A type of application where a dry powder rich in growth factors reduces inflammation or injury

Abstract

To provide a growth factor enriched dry powder making it possible to effectively relieve or treat inflammation or injury.SOLUTION: The invention provides a kind of use of a growth factor enriched dry powder for relieving inflammation or injury, containing PDGF-BB of 9×104 pg or more per gram of the growth factor enriched dry powder.SELECTED DRAWING: Figure 2A

Description

[Technical field]

[0001] The present invention relates to a growth factor enriched dry powder, and more particularly to a use of the growth factor enriched dry powder for alleviating inflammation or injury. [Background technology]

[0002] Inflammation is a defense mechanism of the human body. Inflammation occurs in body parts due to infection, trauma, or hypersensitivity to remove harmful stimuli, eliminate pathogens, and promote tissue repair. Inflammation is classified as acute inflammation and chronic inflammation. Examples include dermatitis (Dermatitis), allergic rhinitis (Allergic Rhinitis), pneumonia (Pneumonia), hepatitis (Hepatitis), and enteritis (Enteritis). Dermatitis is divided into irritant contact dermatitis (ICD) and allergic contact dermatitis (Allergic Contact Dermatitis). Severe pneumonia causes diseases such as chronic obstructive pulmonary disease (COPD) and pulmonary fibrosis. Severe hepatitis causes diseases such as liver cirrhosis and cancer.

[0003] Platelet-Rich Plasma (PRP) is a blood-derived product with a platelet concentration 1.5 to 5 times higher than that of normal blood. Platelets contain platelet-related growth factors, including Platelet-Derived Growth Factor (PDGF), Vascular Endothelial Growth Factor (VEGF), Epidermal Growth Factor (EGF), Fibroblast Growth Factor (FGF), and Transforming Growth Factor Beta 1 (TGF-β1).

[0004] Platelet-rich plasma (PRP) has anti-inflammatory properties (see references 1 and 2). However, clinical studies have shown that the anti-inflammatory effect of platelet-rich plasma (PRP) is insufficient, due to the low concentration of growth factors in PRP, its unstable quality, and its difficulty in storage. In addition, the use of platelet-rich plasma (PRP) in xenotransplantation may cause allergic reactions, increasing the risks of clinical use.

[0005] There is a need in the market for pharmaceutical compositions to alleviate or treat inflammation or injury at various sites that can overcome the limitations of current technology. Summary of the Invention

[0006] In view of the shortcomings of the prior art, the object of the present invention is to provide a growth factor enriched dry powder that can effectively alleviate or treat inflammation and injury.

[0007] Another object of the present invention is to provide a method for producing a growth factor enriched dry powder.

[0008] Another object of the present invention is to provide a use of growth factor enriched dry powder for effectively alleviating inflammation and injury.

[0009] Another object of the present invention is to provide a use for preparing a pharmaceutical composition or dietary supplement for alleviating or treating inflammation or injury.

[0010] A method for producing growth factor enriched dry powder, comprising the steps of: (a) taking a unit of a solution containing platelets and carrying out a process for purifying platelets to obtain an ultra-pure platelet solution; (b) activating the ultra-pure platelet solution to release growth factors and obtain a platelet growth factor extract solution; (c) carrying out a process for removing platelet-associated structures to obtain a growth factor enriched solution; and (d) drying the growth factor enriched solution to obtain a growth factor enriched dry powder.

[0011] In some embodiments, the drying process is a freeze-drying process. In some embodiments, the drying process is a freeze-drying process, which includes multiple manipulation steps that freeze multiple components in the growth factor enriched solution and directly sublimate at least one frozen component into a gas, thereby (1) forming multiple pores (e.g., multiple gas-accommodating pores or multiple gas-evaporating pores) in the growth factor enriched dry powder, or (2) changing the extent to which a physiological response is changed during the transformation from the growth factor enriched solution to the growth factor enriched dry powder. In some embodiments, the frozen components include frozen solutes, frozen platelet-associated growth factors, and multiple frozen other components, and the frozen components that directly sublimate into a gas include (1') frozen solutes or (1'') frozen solutes and at least one frozen other component. In some embodiments, the morphology of the pores (e.g., gas-accommodating pores or gas-evaporation pores) in the growth factor enriched dry powder is observed using an electron microscope or other methods.

[0012] In some embodiments, the drying process is freeze-drying, low temperature drying, or vacuum drying.

[0013] In some embodiments, the process purifying platelets in step (a) includes reducing the number of white blood cells in the ultra-pure platelet solution to 10 6 The method further includes a process of removing white blood cells to reduce the number of white blood cells to less than 100.

[0014] In some embodiments, the process for purifying platelets in step (a) includes the step of: and removing leukocytes such that the number of leukocytes in the solution is less than 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000, 100,000, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, or 800,000.

[0015] In some embodiments, the process purifying platelets in step (a) includes removing plasma such that the ultra-pure platelet solution has an albumin concentration of less than 3 g / dL. In some embodiments, the process purifying platelets in step (a) includes removing plasma such that the ultra-pure platelet solution has a globulin concentration of less than 2 g / dL. In some embodiments, the process purifying platelets in step (a) includes removing plasma such that the ultra-pure platelet solution has a fibrinogen concentration of less than 100 μg / mL.

[0016] In some embodiments, the process purifying platelets in step (a) further comprises removing plasma such that the albumin concentration in the ultra-pure platelet solution is less than 0.25, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, or 2.75 g / dL.

[0017] In some embodiments, the process purifying platelets in step (a) further comprises removing plasma such that the globulin concentration in the ultra-pure platelet solution is less than 0.25, 0.5, 0.75, 1, or 1.25 g / dL.

[0018] In some embodiments, the process purifying platelets in step (a) further comprises removing plasma such that the fibrinogen concentration in the ultra-pure platelet solution is less than 10, 20, 30, 40, 50, 60, 70, 80 or 90 μg / mL.

[0019] In some embodiments, the platelet-containing solution is whole blood, and step (a) includes the steps of: (a1) taking a unit of the platelet-containing solution; (a2) subjecting the platelet-containing solution to a first sedimentation process to separate the platelet-containing solution into a plasma layer, a buffy coat layer, and a red blood cell layer; (a3) ​​removing the platelets from the buffy coat layer and the red blood cell layer to obtain a plasma layer solution; (a4) subjecting the platelets to a sedimentation process to cause them to precipitate and form a pellet; and (a5) removing the supernatant and mixing the pellet with a solvent to obtain an ultra-pure platelet solution.

[0020] In some embodiments, the activation process includes a freezing and then thawing process (i.e., a freeze-thaw procedure), the addition of calcium ions, 2+ ) process, the addition of thrombin process, the addition of collagen process, or the addition of ADP process, or any combination thereof.

[0021] In some embodiments, the activation process is a freezing and then thawing process (i.e., a freeze-thaw procedure) and includes the steps of: (b1) placing a container containing an ultra-pure platelet solution in liquid nitrogen to freeze the ultra-pure platelet solution; and (b2) raising the temperature of the container to thaw the frozen ultra-pure platelet solution.

[0022] In some embodiments, the PDGF-BB content per gram (g) of growth factor enriched dry powder is between 10 pg and 3×10 7 pg.

[0023] In some embodiments, the PDGF-BB content per gram (g) of growth factor enriched dry powder is 10, 20, 30, 40, 50, 60, 70, 80, 90, 1×10 2 , 5×10 2 , 1×10 3 , 5×10 3 , 1×10 4 , 5×10 4 , 1×10 5 , 5×10 5 , 1×10 6 , 5×10 6 , or 1 × 10 7 pg.

[0024] In some embodiments, the VEGF content per gram (g) of growth factor enriched dry powder is greater than or equal to 2×10 2 pg ~3×10 6 pg.

[0025] In some embodiments, the VEGF content per gram (g) of growth factor enriched dry powder is 4×10 2 , 8×10 2 , 1×10 3 , 5×10 3 , 1×10 4 , 5×10 4 , 1×10 5 , 5×10 5 , or 1 × 10 6 pg.

[0026] In some embodiments, the FGF content per gram (g) of growth factor enriched dry powder is between 10 pg and 1×10 4 pg.

[0027] In some embodiments, the FGF content per gram (g) of growth factor enriched dry powder is 10, 20, 30, 40, 50, 60, 70, 80, 90, 1×10 2 , 5×10 2 , 1×10 3 , or 5 × 10 3 pg.

