Bioactive compositions derived from platelet concentrate, and their preparation and use methods

Abstract

This disclosure relates to bioactive compositions derived from platelet concentrate, and methods for their preparation and use. Specifically, this disclosure provides compositions comprising a bioactive fraction derived from platelet concentrate, methods for preparing said bioactive fraction, and culture media supplemented with said bioactive fraction. Preferred bioactive fractions have relatively low fibrinogen concentrations while retaining beneficial amounts and proportions of natural growth factors.

Description

[0001] This application is a divisional application of Chinese patent application No. 201480056186.0, filed on August 27, 2014, entitled "Bioactive Compositions Derived from Platelet Concentrate and Methods for Preparation and Use Thereof". Technical Field

[0002] This invention generally relates to the field of bioactive substances derived from animal platelet products and methods for their preparation and use. Background Technology

[0003] The application of cells or cell-containing compositions for therapeutic treatment is becoming an increasingly popular treatment approach. Such treatments may include, for example, the application of mesenchymal stem cells (MSCs) with the potential to differentiate into mesenchymal cell lineages, including, for example, bone, fat, cartilage, and muscle.

[0004] To obtain therapeutic quantities of cells for transplantation, it is often necessary to expand the cell population from the original population. The culture medium used for expanding the cell population provides the essential nutrients for cell metabolism, growth, and proliferation. Fetal bovine serum (FBS) is frequently used as a supplement to promote population expansion. FBS has been a preferred supplement because of its low antibody content, the presence of many growth factors that stimulate cell growth and proliferation, and its relatively inexpensive production. However, FBS has recognized disadvantages, including the risk of pathogen transmission, such as bovine spongiform encephalopathy.

[0005] Human platelet lysate (hPL) has emerged as a potential, non-heterogeneous alternative to fibrinogen-derived platelets (FBS). hPL is derived from platelets known to contain a variety of growth factors. In addition to growth factors, current hPL separation techniques typically yield compositions that retain high concentrations of fibrinogen (a glycoprotein involved in clot formation). Due to their fibrinogen content and clot-forming tendency, current commercial hPL compositions are often used in conjunction with one or more anticoagulant additives, typically heparin. Anticoagulant additives in hPL increase the cost of hPL production and / or use, and can pose problems if the bioactivity of heparin is detrimental. Furthermore, while various methods for producing hPL have been proposed, attempts to achieve the desired compositional distribution or bioactivity of hPL products often result in process complexity and / or intensive equipment requirements. Widespread adoption of hPL as an alternative to FBS will require an economically viable method that yields the desired and efficient composition.

[0006] Given this background, there is a need for human platelet lysate compositions that are essentially free of fibrinogen, retain growth factors beneficial to cell proliferation, and can be easily and economically manufactured. Summary of the Invention

[0007] In certain aspects of the invention, it has been discovered that advantageous bioactive fractions of blood-derived platelet concentrates (preferably human) can be uniquely processed from the concentrate. The bioactive fractions may have a novel compositional distribution of growth factors and / or other substances present in the initial concentrate, and the processing methods may involve novel techniques for coagulating and separating the concentrate fractions and / or novel deep filtration operations for clarifying the liquid fractions derived from the concentrate.

[0008] In one aspect, a method for processing a platelet lysate composition includes the steps of: lysing platelets from a human blood concentrate containing platelets and plasma to form a lysed platelet preparation, forming a clot gel by converting fibrinogen in the lysed platelet preparation into fibrin, and pressing the clot gel to expel liquid from the clot gel.

[0009] In another aspect, the method includes passing a liquid bioactive fraction of a blood-derived platelet concentrate through at least a first depth filter to remove suspended solids from the bioactive fraction, said liquid bioactive fraction comprising natural components of the platelet concentrate, including fibrinogen, albumin, globulin, and at least one, optionally all, of TGF-β1, EGF, FGF-β, PDGF-AA, PDGF-BB, SDF-1α, and VEGF, wherein fibrinogen is present at a concentration of less than 20,000 ng / mL. The platelet concentrate is preferably human platelet concentrate. In a preferred embodiment, the liquid bioactive fraction is passed through a first depth filter and also through a second depth filter, wherein the second depth filter optionally has a nominal micron scale smaller than that of the first depth filter.

[0010] In another aspect, a composition comprising a bioactive fraction of a human blood-derived platelet concentrate containing human platelets and human plasma is provided. The bioactive fraction comprises natural components of the platelet concentrate, including fibrinogen, albumin, globulin, TGF-β1, EGF, FGF-β, PDGF-AA, PDGF-BB, SDF-1α, and VEGF. The bioactive fraction may have fibrinogen present at a concentration below about 20,000 ng / mL, for example, in the range of about 500 ng / mL to about 20,000 ng / mL, and / or the bioactive fraction or the cell culture medium containing it may be free of or substantially free of added heparin (i.e., heparin not derived from the platelet concentrate starting material). The bioactive fraction may have the growth factor content or proportions disclosed herein. In some embodiments, the bioactive fraction is a liquid bioactive fraction, and the natural components include: Liquid bioactive fractions with concentrations below 20,000 ng / mL, for example, fibrinogen in the range of about 500 ng / mL to about 20,000 ng / mL. Albumin with a concentration of at least 2 mg / dL of liquid bioactive fraction; Globulins with a concentration of at least 1 g / dL of liquid bioactive fraction; TGF-β1 in a liquid bioactive fraction of at least 5000 picograms per milliliter (“pg / mL”); EGF at a concentration of at least 20 pg / mL liquid bioactive fraction; FGF-β at a concentration of at least 5 pg / mL liquid bioactive fraction; PDGF-AA with a content of at least 200 pg / mL liquid bioactive fraction; PDGF-BB with a content of at least 50 pg / mL liquid bioactive fraction; SDF-1α at a concentration of at least 100 pg / mL liquid bioactive fraction; and VEGF at a concentration of at least 10 pg / mL liquid bioactive fraction.

[0011] This disclosure also provides a method for preparing a bioactive composition, comprising the steps of: adding a coagulant to a platelet lysate composition to form a coagulated substance, separating the coagulated solids from the liquid in the coagulated substance, and subjecting the liquid to deep sterile filtration. In some forms, the method further includes packaging the bioactive composition in a sterile container.

[0012] In some embodiments, the bioactive fraction is a liquid bioactive fraction, and the natural component includes: FGF-2 with a concentration between approximately 200 pg / mL and approximately 350 pg / mL; EGF at concentrations between approximately 1800 pg / mL and approximately 3100 pg / mL; PDGF-AA with a content between approximately 24,000 pg / mL and approximately 28,000 pg / mL; PDGF-BB at concentrations between approximately 50 ng / mL and approximately 80 ng / mL; VEGF at concentrations between approximately 500 pg / mL and approximately 800 pg / mL; TGF-b at concentrations between approximately 60 ng / mL and approximately 90 ng / mL; and Fibrinogen content below 2.5 μg / mL.

[0013] Other embodiments disclosed herein relate to the use of the bioactive fractions or compositions described herein for cell culture, cryopreservation, or therapeutic purposes.

[0014] Further implementation methods, features, and advantages will become apparent to those skilled in the art from the description herein. Attached Figure Description

[0015] Figure 1 A method for preparing a liquid bioactive fraction of a human platelet concentrate composition is shown.

[0016] Figure 2 This is a perspective view of one embodiment of the liquid bioactive fraction disclosed herein in aseptic packaging.

