One kind of use of purified exosome dry powder for relieving inflammation or injury

Abstract

To provide purified platelet-derived exosome dry powder and a method for manufacturing the purified platelet-derived exosome dry powder that make it possible to effectively relieve or treat inflammation or injury.SOLUTION: Manufacturing steps in the method comprise: (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; (b) performing an activation process to activate platelets in the pure platelet solution and release exosomes to obtain an activated pure platelet solution, where the activation process is a co-treatment process with collagen and thrombin; (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 purified platelet-derived exosome dry powder containing a plurality of platelet-derived exosomes.SELECTED DRAWING: Figure 2

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, body parts cause an inflammatory response due to infection, trauma, or hypersensitivity, remove harmful stimuli, remove 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 (abbreviation 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 injury (usually accompanied by an inflammatory reaction), a series of wound repair reactions 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 to produce fibrogenesis at the injury site. Furthermore, fibroblasts are the main source of extracellular matrix proteins (ECM), mainly including collagen and fibronectin, form granulation tissue, and provide structural integrity to the wound.

[0004] Exosomes are nano-scale 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 communication and transmit different information between cells by transporting 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, and gastric acid (Reference 3), and plasma and serum contain exosomes 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 shown 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 technical 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 relieve or treat inflammation and injury.

[0008] Another object of the present invention is to provide a method for producing 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 relieving 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 relieving or treating inflammation and injury.

[0011] The present invention provides a method for manufacturing purified platelet-derived exosome dry powder, which includes: (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) 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 to release exosomes and obtain an activated pure platelet solution; here, the activation process refers to a co-treatment process of collagen and thrombin; (c) performing a purification process: (c1) a process of performing a centrifugation process to remove most of the platelet-associated structures, that is, a process of removing the pellet obtained after centrifugation;and (c2) a process of performing a membrane filtration process to completely remove platelet-associated structure pellets 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 such that the albumin concentration 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 such that the globulin concentration 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 fibrinogen concentration 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 includes a buffy coat layer and a red blood cell layer.

[0019] In some embodiments, the activation process includes (b1') mixing collagen and thrombin with the pure platelet solution, and (b2') vortexing at room temperature for 30 minutes.

[0020] In some embodiments, the activation process includes the process of (b1’) mixing collagen and thrombin with a pure platelet solution to obtain a mixed solution, wherein the collagen concentration in the mixed solution is 5-20 μg / mL and the thrombin concentration in the mixed solution is 0.1-2 U / mL; and (b2’) shaking at room temperature for 30 minutes.

[0021] In some embodiments, the activation process includes the process of (b1’) mixing collagen and thrombin with a pure platelet solution to obtain a mixed solution, wherein the collagen concentration in the mixed solution is 7-12 μg / mL and the thrombin concentration in the mixed solution is 0.5-1.5 U / mL; and (b2’) shaking at room temperature for 30 minutes.

[0022] In some embodiments, the activation process includes the process of (b1’) mixing collagen and thrombin with a pure platelet solution to obtain a mixed solution, wherein the collagen concentration in the mixed solution is 8, 9, 10, or 11 μg / mL and the thrombin concentration in the mixed solution is 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, or 1.4 U / mL; and (b2’) shaking at room temperature for 30 minutes.

[0023] In some embodiments, the lyophilization process includes adjusting the temperature to -35°C or lower and adjusting the pressure to 80 mTorr or lower.

[0024] 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.

[0025] In some embodiments, the lyophilization process includes adjusting the pressure to 70, 60, 50, 40, or 30 mTorr or less.

[0026] 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.

[0027] In some embodiments, the solvent in step (a5) is sterile saline, water for injection, 0.45% NaCl, 4.5% hypertonic saline, sterile hot spring water, or isotonic saline; alternatively, the platelet concentration in the pure platelet solution in step (a5) is 1×10 9 cells / mL to 10×10 9 cells / mL.

[0028] 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.

[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 0.45 μm or less.

[0030] 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.

[0031] The present invention further provides a method for manufacturing purified platelet-derived exosome dry powder, which is manufactured by a manufacturing method including: taking one unit of a blood-derived solution containing platelets and performing a process of purifying platelets to obtain a pure platelet solution; wherein 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 to release exosomes and obtain an activated pure platelet solution; here, the activation process is a co-treatment process of collagen and thrombin; (c) performing a purification process: (c1) a 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 and 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.;

[0032] In some embodiments, the drying process is a freeze-drying process.

