Method and system for preparing mononuclear cell-platelet rich fibrin matrix and composition thereof

Abstract

A purified non-naturally occurring wound healing composition is provided that includes platelets, monocytes, and lymphocytes, the composition being substantially free of neutrophils. Also provided are a system for preparing the purified non-naturally occurring wound healing composition, a method of making the composition, and a method of using the composition to treat a wound.

Description

[Technical field]

[0001] The present invention relates generally to blood cell processing techniques and devices. In one embodiment, the present invention relates to a method and system for separating mononuclear cells, such as lymphocytes and monocytes, as well as platelets, from a whole blood sample, and in particular to a blood separation method and system that provides high recovery of platelets and mononuclear cells without significant contamination by red blood cells or by polymorphonuclear granulocytes.

[0002] All publications, patents, patent applications, and other references cited in this application are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, or other reference was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. The citation of a reference herein should not be construed as an admission that it is prior art to the present invention. [Background technology]

[0003] Acute wounds follow an organized wound healing sequence and often heal in 3 to 4 weeks. If a wound still exists 4 weeks after wounding, it is defined as a chronic wound. [Demidova-Rice, TN, et al. (2012) Adv Skin Wound Care. 25(7): 304-14]. Many research studies have been conducted on chronic wound management to address the growing demand for effective and affordable treatments. The healing trajectory of chronic wounds is expected to take 12 weeks [Sibbald, RG, et al. (2011) Adv Skin Wound Care. 24: 415-37]. This period may be prolonged if the wound exhibits an altered molecular environment, chronic inflammation, or fibrosis [Canedo-Dorantes, L, et. al. (2019) Int J Inflam. 2019: 3706315], or unmodified pre-existing systemic factors.

[0004] The most common chronic wounds are diabetic foot ulcers (DFUs), pressure ulcers (PUs), venous leg ulcers (VLUs), and non-healing surgical wounds, which are major healthcare problems. Chronic wounds usually occur in older people with underlying conditions such as diabetes, vascular disease, and obesity [Gould, L, et al. (2015) Wound Repair Regen. 23(1): 1-13]. Reduced immune and nutritional status, as well as chronic mechanical stress, have also been shown to contribute to poor wound healing outcomes [Eming, SA, et al. (2014) Sci Transl Med. 6(265): 265sr6]. Chronic wounds are associated with alarmingly high mortality rates: 5-year mortality rates for ischemic (55% mortality), neuropathic (45%), and neuroischemic (18%) diabetic foot ulcers [Moulik, PK, et al. (2003) Diabetes Care. 26(2): 491-4] are higher than or similar to those associated with breast and prostate cancer (18% and 8%, respectively) [Armstrong, DG, et al. (2007) Int. Wound J. 4(4): 286-287]. Chronic wounds are also associated with high healthcare costs: in the United States, total expenditure estimates for chronic non-healing wounds ranged from US$28.1 billion to US$96.8 billion in 2014 according to a retrospective analysis of the Medicare 5% Limited Data Set [Nussbaum, SR, et al. (2018) Value Health. 21(1): 27-32]. Despite the alarming prevalence and high healthcare costs, efficient treatments remain lacking.

[0005] For example, the complexity and multiplicity of diabetic foot wounds make them an extremely challenging therapeutic target, and the uneven progress seen in mouse models highlights the need for new methods to overcome obstacles from bench to bedside. Clinical progress requires more innovative research strategies that leverage both existing knowledge and the potential for new advances across disciplines [Barakat, M, et al. (2020) Adv Wound Care. https: / / doi.org / 10.1089 / wound2020.1254].

[0006] Platelets are small, bioactive anucleated cells that vary in diameter from 2 to 4 μm and originate from mature megakaryocytes in the bone marrow and lungs [Lefrancais, E, et al. (2017) Nature. 544(7648): 105-109]. Platelets are essential for primary hemostasis but also play important roles in tissue regeneration and inflammation [Etulain J. (2018) Platelets. 29(6): 556-568].

[0007] Platelet α-granules constitute the major granule population in terms of size and number within platelets. These include transforming growth factor-β (TGF-β), platelet-derived growth factor (PDGF), platelet-derived endothelial growth factor (ECGF), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), and insulin-like growth factor (IGF), as well as growth and adhesion factors such as P-selectin, platelet factor 4, fibronectin, beta-thromboglobulin, von Willebrand factor (vWF), fibrinogen, and coagulation factors V and XIII [Eisinger, F, et al. (2018) Front Med. 5: 317].

[0008] Platelets have been used in wound care for decades. The benefits of platelet-rich plasma (PRP) administration are associated with economic advantages, considering that PRP administration does not require complex equipment or training for its implementation. Furthermore, due to their primary autologous origin, concerns about disease prevalence or immunogenic reactions can be ignored [Etulain, J. (2018) Platelets. 29(6): 556-568]. Platelets are readily available in large quantities from blood. Normal platelet counts range from 150,000 to 450,000 per microliter of blood. Platelets contain 50-80 alpha granules that release hundreds of bioactive proteins [Blair, P, et al. Blood Rev. (2009) 23(4): 177-189], including growth factors, adhesion molecules, and serotonin that promote cell survival, proliferation, and migration [Cloutier, N, et al. (2018) PNAS 115(7): E1550-E1559]. Platelet-derived microparticles (PDMs) stimulate the release of cytokines, activate intracellular signaling pathways, promote angiogenesis, and are involved in tissue regeneration [Neumuller, J, et al. In: Khan M, ed. Intechopen. London: Intechopen; (2015). pp. 255-284]. Platelets interact with immune cells [Hu H, et al. Thromb Haemost (2010) 104: 1184-1192] and have analgesic effects [Miyamoto, H, et al. J Oral Max Surgery, Med, and Path. (2020) 32(4): 237-240].Platelets are involved in tissue remodeling [Langer HF, et al. Arterioscler Thromb Vasc Biol. (2007) 27: 1463-1470] and recruit bone marrow-derived progenitor cells [Massberg S, et al. J Exp Med. (2006) 203: 1221-1233] and mesenchymal stem cells [Langer HF, et al. J Mol Cell Cardiol. (2009) 47: 315-325]. Platelets are also important for maintaining vascular integrity [Boulaftali Y, et al. J Clin Invest. (2013) 123: 908-916]. Platelet mediators stimulate extracellular matrix formation and connective tissue remodeling [Xu X, et al. Cells Tissues Organs. (2013) 197: 103-113].

[0009] For the treatment of chronic non-healing skin wounds, techniques have been developed to insert platelet concentrates into the wound to accelerate the wound healing process. Platelets isolated from peripheral blood are a source of autologous growth factors. The term platelet-rich plasma (PRP) was introduced in the 1970s to describe the autologous preparation and concentration of platelets in a plasma concentrate [Pietrzak, WS, et al. (2005) J Craniofac Surg. 16: 1043-1054]. PRP, also known as autologous conditioned plasma, is a concentrate of platelet-rich plasma derived from whole blood, centrifuged to remove red and white blood cells. Autologous PRP gel consists of a fibrin scaffold, cytokines, growth factors, and chemokines derived from the patient's blood [Frykberg, RG, et al. (2010) Ostomy Wound Manage. 56(6): 36-44]. The mechanism of action of PRP gel is thought to be the molecular and cellular induction of normal wound healing responses similar to those seen with platelet activation.

[0010] Monocytes typically circulate in the blood for 1 to 3 days before migrating into tissues where they become macrophages or dendritic cells. Macrophages exhibit plasticity and can adopt proinflammatory, prowound healing, profibrotic, anti-inflammatory, antifibrotic, or tissue regenerative phenotypes. According to their activation state and function, they can be divided into M1 type (classically activated macrophages) and M2 type (alternatively activated macrophages). The balance between M1 and M2 macrophages plays an important role in wound healing [Mosser DM, et al. (2008) Nature Rev Immunol. 8(12): 958-969]. Best characterized, M1 initiates a proinflammatory immune response against pathogen colonization [Porta, C, et al. (2015) Semin Immunol. 27(4): 237-248]. As inflammation is reduced, these alarmins are believed to promote the initiation of an anti-inflammatory or wound healing response characterized by the presence of alternatively activated macrophages or the M2 phenotype.

[0011] Interleukin 4 (IL-4) produced by T helper type 2 lymphocytes (Th2) cells can convert macrophages into M2 type macrophages that suppress inflammation [Abramson, SL, et al. (1990) J Immunol. 144(2): 625-630]. M2 macrophages mainly secrete anti-inflammatory cytokines, which have the function of reducing inflammation and play an important role in wound healing and tissue repair.

[0012] Shifts in metabolic pathway utilization characterize macrophage polarization that produce metabolic and immune outcomes that impact host-pathogen interactions during wound healing [Anders, CB, et al. (2019). Curr Opinion Infect Dis. 32(3): 204-209]. Macrophage plasticity is important for normal tissue repair to ensure the transition from the inflammatory to the proliferative phase of healing.