[0028] In some embodiments, the TGF-β1 content per gram (g) of growth factor enriched dry powder is 2×10 5 pg~1.2×10 7 pg.

[0029] In some embodiments, the TGF-β1 content per gram (g) of growth factor enriched dry powder is 3×10 5 , 5×10 5 , 1×10 6 , 5×10 6 , or 1 × 10 7 pg.

[0030] In some embodiments, the EGF content per gram (g) of growth factor enriched dry powder is 1×10 2 pg~6×10 5 pg.

[0031] In some embodiments, the EGF content in each gram (g) of growth factor enriched dry powder is 2×10 2 , 5×10 2 , 1×10 3 , 5×10 3 , 1×10 4 , 5×10 4 , 1×10 5 , or 5 × 10 5 pg.

[0032] In some embodiments, the freeze-drying process includes adjusting the temperature to below −35° C. and the pressure to below 80 mTorr.

[0033] In some embodiments, the freeze-drying process includes adjusting the temperature to below -40, -45, -50, -60, -65, or -70 °C.

[0034] In some embodiments, the freeze-drying process includes adjusting the pressure to less than or equal to 70, 60, 50, 40, or 30 mTorr.

[0035] In some embodiments, the platelet-containing solution includes whole blood, apheresis platelets, leukocyte-reduced apheresis platelets, platelet-rich plasma (PRP), or any combination thereof; or the platelet-containing solution is derived from an autologous blood sample or an allogeneic blood sample, or a combination thereof.

[0036] In some embodiments, the solvent in step (a5) is sterile saline, water for injection, 45% NaCl, 4.5% hypertonic saline, sterile spring water, or isotonic saline; or the platelet concentration in the ultra-pure platelet solution in step (a5) is 1×10 9 pieces / mL~10×10 9 pieces / mL.

[0037] In some embodiments, the platelet concentration in the ultra-pure platelet solution in step (a5) is 2×10 9 pieces / mL, 3×10 9 pieces / mL, 4×10 9 pieces / mL, 5×10 9 pieces / mL, 6×10 9 pieces / mL, 7×10 9 pieces / mL, 8×10 9 cells / mL, or 9 x 10 9 pieces / mL.

[0038] In some embodiments, step (c) includes (c1) performing a sedimentation process to sediment the platelet-associated structures and form a platelet-associated structure pellet, and (c2) removing the platelet-associated structure pellet to obtain a growth factor enriched solution.

[0039] The present invention further provides a growth factor enriched dry powder produced by a production method, the production method including: (a) taking a unit of a solution containing platelets, and carrying out a process for purifying platelets to obtain an ultra-pure platelet solution; (b) an activation process for activating platelets in the ultra-pure platelet solution to release growth factors, thereby obtaining a platelet growth factor extract solution; (c) carrying out a process for removing platelet-associated structures to obtain a growth factor enriched solution; and (d) drying the growth factor enriched solution to obtain a growth factor enriched dry powder. The freeze-drying process involves controlling the temperature below -30°C and the pressure below 100 mTorr, and produces growth factor enriched dry powder with a PDGF-BB content of 10 pg to 3×10 7 pg.

[0040] In some embodiments, the activation process is a freezing and then thawing process (i.e., a freeze-thaw procedure) and includes the steps of: (b1) placing a container containing an ultra-pure platelet solution in liquid nitrogen to freeze the ultra-pure platelet solution; and (b2) raising the temperature of the container to thaw the frozen ultra-pure platelet solution.

[0041] The present invention further provides use of a growth factor enriched dry powder for preparing a medical or functional composition for administering an effective amount to a site of an individual to relieve inflammation or injury at the site of the individual.

[0042] In some embodiments, the individual is a human or other mammal.

[0043] In some embodiments, the activation process is a freezing and then thawing process (i.e., a freeze-thaw procedure), and the freezing and then thawing process (i.e., a freeze-thaw procedure) further comprises the steps of: (b1) placing a container containing the ultra-pure platelet solution in liquid nitrogen to freeze the ultra-pure platelet solution; and (b2) raising the temperature of the container to thaw the frozen ultra-pure platelet solution.

[0044] In some embodiments, step (a) comprises measuring the white blood cell count in an ultra-pure platelet solution to be less than 10 6 The process of purifying platelets further includes a process of removing white blood cells so that the platelets are less than 10 ...

[0045] In some embodiments, step (a) comprises a process purifying platelets comprising removing plasma such that the ultra-pure platelet solution has an albumin concentration of less than 3 g / dL; or a process purifying platelets comprising removing plasma such that the ultra-pure platelet solution has an albumin concentration of less than 2 g / dL; or a process purifying platelets further comprising removing plasma such that the ultra-pure platelet solution has a fibrinogen concentration of less than 100 μg / mL.

[0046] In some embodiments, the VEGF content per gram (g) of growth factor enriched dry powder is greater than or equal to 2×10 2 pg~3×10 6 pg.

[0047] In some embodiments, the FGF content per gram (g) of growth factor enriched dry powder is between 10 pg and 1×10 4 pg.

[0048] In some embodiments, the TGF-β1 content per gram (g) of growth factor enriched dry powder is 2×10 5 pg~1.2×10 7 pg.

[0049] In some embodiments, the EGF content per gram (g) of growth factor enriched dry powder is 1×10 2 pg~6×10 5 pg.

[0050] In some embodiments, the use is for the preparation of a pharmaceutical composition or dietary supplement for relieving inflammation or injury in a respiratory, skin, muscle, tendon, skeletal, joint, or ligament site in an individual. [Brief description of the drawings]

[0051] [Figure 1] FIG. 1 shows a flow chart of the manufacturing process of growth factor enriched dry powder. [Figure 2A] FIG. 2A shows a bar graph diagram of the effect of different activation methods on fold release of growth factors. [Figure 2B] FIG. 2B shows a bar graph of the effect of different freeze-thaw processes on the fold release of growth factors. [Figure 3A] FIG. 3A shows a bar graph of the effect of growth factor-rich dry powder on the proliferation of human dermal fibroblasts. [Figure 3B] FIG. 3B shows a bar graph of the effect of growth factor-rich dry powder in reducing inflammation in human dermal fibroblasts. [Figure 4] FIG. 4 shows a bar graph of the effect of dry powder solutions enriched with different types of growth factors in reducing inflammation in human dermal fibroblasts. [Diagram 5] FIG. 5 shows a bar graph of the effect of the freeze-drying process on the anti-inflammatory effect of the product. [Figure 6] FIG. 6 shows a bar graph of the effect of platelet concentration during the manufacturing process on the total amount of growth factors. EXAMPLES

[0052] The following describes in detail the embodiments of the present invention, as well as the technology and features of the present invention. However, the present embodiments do not limit the present invention, and those familiar with the technology can make various modifications and improvements within the spirit and scope of the present invention within the scope of the patent of the present invention.

[0053] In the present invention, the term "growth factor enriched dry powder" refers to a dry powder enriched with platelet-related growth factors, such as a dry powder enriched with platelet-derived growth factor (PDGF), a dry powder enriched with vascular endothelial growth factor (VEGF), a dry powder enriched with epidermal growth factor (EGF), a dry powder enriched with fibroblast growth factor (FGF), or a dry powder enriched with transforming growth factor beta 1 (TGF-β1), or any combination thereof.

[0054] In the present invention, an "enriched platelet-related growth factors" dry powder has a higher concentration of platelet-related growth factors than the liquid from which it was isolated.

[0055] In the present invention, a growth factor enriched solution refers to a solution enriched with platelet-related growth factors, such as a solution enriched with platelet-derived growth factor (PDGF), a solution enriched with vascular endothelial growth factor (VEGF), a solution enriched with epidermal growth factor (EGF), a solution enriched with fibroblast growth factor (FGF), or a solution enriched with transforming growth factor beta 1 (TGF-β1), or any combination thereof.

[0056] In the present invention, an "enriched platelet-related growth factors" solution has a higher concentration of platelet-related growth factors than the solution from which they were isolated.

[0057] In the present invention, "one unit" of platelet solution refers to any size of container containing a solution containing platelets. For example, the volume of a bag containing a solution containing platelets may be 250 mL, the volume of a liquid containing platelets contained in a 50 mL centrifuge tube may be 50 mL, or the volume of a liquid containing platelets contained in a 1 mL container may be 1 mL.

