Use of purified platelet-derived exosome dry powder for relieving inflammation or injury

Abstract

To provide purified platelet-derived exosome dry powder that can effectively relieve or treat inflammation or injury.SOLUTION: The present invention provides a method for producing purified platelet-derived exosome dry powder, comprising a step (a) of taking one unit of a solution derived from blood and containing platelets, and performing a platelet purification process to obtain a pure platelet solution; a step (b) of performing an activation process to activate platelets in the pure platelet solution and release exosomes to obtain an activated pure platelet solution; a step (c) of performing a purification process to obtain a purified platelet-derived exosome solution; and a step (d) of performing a drying process on the purified platelet-derived exosome solution to obtain purified platelet-derived exosome dry powder containing a plurality of platelet-derived exosomes.SELECTED DRAWING: Figure 3

Description

Technical Field

[0001] The present invention relates to a purified platelet-derived exosome dry powder, and particularly to a use of the purified platelet-derived exosome dry powder for alleviating inflammation or injury.

Background Art

[0002] Inflammation is a defense mechanism of the human body. In many cases, a body part causes an inflammatory reaction due to infection, trauma, or hypersensitivity to remove harmful stimuli, eliminate pathogens, and promote tissue repair. Inflammation is classified into acute inflammation and chronic inflammation. Examples include dermatitis, allergic rhinitis, pneumonia, hepatitis, and enteritis. Dermatitis is divided into irritant contact dermatitis (ICD) and 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] Fibroblasts play an important role in the repair of many tissues throughout the body. In the repair process after tissue damage (usually accompanied by an inflammatory reaction), a series of wound repair responses such as cell proliferation, cell migration, fibrogenesis, resolution, and remodeling occur (Reference 1). Taking skin wounds as an example, fibroblasts rapidly move to the provisional matrix and rapidly proliferate, generating fibrogenesis at the damaged site. Furthermore, fibroblasts are the main source of extracellular matrix proteins (ECM) mainly containing collagen and fibronectin, forming granulation tissue and providing structural integrity to the wound.

[0004] Exosomes are nanoscale extracellular vesicles (EVs) with a diameter of approximately 30 - 200 nm, secreted from most eukaryotic cells, and contain bioactive molecules such as proteins, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), microRNAs, and metabolites. Therefore, exosomes play an important role in cell - cell communication, transmitting different information between cells by carrying bioactive molecules (References 2, 3, 4). Exosomes are contained in various biological fluids such as plasma, urine, semen, saliva, bronchial fluid, cerebral spinal fluid (CSF), breast milk, serum, amniotic fluid, synovial fluid, tears, lymph, bile, gastric acid, etc. (Reference 3), and plasma and serum contain exosomes derived from white blood cells, red blood cells, and other cells.

[0005] Current research has shown that exosomes derived from specific cells or tissues can achieve the purpose of tissue regeneration and repair by inhibiting inflammation, promoting proliferation, inhibiting apoptosis, and promoting angiogenesis. However, in the isolation and purification process, exosomes are prone to rupture, have unstable quality, and are difficult to preserve, which has been revealed by clinical research to affect the therapeutic effect.

[0006] In the market, there is an urgent need for pharmaceutical compositions to relieve or treat inflammation or damage in each part that can break through the current technological limitations. Summary of the Invention

[0007] In view of the drawbacks of the prior art, an object of the present invention is to provide a purified platelet-derived exosome dry powder that can effectively alleviate or treat inflammation and injury.

[0008] Another object of the present invention is to provide a method for manufacturing the purified platelet-derived exosome dry powder.

[0009] Another object of the present invention is to provide the use of the purified platelet-derived exosome dry powder for effectively alleviating or treating inflammation and injury.

[0010] Another object of the present invention is to provide the use in the preparation of a pharmaceutical composition or a health supplement for alleviating or treating inflammation and injury.

[0011] The present invention provides a method for manufacturing a purified platelet-derived exosome dry powder, which includes the process of taking one unit of a blood-derived solution containing platelets and performing a process purifying platelets to obtain a pure platelet solution.Among them, step (a) includes: (a1) a process of taking one unit of a blood-derived solution containing platelets; (a2) a process of performing a first sedimentation process to separate the blood-derived solution containing platelets into a plasma layer and a blood cell layer; (a3) a process of removing the blood cell layer to obtain a plasma layer solution; (a4) a process of performing a second sedimentation process to sediment platelets to form a pellet; (a5) a process of removing the supernatant and mixing the pellet with a solvent to obtain a pure platelet solution. (b) A process of performing an activation process to activate the platelets in the pure platelet solution, release exosomes, and obtain an activated pure platelet solution. Here, the activation process refers to the process of freezing and then thawing. (c) Performing a purification process: (c1) a process of performing a centrifugation process to remove most of the platelet-associated structures; and (c2) a process of performing a filter membranes filtration process to completely remove the platelet-associated structure pellet and obtain a purified platelet-derived exosome solution. And (d) a process of drying the purified platelet-derived exosome solution to obtain a purified platelet-derived exosome dry powder containing a plurality of platelet exosomes.

[0012] In some embodiments, the drying process is a freeze-drying process.

[0013] In some embodiments, step (a3) includes a process of removing white blood cells so that the number of white blood cells in the pure platelet solution is less than 10 6 per milliliter (mL).

[0014] In some embodiments, step (a5) includes a process of removing plasma so that the concentration of albumin in the pure platelet solution is less than 3 g / dL; alternatively, step (a5) includes a process of removing plasma so that the concentration of globulin in the pure platelet solution is less than 2 g / dL; alternatively, step (a5) includes a process of removing plasma so that the concentration of fibrinogen in the pure platelet solution is less than 100 μg / mL.

[0015] In some embodiments, step (a5) includes a process of removing plasma so that the concentration of albumin in the 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.

[0016] In some embodiments, step (a5) includes a process of removing plasma so that the concentration of globulin in the pure platelet solution is less than 0.25, 0.5, 0.75, 1, 1.25, 1.5, or 1.75 g / dL.

[0017] In some embodiments, step (a5) includes a process of removing plasma such that the concentration of fibrinogen in the pure platelet solution is less than 10, 20, 30, 40, 50, 60, 70, 80 or 90 μg / mL.

[0018] In some embodiments, the blood-derived solution containing platelets is whole blood, and the buffy coat layer and the red blood cells layer are included in the buffy coat layer.

[0019] In some embodiments, the process of freezing and then thawing (i.e., the freeze-thaw procedure) includes: (b1') freezing the pure platelet solution by placing the container containing the pure platelet solution in an environment in the range of -70°C to -196°C; and (b2') raising the temperature of the container to thaw the frozen pure platelet solution; and steps (b1) and (b2) are performed 1, 2, 3, 4, 5 or more than 5 times.

[0020] In some embodiments, the process of freezing and then thawing (i.e., the freeze-thaw procedure) includes: (b1) freezing the pure platelet solution by placing a container containing the pure platelet solution in liquid nitrogen (-196 °C); and (b2') thawing the frozen pure platelet solution by raising the temperature of the container; and steps (b1) and (b2) are performed 1, 2, 3, 4, 5, or more than 5 times.

[0021] In some embodiments, the process of freezing and then thawing (i.e., the freeze-thaw procedure) includes: (b1’) freezing the pure platelet solution by placing a container containing the pure platelet solution in an environment at -80 °C; and (b2') thawing the frozen pure platelet solution by raising the temperature of the container; and steps (b1) and (b2) are performed 1, 2, 3, 4, 5, or more than 5 times.

[0022] In some embodiments, the lyophilization process includes adjusting the temperature to -35 °C or lower and adjusting the pressure to 80 mTorr or lower.

[0023] In some embodiments, the lyophilization process includes adjusting the temperature to -40 °C, -45 °C, -50 °C, -60 °C, -65 °C, or -70 °C or lower.

[0024] In some embodiments, the lyophilization process includes adjusting the pressure to 70, 60, 50, 40, or 30 mTorr or lower.

[0025] In some embodiments, the solution containing platelets includes whole blood, apheresis platelets, leukocytes-reduced platelets apheresis, Platelet-Rich Plasma (PRP), or any combination thereof; alternatively, the solution containing platelets derived from blood is derived from an autologous blood sample or an allogeneic blood sample, or a combination thereof.

[0026] In some embodiments, the solvent in step (a5) is sterile physiological saline, water for injection, 0.45% NaCl, 4.5% hypertonic saline, sterile hot spring water, or isotonic saline; alternatively, the platelet concentration in the pure platelet solution in step (a5) is 1×10 9 cells / mL to 10×10 9 cells / mL.

[0027] In some embodiments, the platelet concentration in the pure platelet solution in step (a5) is 2×10 9 cells / mL, 3×10 9 cells / mL, 4×10 9 cells / mL, 5×10 9 cells / mL, 6×10 9 cells / mL, 7×10 9 cells / mL, 8×10 9 cells / mL, or 9×10 9 cells / mL.

[0028] In some embodiments, the membrane filtration process in step (c2) is a process of obtaining a solution rich in purified platelet exosomes by using membrane filtration with a pore size of 0.45 μm or less.

[0029] In some embodiments, the membrane filtration process in step (c2) is a process of obtaining a solution rich in purified platelet exosomes by using membrane filtration with a pore size of 0.22 μm or less.

[0030] The present invention further provides a method for manufacturing purified platelet-derived exosome dry powder, which is manufactured by a manufacturing method including the process of: (a) taking one unit of a blood-derived solution containing platelets and performing a process of purifying platelets to obtain a pure platelet solution; among which, step (a) includes: (a1) the process of taking one unit of a blood-derived solution containing platelets; (a2) the process of performing a first sedimentation process to separate the blood-derived solution containing platelets into a plasma layer and a blood cell layer; (a3) the process of removing the blood cell layer to obtain a plasma layer solution; (a4) the process of performing a second sedimentation process to sediment platelets to form a pellet; (a5) the process of removing the supernatant and mixing the precipitate with a solvent to obtain a pure platelet solution; (b) performing an activation process to activate the platelets in the pure platelet solution and release exosomes to obtain an activated pure platelet solution; here, the activation process refers to the process of freezing and then thawing; (c) performing a purification process: (c1) the process of performing a centrifugation process to remove most of the platelet-associated structures;and (c2) performing a membrane filtration process to completely remove platelet-associated structure pellets to obtain a purified platelet-derived exosome solution; and (d) performing a drying process on the purified platelet-derived exosome solution to obtain a purified platelet-derived exosome dry powder containing a plurality of platelet exosomes.;

[0031] In some embodiments, the drying process is a freeze-drying process.

[0032] In some embodiments, the process of freezing and then thawing (i.e., the freeze-thaw procedure) includes: (b1') freezing the pure platelet solution by placing the container containing the pure platelet solution in an environment of -80°C; and (b2') raising the temperature of the container to thaw the frozen pure platelet solution; and steps (b1) and (b2) are performed 1, 2, 3, 4, 5, or more than 5 times.