[0033] In some embodiments, the activation process includes: (b1') a process of mixing Collagen and Thrombin with a pure platelet solution to obtain a mixed solution, wherein the Collagen concentration in the mixed solution is 5-20 μg / mL (for example, 10 μg / mL), and the Thrombin concentration in the mixed solution is 0.1-2 U / mL (for example, 1 U / mL); and (b2') a process of vortexing at room temperature for 30 minutes.

[0034] In some embodiments, the activation process includes: (b1’) a process of mixing Collagen and Thrombin with a pure platelet solution to obtain a mixed solution, where the Collagen concentration in the mixed solution is 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 μg / mL, and the Thrombin concentration in the mixed solution is 2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, or 1.9 U / mL; and (b2’) a process of vortexing at room temperature for 30 minutes.

[0035] 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 1 gram (g) of the purified platelet exosome dry powder, at least 1×10 10 platelet exosomes are contained; or, (2) in 1 gram (g) of the purified platelet exosome dry powder, 1×10 11 - 1×10 15 platelet exosomes are contained; or, (3) in 1 gram (g) of the purified platelet exosome dry powder, 2×10 11 , 3×10 11 , 4×10 11 , 5×10 11 , 6×10 11 , 7×10 11 , 8×10 11 , 9×10 11 , 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 , 8×10 13 , 8.5×10 13 , 9×10 13 , 9.5×10 13 , 1×10 14 , 2×10 14 , 3×10 14 , 4×10 14 , 5×10 14 , 6×10 14 , 7×10 14 , 8×10 14 , or 9×10 14 contains the individual platelet exosomes.

[0036] In some embodiments, (1) in the purified platelet exosome dry powder per gram (g), it 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 is.

[0037] In some embodiments, (1) the content of PDGF-BB (platelet-derived growth factor-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 (vascular endothelial growth factor) in 1 gram (g) of the purified platelet-derived exosome powder is 50 pg to 100,000 pg; or, (3) the content of IGF (insulin-like growth factor) in 1 gram (g) of the purified platelet-derived exosome powder is 1 pg to 3,000 pg; or, (4) the content of TGF-β1 (transforming growth factor beta 1) in 1 gram (g) of the purified platelet-derived exosome powder is 50 ng to 20,000 ng; or, (5) the content of EGF (epidermal growth factor) in 1 gram (g) of the purified platelet-derived exosome powder is 0.1 ng to 200 ng.

[0038] In some embodiments, the content of PDGF-BB in 1 gram (g) of the 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 3 ng.

[0039] 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 , 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.

[0040] 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.

[0041] 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.

[0042] In some embodiments, the content of EGF in 1 gram (g) of growth factor enriched dry powder is 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, or 200 ng.

[0043] 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 leukocyte markers; the leukocyte marker is CD45.

[0044] 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 erythrocyte markers; the erythrocyte marker is at least one of CD235ar and Annexin V, or a combination thereof.

[0045] 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.

[0046] 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 at an effective amount at a site of the individual.

[0047] 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.

[0048] 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.

[0049] In some embodiments, the skin site is a skin lesion site.

[0050] The present 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.

[0051] The present invention further provides a method for manufacturing purified platelet-derived exosome dry powder, (a) It is manufactured by a manufacturing method including a process of taking one unit of a blood-derived solution containing platelets and performing a process of purifying platelets to obtain a pure platelet solution; among them, step (a) is (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 a 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 precipitate with a solvent to obtain a pure platelet solution; (b) A process of performing an activation process to activate platelets in a pure platelet solution, release exosomes, and obtain an activated pure platelet solution; here, the activation process is a co-treatment process of collagen and adenosine diphosphate (ADP); (c) A process of performing a purification process to obtain a purified platelet-derived exosome solution; and (d) A process of performing a drying process on a purified platelet-derived exosome solution to obtain a purified platelet-derived exosome dry powder containing a plurality of platelet exosomes.

[0052] In some embodiments, the activation process includes: (b1’) A process of mixing collagen and adenosine diphosphate with a pure platelet solution to obtain a mixed solution, wherein the collagen concentration in the mixed solution is 5 - 20 μg / mL and the adenosine diphosphate concentration in the mixed solution is 40 - 80 μM; and (b2’) A process of vortexing at room temperature for 30 minutes.

[0053] In some embodiments, the activation process includes: (b1’) A process of mixing collagen and adenosine diphosphate with a pure platelet solution to obtain a mixed solution, wherein the collagen concentration in the mixed solution is 7 - 12 μg / mL and the adenosine diphosphate concentration in the mixed solution is 50 - 80 μM; and (b2’) A process of vortexing at room temperature for 30 minutes.

