Patent Description:
Extracellular vesicles (such as exosomes) are released by cells that efficiently transfer their molecular cargo to other cells. The therapeutic effects of extracellular vesicles derive from their cargo (such as miRNAs and proteins) and surface molecules. In addition, extracellular vesicles can be functional components of the extracellular matrix that participate in organization, cell-regulation, and determining the physical properties of connective tissues and bone.

Injections of platelet rich plasma (PRP) and bone marrow concentrate (BMC) are used in clinical applications to promote healing, stimulate tissue regrowth, ameliorate inflammation, and rejuvenate uninjured endogenous tissue. Extracellular vesicles are found in all biofluids, including the blood and marrow, and have been demonstrated to confer many of the effects of the cells that they are produced by. For example, extracellular vesicles from umbilical cord or bone marrow mesenchymal stem cells (MSCs) have been demonstrated to stimulate rejuvenation of human skin, or improve the survival of transplanted fat grafts. It has been demonstrated that extracellular vesicles from bone MSCs exerted similar chondroprotective and anti-inflammatory function and protected mice from developing osteoarthritis, suggesting that extracellular vesicles reproduce the main therapeutic effect of the MSCs. Indeed, recent scientific and clinical evidence suggests that MSCs may not primarily exert their therapeutic functions in a cellular, but rather in a paracrine manner; extracellular vesicles (such as exosomes and microvesicles) have been identified as major mediators of these paracrine effects.

Due to their low density and small size, extracellular vesicles are commonly isolated by filtration, ultra-centrifugation, immunoaffinity, microfluidics, or polymeric precipitation. Current devices employed to partition blood or bone marrow (into fractions such as red blood cells (RBCs), platelet poor plasma (PPP), and BMC or PRP) use low-speed centrifugation, and extracellular vesicles are not effectively isolated or concentrated into one partition. Thus, devices that concentrate whole blood or bone marrow are not concentrating the biological agents, such as extracellular vesicles that are likely to be delivering a substantial portion of the therapeutic effect.

<CIT> discloses a method for isolating extracellular vesicles using an aqueous two-phase system. The method comprises the steps of: (a) mixing a first material and a second material, which are immiscible with each other in an aqueous solution, in bodily fluid or an aqueous solution having extracellular vesicles to prepare an aqueous two-phase system; and (b) isolating extracellular vesicles concentrated in the second material in the aqueous two-phase system.

<NPL>) discloses an extracellular vesicle isolation method using a polyethylene glycol/dextran aqueous two phase system.

In accordance with one exemplary aspect of the present disclosure, a method of isolating extracellular vesicles 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 of the concentration system. The method further includes pumping one or more of bone marrow/platelet rich plasma fractions and platelet poor plasma fractions into a first receptacle of the concentration system, the first receptacle coupled to the centrifuge device, 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 further 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 (i) extracellular vesicles or (ii) extracellular vesicles and platelet rich plasma/bone marrow concentrate fractions. The method also includes pumping one or more of the bone marrow concentrate/platelet rich plasma fractions and isolated extracellular vesicles into a syringe for injection.

According to yet another example of the present disclosure, a system for isolating extracellular vesicles comprises a first input port for receiving one or more of blood or bone marrow, and a centrifuge device coupled to the input port for separating fractions of one or more of red blood cells, platelet poor plasma, and/or bone marrow concentrate/platelet rich plasma. The system further includes a receptacle for collecting one or more of bone marrow concentrate fractions/platelet rich plasma fractions or platelet poor plasma fractions centrifuged from the centrifuge device, the receptacle coupled to the centrifuge device, and a second input port coupled to the receptacle and for receiving an aqueous two-phase solution via a syringe coupled to the second inlet port. The system is configured to perform the following method: after the centrifuge 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 receptacle having one or more of the bone marrow concentrate/platelet rich plasma fractions or platelet poor plasma fractions disposed therein is adapted to receive the aqueous two-phase solution. The aqueous two-phase solution and the one or more of the bone marrow concentrate fractions/platelet rich plasma fractions or platelet poor plasma fractions are adapted to be drawn back into the centrifuge device to for centrifugation to isolate (i) extracellular vesicles or (ii) extracellular vesicles and the bone marrow concentrate/platelet rich plasma fractions for injection.

