Activation of immunocyte

Abstract

To provide a bone marrow container.SOLUTION: A bone marrow container can contain a composition containing nonionic hydrophilic branched polysaccharide at a specific density, where the composition contacts whole blood and then in the case of being centrifuged, the density is sufficient for allowing fluid migration of whole blood through nonionic hydrophilic branched polysaccharide without separating erythrocytes. The nonionic hydrophilic branched polysaccharide can have 20kDa or more of molecular weight. The composition can contain TLR2 ligand. The composition can be contained in a container. The container is aseptic.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] This application claims the benefit of U.S. Provisional Application No. 62 / 691,775, filed Jun. 29, 2018, which is hereby incorporated by reference in its entirety, and claims priority under PCT Rule 4.10 to U.S. National Application No. 16 / 160,006, filed Oct. 15, 2018. All applications identified in the application data sheet as having foreign or domestic priority claims filed with this application are hereby incorporated by reference in this specification under 37 CFR 1.57.

[0002] Some embodiments herein generally relate to methods, compositions, and manufacture for activation of immune cells. According to some embodiments, a composition comprising a nonionic hydrophilic branched polysaccharide and a Toll-like receptor 2 (TLR2) ligand is used to activate bone marrow-derived myeloid cells.

Background Art

[0003] Bone marrow has been used as an easily accessible source of hematopoietic stem cells (HSCs) and other regenerative cell types, such as mesenchymal stromal / stem cells (MSCs). Since bone marrow is a regenerative tissue, it can provide a useful source of renewable cells for regenerative medicine applications, particularly those where autologous cells are preferred.

[0004] Conventionally, bone marrow aspirates have been processed by separating the cellular components. Those methods have conventionally been regarded as valueless and may even be harmful in the context of regenerative medicine applications. By separating red blood cells (RBCs), the bone marrow can be reduced in volume. Generally, those separation methods have utilized the different physical properties of RBCs to enrich in the mononuclear cell fraction containing HSCs and MSCs.

Summary of the Invention

Means for Solving the Problems

[0005] According to some embodiments, a bone marrow container is described. The bone marrow container can include a composition comprising a non-ionic hydrophilic branched polysaccharide at a specific density, wherein when the composition is contacted with whole blood and centrifuged, the density is sufficient to allow fluid movement of the whole blood through the non-ionic hydrophilic branched polysaccharide without separating red blood cells. The non-ionic hydrophilic branched polysaccharide can have a molecular weight of 20 kDa or more. The composition can include a TLR2 ligand. The composition can be contained within the container. The container is sterile. According to some embodiments, the non-ionic hydrophilic branched polysaccharide has a molecular weight of at least 70 kDa. According to some embodiments, the non-ionic hydrophilic branched polysaccharide includes poly(sucrose-co-epichlorohydrin). According to some embodiments, the non-ionic hydrophilic branched polysaccharide has a density of less than 1 g per IL. According to some embodiments, the TLR2 ligand is selected from the group consisting of hyaluronan, hyaluronic acid, monosodium urate crystals, biglycan, endoplasmin, HMGB1, HSP60, HSP70, human heart myosin, zymosan, lipoteichoic acid, plasma-derived lipoproteins, and peptidoglycan containing lipopolysaccharide (LPS). According to some embodiments, the non-ionic hydrophilic branched polysaccharide comprises, consists essentially of, or consists of poly(sucrose-co-epichlorohydrin) at a density of less than 1 g per IL, and the TLR2 ligand comprises, consists essentially of, or consists of hyaluronic acid. According to some embodiments, the container is selected from the group consisting of a bag, a vacutainer, and a syringe. According to some embodiments, the bone marrow container further includes an integrated delivery device. According to some embodiments, the bone marrow container further includes an activation / sedimentation solution. According to some embodiments, the bone marrow container further includes dextran and / or hetastarch. According to some embodiments, dextran is present at a concentration of at least about 4%. According to some embodiments, the bone marrow container further includes a volume configured to receive bone marrow, wherein when bone marrow is present in the volume, the concentration of dextrin in the bone marrow and the composition is at least about 1%. According to some embodiments, the bone marrow container is configured to be received by the separation system described herein.

[0006] According to some embodiments, a separation system is described. The separation system can include a chassis including a cavity configured to receive a bone marrow container as described herein. The chassis can include tracks. The system can include a chassis including a cavity configured to receive the bone marrow container described herein. The chassis can include tracks. The system can include a shaft disposed on the tracks, wherein the shaft is configured to move along the tracks, whereby, when a bone marrow container is disposed within the chassis, the bone marrow container is compressed. According to some embodiments, the shaft is configured to press against the bone marrow container relative to the inner surface of the chassis. According to some embodiments, the shaft is selected from the group consisting of rollers and sliders. According to some embodiments, the shaft is configured to snap into a predetermined position relative to the bone marrow container disposed within the chassis, thereby defining at least two portions of the bone marrow container.

[0007] According to some embodiments, a kit is described. The kit can include a bone marrow container as described herein. The kit can further include a bone marrow aspiration needle, such as a Luer lock. According to some embodiments, the kit can further include a filter. The filter can have a pore size smaller than the diameter of bone marrow cells. According to some embodiments, the pore size is less than or equal to 200 microns. According to some embodiments, the kit can further include an activation / sedimentation solution. According to some embodiments, the kit can further include a separation system as described herein.

[0008] According to some embodiments, a method for activating immune cells of a subject is described. The method includes obtaining bone marrow of the subject. The bone marrow can contain immune cells. The method includes incubating the bone marrow with a nonionic hydrophilic branched polysaccharide and a TLR2 ligand. The nonionic hydrophilic branched polysaccharide can be present at a density such that when the bone marrow is contacted with whole blood and centrifuged, the density is sufficient to allow fluid movement of the whole blood through the nonionic hydrophilic branched polysaccharide without separating red blood cells. The nonionic hydrophilic branched polysaccharide can have a molecular weight of 20 kDa or more. The incubation can be performed until the immune cells are activated. For example, the incubation can be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 hours, including ranges between any two of the recited values. The method can further include administering the activated immune cells to the subject. According to some embodiments, the method further includes identifying a subject in need of activation of the immune cells. According to some embodiments, the subject has cancer. According to some embodiments, the incubation further includes incubating the bone marrow with a cancer cell antigen. According to some embodiments, the nonionic hydrophilic branched polysaccharide has a molecular weight of at least 70 kDa. According to some embodiments, the nonionic hydrophilic branched polysaccharide includes poly(sucrose-co-epichlorohydrin). According to some embodiments, the nonionic hydrophilic branched polysaccharide has a density of less than 1 g per IL. According to some embodiments, it is selected from the group consisting of hyaluronic acid, hyaluronic acid, sodium urate crystals, biglycan, endoplasmin, HMGB1, HSP60, HSP70, human heart myosin, zymosan, lipoteichoic acid, plasma-derived lipoproteins, lipopolysaccharide (LPS), and peptidoglycans including Pam2CSK4 and Pam3CSK4, and combinations of two or more of the recited items. According to some embodiments, the nonionic hydrophilic branched polysaccharide contains, consists essentially of, or consists of poly(sucrose-co-epichlorohydrin) at a density of less than 1 g per liter, and the TLR2 ligand contains, consists essentially of, or consists of hyaluronic acid.According to some embodiments, the non-ionic hydrophilic branched polysaccharide comprises, consists essentially of, or consists of poly(sucrose-co-epichlorohydrin) at a density of less than 1 g per liter, and the TLR2 ligand comprises, consists essentially of, or consists of hyaluronic acid. According to some embodiments, the incubation is performed in a bone marrow container as described herein. According to some embodiments, the method further comprises placing the bone marrow container in the separation system described herein and snapping the shaft into a predetermined position relative to the bone marrow container, thereby defining two portions of the bone marrow container. The method further comprises, after incubation, moving the shaft along a track, thereby compressing the contents in the bone marrow container and extruding immune cells from the bone marrow. According to some embodiments, the method further comprises incubating the bone marrow cells with an activation / sedimentation solution, thereby sedimenting red blood cells from the bone marrow. According to some embodiments, the activated immune cells comprise, consist essentially of, or consist of bone marrow cells. According to some embodiments, the activated immune cells are CD11b. + CD54 + Granulocytes and / or CD66b + including neutrophils. According to some embodiments, the method further comprises separating the activated immune cells from the non-ionic hydrophilic branched polysaccharide and the TLR2 ligand before administering the activated immune cells to a subject. According to some embodiments, the activated immune cells are administered to the subject's musculoskeletal tissue. According to some embodiments, the method does not include leukapheresis. According to some embodiments, the method exists ex vivo.

