container of the centrifugal device, and removing the remaining aqueous two-phase solution and depleted platelet-poor plasma from the container. The method still further includes returning the container from the inverted position to an upright position and adding the second volume of plasma from the transfer device to the container of the centrifugal device. The method also includes resuspending the extracellular vesicles and the platelet-rich plasma fraction with a second volume of plasma in the container by one or more of shaking, inverting, vortexing, and / or centrifuging the container, and finally, inverting the container and removing the platelet-rich plasma containing the extracellular vesicles for placement in an injection device, wherein the volume of platelet-rich plasma and extracellular vesicles is equal to the second volume of plasma in the transfer device.

[0012] Further according to any one or more of the exemplary embodiments, the device for isolating extracellular vesicles or any of the methods of the present disclosure may include any one or more of the following preferred configurations.

[0013] In some aspects, the at least one port may include a first port configured to receive or deliver air and having a filter, and a second port configured to receive or deliver a fluid.

[0014] In another aspect, one or both of the first and second ports may include one or more of a removable cap and a luer lock connection configured to be coupled to a syringe.

[0015] In yet another aspect, the at least one port can include a single port, and the device can further include a Y-connector that is one of fixedly or removably coupled to the single port. The Y-connector can include a first port configured to receive air and include a filter, and a second port having a syringe fitting configured to be coupled to a syringe, such that when the container is in an inverted position, air flows into the first port and extracellular vesicle-depleted fluid flows out of the second port.

[0016] In another embodiment, the container may be cylindrical, such as a tube, and the first end may include a tapered portion and the second end may include an opening.

[0017] In other aspects, the device may include at least one tube disposed within the at least one port and extending into an interior region of the container, the at least one tube may be configured to aid in the addition or removal of fluid to or from the container.

[0018] In still other examples, one or more of: (1) the container may be configured to receive platelet-poor plasma and an aqueous two-phase solution, where the aqueous two-phase solution may be a concentrated aqueous two-phase solution including a concentrated polyethylene glycol-dextran (PEG-DEX) solution; (2) the first fluid may be platelet-poor plasma; and (3) the second fluid may be platelet-rich plasma.

[0019] In still other examples, the first fluid may be a biological fluid or one of the other possible reconstitution fluids. Additionally, the second fluid may be a biological fluid, a sterile solution such as saline, or one of the other possible reconstitution fluids.

[0020] In still other aspects, the centrifugal device may be a first centrifugal device, and prior to transferring the first volume of plasma from the transfer device to the container of the first centrifugal device, the method may further include 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.

[0021] In another example, the method may further include transferring plasma from the container of the second centrifugal device to a transfer device after removing red blood cells from the container of the second centrifugal device.

[0022] Additional optional aspects and features are disclosed, which may be arranged in any functionally suitable manner, alone or in any functionally workable combination, consistent with the teachings of the disclosure. Other aspects and advantages will become apparent upon consideration of the following detailed description. [Brief description of the drawings]

[0023] The present disclosure will be more fully understood from the following description taken in conjunction with the accompanying drawings, in which: Some of the drawings have been simplified by the omission of selected elements in order to more clearly show other elements. The omission of such elements in some drawings does not necessarily indicate the presence or absence of the particular elements in any of the illustrative embodiments, unless expressly stated in the corresponding written description. Additionally, the drawings are not necessarily drawn to scale.