[0033] In some embodiments, the particle size of platelet exosomes in the purified platelet exosome dry powder is 20-150 nm, and the platelet exosomes contain exosome markers and platelet markers; here, the exosome markers are at least one of CD9, CD63, and CD81, or any combination thereof, and the platelet markers are at least one of CD41 and CD42b, or a combination thereof; here, (1) in the purified platelet exosome dry powder per gram (g), at least 1×10 10 platelet exosomes are contained; or, (2) in the purified platelet exosome dry powder per gram (g), 1×10 11 -1×10 15 platelet exosomes are contained; or, (3) in the purified platelet exosome dry powder per gram (g), 1×10 12 , 2×10 12 , 3×10 12 , 4×10 12 , 5×10 12 , 6×10 12 , 7×10 12 , 8×10 12 , 9×10 12 , 1×10 13 , 1.1×10 13 , 1.2×10 13 , 1.3×10 13 , 1.4×10 13 , 1.5×10 13 , 1.6×10 13 , 1.7×10 13 , 1.8×10 13 , 1.9×10 13 , 2×10 13 , 2.5×10 13 , 3×10 13 , 3.5×10 13 , 4×10 13 , 4.5×10 13 , 5×10 13 , 5.5×10 13 , 6×10 13 , 6.5×10 13 , 7×10 13 , 7.5×10 13 , 8×1013 and 8.5×10 13 and 9×10 13 and 9.5×10 13 and 1×10 14 and 1.5×10 14 and 2×10 14 and 2.5×10 14 and 3×10 14 and 3.5×10 14 and 4×10 14 and 4.5×10 14 and 5×10 14 and 5.5×10 14 and 6×10 14 and 6.5×10 14 and 7×10 14 and 7.5×10 14 and 8×10 14 and 8.5×10 14 and 9×10 14 or 9.5×10 14 contains platelet exosomes.

[0034] In some embodiments, (1) the purified platelet exosome dry powder per gram (g) contains at least 50 μg of small molecule ribonucleic acid (microRNA); or (2) the content of low molecular weight ribonucleic acid in the purified platelet exosome dry powder per gram (g) is 100 μg to 2,500 μg.

[0035] In some embodiments, (1) the content of PDGF-BB (platelet-derived growth factor-BB) in purified platelet-derived exosome powder per gram (g) is from 0.01 ng to 2,000 ng; or (2) the content of VEGF (vascular endothelial growth factor) in 1 gram (g) of purified platelet-derived exosome powder is from 50 pg to 100,000 pg; or (3) the content of IGF (insulin-like growth factor) in 1 gram (g) of purified platelet-derived exosome powder is from 1 pg to 3,000 pg; or (4) the content of TGF-β1 (transforming growth factor beta 1) in 1 gram (g) of purified platelet-derived exosome powder is from 50 ng to 20,000 ng; or (5) the content of EGF (epidermal growth factor) in 1 gram (g) of purified platelet-derived exosome powder is from 0.1 ng to 200 ng.

[0036] In some embodiments, the content of PDGF-BB in 1 gram (g) of purified platelet-derived exosome powder is 0.01, 0.1, 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 1×10 2 , 5×10 2 , 1×10 3 , 1.5×10 3 , or 2×10 4 ng.

[0037] In some embodiments, the content of VEGF in 1 gram (g) of purified platelet-derived exosome powder is 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1×10 3 、2×10 3 、3×10 3 、4×10 3 、5×10 3 、6×10 3 、7×10 3 、8×10 3 、9×10 3 、1×10 4 、1.5×10 4 、2×10 4 、2.5×10 4 、3×10 4 、3.5×10 4 、4×10 4 、4.5×10 4 、5×10 4 、6×10 4 、7×10 4 、8×10 4 、9×10 4 、or 1×10 5 pg.

[0038] In some embodiments, the content of IGF in 1 gram (g) of purified platelet-derived exosome powder is 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 1×10 2 、5×10 2 、1×10 3 、1.5×10 3 、2×10 3 、2.5×10 3 、or 3×10 3 pg.

[0039] In some embodiments, the content of TGF-β1 in 1 gram (g) of purified platelet-derived exosome powder is 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1×10 3 、5×10 3 、1×10 4 、1.5×10 4 、or 2×10 4 ng.

[0040] In some embodiments, the content of EGF in 1 gram (g) of purified platelet-derived exosome powder is 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, or 200 ng.

[0041] In some embodiments, among them, (1) no leukocyte exosomes are detected in 1 gram (g) of purified platelet-derived exosome powder; or, (2) the number of leukocyte exosomes in 1 gram (g) of purified platelet-derived exosome powder is 1×10 14 or less; wherein the leukocyte exosomes contain a leukocyte marker; the leukocyte marker is CD45.

[0042] In some embodiments, (1) no erythrocyte exosomes are detected in 1 gram (g) of purified platelet-derived exosome powder; or, (2) the number of erythrocyte exosomes in 1 gram (g) of purified platelet-derived exosome powder is 1×10 14 or less; wherein the erythrocyte exosomes contain an erythrocyte marker; the erythrocyte marker is at least one of CD235ar and Annexin V, or a combination thereof.

[0043] In some embodiments, among them, (1) exosomes other than platelet exosomes in the purified platelet exosome dry powder per gram (g) are not detected; or (2) the number of exosomes other than platelets in the purified platelet exosome dry powder per gram (g) is 1×10 14 is as follows.

[0044] The present invention further provides the use of purified platelet exosome dry powder for preparing a medical composition or a health composition for relieving inflammation or injury of a site of an individual when used at an effective amount at a site of the individual.

[0045] In some embodiments, it is used for preparing a medical composition or a health composition for relieving inflammation or injury in the airway, skin, muscle, tendon, bone, joint, or ligament of the individual.

[0046] The present invention further provides the use of purified platelet exosome dry powder for preparing a pharmaceutical composition or a health composition for enhancing the migration ability of skin fibroblasts to a skin site.

[0047] In some embodiments, the skin site is a skin lesion site.

[0048] The invention further provides the use of purified platelet exosome dry powder for preparing a pharmaceutical composition or a health composition for increasing the expression level of collagen in skin fibroblasts.

Brief Description of the Drawings

[0049]

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Mode for Carrying Out the Invention

[0050] [Example] The following will explain in detail the embodiments of the present invention, as well as the techniques and features of the present invention. However, this example does not limit the present invention, and those skilled in this technology can make various changes and improvements within the scope not exceeding the spirit and scope of the present invention, which are included in the patent scope of the present invention.

[0051] In the present invention, the purified platelet-derived exosome dry powder refers to a dry powder rich in high-purity platelet exosomes. For example, at least 1×10 10 exosomes per gram of the dry powder, and 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% or more are platelet exosomes.

[0052] In the present invention, the “purified platelet-derived exosome” dry powder has a smaller amount of other exosomes than its separation source composition, where “other exosomes” refers to exosomes other than platelet-derived exosomes (“Other exosomes” refer to exosomes other than platelet-derived exosomes).

[0053] In the present invention, the purified platelet-derived exosome solution means a solution rich in high-purity platelet-derived exosomes (pure platelet-derived exosome).

[0054] In the present invention, the "purified platelet-derived exosome solution" has fewer other exosomes than its source solution, where "other exosomes" refer to exosomes other than platelet-derived exosomes.

[0055] In the present invention, the solution containing "1 unit" of platelets refers to the volume of any size of the container that holds the solution containing platelets. For example, when the volume of the blood bag containing the solution with platelets is 250 mL, when the volume of the liquid containing platelets in a 50 mL centrifuge tube is 50 mL, or when the volume of the liquid containing platelets in a 1 mL container is 1 mL.

[0056] In the present invention, the "pure platelet solution" refers to a platelet solution of higher purity obtained by purifying the solution containing platelets.

[0057] In the present invention, the "platelet-associated structure" refers to platelets, platelet fragments, or platelet structures formed by platelet aggregation, or any combination thereof.

[0058] In the present invention, the "sedimentation process" refers to the process of sedimenting and accumulating substances. For example, it is a process of sedimenting and accumulating substances by using gravity, centrifugal force, or electromagnetic force.

[0059] In the present invention, the "health composition" or "health supplement" refers to a composition administered to an individual to promote the maintenance of normal physiological functions of the individual's tissues or a composition administered to an individual to relieve the individual's symptoms.

[0060] Referring to Figure 1, the process of manufacturing purified platelet-derived exosome dry powder includes the following steps: (S11) Take 1 unit of platelet solution and perform a process of purifying platelets to obtain an ultra-pure platelet solution; (S12) Perform an activation process to activate the platelets in the pure platelet solution, release exosomes, and obtain an activated pure platelet solution; (S13) Perform a process of removing platelet-associated structures to obtain a purified platelet-derived exosome solution; and (S14) Dry the purified platelet-derived exosome solution to obtain a purified platelet-derived exosome dry powder containing multiple platelet exosomes.

[0061] In some embodiments, the platelet-containing solution is derived from blood. In some embodiments, the solution containing platelets includes whole blood, apheresis platelets, leukocytes-reduced platelets apheresis, Platelet-Rich Plasma (PRP), or any combination thereof. In some embodiments, the solution containing platelets is derived from an Autologous blood sample or an allogeneic blood sample, or a combination thereof.

[0062] In some embodiments, step (S11) includes sub-steps: (S111) Take one unit of the solution containing platelets; and (S112) Perform at least one sedimentation process to separate the blood-derived solution containing platelets into multiple layers, where the multiple layers include a platelet layer (e.g., the first platelet layer) and a leukocyte layer. Remove the leukocyte layer to obtain a pure platelet solution, and the number of leukocytes per milliliter (mL) of the pure platelet solution is 10 6Cause it to be less than. Alternatively, perform at least one sedimentation process to separate the platelet-containing solution into multiple layers, where the platelet layer (e.g., the second platelet layer) and the plasma and plasma protein layers are included. Remove the plasma and plasma protein layers to obtain a pure platelet solution, such that the albumin concentration in the pure platelet solution is 3 g / dL or less, or the globulin concentration in the pure platelet solution is 2 g / dL or less, or the fibrinogen concentration in the pure platelet solution is 100 μg / mL or less.

[0063] In some embodiments, step (S112) includes sub-steps: (S1121) Perform a primary sedimentation process 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) Remove the buffy coat layer and the red blood cell layer to obtain a plasma layer solution; (S1123) Perform a secondary sedimentation process to sediment the platelets and form a pellet. This pellet is the second platelet layer, and the supernatant is the plasma and plasma protein layer; and (S1124) Remove the supernatant and mix the pellet with a solvent to obtain a pure platelet solution.

[0064] In some embodiments, the solvent is sterile physiological saline, water for injection, 0.45% NaCl, 4.5% hypertonic saline, sterile hot spring water, or isotonic saline, or any combination thereof. In some embodiments, the platelet concentration in the pure platelet solution is 1×10 9 cells / mL to 10×10 9 cells / mL.

[0065] In some embodiments, the activation process of step (S12) is a process of freezing and then thawing (i.e., the freeze - thaw procedure), and step (S12) further includes the following sub - steps: (S121) Place the container containing the pure platelet solution at - 70°C to - 196°C to freeze the pure platelet solution; and (S122) Raise the temperature of the container and thaw the frozen pure platelet solution to activate the platelets in the pure platelet solution and release exosomes.