[0054] In some embodiments, the activation process includes: (b1’) A process of mixing collagen and adenosine diphosphate with a pure platelet solution to obtain a mixed solution, wherein the collagen concentration in the mixed solution is 8, 9, 10, or 11 μg / mL and the adenosine diphosphate concentration in the mixed solution is 40, 45, 50, 55, 60, 65, 70, or 75 μM; and (b2’) A process of vortexing at room temperature for 30 minutes.

Brief Description of the Drawings

[0055]

Figure 1

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DETAILED DESCRIPTION OF THE INVENTION

[0056] The following details the embodiments of the present invention, as well as the technology 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.

[0057] 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 per gram of the dry powder 10exosomes, and 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% or more are platelet exosomes.

[0058] In the present invention, the “purified platelet-derived exosome” dry powder has fewer other exosomes than its source composition, where “other exosomes” refers to exosomes other than platelet-derived exosomes (“Other exosomes” refer to exosomes other than platelet-derived exosomes).

[0059] In the present invention, the purified platelet-derived exosome solution means a solution rich in high-purity platelet-derived exosomes (pure platelet-derived exosome).

[0060] In the present invention, the “purified platelet-derived exosome” solution has fewer other exosomes than its source solution, where “other exosomes” refers to exosomes other than platelet-derived exosomes (“Other exosomes” refer to exosomes other than platelet-derived exosomes).

[0061] In the present invention, a 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 containing 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.

[0062] In the present invention, a pure platelet solution refers to a platelet solution of higher purity obtained by purifying a solution containing platelets.

[0063] In the present invention, a platelet-associated structure refers to platelets, platelet fragments, platelet structures formed by platelet aggregation, or any combination thereof.

[0064] In the present invention, a sedimentation process refers to a process of sedimenting and accumulating substances. For example, it is a process of sedimenting and accumulating substances using gravity, centrifugal force, or electromagnetic force. In the present invention, a "health composition" or "health supplement" refers to a nutritional supplement, 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.

[0065] Referring to FIG. 1, the process of manufacturing purified platelet-derived exosome dry powder includes the following steps: (S11) Take one 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) Execute a process to remove 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 a plurality of platelet exosomes.

[0066] 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.

[0067] In some embodiments, step (S11) includes sub-steps: (S111) Take one unit of the solution containing platelets; and (S112) Execute at least one sedimentation process to separate the blood-derived solution containing platelets into a plurality of layers, where the plurality of layers includes 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 106 Cause it to be less. 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.

[0068] In some embodiments, step (S112) includes sub-steps: (S1121) Perform a first 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 second 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.

[0069] 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 / mL to 10×10 9 / mL.

[0070] In some embodiments, in step (S12), the activation process is a co-treatment process of collagen and thrombin, and step (S12) further includes sub-steps: (S121) Mix collagen and thrombin with the pure platelet solution to obtain a mixed solution, wherein the collagen concentration in the mixed solution is 7-12 μg / mL and the thrombin concentration in the mixed solution is 0.5-1.5 U / mL; and (S122) Vortex at room temperature for 30 minutes to activate the platelets in the pure platelet solution, release exosomes, and obtain an activated pure platelet solution.

[0071] In some embodiments, step (S13) further includes sub-steps: (S131) Perform a centrifugation process to remove most of the platelet-associated structures, that is, remove the pellet obtained after centrifugation; and (S132) Perform a filter membranes filtration process to completely remove platelet associate structures and obtain a purified platelet-derived exosome solution.

[0072] In some embodiments, in step (S14), the drying process is a freeze-drying process. In some embodiments, the freeze-drying 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 freeze-drying process of step (S14) includes adjusting the temperature to -35 °C or lower and adjusting the pressure to 80 mTorr or lower.

[0073] Experiment 1: Production and component analysis of purified platelet-derived exosome dry powder.

[0074] In this experiment, 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 cells layer. The separation situation of blood cells after centrifugation is well known to those skilled in the art, 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.

[0075] (S1122’) By aseptic operation, take out the plasma layer, completely remove white blood cells and red blood cells, and this 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). As a result, platelets form a 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’) Completely remove the supernatant to completely remove plasma and proteins in the plasma, and use an isotonic sodium chloride solution injection to suspend the platelets in the platelet layer (platelet concentration is 1×10 9Adjust it to 10 - 20 mL so that the concentration becomes 1 × 10 cells / mL to obtain a pure platelet solution.

