System and method for isolating extracellular vesicles

Abstract

To provide methods of effectively isolating one or more of extracellular vesicles and platelet-rich plasma fractions from bone marrow or blood.SOLUTION: The method comprises loading one or more of blood or bone marrow into an input port of a concentration system and centrifuging one or more of the blood or bone marrow to separate one or more of red blood cells, platelet poor plasma, or platelet rich plasma / bone marrow concentrate fractions, via a centrifuge device. The method further comprises pumping one or more of bone marrow / platelet rich plasma fractions and platelet poor plasma fractions into a receptacle of the concentration system and adding a concentrated aqueous two-phase solution to one or more of the bone marrow concentrate / platelet rich plasma fractions and platelet poor plasma fractions. The method also includes drawing the concentrated aqueous two-phase solution and one or more of the bone marrow concentrate / platelet rich plasma fractions or platelet poor plasma fractions back into the centrifuge device to isolate extracellular vesicles or extracellular vesicles and platelet rich plasma / bone marrow concentrate fractions.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present disclosure relates to cell isolation methods and devices, and in particular to systems and methods for isolating extracellular vesicles from bone marrow or blood. [Background technology]

[0002] Extracellular vesicles (such as exosomes) are released by cells that efficiently transfer their molecular cargo to other cells. The therapeutic effects of extracellular vesicles arise from their cargo (such as miRNA and proteins) and surface molecules. Furthermore, extracellular vesicles can be functional elements of the extracellular matrix involved in the organization, cell regulation, and determining the physical properties of connective tissue and bone.

[0003] Injections of platelet-rich plasma (PRP) and bone marrow concentrate (BMC) have been used in clinical applications to promote healing, stimulate tissue regrowth, ameliorate inflammation, and rejuvenate intact endogenous tissue. 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 they produce. For example, extracellular vesicles from umbilical cord or bone marrow mesenchymal stem cells (MSCs) have been demonstrated to stimulate human skin rejuvenation and improve engraftment of transplanted fat grafts. It has been demonstrated that extracellular vesicles from bone MSCs 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 qualified as the primary mediators of these paracrine effects.

[0004] Due to their low density and small size, extracellular vesicles are typically 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 (RBCs), platelet poor plasma (PPP), BMC, or PRP) use low-speed centrifugation, and extracellular vesicles are not effectively separated or concentrated into one partition. 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. Summary of the Invention

[0005] According to an exemplary embodiment of the present disclosure, a method for isolating extracellular vesicles includes loading one or more of blood or bone marrow into an input port of an enrichment system, and centrifuging one or more of the blood or bone marrow through a centrifugation device of the enrichment system to separate one or more of red blood cells, platelet-poor plasma, or platelet-rich plasma / bone marrow concentrate fractions. The method further includes pumping one or more of the bone marrow / platelet-rich plasma fraction and the platelet-poor plasma fraction into a first container of the enrichment system, and adding a concentrated aqueous two-phase solution, such as a poly(ethylene glycol)-dextran (PEG-DEX) solution, to one or more of the bone marrow enrichment / platelet-rich plasma fraction and the platelet-poor plasma fraction. The method further includes returning the concentrated aqueous two-phase solution and one or more of the bone marrow enrichment / platelet-rich plasma fraction or the platelet-poor plasma fraction back to the centrifugation device to isolate the extracellular vesicles and one or more of the platelet-rich plasma / bone marrow concentrate fraction. The method also includes pumping one or more of the bone marrow concentrate / platelet-rich plasma fraction and the isolated extracellular vesicles into a syringe for injection.

[0006] According to another aspect of the present disclosure, a method for isolating extracellular vesicles includes disposing a concentrated aqueous two-phase PEG-DEX solution in a syringe or container and adding one or more of a platelet-poor plasma fraction or a bone marrow / platelet-rich plasma fraction to the concentrated aqueous two-phase solution. The method further includes centrifuging the concentrated aqueous two-phase solution disposed in the syringe or container and one or more of the platelet-poor plasma fraction or the bone marrow / platelet-rich plasma fraction to isolate one or more of the extracellular vesicles and the bone marrow / platelet-rich plasma fraction. The method also includes creating a pellet comprising one or more of the extracellular vesicles and the bone marrow / platelet-rich plasma fraction from the centrifugation of the concentrated aqueous two-phase solution and one or more of the platelet-poor plasma fraction and the bone marrow / platelet-rich plasma fraction, the pellet being for injection.

[0007] According to yet another example of the present disclosure, a system for isolating extracellular vesicles includes a first input port for receiving one or more of blood or bone marrow, and a centrifugation device coupled to the input port for separating one or more fractions of red blood cells, platelet-poor plasma, and / or bone marrow concentrate / platelet-rich plasma. The system further includes a container for collecting one or more of the bone marrow concentrate / platelet-rich plasma fraction or platelet-poor plasma fraction centrifuged from the centrifugation device and coupled to the centrifugation device, and a second inlet port coupled to the first container for receiving an aqueous two-phase solution via a syringe coupled to the second inlet port. The outlet port is coupled to the centrifugation device for receiving the extracellular vesicles isolated in the centrifugation device. As configured in this manner, after the centrifugation device separates one or more of the blood and bone marrow into one or more of the red blood cells, platelet-poor plasma, and / or bone marrow concentrate / platelet-rich plasma fraction, the aqueous two-phase solution is added to the first container in which the one or more of the bone marrow concentrate / platelet-rich plasma fraction or platelet-poor plasma is disposed. The aqueous two-phase solution and one or more of the bone marrow concentrate / platelet-rich plasma fraction or the platelet-poor plasma fraction are then returned to the centrifugation device and the extracellular vesicles or one or more of the bone marrow concentrate / platelet-rich plasma fraction are isolated for injection.