In further accordance with any one or more of the exemplary aspects, the system for isolating extracellular vesicles or any method of the present disclosure may include any one or more of the following preferred forms.

In some aspects, the method further comprises premixing the aqueous two-phase solution at a predetermined concentration before adding the concentrated aqueous two-phase solution to one or more of the bone marrow concentrate/platelet rich plasma fractions and platelet poor plasma fractions. In addition, the method may comprise allowing a period of 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 fractions and platelet poor plasma fractions. In addition, the method may comprise pumping the solution and extracellular vesicles-poor plasma into the first receptacle after 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 for centrifugation.

According to other aspects, 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 for centrifugation may comprise isolating extracellular vesicles from the platelet poor plasma fractions, creating an extracellular vesicles pellet for injection. In addition, 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 may comprise adding the concentrated aqueous two-phase solution to one or more of the bone marrow concentrate/platelet rich plasma fractions and platelet poor plasma fractions based upon a volume of one or more of the bone marrow concentrate/platelet rich plasma fractions and platelet poor plasma fractions in the first receptacle.

In still other aspects, pumping one or more of bone marrow/platelet rich plasma fractions and platelet poor plasma fractions into a first receptacle of the concentration system may comprise pumping only the platelet poor plasma fractions into the first receptacle and then pumping the bone marrow/platelet rich plasma fractions into a syringe. In this example, adding a concentrated aqueous two-phase solution to one or more of bone marrow concentrate/platelet rich plasma fractions and platelet poor plasma fractions may comprise adding a concentrated aqueous two-phase solution to only the platelet poor plasma fractions. In addition, 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 for centrifugation may comprise drawing the concentrated aqueous two-phase solution and the platelet poor plasma fractions back into the centrifuge device for centrifugation. Further, pumping one or more of the bone marrow concentrate/platelet rich plasma fractions and extracellular vesicles into a syringe for injection may comprise pumping the extracellular vesicles into the syringe for injection.

In still yet other aspects, the method may further comprise determining a volume of aqueous two-phase solution to be injected based upon the volume of the bone marrow/platelet rich plasma fractions isolated in the syringe, reducing the concentration of aqueous two-phase solution used and minimizing the effect of the aqueous two-phase solution on nucleated cells in the bone marrow/platelet rich plasma fractions. In addition, the method may include determining a volume of the extracellular vesicles based on the volume of the bone marrow/platelet rich plasma fractions isolated in the syringe.

In other aspects, the method may comprise premixing the aqueous two-phase solution at a predetermined concentration before disposing in the syringe or the receptacle. In addition, adding one or more of platelet poor plasma fractions or bone marrow/platelet rich plasma fractions into the concentrated aqueous two-phase solution may comprise adding an amount of platelet poor plasma such that the amount of the concentrated aqueous two-phase solution is diluted, such as diluted to a working solution of about <NUM>% concentrated aqueous two-phase solution in one example. Further, the method may comprise mixing the extracellular vesicle created with biofluid including one or more of platelet rich plasma, bone marrow concentrate or platelet poor plasma.

In still yet other aspects, the system may include a syringe coupled to the second inlet port and, thus, the first receptacle, the syringe including a pre-mixed aqueous two-phase solution to be added to one or more of the bone marrow concentrate fractions/platelet rich plasma fractions or platelet poor plasma fractions disposed within the first receptacle. In addition, an amount of the aqueous solution added to the bone marrow concentrate/platelet rich plasma fractions or platelet poor plasma fractions may be based upon an output volume of one or more of the bone marrow concentrate/platelet rich plasma fractions or platelet poor plasma fractions disposed in the receptacle. The system may further comprise an isolation syringe to be coupled to the outlet port for receiving one or more of the extracellular vesicles isolated or the bone marrow concentrate fractions/platelet rich plasma fractions created after centrifugation in the centrifuge device. Further, the receptacle may include a first receptacle, and the system may further comprise a second receptacle for collecting red blood cell fractions centrifuged from the centrifuge device, the second receptacle coupled to the centrifuge device.