[0076] (S12') Execute the activation process, which includes the following sub - steps: (S121') Mix collagen and thrombin with the pure platelet solution to obtain a mixed solution. The collagen concentration in the mixed solution is 5 - 20 μg / mL, and the thrombin concentration in the mixed solution is 0.1 - 2 U / mL. Here, the platelet concentration in the pure platelet solution is 1 × 10 9 / mL. When the activation process is executed, the pure platelet solution contains 1 × 10 9 platelets. Preferably, the collagen concentration is 7 - 12 μg / mL and the thrombin concentration is 0.5 - 1.5 U / mL.

[0077] (S122’) Vortex at room temperature for 30 minutes (speed 100 rpm) to activate the platelets in the pure platelet solution, release exosomes, and obtain an activated pure platelet solution.

[0078] (S13’) Execute a purification process to remove platelet - associated structures. This process includes the following sub - steps: (S131’) Centrifuge the activated pure platelet solution with a centrifuge (10,000 - 15,000 g, for 5 - 10 minutes), and the platelet-associated structures will precipitate as a platelet-associated structure pellet. The centrifugation conditions can be extended to 8,000 - 20,000 g, for 3 - 15 minutes.

[0079] (S132’) Carefully transfer the supernatant to a specimen bottle and filter it through a filter membrane with a pore size of 0.45 μm (PES material, 25 mm, model C0000296, brand Labfil (R) ) to completely remove the precipitate of platelet-associated structures. The supernatant is the purified platelet-derived exosome solution. Here, the specimen bottle contains approximately 1 mL of the purified platelet-derived exosome solution. This 1 mL of the purified platelet-derived exosome solution contains platelet exosomes released from 1×10 9 platelets. Next, quantitative analysis of the 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 revealed that the particle size was 20 - 150 nm. Subsequently, using a fully automatic exosome fluorescence quantitative analyzer (ExoView, manufactured by Leprechaun), it was analyzed whether these exosomes contained platelet exosome bi-exosome biomarkers. Since these exosomes had exosome markers CD9, CD63, CD81 and platelet markers CD41, CD42b, it was confirmed that they were indeed platelet exosomes; furthermore, these exosomes did not have white blood cell marker CD45, red blood cell marker CD235ar, or annexin V. Therefore, it can be seen that the purified platelet-derived exosome dry powder produced in steps (S11') to (S14') of Experiment 1 of this example did not contain white blood cell exosomes or red blood cell exosomes.

[0080] (S14’) Perform a freeze-drying process, including the following sub-steps: (S141’) Pre-cooling (-30°C 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) was placed in a 1-bottle specimen bottle and freeze-dried to obtain a dry product, which became 1 bottle of purified platelet-derived exosome dry powder. Using a precision electronic balance (model ME204, Mettler brand), the weight of 1 bottle of purified platelet-derived exosome dry powder was measured. The results showed that the average weight of each bottle of purified platelet-derived exosome dry powder was 0.0067 grams (g). According to the calculation results, it was revealed that there were at least 10 13 platelet exosomes per gram of the purified platelet-derived exosome dry powder.

[0081] In this embodiment, the collagen concentration is 10 μg / mL and the thrombin concentration is 1 U / mL.

[0082] 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 adding collagen and thrombin), 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.

[0083] In some embodiments, the purified platelet-derived exosome dry powder contains a low concentration of albumin or does not contain any albumin at all.

[0084] In some embodiments, the purified platelet-derived exosome dry powder contains a low concentration of globulin or does not contain any globulin at all.

[0085] In some embodiments, the purified platelet-derived exosome dry powder contains a low concentration of fibrinogen or does not contain any fibrinogen at all.

[0086] Experiment 2: Influence of the activation process on platelet exosomes and PDGF-BB release.

[0087] Execute step (S11’) of Experiment 1 to produce a pure platelet solution, with the platelet concentration in the pure platelet solution being 1×10 9 cells / mL. Next, divide the pure platelet solution into five groups: a control group, a calcium chloride group, an adenosine diphosphate group, a collagen group, and a thrombin group.

[0088] 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, do not mix the pure platelet solution with collagen and thrombin, and let it stand for 20 - 30 minutes. Next, execute step (S13’) of Experiment 1 again to produce the control group solution.