[0066] (S123) Execute steps (S121) and (S122) 1, 2, 3, 4, or 5 times to obtain the activated pure platelet solution.

[0067] In some embodiments, step (S13) further includes sub - steps: (S131) Execute the centrifugation process to remove most of the platelet - associated structures.

[0068] (S132) Perform a filter membranes filtration process to completely remove platelet associate structures and obtain a purified platelet-derived exosome solution.

[0069] In some embodiments, in step (S14), the drying process is a lyophilization process. In some embodiments, the lyophilization process of step (S14) includes adjusting the temperature to -30°C or lower and adjusting the pressure to 100 mTorr or lower. In some embodiments, the lyophilization process of step (S14) includes adjusting the temperature to -35°C or lower and adjusting the pressure to 80 mTorr or lower.

[0070] Experiment 1: Component analysis of purified platelet-derived exosome dry powder.

[0071] In this experiment, a purified platelet-derived exosome dry powder is produced by the following steps (S11') to (S14'): (S11') Execute a process for producing a pure platelet solution, and the process includes the following sub-steps: (S111') Put 250 mL of human Whole Blood into a blood bag containing an anticoagulant; (S112') Execute a process purifying platelets, and the process includes the following sub-steps: (S1121') Use a centrifuge to centrifuge human whole blood (500 - 1,200 g, 5 - 8 minutes, this is the first centrifugation), and the human whole blood is separated into three layers in order from top to bottom: the plasma layer, the buffy coat layer (where white blood cells are distributed), and the red blood cell layer. The separation situation of blood cells after centrifugation is well-known to those skilled in the art with general knowledge, and the first centrifugation conditions can be extended to 300 - 1,500 g, 3 - 10 minutes. After centrifugation, platelets are distributed in the plasma layer and are located near the buffy coat layer.

[0072] (S1122’) By aseptic operation, take out the plasma layer and completely remove white blood cells and red blood cells, which is called platelet-rich plasma (PRP). In the present invention, it is also called plasma layer solution; (S1123’) Use a fully automatic blood cell analyzer (model XP-300, brand SYSMEX) to measure the total number of platelets in the plasma layer solution. Use a centrifuge to centrifuge the plasma layer solution again (1,000 - 2,500, 5 minutes, this is the second centrifugation). This forms a platelet pellet, and the pellet becomes the platelet layer. The supernatant is the plasma layer and the plasma protein layer. The conditions for the second centrifugation can be extended to 500 - 3,000 g, 5 - 20 minutes, but it is necessary to be faster than the actual centrifugation speed used in the above-mentioned first centrifugation (300 - 1,500 g, 3 - 10 minutes); (S1124’) By completely removing the supernatant, plasma and proteins in the plasma are completely removed, and platelets in the platelet layer are suspended using an Isotonic Sodium Chloride Solution Injection (make it 10 - 20 mL so that the platelet concentration becomes 1×109 cells / mL), thereby obtaining a pure platelet solution.

[0073] (S12') Execute the activation process, including the following sub - steps: (S121’) Place the cryotube containing the pure platelet solution at - 80°C and let it stand for 24 - 36 hours (the freezing time can be extended to 12 - 60 hours) to freeze the pure platelet solution. Here, the cryotube contains 1×10 9 platelets, and the platelet concentration in the pure platelet solution is 1×10 9 cells / mL.

[0074] (S122’) Place the cryotube in a 37°C water bath to thaw the frozen pure platelet solution, activate the platelets in the pure platelet solution, and release exosomes.

[0075] (S123’) Repeat step (S121’) and step (S122’) twice (i.e., a total of 3 freeze - thaw cycles) to obtain this activated pure platelet solution.

[0076] (S13’) Execute a purification process to remove platelet - associated structures, and the process includes the following sub - steps: (S131’) Centrifuge the activated pure platelet solution at 10,000 - 15,000 g for 5 - 10 minutes, so that platelet-associated structures precipitate as a platelet-associated structure pellet. The centrifugation conditions can be extended to 8,000 - 20,000 g for 3 - 15 minutes.

[0077] (S132’) Carefully transfer the supernatant to a specimen bottle and filter it through a 0.45 μm pore size filter membrane (PES material, 25 mm, model C0000296, brand Labfil (R) ) to completely remove the platelet-associated structure pellet. The supernatant is the purified platelet-derived exosome solution. Here, the specimen bottle contains approximately 1 mL of the purified platelet-derived exosome solution, and this 1 mL of the purified platelet-derived exosome solution contains platelet exosomes released from 1×10 9 platelets. Next, quantitative analysis of nanoparticles was performed using a nanoparticle size analyzer (Nano Tracking Analysis, NTA, model NanoSight NS300, brand Malvern). As a result of the analysis, the exosome content in approximately 1 mL of the purified platelet-derived exosome solution is about 1×10 9 - 1×10 12It was clarified that the particle size is 20 - 150 nm. Subsequently, using a fully automated exosome fluorescence quantitative analyzer (ExoView, manufactured by Leprechaun), it was analyzed whether these exosomes contain platelet exosome biomarkers. Since these exosomes have exosome markers CD9, CD63, CD81 and platelet markers CD41, CD42b, it was confirmed that they are indeed platelet exosomes; furthermore, these exosomes do not have the leukocyte marker CD45, the erythrocyte marker CD235ar, or annexin V. Therefore, it can be seen that the purified platelet-derived exosome dry powder produced in steps (S11') - (S14') of Experiment 1 of this example does not contain leukocyte exosomes or erythrocyte exosomes.

[0078] (S14’) Execute the freeze-drying process, including the following sub-steps: (S141’) Pre-cooling (-30 to -50 °C, at least 5 hours); (S142’) Primary drying (the temperature rises from the temperature gradient in step S141' to 10 °C, for 10 - 20 hours, and the vacuum value is 100 mTorr or less); and (S143’) Secondary drying (20 °C, 3 - 5 hours, and the vacuum value is 100 mTorr or less). The purified platelet-derived exosome solution (about 1 mL) is placed in a 1-bottle sample vial and freeze-dried to obtain 1 bottle of purified platelet-derived exosome dry powder. Use a precision electronic balance (model ME204, Mettler brand) to measure the weight of 1 bottle of purified platelet-derived exosome dry powder. The results showed that the average weight of each bottle of purified platelet-derived exosome dry powder is 0.0067 grams (g). According to the calculation results, there are at least 1×10 13 ~10×10 13It was revealed that individual platelet exosomes were contained.

[0079] From the above manufacturing process, the manufacturing process of purified platelet-derived exosome dry powder includes a step of removing white blood cells (WBC) and plasma, a step of activating platelets (for example, by freeze-thawing), a step of removing platelet-associated structures, and a drying step (for example, steps such as freezing, cryogenic methods, or vacuum drying). Therefore, the purified platelet-derived exosome dry powder contains little or no white blood cells (WBC), plasma, and platelets.

[0080] In some embodiments, the purified platelet-derived exosome dry powder contains a low concentration of albumin or does not contain any.

[0081] In some embodiments, the purified platelet-derived exosome dry powder contains a low concentration of globulin or does not contain any.

[0082] In some embodiments, the purified platelet-derived exosome dry powder contains a low concentration of fibrinogen or does not contain any.

[0083] Experiment 2: The effect of freeze-thawing (activation process) on the release amounts of platelet exosomes and PDGF-BB.

[0084] Execute step (S11’) of Experiment 1 to produce a pure platelet solution, with a platelet concentration of 1×10 9 cells / mL in the pure platelet solution. Next, divide the pure platelet solution into three groups: a control group, a -196°C group, and a freeze-thaw 3-cycle group.

[0085] Control group: Collect 1 mL of the pure platelet solution and do not perform the activation process in step (S12') of Experiment 1. That is, after placing the pure platelet solution at -80°C, do not perform 3 cycles of freeze-thawing, but only leave it standing for 20 - 30 minutes. Then, execute step (S13') of Experiment 1 again to produce a control group solution.

[0086] -196°C group: Collect 1 mL of the pure platelet solution and do not perform the activation process in step (S12') of Experiment 1. That is, after placing the pure platelet solution at -80°C, do not perform 3 cycles of freeze-thawing. Instead, place it at -196°C and freeze it for 20 - 30 minutes, then thaw it once (at 37°C). Then, execute step (S13') of Experiment 1 again to produce a -196°C group solution.

[0087] Freeze-thaw 3-cycle group: Collect 1 mL of the pure platelet solution and execute steps (S12’) - (S13’) of Experiment 1 to produce a freeze-thaw 3-cycle group solution (i.e., a purified platelet-derived exosome solution).

[0088] Using a nanoparticle size analyzer (Tunable Resistive Pulse Sensing (TRPS), model Exoid, IZON Science brand), the concentration of platelet exosomes in the solution of each group was analyzed, and using enzyme-linked immunosorbent assay (ELISA), the concentration of growth factor (PDGF-BB) in the solution of each group was analyzed. Next, the release multiples of platelet exosomes and growth factors of each group were calculated using the following formulae.

[0089] Release multiple of platelet exosomes = Concentration of platelet exosomes in the experimental group solution ÷ Concentration of platelet exosomes in the control group solution.

[0090] Release multiple of growth factor = Concentration of growth factor in the experimental group solution ÷ Concentration of growth factor in the control group solution.

[0091] Among them, the experimental group solution is the -196 °C group solution or the freeze-thaw 3-cycle group solution.

[0092] For the experimental results, refer to Table 1 and Figure 2. Figure 2 is a bar graph showing the effects of different activation methods on platelet exosome and PDGF-BB release.

[0093]

Table 1

[0094] From the results in Table 1 and Figure 2, it is shown that the release multiple of platelet exosomes in the -196 °C group is 9 and the release multiple of PDGF-BB is 2.65; the release multiple of platelet exosomes in the freeze-thaw 3-cycle group increased to 29, while the release multiple of PDGF-BB decreased to 0.47. It can be seen that the changing trends of the release multiples of platelet exosomes and PDGF-BB in the solutions produced by different activation methods are independent of each other. Therefore, the inventor further conducted the following experiments to find the best method for producing platelet exosomes.

[0095] Experiment 3: Influence of Different Activation Methods on Exosome Concentration Execute step (S11') of Experiment 1 to produce a pure platelet solution, and the platelet concentration in this pure platelet solution was 1×10 9 cells / mL. Next, divide this pure platelet solution into six experimental groups: a control group, a calcium chloride group, a -196 °C group, an adenosine diphosphate group, a collagen group, a thrombin group, and a freeze-thaw 3-cycle group.

[0096] Control group: Take 1 mL of the pure platelet solution and let it stand for 20 - 30 minutes instead of performing the activation process in step (S12') of Experiment 1-1. Next, execute step (S13') of Experiment 1 again to produce the control group solution.