[0024] [Figure 1] FIG. 1 is a front perspective view of a centrifuge device according to one aspect of the present disclosure. [Diagram 2] 2 is the device of FIG. 1 with a tube attached. [Figure 3A] 1 illustrates steps of a method for isolating extracellular vesicles according to another aspect of the present disclosure. [Figure 3B] 1 illustrates steps of a method for isolating extracellular vesicles according to another aspect of the present disclosure. [Figure 3C] 1 illustrates steps of a method for isolating extracellular vesicles according to another aspect of the present disclosure. [Figure 3D] 1 illustrates steps of a method for isolating extracellular vesicles according to another aspect of the present disclosure. [Figure 3E] 1 illustrates steps of a method for isolating extracellular vesicles according to another aspect of the present disclosure. [Figure 3F] 1 illustrates steps of a method for isolating extracellular vesicles according to another aspect of the present disclosure. [Figure 3G] 1 illustrates steps of a method for isolating extracellular vesicles according to another aspect of the present disclosure. [Figure 4] FIG. 13 is a front perspective view of another centrifuge device according to another aspect of the present disclosure. [Diagram 5] FIG. 5 is an inverted view of the device of FIG. [Figure 6A] 1 shows steps of another method for isolating extracellular vesicles according to another aspect of the present disclosure. [Figure 6B] 1 shows steps of another method for isolating extracellular vesicles according to another aspect of the present disclosure. [Figure 6C] 1 shows steps of another method for isolating extracellular vesicles according to another aspect of the present disclosure. [Figure 6D] 1 shows steps of another method for isolating extracellular vesicles according to another aspect of the present disclosure. [Figure 6E] 1 shows steps of another method for isolating extracellular vesicles according to another aspect of the present disclosure. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] Generally, centrifugal devices, systems, and methods are disclosed for rapidly isolating a fraction of extracellular vesicles from plasma, which can be used in the preparation of platelet-rich plasma.

[0026] Referring now to FIG. 1, a centrifuge device 10 according to one aspect of the present disclosure is shown. The centrifuge device 10 includes a container 12, which may take the form of a tube and / or may be cylindrical. The container 12 includes a body 14 having a first end 16 and a second end 18 disposed opposite the first end 16. A cap 20 is removably coupled to the second end 18 of the container 12 and includes a top surface 22 having at least one port 24 configured to receive or deliver one or more of a fluid, such as a biological fluid, a fluid other than a biological fluid, or air, as will be further described below. The cap 20 may be circular, partially circular, or have any other geometry or shape that aids in the withdrawal of fluid, as will be further described below, and still fall within the scope of the present disclosure.

[0027] As so configured, and described further below with respect to, for example, Figures 3A-3G, the container 12 is movable between an upright position, as shown in Figure 1, and an inverted position, as shown in Figure 3D. The upright position is a position in which a fluid, such as a biological fluid containing platelet-poor plasma, disposed within the container 12 is centrifuged to separate and sediment the at least one extracellular vesicle from the fluid disposed within the container 12. The inverted position is a position in which one or more of the fluids depleted of at least one extracellular vesicle are removed from the container 12, and the platelet-rich plasma mixed with the at least one extracellular vesicle removed from the platelet-poor plasma is withdrawn from the container 12 through at least one port 24 in the cap 20 for injection.

[0028] 1, in one example, the at least one port 24 of the cap 20 includes a first port 26 configured to receive or deliver air and has a filter 27. The at least one port 24 further includes a second port 28 configured to receive or deliver a fluid, such as a biological fluid, as described further below. In addition, each of the first and second ports 26, 28 may include a removable cap 30, and the second port 28 configured to receive or deliver a fluid may include a luer lock connection 32 configured to be coupled to a syringe.

[0029] As further shown in FIG. 1, the first end 16 of the container 12 may include a tapered portion 34, and the second end 18 may be an open end, such as when the cap 20 is removed from the second end 18.

[0030] 2, the centrifuge device 10 may further include at least one tube 36 disposed within the second port 28 and extending to an interior region 38 of the container 12. The at least one tube 36 is configured to aid in the addition or removal of a fluid, such as a biological fluid, to or from the container 12.

[0031] 3A-3G, steps of a method for isolating extracellular vesicles from a fluid are shown, for example, using one or more of the centrifugal devices 10 of FIGS. 1 and 2. Referring first to FIG. 3A, the method includes first adding an aqueous two-phase solution to the container 12 of the centrifugal device 10 through a first port 26 disposed at the second end 18 of the body 12 of the container 12, such as via a syringe 40. The container 12 is shown in an upright position in FIGS. 3A and 3B. In this example, the aqueous two-phase solution is a concentrated aqueous two-phase solution that includes a concentrated polyethylene glycol-dextran (PEG-DEX) solution. The aqueous two-phase solution is required for the purification of biological materials. Additionally, the aqueous two-phase solution may be referred to as Forever Labs, Inc.'s SuperShot™ solution. It will be understood that the aqueous two-phase solution may include other aqueous polymers and still fall within the scope of the present disclosure. Additionally, and in some examples, the container 12 may be made from one of polypropylene and / or polystyrene and may be coated with a hydrophobic layer. During use, containers 12 made from polystyrene have been found to have the lowest residual aqueous biphasic solution. Since minimizing residual aqueous biphasic solution, such as PEG-DEX, is desirable when preparing platelet-rich plasma containing extracellular vesicles for injection, it may be optimal to have a container 12, e.g., a centrifuge tube, made from polystyrene.