[0089] Calcium chloride group: Collect 1 mL of the pure platelet solution and do not perform the activation process in step (S12’) of Experiment 1, that is, do not mix the pure platelet solution with collagen and thrombin, mix it with calcium chloride until the calcium chloride concentration in the mixture reaches 0.45%. Next, execute step (S13’) of Experiment 1 again to produce the calcium chloride group solution.

[0090] Adenosine diphosphate group: Collect 1 mL of the pure platelet solution and do not perform the activation process in step (S12’) of Experiment 1, that is, do not mix the pure platelet solution with collagen and thrombin, mix it with adenosine diphosphate until the adenosine diphosphate concentration in the mixture reaches 60 μM. Next, execute step (S13’) of Experiment 1 again to produce the adenosine diphosphate group solution.

[0091] 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, the pure platelet solution was not mixed with collagen and thrombin, but only with collagen, and the collagen concentration in the mixed solution reached 10 μg / mL. Next, step (S13') of Experiment 1 was executed again to produce the collagen group solution.

[0092] 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, the pure platelet solution was not mixed with collagen and thrombin, but only with thrombin, and the thrombin concentration in the mixed solution reached 1 U / mL. Next, step (S13') of Experiment 1 was executed again to produce the thrombin group solution.

[0093] 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 the enzyme-linked immunosorbent assay (ELISA), the concentration of growth factor (PDGF-BB) in the solution of each group was analyzed.

[0094] From the results of this experiment, it can be seen that the change trends of the platelet exosome and PDGF-BB release multiples 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: The influence of different activation methods on exosome concentration Step (S11') of Experiment 1 was executed to produce a pure platelet solution, and the platelet concentration in this pure platelet solution was 1×109 It was at / mL. Next, this pure platelet solution was divided into a control group and seven experimental groups: a calcium chloride group, an adenosine diphosphate group, a collagen group, a thrombin group, a calcium chloride + adenosine diphosphate group, an adenosine diphosphate + collagen group, and a collagen + thrombin 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. Next, repeat step (S13') of Experiment 1 to produce the control group solution.

[0097] Calcium chloride (CaCl2) group: Take 1 mL of the pure platelet solution and do not perform the activation process in step (S12') of Experiment 1, that is, do not mix the pure platelet solution with collagen and thrombin, but mix it with calcium chloride so that the calcium chloride concentration in the mixture is 0.45%. Place it in a cyclotron shaker (model DSR-D-N1, brand DIGISYSTEM) with a rotation speed of 100 rpm at room temperature and let it act for 20 - 30 minutes. Next, repeat step (S13') of Experiment 1 to produce the calcium chloride group solution.

[0098] Adenosine diphosphate (ADP) group: Take 1 mL of the pure platelet solution and do not perform the activation process in step (S12') of Experiment 1, that is, do not mix the pure platelet solution with collagen and thrombin, but mix it with adenosine diphosphate (ADP) so that the adenosine diphosphate (ADP) concentration in the mixture is 60 μM. Place it in a cyclotron shaker (model DSR-D-N1, brand DIGISYSTEM) with a rotation speed of 100 rpm at room temperature and let it act for 20 - 30 minutes. Next, repeat step (S13') of Experiment 1 to produce the adenosine diphosphate (ADP) group solution.

[0099] Collagen group: Take 1 mL of pure platelet solution, without performing the activation process in step (S12') of Experiment 1, that is, without mixing the pure platelet solution with collagen and thrombin, but only mixing it with collagen so that the collagen concentration in the mixture is 10 μg / mL. Place it in a cyclotron shaker (model DSR-D-N1, brand DIGISYSTEM) with a rotation speed of 100 rpm at room temperature and let it act for 20 - 30 minutes. Next, repeat step (S13') of Experiment 1 to produce the collagen group solution.

[0100] Thrombin group: Take 1 mL of pure platelet solution, without performing the activation process in step (S12') of Experiment 1, that is, without mixing the pure platelet solution with collagen and thrombin, but only mixing it with thrombin so that the thrombin concentration in the mixture is 1 U / mL. Place it in a cyclotron shaker (model DSR-D-N1, brand DIGISYSTEM) with a rotation speed of 100 rpm at room temperature and let it act for 20 - 30 minutes. Next, repeat step (S13') of Experiment 1 to produce the thrombin group solution.