[0013] Studies in mice have shown that certain combinations of growth factors (GFs) enhance the survival, adhesion, and angiogenic potential of mononuclear cells [Jin, E, et al. (2013) J Cell. Mol. Med. 17(12): 1644-1651]. In vivo wound healing results revealed that wounds treated with GFs demonstrated accelerated wound healing on days 7 and 14 compared to untreated wounds. Histological analysis demonstrated that the number of transplanted cells and transdifferentiated keratinocytes in the wounds was significantly higher in subjects treated with GFs. This suggests that priming of mononuclear cells with growth factors released by platelets can enhance cell-based therapies.

[0014] Macrophages enter the wound and produce IL-10, which then triggers cells around the wound to initiate wound closure [Quiros, M, et al. (2017) J Clin Invest. 127(9): 3510-3520]. Studies of mucosal wounds have revealed some of the signaling pathways that IL-10 uses to orchestrate wound repair.

[0015] Lymphocytes have a role in regulating the direction of wound repair, with or without scarring. A subset of T lymphocytes has been shown in mice to attenuate the degree of inflammation and promote associated angiogenesis, thereby lowering the risk of skin scarring [Wang, X, et al. (2019) Adv in Wound Care 8(11): 527-537]. The time course of T lymphocyte infiltration into wounds showed that CD3+ T lymphocytes were present in wounds at day 3, peaked at day 14, and persisted until day 30, suggesting a key T lymphocyte role in skin wound healing and scarring responses. CD4+ T lymphocytes may be a key lymphocyte population that regulates the response to wound injury and repair. There is a balance between inflammation and angiogenesis induced by T lymphocytes, which may be part of the mechanism explaining tissue repair and scar formation.

[0016] The addition of mononuclear cells (lymphocytes and monocytes) to platelets can enhance wound healing. The immune system plays an essential role in successful wound healing. In addition to contributing to host defense, immune cells are important regulators of wound healing through secretion of cytokines, lymphokines, and growth factors [Park, JE, et al. Am J Surg. (2004) 187(5): S11-S16]. Various studies of PRP techniques have revealed that extensive changes in blood components including platelets, red blood cells, white blood cells, pH, and glucose in PRP extraction play a key role in successful wound healing. A high concentration of cells is as important as the white blood cell count in the PRP sample. Non-standardized methods for collecting PRP fractions have been considered unimportant and often ignored by researchers. However, the lack of standardization of PRP preparation for clinical use contributes, at least in part, to the variable clinical effectiveness of PRP use [Fitzpatrick, J, et al. (2017) Orthop J Sports Med. 5(1):2325967116675272].

[0017] Various anticoagulants have been used in blood collection / separation devices, either alone or in conjunction with cell support solutions, to preserve blood samples in a non-coagulated state for a period of time prior to centrifugation and analysis. For example, some common anticoagulants include sodium heparin, K2EDTA, K3EDTA, and sodium citrate in various concentrations. In particular, sodium citrate solutions have been used as anticoagulants for many years. US Pat. No. 5,399,433, which is incorporated by reference, discloses a sodium citrate-based anticoagulant solution having a pH ranging from greater than pH 6.0 to about pH 8.5, and a sodium citrate concentration preferably ranging from about 0.05M to about 0.2M.

[0018] Calcium is known to play a key role in the blood clotting cascade. Sodium citrate solutions prevent calcium's involvement in blood clotting. Typically, these sodium citrate solutions are added to freshly drawn whole blood to prevent clotting. Calcium can then be added back to the whole blood suspension as needed to induce subsequent clotting. Sodium citrate is a particularly advantageous anticoagulant because it offers good buffering capacity over a wide range of pH. In particular, the buffering capacity of sodium citrate can be attributed to the three carboxyl groups present on the corresponding acid of the compound. Since sodium citrate is the corresponding sodium-based salt of citric acid, it is the citric acid / sodium citrate combination that functions to perform the buffering chemistry.

[0019] As mentioned above, citric acid (a hydroxytricarboxylic acid) has three carboxyl groups and therefore three pKas. The first, pKa1, appears at a pH of about 3.06. The second, pKa2, appears at a pH of about 4.76. The third, pKa3, appears at a pH of about 5.4. Thus, sodium citrate performs its most effective buffering function at these pH values ​​and is particularly useful for performing buffering functions when added to cell suspensions in vitro. As a result, sodium citrate is used as an anticoagulant in various blood separation devices due to its buffering capacity over a range of pHs. Citric acid is commonly used as an anticoagulant in three types of solutions. The first type of solution is called buffered sodium citrate. The second type of solution is typically called CPD solution or citrate-phosphate-dextrose. The third type is designated as ACD or acid-citrate-dextrose. The citrate ion concentration in these solutions is typically higher than that required to perform the anticoagulant function.

[0020] US Patent No. 5,399,633, which is incorporated by reference, discloses a method for separating lymphocytes and monocytes from a blood sample. An integral part of this invention is an improved blood separation tube, which has a molecular weight of 1.060 to 1.065 g / cm 3Utilizing a gel-like material having a specific gravity of 0.1 to 1.0 g / ml has been shown to significantly enhance the purity of cell separation while providing acceptable cell yields.

[0021] US Patent No. 5,399,633, incorporated by reference, discloses a method for suppressing the apparent shift in buoyant density of granulocytic leukocytes in a blood sample and / or recovering any loss in buoyant density, thereby ensuring the quality of separation of lymphocytes and monocytes from granulocytes in a blood sample.

[0022] US Patent No. 5,399,633, which is incorporated by reference, discloses a method for preparing a solid fibrin web, which includes drawing blood from a patient, separating plasma from the blood, contacting the plasma with a calcium-coagulation activator, and simultaneously coagulating and centrifuging the plasma to form a solid fibrin web, which is suitable for regenerating body tissue in a living organism.

[0023] US Patent No. 5,399,633, which is incorporated by reference, discloses a system for preparing an autologous solid fibrin web suitable for tissue regeneration in vivo, which includes a sealed primary container having a separation medium and a low-density, high-viscosity liquid.

[0024] Patent Document 6, which is incorporated by reference, discloses a method and apparatus for preparing a solid fibrin web. One method may include drawing blood from a patient, separating plasma from the blood, contacting the plasma with a calcium coagulation activator, and simultaneously coagulating and axially centrifuging the plasma to form a solid fibrin web. The solid fibrin web may be suitable for regenerating body tissue in a living organism.

[0025] Patent Document 7, incorporated by reference, discloses a method and apparatus for preparing a solid fibrin web. One method may include drawing blood from a patient, separating plasma from the blood, contacting the plasma with a calcium coagulation activator, and simultaneously coagulating and axially centrifuging the plasma to form a solid fibrin web. The solid fibrin web may be suitable for regenerating body tissue in a living organism.

[0026] US Patent No. 5,399,633, which is incorporated by reference, discloses a system for preparing an autologous solid fibrin web suitable for tissue regeneration in vivo, which includes a sealed primary container having a separation medium and a low-density, high-viscosity liquid.

[0027] US Patent No. 5,399,633, which is incorporated by reference, discloses a system for obtaining plasma enriched in platelets, which is closed to the atmosphere, and which includes a collection tube having an anticoagulant portion and a separating gel.

[0028] None of the systems have used a thixotropic gel separator and density gradient media to prepare enriched mononuclear (monocytes and lymphocytes) cell preparations with platelets in plasma without the use of thrombin or batroxobin. The advantage of combining mononuclear cells with platelets for wound healing is that it combines the immune functions of mononuclear cells with the cell signaling of platelets. Granulocytes are excluded because they contribute to inflammation. The use of PRP enriched in neutrophils may result in a higher ratio of collagen type III to collagen type I, leading to increased fibrosis and reduced tendon strength [Zhou, Y, et al. (2016) BioMed Res. Int. 2016: 1-8]. Other neutrophil-mediated deleterious effects include the release of inflammatory cytokines and matrix metalloproteinases (MMPs) that promote pro-inflammatory and catabolic effects when applied to tissues [Fedorova, NV, et al. (2018) Mediat Inflamm. 2018: ID 1574928]. Neutrophils can produce extracellular traps (NETs), which are large extracellular web-like structures composed of decondensed chromatin bound to various cytoplasmic and granule proteins. Although primarily recognized as a defense mechanism against pathogens, they can impede regeneration [Wong, SL, et al. (2015) Nat Med. 21(7):815-819]. In addition to the presence of neutrophils immediately after wounding, neutrophils persist in the wound even after the NET barrier is re-established. Taken together, the findings demonstrate that although neutrophils are stimulated by common pro-regenerative cues, their presence and NETs can impede regeneration [Wier, E, et al. bioRxiv (2020.07.06).189910].