[0008] Further according to any one or more of the exemplary embodiments, the system for isolating extracellular vesicles or any of the methods of the present disclosure can include any one or more of the following preferred aspects.

[0009] In some embodiments, the method further comprises premixing the aqueous biphasic solution at a predetermined concentration before adding the concentrated aqueous biphasic solution to one or more of the bone marrow concentrate / platelet-rich plasma fraction and the platelet-poor plasma fraction. Additionally, the method may comprise allowing a time for room temperature incubation after adding the concentrated aqueous biphasic solution to one or more of the bone marrow concentrate / platelet-rich plasma fraction and the platelet-poor plasma fraction. Additionally, the method may comprise pumping the solution and the extracellular vesicle-poor plasma into the first container after returning the concentrated aqueous biphasic solution and one or more of the bone marrow concentrate / platelet-rich plasma fraction or the platelet-poor plasma fraction to the centrifugation device for centrifugation.

[0010] According to other aspects, returning the concentrated aqueous two-phase solution and one or more of the bone marrow concentrate / platelet-rich plasma fraction or the platelet-poor plasma fraction to the centrifugation device for centrifugation may include isolating extracellular vesicles from the platelet-poor plasma fraction to create an extracellular vesicle pellet for injection. Further, adding the concentrated aqueous two-phase solution to one or more of the bone marrow concentrate / platelet-rich plasma fraction and the platelet-poor plasma fraction may include adding the concentrated aqueous two-phase solution to one or more of the bone marrow concentrate / platelet-rich plasma fraction and the platelet-poor plasma fraction based on the amount of one or more of the bone marrow concentrate / platelet-rich plasma fraction and the platelet-poor plasma fraction in the first container.

[0011] In yet another embodiment, pumping one or more of the bone marrow / platelet-rich plasma fraction and the platelet-poor plasma fraction into a first container of the enrichment system may include pumping only the platelet-poor plasma fraction into said first container and then pumping the bone marrow / platelet-rich plasma fraction into a syringe. In this example, adding the concentrated aqueous two-phase solution to one or more of the bone marrow concentrate / platelet-rich plasma fraction and the platelet-poor plasma fraction may include adding the concentrated aqueous two-phase solution to only the platelet-poor plasma. Furthermore, returning the concentrated aqueous two-phase solution and one or more of the bone marrow concentrate / platelet-rich plasma fraction or the platelet-poor plasma fraction to a centrifugation device for centrifugation may include returning the concentrated aqueous two-phase solution and the platelet-poor plasma fraction to a centrifugation device for centrifugation. Furthermore, pumping one or more of the bone marrow concentrate / platelet-rich plasma fraction and the extracellular vesicles into an injection syringe may include pumping the extracellular vesicles into an injection syringe.

[0012] In yet another embodiment, the method may further comprise determining the amount of aqueous two-phase solution to be injected based on the amount of isolated bone marrow / platelet-rich plasma fraction in the syringe, and reducing the concentration of the aqueous two-phase solution used to minimize the effect of the aqueous two-phase solution on nucleated cells in the bone marrow / platelet-rich plasma fraction.Furthermore, the method may comprise determining the amount of extracellular vesicles based on the amount of isolated bone marrow / platelet-rich plasma fraction in the syringe.

[0013] In other embodiments, the method may include premixing the aqueous two-phase solution at a predetermined concentration before placing in the syringe or container. Additionally, adding one or more of the platelet-poor plasma fraction or the bone marrow / platelet-rich plasma fraction to the concentrated aqueous two-phase solution may include adding a volume of platelet-poor plasma such that the volume of the concentrated aqueous two-phase solution is diluted, such as, in one example, to about 1.5% working solution of the concentrated aqueous two-phase solution. Additionally, the method may include mixing the prepared extracellular vesicles with a biological fluid containing one or more of platelet-rich plasma, bone marrow concentrate, or platelet-poor plasma.

[0014] In yet another aspect, the system may include a syringe coupled to the second inlet port and thus the second container, the syringe containing a premixed aqueous two-phase solution that is added to one or more of the bone marrow concentrate / platelet-rich plasma fraction or platelet-poor plasma fraction disposed in the first container. Furthermore, the amount of aqueous solution added to the bone marrow concentrate / platelet-rich plasma fraction or platelet-poor plasma fraction may be based on the output amount of one or more of the bone marrow concentrate / platelet-rich plasma fraction or platelet-poor plasma fraction disposed in the container. The system may further include an isolation syringe coupled to the outlet port and receiving any one or more of the isolated extracellular vesicles or the bone marrow concentrate / platelet-rich plasma fraction created after centrifugation in the centrifuge device. Furthermore, the container may include the first container, and the system may further include a second container for collecting the centrifuged red blood cell fraction from the centrifuge device, the second container being coupled to the centrifuge device.

[0015] 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]

[0016] 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.