[0101] Calcium chloride + adenosine diphosphate group: Take 1 mL of pure platelet solution, without performing the activation process in step (S12') of Experiment 1, that is, without mixing the pure platelet solution with collagen and thrombin, mix it with calcium chloride and adenosine diphosphate (ADP), adjust the calcium chloride concentration in the mixture to 0.45% and the adenosine diphosphate concentration to 60 μM, place it in a cyclotron shaker (model DSR-D-N1, brand DIGISYSTEM) with a rotation speed of 100 rpm at room temperature, and let it act for 20 - 30 minutes. Next, repeat step (S13') of Experiment 1 to produce the calcium chloride + adenosine diphosphate group solution.

[0102] Adenosine diphosphate + collagen group: Take 1 mL of pure platelet solution, without performing the activation process in step (S12') of Experiment 1, that is, without mixing the pure platelet solution with collagen and thrombin, mix it with adenosine diphosphate and collagen, adjust the adenosine diphosphate concentration in the mixture to 60 μM and the collagen concentration to 10 μg / mL, place it in a cyclotron shaker (model DSR-D-N1, brand DIGISYSTEM) with a rotation speed of 100 rpm at room temperature, and let it act for 20 - 30 minutes. Next, repeat step (S13') of Experiment 1 to produce the adenosine diphosphate + collagen group solution.

[0103] Collagen + thrombin group: Take 1 mL of pure platelet solution, perform steps (S12') - (S13') of Experiment 1 to produce the collagen + thrombin group solution (that is, the purified platelet-derived exosome solution in this example).

[0104] Perform quantitative nanoparticle analysis using a nanoparticle size analyzer (Tunable Resistive Pulse Sensing (TRPS), model Exoid, IZON Science brand) to analyze the concentration of exosomes in the solution of each group.

[0105] The experimental results are shown in Table 1 and Figure 2.

[0106]

Table 1

[0107] Refer to Figure 2. Figure 2 is a bar graph showing the effect of different activation processes on platelet exosome concentration.

[0108] The results in Table 1 and Figure 2 show that compared with the control group, activation methods such as calcium chloride, adenosine diphosphate, collagen, thrombin, calcium chloride + adenosine diphosphate could not significantly increase the concentration of platelet exosomes. Unexpectedly, when using activation methods such as adding adenosine diphosphate and collagen, or collagen and thrombin, platelet exosomes are released up to 21 - fold or 22 - fold (Calculation method: 45,200,000,000 ÷ 2,180,000,000 = 20.7 or 47,600,000,000 ÷ 2,180,000,000 = 21.8). That is, the activation process of adding adenosine diphosphate and collagen, or collagen and thrombin significantly increases the release of platelet exosomes, resulting in an unexpected effect.

[0109] Experiment 4: The effect of platelet exosomes obtained by co - treating collagen and thrombin on alleviating inflammatory response and tissue repair.

[0110] Experiment 4 - 1: The effect of the platelet exosome composition obtained by co - treating collagen and thrombin on alleviating inflammatory response Human dermal fibroblasts (purchased from ScienCell Research Laboratories, Inc., product number 2320) 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 replaced with fresh medium. The above-mentioned human dermal fibroblasts (HDF) are adherent cells.

[0111] 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 9 cells / mL. Next, the pure platelet solution was divided into (1) a control group and (2) a collagen + thrombin group.

[0112] Control group: 1 mL of the pure platelet solution was collected and allowed to stand for 20 - 30 minutes instead of performing the activation process in step (S12') of Experiment 1. Next, step (S13') of Experiment 1 was performed again to produce a control group solution. The control group solution was mixed with DMEM culture medium (containing 2% FBS) (the volume ratio of the control solution to the DMEM culture medium was 1:19) to produce a control group's platelet-derived exosome medium.

[0113] Collagen + thrombin loop: Collect 1 mL of pure platelet solution and perform steps (S12’) to (S13’) of Experiment 1 to produce a collagen + thrombin group solution (i.e., in this example, a purified platelet-derived exosome solution). Mix the collagen + thrombin group solution with DMEM culture medium (containing 2% FBS) (the volume ratio of the collagen + thrombin group solution to the DMEM culture medium is 1:19) to prepare a collagen and thrombin group’s platelet-derived exosome medium.

[0114] Divide a 96-well plate into untreated wells, LPS control group wells, control group exosome solution wells, and collagen + thrombin 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 collagen + thrombin group exosome solution wells so that each well contains the same number of cells, and culture them in an incubator at 37°C and 5% carbon dioxide (CO2) for 24 hours.

[0115] Remove the used medium. Suspend the cells in the LPS control group wells, control group exosome solution wells, and collagen + thrombin group exosome solution wells in DMEM culture 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 them in an incubator at 37°C and 5% carbon dioxide (CO2) for 1 hour. Next, perform the following steps for each group.