[0029] Thus, there is a need in the art for purified, non-naturally occurring wound healing compositions comprising platelets, monocytes, and lymphocytes, which are substantially free of neutrophils. Such compositions would be useful, for example, for wound healing in a subject in need of wound healing. [Prior art documents] [Patent documents]

[0030] [Patent Document 1] U.S. Patent No. 5,494,590 [Patent Document 2] U.S. Patent No. 4,640,785 [Patent Document 3] U.S. Patent No. 4,816,168 [Patent Document 4] U.S. Patent No. 6,368,298 [Patent Document 5] U.S. Patent No. 6,979,307 [Patent Document 6] U.S. Patent No. 7,745,106 [Patent Document 7] U.S. Patent No. 8,802,362 [Patent Document 8] U.S. Patent No. 8,491,564 [Patent Document 9] U.S. Patent No. 10,617,812 Summary of the Invention

[0031] In one embodiment of the invention, a purified non-naturally occurring wound healing composition is provided that includes platelets, monocytes, and lymphocytes, the composition being substantially free of neutrophils, hi a further embodiment, the purified non-naturally occurring wound healing composition is a mononuclear-platelet rich fibrin matrix.

[0032] Also provided is a system for preparing a purified non-naturally occurring wound healing composition comprising platelets, monocytes, and lymphocytes, said composition being substantially free of neutrophils, as well as a method for preparing said composition using the system of the invention.

[0033] Further provided is a method of treating a wound, the method comprising administering a purified, non-naturally occurring wound healing composition of the present invention comprising platelets, monocytes, and lymphocytes, and substantially free of neutrophils, to a subject in need of treatment of the wound. [Brief description of the drawings]

[0034] [Figure 1] FIG. 1 illustrates an exemplary sodium citrate-based anticoagulant solution disposed within a blood collection tube and separation assembly. [Diagram 2] FIG. 1 illustrates an exemplary transfer device between a processed blood collection tube for clot formation and a centrifugation assembly. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0035] It should be understood that the figures and descriptions of the present invention are simplified to illustrate elements relevant for a clear understanding of the present invention, but for clarity, many other elements found in typical pharmaceutical compositions and stabilization methods have been omitted. Those skilled in the art will recognize that other elements and / or steps are desirable and / or required in the practice of the present invention. However, because such elements and steps are well known in the art, and because they do not facilitate a better understanding of the present invention, a discussion of such elements and steps is not provided herein. The disclosure herein covers all such variations and modifications to such elements and methods known to those skilled in the art. Moreover, the embodiments specified and illustrated herein are for illustrative purposes only and are not intended to be exclusive or limiting in describing the present invention.

[0036] The present invention generally provides for the separation of mononuclear cells (monocytes and lymphocytes) and platelets from whole blood by centrifugation. In one embodiment, the present invention is a blood collection tube that utilizes a non-Newtonian, thixotropic gel of a specific density. The gel is placed in the blood collection tube to form a stable barrier between a liquid density medium, such as Ficoll® Paque [Sigma Aldrich], placed below the gel barrier, and a liquid anticoagulant, preferably sodium citrate, placed above the gel barrier. The evacuated blood collection tube allows for the collection of a blood sample using a standard venipuncture prior to centrifugation. Upon collection, the blood is mixed with an anticoagulant to prevent blood clotting. Upon centrifugation, blood components are separated by their cellular density, allowing the denser population of red blood cells and granulocytes to migrate below the gel barrier, while the less dense mononuclear cells and platelets remain above the gel barrier. Effective separation and isolation of these cells is often important for various clinical assays as well as laboratory protocols. Therapeutic applications of the isolated cell fractions are also important, such as the use of platelet-rich plasma in wound care.

[0037] Once separation has occurred, one application involves the direct injection of a mononuclear cell-platelet plasma suspension into the site of injury. The mononuclear cells, platelets, and the upper fraction of plasma, i.e., mononuclear cell-platelet rich plasma ("M-PRP"), are transferred aseptically to an injection device, such as a needle and syringe. The platelets collected in the PRP are activated, by way of example, by the addition of calcium gluconate or calcium chloride, which induces the release of factors from the alpha granules. This process increases the concentration of mononuclear cells and platelets, and the concentrated M-PRP is then injected into and around the affected area, jump-starting and greatly enhancing the body's natural healing signals.

[0038] A similar application is the use of M-PRP suspensions as adhesives for split thickness skin grafts, where the cells contained within the M-PRP help to fasten the graft to the substrate and accelerate the "take" of the graft. This use also eliminates the need for stitches or staples to secure the graft to the substrate.

[0039] In another application, once separation has occurred, the mononuclear cells, platelets, and upper fraction of plasma are aseptically transferred to a second vacuum tube or vial containing calcium chloride. Upon contact, calcium ions overcome the anticoagulant effect of citrate and activate plasma fibrinogen to fibrin, trapping the cell suspension in a fibrin clot. Behind this clot formation is the intrinsic coagulation pathway, which is activated at the factor XII level by the tube glass surface and proceeds in the presence of calcium to convert prothrombin to thrombin and subsequently fibrinogen to fibrin, thereby promoting the polymerization and cross-linking of fibrin [Margolis, J. (1956) Nature. 178:805-806].

[0040] Ca 2+ The mechanism of α-induced clot formation involves a fibrin mesh accumulated on platelet aggregates in white thrombi. Platelet aggregates act as nuclei for clot formation and are mainly located near the center or in the deep regions of the clot, which can essentially be classified as white thrombi. In this case, it can be hypothesized that the growth factors stored in platelet α-granules are retained for a relatively long period of time. This type of clot acts as a long-term carrier of growth factors with better regenerative ability.

[0041] As clot formation progresses, the second tube or vial is centrifuged to advantageously generate a mononuclear cell-platelet rich fibrin matrix ("M-PRFM"), which can be placed directly onto the wound after centrifugation. This fibrin matrix allows for easy handling of the M-PRFM and can be sutured into place as needed, such as in certain orthopedic applications.

[0042] In another application, once separation has occurred, the top fraction of mononuclear cells, platelets, and plasma is aseptically transferred to a second evacuated vial containing calcium chloride. This vial may have a flat bottom, which renders the M-PRFM into a flat membrane after centrifugation. This M-PRFM membrane has a large surface area and therefore a large exposure of the wound surface to the M-PRFM.

[0043] Description of Specific Embodiments A "patient" is a mammal, e.g., a human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, or a non-human primate such as a monkey, chimpanzee, baboon, or rhesus monkey, and the terms "patient" and "subject" are used interchangeably herein.

[0044] The term "treating" with respect to a subject refers to improving at least one symptom of a disorder in a subject. Treatment can be curing, ameliorating, or at least partially remitigating a disorder. As used in this disclosure, the terms "administer," "administering," or "administration" refer to administering a composition of the invention to a subject.

[0045] In one embodiment, a fibrin matrix with an autologous fibrin molecular structure is provided, which acts as a biodegradable scaffold to embed mononuclear cells (monocytes and lymphocytes) and platelets and support cell migration and the achievement of microvascularization. This advantageous mononuclear cell-platelet rich fibrin matrix (M-PRFM) acts as a delivery system for cells and growth factors leading to enhanced wound healing. M-PRFM has a majority of the platelets and mononuclear cells present in the initial blood sample. The platelets are mostly activated and act to reinforce the strongly polymerized fibrin matrix. The mononuclear cells (monocytes and lymphocytes) are also trapped within the fibrin network and contribute to the tissue healing process. A large amount of mediators, especially platelet growth factors, are released into the wound to activate the tissue regeneration process.

[0046] M-PRFM may act as a delivery system for cells and growth factors leading to enhanced wound healing during the first 2 weeks. Platelets are largely activated and act as a cement reinforcing the strongly polymerized fibrin matrix. Mononuclear cells are also trapped in the strong native fibrin matrix. The cellular composition of M-PRFM means that this biological material is a blood-derived living tissue and must be handled carefully to keep its cellular content alive and stable. During the formation of M-PRFM, fibrils undergo lateral associations to form branches resulting in a complex fibrous network. The high degree of equilateral fibril branching results in the elasticity of the membrane. The fine nanostructure of fibrin after the gel point has been physicochemically characterized, showing dynamic behavior and complex hierarchy at different scales.

[0047] The complex structure of M-PRFM may provide advantageous mechanical behavior due to the design and elasticity provided by the cross-linked monomer units. The morphology and mechanical behavior of M-PRFM depends on the ratio of fibrinogen and thrombin. For example, lower thrombin concentrations result in clots with thicker fibers, less branching, and larger cavities, and therefore less stable. The diameter of the fibrin fibers affects the surface area available for cell adhesion and interaction during platelet activation. Analysis of an exemplary fibrin network formed according to one embodiment of the present invention using a standard protocol produced M-PRFM with a dense network of fibers approximately 90 nm thick. The microspaces found in the fibrin network are filled by cells and growth factors.