[0058] In the present invention, the term "ultra-pure platelet solution" refers to a platelet solution of higher purity obtained by purifying a solution containing platelets.

[0059] In the present invention, platelet-associated structures refer to platelets, platelet debris, or platelet structures formed by inter-platelet aggregation, or any combination thereof.

[0060] In the present invention, a sedimentation process refers to a process in which materials are allowed to settle and accumulate, for example, by the use of gravity, centrifugal force, or electromagnetic force.

[0061] As used herein, a "health composition" or "health supplement" refers to a dietary supplement, a composition administered to an individual to promote normal physiological function of the individual's tissues, or a composition administered to an individual to alleviate a symptom of the individual.

[0062] Referring to FIG. 1, the process for producing growth factor enriched dry powder includes the following steps: (S11) Take one unit of platelet solution and carry out a process of purifying platelets to obtain an ultra-pure platelet solution; (S12) performing an activation process to activate platelets in the ultra-pure platelet solution and release growth factors to obtain a platelet growth factor extract solution; (S13) performing a process to remove platelet-associated structures to obtain a growth factor enriched solution; and (S14) The growth factor enriched solution is freeze-dried to obtain a growth factor enriched dry powder.

[0063] In some embodiments, the platelet-containing solution is whole blood, apheresis platelets, leukocyte-reduced platelets apheresis, or platelet-rich plasma (PRP), or any combination thereof. In some embodiments, the platelet-containing solution is obtained from an autologous blood sample or an allogeneic blood sample, or any combination thereof.

[0064] In some embodiments, step (S11) includes the substeps: (S111) Take one unit of a solution containing platelets; and (S112) At least one sedimentation process is performed to separate the platelet layer (e.g., the first platelet layer) and the white blood cell layer into multiple layers. The white blood cell layer is removed to obtain a white blood cell count of 10 6 Obtain an ultra-pure platelet solution with less than 100 μg / mL of platelets. Alternatively, perform at least one sedimentation process to separate the platelets into multiple layers, including a platelet layer (e.g., a second platelet layer) and a plasma / plasma protein layer. Remove the plasma / plasma protein layer to obtain an ultra-pure platelet solution with an albumin concentration of 3 g / dL or less, a globulin concentration of 2 g / dL or less, or a fibrinogen concentration of 100 μg / mL or less.

[0065] In some embodiments, step (S112) includes the substeps: (S1121) A first sedimentation process is carried out to separate the solution containing platelets into a first platelet layer (e.g., a plasma layer), a white blood cell layer (e.g., a buffy coat layer), and a red blood cell layer; (S1122) Removing the buffy coat layer and the red blood cell layer to obtain a plasma layer solution; (S1123) performing a second sedimentation process to allow platelets to settle and form a pellet, the pellet being the second platelet layer, and the supernatant being the plasma and plasma protein layer; and (S1124) The supernatant is removed and the precipitate is mixed with a solvent to obtain an ultra-pure platelet solution.

[0066] In some embodiments, the solvent is sterile saline, water for injection, 45% NaCl, 4.5% hypertonic salt water, sterile spring water, or isotonic salt water, or any combination thereof. In some embodiments, the pure platelet solution has a platelet concentration of 1×10 9 / mL~10×10 9 / mL.

[0067] In some embodiments, the disruption process in step (S12) may include a process of freezing and then thawing, addition of calcium ions, etc. 2+ ) process, the addition of thrombin process, the addition of collagen process, or the addition of ADP process, or any combination thereof.

[0068] In some embodiments, the activation process of step (S12) is a freezing and then thawing process (i.e., a freeze-thaw procedure), and step (S12) includes the following sub-steps:

[0069] (S121) placing the container containing the ultra-pure platelet solution in liquid nitrogen to freeze the ultra-pure platelet solution; and

[0070] (S122) The temperature of the container is raised to thaw the frozen ultra-pure platelet solution, activating the platelets in the ultra-pure platelet solution to release growth factors, thereby obtaining a platelet growth factor extract solution.

[0071] In some embodiments, step (S13) includes the following sub-steps:

[0072] (S131) ​​performing a sedimentation process to cause platelet-associated structures to sediment and form a platelet-associated structure pellet; and (S132) The platelet-associated structure pellet is removed to obtain a growth factor enriched solution.

[0073] In some embodiments, the freeze-drying process of step (S14) includes adjusting the temperature to below −30° C. and adjusting the pressure to below 100 mTorr. In some embodiments, the freeze-drying process of step (S14) includes adjusting the temperature to below −35° C. and adjusting the pressure to below 80 mTorr.

[0074] Experiment 1: Preparation of growth factor enriched dry powder and growth factor concentration analysis Experiment 1-1: Production of growth factor enriched dry powder In this experiment, growth factor enriched dry powder was produced by following the steps (S11') to (S14') below.

[0075] (S11') The procedure for preparing a pure platelet solution includes the following substeps: (S111') 250 mL of human whole blood is placed in a blood bag containing anticoagulant; (S112') A process for purifying platelets is carried out. This process includes the following sub-steps: (S1121') Using a centrifuge, human whole blood is centrifuged (500-1,200g, 5-8 minutes; this is the first centrifugation), and the whole blood is separated into three layers, from the top: the plasma layer, the buffer layer (buffy coat layer, in which white blood cells are distributed), and the red blood cell layer (see references 3-6). The separation of blood cells after centrifugation is well known to people in the technical field with general knowledge, and the first centrifugation conditions can be expanded to 300-1,500g, 3-10 minutes. After centrifugation, platelets are distributed in the plasma layer and located near the buffer layer (buffy coat layer). (S1122') Using a sterile technique, the plasma layer is removed, and white blood cells and red blood cells are completely removed. This is called platelet-rich plasma (PRP). In the present invention, this is also called plasma layer solution; (S1123') Measure the total number of platelets in the plasma layer solution using an automated blood cell analyzer (model XP-300, brand SYSMEX). Using a centrifuge, centrifuge the plasma layer solution again (1,000-2,500, 5 min; this is the second centrifugation). This causes the platelets to form a pellet, and the pellet becomes the platelet layer. The supernatant is the plasma layer and plasma protein layer. The second centrifugation conditions can be extended to 500-3,000g, 5-20 min, and should be faster than the actual eccentric speed used in the first centrifugation mentioned above (300-1,500g, 3-10 min); (S1124') The supernatant is completely removed to completely remove plasma and proteins in the plasma, and the platelets in the platelet layer are suspended using Water for Injection (platelet concentration is 1 x 10 9 / m) to obtain an ultra-pure platelet solution.

[0076] (S12') Execute an activation process, which includes the following sub-steps: (S121') Liquid nitrogen is added to the freezing tube containing the ultra-pure platelet solution and left to stand for 20 to 30 minutes (freezing time can be extended to 5 to 120 minutes). For this purpose, 1 x 10 9 platelets, and the platelet concentration in the pure platelet solution is 1 × 10 9 / mL. (S122') A frozen ultra-pure platelet solution is thawed in a water bath at 37°C to activate the platelets in the ultra-pure platelet solution and release growth factors, thereby obtaining a platelet growth factor extract solution.

[0077] (S13') performing a process for removing platelet-associated structures, the process comprising the following sub-steps: (S131') The platelet growth factor extract solution is centrifuged (10,000-15,000g, 5-10 minutes) and the platelet-associated structures are precipitated as a platelet-associated structure pellet. Centrifugation conditions can be expanded to 8,000-20,000g, 3-15 minutes. (S132') The supernatant is carefully transferred to a specimen bottle, and the platelet-associated structure pellet is completely removed. The supernatant is a growth factor enriched solution. The specimen bottle contains about 1 mL of growth factor enriched solution, which contains 1 × 10 9 It contains growth factors released by platelets.

[0078] (S14') Perform a freeze-drying process, including the following substeps: (S141') Pre-cooling (-30 to -50°C, at least 5 hours); (S142') Primary drying (the temperature is increased from the temperature gradient of step S141' to 10°C for 10 to 20 hours, and the vacuum value is 100 mTorr or less); and (S143') Secondary drying (20℃, 3-5 hours, vacuum value 100mTorr or less). The dried material after freeze-drying is a growth factor enriched dry powder.