[0017] Figure 3 This is a perspective view of one embodiment of the liquid bioactive fraction disclosed herein in a dropper bottle. Detailed Implementation

[0018] For the purpose of aiding in the understanding of the principles of the invention, reference will now be made to certain embodiments and they will be described using specific language. However, it should be understood that this is not intended to limit the scope of the invention. Any variations and further modifications to the described embodiments, as well as any further applications of the principles of the invention as described herein, are to be considered as would normally occur to those skilled in the art to which this invention pertains.

[0019] Generally, this disclosure provides bioactive compositions and methods for preparing bioactive compositions, which can be used, for example, as cell culture supplements and / or therapeutic agents. The compositions disclosed herein include human-derived compositions, thereby overcoming the problems associated with heterologous compositions such as FBS. Furthermore, the preferred compositions of this invention, while retaining important growth factors and other chemicals, have low fibrinogen content and do not require the addition of anticoagulants such as heparin to prevent problematic coagulation during use.

[0020] Turn now Figure 1The diagram illustrates an exemplary method 100 for preparing a bioactive fraction from a platelet concentrate composition. The method includes the following steps: obtaining a platelet concentrate 01, freezing the platelet concentrate 02, thawing the platelet concentrate 03, adding a coagulant to the platelet concentrate 04, separating the clotted solids from the liquid 05, filtering the liquid using a first depth filter 06, filtering the liquid using a second depth filter 07, filtering the liquid using a sterile filter 08, and packaging the liquid 09. In some respects, the selection of each described general step is described in detail in the following discussion; however, it should be understood that not all described general steps are necessary for all embodiments herein, and novel methods incorporating features corresponding to one, some, or all of the described steps are considered embodiments of this invention.

[0021] Platelet concentrate compositions and bioactive fractions used as source materials for the disclosed methods can be obtained in any suitable manner. As used herein, the term platelet concentrate refers to a liquid composition containing platelets that have been concentrated from a blood source. The blood source is preferably human blood, such as whole human peripheral blood. The platelet concentrate preferably contains both platelets and plasma proteins and can be provided by platelet units obtained by apheresis from whole peripheral blood of a human donor. Whole blood from other species, such as mammalian species, can also be used as a source of platelet concentrates to be processed as described herein. In some embodiments, at some point during the processing, platelet units from different humans or other donors can be pooled to obtain a bioactive fraction. In typical practice today, each donor apheresis platelet unit has a volume of about 100 to about 500 mL, more typically about 100 to 400 mL, and contains about 100 to 500 × 10⁻⁶ platelets. 9 Platelets and plasma separated from platelets during the apheresis process. Donated apheresis platelet units have a relatively short shelf life for use in healthcare settings, typically about five days. The platelet units used in the methods described herein may be recently expired apheresis platelet units obtained from healthcare settings and may optionally be frozen at any suitable temperature, such as about -20°C, before being used to prepare the bioactive fraction as described herein.

[0022] In the preparation of bioactive fractions, platelet contents can be released by suitable methods. In some ways, platelets are lysed by subjecting them to at least one freeze-thaw cycle for releasing platelet contents, and optionally multiple freeze-thaw cycles (e.g., two or three freeze-thaw cycles). During freeze-thaw cycles, the platelet concentrate can be frozen at any suitable temperature. In some aspects, the platelet concentrate is frozen at a temperature between about -10°C and about -80°C. In a particularly preferred embodiment, the platelet concentrate is frozen at about -20°C. To lyse the platelets, the frozen platelet concentrate is thawed, for example, in a water bath at 37°C or by other effective means, thereby forming a “raw” platelet lysate composition. The raw platelet lysate contains lysed platelet membranes and growth factors, as well as other substances released from the lysed platelets. When the thawed platelet concentrate contains plasma and platelets, the platelet lysate will also contain plasma, including plasma proteins therein. In some aspects of this article, other techniques for releasing platelet contents, such as activation with thrombin, can be used. However, the advantage of freeze-thaw or other mechanical techniques for lysing platelets is that they do not require the addition of non-natural proteins (such as thrombin) to the platelet concentrate, which would both increase costs and result in the presence of at least some thrombin in the material processed downstream.

[0023] Raw platelet lysates contain a variety of growth factors derived from the starting material of platelet concentrate. These growth factors may include, for example, transforming growth factor β1, epidermal growth factor, basic fibroblast growth factor, platelet-derived growth factor AA, platelet-derived growth factor BB, stromal cell-derived factor-1α, and vascular endothelial growth factor.

[0024] Transforming growth factor β1 (TGF-β1) is a multifunctional peptide that controls proliferation, differentiation, and other functions in many cell types. Epidermal growth factor (EGF) stimulates cell proliferation, differentiation, and survival. Basic fibroblast growth factor (FGF-β) promotes angiogenesis and binds to heparin, stimulating a wide variety of cells. Platelet-derived growth factor AA (PDGF-AA) is a diglycoprotein that regulates cell growth and division and promotes angiogenesis. Platelet-derived growth factor BB (PDGF-BB) is a diglycoprotein that regulates cell growth and division and promotes angiogenesis. Stromal cell-derived factor-1α (SDF-1α) activates leukocytes and promotes angiogenesis.

[0025] Vascular endothelial growth factor (VEGF) contributes to angiogenesis and vascularization.

[0026] In some implementations, the raw platelet lysate contains the following growth factors and their amounts (based on the volume of the raw, undiluted platelet concentrate): TGF-β1 of about 50,000 to about 150,000 pg / ml, preferably about 70,000 to about 120,000 pg / ml; and / or EGF of about 100 to about 600 pg / ml, preferably about 200 to about 600 pg / ml; and / or FGF-b at a concentration of about 5 to about 250 pg / ml, preferably about 50 to about 200 pg / ml; and / or PDGF-AA of about 500 to about 20,000 pg / ml, preferably about 5,000 to about 15,000 pg / ml; and / or PDGF-BB at approximately 1,000 to approximately 20,000 pg / ml, preferably at approximately 2,000 to approximately 15,000 pg / ml; and / or SDF-1α of about 400 to 1100 pg / ml, preferably about 500 to about 1000 pg / ml; and / or VEGF of about 10 to about 800 pg / ml, preferably about 100 to about 600 pg / ml.

[0027] In a preferred form, the raw platelet lysate further comprises one or more plasma components derived from the platelet concentrate starting material, including, for example, fibrinogen, globulin, albumin, triglycerides, glucose, sodium, calcium, and / or cholesterol. In a preferred form, the raw platelet lysate comprises the following components and amounts: Globulin at a concentration of approximately 0.5 to 2.5 g / dL, preferably approximately 1.5 to 2.5 g / dL; Albumin at approximately 2 to 5 g / dL, preferably approximately 3 to 4 g / dL; Sodium concentrations of approximately 100 to 200 mmol / L, preferably approximately 120 to approximately 160 mmol / L; Triglycerides of approximately 40 to 200 mg / dL, preferably approximately 50 to 120 mg / dL; Approximately 150 to 300 mg / dL of glucose, preferably approximately 150 to 250 mg / dL of glucose; Calcium at a concentration of approximately 5 to 12 mg / dL, preferably approximately 6 to 10 mg / dL; and / or Fibrinogen of approximately 1 to 3.5 million ng / mL, preferably approximately 1.5 to 2.5 million ng / mL.

[0028] Raw platelet lysates may also contain other bioactive substances, such as one or more interleukins, interferons, and / or tumor necrosis factors. These interleukins, interferons, and / or tumor necrosis factors may include, for example, one, some, or all of interleukin (IL)-1b, IL-6, IL-8, IL-10, IL-13, IL-17, interferon-γ (IFN-γ), and tumor necrosis factor-α (TNF-α).

[0029] In some embodiments described herein, the raw platelet lysate is treated to remove particulate matter, for example, by centrifugation, and sterilized for use as a platelet lysate product. Such sterilization may, for example, involve passing the raw platelet lysate, from which particulate matter has been removed, through a sterile filter.