[0032] In addition, and with reference to FIG. 3B, the method further includes adding a fluid, such as platelet-poor plasma, to the container 12 of the centrifugal device 10, for example, through a first port 26 also disposed at the second end 18 of the container 12, and by a syringe 40. In one example, the platelet-poor plasma added to the container 12 is created after first centrifuging platelet-rich plasma. Specifically, after centrifugation of the platelet-rich plasma, a portion of the platelet-poor plasma is aseptically removed and then combined with the aqueous two-phase solution in the container 12 of the centrifugal device 10. The platelet-rich plasma may be centrifuged using the centrifugal device 10 of the present disclosure or any other centrifugal device or system 11 commonly known to one of skill in the art (e.g., see FIG. 3B).

[0033] Additionally, in another example, while a fluid, such as an aqueous two-phase solution and platelet poor plasma as described above, is being added to the container 12 via the first port 26, air can simultaneously exit through a second port 28 of the cap 20 disposed at the second end 18 of the body 14 of the container 12. Addition of an aqueous two-phase solution, such as a PEG-DEX solution, to the plasma allows for precipitation of small amounts of low density molecules, including extracellular vesicles, under centrifugation.

[0034] After the platelet poor plasma is added to the container 12 having the two-phase aqueous solution AS disposed therein, the method further includes mixing and centrifuging the aqueous two-phase solution and the platelet poor plasma in the container 12, for example, using the centrifugal device 10 and centrifugation system 11 of FIG. 3B. In so doing, a fraction of low density biomolecules including extracellular vesicles (FEV) is formed and concentrated near or at one or more of the first ends 16 of the container 12, as shown in FIG. 3C. The fraction of extracellular vesicles is separated from and / or depleted or removed from the platelet poor plasma (PPP), as also shown in FIG. 3C. Additionally, in another example, centrifuging the aqueous two-phase solution and platelet-poor plasma in container 12 to separate and form a fraction of extracellular vesicles located near the first end 18 of container 12 from the platelet-poor plasma may include removing a fraction of extracellular vesicles from the platelet-poor plasma and separating and forming a fraction of extracellular vesicles located near the first end 18 of container 12 from the platelet-poor plasma in container 12, as also shown in FIG. 3C.

[0035] Now referring to FIG. 3D, the method further includes, for example, moving the container 12 of the centrifugal device 10 from the upright position shown in FIG. 3A to the inverted position shown in FIG. 3D. Once in the inverted position, the method further includes removing the platelet-poor plasma depleted of the extracellular vesicles fraction and the aqueous two-phase solution from the container 12 through at least one port 24 of the cap 20, such as the first port 26 shown in FIG. 3D. In this example, the platelet-poor plasma depleted of the extracellular vesicles fraction is removed from the container 12 via a syringe 40, and the second port 28 receives air flowing into the container 12. However, the platelet-poor plasma depleted of the extracellular vesicles fraction may be removed from the container 12 through the first port 26 using another device different from the syringe 40, such as the tube 36 in FIG. 2, and still fall within the scope of the present disclosure.

[0036] 3E, the method further includes returning the container 12 to an upright position as shown in FIG. 3E, and adding platelet-rich plasma (PRP) to the container 12 having the fraction of extracellular vesicles. The method further includes mixing the platelet-rich plasma with the fraction of extracellular vesicles disposed near the first end 16 of the container 12 and resuspending the extracellular vesicles in the platelet-rich plasma. In one example, returning the container 12 of the centrifugal device 10 to an upright position and adding the platelet-rich plasma to the container 12 includes adding the platelet-rich plasma through a first port 26 of a cap 20 disposed at a second end 18 of the container 12, while the fraction of extracellular vesicles removed from the platelet-poor plasma is disposed near the first end 16 of the container 12.

[0037] 3F, the method still further includes returning the container 12 of the centrifugal device 10 to an inverted position and removing the platelet-rich plasma (PRP) including the fraction of extracellular vesicles (EVF) mixed therein through at least one port 24, such as the first port 26 of the cap 20, via a syringe 40 for injection. In one example, the platelet-rich plasma including one or more of the fractions of extracellular vesicles (EVF) mixed or disposed therein is referred to as SuperShot™ PRP.