[0097] Calcium chloride (CaCl2) group: 1 mL of pure platelet solution was collected, and the activation process in step (S12') of Experiment 1 was not performed. That is, after placing the pure platelet solution at -80°C, the freeze-thaw cycle was not performed 3 times. Instead, it was mixed with calcium chloride so that the calcium chloride concentration in the mixed solution was 0.45%, and then placed in a cyclotron shaker (model DSR-D-N1, brand DIGISYSTEM) with a rotation speed of 100 rpm at room temperature and allowed to act for 20 - 30 minutes. Next, step (S13') of Experiment 1 was performed again to produce the calcium chloride group solution.

[0098] -196°C group: 1 mL of pure platelet solution was collected, and the activation process in step (S12') of Experiment 1 was not performed. That is, after placing the pure platelet solution at -80°C, the freeze-thaw cycle was not performed 3 times. Instead, it was placed at -196°C and frozen for 20 - 30 minutes, and then thawed once (at 37°C). Then, step (S13') of Experiment 1 was performed again to produce the -196°C group solution.

[0099] Adenosine diphosphate (ADP) group: 1 mL of pure platelet solution was collected, and the activation process in step (S12') of Experiment 1 was not performed. That is, after placing the pure platelet solution at -80°C, the freeze-thaw cycle was not performed 3 times. Instead, it was mixed with adenosine diphosphate (ADP), and the concentration of adenosine diphosphate (ADP, catalog number A2754, brand Sigma-Aldrich (R) ) in the mixed solution was adjusted to 60 μM. Then, it was placed in a cyclotron shaker (model DSR-D-N1, brand DIGISYSTEM) with a rotation speed of 100 rpm at room temperature and allowed to act for 20 - 30 minutes. Next, step (S13') of Experiment 1 was performed again to produce the adenosine diphosphate (ADP) group solution.

[0100] Collagen group: 1 mL of pure platelet solution was collected, and the activation process in step (S12') of Experiment 1 was not performed. That is, after placing the pure platelet solution at -80°C, the freeze-thaw cycle was not performed three times. Instead, it was mixed with collagen (C7661, brand Sigma-Aldrich) so that the concentration of collagen in the mixed solution was 10 μg / mL, and then placed in a cyclotron shaker (model DSR-D-N1, brand DIGISYSTEM) with a rotation speed of 100 rpm at room temperature and allowed to act for 20 to 30 minutes. Next, step (S13') of Experiment 1 was repeated to produce the collagen group solution.

[0101] Thrombin group: 1 mL of pure platelet solution was collected, and the activation process in step (S12') of Experiment 1 was not performed. That is, after placing the pure platelet solution at -80°C, the freeze-thaw cycle was not performed three times. Instead, it was mixed with thrombin (Thrombin, catalog number T7326, brand Sigma-Aldrich (R) ) so that the concentration of thrombin in the mixed solution was 1 U / mL, and then placed in a cyclotron shaker (model DSR-D-N1, brand DIGISYSTEM) with a rotation speed of 100 rpm at room temperature and allowed to act for 20 - 30 minutes. Next, step (S13') of Experiment 1 was repeated to produce the thrombin group solution.

[0102] Freeze-thaw 3-cycle group: 1 mL of pure platelet solution was collected, and steps (S12') to (S13') of Experiment 1 were performed to produce the freeze-thaw 3-cycle group solution (that is, purified platelet-derived exosome solution).

[0103] Quantitative nanoparticle analysis was performed using a nanoparticle size analyzer (Tunable Resistive Pulse Sensing, TRPS, Exoid model, IZON Science brand) to analyze the concentration of platelet exosomes in the solution of each group, and the exosome release multiple of each group was calculated using the following formula.

[0104] Exosome release multiple of the experimental group of platelets = Concentration of platelet exosomes in the experimental group solution ÷ Concentration of platelet exosomes in the control group solution.

[0105] The experimental results are shown in Table 2 and Figure 3.

[0106]

Table 2

[0107] Refer to Figure 3. Figure 3 is a bar graph showing the effect of different activation methods on the platelet exosome concentration.

[0108] From the results of Table 2 and Figure 3, it is shown that compared with the control group, activation methods such as calcium chloride, -196°C, adenosine diphosphate, collagen, and thrombin could not significantly increase the concentration of platelet exosomes. Unexpectedly, when using the activation method of repeating freeze-thaw cycles 3 times, platelet exosomes are released up to 29 times. That is, through the activation process of freezing at -80°C and then thawing 3 times repeatedly, the release amount of platelet exosomes is significantly increased, and an unexpected effect is obtained.

[0109] Experiment 4: Effect of the number of freeze-thaw cycles on platelet exosome release.

[0110] Step (S11’) of Experiment 1 was performed to produce a pure platelet solution, and the platelet concentration in this pure platelet solution was 1×10 9It was cells / mL. Next, this pure platelet solution was divided into six experimental groups: a 1-cycle group, a 1-cycle (-196°C) group, a 3-cycle group, a 3-cycle (-196°C) group, a 5-cycle group, and a 5-cycle (-196°C) group.

[0111] 1-cycle group: Take 1 mL of the pure platelet solution and perform steps (S121') to (S122') of Experiment 1, but do not perform the activation process in step (S123') of Experiment 1. That is, place the pure platelet solution at -80°C, freeze it, and then thaw it, but perform only 1 freeze-thaw cycle. Next, repeat step (S13') of Experiment 1 to produce a 1-cycle group solution.

[0112] 1-cycle (-196°C) group: Take 1 mL of the pure platelet solution and do not perform the activation process in step (S12') of Experiment 1. That is, after placing the pure platelet solution at -80°C, do not perform 3 freeze-thaw cycles. Instead, place it at -196°C and freeze it for 20 to 30 minutes, and then thaw it once (at 37°C). Next, repeat step (S13') of Experiment 1 to produce a 1-cycle (-196°C) group solution.

[0113] 3-cycle group: Take 1 mL of the pure platelet solution and perform steps (S12') to (S13') of Experiment 1-1 to produce a 3-cycle group solution (i.e., a purified platelet-derived exosome solution).

[0114] 3-cycle (-196°C) group: Take 1 mL of the pure platelet solution and do not perform the activation process in step (S12') of Experiment 1. That is, the pure platelet After placing the solution at -80°C, instead of performing 3 cycles of freeze-thaw, it was placed at -196°C and frozen for 20 - 30 minutes, and then thawed (37°C) 3 times again. Next, step (S13') of Experiment 1 was re-executed to produce the 3-cycle (-196°C) group solution.

[0115] 5-cycle group: 1 mL of pure platelet solution was collected, and steps (S121') - (S122') of Experiment 1 were executed, but the activation process in step (S123') of Experiment 1 was not performed. That is, after placing the pure platelet solution at -80°C, freeze-thaw was carried out, but instead of 3 cycles, 5 cycles were performed. Next, step (S13') of Experiment 1 was re-implemented to produce the 5-cycle group solution.

[0116] 5-cycle (-196°C) group: 1 mL of pure platelet solution was collected, and the activation process in step (S12') of Experiment 1 was not performed. That is, after placing the pure platelet solution at -80°C, instead of performing 3 cycles of freeze-thaw, it was placed at -196°C and frozen for 20 - 30 minutes, and then thawed (37°C) 5 times. Next, step (S13') of Experiment 1-1 was re-implemented to produce the 5-cycle (-196°C) group solution.

[0117] Quantitative nanoparticle analysis was performed using a nanoparticle size analyzer (Tunable Resistive Pulse Sensing (TRPS), model Exoid, IZON Science brand) to analyze the concentration of platelet exosomes in the solutions of each group.

[0118] The experimental results are shown in Table 3 and Figure 4.

[0119]

Table 3

[0120] Refer to FIG. 4. FIG. 4 is a bar graph showing the effect of the number of freeze-thaw cycles on the platelet exosome concentration.

[0121] From the results in Table 3 and FIG. 4, the platelet exosome concentrations in the solutions of the 1-cycle group, 3-cycle group, and 5-cycle group are higher than those in the solutions of the 1-cycle (-196°C) group, 3-cycle (-196°C) group, and 5-cycle (-196°C) group. It can be seen that the platelet exosome concentration in the solution of the 3-cycle group increases by more than 45% compared with the solution of the 3-cycle (-196°C) group. Regardless of the number of freeze-thaw cycles, the concentration of platelet exosomes obtained by freeze-thawing at -80°C is higher than that of platelet exosomes obtained by freeze-thawing at -196°C, and an unexpected effect is obtained. That is, the activation process of freeze-thawing at -80°C can achieve the highest platelet exosome release amount.

[0122] Experiment 5: Effects of Freeze-Thawing on Platelet Exosome Release Amount and Total Protein Release Amount Execute step (S11') of Experiment 1-1 to produce a pure platelet solution, and the platelet concentration in this pure platelet solution is 1×10 9 cells / mL. Next, the pure platelet solution was divided into a control group and a freeze-thaw 3-cycle group.

[0123] Control group: Take 1 mL of the pure platelet solution and let it stand for 20 - 30 minutes instead of performing the activation process in step (S12') of Experiment 1-1. Next, execute step (S13') of Experiment 1-1 again to produce a control group solution.

[0124] Freeze-thaw 3-cycle group: Take 1 mL of the pure platelet solution and execute steps (S12')-(S13') of Experiment 1-1 to produce a freeze-thaw 3-cycle group solution (i.e., a purified platelet-derived exosome solution).

[0125] Using a nanoparticle size analyzer (Tunable Resistive Pulse Sensing, TRPS, Exoid model, IZON Science brand), the concentrations of platelet exosomes in two solutions were analyzed, and using an automated biochemical analyzer (TOSHIBA TBA-120FR), the total protein was quantitatively analyzed.

[0126] The experimental results of platelet exosome concentration are shown in Table 4 and Figure 5A. The experimental results of total protein amount are shown in Table 4 and Figure 5B. The data are presented as mean ± SD, and the statistical analysis is performed by t-test. The groups marked with "*" show statistical differences from the control group (p < 0.05).

[0127]

Table 4

[0128] See Figures 5A and 5B. Figure 5A is a bar graph showing the effect of freeze-thaw on platelet exosome concentration. Figure 5B is a bar graph showing the effect of freeze-thaw on total protein amount.

[0129] The results in Table 4 and Figure 5A show that the concentration of platelet exosomes in the control group was 9,450,000,000 particles / mL; the platelet exosome concentration in the 3-cycle freeze-thaw group was 17,000,000,000 particles / mL. It can be seen that the platelet exosome concentration in the 3-cycle freeze-thaw group was significantly higher than that in the control group.

[0130] The results in Table 4 and Figure 5B show that the average total protein concentration in the control group was 0.0852 g / dL, and the average total protein concentration in the 3-cycle freeze-thaw group was 0.1125 g / dL.

[0131] Experiment 6: The effect of the composition of platelet exosomes obtained by the freeze-thaw activation process on the alleviation of inflammatory response and tissue repair Experiment 6-1: Influence of the platelet exosome composition obtained by the freeze-thaw activation process on inflammation alleviation Human dermal fibroblasts (purchased from ScienCell Research Laboratories, Inc., product number 2320) are cultured in Eagle's minimum essential medium (Dulbecco's Modified Eagle medium, DMEM, Corning 10-013CV) containing 2% fetal bovine serum (FBS), and then replaced with fresh medium. The above-mentioned human dermal fibroblasts (HDF) are adherent cells.