[0017] [Figure 1] FIG. 1 is a perspective view of a system for isolating extracellular vesicles according to one embodiment of the present disclosure. [Diagram 2] FIG. 2 is another perspective view of a portion of the system of FIG. 1. [Figure 3A] FIG. 2 is a top view of the system of FIG. 1, illustrating an exemplary method for isolating vesicles according to one embodiment of the present disclosure. [Figure 3B] FIG. 2 is another top view of the system of FIG. 1 , illustrating another exemplary method for isolating vesicles according to another embodiment of the present disclosure. [Figure 4] FIG. 2 is a perspective view of a syringe adapted for use with the system of FIG. 1. [Diagram 5] FIG. 5 is a perspective view of the syringe of FIG. 4 with platelet poor plasma added to the syringe. [Figure 6] FIG. 5 is a perspective view of the syringe of FIG. 4 with an extracellular vesicle pellet created after centrifugation in the centrifugation device of the system of FIG. 1. [Figure 7] FIG. 2 is a perspective view of another syringe adapted for use with the system of FIG. 1, the syringe containing another exemplary extracellular vesicle pellet. [Figure 8] FIG. 2 is a perspective view of a syringe adapted for use with the system of FIG. 1, the syringe containing another exemplary extracellular vesicle pellet and one or more of a bone marrow concentrate fraction or a platelet-rich plasma fraction. [Figure 9] FIG. 2 is a perspective view of a container adapted to be coupled to the system of FIG. 1. [Figure 10] FIG. 10 is a perspective view of the container of FIG. 9 after a 10 minute centrifugation process, the container having an extracellular vesicle pellet. [Figure 11] FIG. 2 is a perspective view of a container adapted to be coupled to the system of FIG. 1. [Figure 12] FIG. 10 is a perspective view of the container of FIG. 9, in which the platelet poor plasma has been pre-spun to completely remove residual cells. [Figure 13] Phase contrast photograph showing the resuspended extracellular vesicle pellet at 20x magnification. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] In general, a system and method for isolating extracellular vesicles is disclosed. The system includes a first input port for receiving one or more of blood or bone marrow, and a centrifugation device is coupled to the input port to separate one or more fractions of red blood cells, platelet-poor plasma, and / or bone marrow concentrate / platelet-rich plasma. The system also includes a first container for collecting one or more of the bone marrow concentrate fraction / platelet-rich plasma fraction or the platelet-poor plasma fraction centrifuged from the centrifugation device, and the first container is coupled to the centrifugation device. A second container for collecting the red blood cell fraction centrifuged from the centrifugation device is also included, and the second container is also coupled to the centrifugation device. A second inlet port is coupled to the first container and receives a concentrated aqueous two-phase solution, such as a poly(ethylene glycol)-dextran (PEG-DEX) solution, via a syringe coupled to the second inlet port, and an outlet port is coupled to the centrifugation device to receive the extracellular vesicles isolated in the centrifugation device. As configured in this manner, after the centrifugation device separates one or more of the blood and bone marrow into red blood cells, platelet-poor plasma, and / or one or more of the bone marrow concentrate / platelet-rich plasma fractions, the concentrated aqueous two-phase solution is added to the first container in which the one or more of the bone marrow concentrate / platelet-rich plasma fractions or platelet-poor plasma are disposed. The concentrated aqueous two-phase solution and one or more of the bone marrow concentrate / platelet-rich plasma fractions or platelet-poor plasma fractions are then returned to the centrifugation device, and the one or more of the extracellular vesicles or the bone marrow concentrate / platelet-rich plasma fractions are isolated for injection.

[0019] More specifically, the extracellular vesicles can be isolated from platelet-poor plasma using an aqueous two-phase solution in a centrifugation process, such as a 10 minute centrifugation process. The platelet-poor plasma can be from peripheral blood or from bone marrow. The isolated extracellular vesicles can then be applied directly or suspended in platelet-rich plasma or bone marrow concentrate. This isolation protocol can be used in combination with platelet-rich plasma / bone marrow concentrate systems, greatly expanding the therapeutic potential of these biological therapies.

[0020] Referring now to Figure 1, a system for isolating extracellular vesicles 10, such as a concentration system, is shown. Concentration systems are typically used to concentrate whole blood or bone marrow. For example, bone marrow concentrate fractions can be used for biological injections.

[0021] More specifically, the system 10 includes a compartment 11 and a first input port 12 for receiving one or more of blood or bone marrow adjacent or disposed in a portion of the compartment 11. In one example, the first input port 12 is disposed in a first side 13 of the housing 11 as shown in FIG. 1. The centrifugation device 14 is coupled to the input port 12 and disposed adjacent to a second side portion 15 (FIG. 3A) of the compartment 11. The centrifugation device 14 separates one or more fractions of, for example, red blood cells, platelet poor plasma, and / or bone marrow concentrate / platelet rich plasma that are transferred to the centrifugation device 14 via the first input port 12. Furthermore, the system 10 also includes a first container 16 for collecting one or more of the bone marrow concentrate fraction / platelet rich plasma fraction or platelet poor plasma fraction centrifuged from the centrifugation device 14. The first container 16 is coupled to the centrifugation device 14 and disposed adjacent to the first side 13 of the compartment 11. Similar to the first container 16, a second container 18 is also coupled to the centrifugation device 14 and disposed adjacent to the first side 13 of the compartment 11. The second container 18, for example, collects the centrifuged red blood cell fraction from the centrifugation device 14.

[0022] A second inlet port 20 is coupled to the first container 16 and receives a concentrated aqueous two-phase solution, such as a PEG-DEX solution, as described further below. In one example, as shown in FIG. 2, a syringe 22 is coupled to the second inlet port 20 and contains a concentrated aqueous two-phase solution to be added to one or more of the platelet poor plasma and / or bone marrow concentrate / platelet rich plasma fractions disposed within the first container 16.

[0023] Aqueous two-phase solutions include any solution that allows for the separation and partitioning of microvesicles during centrifugation. More generally, aqueous (or water-based) solutions are polar and immiscible with non-polar organic solvents (chloroform, toluene, hexane, etc.), for example, forming a two-phase system. The formation of different phases is influenced by the pH, temperature, and ionic strength of the two components, and separation occurs when the amount of polymer present exceeds a certain limit concentration determined by these factors. In one example, and as described above, the aqueous two-phase solution includes a concentrated PEG-DEX solution. In this example, the "upper phase" is formed by the more hydrophobic polyethylene glycol (PEG), which is less dense than the "lower phase" consisting of the more hydrophilic, denser dextran solution.