[0038] 3G, a syringe 40 is shown having platelet-rich plasma, including platelet-poor plasma and a fraction of extracellular vesicles (EVFs) centrifuged from the aqueous two-phase solution mixture, disposed therein and ready for injection, as described above. Although a syringe 40 is shown, it will be understood that another injection or drug administration device or system may alternatively and / or additionally be used and still fall within the scope of the present disclosure.

[0039] 4 and 5, another centrifuge device 110 according to another embodiment of the present disclosure is shown. The centrifuge device 110 includes many of the same features as the centrifuge device 10 of FIGS. 1-3G, but instead of the cap 20 having the first port 26 and the second port 28 of the centrifuge device 10, the centrifuge device 110 includes a cap having only a single port with a Y-shaped connector coupled thereto, as described further below. Parts of the centrifuge device 110 that are the same as parts of the centrifuge device 10 are numbered 100 and above and will not be described again here for brevity. As will be understood at least in light of the following description, the centrifuge device 110 can also be operated according to the method described above and illustrated in FIGS. 3A-3G. In other words, each of the centrifuge devices 10, 110 of the present disclosure can be used to employ the method of isolating extracellular vesicles described above. It will be understood that the centrifugal device 110 may be substituted for, for example, the centrifugal device 10 shown in FIGS. 3A-3G and may be used in a manner similarly effective to that of the centrifugal device 10.

[0040] 4, the centrifuge device 110 includes a container 112, which may take the form of a tube and / or may be cylindrical. The container 112 includes a body 114 having a first end 116 and a second end 118 disposed opposite the first end 116. A cap 120 is removably coupled to the second end 118 of the container 112 and includes a top surface 122 having a port 124 configured to receive or deliver one or more of a fluid, such as a biological fluid, or air, as described further below.

[0041] Additionally, the Y-connector 125 is removably coupled to the port 124 of the cap 120 and has a first portion 129, such as a first port for receiving or delivering air and having a filter 129A (FIG. 5A), and a second portion 131, such as a second port for receiving or delivering a fluid, such as a biological fluid.

[0042] As so configured, and similar to the centrifugal device 10 and method shown in Figures 3A-3G, for example, the container 112 is movable between an upright position as shown in Figure 4 and an inverted position as shown in Figure 5. The upright position is a position in which a fluid, such as a biological fluid containing platelet poor plasma, disposed in the container 112 is centrifuged to separate and precipitate at least one extracellular vesicle from the fluid disposed in the container 112. The inverted position is a position in which a fluid depleted of at least one extracellular vesicle is removed from the container 112 through the second port 131 of the Y-shaped connector 125. The inverted position is a position in which, alternatively and / or additionally, the platelet rich plasma mixed with at least one extracellular vesicle removed from the platelet poor plasma is withdrawn from the container 112 through the port 124 of the cap 120 and the second port 131 of the Y-shaped connector 125 for injection. Additionally, the first portion 129, such as the first port, may receive air when the container 112 is in an inverted position, as shown in FIG.

[0043] Additionally, each of the first and second ports 129, 131 may include a removable cap 133 (FIG. 4), and the second port 131, configured to receive or deliver a fluid, may include a syringe fitting 135 (FIG. 4), such as a Luer lock connection, configured to be coupled to a syringe.

[0044] 4 and 5, the first end 116 of the container 112 may include a tapered portion 134, and the second end 118 may be an open end, such as when the cap 120 is removed from the second end 118. Although the first end 116 of the container 112 is shown as having a tapered portion 134 in Figures 4 and 5, the first end 116 may alternatively include a shape different from a tapered portion, such as a flat portion, and still fall within the scope of the present disclosure.

[0045] 6A-6E, another method of isolating extracellular vesicles from a fluid is shown, for example, using one or more of the centrifugal devices 10 of FIGS. 1 and 2. Referring first to FIG. 6A, the method includes transferring a first volume of plasma from a transfer device, such as a syringe 40, to a container 12 of the centrifugal device 10, leaving a second volume of plasma in the transfer device 40. In this example, and as shown in FIG. 6A, the container 12 of the centrifugal device 10 is in an upright position. The method further includes maintaining the second volume of plasma in the transfer device 40, which is later used for injection, as further described below.