[0048] Furthermore, the detected cross-links between fibrin fibers mechanically stabilize the structure of the fibrin network and control the fibrinolytic activity of plasmin. Fibrin has binding sites for integrins, growth factors, and other extracellular matrix components, including fibronectin, so it not only acts as a scaffold for cells to invade, but also provides molecular signals that direct cell function. M-PRFM uses all the fibrinogen available in plasma to convert it into fibrin, thus ensuring maximum fibrin density. Overall, the quality and quantity of fibrin fibers, in addition to growth factors, affect the efficacy and effectiveness of M-PRFM in tissue healing.

[0049] In another embodiment, a system for preparing a purified wound healing composition comprising platelets, monocytes, and lymphocytes is provided, the system comprising: a first container having a sealable open end for receiving a blood sample and a closed end opposite the open end; The first container contains a density separation medium disposed at a first location adjacent the closed end and a density separation medium of about 1.055 to 1.080 g / cm disposed at a second location between the open end and the first location. 3and an anticoagulant solution having a pH in the range of 6.0 to 8.5 at a third location between the open end and the second location; a first container that, when centrifuged, is operable to produce an extractable suspension of separated platelets, monocytes, lymphocytes, and plasma from the blood sample; and a second container having a sealable open end for receiving the extracted suspension of separated platelets, monocytes, lymphocytes, and plasma produced in the first container, the second container having a coagulation activator; a second container that, when centrifuged, is usable to produce a purified wound healing composition from the suspension of separated platelets, monocytes, and lymphocytes; Includes.

[0050] In one embodiment, the system further includes a transfer device adapted to engage with an open end of the first container and to engage with an open end of the second container, the transfer device, when engaged with the open end of the first container, creating a sterile seal for receiving the suspension of separated platelets, monocytes, lymphocytes, and plasma from the first container, and, when engaged with the open end of the second container, creating a sterile environment for transferring the suspension of separated platelets, monocytes, lymphocytes, and plasma to said second container.

[0051] The density separation medium of the system comprises at least one of a non-Newtonian gel and a Newtonian liquid. In one embodiment, the density separation medium comprises an ionic material having a molecular weight of less than about 1500. In another embodiment, the density separation medium is selected from the group comprising sodium diatrizoate, its derivatives, and combinations thereof. In a further embodiment, the density separation medium is selected from the group comprising sucrose or epichlorohydrin polymers having a molecular weight of at least 400,000, its derivatives, and combinations thereof.

[0052] In one embodiment, the system further includes a first closure device for sealing the open end of the first container. The first closure device is adapted to vacuum seal the open end of the first container. In one embodiment, the first closure device is pierceable by a cannula for delivering a blood sample to the first container by pressure differential.

[0053] In one embodiment, the system further includes a second closure device for sealing the open end of the second container. In another embodiment, the second closure device is adapted to vacuum seal the open end of said second container. In one embodiment, the second closure device is pierceable by a cannula for delivering the suspension of separated platelets, monocytes, lymphocytes, and plasma to the second container by pressure differential.

[0054] In another embodiment of the system, the purified wound healing composition comprising platelets, monocytes, and lymphocytes is a monocyte-platelet-rich fibrin matrix.

[0055] In one embodiment, the system further includes a centrifuge adapted to receive at least one of the first container and the second container.

[0056] In a further embodiment, the thixotropic gel of the system has a viscosity of from 1.060 to about 1.065 g / cm 3 In another embodiment, the separation medium has a specific gravity of 1.065 to about 1.085 g / cm 3 and preferably has a specific gravity of about 1.070 to about 1.080 g / cm 3 It has a specific gravity of 1.077 g / cm 3 It has an optimum specific gravity of .

[0057] In another embodiment of the system, the pH is from about 6.5 to about 7.5, preferably from 6.85 to about 7.15, with an optimal pH of 7.0.

[0058] In one embodiment, the concentration of sodium citrate anticoagulant is from about 0.05 M to about 0.20 M, with the preferred sodium citrate concentration being from about 0.08 M to about 0.13 M, and the optimal sodium citrate concentration ranging from about 0.09 M to about 0.11 M. In one embodiment, the pH is 7.0 and the sodium citrate concentration is 0.1 M.

[0059] In a further embodiment, the anticoagulant comprises 294 gm sodium citrate·2H2O, 0.27 gm citric acid·H2O at 0.10 moles per liter, and a pH of 7.0. In one embodiment, the clot activator is calcium chloride (CaCl2·2H2O) clot activator solution at a concentration of 0.05M to 0.3M, preferably 0.1M to about 0.25M, with an optimal concentration of 0.2M.

[0060] In one embodiment, there is also provided a system for preparing a purified wound healing composition comprising platelets, monocytes, and lymphocytes, the system comprising: a first container having a sealable open end for receiving a blood sample and a closed end opposite the open end; the first container having a first density separation medium disposed at a first location adjacent the closed end and a second density separation medium disposed at a second location between the open end and the first location, and an anticoagulant solution having a pH in the range of 6.0 to 8.5 at a third location between the open end and the second location; a first container that, when centrifuged, is operable to produce an extractable suspension of separated platelets, monocytes, lymphocytes, and plasma from the blood sample; and a second container having a sealable open end for receiving the extracted suspension of separated platelets, monocytes, lymphocytes, and plasma produced in the first container, the second container having a coagulation activator; a second container that, when centrifuged, is usable to produce a purified wound healing composition from the suspension of separated platelets, monocytes, lymphocytes, and plasma; Includes.

[0061] In one embodiment of the system, the second density separation medium has a density of about 1.055 to 1.080 g / cm 3 In another embodiment, the first density separation medium comprises at least one of a Newtonian liquid. In a further embodiment, the first density separation medium comprises an ionic material having a molecular weight of less than about 1500. The first density separation medium can be selected from the group including sodium diatrizoate, derivatives thereof, and combinations thereof.

[0062] In another embodiment of the system, the first density separation medium is selected from the group including polymers of sucrose or epichlorohydrin having a molecular weight of at least 400,000, derivatives thereof, and combinations thereof.

[0063] In one embodiment of the present invention, a system is provided in which the purified wound healing composition containing platelets, monocytes, and lymphocytes is a mononuclear cell-platelet rich fibrin matrix.

[0064] In another embodiment of the present invention, a purified non-naturally occurring wound healing composition is provided comprising platelets, monocytes, and lymphocytes, the composition being substantially free of neutrophils, in one embodiment, the concentration of neutrophils is less than 5% of the isolated white blood cells.

[0065] In one embodiment of the present invention, there is also provided a method of producing a purified non-naturally occurring wound healing composition comprising platelets, monocytes, lymphocytes, and plasma from a blood sample, the method comprising: introducing the blood sample into a first container having a sealable open end for receiving the blood sample and a closed end opposite the open end, the first container comprising a density separation medium disposed at a first location adjacent the closed end and a density separation medium of about 1.055 to 1.080 g / cm disposed at a second location between the open end and the first location; 3and an anticoagulant solution having a pH in the range of 6.0 to 8.5 at a third location between the open end and the second location; Centrifuging the first container to produce an extractable suspension of separated platelets, monocytes, lymphocytes, and plasma from the blood sample; extracting an extractable suspension of separated platelets, monocytes, lymphocytes, and plasma in a second container having a coagulation activator, the second container having a sealable open end for receiving the extracted suspension of separated platelets, monocytes, lymphocytes, and plasma produced in the first container; and Centrifuging the second container to produce a purified non-naturally occurring wound healing composition comprising platelets, monocytes, and lymphocytes; Includes.

[0066] In one embodiment of this method, the wound healing composition is substantially free of neutrophils, hi another embodiment, the concentration of neutrophils is less than 5% of the isolated leukocytes.

[0067] In a further embodiment of this method, the separation medium comprises at least one of a non-Newtonian gel and a Newtonian liquid. In one embodiment, the density separation medium comprises an ionic substance having a molecular weight of less than about 1500. In one embodiment, the density separation medium is selected from the group comprising sodium diatrizoate, its derivatives, and combinations thereof. For example, the density separation medium is selected from the group comprising sucrose or epichlorohydrin polymers having a molecular weight of at least 400,000, its derivatives, and combinations thereof.

[0068] In one embodiment, the method provides a purified wound healing composition comprising platelets, monocytes, and lymphocytes, which is a mononuclear cell-platelet rich fibrin matrix. In one embodiment, the thixotropic gel of the method has a viscosity of 1.060 to about 1.065 g / cm 3In another embodiment, the separation medium of the method of the present invention has a specific gravity of 1.065 to about 1.085 g / cm 3 and preferably has a specific gravity of about 1.070 to about 1.080 g / cm 3 It has a specific gravity of 1.077 g / cm 3 In yet another embodiment, the pH of the sodium citrate anticoagulant is from about 6.5 to about 7.5, preferably from 6.85 to about 7.15, with an optimal pH of 7.0.