[0116] 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.

[0117] 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.

[0118] 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.

[0119] Collagen + thrombin group exosome solution well: Remove the old culture medium (used medium). Suspend the cells in the collagen + thrombin group exosome solution well in the collagen + thrombin group exosome culture medium and culture them in an incubator at 37 °C and 5% carbon dioxide (CO2) for 24 hours.

[0120] Transfer the cell culture medium from the untreated well, LPS control group well, control group exosome solution well, and collagen + thrombin group exosome solution well to a microcentrifuge tube 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 to 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 to the concentration of the inflammatory factor IL-6.

[0121] The data indicate that the exosome solution of the collagen + thrombin group can suppress the inflammatory response of human fibroblasts, thereby alleviating the inflammation of skin tissue. The inventor expects that after inducing the inflammatory response of fibroblasts in other human tissues using a stimulant (such as LPS), the exosome solution of the collagen + thrombin group can also suppress the induced inflammatory response, thereby alleviating the inflammation of other tissues.

[0122] Experiment 4-2: Effect of platelet exosomes obtained by co-treatment of collagen and thrombin on cell migration Cell migration and proliferation are beneficial for tissue repair (see Reference 1). This experiment is performed using the same human dermal fibroblasts (HDF) as in Experiment 4-1. 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 a new medium.

[0123] A 96-well plate containing a cell culture insert is divided into untreated wells, control group exosome solution wells, and collagen + thrombin 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 collagen + thrombin group exosome solution wells such 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. The old medium and the cell culture insert of each group are removed, and photos and records are taken with a microscope. In the wells of each group, confirm that the area occupied by the cell culture insert is not covered by cells. Next, perform the following steps for each group.

[0124] Untreated wells: Continue culturing in DMEM medium (containing 2% FBS) in an incubator at 37 °C and 5% carbon dioxide (CO2) for 6 hours.

[0125] Control group exosome solution wells: Suspend the cells in the control group exosome solution wells with the control group’s platelet-derived exosome medium described in Experiment 4-1, and culture in an incubator at 37 °C and 5% carbon dioxide (CO2) for 6 hours.

[0126] Collagen + thrombin group exosome solution wells: Suspend the cells in the collagen + thrombin group exosome solution wells with the collagen and thrombin group’s platelet-derived exosome medium described in Experiment 4-1, and culture in an incubator at 37 °C and 5% carbon dioxide (CO2) for 6 hours.

[0127] 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. The groups marked with "**" show a statistical difference from the control group exosome solution well (p < 0.01).

[0128] The experimental results are shown in Figure 3. Figure 3 is a bar graph of the effect of the platelet exosome composition obtained by co - treating collagen and thrombin on the cell migration of human dermal fibroblasts.

[0129] The results in Figure 3 show that the average cell migration area of the untreated group was 0.5 square millimeters; the average cell migration area of the control group exosome solution group was 1.00 square millimeters; the average cell migration area of the collagen + thrombin group exosome solution group was 2.15 square millimeters, indicating that there was a significant difference in the cell migration area between the collagen + thrombin group exosome solution group and the control group exosome solution group (p < 0.01). In the present invention, it can be seen that the platelet exosome composition obtained by the activation process of co - treating collagen and thrombin promotes the migration reaction of human dermal fibroblasts and thus promotes tissue repair. Furthermore, from Experiment 2, it can be seen that the cell migration reaction shown in Figure 3 is caused by platelet exosomes rather than PDGF - BB.

[0130] Experiment 4 - 3: The effect of the platelet exosome composition obtained by co - treating collagen and thrombin on cell proliferation The experiment was performed using the same human dermal fibroblasts (HDF) as in Experiment 4 - 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.

[0131] The 96-well plate is divided into untreated wells, control group exosome solution wells, and collagen + thrombin group exosome solution wells, and two replicates are created for each well. Human dermal fibroblasts (HDF) are added to the untreated wells, control group exosome solution wells, and collagen + thrombin 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.

[0132] Untreated wells: Remove the old culture medium. Continue culturing in DMEM culture medium (containing 2% FBS) in an incubator at 37°C and 5% carbon dioxide (CO2) for 6 hours.

[0133] Control group exosome solution wells: Remove the old culture medium. Suspend the cells in the control group exosome solution wells with the control group's platelet-derived exosome medium described in Experiment 4-1, and continue culturing in an incubator at 37°C and 5% carbon dioxide (CO2) for 6 hours.