Brief Description of the Drawings

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Figure 1

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Figure 2

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Figure 3-1

[0012]

Figure 3-2

DETAILED DESCRIPTION OF THE INVENTION

[0013] It has been observed herein that nonionic hydrophilic branched polysaccharides, such as poly(sucrose-co-epichlorohydrin), in combination with a TLR2 ligand (e.g., hyaluronic acid), can activate immune cells, such as regenerative cells, in the bone marrow. The activated immune cells comprise, consist essentially of, or consist of myeloid lineages. Described herein according to embodiments are bone marrow containers, compositions, methods, and kits comprising a nonionic hydrophilic branched polysaccharide and a TLR2 ligand. The nonionic hydrophilic branched polysaccharide can have a molecular weight of at least 20 kDa. The nonionic hydrophilic branched polysaccharide can be at a density sufficient to allow fluid movement of whole blood through the nonionic hydrophilic branched polysaccharide without separating red blood cells. In contrast, conventional use of nonionic hydrophilic branched polysaccharides in blood processing typically performs those polysaccharides at higher concentrations (1.0 g / l or greater) for density-mediated separation of red blood cells from the mononuclear fraction (e.g., under centrifugation). According to some embodiments, the bone marrow container comprises a nonionic hydrophilic branched polysaccharide and a TLR2 ligand. Bone marrow aspirate is added to the container, and the immune cells can be activated by incubating the bone marrow aspirate with the nonionic hydrophilic branched polysaccharide and the TLR2 ligand in the container. According to some embodiments, a method of activating immune cells comprises incubating bone marrow (e.g., bone marrow aspirate) with the nonionic hydrophilic branched polysaccharide and the TLR2 ligand described herein.

[0014] As used herein, to "activate" immune cells has its ordinary customary meaning as understood by one of ordinary skill in the art in view of the present disclosure. It refers to the proliferation, maturation, mobilization (e.g., migration), metabolism or catabolism, and / or activity (e.g., cytokine, growth factor, and / or enzyme secretion) of immune cells.

[0015] Nonionic hydrophilic branched polysaccharide : Some embodiments of the bone marrow treatment containers, kits and methods include nonionic hydrophilic branched polysaccharides. Examples of suitable nonionic hydrophilic branched polysaccharides include, but are not limited to, poly(sucrose-co-epichlorohydrin), which is commercially available as FICOLL polysaccharide. It is contemplated herein that large branched polysaccharides having a molecular weight of at least 20 kDa can activate the innate immune system. Without being limited by theory, nonionic hydrophilic branched polysaccharides having a suitably high molecular weight are thought to have a structure that mimics the bacterial cell wall. According to some embodiments, the nonionic hydrophilic branched polysaccharide includes monomers of disaccharides or more, such as at least trisaccharides or more sugars. According to some embodiments, the nonionic hydrophilic branched polysaccharide has a molecular weight of at least 20 kDa, such as at least 20 kDa, 30 kDa, 40 kDa, 50 kDa, 60 kDa, 70 kDa, 80 kDa, 90 kDa, 100 kDa, 150 kDa, 200 kDa or 500 kDa, such as in the range between any two of the recited values, such as 20 - 500 kDa, 20 - 200 kDa, 20 - 100 kDa, 20 - 80 kDa, 50 - 500 kDa, 50 - 200 kDa, 50 - 100 kDa, 50 - 80 kDa, 70 - 500 kDa, 70 - 200 kDa, 70 - 100 kDa, and 70 - 80 kDa.

[0016] Nonionic hydrophilic branched polysaccharides have conventionally been used at high densities (e.g., by centrifugation) to separate red blood cells from blood, but it is contemplated herein that ionic hydrophilic branched polysaccharides may be useful for activating immune cells at much lower densities. According to some embodiments, the nonionic hydrophilic branched polysaccharide is present at a high density such that, when contacted with whole blood and centrifuged, the whole blood can flow through the nonionic hydrophilic branched polysaccharide without separating the red blood cells. According to some embodiments, the nonionic hydrophilic branched polysaccharide has a density of less than 1 g per IL, e.g., 1 g / L, 0.99 g / L, 0.9 g / L, 0.8 g / L, 0.7 g / L, 0.6 g / L, 0.5 g / L, 0.4 g / L, 0.3 g / L, 0.2 g / L, 0.1 g / L, 0.05 g / L, 10 -2 g / L, 2 x 10 -3 g / L, 10 -3 g / L, 10 -5 g / L, 2x 10 -6 g / L, 10 -6 g / L, 10 -7 g / L, 10 -8 g / L, 2 x 10 -9 g / L, or 10 -9 g / L or less, e.g., a range between any two of the recited values, e.g., 10 -9 -10 -6 g / L, 10 - 9 -10 -3 g / L, 10 -9 -10 -2 g / L, 10 -9 -0.1 g / L, 10 -9 -0.5g / L, 10 -9 -0.9g / L, 10 -9 -0.09g / L, 10 -6 -10 -3 g / L, 10 -6 -10 -2 g / L, 10 -6 -0.1g / L, 10 -6 -0.5g / L, 10 -6 -0.9g / L, 10 -6 -0.09g / L, 2x10-6 -10 -2 g / L, 2x10 -6 -0.1 g / L, 2x10 -6 -0.5g / L, 2x10 -6 -0.9g / L, 2x10 -6 -0.09g / L, 10 -3 -10 -2 g / L, 10 -3 -0.1g / L, 10 -3 -0.5g / L, 10 -3 -0.9g / L, 10 -3 -0.09g / L, 0.002 - 0.01g / L, 0.002 - 0.1g / L, 0.002 - 0.5g / L, 0.002 - 0.9g / L, 0.002 - 0.09g / L, 0.01g / L - 0.99g / L, 0.01 g / L - 0.9g / L, 0.01g / L - 0.5g / L, 0.01g / L - 0.2g / L, 0.01g / L - 0.1g / L, 0.1g / L - 0.99g / L, 0.1g / L - 0.9g / L, 0.1 g / L - 0.5g / L, 0.1g / L - 0.2g / L, 0.5g / L - 0.99g / L, 0.5g / L - 0.9g / L, or 0.5g / L - 0.7g / L density.

[0017] TLR2 ligand : TLR2 ligands can activate bone marrow cells. Without being limited by theory, TLR ligands are thought to be able to activate bone marrow cells to improve their therapeutic effects. Accordingly, some embodiments of the bone marrow treatment containers, kits, compositions, and methods include TLR2 ligands. Examples of suitable TLR2 ligands for some of the methods, compositions, bone marrow containers, and kits herein include, but are not limited to: hyaluronan, hyaluronic acid, monosodium urate crystals, biglycan, endoplasmin, HMGB1, HSP60, HSP70, human cardiac myosin, zymosan, lipoteichoic acid, plasma-derived lipoproteins, LPS, peptidoglycans including Pam2CSK4 and Pam3CSK4, and combinations of two or more of the listed items. According to some embodiments, the TLR2 ligand comprises, consists essentially of, or consists of hyaluronic acid. In some embodiments of the bone marrow treatment containers, kits, compositions, and methods, the TLR2 ligand is present at a concentration of at least 1 μg / ml, such as at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 50, 100, or 500 μg / ml, or at least 1 mg / ml, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg / ml, such as in a range between any two of the listed values, such as 1-10 μg / ml, 1-100 μg / ml, 1 μg / ml-1 mg / ml, 1 μg / ml-10 mg / ml, 10-100 μg / ml, 10 μg / ml-1 mg / ml, 10 μg / ml-10 mg / ml, 100 μg / ml-1 mg / ml, 100 μg / ml-10 mg / ml, or 1-10 mg / ml.

[0018] Activation / precipitation solution : Some embodiments include an activation / sedimentation solution. The activation / sedimentation solution can sediment red blood cells from bone marrow, thus reducing the bone marrow and increasing the concentration of activated immune cells. Dextran and / or hydroxyethyl starch (HES) can sediment red blood cells. Thus, according to some embodiments, the activation / sedimentation solution comprises, consists essentially of, or consists of dextran and / or HES. According to some embodiments, the activation / sedimentation solution comprises, consists essentially of, or consists of HES. According to some embodiments, the activation / sedimentation solution comprises, consists essentially of, or consists of dextran. According to some embodiments, dextran and / or HES are present at a concentration such that, based on the addition of bone marrow, the concentration of dextran and / or HES is at least 1%, at least 1%, 2%, 3% or 4%, for example in a range between any two of the recited values, for example 1-4%, 1-3%, 1-2%, 2-3%, 2-4% or 3-4%. The activation / sedimentation solution may be suitable for incubation with bone marrow and a composition (comprising a non-ionic hydrophilic branched polysaccharide and a TLR2 ligand) in a bone marrow container to sediment red blood cells and thus reduce the activated cells. According to some embodiments, dextran and / or HES have a high molecular weight. Dextran and / or HES are of the 510(k)-compatible grade.

[0019] According to some embodiments, the activation / sedimentation solution is separate from the composition comprising the non-ionic hydrophilic branched polysaccharide and the TLR2 ligand. For example, the activation / sedimentation solution is separate from the bone marrow container as described herein. According to some embodiments, the activation / sedimentation solution is part of the composition comprising the non-ionic hydrophilic branched polysaccharide and the TLR2 ligand as described herein.