[0030] In some embodiments described herein, the raw platelet lysate is treated to recover its fraction having a reduced fibrinogen concentration. Fibrinogen can be removed by any suitable technique, including, for example, by conversion to fibrin to form a solid clot that can be separated from the liquid bioactive fraction. Such conversion to fibrin can be induced by adding a coagulant. According to some forms of the disclosed methods, a coagulant, such as calcium chloride, can be added to the raw platelet lysate. Exemplarily, calcium chloride can be added in a concentration of about 0.1 g to 2 g per liter of raw platelet lysate. In a preferred embodiment, about 0.4 g to about 0.75 g of calcium chloride is added per liter of raw platelet lysate. The combined platelet lysate and calcium chloride or other coagulant can be placed on a shaker or otherwise agitated to ensure thorough mixing of the coagulant and concentrate. The resulting mixture is then allowed to form a solid clot, in some embodiments for at least about 8 hours, or at least about 12 hours, typically in the range of about 8 hours to about 36 hours. In a preferred form, at least a major amount (more than 50%) of the generated coagulated material, possibly at least 80% or at least 90%, consists of a substantially homogeneous clot gel. This substantially homogeneous clot gel can exhibit a consistent gel phase throughout the material, with the liquid entrained within a continuous fibrin matrix. These preferred forms of coagulated material differ from coagulated platelet concentrate materials, in which a large number of discrete solid clot particles are suspended in the liquid phase, which is desirable for subsequent centrifuge-based separation techniques.

[0031] Once a clot forms, the liquid substance can be separated from the solid clot. Any suitable technique can be used for this purpose. In a preferred form, the clotted substance is squeezed between two or more surfaces to separate the solidified substance from the liquid. When the clotted substance exhibits the form of a substantially homogeneous clotted gel as discussed herein, such squeezing can expel the liquid from the gelled substance while pressing and concentrating the fibrin matrix of the gel. In some forms, the clotted substance can be squeezed into a flexible container such as a plastic bag. For example, by hand or by applying force with a tool, the clotted gel can be squeezed into one area (e.g., the end) of the bag or other flexible container, while the liquid expelled from the solid fibrin matrix can accumulate in another area (e.g., the end) of the bag or other flexible container. During, during, or after squeezing, a second bag or other container can be connected to the first bag, and the liquid substance can be transferred into the second bag or other container. In other ways, the clotted gel can be located in a rigid container such as a bucket and squeezed by hand or by a tool, thereby expelling the liquid from the solid fibrin matrix and pressing and concentrating the fibrin matrix.

[0032] Following coagulation and separation of the liquid and solid components of the coagulated platelet concentrate, the separated liquid has a reduced fibrinogen concentration compared to the original platelet lysate before coagulation. In a preferred form, the original platelet lysate has a fibrinogen content of at least 1,000,000 ng / mL, typically in the range of about 1,500,000 to 3,500,000 (1.5 million to 3.5 million) ng / mL, and after coagulation and separation, the liquid has a fibrinogen content of less than about 50,000 ng / mL, preferably less than about 20,000 ng / mL, and more preferably less than about 10,000 ng / mL. Exemplarily, such separated liquid may have a fibrinogen content in the range of about 500 ng / mL to about 20,000 ng / mL or about 500 ng / mL to about 10,000 ng / mL. Alternatively, the separated liquid may contain less than about 5%, preferably less than about 2%, more preferably less than about 1%, of fibrinogen present in the platelet concentrate prior to coagulation. Furthermore, the separated liquid may constitute at least about 70%, preferably at least about 75%, typically in the range of about 75% to about 90% of the volume of the original platelet lysate.

[0033] After the original platelet lysate coagulates and undergoes liquid / solid separation, the recovered fibrinogen-free liquid bioactive fraction contains multiple growth factors derived from the original platelet lysate. These growth factors may include TGF-β1, EGF, FGF-β, PDGF-AA, PDGF-BB, SDF-1α, and VEGF. In some embodiments, this fibrinogen-free liquid bioactive fraction contains the following growth factors from the original platelet lysate and their amounts: TGF-β1 of about 50,000 to about 150,000 pg / ml, preferably about 70,000 to about 120,000 pg / ml; EGF of about 20 to about 800 pg / ml, preferably about 400 to about 800 pg / ml; and / or FGF-b at a concentration of about 5 to about 250 pg / ml, preferably about 50 to about 250 pg / ml; and / or PDGF-AA of about 500 to about 25,000 pg / ml, preferably about 5,000 to about 18,000 pg / ml; and / or PDGF-BB at approximately 1,000 to approximately 25,000 pg / ml, preferably at approximately 2,000 to approximately 18,000 pg / ml; and / or SDF-1α of about 400 to 1000 pg / ml, preferably about 500 to about 900 pg / ml; and / or VEGF of about 10 to about 600 pg / ml, preferably about 150 to about 450 pg / ml.

[0034] In other embodiments, the liquid bioactive fraction after fibrinogen removal includes the following growth factors and amounts derived from the original platelet lysate: FGF-2 (i.e., FGF-b) at a concentration between about 200 pg / mL and about 350 pg / mL; and / or EGF at a concentration between about 1800 pg / mL and about 3100 pg / mL; and / or PDGF-AA at a concentration between approximately 24,000 pg / mL and approximately 28,000 pg / mL; and / or PDGF-BB at concentrations between approximately 50 ng / mL and approximately 80 ng / mL; and / or VEGF at concentrations between approximately 500 pg / mL and approximately 800 pg / mL; The concentration of TGF-b is between approximately 60 ng / mL and approximately 90 ng / mL.

[0035] In some forms, the liquid bioactive fraction, excluding fibrinogen, also has a fibrinogen content of less than 3 μg / mL, preferably less than 2.5 μg / mL.

[0036] In a preferred form, this liquid bioactive fraction, after defibrinogen removal, further comprises one or more plasma components derived from the platelet concentrate starting material, including, for example, globulins, albumin, triglycerides, glucose, sodium, and / or calcium. When calcium chloride is used to coagulate the original platelet lysate, the calcium present in the separated liquid bioactive fraction can be derived from both the lysate and the added calcium salt. In some embodiments, this separated liquid bioactive fraction comprises the following components and amounts derived from the original platelet lysate: Globulin at a concentration of approximately 0.5 to 2.5 g / dL, preferably approximately 1 to 2 g / dL; Albumin at approximately 2 to 5 g / dL, preferably approximately 3 to 4 g / dL; Sodium concentrations of approximately 100 to 200 mmol / L, preferably approximately 120 to approximately 160 mmol / L; Triglycerides of approximately 40 to 70 mg / dL, preferably approximately 50 to 65 mg / dL; and / or Approximately 150 to 300 mg / dL of glucose, preferably approximately 150 to 250 mg / dL of glucose.

[0037] Furthermore, when calcium chloride salt is used as a coagulant for the original platelet lysate, in some forms, this separated liquid bioactive fraction may contain calcium at a concentration of about 15 to 35 mg / dL, preferably about 15 to 25 mg / dL.

[0038] According to some embodiments of the invention, the liquid bioactive fraction is passed through a sterile filter. In a preferred embodiment, the sterile filter comprises a 0.2 μm sterile filter. After passing through the sterile filter, the liquid bioactive fraction can be sealed in a sterile container.