Additional optional aspects and features are disclosed, which may be arranged in any functionally appropriate manner, either alone or in any functionally viable combination, consistent with the teachings of the disclosure. Other aspects and advantages will become apparent upon consideration of the following detailed description.

It is believed that the disclosure will be more fully understood from the following description taken in conjunction with the accompanying drawings. Some of the drawings may have been simplified by the omission of selected elements for the purpose of more clearly showing other elements. Such omissions of elements in some drawings are not necessarily indicative of the presence or absence of particular elements in any of the example embodiments, except as may be explicitly delineated in the corresponding written description. Also, none of the drawings are necessarily to scale.

Generally, a system and methods of isolating extracellular vesicles are disclosed. The system includes a first input port for receiving one or more of blood or bone marrow, and a centrifuge device is coupled to the input port for separating fractions of one or more of red blood cells, platelet poor plasma, and/or bone marrow concentrate/platelet rich plasma. The system also includes a first receptacle for collecting one or more of bone marrow concentrate fractions/platelet rich plasma fractions or platelet poor plasma fractions centrifuged from the centrifuge device, and the first receptacle is coupled to the centrifuge device. A second receptacle for collecting red blood cell fractions centrifuged from the centrifuge device is also included, and the second receptacle is likewise coupled to the centrifuge device. A second inlet port is coupled to the first receptacle 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 centrifuge device for receiving extracellular vesicles isolated in the centrifuge device. So configured, after the centrifuge 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 concentrated aqueous two-phase solution is added to the first receptacle having one or more of the bone marrow concentrate/platelet rich plasma fractions or platelet poor plasma fractions disposed therein. The concentrated aqueous two-phase solution and the one or more of the bone marrow concentrate fractions/platelet rich plasma fractions or platelet poor plasma fractions are then drawn back into the centrifuge device to isolate one or more of extracellular vesicles or the bone marrow concentrate/platelet rich plasma fractions for injection.

More specifically, using an aqueous two-phase solution, the extracellular vesicles can be isolated from platelet poor plasma in a centrifugation process, such as a <NUM>-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 within platelet rich plasma or bone marrow concentrate and then applied. This isolation protocol may be used in conjunction with platelet rich plasma/bone marrow concentrate systems, significantly extending the therapeutic potential of these biological treatments.

Referring now to <FIG>, a system for isolating extracellular vesicles <NUM>, such as concentration system, is depicted. The concentration system is typically used to concentrate whole blood or bone marrow. For example, the bone marrow concentrate fraction may be used for the biologic injection.

More specifically, the system <NUM> includes a compartment <NUM> and a first input port <NUM> for receiving one or more of blood or bone marrow that is one or more of adjacent to or disposed on a portion of the compartment <NUM>. In one example, the first input port <NUM> is disposed on a first side portion <NUM> of the housing <NUM>, as depicted in <FIG>. A centrifuge device <NUM> is coupled to the input port <NUM> and disposed adjacent to a second side portion <NUM> (<FIG>) of the compartment <NUM>. The centrifuge device <NUM> separates fractions of one or more of red blood cells, platelet poor plasma, and/or bone marrow concentrate/platelet rich plasma directed to the centrifuge device <NUM> via the first input port <NUM>, for example. In addition, the system <NUM> also includes a first receptacle <NUM> for collecting one or more of bone marrow concentrate fractions/platelet rich plasma fractions or platelet poor plasma fractions centrifuged from the centrifuge device <NUM>. The first receptacle <NUM> is coupled to the centrifuge device <NUM> and is disposed adjacent to the first side portion <NUM> of the compartment <NUM>. Like the first receptacle <NUM>, the second receptacle <NUM> is also coupled to the centrifuge device <NUM> and disposed adjacent to the first side portion <NUM> of the compartment <NUM>. The second receptacle <NUM> collects red blood cell fractions centrifuged from the centrifuge device <NUM>, for example.