[0020] Composition : According to some embodiments, the composition comprises a non-ionic hydrophilic branched polysaccharide as described herein and a TLR2 ligand as described herein. The non-ionic hydrophilic branched polysaccharide can have a molecular weight of at least 20 kDa, as described herein. The non-ionic hydrophilic branched polysaccharide can have a density of 1 g / L or less, as described herein. According to some embodiments, the non-ionic hydrophilic branched polysaccharide comprises, consists essentially of, or consists of poly(sucrose-co-epichlorohydrin). According to some embodiments, the TLR2 ligand comprises, consists essentially of, or consists of hyaluronic acid. According to some embodiments, the composition is included in a solution (which can comprise, consist essentially of, or consist of the composition), for example an aqueous solution. According to some embodiments, the composition is a solution, for example an aqueous solution. According to some embodiments, the non-ionic hydrophilic branched polysaccharide has a density of less than 1 g per IL, for example 1 g / L, 0.99 g / L, 0.9 g / L, 0.8 g / L, 0.7 g / L, 0.6 g / L, 0.5 g / L, 0.4 g / L, 0.3 g / L, 0.2 g / L, 0.1 g / L, 0.05 g / L, 10 -2 g / L, 2 x 10 -3 g / L, 10 -3 g / L, 10 -5 g / L, 2x 10 -6 g / L, 10 -6 g / L, 10 -7 g / L, 10 -8 g / L, 2 x 10 -9 g / L, or 10 -9 g / L or less, for example a range between any two of the listed values, for example 10 -9 -10 -6 g / L, 10 - 9 -10 -3 g / L, 10 -9 -10 -2 g / L, 10 -9 -0.1 g / L, 10 -9 -0.5g / L, 10 -9 -0.9g / L, 10 -9 -0.09g / L, 10-6 -10 -3 g / L, 10 -6 -10 -2 g / L, 10 -6 -0.1 g / L, 10 -6 -0.5 g / L, 10 -6 -0.9 g / L, 10 -6 -0.09 g / L, 2x10 -6 -10 -2 g / L, 2x10 -6 -0.1 g / L, 2x10 -6 -0.5 g / L, 2x10 -6 -0.9 g / L, 2x10 -6 -0.09 g / L, 10 -3 -10 -2 g / L, 10 -3 -0.1 g / L, 10 -3 -0.5 g / L, 10 -3 -0.9 g / L, 10 -3 -0.09 g / L, 0.002 - 0.01 g / L, 0.002 - 0.1 g / L, 0.002 - 0.5 g / L, 0.002 - 0.9 g / L, 0.002 - 0.09 g / L, 0.01 g / L - 0.99 g / L, 0.01 g / L - 0.9 g / L, 0.01 g / L - 0.5 g / L, 0.01 g / L - 0.2 g / L, 0.01 g / L - 0.1 g / L, 0.1 g / L - 0.99 g / L, 0.1 g / L - 0.9 g / L, 0.1 g / L - 0.5 g / L, 0.1 g / L - 0.2 g / L, 0.5 g / L - 0.99 g / L, 0.5 g / L - 0.9 g / L, or 0.5 g / L - 0.7 g / L density. According to some embodiments, the TLR2 ligand is present at a concentration of at least 1 μg / ml, such as at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 50, 100 or 500 μg / ml, or at least 1 mg / ml, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg / ml, such as in a range between any two of the recited values, such as 1 - 10 μg / ml, 1 - 100 μg / ml, 1 μg / ml - 1 mg / ml, 1 μg / ml - 10 mg / ml, 10 - 100 μg / ml, 10 μg / ml - 1 mg / ml, 10 μg / ml - 10 mg / ml, 100 μg / ml - 1 mg / ml, 100 μg / ml - 10 mg / ml or 1 - 10 mg / ml.

[0021] Although not limited by theory, nonionic hydrophilic branched polysaccharides can activate the innate immune response, and TLR2 ligands are thought to be able to activate myeloid cells. According to some embodiments, the composition comprises a nonionic hydrophilic branched polysaccharide in an amount sufficient to activate the innate immune response. According to some embodiments, the composition comprises a TLR2 ligand in an amount sufficient to activate myeloid cells. According to some embodiments, the composition comprises a nonionic hydrophilic branched polysaccharide and a YLR2 ligand in an amount sufficient to activate myeloid cells and stimulate the innate immune response. According to some embodiments, the composition is CD66b + comprises a nonionic hydrophilic branched polysaccharide in an amount sufficient to activate neutrophils.

[0022] According to some embodiments, the product further comprises an activation / precipitation solution as described herein.

[0023] According to some embodiments, the composition is for use in activating immune cells as described herein, such as myeloid immune cells. According to some embodiments, the use is an in vitro use.

[0024] Bone marrow container : It is thought that a bone marrow container containing a nonionic hydrophilic branched polysaccharide and a TLR2 ligand as described herein can activate immune cells in the bone marrow. The bone marrow container can be useful for stimulating the immune response in the autologous bone marrow of a patient in need of an immune response and / or for hydrating a transplantable gel or pate (e.g., demineralized bone matrix (DBM)) prior to transplantation. Thus, according to some embodiments, bone marrow containers are described. The bone marrow container can comprise a composition comprising a nonionic hydrophilic branched polysaccharide as described herein and a TLR2 ligand as described herein. The nonionic hydrophilic branched polysaccharide can be present at a density sufficient to allow fluid movement of whole blood through the nonionic hydrophilic branched polysaccharide without separating red blood cells when the composition is contacted with whole blood and centrifuged. The reference centrifugation parameters can be, for example, at least 50 g, for example, at least 100 g, 200 g, 300 g or 400 g. The nonionic hydrophilic branched polysaccharide can have a molecular weight of 20 kDa or more, for example, at least 20 kDa, for example, at least 20 kDa, 30 kDa, 40 kDa, 50 kDa, 60 kDa, 70 kDa, 80 kDa, 90 kDa, 100 kDa, 150 kDa, 200 kDa or 500 kDa, for example, in the range between any two of the recited values, for example, 20 - 50 kDa, 20 - 100 kDa, 20 - 200 kDa, 20 - 500 kDa, 50 - 100 kDa, 50 - 200 kDa, 50 - 500 kDa, 70 - 100 kDa, 70 - 200 kDa, 70 - 500 kDa, 100 - 200 kDa or 100 - 500 kDa. The composition can be contained in the bone marrow container. The bone marrow container can be sterile. According to some embodiments, the bone marrow container is configured to be inserted into a separation system as described herein. According to some embodiments, the nonionic hydrophilic branched polysaccharide is present at a density of less than 1 g per liter. According to some embodiments, the nonionic hydrophilic branched polysaccharide comprises poly(sucrose-co-epichlorohydrin) at a density of less than 1 g per liter, and the TLR2 ligand comprises hyaluronic acid.

[0025] Exemplary bone marrow containers of some embodiments are shown in FIG. 3A. The bone marrow container 100 can include, consist essentially of, or consist of a composition 110 comprising a nonionic hydrophilic branched polysaccharide and a TLR2 ligand as described herein. The composition 110 can be disposed inside the bone marrow container 100. The interior of the bone marrow container can be sterile. The bone marrow container 100 can further include an inlet 120 and an outlet 130. The inlet and outlet can be arranged to fluidly communicate the interior of the bone marrow container 100 with the exterior. According to some embodiments, the inlet 120 includes a channel and / or a valve. According to some embodiments, the inlet 120 can be sealed by pressure, friction, or an adhesive. According to some embodiments, the outlet 130 includes a channel and / or a valve. According to some embodiments, the outlet 130 can be sealed, for example, by pressure, friction, or an adhesive. According to some embodiments, the inlet 120 and the outlet 130 have the same structure. According to some embodiments, the inlet 120 and the outlet 130 have different structures. According to some embodiments, the bone marrow container 100 further includes an integrated delivery device (not shown), such as an intravenous needle. The integrated delivery device can be in fluid communication with the outlet 130 and can be configured to administer activated immune cells from the container to the subject in need thereof.

[0026] According to some embodiments, the bone marrow container is selected from the group consisting of a bag, a vacutainer, and a syringe. According to some embodiments, after the bone marrow is incubated in the bone marrow container to activate immune cells as described herein, the activated immune cells are administered to the subject in need thereof. The immune cells can be autologous. Thus, according to some embodiments, the bone marrow container further includes a delivery device, such as a needle, such as an intravenous needle. According to some embodiments, the delivery device is essential to the bone marrow container. According to some embodiments, the delivery device is configured to be attached to and optionally removed from the bone marrow container. For example, the delivery device can be configured to be disposed in fluid communication with the outlet of the bone marrow container.