[0039] Following the coagulation and liquid / solid separation steps, certain embodiments of the invention include filtering the recovered liquid bioactive fraction to remove suspended solids, such as any residual platelet fragments, cell debris, and clotted solids. In a preferred embodiment, such filtration involves treating the liquid bioactive fraction with at least one depth filter, preferably multiple depth filters such as two or three depth filters of possibly different micron-scales. In this context, as known and as used herein, "depth filter" or "depth filtration" refers to a filter and filtration that utilizes a porous filter medium to retain particles throughout the medium, rather than merely on the surface of the medium. Furthermore, as known and as used herein, the "nominal micron-scale" applied to a filter refers to a particle size above which, across the filter's rated capacity, 98% of all suspended solids will be removed. Certain variations of the invention include filtration through at least one depth filter followed by at least one sterile filter. Further variations of the invention include filtration through at least two depth filters followed by at least one sterile filter. In a preferred form, the depth filters or multiple depth filters used have filter media with a positive surface charge.

[0040] In some embodiments, a first depth filter and a second depth filter are used in the depth filtration of the liquid bioactive fraction. The first depth filter has a nominal micron scale larger than that of the second depth filter. In some forms, the first depth filter has a nominal micron scale between about 10 and 0.1 microns. In a preferred embodiment, the first depth filter has a nominal micron scale between 5 and 0.1 microns, and even more preferably between about 3 and 0.2 microns. In some embodiments, the first depth filter has a cellulose membrane and a filter media with a positive surface charge, composed of cellulose fibers and inorganic filter aids such as diatomaceous earth.

[0041] In some embodiments, the second depth filter has a nominal micrometer scale smaller than that of the first depth filter, for example, less than about 0.5 micrometers in some forms. In preferred embodiments, the second depth filter has a nominal micrometer scale between 0.5 and 0.001 micrometers, and even more preferably between about 0.1 and 0.001 micrometers. In some embodiments, the first depth filter has a cellulose membrane and a filter media with a positive surface charge, composed of cellulose fibers and inorganic filter aids such as diatomaceous earth.

[0042] In a preferred embodiment, after deep filtration or other filtration to remove suspended solids, the liquid bioactive fraction still contains a variety of growth factors derived from the original platelet lysate. These growth factors may include TGF-β1, EGF, FGF-β, PDGF-AA, PDGF-BB, SDF-1α, and VEGF. In some embodiments, this filtered liquid bioactive fraction contains the following growth factors derived from the original platelet lysate and their amounts: TGF-β1 of about 5,000 to about 75,000 pg / ml, preferably TGF-β1 of about 5,000 to about 60,000 pg / ml; EGF of about 20 to 300 pg / ml, preferably about 50 to about 250 pg / ml.

[0043] FGF-β of about 5 to about 150 pg / ml, preferably about 30 to 130 pg / ml; PDGF-AA of about 200 to about 4000 pg / ml, preferably about 1000 to about 3000 pg / ml; PDGF-BB at approximately 50 to approximately 1000 pg / ml, preferably at approximately 100 to approximately 500 pg / ml; SDF-1α of about 100 to 700 pg / ml, preferably about 300 to about 600 pg / ml; and / or VEGF of about 10 to about 400 pg / ml, preferably about 40 to about 200 pg / ml.

[0044] In a preferred form, this deep-filtered or otherwise filtered liquid bioactive fraction further comprises one or more plasma components derived from the platelet concentrate starting material, including, for example, globulins, albumin, triglycerides, glucose, sodium, and / or calcium. Furthermore, when calcium chloride is used to coagulate the raw platelet lysate, the calcium present in the filtered liquid bioactive fraction can be derived from both the lysate and the added calcium salt. In some embodiments, this filtered bioactive liquid fraction comprises the following components and amounts derived from the raw platelet lysate: Globulin at a concentration of approximately 0.5 to 2.5 g / dL, preferably approximately 1 to 2 g / dL; Albumin at approximately 2 to 5 g / dL, preferably approximately 3 to 4 g / dL; Sodium concentrations of approximately 100 to 200 mmol / L, preferably approximately 120 to approximately 160 mmol / L; Triglycerides of approximately 50 to 120 mg / dL, preferably approximately 60 to 110 mg / dL; and / or Approximately 150 to 300 mg / dL of glucose, preferably approximately 150 to 250 mg / dL of glucose.

[0045] Furthermore, when calcium chloride salt is used as a coagulant for the original platelet lysate, in some forms, this separated bioactive liquid fraction may contain calcium at a concentration of about 15 to 60 mg / dL, preferably about 20 to 50 mg / dL.

[0046] The bioactive liquid fraction may also contain other bioactive substances, such as one or more interleukins, interferons, and / or tumor necrosis factors. These interleukins, interferons, and / or tumor necrosis factors may include, for example, one, some, or all of interleukin (IL)-1b, IL-6, IL-8, IL-10, IL-13, IL-17, interferon-γ (IFN-γ), and tumor necrosis factor-α (TNF-α).

[0047] As described above, in some embodiments of the methods herein, it is preferable to pass the liquid bioactive fraction through at least one sterile filter after removing the suspended solids discussed above by a depth filter or other filter. Various sterile filters and related methods are known and can be used. Exemplary contaminants removed by sterile filters include, for example, Staphylococcus aureus, Pseudomonas aeruginosa, Clostridium sporogenes, Candida albicans, Aspergillus niger, Mycoplasma, and / or Bacillus subtilis. Sterile filters exhibiting relatively low protein binding can be selected. Following sterile filtration, in a preferred form, the sterile-filtered liquid bioactive fraction may have the same composition as described above for liquid bioactive fractions after depth filtration or other filtration, and also have the content of those components within the range described above for liquid bioactive fractions after depth filtration or other filtration. However, it should be understood that the content of some or all components may be reduced during aseptic filtration.

[0048] In some preferred embodiments, the sterile liquid bioactive fraction composition obtained by the above-described steps of platelet lysis, fibrinogen removal, and deep filtration or other filtration to remove suspended particles comprises: Liquid bioactive fractions with concentrations below 20,000 ng / mL, for example, fibrinogen in the range of about 500 ng / mL to about 20,000 ng / mL. Albumin with a concentration of at least 2 mg / dL of liquid bioactive fraction; Globulins with a concentration of at least 1 g / dL of liquid bioactive fraction; TGF-β1 at a concentration of at least 5000 pg / mL liquid bioactive fraction; EGF at a concentration of at least 20 pg / mL liquid bioactive fraction; FGF-β at a concentration of at least 5 pg / mL liquid bioactive fraction; PDGF-AA with a content of at least 200 pg / mL liquid bioactive fraction; PDGF-BB with a content of at least 50 pg / mL liquid bioactive fraction; SDF-1α at a concentration of at least 100 pg / mL liquid bioactive fraction; and VEGF at a concentration of at least 10 pg / mL liquid bioactive fraction.

[0049] In some forms, the liquid bioactive fraction compositions of this disclosure also have the following characteristics: Endotoxin levels below approximately 10 EU / ml; Hemoglobin levels below approximately 25 mg / dL; Approximately 4 to 6 g / dL of total protein; A molar osmolar concentration of approximately 260 to 340 mmol / kg; and / or pH between 6.8 and 7.8.

[0050] These characteristics may be present in the original platelet lysate composition (and possibly after solids removal by centrifugation or other methods and sterilization), in the defibrinogen-free liquid bioactive fraction recovered after coagulation and liquid / solid separation (and possibly sterilization), or in the defibrinogen-free liquid bioactive fraction after deep filtration and / or other filtration to remove suspended solids (and possibly sterilization), as described above. Meanwhile, since the preferred processing methods do not require the use of detergents as processing aids, these compositions may be detergent-free or substantially detergent-free.