A second inlet port <NUM> is coupled to the first receptacle <NUM> and receives a concentrated aqueous two-phase solution, such as a PEG-DEX solution, as explained more below. In one example, and as depicted in <FIG>, a syringe <NUM> is coupled to the second inlet port <NUM> and includes a concentrated two-phase solution to be added to one or more of the platelet poor plasma and/or the bone marrow concentrate/platelet rich plasma fractions disposed within the first receptacle <NUM>.

The aqueous two-phase solution includes any solution that enables separation and partitioning of microvesicles during centrifugation. More generally, aqueous (or water-based) solutions, being polar, are immiscible with non-polar organic solvents (chloroform, toluene, hexane etc.) and form a two-phase system, for example. The formation of the distinct phases is affected by the pH, temperature, and ionic strength of the two components, and separation occurs when the amount of a polymer present exceeds a certain limiting concentration, which is determined by these factors. In one example, and as noted above, the aqueous two-phase solution includes the concentrated PEG-DEX solution. In this example, an "upper phase" is formed by the more hydrophobic polyethylene glycol (PEG), which is of lower density than a "lower phase," consisting of the more hydrophilic and denser dextran solution.

Referring back to <FIG>, an outlet port <NUM> is disposed on a top portion <NUM> (<FIG>) of the compartment <NUM> and is coupled to the centrifuge device <NUM>. In one example, an isolation syringe <NUM> is coupled to the outlet port <NUM>. The isolation syringe <NUM> receives one or more of the extracellular vesicles isolated at least by the centrifuge device <NUM> according to one of the centrifuge processes described below and/or the bone marrow concentrate fractions/platelet rich plasma fractions created after centrifugation in the centrifuge device.

So configured, upon loading one or more of blood or bone marrow into the input port <NUM>, the centrifuge device <NUM> separates the blood into red blood cells and separates the bone marrow into one or more of platelet poor plasma and/or bone marrow concentrate/platelet rich plasma fractions. One or more of the platelet poor plasma or the bone marrow concentrate/platelet rich plasma fractions is pumped into the first receptacle <NUM> and the red blood cells are directed, such as pumped, into the second receptacle <NUM>. As explained more below, a concentrated aqueous two-phase solution is then 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 receptacle <NUM>. Thereafter, the aqueous two-phase solution and the one or more of the bone marrow concentrate fractions/platelet rich plasma fractions or platelet poor plasma fractions are drawn back into the centrifuge device <NUM> to isolate one or more of extracellular vesicles or the bone marrow concentrate/platelet rich plasma fractions for injection. Generally, and in one example, an amount of the aqeuous two-phase solution added to the bone marrow concentrate/platelet rich plasma fractions or platelet poor plasma fractions is based upon an output volume of one or more of the bone marrow concentrate/platelet rich plasma fractions or platelet poor plasma fractions disposed in the first receptacle <NUM>, as also explained more below.

Referring now to <FIG>, a top view of the system <NUM> of <FIG> and <FIG> is depicted, illustrating an exemplary method <NUM> of isolating vesicles in accordance with one aspect of the present disclosure. More specifically, the method <NUM> of isolating extracellular vesicles comprises loading one or more of blood or bone marrow into the input port <NUM> of concentration system <NUM>, as indicated at point <NUM> of <FIG>, for example. The method <NUM> next includes 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 the centrifuge device <NUM> of the concentration system <NUM>, as indicated at point <NUM>. More specifically, one or more of the blood or bone marrow is drawn from the input port <NUM> and into the centrifuge device <NUM> via tubing <NUM>, as depicted in <FIG>, for example. While tubing <NUM> is depicted, one of ordinary skill in the art will appreciate that other mechanisms and/or processes may alternatively and/or additionally be used to direct one or more of the blood and/or bone marrow into the centrifuge device <NUM> and still fall within the scope of the present disclosure.

In addition, the method <NUM> further includes pumping one or more of the bone marrow/platelet rich plasma fractions and the platelet poor plasma fractions created after centrifugation within the centrifuge device <NUM> into the first receptacle <NUM> of the concentration system <NUM>, as indicated at point <NUM> in <FIG>. In a similar manner, the method may further include pumping red blood cell fractions into the second receptacle <NUM> of the concentration system <NUM> as indicated at point <NUM>, for example.