[0079] From the above manufacturing process, it can be seen that the process of producing growth factor enriched dry powder includes a white blood cell (WBC) and plasma removal step, a platelet stimulation step (e.g., via freezing and thawing), a platelet-associated structure removal step, and a drying step (e.g., freezing, cryogenic, or vacuum drying step). Thus, the growth factor enriched dry powder contains low or no white blood cells (WBC), plasma, or platelets.

[0080] In some embodiments, the growth factor enriched dry powder contains low levels or no albumin.

[0081] In some embodiments, the growth factor enriched dry powder contains low levels or no globulin.

[0082] In some embodiments, the growth factor enriched dry powder contains low levels or no fibrinogen.

[0083] Experiment 1-2: Analysis of components of growth factor enriched dry powder The weight of three bottles of growth factor enriched dry powder was measured using a precision electronic balance (Model ME204, Mettler). Three bottles of growth factor enriched dry powder were mixed with 1 mL of saline to create three bottles of growth factor enriched dry power solution / suspension. The white blood cell (WBC) counts in the growth factor enriched dry power solution / suspension were analyzed using an automated blood cell analyzer (Model XP-300, SYSMEX), the platelet counts in the growth factor enriched dry power solution / suspension were detected using a microscope and counting method, and the concentrations of various growth factors (including PDGF-BB, VEGF, EGF, FGF, and TGF-β1) in the growth factor enriched dry power solution were analyzed using enzyme-linked immunosorbent assay (ELISA). Next, the weight of each growth factor, the number of white blood cells (WBC), and the number of platelets in the growth factor enriched dry power solution / suspension per gram (g) were estimated using the concentrations of each growth factor, the concentration of white blood cells (WBC), and the number of platelets in the growth factor enriched dry power solution / suspension. The experimental results are shown in Table 1.

[0084] [Table 1]

[0085] From Table 1, the average weight of each growth factor enriched dry powder is 0.0030g, and each gram (g) of growth factor enriched dry powder contains an average of 3,446,200pg of PDGF-BB, 379,787.01pg of VEGF, 253,430pg of EGF, 4,515.05pg of FGF, and 819,530pg of TGF-β1. Also, each nanogram (ng) of growth factor enriched dry powder contains undetectable white blood cells (WBC) (or contains about 0-100 WBC) or undetectable platelets (or contains about 0-1,000 platelets).

[0086] Experiment 2: Effect of freezing and thawing (activation process) on fold growth factor release Execute the procedure (S11') of Experiment 1-1 to prepare a pure platelet solution, and the platelet concentration in the pure platelet solution is 1 × 10 9 The pure platelet solution was divided into a refrigerated group and a frozen-thawed group.

[0087] Freeze-thaw group: 1 mL of pure platelet solution was taken, and steps (S12') to (S14') of Experiment 1-1 were performed to create a dry powder in the freeze-thaw group (i.e., the growth factor enriched dry powder of the present invention).

[0088] Refrigerated group: 1 mL of the pure platelet solution was taken and refrigerated to 4°C without carrying out step (S12') of experiment 1-1 of the activation process, i.e., the ultra-pure platelet solution was placed in liquid nitrogen and not frozen or thawed. Then, steps (S13') and (S14') of experiment 1-1 were carried out to create dry powder in the refrigerated group.

[0089] The refrigerated group dry powder and the freeze-thawed group dry powder were each mixed with 1 mL of water for injection to prepare a refrigerated group dry powder solution and a freeze-thawed group dry powder solution. The growth factor concentration in each solution was analyzed using enzyme-linked immunosorbent assay (ELISA), and the growth factor release fold for each group was calculated using the following formula:

[0090] Growth factor release fold of the freeze-thaw group = (growth factor concentration of the freeze-thaw group solution × volume of the freeze-thaw group solution) ÷ (growth factor concentration of the refrigerated group solution × volume of the refrigerated group solution). Growth factor release fold of the refrigerated group = (growth factor concentration of the refrigerated group solution × volume of the refrigerated group solution) ÷ (growth factor concentration of the refrigerated group solution × volume of the refrigerated group solution)

[0091] [Table 2]

[0092] See the experimental results in Table 2. From Table 2, it can be seen that the total amount of growth factors in the dried powder solution of the freeze-thaw group was 1.8-15 times that of the dried powder solution of the refrigerated group. This shows that the activation process of freezing and then thawing significantly increases the amount of growth factors released by 1.8-15 times, which can be said to have an unexpected effect.

[0093] Experiment 3: Effect of different activation methods on the fold increase in growth factor release Experiment 3-1: Effect of different activation methods on fold increase in growth factor release Execute the procedure (S11') of Experiment 1-1 to confirm that the platelet concentration is 1 × 10 9A pure platelet solution was prepared at 100 μg / mL. This pure platelet solution was then divided into four experimental groups: a control group, a calcium ion group, a collagen group, an adenosine diphosphate (ADP) group, and a -196°C (liquid nitrogen) group.

[0094] Control group: 1 mL of pure platelet solution was taken, and the activation process of step (S12') of experiment 1-1 was not carried out. Instead, steps (S13') to (S14') of experiment 1-1 were carried out to prepare unactivated growth factor powder, which was then mixed with 1 mL of water for injection, and used as the control group solution.

[0095] -196°C (liquid nitrogen) group: 1 mL of pure platelet solution was taken, and steps (S12') to (S14') of Experiment 1-1 were performed to prepare -196°C (liquid nitrogen) group powder (i.e., growth factor enriched dry powder of the present invention), which was then mixed with 1 mL of water for injection to prepare the -196°C (liquid nitrogen) group solution.

[0096] Calcium ion group: Take 1 mL of pure platelet solution, without performing the activation process of step (S12') of experiment 1-1, i.e., the ultra-pure platelet solution is not frozen and thawed in liquid nitrogen, and is mixed with calcium chloride so that the calcium ion concentration in the mixture becomes 4.54 mg / mL, and is left to stand for 20 to 30 minutes (the activation time can be extended from 5 to 120 minutes, and the standing time is the same as when the -196 ° C (liquid nitrogen) group performed step (S121')). Next, perform steps (S13') and (S14') of experiment 1-1 to prepare calcium ion group powder, and mix it with 1 mL of water for injection to prepare calcium ion group solution.

[0097] Collagen group: 1 mL of pure platelet solution was taken, and the activation process of step (S12') in experiment 1-1 was not performed. The ultra-pure platelet solution was not frozen and thawed in liquid nitrogen, and mixed with collagen (C7661, Sigma-Aldrich) so that the collagen concentration in the mixture was 25 μg / mL, and left to stand for 20 to 30 minutes (the activation time can be extended from 5 to 120 minutes, and the standing time is the same as when the -196 ° C (liquid nitrogen) group performed step (S121')). Next, steps (S13') and (S14') in experiment 1-1 were performed to prepare collagen group powder, which was mixed with 1 mL of water for injection to prepare collagen group solution.

[0098] Adenosine diphosphate (ADP) group: 1 mL of pure platelet solution was taken, and without carrying out the activation process of step (S12') of experiment 1-1, the ultra-pure platelet solution was frozen and thawed in liquid nitrogen, and mixed with adenosine diphosphate (ADP) so that the adenosine diphosphate (ADP) concentration in the mixture was 0.8 mM, and left to stand for 20 to 30 minutes (the activation time can be extended from 5 to 120 minutes, and the standing time is the same as when the -196 ° C (liquid nitrogen) group carried out step (S121')). Next, steps (S13') and (S14') of experiment 1-1 were carried out to prepare adenosine diphosphate (ADP) group powder, which was then mixed with 1 mL of water for injection to prepare adenosine diphosphate (ADP) group solution.

[0099] The growth factor concentration in each solution was analyzed using enzyme-linked immunosorbent assay (ELISA), and the growth factor release fold for each set was calculated using the following formula:

[0100] Fold growth factor release from the control group = (growth factor concentration in control solution x volume of control solution) ÷ (growth factor concentration in control solution x volume of control solution).

[0101] Fold growth factor release of the experimental group = (growth factor concentration of the experimental solution x volume of the experimental solution) ÷ (growth factor concentration of the control solution x volume of the control solution).

[0102] The experimental results are shown in Table 3 and FIG. 2A.

[0103] [Table 3]

[0104] See Figure 2A, which is a bar graph of the effect of different activation methods on fold growth factor release.