[0069] In one embodiment, the concentration of sodium citrate anticoagulant is from about 0.05 M to about 0.20 M, with a preferred sodium citrate concentration of from about 0.08 M to about 0.13 M and an optimal sodium citrate concentration ranging from about 0.09 M to about 0.11 M. In a further embodiment, the pH is 7.0 and the sodium citrate concentration is 0.1 M.

[0070] In a further embodiment, the anticoagulant comprises 294 gm sodium citrate·2H2O, 0.27 gm citric acid·H2O at 0.10 moles per liter, and a pH of 7.0. In a particular embodiment, the clot activator is calcium chloride (CaCl2·2H2O) clot activator solution at a concentration of 0.05M to 0.3M, preferably 0.1M to about 0.25M, with an optimal concentration of 0.2M.

[0071] In another embodiment of the invention, there is provided a purified non-naturally occurring wound healing composition comprising platelets, monocytes and lymphocytes produced by the method described above, said composition being substantially free of neutrophils. In a further embodiment, the purified non-naturally occurring wound healing composition is a monocyte-platelet rich fibrin matrix.

[0072] In yet another method according to the invention, a method of treating a wound is provided comprising administering to a subject in need of wound treatment a purified, non-naturally occurring wound healing composition of the invention comprising platelets, monocytes, and lymphocytes, and substantially free of neutrophils. The M-PRFM is removed from the second vial and applied directly to the wound surface. The wound is then covered with a suitable dressing and bandage to support the M-PRFM on the wound surface for several days.

[0073] According to another embodiment of the present invention, a sodium citrate-based anticoagulant solution is prepared in the following manner: Trisodium citrate·2H2O and citric acid·HO are dissolved in a sufficient amount of water to obtain a sodium citrate solution with the desired concentration and pH within the ranges described below. For example, a 0.1M sodium citrate solution with a pH of 7.0 (+0.15) can be prepared by dissolving 29.4g Na citrate·2H2O and 0.27g citric acid·HO in 1 liter of H2O. The concentration of the sodium citrate-based solution should be sufficient to prevent clotting of blood samples that are added to blood separation / collection devices or involved in some other experimental / clinical techniques. In particular, the concentration of sodium citrate should range from about 0.05M to about 0.2M, preferably from about 0.08M to about 0.13M. The most preferred range is from about 0.09M to about 0.11M. In addition, the pH of the final sodium citrate-based solution ranges from greater than pH 6.0 to about pH 8.5, preferably from about pH 6.5 to about pH 7.5. In the most preferred embodiment, the pH ranges from about pH 6.85 to about pH 7.15, and ideally is pH 7.0.

[0074] The anticoagulant solution may alternatively include, for example, the disodium, dipotassium and tripotassium salts of ethylenediaminetetraacetic acid, and combinations thereof. Other suitable citrate-based anticoagulant formulations include, for example, buffered sodium citrate, containing, per liter: 0.109 mol 0.129 mol, 24.7 gm 32.0 gm sodium citrate·2H2O, and 4.42 gm 4. ... citrate phosphate dextrose (CPDA-1 contains 0.275 gm adenine) containing, for example, per litre: 26.3 gm sodium citrate·2H2O, 3.27 gm citric acid·HO, 2.22 gm sodium phosphate monobasic·H2O, 25.5 gm dextrose·HO, pH 5.8; Alsever's solution containing, for example, per litre: 8.0 gm sodium citrate·2H2O, 22.6 gm dextrose·H2O, 4.2 gm sodium chloride, and citric acid to adjust the pH to 6.1; and M-PRFM citrate containing, for example, per litre: 294 gm sodium citrate·2H2O and 0.27 gm citric acid·HO at 0.10 molar, pH 7.0.

[0075] Once the solution of the present invention is prepared according to the steps described above, it may be utilized in some laboratory techniques or added to any of several blood separation and / or collection tubes available in the art for separating lymphocytes, monocytes, and platelets from the heavier phase of whole blood or its pre-treated cellular fraction. Although the sodium citrate-based anticoagulant solution of the present invention may be used in any blood separation device, it provides the greatest advantages when used with those devices that utilize a thixotropic gel layer utilized as a cell density separation medium or as a barrier means to isolate various components of the device prior to centrifugation. In particular, the solution of the present invention may be utilized in the construction of an improved blood separation assembly. A preferred embodiment of the assembly includes a container having a closed end and an open end. The container is preferably of a type known in the art that can collect a blood sample and undergo subsequent centrifugation to separate the sample.

[0076] 1, a blood collection and separation system 10 according to an illustrative embodiment of the present invention is shown. System 10 includes a container or tube 12, a layer of a high viscosity, low density immiscible gel 14, such as a thixotropic gel, disposed within the tube at a first location, and a layer of a liquid density separation medium 16 disposed immediately below the thixotropic gel 14, with a layer of anticoagulant solution 18 disposed above the gel 14. Reference numeral 26 collectively denotes the layered portions including the anticoagulant solution 18, the thixotropic gel 14, and the liquid density separation medium layer 16. Typically, the liquid density separation medium 16 is of a suitable type known in the art for separating mononuclear cells and platelets from whole blood, such as, for example, the commercially available liquid density gradient separation medium Ficoll® Paque.

[0077] Depending on the desired operation to be performed, various thixotropic gels known in the art can be used for the thixotropic gel 14. For example, if the thixotropic gel is utilized as a separation medium as well as a barrier means, the gel may have a viscosity of 1.055 g / cm 3to about 1.080 g / cm 3 Specific gravity of about 1.060 g / cm 3 to about 1.065 g / cm 3 When a thixotropic gel is utilized with a liquid density gradient material, the gel functions primarily as a temporary barrier means prior to centrifugation. In such an assembly, the gel maintains the isolation of the blood sample delivered to the tube from the liquid density gradient material present within the tube until analysis can be performed at a later time. In such circumstances, the specific gravity of the thixotropic gel should be in a range sufficient to allow adequate separation of the mononuclear and platelet cell layers from other components of the blood sample. Preferably, the thixotropic gel utilized in such an assembly has a specific gravity of about 1.055 g / cm 3 to about 1.075 g / cm 3 Thixotropic gels have specific gravities ranging from 0.1 to 1.0 mm. Thixotropic gels are well known in the art and are typically water insoluble and chemically inert to blood. They are commonly prepared from dimethylpolysiloxane or polyester and precipitated methylated silica, the methylation rendering the material hydrophobic. A preferred embodiment of the improved blood separation assembly of the present invention also includes a suitable liquid density separation medium utilized within the container at a second location further from the open end of the container than the thixotropic gel layer.

[0078] An illustrative method according to one embodiment of the present invention includes introducing a sample of whole blood or a cell fraction of pretreated blood into a container 12 having a layered portion 26. Subsequent centrifugation of the assembly 10 then causes the thixotropic gel layer 14 to move from a first position toward the top end 15 of the container 12. As the centrifugation proceeds, the red blood cells and granulocytes separate from the mononuclear cell and platelet fraction and become concentrated in the layer immediately above the thixotropic gel. As the thixotropic gel moves toward a new position in the tube, the red blood cells and granulocytes move through the gel and displace the liquid density separation medium below. As the liquid density separation medium is displaced, they move upward through the thixotropic gel and mix with the mononuclear cell and platelet fraction / anticoagulant solution. The red blood cells and granulocytes pellet toward the bottom of the tube, while the lymphocytes, monocytes, and platelets form a layer of highly purified mononuclear cell-platelet cells just above the thixotropic gel layer, thereby facilitating the isolation and subsequent removal of the mononuclear and platelet cells.

[0079] Finally, an anticoagulant solution 18 is placed on top of the thixotropic gel layer 14 so that it can adequately contact the whole blood sample introduced into the tube for centrifugation and subsequent isolation of the mononuclear and platelet cell layers. In FIG. 1, the anticoagulant solution 18 is shown placed on top of the thixotropic gel layer 14 at a location closer to the open end of the separation tube 10 than the gel. This anticoagulant solution 18 may, for example, have an effective concentration of sodium citrate sufficient to prevent clotting of the blood sample when it is subsequently added to the tube for centrifugation and subsequent analysis. The anticoagulant solution may have a pH ranging from greater than pH 6.0 to about pH 8.5, preferably from about pH 6.5 to about pH 7.5. Most preferably, the pH of the solution should range from about pH 6.85 to about pH 7.15. Optimally, the pH should be 7.0. In one embodiment of the improved blood separation 10, the concentration of sodium citrate should range from about 0.05 M to about 0.2 M, preferably from about 0.08 M to about 0.13 M. Most preferably, the concentration of sodium citrate should be from about 0.09 M to about 0.11 M. Optimally, the concentration should be 0.1 M.