[0051] In certain operating methods, the process used to prepare the fibrinogen-free, filtered (e.g., deep-filtration) liquid bioactive fraction composition of this disclosure reduces the content of growth factors, interleukins, interferons, and / or tumor necrosis factor as defined herein. As an example, in some embodiments, the fibrinogen-free fraction is subjected to deep filtration or other filtration to remove suspended solids, and such that: The levels of one, some, or all of TGF-β-1, EGF, FGF-b, PDGF-AA, PDGF-BB, SDF-1α, and VEGF (e.g., pg / mL) are reduced by at least 20%; and / or The content of TGF-β-1 (e.g., pg / mL) is reduced by at least 50%; and / or The EGF content (e.g., pg / mL) is reduced by at least 30%; and / or The content of FGF-b (e.g., pg / mL) is reduced by at least 20%; and / or The PDGF-AA content (e.g., pg / mL) is reduced by at least 50%; and / or The PDGF-BB content (e.g., pg / mL) is reduced by at least 50%; and / or The content of SDF-1α (e.g., pg / mL) is reduced by at least 20%; and / or The VEGF content (e.g., pg / mL) decreased by at least 30%.

[0052] Additionally or alternatively, depth filtration or other filtration can result in the removal of extremely significant amounts of interleukin-17 (IL-17). In some embodiments, the IL-17 content after particle removal by depth filtration or other filtration, and possibly in the final, sterilized liquid bioactive fraction product, is less than about 1 pg / mL, more preferably less than about 0.75 pg / mL, and even more preferably less than about 0.5 pg / mL. IL-17 is an inflammatory cytokine and also triggers the release of other inflammatory cytokines in a cascade. Preferred products having low IL-17 content as defined herein can be used in a manner that results in little or no inflammatory activity due to the presence of IL-17.

[0053] Alternatively, deep filtration or other filtration of the fraction from which fibrinogen has been removed to remove suspended solids can produce a liquid bioactive fraction product with PDGF-BB concentrations below 1000 pg / mL, PDGF-AA concentrations below 3000 pg / mL, TGF-β1 concentrations of at least 5000 pg / mL, and / or VEGF concentrations below 300 pg / mL. These values ​​may also be present in sterile products prepared after deep filtration or other filtration to remove suspended solids (e.g., by aseptic filtration).

[0054] According to some of the methods disclosed herein, the liquid bioactive fraction described herein can be processed to beneficially retain plasma components. Plasma components that can be retained by the liquid bioactive fraction include: globulins, albumins, triglycerides, glucose, sodium, and / or calcium. In some forms, the liquid bioactive fraction contains one or more of these plasma components, possibly all, and also has the following characteristics: FGF-2 with a concentration of at least 200 pg / mL liquid bioactive fraction; EGF at a concentration of at least 1800 pg / mL liquid bioactive fraction; PDGF-AA with a content of at least 24,000 pg / mL liquid bioactive fraction; PDGF-BB at a concentration of at least 50 ng / mL liquid bioactive fraction; VEGF at a concentration of at least 500 pg / mL liquid bioactive fraction; TGF-β1 at a concentration of at least 60 ng / mL liquid bioactive fraction; and Fibrinogen with a content of less than 2.5 μg / mL in the liquid bioactive fraction.

[0055] In some forms, the liquid bioactive fraction compositions of this disclosure can be packaged in sterile packaging for storage or delivery. The liquid bioactive fraction can be packaged at its fully recoverable concentration, or it can be diluted with water or an aqueous medium for packaging and subsequent use, for example, to 90% to 10% of the initial concentration of the liquid bioactive fraction, and such diluted compositions, and the resulting corresponding reductions in the content of the components described herein, form other embodiments disclosed herein. Figure 2 One embodiment of such packaging is shown. According to some forms of implementing this disclosure, composition 200 is stored in a sterile culture medium bottle 210. The sterile culture medium bottle may, for example, have a volumetric capacity ranging from 50 mL to 5000 mL. As examples, bottles of 60 mL, 125 mL, 250 mL, 500 mL, 1000 mL, or 2000 mL can be used. In some forms, the cap 220 of the sterile culture medium bottle 210 is protected by a shrink wrap 230. In some forms, the bottle is shrink-wrapped. In some embodiments, the bottle is affixed with an article label 240. In some forms, the bottle is placed in a product box containing dry ice.

[0056] In some embodiments, the liquid bioactive fraction composition of this disclosure can be combined with other components to form a cell culture medium. Such a cell culture medium comprises the liquid bioactive fraction of this disclosure mixed with other nutrients or culture media for cell culture, including those present in, for example, known cell culture media such as Limit Essential Medium (MEM) or Dulbecco's Modified Eagle Medium (DMEM). The cell culture medium according to this disclosure is formulated to provide nutrients (e.g., growth factors, etc.) necessary for cell growth or maintenance, including, for example, stem cells and / or progenitor cells, such as mesenchymal stem cells. In a preferred form, such a cell culture medium is free of added heparin and, despite this, free of any clumping material (e.g., this would be demonstrated by the presence of clumping particles visible to the naked eye without magnification).

[0057] In other embodiments, the liquid bioactive fraction composition or fraction thereof disclosed herein can be used as a therapeutic substance. For example, the composition can be used for medical treatment, including for treating diseased or damaged tissues such as nerves, tendons, bones, muscles, skin (e.g., wound healing), connective tissue, eye, and / or cardiovascular (e.g., heart or aorta) tissues. The liquid bioactive fraction described herein, or a composition containing it, can be delivered to these or other tissues by any suitable means, including, for example, injection or other surgical implantation. In some uses, in the treatment of ocular tissues, the liquid bioactive composition or a composition containing it is applied to the ocular surface (e.g., in the form of droplets), for example in the treatment of ocular surface defects or diseases such as graft-versus-host disease of the eye (GVHD of the eye), corneal ulcers, dry eye (Keratoconjunctivitis Sicca), or corneal repair after surgery or injury.

[0058] In other embodiments, the liquid bioactive fraction composition or fraction thereof of the present invention can be used as a therapeutic substance. For example, the composition can be used as a therapeutic substance for medical treatment, including for treating diseased or damaged tissues such as nerves, tendons, bones, muscles, skin (e.g., wound healing), connective tissue, eye, and / or cardiovascular (e.g., heart or aorta) tissues. The liquid bioactive fraction described herein or a composition containing it can be delivered to these or other tissues by any suitable means, including, for example, injection or other surgical implantation. In some uses, in the treatment of ocular tissues, the liquid bioactive composition or a composition containing it is applied to the ocular surface (e.g., in the form of droplets), for example in the treatment of ocular surface defects or diseases such as graft-versus-host disease of the eye (GVHD of the eye), corneal ulcers, dry eye (keratoconjunctivitis sicca), or corneal repair after surgery or injury.

[0059] According to certain variations of the present invention, the liquid bioactive fraction of this disclosure is used to treat mammalian patients (e.g., humans, dogs, cats, horses, etc.). In some embodiments, the liquid bioactive fraction for the target patient is allogeneic; in other embodiments, the liquid bioactive fraction for the target patient is heterogeneous. For example, in some embodiments, platelet lysate compositions derived from human platelets can be used to treat canine patients. It is also contemplated that platelet lysate compositions derived from canine platelets can be used to treat canine patients, and that platelet lysate compositions derived from human platelets can be used to treat human patients. In some forms, the patient suffers from keratoconjunctivitis sicca. According to certain variations of this disclosure, the liquid bioactive fraction of the present invention is used to treat canine patients suffering from keratoconjunctivitis sicca. Canine patients can be any breed of canine, but breeds commonly affected by dry keratoconjunctivitis include: Cavalier King Charles Spaniel, Bulldog, Chinese Shar Pei, Lhasa Apso, Shih Tzu, West Highland White Terrier, Pug, Bloodhound, Cocker Spaniel, Pekingese, Boston Terrier, Miniature Schnauzer, and Samoyed.