[0027] According to some embodiments, the bone marrow container is configured to receive a dose of 2 to 4 ml in a single aspiration and up to about 25 ml in a single aspiration. Thus, the bone marrow container can have a volume for containing the aspirated bone marrow and a composition comprising a non-ionic hydrophilic branched polysaccharide and a TLR2 ligand. Thus, according to some embodiments, the bone marrow container has a volume of at least 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90 or 100 ml, for example in a range between any two of the recited values, such as 2 - 25 ml, 2 - 30 ml, 2 - 40 ml, 2 - 50 ml, 2 - 70 ml, 2 - 100 ml, 5 - 25 ml, 5 - 30 ml, 5 - 40 ml, 5 - 50 ml, 5 - 70 ml, 5 - 100 ml, 10 - 25 ml, 10 - 30 ml, 10 - 40 ml, 10 - 50 ml, 10 - 70 ml, 10 - 100 ml, 20 - 25 ml, 20 - 30 ml, 20 - 40 ml, 20 - 50 ml, 20 - 70 ml, 20 - 100 ml, 50 - 70 ml or 50 - 100 ml.

[0028] It is advantageous to remove some or all of the red blood cells from the bone marrow containing the activated immune cells described herein, for example to increase the concentration of activated mononuclear immune cells in the bone marrow. According to some embodiments, the bone marrow container is further configured for sedimentation of red blood cells. Thus, according to some embodiments, the bone marrow container contains an activation / sedimentation solution as described herein. The activation / sedimentation solution can comprise dextran and / or hespan. According to some embodiments, the bone marrow container contains a volume configured to receive the bone marrow, and when the bone marrow is present in that volume, the concentration of dextran and / or hespan in the combined bone marrow and activation / sedimentation solution is at least about 1%.

[0029] According to some embodiments, the bone marrow container is a point-of-care device. Thus, according to some embodiments, the bone marrow container is portable.

[0030] Separation system : The separation system may be useful in combination with a bone marrow container as described herein to prepare concentrated activated immune cells after the bone marrow has been incubated in the bone marrow container.

[0031] Exemplary separation systems of some embodiments are shown in FIGS. 3B and 3C. FIG. 3B shows the separation system 200 in a first configuration (prior to separation of the bone marrow within the bone marrow container 100). FIG. 3C shows the separation system 200 in a second configuration (after separation of the bone marrow within the bone marrow container 100).

[0032] Referring to FIG. 3B, the separation system 200 is configured to receive bone marrow as described herein. The separation system 200 can include a chassis 205 that includes a cavity 210 configured to contain the bone marrow container 100. The chassis 205 can further include a track 220. The track 220 can be disposed along the axis of the cavity 210. A shaft 230 can be disposed on the track. Optionally, since the shaft 230 is in mechanical communication with a handle 235, the shaft 230 can be moved along the track 220 via the handle 235. Thus, when the shaft 230 moves along the track 220, the shaft 230 can pass through the cavity. According to some embodiments, the shaft 230 is selected from the group consisting of a roller and a slider. According to some embodiments, the shaft 230 is configured to snap into a predetermined position relative to the bone marrow container 100 disposed within the chassis 205, thus defining at least two portions of the bone marrow container. According to some embodiments, the shaft 230 is configured to compress the bone marrow container 100 against the inner surface of the chassis 205. According to some embodiments, the separation system 200 includes a cavity 210 configured to receive the bone marrow container 100 as described herein, and the chassis includes a track 220. The separation system further includes a shaft 230 disposed on the track 220, where the shaft 230 is configured to move along the track 220, and thus compress the bone marrow container 100 when the bone marrow container 100 is disposed in the chassis 205. According to some embodiments, the chassis 205 is plastic.

[0033] Referring to FIG. 3C, when the bone marrow container 100 is disposed within the cavity 220, the inlet 120 and the outlet 130 may remain accessible. As the shaft 230 moves along the track 220 and passes through the cavity 210, the shaft 230 can compress the bone marrow container 100 therein. Movement of the shaft 230 along the track towards the outlet 130 can extrude the activated immune cells from the bone marrow container 100 through the outlet. According to some embodiments, an activation / sedimentation solution (which can include, consist essentially of, or consist of dextran and / or hetastarch) can be added through the inlet 120, mixed with the composition 110, and incubated to enable activation of immune cells as described herein and also sedimentation of other components such as red blood cells. Next, the shaft 230 moves along the track 220, for example, by pulling a handle, thus compressing the bone marrow container 100 and extruding the activated immune cells through the outlet 130. According to some embodiments, the incubation is at least 0.5 hour, 1 hour, 1.5 hours or 2 hours.

[0034] Kit : According to some embodiments, a kit is described. The kit can include a bone marrow container as described herein. The bone marrow container can include a composition comprising a nonionic hydrophilic branched polysaccharide as described herein and a TLR2 ligand as described herein. The kit can further include a bone aspiration needle, such as a Luer lock. The bone aspiration needle is suitable for administering a bone marrow aspirate through the inlet of the bone marrow container.

[0035] According to some embodiments, the kit further includes a filter. The filter may be suitable for separating bone marrow cells from other components of the bone marrow. Thus, according to some embodiments, the filter has a pore size smaller than the diameter of the bone marrow cells. According to some embodiments, the filter is 200 microns, 150 microns, 100 microns or 50 microns or less, or equal, for example, in the range between any two of the listed values, such as 50 - 200 microns, 50 - 150 microns, or 100 - 200 microns. According to some embodiments, the filter can be used to retain bone marrow cells while disposing of the bone marrow container and smaller components of the bone marrow (e.g., the filter is disposed in a liquid path including the interior and outlet of the bone marrow container, such that substances smaller than the bone marrow cells can be discharged from the outlet while retaining the bone marrow cells).

[0036] According to some embodiments, the kit further includes an activation / sedimentation solution. According to some embodiments, the activation / sedimentation solution is disposed inside the bone marrow container of the kit. According to some embodiments, the activation / sedimentation solution is disposed separately from the bone marrow container, for example, in a separate container. A dried and / or lyophilized precursor to the activation / sedimentation solution can also be appropriately reconstituted as the activation / sedimentation solution based on the addition of a suitable fluid, such as bone marrow. Thus, the activation / sedimentation "solution" is referred to herein as an ellipsis, but it will also be understood that suitable dried and / or lyophilized precursors are also contemplated.

[0037] According to some embodiments, the kit further includes plasmin. Plasmin is useful for the coagulation and delivery of activated immune cells.

[0038] According to some embodiments, the kit further includes another system as described herein.

[0039] Method for activating immune cells : Some embodiments include a method of activating immune cells of a subject. The method includes obtaining bone marrow of the subject. The bone marrow can contain immune cells. The method can include incubating the bone marrow with a composition comprising a nonionic hydrophilic branched polysaccharide and a TLR2 ligand, such as a composition comprising a nonionic hydrophilic branched polysaccharide and a TLR2 ligand described herein. The nonionic hydrophilic branched polysaccharide can have a molecular weight of 20 kDa or greater. The nonionic hydrophilic branched polysaccharide can be present at a density sufficient to allow fluid movement of whole blood through the nonionic hydrophilic branched polysaccharide without separating red blood cells when the bone marrow is contacted with whole blood and centrifuged. Incubation can be performed until the immune cells are activated. For example, the incubation can be for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 hours, such as in a range between any two of the recited values. For example, according to some embodiments, it has been shown that incubating bone marrow overnight with a nonionic hydrophilic branched polysaccharide and a TLR2 ligand can activate immune cells (see Example 1). The method can include administering the activated immune cells to the subject. Thus, the method of some embodiments delivers autologous activated immune cells to the subject. According to some embodiments, the density of the nonionic hydrophilic branched polysaccharide is 1 g or less per liter. According to some embodiments, the nonionic hydrophilic branched polysaccharide and the TLR2 ligand are placed in a bone marrow container as described herein, and the incubation is performed in the bone marrow container. According to some embodiments, the activated immune cells comprise, consist essentially of, or consist of bone marrow cells. According to some embodiments, the activated immune cells comprise, consist essentially of, or consist of CD66b + positive neutrophils. According to some embodiments, the bone marrow (and the activated immune cells) are autologous to the subject. According to some embodiments, the bone marrow (and the activated immune cells) are allogeneic, for example, if the patient is deficient in the production and / or activity of bone marrow and / or immune cells.

[0040] According to some embodiments, the method further includes identifying a subject in need of immune cell activation. According to some embodiments, the subject has cancer. Activation of immune cells in cancer patients may be useful in initiating an immune response against the cancer. Furthermore, activation of immune cells in the presence of cancer cell antigens is thought to be able to activate an immune response targeting those tumor cells. Thus, according to some embodiments, the method includes identifying a subject as having cancer. The method further includes incubating bone marrow with cancer cell antigens, such as cancer cell antigens present on the tumor cells of the subject's cancer. The incubation can be performed in a bone marrow container as described herein. According to some embodiments, the cancer cell antigen is constituted by a composition that also includes a nonionic hydrophilic branched polysaccharide and a TLR2 ligand. Next, the activated immune cells can be administered to a subject having cancer. According to some embodiments, the activated immune cells are administered to the subject's musculoskeletal tissue.