[0105] From the results in Table 3 and Figure 2A, the total amount of growth factors in the calcium ion solution, collagen solution, and adenosine diphosphate solution was 0.63 times, 0.23 times, and 0.19 times that of the control solution, respectively, indicating that these treatment methods significantly reduced the total amount of growth factors. What was unexpected was that the total amount of growth factors in the -196 ° C (liquid nitrogen) solution was 1.8 times that of the control solution. In other words, there is an unexpected effect that refreezing and thawing after freezing at -196 ° C (liquid nitrogen) can significantly increase the amount of growth factors released from the calcium ion solution, collagen solution, and adenosine diphosphate solution by 2.9 to 9.5 times (1.8 ÷ 0.63 ≒ 2.9, 1.8 ÷ 0.19 ≒ 9.5).

[0106] Experiment 3-2: Effects of different freeze-thaw processes on growth factor release fold Execute the procedure (S11') of Experiment 1-1 to confirm that the platelet concentration is 1 × 10 9 The pure platelet solution was then divided into five experimental groups: a control group and a -20°C group, a -30°C group, a -40°C group, a -80°C group, and a -196°C (liquid nitrogen) group.

[0107] Control group: 1 mL of pure platelet solution was taken, and without carrying out the activation process of step (S12') of experiment 1-1, steps (S13') to (S14') of experiment 1-1 were directly carried out to create growth factor dry powder without the activation process, and this was mixed with 1 mL of water for injection, which was used as the control group solution.

[0108] -196°C (liquid nitrogen) group: 1 mL of pure platelet solution was taken, and steps 1-1 (S12') to (S14') were carried out to prepare a -196°C (liquid nitrogen) group dry powder (i.e., the growth factor enriched dry powder of the present invention).

[0109] -20°C group: 1 mL of pure platelet solution was taken and frozen and thawed in a -20°C freezer without carrying out the activation process of step (S12') in experiment 1-1, i.e., the ultra-pure platelet solution was not frozen and thawed in liquid nitrogen. Then, steps (S13') and (S14') in experiment 1-1 were carried out to prepare the -20°C group dry powder.

[0110] -30°C group: 1 mL of pure platelet solution was taken and frozen and thawed in a -30°C freezer without carrying out the activation process of step (S12') in experiment 1-1, i.e., the ultra-pure platelet solution was not frozen and thawed in liquid nitrogen. Then, steps (S13') and (S14') in experiment 1-1 were carried out to prepare the -30°C group dried powder.

[0111] -40°C group: 1 mL of pure platelet solution was taken and frozen and thawed in a -40°C freezer without carrying out the activation process of step (S12') in experiment 1-1, i.e., the ultra-pure platelet solution was not frozen and thawed in liquid nitrogen. Then, steps (S13') and (S14') in experiment 1-1 were carried out to prepare the -40°C group dry powder.

[0112] -80°C group: 1 mL of pure platelet solution was taken and frozen and thawed in a -80°C freezer without carrying out the activation process of step (S12') in experiment 1-1, i.e., the ultra-pure platelet solution was not frozen and thawed in liquid nitrogen. Then, steps (S13') and (S14') in experiment 1-1 were carried out to prepare the -80°C group dried powder.

[0113] The -20°C group dry powder, -30°C group dry powder, -40°C group dry powder, -80°C group dry powder, and -196°C (liquid nitrogen) group dry powder were each mixed with 1 mL of water for injection, and the growth factor concentration in each solution was analyzed using enzyme-linked immunosorbent assay (ELISA). The growth factor release fold for each group was calculated using the following formula.

[0114] Fold release of growth factor in control group=(concentration of growth factor in control solution×volume of control solution)÷(concentration of growth factor in control solution×volume of control solution).

[0115] Fold release of growth factor in experimental group=(concentration of growth factor in experimental solution×volume of experimental solution)÷(concentration of growth factor in control solution×volume of control solution).

[0116] The experimental results are shown in Table 4 and FIG. 2B.

[0117] [Table 4]

[0118] See Figure 2B, which is a bar graph showing the effect of different freeze-thaw processes on fold growth factor release.

[0119] The results in Table 4 and FIG. 2B show that the total amount of growth factors in the -20°C group solution, the -30°C group solution, the -40°C group solution, and the -80°C group solution is 1.04 times, 0.97 times, 0.88 times, and 1.02 times that of the control group solution, respectively. That is, the total amount of growth factors obtained by freezing and thawing at -20°C to -80°C is 1±0.12 times that of the non-frozen and thawed group (control group), and the freezing and thawing process at -20°C to -80°C cannot significantly increase the total amount of growth factors. Unexpectedly, the total amount of growth factors in the -196°C (liquid nitrogen) group is 1.8 times that of the control group solution. That is, the activation process after freezing at -196°C (liquid nitrogen) can significantly increase the amount of growth factors released from 1 to 1.8 times, which has an unexpected effect.

[0120] Experiment 4: Effect of growth factor-rich dry powder on tissue repair and anti-inflammatory effects Experiment 4-1: Effect of dry powder rich in growth factors on tissue repair Human dermal fibroblasts (HDF) (purchased from ScienCell Sesearch Laboratories, Inc., product number 2320) were cultured in Dulbecco's Modified Eagle medium (DMEM, Corning 10-013CV) containing 1% human platelet lysate (HPL), and then the medium was replaced with fresh medium. The above-mentioned dermal fibroblasts (HDF) are adherent cells.

[0121] A 96-well plate was divided into untreated wells (control wells) and wells (RD2201 wells) containing the growth factor-rich dry powder prepared in Experiment 1-1 in duplicate. Human dermal fibroblasts (HDFs) were added to the untreated wells (control wells) and the RD2201 wells, respectively, so that each well contained the same number of cells, and the wells were incubated at 37°C in a 5% carbon dioxide (CO2) incubator for 24 hours.

[0122] The used medium was removed. One bottle of the growth factor-rich dry powder from Experiment 1-2 (containing 0.0030 grams (g) of growth factor-rich dry powder per bottle on average) was mixed with 1 mL of DMEM medium (containing 1% HPL) to obtain a high-concentration mixture. The high-concentration mixture was then mixed with DMEM medium (containing 1% HPL) (the volume ratio of the high-concentration mixture to the DMEM medium was 1:19) to prepare a growth factor-rich dry powder culture medium. The cells in the RD2201 well were suspended in the growth factor-rich dry powder culture medium, and the cells in the untreated well (control well) were suspended in the DMEM medium (containing 1% HPL), and the cells were cultured in an incubator at 37°C with 5% carbon dioxide (CO2) for 24 hours.

[0123] The used medium was removed. 90 μL of DMEM and 10 μl of CCK-8 buffer (DOJINDO, product number CK04) were added to each well. The cells were cultured for 3 hours in an incubator at 37°C with 5% carbon dioxide (CO2). The absorbance was set at 450 nm and analyzed using a multifunctional microplate spectrophotometer (model Varioskan LUX, Thermo Scientific). A regression curve of absorbance and cell number was created according to the product instructions for the CCK-8 buffer, and the absorbance was converted to the number of viable cells.

[0124] Data are presented as mean ± SD and statistical analysis was performed using T TEST. Groups marked with "*" are statistically significantly different (p < 0.05) from untreated wells (control wells).

[0125] Please refer to Figure 3A. Figure 3A is a bar graph showing the effect of the growth factor-rich dry powder of the present invention on the proliferation of human dermal fibroblasts. From Figure 3A, the number of cells in the control well was 5,000, and the number of cells in the RD2201 well was 6,203, with a significant difference (p < 0.05) between the two. Thus, the growth factor-rich dry powder promotes the proliferation of dermal fibroblasts and promotes skin tissue repair. The inventors predicted that the growth factor-rich dry powder could promote the proliferation of fibroblasts in other human tissues, thereby promoting the repair of the other tissues.

[0126] Experiment 4-2: The effect of dry powder rich in growth factors on alleviating inflammatory responses The same human dermal fibroblasts (HDF) as in Experiment 4-1 were used in this experiment. Human dermal fibroblasts were cultured in Dulbecco's Modified Eagle medium (DMEM, Corning 10-013CV) containing 2% fetal bovine serum (FBS), and then the medium was replaced with fresh medium.

[0127] A 96-well plate was divided into untreated wells (control wells) and growth factor-enriched dry powder wells (RD2201 wells), each of which was replicated in triplicate. The triplicate samples were from three different human whole blood samples. Human dermal fibroblasts (HDFs) were added to the untreated wells (control wells) and the RD2201 wells, respectively, to contain the same cell numbers, and incubated for 24 hours in a 37°C, 5% carbon dioxide (CO2) incubator.