[0024] 1, outlet port 24 is located at an upper portion 25 (FIG. 3A) of compartment 11 and is coupled to centrifugation device 14. In one example, isolation syringe 26 is coupled to outlet port 24. Isolation syringe 26 receives at least the extracellular vesicles separated by centrifugation device 14 and / or one or more of the bone marrow concentrate fraction / platelet-rich plasma fraction created after centrifugation in the centrifugation device according to one of the centrifugation processes described below.

[0025] Thus configured, upon loading one or more of blood or bone marrow into the input port 12, the centrifuge device 14 separates the blood into red blood cells and the bone marrow into one or more of platelet poor plasma and / or bone marrow concentrate / platelet rich plasma fractions. The one or more of the platelet poor plasma or bone marrow concentrate / platelet rich plasma fractions are pumped into the first container 16, and the red blood cells are moved, e.g., pumped, into the second container 18. As described further below, a concentrated aqueous two-phase solution is added to the one or more of the bone marrow concentrate / platelet rich plasma fractions and / or platelet poor plasma fractions disposed in the first container 16. The aqueous two-phase solution and one or more of the bone marrow concentrate / platelet rich plasma fractions or platelet poor plasma fractions are then returned to the centrifuge device 14, and the extracellular vesicles or one or more of the bone marrow concentrate / platelet rich plasma fractions are isolated for injection. Generally, and in one example, as further described below, the amount of aqueous two-phase solution added to the bone marrow concentrate / platelet-rich plasma fraction or platelet-poor plasma fraction is based on the output amount of one or more of the bone marrow concentrate / platelet-rich plasma fraction or platelet-poor plasma fraction placed in the first container 16.

[0026] 3A, a top view of the system 10 of FIGS. 1 and 2 is shown illustrating an exemplary method 100 of isolating vesicles according to one embodiment of the present disclosure. More specifically, the method 100 of isolating extracellular vesicles includes loading one or more of blood or bone marrow into the input port 12 of the enrichment system 10, for example, as shown at point 1 in FIG. 3A. The method 100 then includes centrifuging the one or more of blood or bone marrow by the centrifugation device 14 of the enrichment system 10 to separate one or more of red blood cells, platelet-poor plasma, or platelet-rich plasma / bone marrow concentrate fractions, as shown at point 2. More specifically, for example, as shown in FIG. 3A, one or more of blood or bone marrow is drawn from the input port 12 into the centrifugation device 14 via tube 17. Although tube 17 is shown, one skilled in the art will understand that other mechanisms and / or processes may alternatively and / or additionally be used to move one or more of the blood and / or bone marrow to the centrifuge device 14 and still fall within the scope of the present disclosure.

[0027] Additionally, the method 100 further includes pumping one or more of the bone marrow / platelet-rich plasma fraction and the platelet-poor plasma fraction produced after centrifugation in the centrifuge device 14 into a first container 16 of the enrichment system 10, as shown at point 3 in FIG. 3A. Similarly, the method may further include pumping a red blood cell fraction into a second container 18 of the enrichment system 10, as shown at point 4, for example.

[0028] The method 100 then includes adding the concentrated aqueous two-phase solution to one or more of the bone marrow concentrate / platelet-rich plasma fraction and the platelet-poor plasma fraction disposed in the first container 16, as shown at point 5. The method further includes returning the concentrated aqueous two-phase solution and one or more of the bone marrow concentrate / platelet-rich plasma fraction or the platelet-poor plasma fraction to the centrifugation device 14 to isolate the extracellular vesicles and one or more of the platelet-rich plasma / bone marrow concentrate fraction, as shown at point 6. In one example, the method 100 may further include pumping the aqueous two-phase solution and the extracellular vesicle-poor plasma (EPP) back to the first container 16, as shown at point 7, for example in FIG. 3A. The method 100 then includes pumping one or more of the bone marrow concentrate / platelet-rich plasma fraction and the isolated extracellular vesicles into the injection syringe 26, as shown at point 8.

[0029] Referring now to FIG. 3B, a top view of the system 10 of FIG. 1 is shown illustrating an exemplary method 200 of isolating vesicles according to another embodiment of the present disclosure. In general, and as an alternative to the method 100 described above in connection with FIG. 3A, one or more of the bone marrow concentrate or platelet-rich plasma concentrate may be isolated prior to isolation of the extracellular vesicles in order to minimize the amount of aqueous two-phase solution injected. Advantageously, in this example, the amount of extracellular vesicles can be determined, the concentration of the aqueous two-phase solution is reduced, and the effect of the aqueous two-phase solution on the cells in the bone marrow concentrate / platelet-rich plasma fraction is minimized.

[0030] More specifically, another method 200 of isolating vesicles using the system 10 of the present disclosure is described below. Similar to the method 100 described above, the method 200 includes loading one or more of blood or bone marrow into the input port 12 of the enrichment system 10, as shown in part 1 of FIG. 3A. The method 200 then includes centrifuging the one or more of blood or bone marrow via the centrifugation device 14 of the enrichment system 10 to separate one or more of red blood cells, platelet poor plasma, or platelet rich plasma / bone marrow concentrate fractions for a first centrifugation, as shown in part 2. Unlike the method 100, the method 200 then includes pumping only the bone marrow / platelet rich plasma fraction into a syringe, such as the isolation syringe 26, located adjacent to the top 25 of the compartment 11, as shown in part 3 of FIG. 3A. One skilled in the art will appreciate that only the bone marrow / platelet-rich plasma fraction created after the first centrifugation may be pumped into another syringe or container, for example, different from isolation syringe 26, and still fall within the scope of the present disclosure. Additionally, method 200 then includes pumping only the platelet-poor plasma fraction into first container 16 of enrichment system 10, as shown in portion 4. In some examples, method 200 may further include pumping the red blood cell fraction into second container 18, as shown in portion 5.