[0041] In the method of some embodiments, the nonionic hydrophilic branched polysaccharide has a molecular weight of at least 20 kDa, 30 kDa, 40 kDa, 50 kDa, 60 kDa, 70 kDa, 80 kDa, 90 kDa, or 100 kDa, such as in the range of any two of the recited values. According to some embodiments, the nonionic hydrophilic branched polysaccharide comprises, consists essentially of, or consists of poly(sucrose-co-epichlorohydrin). According to some embodiments, the nonionic hydrophilic branched polysaccharide comprises, consists essentially of, or consists of FICOLL polysaccharide. In the method of some embodiments, the nonionic hydrophilic branched polysaccharide is 1 g or less per liter, such as 1 g / L, 0.99 g / L, 0.9 g / L, 0.8 g / L, 0.7 g / L, 0.6 g / L, 0.5 g / L, 0.4 g / L, 0.3 g / L, 0.2 g / L, 0.1 g / L, 0.05 g / L, 10 -2 g / L, 2 x 10 -3 g / L, 10 -3 g / L, 10 -5 g / L, 2x 10 -6g / L, 10 -6 g / L, 10 -7 g / L, 10 -8 g / L, 2 x 10 -9 g / L, or 10 -9 g / L or less, for example, the range between any two of the listed values, for example 10 -9 -10 -6 g / l, 10 -9 -10 -3 g / l, 10 -9 -10 -2 g / l, 10 -9 -0.1 g / l, 10 -9 -0.5 g / l, 10 -9 -0.9 g / l, 10 -9 -0.09 g / l, 10 -6 -10 -3 g / l, 10 -6 -10 -2 g / l, 10 -6 - 0.1 g / l, 10 -6 -0.5 g / l, 10 -6 -0.9 g / l, 10 -6 -0.09 g / l, 10 -3 -10 -2 g / l, 10 -3 -0.1g / l, 10 -3 -0.5 g / l, 10 -3 -0.9 g / l, 10 -3 -0.09 g / l, 0.01 g / l - 0.99 g / l, 0.01 g / l - 0.9 g / l, 0.01 g / l - 0.5 g / l, 0.01 g / l - 0.2 g / l, 0.01 g / l - 0.1 g / l, 0.1 g / l - 0.99 g / l, 0.1 g / l - 0.9 g / l, 0.1 g / l - 0.5 g / l, 0.1 g / l - 0.2 g / l, 0.5 g / l - 0.99 g / l, 0.5 g / l - 0.9 g / l and 0.5 g / l - 0.7 g / l.

[0042] In the methods of some embodiments, the TLR2 ligand is selected from the group consisting of hyaluronan, hyaluronic acid, monosodium urate crystals, biglycan, endoplasmin, HMGB1, HSP60, HSP70, human cardiac myosin, zymosan, lipoteichoic acid, plasma-derived lipoproteins, LPS, and peptidoglycans including Pam2CSK4 and Pam3CSK4, and combinations of two or more of the listed items.

[0043] In the methods of some embodiments, the nonionic hydrophilic branched polysaccharide and the TLR2 ligand include hyaluronic acid. In the methods of some embodiments, the nonionic hydrophilic branched polysaccharide is included at a density of 1 g or less per liter, and the TLR ligand includes hyaluronic acid.

[0044] According to some embodiments, the incubation is performed in a bone marrow container as described herein. According to some embodiments, the incubation is for at least 0.5 hour, 1 hour, 1.5 hours, 2 hours, or 3 hours, such as a time in the range between any two of the recited values, such as 0.5 - 1, 0.5 - 2, 0.5 - 3, 1 - 2, 1 - 3, 1.5 - 2, 1.5 - 3, or 2 - 3 hours.

[0045] According to some embodiments, the method further includes placing the bone marrow container in a separation system as described herein. The method further includes snapping a shaft into a predetermined position relative to the bone marrow container, thus defining two portions of the bone marrow container. After incubation, the method includes moving the shaft along a track, thus compressing the contents of the bone marrow container and extruding immune cells from the bone marrow. According to some embodiments, the immune cells are extruded through an outlet of the bone marrow container. Next, the extruded immune cells can be administered to the desired subject.

[0046] According to some embodiments, the method further comprises contacting the bone marrow with an activation / sedimentation solution and thus sedimenting red blood cells from the bone marrow. The activation / sedimentation solution comprises, consists essentially of, or consists of dextran and / or hetastarch, as described herein. According to some embodiments, the incubation is performed in a bone marrow container as described herein, and the activation / sedimentation solution is present in the bone marrow container at the time it is placed therein. According to some embodiments, the incubation is performed in a bone marrow container as described herein, and the activation / sedimentation solution is added to the bone marrow container simultaneously with the bone marrow aspirate or after the bone marrow aspirate has been placed in the container.

[0047] According to some embodiments, the activated immune cells of the bone marrow are bone marrow cells such as CD11b + CD54 + granulocytes and / or CD66b + neutrophils, comprising, consisting essentially of, or consisting of the same. According to some embodiments, the activated immune cells are CD11b + CD54 + granulocytes, comprising, consisting essentially of, or consisting of the same. According to some embodiments, the activated immune cells are CD66b + neutrophils, comprising, consisting essentially of, or consisting of the same. According to some embodiments, the activated immune cells are CD11b + CD54 + granulocytes and CD66b + neutrophils, comprising, consisting essentially of, or consisting of the same.

[0048] According to some embodiments, the method further comprises separating the activated immune cells from a nonionic hydrophilic branched polysaccharide and a TLR2 ligand before administering the activated immune cells to a subject. According to some embodiments, the separation is through filtration. According to some embodiments, the separation is through a separation system as described herein.

[0049] According to some embodiments, the method does not include leukapheresis.