[0128] Remove the used medium. Mix one bottle of the growth factor-rich dry powder from Experiment 1-2 (containing 0.0030 grams (g) of growth factor-rich dry powder per bottle on average) with 1 mL of DMEM medium (containing 2% HPL) to obtain a high-concentration mixture. Then, mix the high-concentration mixture with DMEM medium (containing 2% HPL) (volume ratio of high-concentration mixture to DMEM medium is 1:19) to prepare a growth factor-rich dry powder culture medium. Suspend the cells in the RD2201 well with the growth factor-rich dry powder culture medium, and suspend the cells in the untreated well (control well) with DMEM medium (containing 2% HPL), and incubate them at 37°C with 5% carbon dioxide (CO2) for 24 hours.

[0129] The cell culture medium from the untreated wells (control group wells) and RD2201 wells was transferred to a microcentrifuge tube, and the human interleukin (IL-6, an inflammatory factor) ELISA reagent kit (Human IL-6 ELISA Kit) (product number ab178013, manufactured by Abcam) was used to measure the human interleukin (IL-6) concentration in the cell culture medium. The absorbance value was set at 450 nm and analyzed using a multifunctional microplate spectrophotometer (model Varioskan LUX, brand Thermo Scientific). According to the product instructions of the interleukin (IL-6) ELISA reagent kit, a regression curve of the absorbance value and the inflammatory factor IL-6 concentration was created, and the inflammatory factor release rate of each group was calculated by the following formula:

[0130] Control group inflammatory factor release rate = (control group inflammatory factor IL-6 concentration ÷ control group inflammatory factor IL-6 concentration) × 100%.

[0131] Inflammatory factor release rate in RD2201 group = (inflammatory factor IL-6 concentration in RD2201 group ÷ inflammatory factor IL-6 concentration in control group) × 100%.

[0132] Data are presented as mean ± SD and statistical analysis was performed using T TEST. Groups marked with "*" are statistically significantly different (p < 0.05) from untreated wells (control wells).

[0133] [Table 5]

[0134] Please refer to Table 5 and Figure 3B. Figure 3B is a bar graph showing the effect of the growth factor-rich dry powder of the present invention on alleviating the inflammatory response of human skin fibroblasts. From the results of Table 5 and Figure 3B, the average inflammatory factor release rate of the control group was 100%, and the average inflammatory factor release rate of the RD2201 group was 36.98%, which was significantly different from the control group (p<0.05), and the reduction rate reached 63%. This shows that the growth factor-rich dry powder can inhibit the inflammatory response of human skin fibroblasts and alleviate skin inflammation. The inventor predicted that after inducing the inflammatory response of human fibroblasts in other tissues by a stimulant (e.g., LPS), the growth factor-rich dry powder can alleviate the induced inflammatory response and alleviate inflammation in other tissues.

[0135] Experiment 5: The effect of dry powders rich in different types of growth factors on alleviating inflammatory responses The required human dermal fibroblasts were prepared according to the procedure of Experiment 4-2. A 96-well plate was divided into untreated wells (control wells), wells with growth factor-rich dry powder (RD2201 wells), and wells with a mixture of plasma and RD2201, each of which was repeated three times. The triplicate samples were from three different human whole blood samples. Human dermal fibroblasts (HDFs) were added to the untreated wells (control wells), RD2201 wells, and wells with a mixture of plasma and RD2201, respectively, to contain the same cell number, and incubated in a 37°C, 5% carbon dioxide (CO2) incubator for 24 hours.

[0136] Remove the used medium. Take a blood sample and obtain two bottles of the same weight of growth factor-rich dry powder by the procedure of Experiment 1-1. Mix one bottle of growth factor-rich dry powder with 1 mL of plasma (i.e., the solution of plasma and plasma protein layer obtained by the procedure of S1123') to prepare a growth factor-rich plasma solution. Mix the other bottle of growth factor-rich dry powder with 1 mL of DMEM medium (containing 2% FBS) to prepare a growth factor-rich medium. Next, the following procedures were performed for each group.

[0137] Untreated wells (control wells): The cells in the untreated wells (control wells) were suspended in DMEM medium (containing 2% FBS) and cultured in an incubator at 37°C with 5% carbon dioxide (CO2) for 24 hours.

[0138] RD2201 well: Mix the growth factor-rich medium and DMEM medium (containing 2% FBS) (volume ratio of growth factor-rich medium to DMEM medium is 1:19), and add the growth factor-rich dry powder medium (growth factor dry powder concentration is 1.5 × 10 -4 The cells in the RD2201 wells were suspended in the growth factor-rich dry powder medium and cultured for 24 hours in an incubator at 37°C with 5% carbon dioxide (CO2).

[0139] Wells containing a mixture of plasma and RD2201: A plasma solution rich in growth factors was mixed with DMEM medium (containing 2% FBS) (the volume ratio of the plasma solution rich in growth factors to the DMEM medium was 1:19) to create a medium containing dry powder and plasma rich in growth factors (the concentration of dry powder of growth factors was 1.5 × 10 -4 The cells from the wells containing the mixture of plasma and RD2201 (g / mL) were suspended in a medium containing the dried powder and plasma rich in growth factors and cultured for 24 hours in an incubator at 37°C with 5% carbon dioxide (CO2).

[0140] The cell culture fluids from the untreated wells (control wells), RD2201 wells, and wells containing a mixture of plasma and RD2201 were transferred to microcentrifuge tubes, respectively, and the human IL-6 concentration in the cell culture fluid was measured using an interleukin (IL-6, a type of inflammatory factor) ELISA reagent kit (Human IL-6 ELISA Kit) (product number ab178013, manufactured by Abcam). It was then analyzed using a multifunctional microplate spectrometer (model Varioskan LUX, manufactured by Thermo Scientific), and the absorbance value was set at 450 nm. According to the product instructions of the interleukin (IL-6) ELISA reagent kit, a regression curve of the absorbance value and the inflammatory factor IL-6 concentration was created, the absorbance value was converted to the concentration of the inflammatory factor IL-6, and the inflammatory factor release rate of each group was calculated by the following formula:

[0141] Inflammatory factor release rate in the control group = (inflammatory factor IL-6 concentration in the control group ÷ inflammatory factor IL-6 concentration in the control group) × 100%.

[0142] Inflammatory factor release rate in the RD2201 group = (inflammatory factor IL-6 concentration in the RD2201 group ÷ inflammatory factor IL-6 concentration in the control group) × 100%.

[0143] Inflammatory factor release rate of the mixture of plasma and RD2201 = (inflammatory factor IL-6 concentration of the mixture of plasma and RD2201 ÷ inflammatory factor IL-6 concentration of the control group) × 100%.

[0144] Data are presented as mean ± SD and statistical analysis was performed using T TEST. Groups marked with "+" are statistically significantly different (p < 0.05) from growth factor-enriched dry powder wells (RD2201). Groups marked with "*" are statistically significantly different (p < 0.05) from untreated wells (control wells).

[0145] [Table 6]

[0146] Please refer to Figure 4. Figure 4 is a bar graph showing the effect of dry powder solutions containing different types of growth factors on alleviating inflammation in human skin fibroblasts. The results in Figure 4 showed that the average inflammatory factor release rate of the control group was 100%, the average inflammatory factor release rate of the RD2201 group was 36.98%, and the average inflammatory factor release rate of the plasma and RD2201 mixed group was 494.46%. Compared with the control group, the inflammatory factor release rate of the RD2201 group was significantly decreased (p < 0.05), with the decrease rate reaching 63%. Meanwhile, the inflammatory factor release rate of the plasma and RD2201 mixed group was significantly increased (p < 0.05), with the increase rate reaching 394%. Compared with the RD2201 group, the inflammatory factor release rate of the plasma and RD2201 mixed group was also significantly increased (p < 0.05), with the increase rate reaching 457%. From the above experiments, it was found that the dry powder solution rich in growth factors containing plasma and plasma protein layer could not effectively alleviate the inflammation of human skin fibroblasts, and even significantly aggravated the inflammation. Therefore, the removal of plasma and plasma protein is an important technical feature in this case, and it shows that the product can significantly alleviate the inflammatory response, with unexpected effects.