[0031] 3B, the method 200 also includes, for example, adding the concentrated aqueous two-phase solution to the platelet-poor plasma fraction disposed in the first container 16, as shown in part 6, and then returning the concentrated aqueous two-phase solution and the platelet-poor plasma to the centrifugation device 14 to isolate one or more of the extracellular vesicles, as shown in part 7. In one example, returning the concentrated aqueous two-phase solution and the platelet-poor plasma fraction to the centrifugation device 14 for a second centrifugation includes isolating the extracellular vesicles from the platelet-poor plasma fraction and creating an extracellular vesicle pellet for injection, as described further below.

[0032] Next, method 200 (similar to method 100) includes returning the concentrated aqueous two-phase solution and one or more of the bone marrow concentrate / platelet-rich plasma fraction or the platelet-poor plasma fraction to the centrifugation device for centrifugation, as shown in portion 8, and then pumping the aqueous two-phase solution and the extracellular vesicle-poor plasma into the first container 16.

[0033] Additionally, the method 200 also includes pumping only the isolated extracellular vesicles into a syringe 26 positioned adjacent to the top 25 of the compartment 11 for injection, as shown in part 9 of FIG. 3B. More specifically, the extracellular vesicles created by centrifugation of the concentrated aqueous two-phase solution and the platelet-poor plasma fraction returned to the centrifugation device 14 for, for example, a second centrifugation may be pumped into a syringe filled with the bone marrow concentrate / platelet-rich plasma fraction from the first centrifugation. In one example, this syringe may be the isolation syringe 26 positioned adjacent to the top 25 of the compartment 11, as shown in FIGS. 3A and 3B. Thus, in this example, the concentrated aqueous two-phase solution is only added together with the extracellular vesicles, so that the residual concentrated aqueous two-phase in the isolation syringe 26 is significantly reduced.

[0034] As an example, adding 1 mL of extracellular vesicles to 4 mL of bone marrow concentrate already placed in the isolation syringe 36 (e.g., to be injected) can reduce the residual concentrated aqueous biphasic solution, such as a PEG-DEX solution, by a factor of 5. Thus, by adding a concentrated aqueous biphasic solution, such as a PEG-DEX solution, to only the platelet poor plasma (as in part 6 of FIG. 3B), and then adding extracellular vesicles created in a second centrifugation, such as platelet poor plasma extracellular vesicles, to the bone marrow concentrate / platelet rich plasma fraction pumped into the syringe 26 (as in part 3 of FIG. 3B), the residual concentrated aqueous biphasic solution is reduced. Furthermore, since extracellular vesicles, such as platelet poor plasma extracellular vesicles, are added to the isolation syringe 26, the amount of additional extracellular vesicles can be easily determined.

[0035] In another example, method 200 may further include determining the amount of PEG-DEX solution to inject based on the amount of isolated bone marrow / platelet-rich plasma fraction in syringe 26. As a result, the concentration of the aqueous two-phase solution used is reduced, e.g., minimizing the effect of the aqueous two-phase solution on nucleated cells in the bone marrow / platelet-rich plasma fraction.

[0036] 4-6, there are shown perspective views of a syringe adapted for use with the system 10 described above. More specifically, and in one example, the syringe of FIGS. 4-6 may be the syringe 22 shown in FIG. 2, which is coupled to the first container 16 as described above. In this example, a concentrated aqueous two-phase solution is first placed in the syringe 22, as shown in FIG. 4. Further, as described above in connection with the method 200, for example, platelet poor plasma (PPP) is added to the syringe 22 having the aqueous two-phase solution, and an aqueous two-phase solution, such as a PEG-DEX solution, and a PPP solution 32 are created in the syringe 22, as shown in FIG. 5. More specifically, and in one example, 9 mL of platelet poor plasma is drawn into the syringe 22 for a total volume of 10 mL when 1 mL of 15% PEG-DEX is in the syringe 22 to obtain a final PEG-DEX concentration of 1.5%. More generally, a volume of platelet poor plasma can be added to the concentrated aqueous biphasic solution, such as by being drawn into syringe 22, to dilute the volume of concentrated aqueous biphasic PEG-DEX solution to about a 1.5% working solution of the concentrated aqueous biphasic solution. In addition, and in another example, as described further below, the concentrated aqueous biphasic solution is added to the platelet poor plasma and allowed to incubate at room temperature for about 5 minutes.

[0037] 6, the aqueous two-phase solution and PPP solution 32 in the syringe 22 are centrifuged in a centrifuge device to create a pellet 34 of extracellular vesicles that can be, for example, injected or added to one or more of platelet rich plasma or bone marrow concentrate. More specifically, and in one example, the syringe 22 with the aqueous two-phase and PPP solution is centrifuged in a centrifuge device capable of holding the syringe 22 at 200×g for about 10 minutes, thus isolating the extracellular vesicles and creating the extracellular vesicle pellet 34.

[0038] More generally, another method 300 of isolating extracellular vesicles using, for example, the system 10 and syringe 22 shown in Figures 4-6 includes disposing a concentrated aqueous two-phase solution in one or more of the syringes or containers of the system 10, such as the syringe 22. The method 300 further includes adding one or more of a platelet-poor plasma fraction or a bone marrow / platelet-rich plasma fraction to the concentrated aqueous two-phase solution, for example, as shown in the syringe 22 of Figure 5. The method 300 also includes centrifuging the solution disposed in the syringe 22 and one or more of the platelet-poor plasma fraction or the bone marrow / platelet-rich plasma fraction to isolate the extracellular vesicles. As shown in Figure 6, the method 300 further includes creating a pellet 34 having isolated extracellular vesicles, the pellet 34 being for injection. In another example, the extracellular vesicle pellet 34 may be added to, for example, one or more of the bone marrow / platelet-rich plasma fraction.