[0046] More generally, and in one example, the second volume of plasma maintained within the transfer device 40 is equal to the desired volume of the final injection. Thus, if a user, such as a physician, desires to inject 5 mL of platelet-rich plasma and has 20 mL of plasma, the physician adds 15 mL of plasma to the container 12 of the centrifugal device 10 and retains 5 mL of plasma in the transfer device 40, such as the transfer syringe 40. Thus, in this example, the first volume of plasma transferred from the transfer device 40 to the container 12 is 15 mL of plasma, and the second volume of plasma maintained within the transfer device 40 is 5 mL. It will be understood that various other combinations of first and second volumes of plasma may alternatively be desired and thus selected by the user and / or physician, for example, allowing flexibility and ease of injection volume, and still falling within the scope of the present disclosure. In addition, it will be understood that various other types of transfer devices different from the transfer device 40, such as the syringe 40, may alternatively and / or additionally be used, and also still falling within the scope of the present disclosure.

[0047] Additionally, in one example, prior to transferring the first volume of plasma from the transfer device 40 to the container 12 of the centrifugal device 10, the method may also include adding whole blood to a container of another centrifugal device, such as a second centrifugal device (not shown). In this example, the centrifugal device 10 of FIG. 6A is the first centrifugal device 10. The method then also includes centrifuging the whole blood and removing red blood cells from the container of the second centrifugal device. The plasma from the container of the second centrifugal device is then transferred to a transfer device 40, such as a syringe 40, after removing the red blood cells.

[0048] Still referring to FIG. 6A, the method then includes adding the aqueous two-phase solution to the container 12 of the centrifugal device 10, such as through at least one port 26 of the container 12. In this manner, the aqueous two-phase solution is combined with the plasma transferred from the transfer device 40. In some examples, while the aqueous two-phase solution is being added to the container 12, air can simultaneously exit the container 14, such as through a second port 28. In this example, the aqueous two-phase solution is also a concentrated aqueous two-phase solution that includes a concentrated polyethylene glycol-dextran (PEG-DEX) solution. As mentioned above, the aqueous two-phase solution is necessary for the purification of biological materials and can be referred to as Forever Labs, Inc.'s SuperShot™ solution. It will be understood that the aqueous two-phase solution may include other aqueous polymers and still fall within the scope of the present disclosure.

[0049] The method further includes centrifuging the aqueous two-phase solution and plasma in the container 12 to form a fraction of extracellular vesicles and platelet-rich plasma. In one example, and as shown in FIG. 6B, the fraction of extracellular vesicles and platelet-rich plasma is separated from the platelet-poor plasma and remaining aqueous two-phase solution and disposed near the first end 16 of the container 12 of the centrifugal device 10.

[0050] 6B , the method further includes inverting the container 12 of the centrifugal device 10 and removing the remaining aqueous two-phase solution and the depleted platelet poor plasma from the container 12. In one example, inverting the container 12 of the centrifugal device 10 and removing the remaining aqueous two-phase solution and the depleted platelet poor plasma from the container 12 includes removing the remaining aqueous two-phase solution and the depleted platelet poor plasma from the container 12 through the first port 26 via a transfer device 40, such as a syringe 40, while air flows into the second port 28 of the container 12.

[0051] 6C, the method still further includes returning the container 12 to an upright position from an inverted position (FIG. 6B) and adding the second volume of plasma maintained in the transfer device 40, such as a syringe 40, to the container 12 of the centrifugal device 10. In one example, returning the container 12 to an upright position from an inverted position and adding the second volume of plasma maintained in the transfer device 40 to the container 12 of the centrifugal device 10 includes adding the second volume of plasma from the transfer device 40 through a first port 26 disposed at a second end 18 of the container 12 while the fraction of extracellular vesicles and platelet-rich plasma is disposed at or near the first end 16 of the container 12, as shown in FIG. 6C. The method still further includes resuspending the fraction of extracellular vesicles and platelet-rich plasma with the second volume of plasma in the container 12, for example, by one or more of shaking, inverting, vortexing, and / or centrifuging the container 12.