[0147] The inventors have performed experiments inducing inflammatory responses in other human tissues using irritants (e.g. LPS) and have predicted similar results, namely that by removing plasma and plasma proteins in the present invention, the product will be able to significantly reduce the inflammatory response induced.

[0148] Experiment 6: Effect of the freeze-drying process on the anti-inflammatory effect of the product Human dermal fibroblasts were prepared according to the procedure of experiment 4-2. A 96-well plate was divided into untreated wells (control wells), growth factor-rich dry powder wells (RD2201 wells), and growth factor-rich solution wells (RD2201 non-lyophilized wells), each of which was replicated three times. The triplicate samples were from three different human whole blood samples. Human dermal fibroblasts (HDFs) were added to the untreated wells (control wells), RD2201 wells, and RD2201 non-lyophilized wells, respectively, to contain the same cell number, and incubated in a 37°C, 5% carbon dioxide (CO2) incubator for 24 hours.

[0149] Remove the used medium. Follow steps (S11') to (S13') of Experiment 1-1 to obtain a solution rich in growth factors. Next, each group performed the following steps.

[0150] Untreated wells (control wells): The cells in the untreated wells (control wells) were suspended in DMEM medium (containing 2% FBS) and cultured in an incubator at 37°C with 5% carbon dioxide (CO2) for 24 hours.

[0151] RD2201 well: Take 1 mL of the solution rich in growth factors and perform the procedure (S14') of Experiment 1-1 to obtain a dry powder rich in growth factors. Mix the dry powder with 1 mL of water for injection to prepare a dry powder solution rich in growth factors. Mix the dry powder solution with DMEM medium (containing 2% FBS) to prepare a dry powder medium solution rich in growth factors (the dry powder concentration of growth factors is 1.5 × 10 -4 The cells in the RD2201 well were suspended in the dried powder medium solution, and cultured for 24 hours in an incubator at 37°C with 5% carbon dioxide (CO2).

[0152] RD2201 non-lyophilized well: Mix 1 mL of growth factor-rich solution with DMEM medium (containing 2% FBS) to prepare a growth factor-rich mixture (the dry powder concentration of growth factors is 1.5 × 10-4 The mixture was used to suspend the cells in the RD2201 non-freeze-dried wells, and the cells were cultured at 37°C in a 5% carbon dioxide (CO2) culture box for 24 hours.

[0153] The cell culture fluids from the untreated wells (control wells), RD2201 wells, and RD2201 non-lyophilized wells were transferred to microcentrifuge tubes, respectively, and the human IL-6 concentration in the cell culture fluid was measured using an interleukin (IL-6, a type of inflammatory factor) ELISA kit (Human IL-6 ELISA Kit) (product number ab178013, Abcam). Analysis was performed using a multifunctional microplate spectrophotometer (model Varioskan LUX, Thermo Scientific), and the absorbance value was set at 450 nm. According to the product instructions of the interleukin (IL-6) ELISA kit, a regression curve of the absorbance value and the inflammatory factor IL-6 concentration was created, and the absorbance value was converted to IL-6 concentration, and the inflammatory factor release rate of each group was calculated by the following formula:

[0154] Control group inflammatory factor release rate = (control group inflammatory factor IL-6 concentration ÷ control group inflammatory factor IL-6 concentration) × 100%.

[0155] Inflammatory factor release rate in RD2201 group = (inflammatory factor IL-6 concentration in RD2201 group ÷ inflammatory factor IL-6 concentration in control group) × 100%.

[0156] Inflammatory factor release rate in the non-lyophilized RD2201 group = (inflammatory factor IL-6 concentration in the non-lyophilized RD2201 group ÷ inflammatory factor IL-6 concentration in the control group) x 100% Data are presented as mean ± SD and statistical analysis was performed using T TEST. Groups marked with "*" are statistically significantly different (p < 0.05) from untreated wells (control wells).

[0157] [Table 7]

[0158] Please refer to Table 7 and Figure 5. Figure 5 is a bar graph of the effect of freeze-drying process on the anti-inflammatory effect of the product. The experimental results in Figure 5 show that the average inflammatory factor release rate of the control group is 100%, while the average inflammatory factor release rate of the RD2201 group is 36.98%, and the average inflammatory factor release rate of the RD2201 non-freeze-dried group is 75.69%. Compared with the control group, the inflammatory factor release rate of the RD2201 group is significantly reduced (p < 0.05), and the reduction rate reaches 63%. Meanwhile, compared with the control group, the inflammatory factor release rate of the RD2201 non-freeze-dried group is not significantly reduced (p > 0.05). From the above experiment, it can be seen that the RD2201 non-freeze-dried group cannot effectively alleviate the inflammation of human skin fibroblasts. Therefore, since the RD2201 non-freeze-dried group cannot effectively alleviate the inflammation of human skin fibroblasts, the freeze-drying step is an important technical feature in the present invention, which allows the product to significantly alleviate inflammatory responses and has an unexpected effect.

[0159] The inventors have performed experiments inducing an inflammatory response in other human tissues using irritants (e.g. LPS) and have predicted similar results, i.e., that the freeze-drying step of the present invention will enable the product to significantly reduce the inflammatory response induced and reduce inflammation in the tissue.

[0160] Experiment 7: Effect of platelet concentration on total growth factor content during formulation procedure In this experiment, subjects were divided into groups based on the platelet concentration: low, medium, and high. A pure platelet solution was prepared according to the procedure in Experiment 1-1 (S11'), and the platelet concentration in the pure platelet solution was 1 x 10 9 Next, dry powders for each group were prepared according to the following procedure.

[0161] High-concentration group: 10 mL of pure platelet solution was taken and centrifuged (1,000-2,500, 5 minutes) using a centrifuge to allow the platelets to settle and form a pellet. The supernatant was removed and 1 mL of water for injection was added to prepare the pure platelet solution for the high-concentration group (platelet concentration was 10 × 10 9Next, dry powders for the high concentration group were prepared according to the procedures (S12') to (S14') of Experiment 1-1.

[0162] Medium concentration group: 10 mL of pure platelet solution was taken and centrifuged (1,000-2,500, 5 minutes) using a centrifuge to allow the platelets to settle and form a pellet. The supernatant was removed and 2 mL of water for injection was added to the medium concentration group pure platelet solution (platelet concentration was 5 × 10 9 Next, dry powders of the medium concentration group were prepared according to the procedures (S12') to (S14') of Experiment 1-1.

[0163] Low concentration group: 10 mL of pure platelet solution was taken and centrifuged (1,000-2,500, 5 minutes) using a centrifuge to allow the platelets to settle and form a pellet. The supernatant was removed and 3 mL of water for injection was added to the medium concentration group pure platelet solution (platelet concentration was 3.3 × 10 9 Next, dry powders of the medium concentration group were prepared according to the procedures (S12') to (S14') of Experiment 1-1.

[0164] The high-, medium-, and low-concentration solutions were mixed with 1, 2, and 3 mL of water for injection, respectively, to obtain high-, medium-, and low-concentration solutions. Enzyme-linked immunosorbent assay (ELISA) was then used to analyze the growth factor concentrations in the three groups, and the total amount of growth factor released in each group was calculated using the following formula: Total amount of growth factor released in the high concentration group = Growth factor concentration in the high concentration group solution × Volume of the high concentration group solution Total amount of growth factor released in the medium concentration group = Growth factor concentration in the medium concentration group solution × Volume of the medium concentration group solution Total amount of growth factor released in the low concentration group = Growth factor concentration in the low concentration group solution × Volume of the low concentration group solution

[0165] [Table 8]

[0166] Please refer to Figure 6. Figure 6 is a bar graph showing the effect of platelet concentration on the total amount of growth factors during the formulation procedure. The results of Figure 6 show that the platelet concentration in the pure platelet solution was 3.3 x 10 9 / mL, the total amount of growth factor released is 109.5ng, and the platelet concentration in the pure platelet solution is 5 × 10 9 / mL, the total amount of growth factor released was 126.3 ng, and the platelet concentration in the pure platelet solution was 10 × 10 9 When the platelet concentration in the pure platelet solution was 5 × 10 9 / mL~10×10 9 At / mL, the total amount of growth factors released was significantly increased, with the increase reaching 15%-141%, demonstrating unexpected efficacy.