[0039] As shown in Figures 7 and 8, an alternative syringe 40 may be designed to accommodate either or both of an extracellular vesicle pellet alone, as shown in Figure 7, or a combination of an extracellular vesicle pellet and bone marrow concentrate or platelet-rich plasma, as shown in Figure 8. More specifically, with reference to Figure 7, the syringe 40 may include a body 42 having a distal end 44 and a proximal end 46. A protrusion 48, such as a cylindrical protrusion, extends from the distal end 44 of the body 42 and contains an extracellular vesicle pellet 50 formed by the centrifugation process described above. In this example, unlike the syringe 22 of Figures 4-6, the syringe 40 of Figures 7 and 8 receives the extracellular vesicle pellet 50 within the protrusion 48 disposed on the outside of the body 42 of the syringe 40, rather than within the body 42, as shown in Figures 4-6, for example. Similarly, Figure 8 illustrates the protrusion 48 of the syringe 40 having a pellet 52 containing both extracellular vesicles and one or more of bone marrow concentrate or platelet-rich plasma. By placing the extracellular pellets 50, 52 within the projections 48 of the syringe 40 rather than within the body 42, the extracellular vesicle concentrate can be more easily expelled from the syringe 40 while minimizing the amount of aqueous two-phase solution, e.g., extracellular vesicle-poor plasma and PEG-DEX, expelled from the syringe 40.

[0040] All of the above methods 100, 200, 300 may further include premixing a PEG-DEX solution at a predetermined concentration before adding the concentrating biphasic solution to one or more of the bone marrow concentrate / platelet-rich plasma fraction and the platelet-poor plasma fraction. In one example, premixing a PEG-DEX solution at a predetermined concentration includes premixing a PEG-DEX solution at a 10x concentration. In another example, premixing a PEG-DEX solution at a predetermined concentration includes premixing a PEG-DEX solution at a 5x concentration. In yet another example, premixing a PEG-DEX solution at a predetermined concentration includes premixing a PEG-DEX solution at an 8x concentration. In other examples, and as one of skill in the art will appreciate, the predetermined concentration may be anywhere in the range of 3x concentration to 15x concentration and still fall within the scope of the present disclosure. In some examples, premixing a PEG-DEX solution is essential for rapid extracellular vesicle isolation. Additionally, the methods 100, 200, 300 may include allowing a time for room temperature incubation after adding the concentrated aqueous two-phase solution to one or more of the bone marrow concentrate / platelet-rich plasma fraction and the platelet-poor plasma fraction. In one example, the time for room temperature incubation is about 5 minutes. One skilled in the art will understand that the time may be longer than 5 minutes or slightly shorter, such as 3 minutes, 4 minutes, or 4 1 / 2 minutes, and still fall within the scope of the present disclosure.

[0041] Further, in each of methods 100, 200, 300, adding the concentrated aqueous two-phase solution to one or more of the bone marrow concentrate / platelet-rich plasma fraction and the platelet-poor plasma fraction may include adding the concentrated aqueous two-phase solution to one or more of the bone marrow concentrate / platelet-rich plasma fraction and the platelet-poor plasma fraction based on the amount of the one or more of the bone marrow concentrate / platelet-rich plasma fraction and the platelet-poor plasma fraction in the first container 16.

[0042] Referring now to Figures 9-12, various experimental results of the system 10 and methods 100, 200, and 300 of the present disclosure are shown. More specifically, Figure 9 is a perspective view of a combination of an aqueous two-phase solution and platelet poor plasma in a container 60 prior to centrifugation. As shown therein, prior to centrifugation, extracellular vesicles were not isolated. In this example, the aqueous two-phase solution is PEG-DEX. Furthermore, a PEG-DEX concentration of 1.5% when added to platelet poor plasma allowed for the isolation of extracellular vesicles using, for example, a tabletop centrifuge, such as a centrifugation device capable of holding a syringe. Additionally, dextran with a molecular weight of 450k-650k was used, and the centrifugation device was run at 1000 x g for 10 minutes. As will be appreciated, various other percentage amounts of PEG-DEX concentration, or more generally, the first phase of the aqueous two-phase solution, may be used instead and still fall within the scope of the present disclosure. Similarly, various other weights of dextran, or more generally, the second phase of the aqueous two-phase solution, can be used and still fall within the scope of the present disclosure. In other words, various combinations of PEG-DEX solutions can be added to platelet poor plasma to isolate extracellular vesicles during centrifugation. More generally, various combinations of the first phase of the aqueous two-phase solution and the second phase of the aqueous two-phase solution can be used to isolate extracellular vesicles during centrifugation and still fall within the scope of the present disclosure.

[0043] 10, there is shown a perspective view of the container 60 of FIG. 9 after, for example, a 10 minute centrifugation process. By the above process and the experimental parameters set forth above, the extracellular vesicles were isolated and an extracellular vesicle pellet 62 was created.

[0044] 11, a container 60 is shown with a combination of PEG-DEX concentration and ddH2O placed therein. Centrifugation was performed and no extracellular vesicle pellet was produced.

[0045] Referring now to FIG. 12, the container 60 is shown after centrifugation. In this example experiment, a combination of the PEG-DEX enriched solution and platelet poor plasma was again placed in the container 60 prior to centrifugation. Prior to placing the platelet poor plasma with the PEG-DEX enriched solution, the platelet poor plasma was pre-spun, for example, at 1900×g for 30 minutes to remove any residual cells. After centrifugation, an extracellular vesicle pellet 64 was also created and collected, as shown in FIG.