[0052] Now referring to FIG. 6D, the method also includes inverting the container 12 and removing the platelet-rich plasma containing the extracellular vesicles for placement in an injection device 42, such as an injection syringe 42 (see also FIG. 6E). The volume of the platelet-rich plasma and the extracellular vesicles is equal to the second volume of plasma in the transfer device 40. In one example, inverting the container 12 and removing the platelet-rich plasma containing the extracellular vesicles for placement in the injection device 42 includes removing the platelet-rich plasma containing the extracellular vesicles through a first port 26 of the container 12 via the injection syringe 42. In another example, inverting the container 12 and removing the platelet-rich plasma containing the extracellular vesicles for placement in the injection device 42 includes removing the platelet-rich plasma containing the extracellular vesicles through a port of a Y-shaped connector 125 (see, e.g., FIGS. 4 and 5) coupled to the first port 26 of the container 12 of the centrifugal device 10 via the injection syringe 42.

[0053] In view of at least the foregoing, it will be appreciated that the disclosed centrifugal devices 10, 110 and methods for isolating extracellular vesicles include several advantages. For example, the methods and devices 10, 110 reduce the amount of extracellular vesicles remaining after, for example, an injection of platelet-rich plasma. In addition, the disclosed methods and devices 10, 110 also increase the overall concentration of extracellular vesicles within the platelet-rich plasma, improving the overall therapeutic effect of the platelet-rich plasma.

[0054] The following additional considerations apply to the above discussion: Throughout this specification, multiple instances may implement components, operations, or structures described as a single instance. Although individual operations of one or more methods are illustrated and described as separate operations, one or more of the individual operations may be performed simultaneously, and the operations need not be performed in the order illustrated. Structures and functions presented as separate components in an example configuration may be implemented as a combined structure or component. Similarly, structures and functions presented as a single component may be implemented as separate components. These and other variations, modifications, additions, and improvements are included within the scope of the subject matter of this specification.

[0055] Some implementations may be described using the term "coupled" along with derivatives. For example, some implementations may be described using the term "coupled" to indicate that two or more elements are in direct physical or electrical contact with each other. However, the term "coupled" may also mean that two or more elements are not in direct contact with each other, but yet still cooperate or interact with each other. The above implementations are not intended to be limiting in this context.

[0056] As used herein, the terms "comprises," "comprising," "includes," "including," "has," "having," or any other variations thereof, are intended to cover non-exclusive inclusions. For example, a process, method, article, or apparatus that includes a list of elements is not necessarily limited to only those elements and may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Furthermore, unless expressly stated to the contrary, "or" refers to an inclusive or, not an exclusive or. For example, a condition A or B is satisfied by any one of A being true (or present) and B being false (or absent), A being false (or absent) and B being true (or present), and both A and B being true (or present).

[0057] In addition, the use of "a" or "an" is used to describe elements and components of implementations herein. This is done merely for convenience and to give a general idea of ​​the invention. This description should be read to include one or at least one, and the singular also includes the plural unless otherwise clearly meant.

[0058] Moreover, while specific implementations and applications have been illustrated and described, it should be understood that the disclosed implementations are not limited to the precise structure and components disclosed herein. Various modifications, changes and variations, which will be apparent to those skilled in the art, may be made in the arrangement, operation and details of the methods and apparatus disclosed herein without departing from the spirit and scope, as defined in the appended claims.

Claims

1. 1. A centrifugal device comprising: a container having a body with a first end and a second end disposed opposite the first end; a cap coupled to the second end of the container, the cap including a top surface having at least one port configured to receive or deliver one or more of air or a fluid; A centrifugal device, wherein the container is movable between an upright position in which a first fluid placed in the container is centrifuged to separate and precipitate at least one extracellular vesicle from the first fluid, and an inverted position in which one or more of the first fluids depleted of at least one extracellular vesicle are removed from the container, and a second fluid mixed with the removed at least one extracellular vesicle is withdrawn from the container for injection.

2. 10. The device of claim 1, wherein the at least one port comprises a first port configured to receive or deliver air and having a filter, and a second port configured to receive or deliver a fluid.

3. 3. The device of claim 2, wherein one or both of the first and second ports includes one or more of a removable cap and a luer lock connection configured to be coupled to a syringe.