[0080] Although anticoagulant solutions suitable for use as solution 18 are primarily comprised of sodium citrate, additional reagents, such as a cell maintenance solution or other reagents, may be added to impart additional properties to the solution. A preferred embodiment of the improved blood separation assembly of the present invention also includes a free space adjacent the open end of the container or tube that is of sufficient volume to receive a sample of whole blood or a portion thereof, either alone or with added reagents. Figure 1 shows a free space 20 positioned above the anticoagulant solution 16 to provide a suitable space to accommodate the blood sample to be separated.

[0081] In addition, the assembly of the present invention may optionally include a closure element for sealing the open end of the container or tube. Typically, the closure element is adapted to provide a vacuum seal of the open end of the container as well as to be penetrable by a needle to make the container suitable for taking a blood sample from a test subject. In FIG. 1, a closure element 22, e.g., a seal, is provided at the open end of the container or tube to provide a vacuum seal of the container as described above. When a blood sample is added to the assembly 10, mixing of the sample with the anticoagulant solution 18 occurs, typically by manual inversion of the container 12. The thixotropic gel layer 14 remains in a temporarily fixed first position within the tube to act as a barrier to isolate the blood sample / anticoagulant solution suspension from any contact with other components of the assembly, such as the liquid density separation medium 16.

[0082] The present invention further includes a method for separating lymphocytes, monocytes, and platelets from a heavier phase of a sample of whole blood or a pretreated cellular fraction thereof. The method includes the step of providing a container having an open end and a closed end. Preferably, the container is a blood collection / separation tube of the type described above. The method includes introducing a first layer of a thixotropic gel-like substance into the container or tube at a first location. The method further includes introducing an anticoagulant solution into the container at a second location closer to the open end of the container than the first thixotropic gel layer. The method also includes the steps of limiting the pH of the solution to one of the preferred ranges described above, and introducing an anticoagulant solution of the present invention into the container at an effective concentration of sodium citrate sufficient to prevent clotting of the blood sample. In addition, the method includes the step of limiting the concentration of sodium citrate in the solution to one of the ranges described above in the description of the anticoagulant solution.

[0083] The method of the present invention also includes the step of introducing a sample of whole blood or a cellular fraction of pretreated blood into a container, and the subsequent step of centrifuging the container to induce separation of lymphocytes, monocytes, and platelets from the heavier phase of the sample.

[0084] Many methods and systems require the transfer of fluids from one container to another. A common practice is to remove the closures of the two containers and pipette the liquid in one container into the other. However, this practice exposes the sample to environmental contaminants. For example, this technique is used to transfer plasma separated from red blood cells in a blood sample. However, in many cases, a different technique is required to remove the plasma at the interface meniscus. Often, the high density and undesirable low fraction of red blood cells contaminate the aspirated sample. To avoid this problem, the pipette can be kept at a safe distance from the meniscus (i.e., the interface between the plasma and the red blood cells), which can result in incomplete transfer of the sample. Incomplete transfer of the desired fraction results in less than optimal volumetric yields and non-stoichiometric ratios of the sample reagents and their mixtures in the second container. This second condition can be a serious source of product performance variability. This is the case in many enzymatic reactions where the reaction rate is maximized at a particular stoichiometric ratio and decreases rapidly at higher or lower ratios. The use of sterile needles and syringes does not improve the ability to recover the various layers of plasma or cells, as the technique remains highly sensitive to the operator's ability and experience to accurately see the various layers.

[0085] Wound care is one of the most important problems in medicine, especially with regard to chronic ulcers. This problem is important not only because of the high cost of management, but also because of the variable success rate. Other problems related to wound care include the loss of fluid and the possibility of infection. Synthetic or animal-derived membranes have been used in wound care as dressings or to separate bone cavities from soft tissues in the process of re-ossification.

[0086] One treatment for wound care may involve the application of biological tissue or animal-derived sponges (generally protein-based), such as collagen, fibrin, albumin, etc., to the wound site. However, allergic and immunological reactions are common with these applications. Most of these cases do not resolve with a single application and multiple applications may be required.

[0087] Alternative treatments include skin grafts, which are performed for the most difficult cases. However, skin grafts are expensive and significantly increase the overall cost of treatment. A mesh of modified animal collagen has been used to support the new autologous tissue. Its application is a difficult process that can take up to 20 days to culture the skin tissue, and there is a risk of the device becoming contaminated.

[0088] Overall, methods and systems for preparing autologous PRP or M-PRP or a solid fibrin matrix capable of regenerating tissue in vivo are desired.

[0089] The present invention also provides systems and methods for forming a solid fibrin matrix or autologous fibrin membrane capable of regenerating tissue in vivo. In these methods and systems, anticoagulated plasma with mononuclear cells and platelets is obtained by centrifugation of a blood sample. The transfer device described herein allows the cell-plasma suspension to be transferred to a second container with a calcium coagulant and then immediately centrifuged to obtain a stable and dense autologous fibrin mononuclear cell-platelet network. The transfer device described herein can also be used to transfer other liquids in other applications. In other words, the methods, transfer devices, and systems described herein allow simultaneous centrifugation and coagulation.

[0090] By using these systems and methods, at least one of the following can be achieved: (1) the sample is manipulated in a manner that maintains sterility; (2) the total amount of plasma is transferred to maximize the complete yield of clots; (3) the stoichiometric ratio of anticoagulant to calcium coagulant is maintained within a narrow range to minimize clotting time; (4) the pH of the applied matrix is ​​close to the normal pH of human tissue, thus avoiding a stinging sensation; (5) the transfer is completed rapidly and can be performed within the half-life of platelet-derived growth factor during surgery; (6) healthcare providers (e.g., nurse practitioners, etc.) who do not normally perform these manipulations can easily perform these methods and operate the system; (7) the equipment is single-use to prevent reuse and possible contamination with blood-borne pathogens.

[0091] Generally speaking, the present invention provides an integrated system and method for preparing a solid fibrin matrix or an autologous fibrin membrane that can be used to regenerate tissue in vivo. In one embodiment depicted in Figure 2, the system includes a primary container 10, such as the container 10 of Figure 1, a secondary container 48 (or alternative container 38), and a transfer device 18. Preferably, the primary and secondary containers 10, 48 are tubes or flat-bottomed vials 38, more particularly test tubes or vials, although any container capable of holding a fluid or liquid and capable of being centrifuged is suitable for use in the present invention. Preferably, the containers 10, 48, and 38 are made of glass or plastic.

[0092] The primary container 10 should have the ability to draw blood therein using standard venipuncture techniques. Preferably, the primary container 10 is sealed with a seal 22 to prevent contamination while blood is being drawn, although the container 10 may be sealed immediately thereafter. A variety of seals 22 can be used to seal the primary container 10, such as, for example, rubber stoppers, caps, foams, elastomers, or other composite materials. The seal 22 should be pierceable or puncturable, thus rubber and silicone are preferred materials from which the seal is made, although any material that provides a seal and is pierceable can be used. The primary container 10 may have a layered portion 26 of FIG. 1.

[0093] In operation, the anticoagulant 18 tends to slightly dilute the blood collected in the primary container 10 to condition it for centrifugation. In addition, the primary container contains a density gradient separation medium 26, air 27, and a high viscosity, low density gel 28.

[0094] The transfer device 18 may include two parts, as depicted in FIG. 2. The transfer device 18 includes a cannula having a first end 42 with a first opening and a second end 50 with a second opening. The cannula ends 42, 50 are sharp or pointed (or even have a bevel ground on them) so that they can pierce or penetrate the seals 22, 24 of the primary and secondary containers 10, 48, or 38. The cannula is recessed and coaxially mounted within the housing to prevent accidental finger sticks during manipulation of the containers. The housing 58 has two cylindrical opposing guides 62, 64 centered with the cannula and axially oriented. The guides 62, 64 serve to guide the primary container 10 and secondary container 48 or 38 onto the first and second ends 42, 50 of the transfer device 18.

[0095] The cannula ends 42, 50 may be encompassed or covered by a safety valve, sheath, or elastomeric sleeve 68, 72 that forms a hermetic seal. The safety sheath 68, 72 also covers the first and second openings 46, 54. When the first and second ends 42, 50 pierce the elastomeric sleeve 68, 72, the sleeve 68, 72 retracts accordingly. The ends 42, 50 extend long enough to completely pierce the seal 22, 24, but do not extend further into the container 10, 48, or 38. This allows for maximum volume transfer of the liquid volume of the inverted primary container 10 to the secondary container 48 or 38. The elastomeric sleeve 68, 72 prevents the flow of gas or liquid when not pierced. Suitable materials for the sleeve 68, 72 include, but are not limited to, rubber types and thermoplastic elastomers.

[0096] Following centrifugation of the blood collection tubes 10, the sealed primary holder 10 is inverted before piercing the seal 22 using the transfer device 18. In other words, the primary container 10 is inverted so that the sealed opening is in the lowest vertical position. Inverting the primary container changes the order in which the layers are arranged. Above the seal 22 are the following layers, from bottom to top: mononuclear cell-platelet rich plasma, gel 14 (shown in FIG. 1), residual gas, separation media 16, and red blood cells.