[0050] Additional embodiments : In addition to the above items, the following options are shown: 1. A composition comprising: A nonionic hydrophilic branched polysaccharide at a specific density, where when the composition is contacted with whole blood and centrifuged, the density is sufficient to allow fluid movement of the whole blood through the nonionic hydrophilic branched polysaccharide without separating red blood cells; A nonionic hydrophilic branched polysaccharide having a molecular weight of 20 kDa or more; and A TLR2 ligand, A bone marrow container aseptically containing therein the composition comprising the above. 2. The bone marrow container of option 1, wherein the nonionic hydrophilic branched polysaccharide has a molecular weight of at least 70 kDa. 3. The bone marrow container of option 1 or 2, wherein the nonionic hydrophilic branched polysaccharide comprises poly(sucrose-co-epichlorohydrin). 4. The nonionic hydrophilic branched polysaccharide is less than 1 g per liter, for example 1 g / L, 0.99 g / L, 0.9 g / L, 0.8 g / L, 0.7 g / L, 0.6 g / L, 0.5 g / L, 0.4 g / L, 0.3 g / L, 0.2 g / L, 0.1 g / L, 0.05 g / L, 10 -2 g / L, 2 x 10 -3 g / L, 10 -3 g / L, 10 -5 g / L, 2x 10 -6 g / L, 10 -6 g / L, 10 -7 g / L, 10 -8 g / L, 2 x 10 -9 g / L, or less than 10 -9 g / L, for example a range between any two of the listed values, for example 10 -9 -10 -6 g / L, 10 - 9 -10 -3 g / L, 10 -9 -10 -2g / L, 10 -9 -0.1 g / L, 10 -9 -0.5 g / L, 10 -9 -0.9 g / L, 10 -9 -0.09 g / L, 10 -6 -10 -3 g / L, 10 -6 -10 -2 g / L, 10 -6 -0.1 g / L, 10 -6 -0.5 g / L, 10 -6 -0.9 g / L, 10 -6 -0.09 g / L, 2x10 -6 -10 -2 g / L, 2x10 -6 -0.1 g / L, 2x10 -6 -0.5 g / L, 2x10 -6 -0.9 g / L, 2x10 -6 -0.09 g / L, 10 -3 -10 -2 g / L, 10 -3 -0.1 g / L, 10 -3 -0.5 g / L, 10 -3 -0.9 g / L, 10 -3 -0.09 g / L, 0.002 - 0.01 g / L, 0.002 - 0.1 g / L, 0.002 - 0.5 g / L, 0.002 - 0.9 g / L, 0.002 - 0.09 g / L, 0.01 g / L - 0.99 g / L, 0.01 g / L - 0.9 g / L, 0.01 g / L - 0.5 g / L, 0.01 g / L - 0.2 g / L, 0.01 g / L - 0.1 g / L, 0.1 g / L - 0.99 g / L, 0.1 g / L - 0.9 g / L, 0.1 g / L - 0.5 g / L, 0.1 g / L - 0.2 g / L, 0.5 g / L - 0.99 g / L, 0.5 g / L - 0.9 g / L, or 0.5 g / L - 0.7 g / L density, any one of Option 1 - 3 bone marrow containers. 5. The TLR2 ligand is selected from the group consisting of hyaluronic acid, hyaluronic acid, monosodium urate crystals, biglycan, endoplasmin, HMGB1, HSP60, HSP70, human heart myosin, zymosan, lipoteichoic acid, plasma-derived lipoproteins, lipopolysaccharide (LPS), and peptidoglycans including Pam2CSK4 and Pam3CSK4, any one of Option 1 - 4 bone marrow containers. 6. The bone marrow container of Option 1, wherein the nonionic hydrophilic branched polysaccharide contains poly(sucrose-co-epichlorohydrin) at a density of less than 1 g per liter, and the TLR2 ligand contains hyaluronic acid. 7. The bone marrow container of any one of Options 1 to 6, wherein the container is selected from the group consisting of a bag, a vacutainer, and a syringe. 8. The bone marrow container of any one of Options 1 to 7, further comprising an integrated delivery device. 9. The bone marrow container of any one of Options 1 to 8, further comprising dextran. 10. The bone marrow container of Option 9, wherein the dextran is present at a concentration of at least about 4%. 11. The bone marrow container of any one of Options 9 to 10, further comprising a volume configured to receive bone marrow, wherein when bone marrow is present in the volume, the concentration of dextrin in the bone marrow and the composition is at least about 1%. 12. The bone marrow container of any one of Options 9 to 11, wherein the container is configured to be received by a separation system. 13. A chassis including a cavity configured to receive the bone marrow container of any one of Options 1 to 12, the chassis including a track; and A shaft disposed on the track, wherein the shaft is configured to move along the track, whereby when the bone marrow container is disposed within the chassis, the bone marrow container is compressed, a separation system including. 14. The separation system of Option 13, wherein the shaft is configured to press the bone marrow container against the inner surface of the chassis. 15. The separation system of Option 13 or 14, wherein the shaft is selected from the group consisting of a roller and a slider. 16. The separation system of Option 13 or 14, wherein the shaft is configured to snap into a predetermined position with respect to the bone marrow container disposed within the chassis, thereby defining at least two portions of the bone marrow container. 17. A kit including the bone marrow container of any one of Options 1 to 12 and a bone marrow aspiration needle such as a Luer lock. The kit of option 17, further comprising a filter having a pore size smaller than the diameter of the bone marrow cells. 19. The kit of option 18, wherein the pore size is less than or equal to 200 microns. 20. The kit of any one of options 17 - 19, further comprising an activation / precipitation solution. 21. The kit of any one of options 17 - 20, further comprising a separation system of any one of options 13 - 16. 22. A method for activating immune cells of a subject, comprising: obtaining bone marrow containing immune cells from the subject; incubating the bone marrow with a non - ionic hydrophilic branched polysaccharide and a TLR2 ligand, wherein when the bone marrow is contacted with whole blood and centrifuged, the density is sufficient to allow fluid movement of the whole blood through the non - ionic hydrophilic branched polysaccharide without separating red blood cells, the non - ionic hydrophilic branched polysaccharide has a molecular weight of 20 kDa or more, and the incubation is carried out until the immune cells are activated; and administering the activated immune cells to the subject. 23. The method of option 22, further comprising identifying a subject in need of activation of immune cells. 24. The method of option 23, wherein the subject has cancer. 25. The method of option 24, further comprising incubating the bone marrow with a cancer cell antigen. 26. The method of any one of options 22 - 25, wherein the non - ionic hydrophilic branched polysaccharide has a molecular weight of at least 70 kDa. 27. The method of any one of options 22 - 26, wherein the non - ionic hydrophilic branched polysaccharide comprises poly(sucrose - co - epichlorohydrin). 28. The method of any one of options 22 - 27, wherein the non - ionic hydrophilic branched polysaccharide has a density of less than 1 g per IL. 29. The method according to any one of options 22 to 28, wherein the TLR2 ligand is selected from the group consisting of hyaluronic acid, hyaluronic acid, sodium urate crystals, biglycan, endoplasmin, HMGB1, HSP60, HSP70, human heart myosin, zymosan, lipoteichoic acid, plasma-derived lipoproteins, lipopolysaccharide (LPS), and peptidoglycans including Pam2CSK4 and Pam3CSK4. 30. The method of option 22, wherein the nonionic hydrophilic branched polysaccharide contains poly(sucrose-co-epichlorohydrin) at a density of less than 1 g per liter, and the TLR2 ligand contains hyaluronic acid. 31. The method according to any one of options 22 to 28, wherein the nonionic hydrophilic branched polysaccharide contains poly(sucrose-co-epichlorohydrin) at a density of less than 1 g per liter, and the TLR2 ligand contains hyaluronic acid. 32. The method according to any one of options 22 to 31, wherein the incubation is carried out in a bone marrow container according to any one of options 1 to 16. 33. Place the bone marrow container in a separation system according to any one of options 17 to 21; Snap the shaft into a predetermined position relative to the bone marrow container, thereby defining two parts of the bone marrow container; The method of option 32, further comprising moving the shaft along the track after the incubation, thereby compressing the contents in the bone marrow container and extruding immune cells from the bone marrow. 34. The method according to any one of options 22 to 33, further comprising incubating the bone marrow cells with an activation / sedimentation solution, thereby sedimenting red blood cells from the bone marrow. 35. The method according to any one of options 22 to 34, wherein the activated immune cells contain bone marrow cells. 36. The activated immune cells are CD11b + CD54 + Granulocytes and / or CD66b + The method of option 35, including neutrophils. Any of the methods of options 22 - 36, further comprising separating activated immune cells from a nonionic hydrophilic branched polysaccharide and a TLR2 ligand before administration to a subject. 38. Any of the methods of options 22 - 37, wherein the activated immune cells are administered to the subject's musculoskeletal tissue. 39. Any of the methods of options 22 - 38, which does not include leukapheresis therapy. 40. Any of the methods of options 22 - 39, wherein the method is ex vivo.

[0051] Generally, conventional separation methods utilized different physical properties of RBCs to enrich in the mononuclear cell fraction containing HSC and MSC. Density gradient separation conventionally used FICOLL - HIPAQUE polysaccharides at a density of 1.0 or above and applied centrifugal force for physical separation. This reduced the bone marrow by removing RBCs and granulocytes, enabling the use of a mononuclear cell fraction that contains regenerative cells in a smaller and more useful amount.

[0052] Without being limited by theory, it is contemplated that in some embodiments, the nonionic hydrophilic branched polysaccharide can activate regenerative cells in the bone marrow using a mechanism similar to that of bacterial cell wall polysaccharides that can activate the innate arm of the human immune system. Without being limited by theory, it is contemplated that in some embodiments, the TLR2 ligand can activate bone marrow cells, and the activation of bone marrow cells can enhance their therapeutic effects. Thus, the activation of immune cells using a nonionic hydrophilic branched polysaccharide and a TLR2 ligand according to the compositions, bone marrow containers, kits, and methods described herein is thought to be able to enhance the effectiveness of treating a patient with autologous bone marrow cells.

Examples

[0053] Example 1 : Flow cytometry analysis of bone marrow dendritic cell activation after exposure of bone marrow aspirates to RICOLL polysaccharide and the following TLR2 ligands: plasma-derived lipoproteins, synthetic PAM-3CSR4, and lipopolysaccharide (LPS). Samples of peripheral blood (PB) and bone marrow (BM) were obtained from a single donor, and all samples were treated with hypotonic saline solution (FACS lysing solution, Becton Dickinson) to remove red blood cells and then stained using no wash. Next, appropriate fluorescent dye-conjugated monoclonal antibodies were incubated on ice for 30 minutes in PBS with 2% bovine serum albumin according to the manufacturer's instructions, and data were immediately collected on >50,000 events. Gates were set using isotype-matched controls. Note that the PB control provides a positive control for relatively rare mature dendritic cells. Dendritic cells were compared between bone marrow cells that matched negative control bone marrow cells after exposure to TLR ligands and Ficoll diluted 1 / 100 (v / v).

[0054] Figure 1A and Figure 1D (peripheral blood sample positive control, PB), Figure 1B and Figure 1E (total bone marrow negative control before exposure to FICOLL polysaccharide and TLR2 agonist), and Figure 1C and Figure 1F (bone marrow aspirate mononuclear cells (MNC) after exposure to Ficoll and TLR2 agonist. Classical dendritic cells in lineage-negative, CD14-negative, bone marrow size gate (Figure 1A, Figure 1B, and Figure 1C; white arrows 1, 2, and 3 indicate dendritic cells in regions 1, 2, and 3), and gated dendritic cells were analyzed for activation by measuring the percentage of cells activated for HLA-DR (a marker of maturity) expression by the percentage of positive and relative fluorescence intensity (Figure 1D, Figure 1E, and Figure 1F).