[0060] In some embodiments, human platelet lysate containing a liquid bioactive fraction is stored in a liquid delivery device configured to deliver the liquid bioactive fraction to a patient's eye. In some forms, the liquid delivery device is a sterile container. Figure 3 An embodiment of a liquid delivery device is illustrated. In the illustrated embodiment, a liquid bioactive fraction is stored within the device 300. The device 300 has a storage section 310 and a dispersion section 320. In the illustrated embodiment, the dispersion section 320 may optionally be covered with a cap 322. The dispersion section 320 may be configured to dispense a portion of the liquid bioactive fraction (e.g., dropwise). In some forms, the storage section 310 comprises a deformable plastic material that can be squeezed by the user. Other suitable liquid delivery devices include, but are not limited to, eye drops and pipettes.

[0061] According to certain embodiments, the liquid bioactive grade of the present invention is dispensed into an ointment. In some forms, the ointment containing hPL is applied topically to the affected area (e.g., a patient's eye).

[0062] Liquid bioactive compositions can also be used for other purposes, including, for example, as cryoprotectants for cells. In such cryoprotectant use, the liquid bioactive composition can be incorporated into a cell suspension composition, which can then be cryopreserved to maintain cell viability. The cells can be any type of cell, including stem cells such as mesenchymal stem cells, progenitor cells, or other cells. Cryopreservation can be performed in suitable containers such as bags or vials.

[0063] In addition to derivatives derived from the recovered liquid bioactive fraction from lysed platelet concentrate, valuable products can also be prepared from the solid clot material formed during coagulation and liquid-solid separation processes. In some ways, the separated solid clot material has been found to be rich in growth factors and contains sufficient amounts of fibrinogen and coagulation factors for use as a coagulable carrier, for example in bioadhesives, and / or as a hemostatic material for medical applications. For these or other purposes, the recovered solid clot material can be stored under refrigerated or frozen conditions and / or can be freeze-dried to form a dry material that can optionally be pulverized into powder form. For medical, diagnostic, research, or other applications, the resulting solid clot material or fraction can be sterilized by any suitable means, including, for example, by exposure to radiation or a chemical disinfectant (e.g., ethylene oxide).

[0064] In addition to recovering the liquid bioactive fraction and possibly the products prepared from the solid clumps formed during the coagulation and liquid-solid separation processes discussed above, bioactive substances such as growth factors or other proteins can be recovered individually or in mixtures from one or more filters used to process platelet lysates. This recovers added value from the original starting materials. Exemplarily, a depth filter (e.g., a depth filter as described above) used to filter the platelet lysate composition can then be treated to recover one or more growth factors or other bioactive substances captured on the filter. This can be done in any suitable manner. Exemplarily, one or more proteins, such as growth factors, can be eluted from the filter by passing the eluent through the filter to overcome the attraction of the protein to the filter medium, thereby generating an eluent stream containing the protein. In the case of a charged (e.g., positively charged) depth filter that retains proteins based at least in part on the charge interaction between the protein and the charged filter medium, the protein can be recovered from the filter medium by elution with a salt solution, a pH change of the eluent (relative to that used in the initial filtration process), or with an affinity elution medium containing a ligand of the protein to be eluted. Gradient elution (e.g., by a salt or pH gradient) can be used to sequentially elute fractions that are purified to obtain a specific protein or protein of interest, or fractions enriched with a specific protein or protein of interest. The recovered protein or proteins can be, for example, any of those defined herein, preferably one or more of the growth factors, interleukins, interferons, and / or tumor necrosis factors defined herein. These proteins can be used for, for example, therapeutic, diagnostic, or research purposes. For these or other purposes, they may optionally be purified and / or sterilized after recovery from the filter medium.

[0065] To aid in a further understanding of the aspects, features, and advantages of this disclosure, the following specific embodiments are provided. It should be understood that these embodiments are exemplary and not intended to limit the implementation of the invention.

[0066] Example Example 1 Preparation of human platelet lysate composition Collect platelet units (obtained from peripheral blood) from apheresis patients whose shelf life has just expired after 5 days and freeze them at -20°C until use. Remove a number of units (e.g., about 10 units) from the freezer and thaw them at room temperature to lyse the platelets and form a "raw hPL" composition. Collect the raw hPL from the selected units into a bag. Add calcium chloride at a concentration of 0.7 g / L (about 6 mM CaCl2) to the collected raw hPL and mix thoroughly with the raw hPL on a shaker for 2 hours at room temperature. After mixing, allow the CaCl2-treated raw hPL to coagulate overnight at room temperature, during which time a strong, substantially homogeneous coagulated gel clump forms from the volume of the raw hPL.

[0067] While keeping the bag sealed, manually squeeze the bag containing the original hPL gel clump to expel the liquid from the gel clump. Perform this squeezing thoroughly so that the solid clump remains at one end of the bag, and the separated liquid volume is located at the other end of the bag adjacent to the outlet nozzle. The separated liquid comprises approximately 75-80% of the initial, collected volume of the original hPL, with the solidified material making up the remainder. Transfer the liquid from the bag to a second refrigerated bag with a 100L capacity. Perform a sufficient number of such thaw-collect-coagulate-squeeze runs to fill the 100L refrigerated bag with liquid.

[0068] The liquid in the 100L bag is aseptically connected to and processed through a filter assembly consisting of a first depth filter with a positively charged filter media and a nominal micron class between 3 and 0.2 microns, and a second depth filter with a positively charged filter media and a nominal micron class between 0.1 and 0.001 microns. Filtration is performed at a filtrate flow rate of approximately 100 liters per square meter of filter surface area per hour (“LMH”). The first depth filter is supplied by Millistack Pod Filter, C0 series HC depth filter, and the second depth filter is supplied by Millistack Pod Filter, XO series HC depth filter, both commercially available from Millipore. Each of these filters has a membrane composed of a mixture of cellulose esters and a filter media composed of cellulose fibers with an inorganic filter aid (diatomaceous earth). The filter assembly is pre-filled with sterile distilled water before processing the 100L bag material. The hPL liquid exiting the filter assembly is collected into the 100L second bag.

[0069] A second 100L hPL bag is aseptically attached to a sterile filter and pumped through the filter into a smaller container, such as a 100mL or 500mL wide-mouth bottle (e.g., Nalgene wide-mouth bottle). This can be done under aseptic filling conditions. The wide-mouth bottles can be shrink-packed to cover their capped ends and labeled.

[0070] The hPL product prepared according to this embodiment has the compositional distribution as described herein and can be used as a supplement to cell culture medium without the need for heparin to prevent clot formation. Adding this hPL product to cell culture medium produces a substantially clot-free medium, even without the addition of heparin. The cell culture medium thus prepared exhibits excellent performance in cell culture, and in preferred applications, a relatively high cell count or confluence percentage can be obtained after a given culture period. These cells include, but are not limited to, bone marrow mesenchymal cells, adipocyte stem cells, placental mesenchymal stem cells, and muscle-derived stem cells or progenitor cells.

[0071] Example 2 Preparation of human platelet lysate composition Collect platelet units (obtained from peripheral blood) from apheresis patients whose shelf life has just expired after 5 days and freeze them at -20°C until use. Remove a number of units (e.g., about 23 units) from the freezer and thaw them at room temperature to lyse the platelets and form a "raw hPL" composition. Collect the raw hPL from the selected units into a bag. Add calcium chloride at a concentration of 0.75 g / L (about 6 mM CaCl2) to the collected raw hPL and mix thoroughly with the raw hPL on a shaker at room temperature for 2 hours. After mixing, allow the CaCl2-treated raw hPL to coagulate overnight at room temperature, during which time a strong, substantially homogeneous coagulated gel clump forms from the volume of the raw hPL.