4. 2. The device of claim 1, wherein the at least one port includes a single port, and the device further comprises a Y-connector that is one of fixedly or removably coupled to the single port, the Y-connector including a first port configured to receive air and include a filter, and a second port having a syringe fitting configured to be coupled to a syringe, such that when the container is in the inverted position, air flows into the first port and the fluid depleted of extracellular vesicles flows out of the second port.

5. 10. The device of claim 1, wherein the container is cylindrical, the first end includes a tapered portion, the second end includes an opening, and the container comprises one of polypropylene or polystyrene.

6. 10. The device of claim 1, further comprising at least one tube disposed within the at least one port and extending into an interior region of the container, the at least one tube configured to facilitate the addition or removal of fluid to or from the container.

7. 10. The device of claim 1, wherein the device is one or more of: (1) the container is configured to receive platelet-poor plasma and an aqueous two-phase solution, the aqueous two-phase solution being a concentrated aqueous two-phase solution comprising a concentrated polyethylene glycol-dextran (PEG-DEX) solution; (2) the first fluid is platelet-poor plasma; and (3) the second fluid is platelet-rich plasma.

8. 1. A method for isolating extracellular vesicles from a fluid, comprising: adding the aqueous two-phase solution and the first fluid to a container of a centrifugal device in an upright position; Centrifuging the aqueous two-phase solution and the first fluid in the container to separate and form a fraction of extracellular vesicles located near a first end of the container from the first fluid; Moving the container of the centrifugal device from an upright position to an inverted position and removing the first fluid depleted of extracellular vesicles from the container through at least one port of the container; returning the container 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; returning the container to the inverted position and removing the second fluid containing the extracellular vesicle fraction through the at least one port of the container for injection.

9. 9. The method of claim 8, further comprising adding the aqueous two-phase solution and the first fluid to a container of the centrifugal device after centrifuging the platelet-rich plasma and aseptically removing a portion of the platelet-poor plasma from the platelet-rich plasma.

10. 9. The method of claim 8, wherein adding the aqueous two-phase solution and the first fluid to the container of the centrifugal device comprises adding a concentrated aqueous two-phase solution comprising a concentrated polyethylene glycol-dextran (PEG-DEX) solution and platelet-poor plasma to the container through at least one port disposed at a second end of the container of the centrifugal device.

11. 11. The method of claim 10, wherein adding the aqueous two-phase solution and the first fluid to the container through 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 through a first port of the container and allowing air to simultaneously exit through a second port of the container.

12. 9. The method of claim 8, wherein centrifuging the aqueous two-phase solution and the first fluid in the container to separate and form a fraction of extracellular vesicles located near the first end of the container from the first fluid comprises: removing the fraction of extracellular vesicles from platelet-poor plasma; and separating and forming the fraction of extracellular vesicles located near the first end of the container from the platelet-poor plasma in the container.

13. 9. The method of claim 8, further comprising moving the container of the centrifugal device from an upright position to an inverted position, and removing the first fluid depleted of extracellular vesicles from the container via a syringe through a first port of the container while air is flowing into a second port of the container.

14. 9. The method of claim 8, wherein returning the container to the upright position and adding a second fluid to the container comprises adding platelet-rich plasma through a first port disposed at a second end of the container while the fraction of extracellular vesicles removed from the first fluid is disposed at the first end of the container.

15. 9. The method of claim 8, wherein returning the container to the inverted position and removing the second fluid containing the extracellular vesicle fraction through the at least one port of the container for injection comprises one or more of: (1) removing the platelet-rich plasma containing the extracellular vesicle fraction through a first port of the container via a syringe for injection; or (2) removing the platelet-rich plasma containing the extracellular vesicle fraction through a port of a Y-connector coupled to the at least one port of the container of the centrifugal device for injection.

16. 1. A centrifugal device comprising: a container having a body with a first end and a second end disposed opposite the first end; a cap coupled to the second end of the container, the cap including a top surface having a port configured to receive or deliver one or more of air or a fluid; a Y-connector coupled to the port of the cap, the Y-connector having a first port for receiving or delivering air and a second port for receiving or delivering a fluid; A centrifugal device, wherein the container is movable between an upright position in which a first fluid and an aqueous two-phase solution disposed in the container are centrifuged to precipitate at least one extracellular vesicle, and an inverted position in which one or more of the first fluids depleted of at least one extracellular vesicle are removed from the container through the second port, and a second fluid mixed with the at least one extracellular vesicle is removed from the container through the second port for injection.