The method <NUM> next includes 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 disposed within the first receptacle <NUM>, for example, as indicated at point <NUM>. The method further 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 <NUM> to isolate one or more of extracellular vesicles and platelet rich plasma/bone marrow concentrate fractions, as indicated at point <NUM>. In one example, the method <NUM> may further include pumping the aqeuous two-phase solution and extracellular vesicles poor plasma (EPP) back into the first receptacle <NUM>, as indicated at point <NUM>, for example in <FIG>. The method <NUM> then includes pumping one or more of the bone marrow concentrate/platelet rich plasma fractions and isolated extracellular vesicles into the syringe <NUM> for injection, as indicated at point <NUM>.

Referring now to <FIG>, another top view of the system <NUM> of <FIG> is depicted, illustrating another exemplary method <NUM> of isolating vesicles in accordance with another aspect of the present disclosure. Generally, and as alternative to the method <NUM> described above relative to <FIG>, to minimize a volume of aqueous two-phase solution being injected, one or more of the bone marrow concentrate or the platelet rich plasma concentrate may be isolated before the extracellular vesicles isolation. Advantageously, in this example, the volume of the extracellular vesicles can be determined, the concentration of aqueous two-phase solution reduced, and the effect of the aqueous two-phase solution upon the cells in the bone marrow concentrate/platelet rich plasma fractions is minimized.

More specifically, another method <NUM> of isolating vesicles using the system <NUM> of the present disclosure is described below. Like the method <NUM> described above, the method <NUM> includes loading one or more of blood or bone marrow into the input port <NUM> of the concentration system <NUM>, as indicated in part <NUM> of <FIG>. The method <NUM> then includes 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 the centrifuge device <NUM> of the concentration system <NUM>, as indicated in part <NUM>, for a first centrifugation. Unlike the method <NUM>, the method <NUM> then includes pumping only the bone marrow/platelet rich plasma fractions into a syringe, such as the isolation syringe <NUM> disposed adjacent to the top portion <NUM> of the compartment <NUM>, as indicated in part <NUM> of <FIG>. One of ordinary skill in the art will appreciate that only the bone marrow/platelet rich plasma fractions created after the first centrifugation may alternatively be pumped into another syringe or a receptacle different from the isolation syringe <NUM>, for example, and still fall within the scope of the present disclosure. In addition, the method <NUM> then includes pumping only the platelet poor plasma fractions into the first receptacle <NUM> of the concentration system <NUM>, as indicated in part <NUM>. In some examples, the method <NUM> may also further comprises pumping the red blood cells fractions into the second receptacle <NUM>, as indicated in part <NUM>.

Still referring to <FIG>, the method <NUM> also includes adding a concentrated aqueous two-phase solution to the platelet poor plasma fractions disposed within the first receptacle <NUM>, as indicated in part <NUM>, and then drawing the concentrated aqueous two-phase solution and the platelet poor plasma fractions back into the centrifuge device <NUM> to isolate one or more of extracellular vesicles, as indicated in part <NUM>, for example. In one example, drawing the concentrated aqueous two-phase solution and the platelet poor plasma fractions back into the centrifuge device <NUM> for a second centrifugation comprises isolating extracellular vesicles from the platelet poor plasma fractions, creating an extracellular vesicles pellet for injection, as explained more below.

Next, the method <NUM> (like the method <NUM>) may also include pumping the aqueous two-phase solution and extracellular vesicles-poor plasma into the first receptacle <NUM> after 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 for centrifugation, as indicated in part <NUM>.

In addition, the method <NUM> also includes pumping only the isolated extracellular vesicles into the syringe <NUM> disposed adjacent to the top portion <NUM> of the compartment <NUM> for injection, as indicated in part <NUM> of <FIG>. More specifically, the extracellular vesicles created from the centrifugation of the concentrated aqueous two-phase solution and the platelet poor plasma fractions drawn back into the centrifuge device <NUM>, for example, e.g., the second centrifugation, may be pumped into a syringe that was filled with the bone marrow concentrate/platelet rich plasma in the first centrifugation. In one example, this syringe may be the isolation syringe <NUM> disposed adjacent to the top portion <NUM> of the compartment <NUM>, as depicted in <FIG> and <FIG>. Thus, in this example, the concentrated aqueous two-phase solution is only added with the extracellular vesicles, greatly reducing any residual concentrated aqueous two-phase in the isolation syringe <NUM>.