[0046] Referring now to Figure 13, a phase contrast photograph of an exemplary extracellular vesicle pellet is shown. Specifically, the extracellular vesicle pellet is resuspended at 20x magnification. No cells are shown in this view, but extracellular vesicle clusters 66 can be seen.

[0047] In view of the foregoing, one skilled in the art will appreciate the following advantages of the system 10 and method 100, 200, 300 of the present disclosure described above. For example, the system 10 and method 100, 200, 300 can rapidly isolate extracellular vesicles from platelet poor plasma in a clinical setting. Rapid isolation of extracellular vesicles allows for application of the extracellular vesicles within the same clinical procedure involving the collection of one or more of blood or bone marrow, which is important for practical, therapeutic, and regulatory reasons. For example, the extracellular vesicles isolated from blood or bone marrow can be used to enhance the efficacy of biological injections or as the sole biological therapy.

[0048] Furthermore, the system 10 allows for the collection of extracellular vesicles from the platelet-poor plasma fraction, which is not normally used but is a significant portion of the output of the centrifugation process in terms of volume in conventional systems. Furthermore, for example, the syringes 22, 26, 40 of the system 10 may be designed such that the syringes 22, 26, 40 are loaded into the centrifugation device 14, eliminating the need to transfer the platelet-poor plasma and aqueous two-phase solution, such as a PEG-DEX solution, or the extracellular vesicle concentrate, to a centrifuge tube. This arrangement minimizes the risk of contamination, reduces the risk of errors and sample loss, and speeds up the procedure.

[0049] Furthermore, the system 10 is designed such that, as further explained above, only one centrifugation device 14 is used even if the biological sample is subjected to two centrifugation cycles. As a result, an additional centrifugation device and an additional centrifugation syringe are not required. Furthermore, the risk of contamination is further reduced and the isolation procedure is expedited.

[0050] 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 have been 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 within the scope of the subject matter of this specification.

[0051] 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. Such implementations are not limited in this context.

[0052] 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" means 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).

[0053] In addition, the use of "a" or "an" is used to describe elements and components of the implementations of this specification. 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 it is clear that it is meant otherwise.

[0054] 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 structures 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 method for isolating extracellular vesicles, comprising: loading one or more of blood or bone marrow into an input port of the enrichment system; centrifuging the blood or bone marrow with a centrifugation device of the enrichment system to separate one or more of red blood cells, platelet poor plasma, or platelet rich plasma / bone marrow concentrate fractions; pumping one or more of a bone marrow / platelet-rich plasma fraction and a platelet-poor plasma fraction into a first container of said enrichment system; adding a concentrated aqueous two-phase solution to one or more of the bone marrow concentrate / platelet-rich plasma fraction and the platelet-poor plasma fraction; returning the concentrated aqueous two-phase solution and the bone marrow concentrate / platelet-rich plasma fraction or the platelet-poor plasma fraction to the centrifugation device to isolate extracellular vesicles and the platelet-rich plasma / bone marrow concentrate fraction; and pumping one or more of the bone marrow concentrate / platelet-rich plasma fraction and isolated extracellular vesicles into a syringe for injection.

2. 2. The method of claim 1, wherein adding a concentrated aqueous two-phase solution to one or more of the bone marrow concentrate / platelet-rich plasma fraction and platelet-poor plasma fraction comprises adding a concentrated PEG-DEX solution to one or more of the bone marrow concentrate / platelet-rich plasma fraction and platelet-poor plasma fraction, the method further comprising premixing the concentrated aqueous two-phase solution at a predetermined concentration prior to adding the concentrated aqueous two-phase solution to one or more of the bone marrow concentrate / platelet-rich plasma fraction and platelet-poor plasma fraction.

3. 3. The method of claim 1 or 2, further comprising allowing time for room temperature incubation after adding the concentrated aqueous two-phase solution to one or more of the bone marrow concentrate / platelet-rich plasma fraction and the platelet-poor plasma fraction.

4. 4. The method of claim 1, further comprising pumping the concentrated aqueous two-phase solution and the bone marrow concentrate / platelet-rich plasma fraction or the platelet-poor plasma fraction back into the centrifugation device for centrifugation, and then pumping the concentrated aqueous two-phase solution and extracellular vesicle-poor plasma back into the first container.

5. 5. The method of any one of claims 1 to 4, wherein returning the concentrated aqueous two-phase solution and one or more of the bone marrow concentrate / platelet-rich plasma fraction or platelet-poor plasma fraction to the centrifugation device for centrifugation comprises isolating extracellular vesicles from the platelet-poor plasma fraction to create an extracellular vesicle pellet for injection.

6. 6. The method of claim 1, wherein adding a concentrated aqueous two-phase solution to one or more of the bone marrow concentrate / platelet-rich plasma fraction and platelet-poor plasma fraction comprises adding the concentrated aqueous two-phase solution to one or more of the bone marrow concentrate / platelet-rich plasma fraction and platelet-poor plasma fraction based on an amount of the one or more of the bone marrow concentrate / platelet-rich plasma fraction and platelet-poor plasma fraction in the first container.

7. 7. The method of any one of claims 1 to 6, wherein pumping one or more of the bone marrow / platelet-rich plasma fraction and the platelet-poor plasma fraction into a first container of the enrichment system comprises pumping only the platelet-poor plasma fraction into the first container and then pumping the bone marrow / platelet-rich plasma fraction into a syringe.

8. 8. The method of claim 7, wherein adding the concentrated aqueous two-phase solution to one or more of the bone marrow concentrate / platelet-rich plasma fraction and the platelet-poor plasma fraction comprises adding the concentrated aqueous two-phase solution only to the platelet-poor plasma fraction.