17. 17. The device of claim 16, wherein the container is cylindrical, the first end includes a tapered portion, the second end includes an opening, and the container comprises one of polypropylene or polystyrene.

18. 17. The device of claim 16, wherein one or more of: (1) the second port includes a syringe fitting configured to be coupled to a syringe; and (2) the first port receives air when the container is in the inverted position.

19. 17. The device of claim 16, wherein the first and second ports of the Y-connector each include a cap, and the first port includes a filter.

20. 17. The device of claim 16, wherein one or more of: (1) the container is configured to receive platelet-poor plasma and an aqueous two-phase solution, the aqueous two-phase solution being a concentrated aqueous two-phase solution comprising a concentrated polyethylene glycol-dextran (PEG-DEX) solution; (2) the first fluid is platelet-poor plasma; and (3) the second fluid is platelet-rich plasma.

21. 1. A method for isolating extracellular vesicles from a fluid, comprising: transferring a first volume of plasma from the transfer device to a container of a centrifugal device and 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 plasma in the container to form an extracellular vesicle and platelet-rich plasma fraction; inverting the container of the centrifugal device to remove the remaining aqueous two-phase solution and depleted platelet-poor plasma from the container; returning the container from the inverted position to an upright position and adding the second volume of plasma from the transfer device to the container of the centrifugal device; resuspending the extracellular vesicles and platelet-rich plasma fraction with the second volume of plasma in the container by one or more of shaking, inverting, vortexing, and / or centrifuging the container; inverting the container and removing platelet-rich plasma containing extracellular vesicles for placement in an injection device, wherein a volume of the platelet-rich plasma and extracellular vesicles is equal to the second volume of plasma in the transfer device.

22. 22. The method of claim 21, wherein the centrifugal device is a first centrifugal device, and prior to 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.

23. 23. The method of claim 22, further comprising transferring 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.

24. 22. The method of claim 21, wherein adding an aqueous two-phase solution to the container of the centrifugal device comprises adding a concentrated aqueous two-phase solution comprising a concentrated polyethylene glycol-dextran (PEG-DEX) solution to the container through at least one port of the container.

25. 25. The method of claim 24, wherein adding the aqueous two-phase solution to the container through at least one port comprises adding the aqueous two-phase solution to the container through a first port of the container and simultaneously allowing air to exit through a second port of the container.

26. 22. The method of claim 21, wherein centrifuging the aqueous two-phase solution and the plasma in the container to form a fraction of extracellular vesicles and platelet-rich plasma comprises centrifuging the aqueous two-phase solution and the plasma in the container to separate and form the fraction of extracellular vesicles and platelet-rich plasma located near a first end of the container from depleted platelet-poor plasma and the remaining aqueous two-phase solution.

27. 22. The method of claim 21, wherein inverting the container of the centrifugal device and removing the 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 via a syringe through a first port of the container while air is flowing into a second port of the container.

28. 22. The method of claim 21, wherein returning the container from an inverted position to an upright position and adding the second volume of plasma from the transfer device to the container of the centrifugal device comprises adding the second volume of plasma from the transfer device and through a first port disposed at a second end of the container while the extracellular vesicles and platelet-rich plasma fraction is disposed at a first end of the container.

29. 22. The method of claim 21, wherein inverting the container and removing platelet-rich plasma containing extracellular vesicles for injection comprises one or more of: (1) removing platelet-rich plasma containing extracellular vesicles for injection via a syringe through a first port of the container; or (2) removing platelet-rich plasma containing extracellular vesicles for injection via a syringe through a port of a Y-connector coupled to the at least one port of the container of the centrifugal device.

30. 1. A method for isolating extracellular vesicles from a fluid, comprising: Transferring a first volume of plasma from the transfer device to a container of a centrifugal device; adding an aqueous two-phase solution to the vessel; centrifuging the aqueous two-phase solution and the plasma in the container to form an extracellular vesicle and platelet-rich plasma fraction; inverting the container of the centrifugal device to remove the 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 to the container; resuspending the extracellular vesicles and platelet-rich plasma fraction with the second volume of plasma in the container; inverting the container and removing the platelet-rich plasma containing extracellular vesicles for placement into an injection device.

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