As an example, <NUM> of extracellular vesicles may be added to <NUM> of bone marrow concentrate already disposed in the isolation syringe <NUM> (to be injected, for example), reducing the residual concentrated aqueous two-phase solution, such as PEG-DEX solution, <NUM>-fold. As such, by adding the concentrated aqueous two-phase solution, such as the PEG-DEX solution, to only the platelet poor plasma (as in part <NUM> of <FIG>), and then adding the extracellular vesicles created in the second centrifugation, e.g., platelet poor plasma-extracellular vesicles, to the bone marrow concentrate/platelet rich plasma fractions pumped into the syringe <NUM> (as in part <NUM> of <FIG>), the residual concentrated aqueous two-phase is reduced. Moreover, as the extracellular vesicles, e.g., platelet poor plasma-extracellular vesicles, are added to the isolation syringe <NUM>, the additional extracellular vesicles volume can be easily determined.

In another example, the method <NUM> may further comprise determining a volume of PEG-DEX solution to be injected based upon the volume of the bone marrow/platelet rich plasma fractions isolated in the syringe <NUM>. As a result, the concentration of aqueous two-phase solution used is reduced, minimizing the effect of the aqueous two-phase solution on nucleated cells in the bone marrow/platelet rich plasma fractions, for example.

Referring now to <FIG>, a perspective view of a syringe adapted to be used with the system <NUM> described above is depicted. More specifically, and in one example, the syringe of <FIG> may be the syringe <NUM> depicted in <FIG>, which is coupled to the first receptacle <NUM>, as described above. In this example, a concentrated aqueous two-phase aqueous two-phase solution is first disposed in the syringe <NUM>, as depicted in <FIG>. In addition, and as described above relative to method <NUM>, for example, the platelet poor plasma (PPP) is added to the syringe <NUM> having the aqueous two-phase solution, creating an aqueous two-phase solution, such as a PEG-DEX solution, and PPP solution <NUM> within the syringe <NUM>, as depicted in <FIG>. More specifically, and in one example, to get a final <NUM>% PEG-DEX concentration, if <NUM> of <NUM>% PEG-DEX is in the syringe <NUM>, <NUM> of platelet poor plasma is drawn into the syringe <NUM> for a total volume of <NUM>. More generally, an amount of platelet poor plasma may be added to the concentrated aqueous two-phase solution, such as drawn into the syringe <NUM>, such that the amount of the concentrated aqueous two-phase PEG-DEX solution is diluted to a working solution of about <NUM>% concentrated aqueous two-phase solution. In addition, and in another example, concentrated aqueous two-phase is added to the platelet poor plasma and allowed about five minutes of room temperature incubation, as described more below.

As depicted in <FIG>, the aqueous two-phase solution and PPP solution <NUM> within the syringe <NUM> is centrifuged in a centrifuge device, creating a pellet <NUM> of extracellular vesicles for injection or addition to one or more of platelet rich plasma or bone marrow concentrate for example. More specifically, and in one example, the syringe <NUM> with the aqueous two-phase and PPP solution is centrifuged within a centrifuge device able to hold the syringe <NUM> for a period of about <NUM> minutes at <NUM> x g, resulting in the isolation of the extracellular vesicles and creation of the extracellular vesicles pellet <NUM>.

More generally, another method <NUM> of isolating extracellular vesicles using the system <NUM> and the syringe <NUM> depicted in <FIG>, for example, includes disposing a concentrated aqueous two-phase solution in one or more of a syringe, such as the syringe <NUM>, or a receptacle of the system <NUM>. The method <NUM> further includes adding one or more of platelet poor plasma fractions or bone marrow/platelet rich plasma fractions into the concentrated aqueous two-phase solution, as depicted in the syringe <NUM> of <FIG>, for example. The method <NUM> also includes centrifuging the solution and one or more of the platelet poor plasma fractions or the bone marrow/platelet rich plasma fractions disposed in the syringe <NUM> to isolate the extracellular vesicles. As depicted in <FIG>, the method <NUM> still further includes creating a pellet <NUM> having the isolated extracellular vesicles, the pellet <NUM> for injection. In another example, the extracellular vesicles pellet <NUM> may be added to one or more of the bone marrow/platelet rich plasma fractions, for example.