[0072] While maintaining a closed seal, squeeze the bag containing the original hPL gel clump to expel the liquid from the gel clump. Perform this squeezing thoroughly so that the solid clump remains at one end of the bag, and the separated liquid volume is located at the other end of the bag adjacent to the outlet nozzle. The separated liquid comprises approximately 75-80% of the initial, collected volume of the original hPL, with the solidified material making up the remainder. Transfer the liquid from the bag to a second filler bag with a 25L volume. Perform a sufficient number of such thaw-collect-coagulate-squeeze runs to fill the 25L filler bags with liquid.

[0073] Store the 25L filler bag overnight in a laboratory freezer at 4°C. Use a peristaltic pump to transfer the fluid from the 25L filler bag through a pre-filter (25μm) and a sterile filter (0.2μm). Collect the filtered liquid into a collection bag. Then aliquot the filtered liquid (hPl) into sterile containers (e.g., 100mL or 500mL Nalgene wide-mouth bottles). This can be done under aseptic filling conditions. The wide-mouth bottles can be shrink-wrapped to cover their capped ends and labeled.

[0074] The hPL product prepared according to this embodiment has the compositional distribution as described herein and can be used as a supplement to cell culture media without the need for heparin to prevent clot formation. It can also be used in other applications of the liquid bioactive fraction defined herein. Adding this hPL product to cell culture media, even without the addition of heparin, produces a substantially clot-free medium. The cell culture media thus prepared exhibit excellent performance in cell culture, and in preferred applications, relatively high cell counts or confluence percentages can be obtained after a given culture period, said cells including, but not limited to, bone marrow mesenchymal cells, adipocyte stem cells, placental mesenchymal stem cells, and muscle-derived stem cells or progenitor cells.

[0075] Example 3 Preparation of canine platelet lysate compositions Canine platelet lysate (CPL) compositions can be prepared essentially as described above. Generally, the preparation of the CPL composition begins with the preparation of canine platelet units obtained from peripheral blood via a screening apheresis. As detailed above, such platelet units can be freshly prepared or obtained from expired platelet units. The platelet units are frozen at -20°C until use. A number of units (e.g., about 10 units) are removed from the freezer and thawed at room temperature, thereby lysing the platelets and forming a “raw CPL” composition. The raw CPL from the selected units is collected into a bag. Calcium chloride at a concentration of 0.7 g / L (about 6 mM CaCl2) is added to the collected raw CPL, and then thoroughly mixed with the raw CPL on a shaker at room temperature for 2 hours. After mixing, the CaCl2-treated raw CPL is allowed to coagulate overnight at room temperature, during which time a strong, substantially homogeneous coagulated gel clump forms from the volume of the raw CPL.

[0076] While keeping the bag sealed, manually squeeze the bag containing the original CPL gel clump to expel the liquid from the gel clump. Perform this squeezing thoroughly so that the solid clump remains at one end of the bag, and the separated liquid volume is located at the other end of the bag adjacent to the outlet nozzle. The separated liquid comprises approximately 75-80% of the initial, collected volume of the original CPL, with the solidified material making up the remainder. Transfer the liquid from the bag to a second refrigerated bag with a 100L capacity. Perform a sufficient number of such thaw-collect-coagulate-squeeze runs to fill the 100L refrigerated bag with liquid.

[0077] The collected liquid (e.g., liquid bioactive fraction) is then passed through a sterile filter. In some forms, the collected liquid is passed through a series of depth filters as described in Example 1. The filtered liquid (cPL) is then aliquoted into sterile containers (e.g., 100 mL or 500 mL Nalgene wide-mouth bottles). This can be done under aseptic filling conditions. The wide-mouth bottles can be shrink-packed to cover their capped ends and labeled.

[0078] The CPL product prepared according to this embodiment has the compositional distribution as described herein and can be used as a supplement to cell culture media without the need for heparin to prevent clot formation. It can also be used in other applications of the liquid bioactive fraction defined herein. Adding this CPL product to cell culture media, even without the addition of heparin, produces a substantially clot-free medium. The cell culture media thus prepared exhibit excellent performance in cell culture, and in preferred applications, relatively high cell counts or confluence percentages can be obtained after a given culture period, said cells including, but not limited to, bone marrow mesenchymal cells, adipocyte stem cells, placental-derived mesenchymal stem cells, and muscle-derived stem cells or progenitor cells. As described herein, the CPL solution can also be used as a therapeutic agent.

[0079] Unless otherwise specified herein or obviously contradicted by the context, the terms “a,” “an,” “the,” and similar designations used in the context of describing the invention, particularly in the context of the following claims, shall be construed as covering both the singular and plural. Unless otherwise indicated herein, the examples of numerical ranges herein are intended only as a shorthand method of referring separately to each individual numerical value falling within that range, and the range of each individual numerical value is incorporated into the specification as if individually listed herein. Unless otherwise stated herein or obviously contradicted by the context, all methods described herein may be performed in any suitable order. Unless otherwise stated, any and all embodiments or exemplary language (e.g., “such”) provided herein are used merely to better illustrate the invention and do not constitute a limitation on the scope of the invention. No language in the specification should be construed as indicating that any element not claimed is essential to the implementation of the invention.

[0080] Just as each individual publication or patent application is specifically and individually incorporated by reference, all publications and patent applications referenced in this specification are incorporated herein by reference. Furthermore, any theories, mechanisms of operation, evidence, or findings described herein are intended to further enhance the understanding of the invention and are not intended to limit the invention in any way to such theories, mechanisms of operation, evidence, or findings. While the invention has been shown and described in detail in the accompanying drawings and the foregoing description, it should be considered that the invention is exemplary rather than restrictive in its characterization, and it is understood that only selected embodiments have been shown and described, and protection is expected to be provided for all equivalents, variations, and modifications as defined herein or in the following claims that fall within the spirit of the invention.

Claims

1. A composition comprising: A bioactive fraction of human blood-derived platelet concentrate, the platelet concentrate containing human platelets and human plasma, the bioactive fraction comprising natural components of the platelet concentrate including fibrinogen, albumin, globulin, and at least one of TGF-β1, EGF, basic FGF, PDGF-AA, PDGF-BB, SDF-1α, and VEGF, wherein the fibrinogen is present in a concentration ranging from 500 ng / mL to 20,000 ng / mL, and wherein the composition is free of or substantially free of added heparin.

2. The composition of claim 1, wherein the composition comprises each of the following: TGF-β1, EGF, basic FGF, PDGF-AA, PDGF-BB, SDF-1α, and VEGF.

3. The composition of claim 1, wherein the composition is heparin-free.

4. The composition of claim 1, wherein the bioactive fraction further comprises at least one of IL-1b, IL-6, IL-8, IL-10, IL-13, IL-17, IFN-γ and TNF-α originating from the platelet concentrate.

5. The composition of claim 1, wherein the bioactive fraction is a liquid bioactive fraction, and wherein the composition comprises: 0.5 to 2.5 g / dL of globulin; Albumin at levels of 2 to 5 g / dL; Sodium concentrations of 100 to 200 mmol / L; Triglycerides of 50 to 120 mg / dL; and / or 150 to 300 mg / dL of glucose.

6. The composition of claim 5, wherein the globulin is 1 to 2 g / dL, or the albumin is 3 to 4 g / dL, or the sodium is 120 to about 160 mmol / L, or the triglycerides are 60 to 110 mg / dL, or the glucose is 150 to 250 mg / dL.