[0097] The secondary container 48 or 38 is then placed in a vertical position with its sealed opening 24 in the uppermost position as shown in FIG. 2. This positions the secondary container 48 or 38 to transfer the contents of the primary holder therein. The guide 64 of the transfer device is then placed over the secondary container 48 or 38 to guide the secondary container 48 or 38 therein, and then the inverted primary container 10 is placed in the other guide 62 (or vice versa). In other words, either end 42, 50 of the cannula can be used to pierce either seal 22, 24. The transfer device 18 is symmetrical at either end, providing the user with a degree of foolproof operation. The user then forces the containers together to pierce both seals 22, 24 with the respective cannula ends 42, 50. The two valve sleeves 68, 72 covering the ends 42, 50 further enhance the foolproof operation. First, if the first end 42 pierces the primary seal 22 (again, either end can be used to pierce either seal), the unpierced sleeve 72 covering the other end 50 contains the fluid, thereby preventing it from spilling out. On the other hand, if the other end 50 first pierces the other seal 24 (and correspondingly the sleeve 72), a vacuum is maintained by the sleeve 68 covering the first end 42.

[0098] When ends 42, 50 pierce both sleeves 68, 72 and seals 22, 24 as shown, the desired fluid is transferred by pressure differential from primary container 10 to secondary container 48, or 38, if used. In other words, the pressure in secondary container 48 or 38 has been vented so that the contents of primary container 10 (more specifically, mononuclear cell-platelet rich plasma) flows into secondary container 48, or 38, if used. The pressure in primary container 10, originally at atmospheric pressure, decreases as the liquid level decreases and the gas volume expands. However, at no point is the pressure zero. Because secondary container 48 or 38 has been completely evacuated to a pressure equal to or slightly greater than zero, there is little or no gas to compress and so the pressure therein does not increase as the tube fills.

[0099] Due to the sequential arrangement of layers in the primary container 10, the mononuclear cell-platelet rich plasma is easily transferred via the transfer device 18 to the secondary container 48 or 38. In addition, the primary container 10 is also preset to an evacuation level so that the container is only partially filled after blood collection. This allows the gas in the "headspace" to remain significantly above zero during transfer as its volume expands, thereby allowing for rapid and complete transfer to the secondary container 48 or 38, if used. This is dictated by the ideal gas law and the Poiseuille-Hagen equation.

[0100] Transfer of the mononuclear cell-platelet-plasma fraction to the secondary container 48 or 38 is completed, thereby allowing for maximum yield and maintaining proper stoichiometric ratios of reagents. The mononuclear cell-platelet-plasma then contacts the clot activator 36 in the secondary container 48 or 38, thereby creating a mixture that can be immediately centrifuged to form a solid fibrin web. The pressure differential between the primary container 10 and the secondary container 48 or 38 is substantially maintained during transfer, allowing for rapid transfer. The transfer device 18 is not sensitive to the order of tube engagement, making the system virtually foolproof. Finally, transfer may occur without venting, maintaining sterility and non-contamination of the sample.

[0101] In the secondary container 48 or 38, the mononuclear cell-platelet-plasma suspension may contact the calcium-coagulation activator 36 immediately followed by simultaneous coagulation and centrifugation of the plasma to form a solid fibrin web. The solid fibrin web is suitable for regeneration of body tissue in vivo. Such a method alleviates the need to first pre-concentrate the plasma by removing water therefrom before it contacts the calcium-coagulation activator 36. Additionally, the transfer device 18 may be used to transfer blood or other fluids in a wide variety of applications.

[0102] The use of secondary container 48 allows the resulting mononuclear cell-platelet fibrin matrix formed after centrifugation to take on the shape of the bottom of secondary tube 14, thus forming a mononuclear cell-platelet rich fibrin matrix (M-PRFM). Alternatively, secondary vial 38 is used such that after centrifugation, the resulting mononuclear cell-platelet rich fibrin matrix forms an M-PRFM membrane of the same diameter as secondary vial 38.

[0103] In a further embodiment, the present invention also provides a ready-to-use kit that includes one or more of primary container 10, secondary container 48 or 38, transfer device 18, alcohol swabs for cleaning the venipuncture site, a multi-sample blood collection needle (21 gauge x 1"), a safety holder, elastic dressing, and a sterile culture dish for receiving the M-PRFM matrix or membrane. These components can be arranged in a variety of ways within the kit.

[0104] The present invention will now be further described in the following examples, which are intended as illustrative only and not as limiting the scope of the invention. EXAMPLES

[0105] A relatively concentrated cell-plasma suspension is obtained by separating mononuclear cells and platelets from whole blood. Reproducibility testing of recovery, purity, viability, red blood cell contamination, and granulocyte contamination was performed using evacuated blood collection tubes with density gradient fluid, thixotropic gel, and citrate anticoagulant. Ten blood samples of approximately 8.0 mL per tube containing 1.0 mL citrate anticoagulant at pH 7.0 were collected from one donor. The tubes were centrifuged at 1500 x g for 20 min. The resulting mononuclear cell-platelet fraction was inverted seven times to resuspend the cells in plasma, creating mononuclear cell-platelet rich plasma (M-PRP). M-PRP was analyzed in duplicate using an automated hematology analyzer. No final wash step was performed. Samples were resuspended to approximately equal final volumes. Tube-to-tube variation was calculated by taking the difference in the mean of duplicate readings for each tube.

[0106] [Table 1] Table 1. Reproducibility study of recovery, viability, RBC contamination, and PMN (polymorphonuclear cells or granulocytes) contamination from whole blood using a vacuum cell separator with density gradient liquid, thixotropic gel, and citrate anticoagulant (pH 7.0). EXAMPLES

[0107] A whole blood sample is collected using a blood collection tube with sodium citrate anticoagulant, thixotropic gel, and density gradient liquid. The tube is gently inverted seven times to mix the anticoagulant with the whole blood. The tube is placed in a swing-out bucket rotor and centrifuged at 1500xg for 20 minutes. Once the M-PRP is separated from the whole blood sample in the gel separation tube, the M-PRP is aseptically transferred to a second vial with sodium citrate. The solution is gently mixed and the second vial is centrifuged at 3500xg for 25 minutes. Once the second centrifugation step is complete, the vial is uncapped and the M-PRFM is retrieved by inverting the vial and emptying the contents onto a sterile receiving device such as a tissue culture dish. The M-PRFM is retrieved, for example, by sterile forceps and placed directly on the wound surface. The wound is then covered with an appropriate covering and dressing to support the M-PRFM within the wound for several days. This treatment can be repeated weekly as necessary.

[0108] References Demidova-Rice, TN, et al. (2012) Adv Skin Wound Care. 25(7): 304-14 Sibbald, RG, et al. (2011) Adv Skin Wound Care. 24: 415-37 Canedo-Dorantes, L, et. al. (2019) Int J Inflam. 2019: 3706315 Gould, L, et al. (2015) Wound Repair Regen. 23(1): 1-13 Eming, SA, et al. (2014) Sci Transl Med. 6(265): 265sr6 Moulik, PK, et al. (2003) Diabetes Care. 26(2): 491-494 Armstrong, DG, et al. (2007) Int. Wound J. 4(4): 286-287 Nussbaum, SR, et al. (2018) Value Health. 21(1): 27-32 Barakat, M, et al. (2020) Adv Wound Care. https: / / doi.org / 10.1089 / wound2020.1254 Lefrancais, E, et al. (2017) Nature. 544(7648): 105-109 Etulain J. (2018) Platelets. 29(6): 556-568 Eisinger, F, et al. (2018) Front Med. 5: 317 Blair, P, et al. Blood Rev. (2009) 23(4): 177-189 Cloutier, N, et al. (2018) PNAS 115(7): E1550-E1559 Neumuller, J, et al. In: Khan M, ed. Intechopen. London: Intechopen; (2015). pp. 255-284 Hu H, et al. Thromb Haemost (2010) 104: 1184-1192 Miyamoto, H, et al. J Oral Max Surgery, Med, and Path. (2020) 32(4): 237-240 Langer HF, et al. Arterioscler Thromb Vasc Biol. (2007) 27: 1463-1470 Massberg S, et al. J Exp Med. (2006) 203: 1221-1233 Langer HF, et al. J Mol Cell Cardiol. (2009) 47: 315-325 Boulaftali Y, et al. J Clin Invest. (2013) 123: 908-916 Xu X, et al. Cells Tissues Organs. (2013) 197:103-113 Pietrzak, W.S., et al. (2005) J Craniofac Surg. 16:1043-1054 Frykberg, RG, et al. (2010) Ostomy Wound Manage. 56(6): 36-44 Zhou, Y, et al. (2016) BioMed Res. Int. 2016: 1-8 Fedorova, N.V., et al. (2018) Mediat Inflamm. 2018: ID 1574928 Wong, SL, et al. (2015) NatMed. 21(7):815-819 Wier, E, et al. bioRxiv (2020.07.06).189910 Mosser DM, et al. (2008) Nature Rev Immunol. 8(12): 958-969 Porta, C, et al. (2015) Semin Immunol. 27(4): 237-248 Abramson, SL, et al. (1990) J Immunol. 144(2): 625-630 Anders, CB, et al. (2019). Curr Opinion Infect Dis. 32(3): 204-209 Jin, E, et al. (2013) J Cell. Mol. Med. 17(12): 1644-1651 Quiros, M, et al. (2017) J Clin Invest. 127(9): 3510-3520 Wang, X, et al. (2019) Adv in Wound Care 8(11): 527-537 Park, JE, et al. Am J Surg. (2004) 187(5): S11-S16 Fitzpatrick, J, et al. (2017) Orthop J Sports Med. 5(1):2325967116675272 Margolis, J. (1956) Nature. 178: 805-806 It is to be understood that the invention is not limited to the specific embodiments of the invention described above, as variations of the specific embodiments can be made and still fall within the scope of the appended claims.