As depicted in <FIG>, an alternative syringe <NUM> may be designed to accommodate one or both of only the extracellular vesicles pellet, as depicted in <FIG>, or the combination of the extracellular vesicles pellet and the bone marrow concentrate or platelet rich plasma, as depicted in <FIG>. More specifically, and referring now to <FIG>, the syringe <NUM> may include a body <NUM> having a distal end <NUM> and a proximal end <NUM>. A projection <NUM>, such as a cylindrical projection, extends from the distal end <NUM> of the body <NUM> and includes the extracellular vesicles pellet <NUM> formed by the centrifuging process described above. In this example, and unlike the syringe <NUM> of <FIG>, the syringe <NUM> of <FIG> receives the extracellular vesicles pellet <NUM> in the projection <NUM> disposed outside of the body <NUM> of the syringe <NUM> and not within the body <NUM>, as depicted in <FIG>, for example. Likewise, <FIG> depicts the projection <NUM> of the syringe <NUM> having a pellet <NUM> including both the extracellular vesicles and one or more of the bone marrow concentrate or platelet rich plasma. By having the extracellular pellets <NUM>, <NUM> disposed in the projection <NUM> of the syringe <NUM> and not the main body <NUM>, the extracellular vesicles concentrate can be more easily expelled from the syringe <NUM> while minimizing the volume of the extracellular vesicles-poor plasma and the aqueous two-phase solution, such as PEG-DEX, that is expelled from the syringe <NUM>, for example.

All of the methods <NUM>, <NUM>, <NUM> described above may further include premixing the PEG-DEX solution at a predetermined concentration before adding the concentrated aqueous two-phase solution to one or more of the bone marrow concentrate/platelet rich plasma fractions and platelet poor plasma fractions. In one example, premixing the PEG-DEX solution at a predetermined concentration includes premixing the PEG-DEX solution at a 10x concentration. In another example, premixing the PEG-DEX solution at a predetermined concentration includes premixing the PEG-DEX solution at a 5x concentration. In yet another example, premixing the PEG-DEX solution at a predetermined concentration includes premixing the PEG-DEX solution at an 8x concentration. In other examples, and as one of ordinary skill in the art will understand, the predetermined concentration may be any concentration within the range of 3x concentration to 15x concentration and still fall within the scope of the present disclosure. In some examples, premixing the PEG-DEX solution is essential for quick extracellular vesicles isolation. In addition, the methods <NUM>, <NUM>, <NUM> may include allowing a period of 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 fractions and platelet poor plasma fractions. In one example, the period of time for room temperature incubation is about five minutes. One of ordinary skill in the art will appreciate that the period of time may be more or slightly less than five minutes, such as three, four or four in a half minutes, and still fall within the scope of the present disclosure.

In addition, in each of the methods <NUM>, <NUM>, <NUM>, 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 may comprise adding the concentrated aqueous two-phase solution to one or more of the bone marrow concentrate/platelet rich plasma fractions and platelet poor plasma fractions based upon a volume of one or more of the bone marrow concentrate/platelet rich plasma fractions and platelet poor plasma fractions in the first receptacle <NUM>.

Referring now to <FIG>, various experimental results of the system <NUM> and methods <NUM>, <NUM>, and <NUM> of the present disclosure are depicted. More specifically, <FIG> is a perspective view of an aqueous two-phase solution and platelet poor plasma combination in a receptacle <NUM> before centrifugation. As indicated therein, before centrifugation no extracellular vesicles have been isolated. In this example, the aqueous two-phase solution is PEG-DEX. In addition, a <NUM>% PEG-DEX concentration when added to the platelet poor plasma enabled the extracellular vesicles isolation with a tabletop centrifuge, such as a centrifuge device able to hold the syringe, for example. In addition, <NUM>-<NUM> molecular weight dextran was employed and the centrifuge device was run for <NUM> minutes at <NUM> x g. As will be appreciated, various other percentage amounts of PEG-DEX concentration and, more generally, a first phase of the aqueous two-phase solution may alternatively be used and still fall within the scope of the present disclosure. Likewise, various other weights of dextran and, more generally, a second phase of the aqueous two-phase solution, may also be used and still fall within the scope of the present disclosure. Said another way, various combinations of PEG-DEX solution may be added to the platelet poor plasma to enable extracellular vesicle isolation 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 may be used to enable the extracellular vesicle isolation during centrifugation and still fall within the scope of the present disclosure.