7. The composition of claim 1, wherein the composition is free of detergent residue.

8. The composition of claim 1, wherein the bioactive fraction is a liquid bioactive fraction and wherein the concentration of PDGF-BB is less than 1000 pg / mL, or the bioactive fraction is a liquid bioactive fraction and wherein the concentration of PDGF-AA is less than 3000 pg / mL, or the bioactive fraction is a liquid bioactive fraction and wherein the concentration of TGF-β1 is at least 5000 pg / mL, or the bioactive fraction is a liquid bioactive fraction and wherein the concentration of VEGF is less than 300 pg / mL.

9. The composition of claim 1, wherein the bioactive fraction is a liquid bioactive fraction, and wherein the natural component comprises: Fibrinogen with a content of less than 20,000 ng / mL of the liquid bioactive fraction; Albumin in a liquid bioactive fraction with a concentration of at least 2 mg / dL; Globulins in a liquid bioactive fraction at a concentration of at least 1 g / dL; The content of TGF-β1 in the liquid bioactive fraction is at least 5000 pg / mL; EGF at a concentration of at least 20 pg / mL of the liquid bioactive fraction; FGF-β at a concentration of at least 5 pg / mL of the liquid bioactive fraction; The liquid bioactive fraction of PDGF-AA contains at least 200 pg / mL; PDGF-BB at a concentration of at least 50 pg / mL of the liquid bioactive fraction; SDF-1α at a concentration of at least 100 pg / mL of the liquid bioactive fraction; and The liquid bioactive fraction of VEGF contains at least 10 pg / mL.

10. The composition of claim 1, wherein: The composition has a molar osmotic pressure concentration between 260 and 340 mmol / kg.

11. The composition of claim 1, wherein: The composition has a pH in the range of 6.8 to 7.

8.

12. The composition of claim 1, wherein: The composition does not contain heparin that is not derived from the platelet concentrate.

13. A cell culture medium that is heparin-free, said cell culture medium comprising the composition according to any one of claims 1 to 12.

14. A method for preparing a bioactive composition, the method comprising: Adding a coagulant to the platelet lysate composition forms a coagulant substance; Pressing the condensed substance separates the liquid from the condensed solids in the condensed substance; as well as The liquid is deep filtered, wherein the composition contains no or substantially no added heparin.

15. The method of claim 14, wherein: The deep filtration is performed using a deep filtration medium with a positive surface charge.

16. The method of claim 14 or 15, wherein: The coagulant includes calcium chloride salt.

17. The method of claim 14 or 15, further comprising: The liquid is then subjected to aseptic filtration.

18. The method of claim 14 or 15, wherein: The platelet lysate composition is prepared by a method comprising freezing and thawing a platelet concentrate composition to lyse the platelets therein.

19. The method of claim 18, wherein: The platelet concentrate composition includes an apheresis platelet composition containing platelets and plasma.

20. The method of claim 14 or 15, further comprising: The bioactive composition is packaged in a sterile container.

21. Use of a therapeutic substance in the preparation of a medicament for treating a patient, wherein the therapeutic substance comprises: The composition according to any one of claims 1 to 12.

22. The use as described in claim 21, wherein the patient is a human patient.

23. A method for preparing a bioactive composition, the method comprising: Platelets from a human blood concentrate containing platelets and plasma are lysed to form a lysed platelet preparation; By converting fibrinogen in the lysed platelet preparation into fibrin, a gel clot of the lysed platelet preparation is formed. The gel mass is pressed between two or more surfaces to expel liquid from the gel mass, resulting in a solid mass and a liquid volume; and The liquid and solid of the gel are separated, wherein the composition contains no or substantially no added heparin.

24. A method for processing a platelet lysate composition, the method comprising: Platelets from a human blood concentrate containing platelets and plasma are lysed to form a lysed platelet preparation; In-container pressing involves converting fibrinogen in lysed platelet preparations into a gel block formed from fibrin to expel liquid from the gel block, wherein the pressing is performed on the gel block between and in contact with a first flexible surface and a second surface of the container, wherein the composition contains no or substantially no added heparin.

25. A method for processing a platelet lysate composition, the method comprising: The liquid bioactive fraction of human blood-derived platelet concentrate is passed through at least a first depth filter to remove suspended solids from the liquid bioactive fraction, the liquid bioactive fraction of human blood-derived platelet concentrate comprising natural components of the platelet concentrate, including fibrinogen, albumin, globulin, TGF-β1, EGF, FGF-β, PDGF-AA, PDGF-BB, SDF-1α, and VEGF, wherein the fibrinogen is present in a range of 500 ng / mL to 20,000 ng / mL, and wherein the composition is free of or substantially free of added heparin.

26. The method of claim 25, further comprising obtaining the liquid bioactive fraction of the blood-derived platelet concentrate without performing a centrifugation step that separates the coagulation fraction of the concentrate from the liquid bioactive fraction of the concentrate.

27. The method of claim 26, wherein obtaining comprises: The platelets in the concentrate are lysed to form a lysed preparation; The fibrinogen of the lysed preparation is converted into fibrin to form a gel block; as well as The gel block is pressed without centrifugation in order to extract the liquid bioactive fraction from the gel block.

28. The method of claim 27, wherein the pressing comprises pressing a gel block between and in contact with the first and second surfaces to expel the liquid bioactive fraction from the gel block and leave a solid clump.

29. The method of any one of claims 25-28, wherein the process involves: The levels of one, some, or all of TGF-β-1, EGF, FGF-b, PDGF-AA, PDGF-BB, SDF-1α, and VEGF are reduced by at least 20%; and / or The content of TGF-β-1 is reduced by at least 50%; and / or EGF levels are reduced by at least 30%; and / or The content of FGF-b is reduced by at least 20%; and / or The content of PDGF-AA is reduced by at least 50%; and / or The content of PDGF-BB is reduced by at least 50%; and / or The content of SDF-1α is reduced by at least 20%; and / or The VEGF content decreased by at least 30%.

30. A method for cryopreserving cells, the method comprising: The cell composition containing the cells and the composition according to any one of claims 1-12 are subjected to cryopreservation conditions.

31. The composition of claim 1, wherein the composition is stored in a liquid delivery device.

32. The composition of claim 31, wherein the liquid delivery device comprises an eye dropper.

33. The composition of claim 1, wherein the composition is effective in treating diseases or lesions in canine patients.

34. The composition of claim 33, wherein the lesion includes keratoconjunctivitis sicca.

35. The composition of claim 1, wherein the bioactive fraction is a liquid bioactive fraction, and wherein the composition comprises: FGF-2 with a concentration between 200 pg / mL and 350 pg / mL; EGF with concentrations between 1800 pg / mL and 3100 pg / mL; PDGF-AA with a content between 24,000 pg / mL and 28,000 pg / mL; PDGF-BB with a concentration between 50 ng / mL and 80 ng / mL; VEGF with a concentration between 500 pg / mL and 800 pg / mL; TGF-b at concentrations between 60 ng / mL and 90 ng / mL; and Fibrinogen content below 2.5 μg / mL.

36. A method for preparing a bioactive composition, the method comprising: Adding a coagulant to the platelet lysate composition forms a coagulant substance; The solidified material in the condensate is separated from the liquid by pressing the condensate within the container, wherein the pressing is performed on the condensate between and in contact with the first flexible surface and the second surface of the container. as well as The liquid is filtered, wherein the composition contains no or substantially no added heparin.

37. The method of claim 36, wherein: The coagulant includes calcium chloride salt.

38. The method of claim 36, wherein: The platelet lysate composition is prepared by a method comprising freezing and thawing a platelet concentrate composition to lyse the platelets therein.

39. The method of claim 36, wherein: The platelet concentrate composition includes an apheresis platelet composition containing platelets and plasma.

40. The method of claim 36, further comprising: The bioactive composition is packaged in a sterile container.

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