9. 9. The method of claim 8, wherein returning the concentrated aqueous two-phase solution and one or more of the bone marrow concentrate / platelet-rich plasma fraction or the platelet-poor plasma fraction to the centrifugation device for centrifugation comprises returning the concentrated aqueous two-phase solution and the platelet-poor plasma fraction to the centrifugation device for centrifugation and centrifuging the concentrated aqueous two-phase solution and the platelet-poor plasma fraction.

10. 10. The method of claim 9, wherein pumping one or more of the bone marrow concentrate / platelet-rich plasma fraction and extracellular vesicles into the injection syringe comprises pumping the extracellular vesicles into the injection syringe, the syringe containing the blood concentrate / platelet-rich plasma after a first centrifugation.

11. 11. The method of claim 10, wherein the concentrated aqueous two-phase solution is added to the platelet-poor plasma only, and then the extracellular vesicles are pumped into the injection syringe, the syringe containing the blood concentrate / platelet-rich plasma, thereby diluting any remaining concentrated aqueous two-phase solution in the syringe.

12. 8. The method of claim 7, further comprising determining the amount of aqueous two-phase solution to inject based on the amount of isolated bone marrow / platelet-rich plasma fraction in the syringe, and using a reduced concentration of aqueous two-phase solution to minimize the effect of the aqueous two-phase solution on nucleated cells in the bone marrow / platelet-rich plasma fraction.

13. 13. The method of claim 12, further comprising determining the amount of extracellular vesicles based on the amount of the isolated bone marrow / platelet-rich plasma fraction in the syringe.

14. 1. A method for isolating extracellular vesicles, comprising: placing the concentrated aqueous two-phase solution in a syringe or container; adding one or more of a platelet-poor plasma fraction or a bone marrow / platelet-rich plasma fraction to said concentrated aqueous two-phase solution; Centrifuging the concentrated aqueous two-phase solution disposed in the syringe or container and one or more of the platelet-poor plasma fraction or the bone marrow / platelet-rich plasma fraction to isolate extracellular vesicles and one or more of the bone marrow / platelet-rich plasma fraction; and producing a pellet comprising extracellular vesicles and one or more of a bone marrow / platelet-rich plasma fraction from centrifugation of the concentrated aqueous two-phase solution and one or more of the platelet-poor plasma fraction and the bone marrow / platelet-rich plasma fraction, wherein the pellet is for injection.

15. 15. The method of claim 14, wherein disposing the concentrated aqueous two-phase solution in a syringe or container comprises disposing a concentrated PEG-DEX solution in the syringe or container.

16. 16. The method of claim 14, wherein adding one or more of a platelet-poor plasma fraction or a bone marrow / platelet-rich plasma fraction to the concentrated aqueous two-phase solution comprises adding a volume of platelet-poor plasma such that the volume of the concentrated aqueous two-phase solution is diluted.

17. 17. The method of any one of claims 14 to 16, further comprising allowing time for room temperature incubation after adding one or more of a platelet poor plasma fraction or a bone marrow / platelet rich plasma fraction to the concentrated aqueous two-phase solution.

18. 19. The method of any one of claims 14 to 18, further comprising mixing the created extracellular vesicles with a biological fluid comprising one or more of platelet rich plasma, bone marrow concentrate, or platelet poor plasma.

19. A system for isolating extracellular vesicles, comprising: a first input port for receiving one or more of blood or bone marrow; a centrifugation device coupled to the first input port for separating one or more fractions of red blood cells, platelet poor plasma, and / or bone marrow concentrate / platelet rich plasma; a container for collecting one or more of the bone marrow concentrate fraction / platelet-rich plasma fraction or platelet-poor plasma fraction centrifuged from the centrifugation device, the container being coupled to the centrifugation device; a second input port coupled to the first container for receiving an aqueous two-phase solution using a syringe coupled to the second input port; After the centrifugation device separates one or more of the blood and the bone marrow into one or more of red blood cells, platelet-poor plasma, and / or bone marrow concentrate / platelet-rich plasma fractions, the aqueous two-phase solution is added to the first container in which the one or more of the bone marrow concentrate / platelet-rich plasma fractions or platelet-poor plasma fractions are disposed, and the aqueous two-phase solution and the one or more of the bone marrow concentrate / platelet-rich plasma fractions or platelet-poor plasma fractions are returned to the centrifugation device for centrifugation, and extracellular vesicles or the one or more of the bone marrow concentrate / platelet-rich plasma fractions are isolated for injection.

20. 20. The system of claim 19, further comprising a syringe coupled to the second inlet port and thus to the first container, the syringe containing a premixed aqueous two-phase solution that is added to one or more of the bone marrow concentrate fraction / platelet-rich plasma fraction or platelet-poor plasma fraction disposed within the first container.

21. 21. The system of claim 19 or 20, wherein the amount of the aqueous two-phase solution added to the bone marrow concentrate / platelet-rich plasma fraction or platelet-poor plasma fraction is based on an output amount of one or more of the bone marrow concentrate / platelet-rich plasma fraction or platelet-poor plasma fraction placed into the container.

22. The system of any one of claims 19 to 21, further comprising an isolation syringe for receiving one or more of the isolated extracellular vesicles or the bone marrow concentrate fraction / platelet-rich plasma fraction prepared after centrifugation.

23. 23. The system of any one of claims 19 to 22, wherein the container is a first container, the system further comprising a second container for collecting the centrifuged red blood cell fraction from the centrifugation device, the second container being coupled to the centrifugation device.

24. 24. The system of any one of claims 19 to 23, further comprising an outlet port coupled to the centrifugation device and an isolation syringe coupled to the outlet port for receiving one or more of isolated extracellular vesicles or the bone marrow concentrate / platelet-rich plasma fraction created after centrifugation.

25. The system of any one of claims 19 to 24, wherein the aqueous two-phase solution is a concentrated PEG-DEX solution.

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