Claims

1. 1. A system for preparing a purified, non-naturally occurring wound healing composition comprising platelets, monocytes, and lymphocytes, and substantially free of neutrophils, comprising: a first container having a sealable open end for receiving a blood sample and a closed end opposite the open end; the first container having a first density separation medium disposed at a first position adjacent the closed end, a second density separation medium disposed at a second position between the open end and the first position, and an anticoagulant solution having a pH in the range of 6.0 to 8.5 at a third position between the open end and the second position; a first container that, when centrifuged, is operable to produce an extractable suspension of separated platelets, monocytes, lymphocytes, and plasma from a blood sample; a second container having a sealable open end for receiving the extracted suspension of separated platelets, monocytes, lymphocytes, and plasma produced in the first container, the second container having a coagulation activator; a second container that, when centrifuged, is usable to produce a wound healing composition from the suspension of separated platelets, monocytes, lymphocytes, and plasma; A system including:

2. The second density separation medium has a density of about 1.055 to 1.080 g / cm ３ 10. The system of claim 1, wherein the gel is a thixotropic gel having a density of

3. The system of claim 1 or claim 2, wherein the first density separation medium comprises at least one of a Newtonian liquid.

4. 3. The system of claim 1 or claim 2, wherein the first density separation medium comprises an ionic material having a molecular weight of less than about 1500, and optionally, the first density separation medium is selected from the group comprising sodium diatrizoate, derivatives thereof, and combinations thereof.

5. 3. The system of claim 1 or claim 2, wherein the first density separation medium is selected from the group consisting of polymers of sucrose or epichlorohydrin having a molecular weight of at least 400,000, derivatives thereof, and combinations thereof.

6. 3. The system of claim 1 or claim 2, wherein the purified wound healing composition comprising platelets, monocytes, and lymphocytes is a mononuclear cell-platelet-rich fibrin matrix.

7. The system described in claim 2, further comprising a transfer device adapted to engage with the open end of the first container and the open end of the second container, wherein the transfer device, when engaged with the open end of the first container, creates a sterile seal for receiving the suspension of separated platelets, monocytes, lymphocytes, and plasma from the first container, and, when engaged with the open end of the second container, creates a sterile environment for transferring the suspension of separated platelets, monocytes, lymphocytes, and plasma to the second container.

8. The system described in claim 2, further comprising a first closure device for sealing the open end of the first container, optionally the first closure device being adapted to vacuum seal the open end of the first container, and optionally the first closure device being pierceable by a cannula for supplying the blood sample to the first container by pressure differential.

9. The system of claim 2, further comprising a second closure device for sealing the open end of the second container, optionally adapted to vacuum seal the open end of the second container, and optionally, the second closure device being pierceable by a cannula for supplying the suspension of separated platelets, monocytes, lymphocytes, and plasma to the second container by a pressure differential.

10. The system described in claim 2, further comprising a centrifuge adapted to receive at least one of the first container and the second container.

11. (i) The thixotropic gel has a specific gravity of 1.060 to about 1.065 g / cm 3 . (ii) the first density separation medium has a specific gravity of 1.065 to about 1.085 g / cm 3 , preferably about 1.070 to about 1.080 g / cm 3 , with an optimum specific gravity of 1.077 g / cm 3 ; (iii) the pH is from about 6.5 to about 7.5, preferably from 6.85 to about 7.15, with an optimum pH of 7.0; (iv) the anticoagulant solution comprises sodium citrate at a concentration of about 0.05M to about 0.20M, with a preferred sodium citrate concentration being about 0.08M to about 0.13M, and an optimal sodium citrate concentration ranging from about 0.09M to about 0.11M; (v) the pH is 7.0 and the anticoagulant solution comprises sodium citrate at a concentration of 0.1 M; (vi) the anticoagulant solution comprises 294 gm sodium citrate·2H 2 O, 0.27 gm citric acid·H 2 O at 0.10 moles per liter, and a pH of 7.0; (vii) the anticoagulant solution comprises at least one of ethylenediaminetetraacetic acid disodium salt, ethylenediaminetetraacetic acid dipotassium salt, or tripotassium salt; or (viii) The system of claim 2, wherein the clot activator is calcium chloride (CaCl2.2H2O) clot activator solution at a concentration of 0.05M to 0.3M, preferably 0.1M to about 0.25M, with an optimal concentration of 0.2M.

12. 1. A method for producing a purified non-naturally occurring wound healing composition comprising platelets, monocytes, lymphocytes, and plasma from a blood sample, comprising: introducing the blood sample into a first container having a sealable open end for receiving the blood sample and a closed end opposite the open end, the first container containing a density separation medium disposed at a first position adjacent the closed end and a density separation medium of about 1.055 to 1.080 g / cm 3 disposed at a second position between the open end and the first position; ３ and an anticoagulant solution having a pH in the range of 6.0 to 8.5 at a third location between the open end and the second location; centrifuging the first container to produce an extractable suspension of separated platelets, monocytes, lymphocytes, and plasma from the blood sample; extracting the extractable suspension of separated platelets, monocytes, lymphocytes, and plasma in a second container having a coagulation activator, the second container having a sealable open end for receiving the extracted suspension of separated platelets, monocytes, lymphocytes, and plasma produced in the first container; centrifuging the second container to produce a purified non-naturally occurring wound healing composition comprising platelets, monocytes, and lymphocytes; A method comprising: (i) the wound healing composition is substantially free of neutrophils, and optionally, the concentration of neutrophils is less than 5% of the isolated leukocytes. (ii) the density separation medium comprises at least one of a non-Newtonian gel and a Newtonian liquid; (iii) the density separation medium comprises an ionic material having a molecular weight of less than about 1500, and optionally the density separation medium is selected from the group comprising sodium diatrizoate, derivatives thereof, and combinations thereof; (iv) the density separation medium is selected from the group consisting of polymers of sucrose or epichlorohydrin having a molecular weight of at least 400,000, derivatives thereof, and combinations thereof; (v) The purified wound healing composition comprising platelets, monocytes, and lymphocytes is a mononuclear cell-platelet-rich fibrin matrix. (vi) the thixotropic gel has a specific gravity of 1.060 to about 1.065 g / cm 3 ; (vii) the density separation medium has a specific gravity of 1.065 to about 1.085 g / cm 3 , preferably about 1.070 to about 1.080 g / cm 3 , with an optimum specific gravity of 1.077 g / cm 3 ; (viii) the pH is from about 6.5 to about 7.5, preferably from 6.85 to about 7.15, with an optimum pH of 7.0; (ix) the anticoagulant solution comprises sodium citrate at a concentration of about 0.05M to about 0.20M, with a preferred sodium citrate concentration being about 0.08M to about 0.13M, and an optimal sodium citrate concentration ranging from about 0.09M to about 0.11M; (x) the pH is 7.0 and the anticoagulant solution comprises sodium citrate at a concentration of 0.1 M; (xi) the anticoagulant solution comprises 294 gm sodium citrate·2H 2 O, 0.27 gm citric acid·H 2 O at 0.10 moles per liter, and a pH of 7.0; or (xii) The method of claim 12, wherein the clot activator is calcium chloride (CaCl2.2H2O) clot activator solution at a concentration of 0.05M to 0.3M, preferably 0.1M to about 0.25M, with an optimal concentration of 0.2M.

14. A purified, non-naturally occurring wound healing composition comprising platelets, monocytes, and lymphocytes, and substantially free of neutrophils.

15. The purified, non-naturally occurring wound healing composition of claim 14, wherein: (i) the concentration of neutrophils is less than 5% of the isolated leukocytes; (ii) the composition is a mononuclear cell-platelet rich fibrin matrix; or (iii) the composition is produced by the method of claim 12.

16. A purified, non-naturally occurring wound healing composition as described in claim 14 for use in a method for treating a wound in a subject in need of treatment.

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