[0055] Figure 1D (PB positive control), Figure 1E (BM negative control). The white arrow numbered 5 indicates the control level of bone marrow cell activation for the total bone marrow negative control, while the arrow 6 in Figure 1F indicates the upregulation of HLA-DR in cell positivity (1.9 vs 15.9%) and the ratio of relative mean fluorescence intensity after overnight exposure to TLR-2 ligand and ficoll at 1.0 g / L or less. The white arrow 4 in Figure 1D indicates the control level of bone marrow cell activation for the PB control.

[0056] Therefore, according to some embodiments herein, it can be concluded that immune cells were activated by incubating bone marrow with FICOLL polysaccharide and TLR ligand according to some embodiments herein. Furthermore, the maturation of dendritic cells from bone marrow progenitor cells is enhanced by activation.

[0057] Example 2 : Further analysis of activated bone marrow cells (activated by the FICOLL polysaccharide and TLR ligand of Example 1) was performed. The gating scheme is shown in Figures 2A-I. Cells were gated for size (Figures 2A-C), viability (Cytotox blue staining) (Figures 2D-F), and singlets (Figures 2G-I).

[0058] Phenotypic characterization of the cells showed that the population of activated immune cells included immature (CD33-low, CD11b-low) bone marrow cells (Figures 4A-I). CD66b is expressed by neutrophils, which indicates a novel DC population (see Figures 4A, 4D, 4G). Therefore, immature bone marrow cells, such as neutrophils, can be concluded to be activated according to some embodiments herein.

[0059] Example 3 : Aspirate the patient and process approximately 25 ml of total bone marrow with either a Terumo BCT SmartPrep Bone Marrow Aspirate Concentrate (BMAC) or a Biomet Marrow Stimulation System according to the instructions of their manufacturers (however, in this specification, according to some embodiments, currently commercially available devices can be used for bone marrow processing and reduction). Next, inject the obtained bone marrow concentrate into a sterile bone marrow container containing a sufficient amount of 100-fold concentrated Ficoll-Hypaque and hyaluronic acid, enhance CD11b expression by flow cytometry or gene expression analysis, and then incubate at the ambient concentration at the point of care for at least 10 minutes to 8 hours. Next, filter the cells so that the cells are retained, reduce the concentration of the activated portion, and then deliver the activated cells to the patient. Surgical procedures involving the implantation of medical devices, including allogeneic biological tissues, artificial bone fillers, spine-related hardware, and stainless steel, titanium, PEEK devices, etc. used to support the regeneration of musculoskeletal tissues, are improved and the results are enhanced compared to bone marrow concentrates not treated with the disclosed compositions and methods of the embodiments herein.

[0060] According to some embodiments, a method, use, or composition includes various steps or features that exist as a single step or feature (as opposed to multiple steps or features). For example, according to some embodiments, the method includes a single incubation of bone marrow with a non-ionic hydrophilic branched polysaccharide and a TLR2 ligand. The non-ionic hydrophilic branched polysaccharide and the TLR ligand can be present in an amount effective to activate immune cells, such as bone marrow cells. A method, bone marrow container, or kit can include a single amount of a non-ionic hydrophilic branched polysaccharide and a TLR2 ligand effective to activate immune cells as described herein. Multiple features or components are provided in alternative embodiments.

[0061] Although various aspects and embodiments are disclosed herein, other aspects and embodiments will be apparent to those of ordinary skill in the art. The various aspects and embodiments disclosed herein are for purposes of illustration and not of limitation, and the true scope and spirit are indicated by the following claims. With respect to each method described herein, the related components for use in that method are clearly contemplated, as are the use of the compositions in that method and, where applicable, the method of manufacture of the agents for use in that method. For example, with respect to the methods for activating immune cells described herein, compositions (and bone marrow containers) comprising a nonionic hydrophilic branched polysaccharide and a TLR2 ligand for use in the corresponding methods are also contemplated. Further, in vitro methods including the treatment of patients, e.g., receiving a bone marrow aspirate and incubating the bone marrow aspirate with a nonionic hydrophilic branched polysaccharide and a TLR2 ligand, and thus activating immune cells in vitro, are contemplated. Those of ordinary skill in the art will understand that, with respect to this and other steps and methods disclosed herein, the functions performed in those steps and methods may be carried out in a different order. Further, the steps and operations outlined are provided by way of example only, and some of those steps and operations are optional and may be combined into fewer steps and operations or expanded to additional steps and operations without detracting from the essence of the disclosed embodiments.

[0062] With respect to substantially any plural and / or singular usage herein, those of ordinary skill in the art can translate from the plural to the singular and / or from the singular to the plural as appropriate to the context and / or application. Various permutations of the singular / plural forms can be explicitly set forth herein for clarity.

[0063] Generally, as used herein, and especially in the appended claims (e.g., in the body of the appended claims), terms are generally intended to be open terms (e.g., the term "including" shall be construed to mean "including but not limited to", the term "having" shall mean "having at least", and the term "include" shall mean "including but not limited to", as will be understood by those skilled in the art. Where an intent to introduce a specific number of recited claims is intended, such intent will be clearly recited in the claim, and it will be further understood by those skilled in the art that where such recitation is absent, such intent does not exist. For example, for purposes of illustration, the following appended claims may include the use of introductory phrases "at least one" and "one or more than one" to introduce the claim recitation. However, the use of such phrases should not be construed to mean that the introduction of a claim recitation by the indefinite article "a" or "an" limits a particular claim that includes such introduced claim recitation to embodiments that include only one such recitation. Further, even where the same claim includes both an introductory phrase "one or more than one" or "at least one", and an indefinite article, e.g., "a" or "an" (e.g., "a" and / or "an" shall be construed to mean "at least one" or "one or more than one"), the same is true; the same also applies to the use of a definite article used to introduce a claim recitation. Further, even where a specific number of introduced claim recitations are clearly recited, those skilled in the art will recognize that such recitation should be construed to mean at least the recited number (e.g., a bare recitation of "one recitation" without other modifiers means at least two recitations, or two recitations). Further, where rules similar to "at least one of A, B, and C" are used, generally such constructs are intended in the sense that those skilled in the art will understand the rule (e.g., "a system having at least one of A, B, and C" is not limited to a system having only A, only B, only C, A and B together, A and C together, B and C together, and / or A, B, and C together).When a rule similar to "at least one of A, B, and C, etc." is used, generally, such a construction is intended in the sense that one of ordinary skill in the art would understand the rule (e.g., "a system having at least one of A, B, and C" is not limited to a system having only A, only B, only C, A and B together, A and C together, B and C together, and / or A, B, and C together. It should be further understood by one of ordinary skill in the art that in virtually every disjunctive word and / or clause presenting two or more alternative terms, in any of the description of the specification, the claims, and the drawings, the possibility of including the term "one", the term "any", or both terms is contemplated. For example, the clause "A or B" would be understood to include the possibility of "A", or "B", or "A and B").

[0064] Furthermore, when a disclosed feature or aspect is described with respect to a Markush group, one of ordinary skill in the art will understand thereby that the disclosure also describes the individual members of the Markush group as well as subgroups thereof.

[0065] As will be understood by those skilled in the art, for any and all purposes, for example with respect to providing a written description, all ranges disclosed herein also include any and all possible sub-ranges, and combinations of such sub-ranges. Any recited range can be readily understood to be sufficiently described so as to be divisible into at least equal halves, thirds, quarters, fifths, tenths, etc. of the same range. By way of non-limiting example, each range discussed herein can be readily divided into lower one-third, middle one-third, and upper one-third, etc. As will also be understood by those skilled in the art, all language such as "up to", "at least", etc. includes the recited number and refers to a range that can later be divided into sub-ranges as described above. For example, "about 5" will include the number 5. Finally, as will be understood by those skilled in the art, ranges include individual members. Thus, for example, a group having 1 to 3 cells refers to a group having 1, 2, or 3 cells. The term, a group having 1 to 5 cells refers to a group having 1, 2, 3, 4, or 5 cells. Terms such as "approximately", "about", and "substantially" as used herein before a number include the recited number (e.g., about 10% = 10%) and also represent an amount close to the recited amount that still performs the desired function and achieves the desired result. The terms "approximately", "about", and "substantially" refer to an amount within less than 10%, less than 5%, less than 1%, and less than 0.01% of the recited amount.

[0066] From the foregoing, it will be understood that various embodiments of the present disclosure are described herein for purposes of illustration, and that various modifications can be made without departing from the scope and spirit of the present disclosure. Accordingly, the various embodiments disclosed herein are not intended to be limiting, and the true scope and spirit are indicated by the following claims.