[0167] The above are only preferred embodiments of the present invention, and do not limit the scope of the patent of the present invention. Therefore, any other changes or modifications that do not depart from the spirit of the present invention should be included in the scope of the patent.

[0168] [Table 9]

Claims

1. A method for producing a growth factor enriched dry powder comprising at least one growth factor released by platelets, the method comprising: (a) a process for obtaining an ultra-pure platelet solution by taking a solution containing platelets and carrying out a process purifying platelets, wherein step (a) comprises: (a1) obtaining a solution containing platelets; (a2) performing a primary sedimentation process to separate the platelet-containing solution into a plasma layer, a buffy coat layer, and a red blood cell layer; (a3) removing the buffy coat layer and the red blood cell layer to obtain a plasma layer solution; (a4) subjecting the plasma layer solution to a secondary sedimentation process to cause platelets to sediment and form a pellet, and to form a supernatant of plasma and plasma proteins; and (a5) removing the plasma and plasma protein supernatant and mixing the pellet with a solvent to obtain the pure platelet solution, such that the albumin concentration in the pure platelet solution is less than 0.75 g / dL and the platelet concentration in the pure platelet solution is between 2×10 9 cells / mL and 10×10 9 cells / mL; (b) activating the ultra-pure platelet solution to release growth factors to obtain a platelet growth factor extract solution; (c) carrying out a process for removing platelet-associated structures to obtain a growth factor enriched solution; and (d) a drying process comprising drying the growth factor enriched solution to obtain a growth factor enriched dry powder comprising at least one growth factor released by the platelets.

2. The method of claim 1, wherein the drying is freeze-drying.

3. In the step (a3), the leukocyte concentration in the ultra-pure platelet solution is 10 ６ The method of claim 1, further comprising a process of removing white blood cells to less than 100 cells.

4. 2. The method of claim 1, further comprising: in step (a5), an albumin concentration in the ultra-pure platelet solution is less than 0.5 g / dL; or in step (a5), a globulin concentration in the ultra-pure platelet solution is less than 2 g / dL; or in step (a5), a fibrinogen concentration in the ultra-pure platelet solution is less than 100 μg / mL.

5. The method of claim 1, wherein the platelet-containing solution comprises whole blood, apheresis platelets, leukocyte-depleted apheresis platelets, platelet-rich plasma, or any combination thereof.

6. The activation process includes a freezing and then thawing process, addition of calcium ions, and ２＋ 2. The method of claim 1, wherein the process is a process of addition of thrombin, a process of addition of collagen, or a process of addition of adenosine diphosphate, or any combination thereof.

7. The activation process is a freezing and then thawing process (i.e., a freeze-thaw procedure), and the steps of the freezing and then thawing process (freeze-thaw procedure) include: (b1) placing a container containing the ultra-pure platelet solution in liquid nitrogen to freeze the ultra-pure platelet solution; and (b2) Raising the temperature of the container to thaw the frozen ultra-pure platelet solution. The method of claim 1 , comprising:

8. A method for producing the growth factor enriched dry powder, comprising: (1) The content of PDGF-BB in the growth factor enriched dry powder per gram (g) is 10 pg to 3 x 10 ７ pg; or (2) The VEGF content in the growth factor enriched dry powder per gram (g) is 2×10 ２ pg ~3×10 ６ pg; or (3) The content of FGF in the growth factor enriched dry powder per gram (g) is 10 pg to 1 x 10 ４ pg; or (4) The content of TGF-β1 in the growth factor enriched dry powder per gram (g) is 2×10 ５ pg ~ 1.2 x 10 ７ pg; or (5) The content of EGF in the growth factor enriched dry powder per gram (g) is 1 x 10 ２ pg ~6×10 ５ pg.

2. The method of claim 1 .

9. 3. The method of claim 2, wherein the freeze-drying process includes adjusting the temperature to below -35°C and the pressure to below 80 mTorr.

10. The method of claim 1, wherein the platelet-containing solution is an autologous blood sample, or an allogeneic blood sample, or a combination thereof.

11. The solvent in step (a5) is sterile saline, water for injection, 0.45% NaCl, 4.5% hypertonic saline, sterile hot spring water, or isotonic saline; or the platelet concentration in the ultra-pure platelet solution in step (a5) is 3.3×10 ９ pieces / mL ~ 10×10 ９ The method of claim 1, wherein the concentration is 1 / mL.

12. The step (c) includes the steps of: (c1) performing a sedimentation process to sediment platelet-associated structures to form a platelet-associated structure pellet; (c2) removing the platelet-associated structure pellet to obtain a growth factor-rich solution; The method of claim 1 , comprising:

13. A growth factor enriched dry powder, comprising: The growth factor-rich dry powder comprises: (a) a process for obtaining an ultra-pure platelet solution by taking a solution containing platelets and carrying out a process purifying platelets, wherein step (a) comprises: (a1) obtaining a solution containing platelets; (a2) performing a primary sedimentation process to separate the platelet-containing solution into a plasma layer, a buffy coat layer, and a red blood cell layer; (a3) removing the buffy coat layer and the red blood cell layer to obtain a plasma layer solution; (a4) subjecting the plasma layer solution to a secondary sedimentation process to cause platelets to sediment and form a pellet, and to form a supernatant of plasma and plasma proteins; and (a5) removing the plasma and plasma protein supernatant and mixing the pellet with a solvent to obtain the pure platelet solution, such that the albumin concentration in the pure platelet solution is less than 0.75 g / dL and the platelet concentration in the pure platelet solution is between 2×10 9 cells / mL and 10×10 9 cells / mL; (b) activating the ultra-pure platelet solution to release growth factors to obtain a platelet growth factor extract solution; (c) carrying out a process for removing platelet-associated structures to obtain a growth factor enriched solution; and (d) freeze-drying the growth factor enriched solution to obtain a growth factor enriched dry powder; A growth factor-enriched dry powder, wherein the PDGF-BB content in said growth factor-enriched dry powder per gram (g) is between 10 pg and 3×10 7 pg.

14. The activation process is a freezing and then thawing process (i.e., a freeze-thaw procedure), and the steps of the freezing and then thawing process (freeze-thaw procedure) include: (b1) placing a container containing the ultra-pure platelet solution in liquid nitrogen to freeze the ultra-pure platelet solution; and (b2) Raising the temperature of the container to thaw the frozen ultra-pure platelet solution.

14. The growth factor enriched dry powder of claim 13, comprising:

15. Claims for the manufacture of a pharmaceutical composition or dietary supplement for administering an effective amount to a portion of an individual to relieve the degree of inflammation or injury to the portion of the individual.

14. Use of a growth factor enriched dry powder according to claim 13.

16. The activation process is a freezing and then thawing process (i.e., a freeze-thaw procedure), and the freezing and then thawing process (freeze-thaw procedure) comprises: (b1) placing a container containing the ultra-pure platelet solution in liquid nitrogen to freeze the ultra-pure platelet solution; and (b2) Raising the temperature of the container to thaw the frozen ultra-pure platelet solution.

16. Use of the growth factor enriched dry powder of claim 15, comprising:

17. In the step (a3), the leukocyte concentration in the ultra-pure platelet solution is 10 ６ The use of claim 15, further comprising a process for removing white blood cells to less than 100 cells.

18. The use according to claim 15, further comprising: in said step (a5), an albumin concentration in said ultra-pure platelet solution is less than 0.5 g / dL; or in said step (a5), a globulin concentration in said ultra-pure platelet solution is less than 2 g / dL; or in said step (a5), a fibrinogen concentration in said ultra-pure platelet solution is less than 100 μg / mL.

19. Use of the growth factor enriched dry powder, comprising: (1) The VEGF content in the growth factor enriched dry powder per gram (g) is 2×10 ２ pg ~3×10 ６ pg; or (2) The content of FGF in the growth factor enriched dry powder per gram (g) is 10 pg to 1 x 10 ４ pg; or (3) The content of TGF-β1 in the growth factor enriched dry powder per gram (g) is 2×10 ５ pg ~ 1.2 x 10 ７ pg; or (4) The content of EGF in the growth factor enriched dry powder per gram (g) is 1 x 10 ２ pg ~6×10 ５ pg.

16. The use according to claim 15.

20. 16. Use according to claim 15 for the manufacture of a pharmaceutical composition or dietary supplement for relieving inflammation or injury in a respiratory, skin, muscle, tendon, skeletal, joint or ligament area of ​​an individual.

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