[0132] Perform step (S11') of Experiment 1 to produce a pure platelet solution, and the platelet concentration in this pure platelet solution was 1×109 cells / mL. Next, the pure platelet solution was divided into a control group and a freeze-thaw 3-cycle group.

[0133] Control group: Take 1 mL of the pure platelet solution and let it stand for 20 - 30 minutes instead of performing the activation process in step (S12') of Experiment 1. Next, perform step (S13') of Experiment 1 again to produce a control group solution. Mix the control group solution and DMEM culture medium (containing 2% FBS) (the volume ratio of the control solution to the DMEM culture medium is 1:19) to produce a control group's platelet-derived exosome medium.

[0134] Three cycles of freezing and thawing group: Collect 1 mL of pure platelet solution and perform steps (S12’) to (S13’) of Experiment 1 to produce a solution of three cycles of freezing and thawing group (i.e., in this example, purified platelet-derived exosome solution). Mix the solution of three cycles of freezing and thawing group with DMEM medium (containing 2% FBS) (the volume ratio of the solution of three cycles of freezing and thawing group to DMEM medium is 1:19) to produce a platelet-derived exosome medium of three cycles of freezing and thawing group.

[0135] Divide a 96-well plate into untreated wells, LPS control group wells, control group exosome solution wells, and three cycles of freezing and thawing group exosome solution wells, and create two replicates for each well. Add human dermal fibroblasts (HDF) to the untreated wells, LPS control group wells, control group exosome solution wells, and three cycles of freezing and thawing group exosome solution wells so that the same number of cells are contained in each well, and culture in an incubator at 37°C and 5% carbon dioxide (CO2) for 24 hours.

[0136] Remove the used medium. Suspend the cells in the LPS control group wells, control group exosome solution wells, and three cycles of freezing and thawing group exosome solution wells in DMEM medium (containing 2% FBS) containing 5 μg / mL LPS, and suspend the cells in the untreated wells in DMEM culture solution (containing 2% FBS) without LPS. Culture in an incubator at 37°C and 5% carbon dioxide (CO2) for 1 hour. Next, perform the following steps for each group.

[0137] Untreated well: Remove the old culture medium (used medium). Suspend the cells in the untreated well (control group well) in DMEM culture medium (containing 2% FBS) and culture them in an incubator at 37°C and 5% carbon dioxide (CO2) for 24 hours.

[0138] LPS control group well: Remove the old culture medium (used medium). Suspend the cells in the LPS control group well in DMEM culture medium (containing 2% FBS) and culture them in an incubator at 37°C and 5% carbon dioxide (CO2) for 24 hours.

[0139] Control group exosome solution well: Remove the old culture medium (used medium). Suspend the cells in the control group exosome solution well in the control group exosome solution and culture them in an incubator at 37°C and 5% carbon dioxide (CO2) for 24 hours.

[0140] Freeze-thaw 3-cycle group exosome solution well: Remove the old culture medium (used medium). Suspend the cells in the freeze-thaw 3-cycle group exosome solution well in the freeze-thaw 3-cycle group exosome culture solution and culture them in an incubator at 37°C and 5% carbon dioxide (CO2) for 24 hours.

[0141] Transfer the cell culture medium from the untreated well, LPS control group well, control group exosome solution well, and freeze-thaw 3-cycle group exosome solution well to microcentrifuge tubes respectively. The concentration of human interleukin (Human IL-6) in the cell culture medium is measured using an interleukin (IL-6, inflammatory factor) ELISA kit (Human IL-6 ELISA Kit) (product number ab178013, manufacturer Abcam). Next, analyze using a multifunctional microplate spectrophotometer (model Varioskan LUX, manufactured by Thermo Scientific), and set the absorbance value at 450 nm. According to the product manual of the interleukin (IL-6) ELISA reagent kit, create a regression curve of the absorbance value and the concentration of inflammatory factor IL-6, and convert the absorbance value into the concentration of inflammatory factor IL-6.

[0142] The data shows that the exosome solution of the freeze-thaw 3-cycle group can suppress the inflammatory reaction of human fibroblasts, thereby alleviating the inflammation of skin tissue. The inventor expects that after inducing the inflammatory reaction of fibroblasts in other human tissues using a stimulant (such as LPS), the exosome solution of the freeze-thaw 3-cycle group can also suppress the induced inflammatory reaction, thereby alleviating the inflammation of other tissues.

[0143] Experiment 6-2: The effect of the platelet exosome composition obtained by the freeze-thaw activation process on cell migration Cell migration and proliferation are beneficial for tissue repair. Refer to Reference 1. This experiment is carried out using the same human dermal fibroblasts (HDF) as in Experiment 6-1. After culturing human dermal fibroblasts in Eagle's minimum essential medium (Dulbecco’s Modified Eagle medium, DMEM, Corning 10-013CV) containing 2% fetal bovine serum (FBS), replace them with fresh medium.

[0144] A 96-well plate containing a cell culture insert is divided into untreated wells, control group exosome solution wells, and freeze-thaw 3-cycle group exosome solution wells, and three replicates are created for each well. Human dermal fibroblasts (HDF) are added to the untreated wells, control group exosome solution wells, and freeze-thaw 3-cycle group exosome solution wells so that the same number of cells are contained in each, and they are cultured in an incubator at 37°C and 5% carbon dioxide (CO2) for 24 hours. The old medium and the cell culture insert of each group are removed, and photos and records are taken with a microscope. It is confirmed that the area occupied by the cell culture insert in the wells of each group is not covered by cells. Next, the following steps are performed for each group.

[0145] Untreated wells: Continue culturing in DMEM medium (containing 2% FBS) in an incubator at 37°C and 5% carbon dioxide (CO2) for 6 hours.

[0146] Control group exosome solution wells: Suspend the cells in the control group exosome solution wells with the control group exosome culture medium (control group’s platelet-derived exosome medium) described in Experiment 6-1, and culture in an incubator at 37°C and 5% carbon dioxide (CO2) for 6 hours.

[0147] Freeze-thaw 3-cycle group exosome solution wells: Similar to Experiment 6-1, suspend the cells in the 3-cycle group exosome solution wells with the 3-cycle group freeze-thaw exosome culture medium (three cycles of freezing and thawing group’s platelet-derived exosome medium), and continue culturing in an incubator at 37°C and 5% carbon dioxide (CO2) for 6 hours.

[0148] Take photos and records with a microscope, and calculate the cell migration area for each group. The data are presented as mean ± SD, and statistical analysis is performed using a t test. Groups marked with "***" show a statistical difference from the control group (p < 0.001).

[0149] The experimental results are shown in Fig. 6A. Fig. 6A is a bar graph showing the effect of the platelet exosome composition obtained by the freeze-thaw activation process in the present invention on the cell migration of human dermal fibroblasts.

[0150] The results in Fig. 6A show that the average cell migration area of the untreated group was 0.21 square millimeters; the average cell migration area of the control group exosome solution group was 0.46 square millimeters; the average cell migration area of the freeze-thaw 3-cycle group exosome solution group was 1.42 square millimeters, indicating that there was a significant difference in the cell migration area between the freeze-thaw 3-cycle group exosome solution group and the control group exosome solution group (p < 0.001). In the present invention, it can be seen that the platelet exosome composition obtained by the -80°C freeze-thaw activation process promotes the migration reaction of human dermal fibroblasts and thus promotes tissue repair. Furthermore, from the experimental results of the control group and the freeze-thaw 3-cycle group in Fig. 2, it can be seen that the cell migration reaction shown in Fig. 6A is caused by platelet exosomes rather than PDGF-BB.

[0151] Experiment 6-3: Effect of the platelet exosome composition obtained by the freeze-thaw activation method on cell proliferation The experiment was performed using the same human dermal fibroblasts (HDF) as in Experiment 6-1. After culturing human dermal fibroblasts in Eagle's minimum essential medium (Dulbecco's Modified Eagle medium, DMEM, Corning 10-013CV) containing 2% fetal bovine serum (FBS), the medium was replaced with fresh medium.

[0152] The 96-well plate is divided into untreated wells, control group exosome solution wells, and freeze-thaw 3-cycle group exosome solution wells, and three replicates are created for each well. Human dermal fibroblasts (HDF) are added to the untreated wells, control group exosome solution wells, and freeze-thaw 3-cycle group exosome solution wells so that the same number of cells are contained in each, and cultured in an incubator at 37 °C and 5% carbon dioxide (CO2) for 24 hours. Next, the following steps are performed for each group.

[0153] Untreated wells: Remove the old culture medium. Continue culturing in a DMEM culture medium (containing 2% FBS) in an incubator at 37 °C and 5% carbon dioxide (CO2) for 6 hours.

[0154] Control group exosome solution wells: Remove the old culture medium. Suspend the cells in the control group exosome solution wells with the control group exosome culture medium (control group’s platelet-derived exosome medium) described in Experiment 6-1, and continue culturing in an incubator at 37 °C and 5% carbon dioxide (CO2) for 6 hours.

[0155] Freeze-thaw 3-cycle group exosome solution wells: Similar to Experiment 6-1, suspend the cells in the 3-cycle group exosome solution wells with the 3-cycle group freeze-thaw exosome culture medium (three cycles of freezing and thawing group’s platelet-derived exosome medium), and continue culturing in an incubator at 37 °C and 5% carbon dioxide (CO2) for 6 hours.

[0156] Remove the old culture medium. Add 90 μL of DMEM culture medium and 10 μL of CCK-8 buffer (DOJINDO, product number CK04) to each well, and continue culturing in a 37 °C, 5% carbon dioxide (CO2) incubator for 4 hours. Analyze using a multifunctional microplate spectrophotometer (model Varioskan LUX, manufactured by Thermo Scientific), and set the absorbance value at 450 nm. According to the product manual of the CCK-8 buffer, create a regression curve of the absorbance value and the number of cells, and convert the absorbance value into the number of viable cells. The data are presented as mean ± SD, and statistical analysis is performed using a t-test. The group marked with "*" indicates a statistical difference from the control group exosome solution well (p < 0.05). The experimental results are shown in Figure 6B. Figure 6B is a bar graph of the effect of the platelet exosome composition obtained by the freeze-thaw activation process on the cell proliferation of human skin fibroblasts.

[0157] The results in Figure 6B show that the average number of cells in the untreated group increased to 17,010 cells per milliliter; the average number of cells in the control group exosome solution group increased to 20,425 cells per milliliter; the average number of cells in the freeze-thaw 3-cycle group exosome solution group increased to 24,836 cells per milliliter. There was a significant difference in the number of cells (p < 0.05) between the freeze-thaw 3-cycle group exosome solution group and the control group exosome solution group. In the present invention, it can be seen that the platelet exosome composition obtained by the -80 °C freeze-thaw activation process promotes the proliferation of human skin fibroblasts and thus promotes tissue repair.