Claims

1. Includes: a non-ionic hydrophilic branched polysaccharide at a particular density, wherein when said composition is contacted with whole blood and centrifuged, said density is sufficient to permit fluid movement of the whole blood through the non-ionic hydrophilic branched polysaccharide without separating red blood cells; A non-ionic hydrophilic branched polysaccharide having a molecular weight of 20 kDa or more; and TLR2 ligands, A bone marrow container containing the composition therein which is sterile.

2. 2. The bone marrow container of claim 1, wherein the non-ionic hydrophilic branched polysaccharide has a molecular weight of at least 70 kDa.

3. 3. The bone marrow container of claim 1 or 2, wherein the non-ionic hydrophilic branched polysaccharide comprises poly(sucrose-co-epichlorohydrin).

4. The non-ionic hydrophilic branched polysaccharide is present at a concentration of less than 1 g per liter, e.g., 1 g / L, 0.99 g / L, 0.9 g / L, 0.8 g / L, 0.7 g / L, 0.6 g / L, 0.5 g / L, 0.4 g / L, 0.3 g / L, 0.2 g / L, 0.1 g / L, 0.05 g / L, 10 －２ g / L, 2 x 10 －３ g / L, 10 －３ g / L, 10 －５ g / L, 2 x 10 －６ g / L, 10 －６ g / L, 10 －７ g / L, 10 －８ g / L, 2 x 10 －９ g / L, or 10 －９ g / L, e.g., a range between any two of the listed values, e.g., 10 －９ -10 －６ g / L, 10 － ９ -10 －３ g / L, 10 －９ -10 －２ g / L, 10 －９ -0.1 g / L, 10 －９ -0.5g / L, 10 －９ -0.9g / L, 10 －９ -0.09g / L, 10 －６ -10 －３ g / L, 10 －６ -10 －２ g / L, 10 －６ -0.1g / L, 10 －６ -0.5g / L, 10 －６ -0.9g / L, 10 －６ -0.09g / L, 2x10 －６ -10 －２ g / L, 2x10 －６ -0.1 g / L, 2x10 －６ -0.5g / L, 2x10 －６ -0.9g / L, 2x10 －６ -0.09g / L, 10 －３ -10 －２ g / L, 10 －３ -0.1g / L, 10 －３ -0.5g / L, 10 －３ -0.9g / L, 10 －３ -0.09g / L, 0.002-0.01g / L, 0.002-0.1g / L, 0.002-0.5g / L, 0.002 -0.9g / L, 0.002-0.09g / L, 0.01g / L-0.99g / L, 0.01 g / L - 0.9g / L, 0.01g / L - 0.5g / L, 0.01g / L -0.2g / L, 0.01g / L - 0.1g / L, 0.1g / L - 0.99g / L, 0.1g / L - 0.9g / L, 0.1 4. The bone marrow container of claim 1, having a density of 0.5 g / L, 0.1 g / L to 0.2 g / L, 0.5 g / L to 0.99 g / L, 0.5 g / L to 0.9 g / L, or 0.5 g / L to 0.7 g / L.

5. 5. The bone marrow container of any one of claims 1 to 4, wherein the TLR2 ligand is selected from the group consisting of hyaluronon, hyaluronic acid, monosodium urate crystals, biglycan, endoplasmin, HMGB1, HSP60, HSP70, human cardiac myosin, zymosan, lipoteichoic acid, plasma-derived lipoproteins, lipopolysaccharide (LPS), and peptidoglycans, including Pam2CSK4 and Pam3CSK4.

6. 2. The bone marrow container of claim 1, wherein the non-ionic hydrophilic branched polysaccharide comprises poly(sucrose-co-epichlorohydrin) at a density of less than 1 g per liter and the TLR2 ligand comprises hyaluronic acid.

7. The bone marrow container of any one of claims 1 to 6, wherein the container is selected from the group consisting of a bag, a vacutainer, and a syringe.

8. The bone marrow container of any one of claims 1 to 7, further comprising an integral delivery device.

9. The bone marrow container of any one of claims 1 to 8, further comprising dextran.

10. 10. The bone marrow container of claim 9, wherein the dextran is present at a concentration of at least about 4%.

11. The bone marrow container of any one of claims 9 to 10, further comprising a volume configured to receive bone marrow, wherein when bone marrow is present in the volume, the concentration of dextrin in the bone marrow and composition is at least about 1%.

12. The bone marrow container of any one of claims 9 to 11, wherein the container is configured to be received by a separation system.

13. A chassis including a cavity configured to receive a bone marrow container according to any one of claims 1 to 12, the chassis including a track; and A shaft disposed on the track, wherein the shaft is configured to move along the track, thereby compressing the bone marrow container when the bone marrow container is disposed within the chassis.

14. The isolation system of claim 13 , wherein the shaft is configured to press the bone marrow container against an inner surface of the chassis.

15. 15. The separation system according to claim 13 or 14, wherein the shaft is selected from the group consisting of a roller and a slider.

16. 15. The separation system of claim 13 or 14, wherein the shaft is configured to snap into place against a bone marrow container disposed within a chassis, thereby defining at least two portions of the bone marrow container.

17. A kit comprising the bone marrow container according to any one of claims 1 to 12 and a bone marrow aspiration needle such as a Luer lock.

18. 18. The kit of claim 17, further comprising a filter having a pore size smaller than the diameter of the bone marrow cells.

19. 20. The kit of claim 18, wherein the pore size is less than or equal to 200 microns.

20. The kit according to any one of claims 17 to 19, further comprising an activation / precipitation solution.

21. A kit according to any one of claims 17 to 20, further comprising a separation system according to any one of claims 13 to 16.

22. 1. A method for activating immune cells in a subject, comprising: Obtaining bone marrow containing immune cells from a subject; incubating the bone marrow with a non-ionic hydrophilic branched polysaccharide and a TLR2 ligand; wherein when said bone marrow is contacted with whole blood and centrifuged, said density is sufficient to permit fluid movement of the whole blood through the non-ionic hydrophilic branched polysaccharide without separating red blood cells; the non-ionic hydrophilic branched polysaccharide has a molecular weight of 20 kDa or more; and The incubation is performed until the immune cells are activated; and A method comprising administering activated immune cells to a subject.

23. 23. The method of claim 22, further comprising identifying a subject in need of immune cell activation.

24. 24. The method of claim 23, wherein the subject has cancer.

25. 25. The method of claim 24, further comprising incubating the bone marrow with a cancer cell antigen.

26. 26. The method of any one of claims 22 to 25, wherein the non-ionic hydrophilic branched polysaccharide has a molecular weight of at least 70 kDa.

27. 27. The method of any one of claims 22 to 26, wherein the non-ionic hydrophilic branched polysaccharide comprises poly(sucrose-co-epichlorohydrin).

28. 28. The method according to any one of claims 22 to 27, wherein the non-ionic hydrophilic branched polysaccharide has a density of less than 1 g per IL.

29. 29. The method of any one of claims 22 to 28, wherein the TLR2 ligand is selected from the group consisting of hyaluronon, hyaluronic acid, monosodium urate crystals, biglycan, endoplasmin, HMGB1, HSP60, HSP70, human cardiac myosin, zymosan, lipoteichoic acid, plasma-derived lipoproteins, lipopolysaccharide (LPS), and peptidoglycans, including Pam2CSK4 and Pam3CSK4.

30. 23. The method of claim 22, wherein the non-ionic hydrophilic branched polysaccharide comprises poly(sucrose-co-epichlorohydrin) at a density of less than 1 g per liter and the TLR2 ligand comprises hyaluronic acid.

31. 29. The method of any one of claims 22 to 28, wherein the non-ionic hydrophilic branched polysaccharide comprises poly(sucrose-co-epichlorohydrin) at a density of less than 1 g per liter and the TLR2 ligand comprises hyaluronic acid.

32. The method according to any one of claims 22 to 31, wherein said incubation is carried out in a bone marrow container according to any one of claims 1 to 16.

33. Placing the bone marrow container in a separation system according to any one of claims 17 to 21; snapping the shaft into place against the bone marrow container, thereby defining two portions of the bone marrow container; 33. The method of claim 32, further comprising moving the shaft along the track after said incubation, thereby compressing the contents in the bone marrow container and pushing the immune cells out of the bone marrow.

34. The method of any one of claims 22 to 33, further comprising incubating the bone marrow cells with an activation / sedimentation solution, thereby sedimenting red blood cells from the bone marrow.

35. The method of any one of claims 22 to 34, wherein the activated immune cells comprise bone marrow cells.

36. The activated immune cells are ＋ CD54 ＋ Granulocytes and / or CD66b ＋ 36. The method of claim 35, comprising neutrophils.

37. 37. The method of any one of claims 22 to 36, further comprising separating the activated immune cells from the non-ionic hydrophilic branched polysaccharide and the TLR2 ligand prior to administering the activated immune cells to a subject.

38. The method of any one of claims 22 to 37, wherein the activated immune cells are administered to a musculoskeletal tissue of a subject.

39. The method according to any one of claims 22 to 38, which does not include leukapheresis.

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