Referring now to <FIG>, a perspective view of the receptacle <NUM> of <FIG> after a <NUM> minute centrifugation process, for example, is depicted. The process and the foregoing experimental parameters noted resulted in the isolation of extracellular vesicles, and the creation of the extracellular vesicles pellet <NUM>.

Referring now to <FIG>, the receptacle <NUM> is depicted with a PEG-DEX concentration and ddH<NUM>O combination disposed therein. Centrifugation was conducted, and no creation of extracellular vesicle pellets resulted.

Referring now to <FIG>, the receptacle <NUM> is depicted after centrifugation. In this experimental example, a PEG-DEX concentrate solution and platelet poor plasma combination was again disposed within the receptacle <NUM> before centrifugation. Before disposing the platelet poor plasma with the PEG-DEX concentrate solution, the platelet poor plasma was prespun for <NUM> minutes at <NUM> x g to completely remove any residual cells, for example. After centrifugation, an extracellular vesicles pellet <NUM> was still created and recovered, as depicted in <FIG>.

Referring now to <FIG>, a phase-contrast photograph of an exemplary extracellular vesicles pellet is depicted. Specifically, the extracellular vesicles pellet is resuspended at 20x magnification. No cells are depicted in this view, but extracellular vesicle clusters <NUM> can be seen.

In view of the foregoing, one of ordinary skill in the art will appreciate the following advantages of the system <NUM> and methods <NUM>, <NUM>, <NUM> of the present disclosure described above. For example, the system <NUM> and methods <NUM>, <NUM>, <NUM> can isolate extracellular vesicles from platelet poor plasma quickly and in a clinical setting. By isolating the extracellular vesicles quickly, the extracellular vesicles can be applied within the same clinical procedure that included 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 employed to enhance the efficacy of biological injections, or as a stand-alone biological therapeutic.

In addition, the system <NUM> enables the collection of extracellular vesicles from the platelet poor plasma fraction, which is typically unused, but is a substantial portion of the output of the centrifugation process by volume in conventional systems. Moreover, the syringes <NUM>, <NUM>, <NUM>, for example, of the system <NUM> may be designed such that the syringe <NUM>, <NUM>, <NUM> may be loaded into the centrifuge device <NUM> so no transfer of platelet poor plasma and the aqueous two-phase solution, such as the PEG-DEX solution, or the extracellular vesicles concentrate to a centrifuge tube is necessary. So configured, the risk of contamination is minimized, the risk for error or sample loss is reduced, and the procedure is faster.

Moreover, the system <NUM> is designed such that only one centrifuge device <NUM> is used, even though the biological sample is run through two centrifugation cycles, as explained more above. As a result, the need for an additional centrifugation device and additional centrifuge syringes is eliminated. In addition, the risk of contamination is further reduced, and the isolation procedure is faster.

Claim 1:
An ex vivo method of isolating extracellular vesicles comprising: loading one or more of blood or bone marrow into an input port (<NUM>) of a concentration system (<NUM>);
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 (<NUM>) of the concentration system;
pumping one or more of bone marrow/platelet rich plasma fractions and platelet poor plasma fractions into a first receptacle (<NUM>) of the concentration system, the first receptacle coupled to the centrifuge device;
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;
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 (i) extracellular vesicles or (ii) extracellular vesicles and platelet rich plasma/bone marrow concentrate fractions; and
pumping one or more of the bone marrow concentrate/platelet rich plasma fractions and isolated extracellular vesicles into a syringe (<NUM>) for injection.