[0158] Experiment 7: Effect of exosomes in different biological solutions on anti-inflammation Experiment 7-1: Analysis of exosome compositions in different biological solutions This experiment is divided into two groups, the pure platelet group and the platelet-rich plasma group, and the following steps are performed for each group.

[0159] Pure platelet group: Collect 1 mL of pure platelet solution and perform steps (S12’) to (S13’) of Experiment 1-1 to produce a pure platelet group exosome solution (i.e., purified platelet-derived exosome solution).

[0160] Platelet-rich plasma group: Collect 1 mL of the high-concentration platelet-rich plasma (PRP) described in Experiment 1. Next, directly perform steps (S12’) to (S13’) of Experiment 1 to produce a platelet-rich plasma group exosome solution.

[0161] Use a nanoparticle size analyzer (Tunable Resistive Pulse Sensing (TRPS), model Exoid, IZON Science brand) to analyze the concentration of exosomes in the solutions of each group. Next, use a fully automated exosome fluorescence quantitative analyzer (ExoView) (trade name Leprechaun) to individually analyze the biomarkers of exosomes in the pure platelet group exosome solution or the platelet-rich plasma group exosome solution. As a result of the experiment, it is shown that the exosomes in the pure platelet group exosome solution have exosome markers CD9, CD63, or CD81, and also have platelet markers CD41 and CD42b, but do not have at least one of the leukocyte marker CD45 and the erythrocyte exosome markers CD235ar and Annexin V. Therefore, it was found that platelet exosomes account for 100% of the pure platelet group exosomes. On the other hand, the exosomes in the platelet-rich plasma group exosome solution have at least one of the erythrocyte markers CD235ar and Annexin V, or the leukocyte marker CD45. As a result of the experiment, it was found that only 41% of the exosomes in the platelet-rich plasma group exosomes are platelet exosomes, and exosomes derived from other cells other than platelets account for 59%. For the experimental results, refer to Table 5 and Figure 7. Figure 7 is a bar graph showing exosome compositions in different biological solutions (platelets and plasma).

[0162]

Table 5

[0163] Experiment 7-2: Influence of exosomes in different biological solutions on alleviating inflammation Using an isotonic sodium chloride solution injection, adjust the concentration of the pure platelet group exosome solution prepared in Experiment 7-1 and the concentration of the platelet plasma group exosome solution prepared in Experiment 7-1 so that the exosome concentrations of both are the same (the total number of exosomes per unit volume is the same). Next, collect 1 milliliter (mL) each and produce pure platelet group exosome dry powder and platelet plasma group exosome dry powder according to Experiment 1 step (S14’).

[0164] The experiment was conducted using the same human dermal fibroblasts (HDF) as in Experiment 6-1. After culturing human dermal fibroblasts in Eagle's minimum essential medium (Dulbecco’s Modified Eagle medium, DMEM, Corning 10-013CV) containing 2% fetal bovine serum (FBS), change to a new medium.

[0165] The 96-well plates are divided into untreated wells, LPS control group wells, platelet plasma experimental group wells, and purified platelet experimental group wells, and two replicates are created for each well. Human dermal fibroblasts (HDF) are added to the untreated wells, LPS control group wells, platelet plasma experimental group wells, and purified platelet experimental group wells so that each contains the same number of cells, and they are cultured in an incubator at 37°C and 5% carbon dioxide (CO2) for 24 hours.

[0166] Remove the used medium. The cells in the LPS control group wells, platelet plasma experimental group wells, and purified platelet experimental group wells are suspended in DMEM medium (containing 2% FBS) with 5 μg / mL LPS, and the cells in the untreated wells are suspended in DMEM culture solution (containing 2% FBS) without LPS. Culture them in an incubator at 37°C and 5% carbon dioxide (CO2) for 1 hour. Next, perform the following steps for each group.

[0167] Untreated wells: Remove the used medium. Suspend the cells in the untreated wells in DMEM medium (containing 2% FBS) and culture them in an incubator at 37°C and 5% carbon dioxide (CO2) for 24 hours.

[0168] LPS control group wells: Remove the used medium. Suspend the cells in the LPS control group wells in DMEM medium (containing 2% FBS) and culture them in an incubator at 37°C and 5% carbon dioxide (CO2) for 24 hours.

[0169] Platelet Plasma Experimental Group Wells: Dissolve the platelet plasma group exosome dry powder in 1 mL of DMEM culture medium (containing 2% FBS) in reverse to produce the platelet plasma group exosome culture solution. Next, mix this with DMEM culture medium (containing 2% FBS) (the volume ratio of the platelet plasma group exosome culture solution to the DMEM culture medium is 1:19) to produce the platelet plasma experimental group culture solution. Remove the old culture medium (used medium). Suspend the cells in the platelet plasma experimental group wells in the platelet plasma experimental group culture solution and culture them in an incubator at 37°C and 5% carbon dioxide (CO2) for 24 hours.

[0170] Pure Platelet Experimental Group Wells: Dissolve the pure platelet group exosome dry powder in 1 mL of DMEM culture medium (containing 2% FBS) in reverse to produce the pure platelet group exosome culture solution. Next, mix this with DMEM culture medium (containing 2% FBS) (the volume ratio of the pure platelet group exosome culture solution to the DMEM culture medium is 1:19) to produce the pure platelet experimental group culture solution. Remove the old culture medium (used medium). Suspend the cells in the pure platelet experimental group wells in the pure platelet experimental group culture solution and culture them in an incubator at 37°C and 5% carbon dioxide (CO2) for 24 hours.

[0171] Transfer the cell culture solutions from the untreated wells (control group wells), LPS control group wells, platelet plasma experimental group wells, and pure platelet experimental group wells to microcentrifuge tubes respectively. The concentration of human interleukin (Human IL-6) in the cell culture solution is measured using an interleukin (IL-6, inflammatory factor) ELISA kit (Human IL-6 ELISA Kit) (product number ab178013, manufacturer Abcam). Next, analyze using a multifunctional microplate spectrophotometer (model Varioskan LUX, manufactured by Thermo Scientific) and set the absorbance value at 450 nm. According to the product manual of the interleukin (IL-6) ELISA reagent kit, create a regression curve of the absorbance value and the concentration of the inflammatory factor IL-6, and convert the absorbance value into the concentration of the inflammatory factor IL-6.

[0172] Data are presented as mean ± SD, and statistical analysis was performed using a t test. Groups marked with "##" showed a statistical difference from the platelet plasma experimental group (p<0.01), and groups marked with "*" showed a statistical difference from the LPS control group (p<0.05).

[0173] The experimental results are shown in Figure 8. Figure 8 is a bar graph showing the effect of exosomes in different biological solutions (platelets and plasma) on the anti-inflammatory effect of human skin fibroblasts.

[0174] Figure 8 shows that the average inflammatory factor release in the untreated group was 9 pg / mL; the average inflammatory factor release in the LPS control group was 396.7 pg / mL; the average inflammatory factor release in the platelet plasma group was 394.9 pg / mL; and the average inflammatory factor release in the pure platelet group was 261.7 pg / mL. There was a significant difference in the inflammatory factor release between the pure platelet experimental group and the LPS control group (p<0.05), and there was also a significant difference between the pure platelet experimental group and the platelet plasma experimental group (p<0.01). It can be seen that highly purified platelet exosomes (i.e., exosomes removed from cells other than platelets) effectively alleviate the pro-inflammatory response and bring unexpected effects.

[0175] Experiment 8: The effect of the freeze-drying process on the anti-inflammatory effect of the product.

[0176] According to the steps (S11’) to (S13’) of Experiment 1, a purified platelet exosome solution was prepared.

[0177] Prepare human dermal fibroblasts required for this experiment according to the experimental procedure of Experiment 6-1. A 96-well plate was divided into untreated wells, LPS control group wells, non-lyophilized group wells, and lyophilized group wells, and two replicates were created for each well. Human dermal fibroblasts (HDF) were added to the untreated wells, LPS control group wells, non-lyophilized group wells, and lyophilized group wells so that each contained the same number of cells, and cultured in an incubator at 37 °C and 5% carbon dioxide (CO2) for 24 hours.

[0178] Remove the old culture medium. The cells in the LPS control group wells, non-lyophilized group wells, and lyophilized group wells were suspended in DMEM medium (containing 2% FBS) containing 5 μg / mL LPS, and the cells in the untreated wells were suspended in DMEM culture solution (containing 2% FBS) without LPS. Cultured in an incubator at 37 °C and 5% carbon dioxide (CO2) for 1 hour. Next, perform the following steps for each group.

[0179] Untreated wells: Remove the old culture medium. Add DMEM medium (containing 2% FBS) to the untreated wells and culture in an incubator at 37 °C and 5% carbon dioxide (CO2) for 24 hours.

[0180] LPS control group wells: Remove the old culture medium. Add DMEM medium (containing 2% FBS) to the LPS control group wells and culture in an incubator at 37 °C and 5% carbon dioxide (CO2) for 24 hours.

[0181] Non-lyophilized group wells: Remove the old culture medium (used medium). Collect 1 mL of the purified platelet exosome solution, mix it with DMEM culture medium (containing 2% FBS) (the volume ratio of the purified platelet exosome solution to the DMEM culture medium is 1:19) to obtain a purified platelet exosome culture medium. Add this purified platelet exosome culture medium to the non-lyophilized group wells and culture them in an incubator at 37 °C and 5% carbon dioxide (CO2) for 24 hours.

[0182] Lyophilized group wells: Remove the old culture medium (used medium). Collect 1 mL of the purified platelet exosome solution and perform step (S14') of Experiment 1 to produce purified platelet-derived exosome dry powder. Reverse dissolve the purified platelet exosome dry powder in physiological saline injection solution to produce 1 mL of purified platelet-derived exosome dry powder solution. Collect 1 mL of the purified platelet exosome dry powder solution, mix it with DMEM culture medium (containing 2% FBS) (the volume ratio of the purified platelet exosome dry powder solution to the DMEM culture medium is 1:19) to obtain a purified platelet exosome dry powder culture medium. Add this purified platelet exosome dry powder culture medium to the lyophilized wells and culture them in an incubator at 37 °C and 5% carbon dioxide (CO2) for 24 hours.

[0183] Transfer the cell culture medium from the untreated well, LPS control group well, non-lyophilized group well, and lyophilized group well into microcentrifuge tubes respectively. The concentration of human interleukin (Human IL-6) in the cell culture medium is measured using an interleukin (IL-6, inflammatory factor) ELISA kit (Human IL-6 ELISA Kit) (product number ab178013, manufacturer Abcam). Next, analyze using a multifunctional microplate spectrophotometer (model Varioskan LUX, manufactured by Thermo Scientific), and set the absorbance value at 450 nm. According to the product manual of the interleukin (IL-6) ELISA reagent kit, create a regression curve of the absorbance value and the concentration of the inflammatory factor IL-6, and convert the absorbance value into the concentration of the inflammatory factor IL-6.

[0184] Data are presented as mean ± SD, and statistical analysis is performed using a t test. Groups marked with "*" or "**" indicate statistical differences from the LPS control group (p < 0.05 or p < 0.01); groups marked with "##" indicate statistical differences from the non-lyophilized group (p < 0.01).

[0185] Refer to Table 6 and Figure 9 for the experimental results. Figure 9 is a bar graph showing the effect of the lyophilization process on the anti-inflammatory effect of the product.

[0186]

Table 6

[0187] The experimental results in Figure 9 showed that the average inflammatory factor release in the untreated group was 9 pg / mL; the average inflammatory factor release in the LPS control group was 396.7 pg / mL; the average inflammatory factor release in the non-lyophilized group was 261.7 pg / mL; and the average inflammatory factor release in the lyophilized group was 69.2 pg / mL. Compared with the LPS control group, the inflammatory factor release in the non-lyophilized group and the lyophilized group decreased significantly (p < 0.05 or p < 0.01). On the other hand, compared with the inflammatory factor release in the non-lyophilized group (261.7 pg / mL), the inflammatory factor release in the lyophilized group was significantly decreased to 69.2 pg / mL (decreased from 100% to 26%; calculation formula: 69.2 ÷ 261.7 × 100% = 26%) (p < 0.01). From the above experiments, it can be seen that the lyophilization step of the present invention is an important technical feature and brings unexpected effects.

[0188] After the inventors induced an inflammatory response in other human tissues with a stimulant (e.g., LPS), they predicted that the platelet exosome dry powder obtained from the lyophilization step of the present invention could also suppress the induced inflammatory response and thereby relieve the inflammation of other human tissues.

[0189] Experiment 9: Comparison of Platelet Exosomes and Mesenchymal Stem Cell Exosomes on Tissue Repair Experiment 9-1: Comparison of Platelet Exosomes and Mesenchymal Stem Cell Exosomes on Cell Migration The supernatant of mesenchymal stem cells was purified using a tangential flow filtration (TFF) system to obtain a mesenchymal stromal / stem cell (MSC)-derived exosome solution. Next, quantitative analysis of nanoparticles was performed using a nanoparticle size analyzer, and the exosome concentration was adjusted to 10 per milliliter by injecting isotonic sodium chloride solution. 7It was adjusted to a mesenchymal stromal / stem cell, MSC-derived exosome solution containing 7 exosomes. The purified platelet exosome solution produced in steps (S11') to (S13') of Experiment 1 was collected, and quantitative analysis of the nanoparticles was performed using a nanoparticle size analyzer. Isotonic Sodium Chloride Solution Injection was injected, and the exosome concentration was adjusted to 10

[0190] The experiment was conducted using the same human dermal fibroblast (HDF) as in Experiment 6-1. Human dermal fibroblasts were cultured in Eagle's minimum essential medium (Dulbecco’s Modified Eagle medium, DMEM, Corning 10-013CV) containing 2% fetal bovine serum (FBS), and then the medium was replaced with fresh medium.

[0191] A 96-well plate containing cell culture inserts was divided into untreated wells, wells of the exosome solution for the freeze-thaw 3-cycle group, and wells of the mesenchymal stromal / stem cell exosome solution, and three replicates were created for each well. Human dermal fibroblasts (HDF) were added to the untreated wells, the wells of the exosome solution for the freeze-thaw 3-cycle group, and the wells of the mesenchymal stromal / stem cell exosome solution so that each well contained the same number of cells, and they were cultured in an incubator at 37°C and 5% carbon dioxide (CO2) for 24 hours. The old medium and the cell culture inserts of each group were taken out, and photos and records were taken with a microscope. Next, the following steps were performed for each group.

[0192] Untreated wells: Add DMEM culture medium (containing 2% FBS) and continue culturing in an incubator at 37°C and 5% carbon dioxide (CO2) for 6 hours.

[0193] Freeze - thaw 3 - cycle group exosome solution well: Mix the purified platelet - derived exosome solution and DMEM culture medium (containing 2% FBS) (volume ratio 1:19) to produce a purified platelet - derived exosome medium. Next, add the purified platelet - derived exosome medium to the freeze - thaw 3 - cycle group exosome solution well and continue culturing in an incubator at 37°C and 5% carbon dioxide (CO2) for 6 hours.

[0194] Mesenchymal stem cell exosome solution well: Mix the mesenchymal stem cell exosome solution and DMEM culture medium (containing 2% FBS) (volume ratio 1:19) to produce a mesenchymal stromal / stem cell - derived exosome medium (MSC - derived exosome medium). Next, add the mesenchymal stem cell exosome medium to the freeze - thaw 3 - cycle group exosome solution well and continue culturing in an incubator at 37°C and 5% carbon dioxide (CO2) for 6 hours.

[0195] Take photos and records with a microscope, and calculate the cell migration area for each group.

[0196] Data are presented as mean ± SD, and statistical analysis is performed using a t - test. The groups marked with "*" show a statistical difference (p < 0.05) from the mesenchymal stem cell exosome solution well. The experimental results are shown in Figure 10A. Figure 10A is a bar graph showing the effects of platelet exosomes and mesenchymal stem cell exosomes on the cell migration of human dermal fibroblasts.

[0197] From Figure 10A, it was found that the average cell migration range area of the untreated group was 0.21 square millimeters, the average cell migration range area of the exosome solution group in the freeze-thaw 3-cycle group was 0.49 square millimeters, and the average cell migration range area of the mesenchymal stem cell exosome solution group was 0.34 square millimeters. There was a significant difference (p < 0.05) in the number of cells between the exosome solution group in the freeze-thaw 3-cycle group and the mesenchymal stem cell exosome solution group. In the present invention, it can be seen that the platelet exosome composition has a better cell migration reaction than the mesenchymal stem cell exosome, and thus promotes tissue repair.

[0198] Experiment 9-2: Effects of platelet exosomes and mesenchymal stem cell exosomes on cell proliferation Manufacture a mesenchymal stromal / stem cell, MSC-derived exosome solution and a purified platelet exosome solution by the method described in Experiment 9-1, and adjust the exosome concentration in the mesenchymal stromal / stem cell, MSC-derived exosome solution to 10 6 per mL; adjust the exosome concentration in the purified platelet exosome solution to 10 6 per mL.

[0199] The experiment was conducted using the same human dermal fibroblasts (HDF) as in Experiment 9-1. A 96-well plate was divided into untreated wells, wells with exosome solution of the freeze-thaw 3-cycle group, and wells with mesenchymal stem cell exosome solution, and three replicates were created for each well. Human dermal fibroblasts (HDF) were added to the untreated wells, wells with exosome solution of the freeze-thaw 3-cycle group, and wells with mesenchymal stem cell exosome solution so that each well contained the same number of cells, and cultured in an incubator at 37°C and 5% carbon dioxide (CO2) for 24 hours. Next, the following steps are performed for each group.

[0200] Untreated wells: Remove the old culture medium. Add DMEM medium (containing 2% FBS) to the untreated wells and continue culturing in an incubator at 37°C and 5% carbon dioxide (CO2) for 6 hours.

[0201] Wells with exosome solution of the freeze-thaw 3-cycle group: Remove the old culture medium. Mix the purified platelet exosome solution with DMEM culture medium (containing 2% FBS) at a volume ratio of 1:19 to produce purified platelet-derived exosome medium. Next, add the purified platelet-derived exosome culture medium to the wells with exosome solution of the freeze-thaw 3-cycle group and continue culturing in an incubator at 37°C and 5% carbon dioxide (CO2) for 6 hours. platelet derived exosome medium) was added, and the culture was continued in an incubator at 37°C and 5% carbon dioxide (CO2) for 6 hours.

[0202] Mesenchymal stem cell exosome solution well: Remove the old culture medium. Mix the mesenchymal stem cell exosome solution with DMEM culture medium (containing 2% FBS) at a volume ratio of 1:19 to produce a mesenchymal stem cell exosome culture medium (Mesenchymal stromal / stem cell, MSC-derived exosome medium). Next, add the mesenchymal stem cell exosome culture medium (Mesenchymal stromal / stem cell, MSC-derived exosome medium) to the mesenchymal stem cell exosome solution well and continue culturing in an incubator at 37 °C and 5% carbon dioxide (CO2) for 6 hours.

[0203] Remove the old culture medium. Add 90 μL of DMEM culture medium and 10 μL of CCK-8 buffer (DOJINDO, product number CK04) to each well and continue culturing in an incubator at 37 °C and 5% carbon dioxide (CO2) for 4 hours. Analyze using a multifunctional microplate spectrophotometer (model Varioskan LUX, manufactured by Thermo Scientific), and set the absorbance value at 450 nm. According to the product manual of the CCK-8 buffer, create a regression curve of the absorbance value and the number of cells, and convert the absorbance value into the number of viable cells. The experimental results are shown in Figure 10B. Figure 10B is a bar graph showing the effects of platelet exosomes and mesenchymal stem cell exosomes on the cell proliferation of human dermal fibroblasts.

[0204] From Figure 10B, it was found that the average cell amount of the untreated group was 17,010, the average cell amount of the exosome solution group of the freeze-thaw 3-cycle group was 20,157, and the average cell amount of the mesenchymal stem cell exosome solution group was 17,972. That is, the average cell amount of the exosome solution group of the freeze-thaw 3-cycle group was more than that of the mesenchymal stem cell exosome solution group. Therefore, it can be seen that the platelet exosome composition of the present invention has the effect of promoting tissue repair, and its effect is superior to that of mesenchymal stem cell exosomes.

[0205] Experiment 9-3: Effects of Platelet Exosomes and Mesenchymal Stem Cell Exosomes on Collagen Expression Level A mesenchymal stromal / stem cell (MSC)-derived exosome solution and a purified platelet exosome solution were prepared by the method described in Experiment 9-1.

[0206] The experiment was conducted using the same human dermal fibroblast (HDF) as in Experiment 9-1. A 96-well plate was divided into untreated wells, exosome solution wells of the freeze-thaw 3-cycle group, and mesenchymal stem cell exosome solution wells, and three replicates were created for each well. Human dermal fibroblasts (HDF) were added to the untreated wells, exosome solution wells of the freeze-thaw 3-cycle group, and mesenchymal stem cell exosome solution wells so that the same number of cells were contained in each, and cultured in an incubator at 37°C and 5% carbon dioxide (CO2) for 24 hours. Next, the following steps were performed for each group.

[0207] Untreated wells: Remove the old culture medium. Add DMEM medium (containing 2% FBS) to the untreated wells and culture in an incubator at 37°C and 5% carbon dioxide (CO2) for 24 hours.

[0208] Exosome solution wells of the freeze-thaw 3-cycle group: Remove the old culture medium. Mix the purified platelet exosome solution with DMEM culture medium (containing 2% FBS) (volume ratio 1:19) to prepare a purified platelet-derived exosome medium. Next, add the purified platelet-derived exosome culture medium to the exosome solution wells of the freeze-thaw 3-cycle group (purified Add platelet-derived exosome medium and culture for 24 hours in an incubator at 37°C with 5% carbon dioxide (CO2).

[0209] Mesenchymal stem cell exosome solution well: Remove the old culture medium. Mix the mesenchymal stem cell exosome solution with DMEM culture medium (containing 2% FBS) at a volume ratio of 1:19 to prepare mesenchymal stromal / stem cell, MSC-derived exosome medium. Next, add mesenchymal stromal / stem cell, MSC-derived exosome medium to the mesenchymal stem cell exosome solution well and culture for 24 hours in an incubator at 37°C with 5% carbon dioxide (CO2).

[0210] Use enzyme-linked immunosorbent assay (ELISA) to analyze the collagen content in the solutions of each group. The data are presented as mean ± SD, and the statistical analysis is performed using a t test. The groups marked with "**" show a statistical difference (p < 0.01) compared to the mesenchymal stem cell exosome solution. The experimental results are shown in Figure 10C. Figure 10C is a bar graph showing the effects of platelet exosomes and mesenchymal stem cell exosomes on the expression level of collagen in human skin fibroblasts.

[0211] From Figure 10C, it was found that the average collagen expression level in the untreated group was 22,617.67 pg / mL, the average collagen expression level in the exosome solution group of the freeze-thaw 3-cycle group was 43,311.33 pg / mL, and the average collagen expression level in the mesenchymal stem cell exosome solution group was 38,317.67 pg / mL. That is, the average collagen expression level in the exosome solution group of the freeze-thaw 3-cycle group was higher than that in the mesenchymal stem cell exosome solution group, and there was a significant difference (p<0.01). Therefore, it can be seen that the platelet exosome composition of the present invention has the effect of promoting tissue repair, and its effect is superior to that of mesenchymal stem cell exosomes.

[0212] The above is only a preferred embodiment of the present invention and does not limit the patent scope of the present invention. Therefore, all other changes or modifications that do not deviate from the spirit disclosed by the present invention should be included in the patent scope of this case.

[0213] References

[0214]

Table 7

Claims

1. A method for producing purified platelet-derived exosome dry powder, comprising in the production process: (a) taking one unit of a blood-derived solution containing platelets and performing a process purifying platelets to obtain a pure platelet solution; wherein step (a) comprises: (a1) taking one unit of a blood-derived solution containing platelets; (a2) performing a first sedimentation process to separate the blood-derived solution containing platelets into a plasma layer and a blood cell layer; (a3) removing the blood cell layer to obtain a plasma layer solution; (a4) performing a second sedimentation process to sediment platelets to form a pellet; (a5) removing the supernatant and mixing the pellet with a solvent to obtain the pure platelet solution; (b) performing an activation process to activate the platelets in the pure platelet solution to release exosomes and obtain an activated pure platelet solution; wherein the activation process is a process of freezing and then thawing; (c) performing a purification process to obtain a purified platelet-derived exosome solution; and (d) performing a drying process on the purified platelet-derived exosome solution to obtain a purified platelet-derived exosome dry powder containing a plurality of platelet exosomes.

2. The method according to claim 1, wherein the drying process is a freeze-drying process.

3. In step (a3), the pure platelet solution per 1 milliliter (mL) The method according to claim 1, further comprising a process of removing white blood cells so that the number of white blood cells in the 6 solution) is less than 10.

4. Step (a5) is a process of removing plasma so that the albumin concentration in the pure platelet solution is less than 3 g / dL; or step (a5) is a process of removing plasma so that the globulin concentration in the pure platelet solution is less than 2 g / dL; or step (a5) further includes a process of removing plasma so that the fibrinogen concentration in the pure platelet solution is less than 100 μg / mL. The method according to claim 1.

5. The solution containing platelets derived from the blood is whole blood, and the blood cell layer includes a Buffy coat layer and a Red blood cells layer. The method according to claim 1. coat layer) and a Red blood cells layer. The method according to claim 1.

6. The process of freezing and then thawing (hereinafter referred to as the freeze-thaw process) after freezing includes (b1’) Placing the container containing the pure platelet solution in an environment of -70°C to -196°C to freeze the pure platelet solution; and (b2') Thawing the frozen pure platelet solution by raising the temperature of the container; And steps (b1’) and (b2’) are performed once, twice, three times, four times, five times, or more than five times. The method according to claim 1.

7. The process of freezing and then thawing (hereinafter referred to as the freeze-thaw procedure) after freezing includes (b1) Placing a container containing the pure platelet solution in liquid nitrogen (-196 °C) to freeze the pure platelet solution; and (b2) thawing the frozen pure platelet solution by raising the temperature of the container; and the method according to claim 1, wherein steps (b1) and (b2) are carried out once, twice, three times, four times, five times, or more than five times.

8. The process of freezing and then thawing (hereinafter referred to as the freeze-thaw procedure) includes: (b1’) Placing a container containing the pure platelet solution in an environment at -80 °C to freeze the pure platelet solution; and (b2') thawing the frozen pure platelet solution by raising the temperature of the container; and the method according to claim 1, wherein steps (b1’) and (b2’) are carried out once, twice, three times, four times, five times, or more than five times.

9. The freeze-drying process according to claim 2 includes adjusting the temperature to -35 °C or lower and adjusting the pressure to 80 mTorr or lower.

10. The solution derived from blood and containing platelets is whole blood, apheresis platelets, leukocytes-reduced platelets apheresis, Platelet-Rich Plasma (PRP), or any combination thereof; or the solution derived from blood and containing platelets is derived from an autologous blood sample, an allogeneic blood sample, a mammalian blood sample, or a combination thereof. The method according to claim 1.

11.

12. In step (a5), the solvent is sterile physiological saline, water for injection, 0.45% NaCl, 4.5% hypertonic saline, sterile hot spring water, or isotonic saline; or, in step (a5), the platelet concentration in the pure platelet solution is 1×10 9 cells / mL to 10×10 9 cells / mL. The method according to claim 1. ​ The purification process includes: (c1) performing a centrifugation process to remove most platelet-associated structures; and (c2) performing a membrane filtration process to completely remove platelet-associated structures. The membrane filtration process in step (c2) uses membrane filtration with a pore size of 0.45 μm or less to obtain a solution rich in purified platelet exosomes. The method according to claim 1.

13. A purified platelet-derived exosome dry powder, which is produced by a manufacturing method including the following steps. (a) Taking one unit of a solution derived from blood and containing platelets, and performing a process of purifying platelets to obtain a pure platelet solution; among which, step (a) includes (a1) Taking one unit of a solution derived from blood and containing platelets; (a2) Performing a first sedimentation process to separate the solution derived from blood and containing platelets into a plasma layer and a blood cell layer; (a3) Removing the blood cell layer to obtain a plasma layer solution; (a4) Performing a second sedimentation process to sediment platelets and form a pellet; (a5) Removing the supernatant, and mixing the pellet with a solvent to obtain a pure platelet solution; (b) Performing an activation process to activate the platelets in the pure platelet solution and release exosomes to obtain an activated pure platelet solution; here, the activation process is a freezing-thaw process; (c) Performing a purification process to obtain a purified platelet-derived exosome solution; and (d) Performing a drying process on the purified platelet-derived exosome solution to obtain a purified platelet-derived exosome dry powder containing a plurality of platelet exosomes.

14. The purified platelet-derived exosome dry powder according to claim 13, wherein the drying process is a freeze-drying process.

15. The process of freezing and then thawing (i.e., the freeze-thaw procedure) after freezing includes: (b1’) Placing the container containing the pure platelet solution in an environment of -80°C to freeze the pure platelet solution; and (b2’) Thawing the frozen pure platelet solution by raising the temperature of the container; and the steps (b1’) and (b2’) are performed once, twice, three times, four times, five times, or more than five times. The purified platelet-derived exosome dry powder according to claim 13.

16. In the purified platelet-derived exosome dry powder, the particle size of the platelet exosomes is 20 to 150 nm, and the platelet exosomes contain exosome markers and platelet markers; the exosome markers are at least one of CD9, CD63, and CD81, or any combination thereof, and the platelet markers are at least one of CD41 and CD42b, or a combination thereof; where the purified platelet-derived exosome dry powder according to any one of claims 13 to 15. (1) The purified platelet exosome dry powder per 1 gram (g) contains at least 1×10 10 exosomes; or, (2) Purified platelet exosome dry powder per 1 gram (g) contains 1×10 11 ~1×10 15 platelet exosomes

17. (1) The purified platelet-derived exosome dry powder per gram (g) contains at least 50 μg of microRNA; or ​ (2) The content of microRNA in the purified platelet-derived exosome dry powder per gram (g) is 100 μg to 2,500 μg. The purified platelet-derived exosome dry powder according to any one of claims 13 to 15.

18. (1) The content of PDGF-BB in the purified platelet-derived exosome powder per gram (g) is 0.01 ng to 2,000 ng; or, (2) The content of VEGF in the purified platelet-derived exosome powder per gram (g) is 50 pg to 100,000 pg; or, (3) The content of IGF in the purified platelet-derived exosome powder per gram (g) is 1 pg to 3,000 pg; or, (4) The content of TGF-β1 in the purified platelet-derived exosome powder per gram (g) is 50 ng to 20,000 ng; or, (5) The content of EGF in the purified platelet-derived exosome powder per gram (g) is 0.1 ng to 200 ng. The purified platelet-derived exosome dry powder according to any one of claims 13 to 15.

19. (1) In the purified platelet-derived exosome dry powder per gram (g), leukocyte exosomes are not detected; or, (2) The number of leukocyte exosomes in the purified platelet exosome dry powder per 1 gram (g) is 1 × 10 14 or less; Here, the leukocyte exosomes contain leukocyte markers; the leukocyte marker is CD45. The purified platelet-derived exosome dry powder according to any one of claims 13 to 15.

20. (1) In the purified platelet-derived exosome dry powder per gram (g), erythrocyte exosomes are not detected; or, (2) The number of red blood cell exosomes in the purified platelet exosome dry powder per 1 gram (g) is 1 × 10 14 or less; Here, the erythrocyte exosomes contain erythrocyte markers; the erythrocyte marker is at least one of CD235ar and Annexin V, or a combination thereof. The purified platelet exosome dry powder according to any one of claims 13 to 15.

21. (1) In the purified platelet exosome dry powder per gram (g), exosomes other than platelet exosomes are not detected; or, (2) The number of exosomes other than platelets in the purified platelet exosome dry powder per 1 gram (g) is 1 × 10 14 or less, The purified platelet exosome dry powder according to any one of claims 13 to 15.

22. Use of the purified dry platelet exosome powder according to any one of claims 13 to 15 for preparing a pharmaceutical composition or a health composition for administering to a site of an individual in an effective amount to relieve the degree of inflammation or injury of the site of the individual.

23. Use according to claim 22 for preparing a pharmaceutical composition or a health composition for relieving the degree of inflammation or injury in the airway site, skin site, muscle site, tendon site, bone site, joint site, or ligament site of the individual.

24. Use of the purified platelet exosome dry powder according to any one of claims 13 to 15 for manufacturing a pharmaceutical composition or a health composition for enhancing the migration ability of skin fibroblasts to the skin site.

25. Use according to claim 24, wherein the skin site is a skin lesion site.

26. Use of the purified platelet exosome dry powder according to any one of claims 13 to 15 for manufacturing a pharmaceutical composition or a health composition for increasing the expression level of collagen in skin fibroblasts.

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