[0134] Collagen + thrombin group exosome solution wells: Suspend the cells in the collagen + thrombin group exosome solution wells with the collagen and thrombin group's platelet-derived exosome medium described in Experiment 4-1, and culture in an incubator at 37°C and 5% carbon dioxide (CO2) for 6 hours.

[0135] 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 to 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 to the number of viable cells.

[0136] From the experimental results, it can be seen that in the present invention, the platelet exosome composition obtained by co-treating collagen and thrombin promotes the proliferation of human skin fibroblasts and thus promotes tissue repair.

[0137] Experiment 5: Effects of exosomes in different biological solutions on anti-inflammation, cell migration, and cell proliferation of human skin fibroblasts Experiment 5-1: 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.

[0138] Pure platelet group: Collect 1 mL of pure platelet solution, and perform steps (S12’) to (S13’) of Experiment 1 to produce a pure platelet group exosome solution (that is, a purified platelet-derived exosome solution).

[0139] 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.

[0140] Using a nanoparticle size analyzer (Tunable Resistive Pulse Sensing, TRPS), model Exoid, IZON Science brand), the concentration of exosomes in the solution of each group was analyzed. Next, using a fully automated exosome fluorescence quantitative analyzer (ExoView) (trade name Leprechaun), the biomarkers of exosomes in the pure platelet group exosome solution or the platelet plasma group exosome solution were individually analyzed. As a result of the experiment, it was shown that 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 the leukocyte marker CD45 and at least one of 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, exosomes in the platelet plasma group exosome solution have at least one of the erythrocyte markers CD235ar and Annexin V, or the leukocyte marker CD45. For the experimental results, refer to Table 2 and Figure 4. Figure 4 is a bar graph of exosomes in different biological solutions. As a result of the experiment, it was found that among the platelet plasma group exosomes, platelet exosomes accounted for only 2.23%, and exosomes derived from other cells other than platelets accounted for 97.77%.

[0141]

Table 2

[0142] Experiment 5-2: Effects of exosomes in different biological solutions on alleviating inflammation of human dermal fibroblasts Using physiological saline injection, adjust the concentration of the pure platelet group exosome solution prepared in Experiment 5-1 and the concentration of the platelet plasma group exosome solution prepared in Experiment 5-1 so that the exosome concentrations of both are the same (the total number of exosomes per unit volume is the same).

[0143] The experiment was performed using the same human dermal fibroblasts (HDF) as in Experiment 4-1. 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 is replaced with fresh medium.

[0144] Divide a 96-well plate into untreated wells, LPS control group wells, platelet plasma experimental group wells, and purified platelet experimental group wells, and create two replicates for each well. Add the same number of human dermal fibroblasts (HDF) to the untreated wells, LPS control group wells, platelet plasma experimental group wells, and purified platelet experimental group wells, and culture them in an incubator at 37°C and 5% carbon dioxide (CO2) for 24 hours.

[0145] Remove the used medium. Suspend the cells in the LPS control group wells, platelet plasma experimental group wells, and purified platelet experimental group wells in DMEM culture medium (containing 2% FBS) containing 5 μg / mL LPS, and suspend the cells in the untreated wells in DMEM culture medium (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.

[0146] Untreated well: Remove the old culture medium (used medium). Suspend the cells in the untreated 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.

[0147] LPS control group well: Remove the old culture medium (used medium). Suspend the cells in the untreated 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.

[0148] Platelet-rich plasma experimental group well: Mix the platelet-rich plasma group exosome solution with DMEM culture medium (containing 2% FBS) (the volume ratio of the platelet-rich plasma group exosome solution to the DMEM culture medium is 1:19) to prepare the platelet-rich plasma group culture medium. Remove the old culture medium (used medium). Suspend the cells in the platelet-rich plasma experimental group well in the platelet-rich plasma experimental group culture medium and culture them in an incubator at 37°C and 5% carbon dioxide (CO2) for 24 hours.

[0149] Pure platelet experimental group well: Mix the pure platelet group exosome solution with DMEM culture medium (containing 2% FBS) (the volume ratio of the pure platelet group exosome solution to the DMEM culture medium is 1:19) to prepare the pure platelet experimental culture medium. Remove the old culture medium (used medium). Suspend the cells in the pure platelet experimental group well in the pure platelet experimental culture medium and culture them in an incubator at 37°C and 5% carbon dioxide (CO2) for 24 hours.

[0150] Transfer the cell culture medium from the untreated well (control group well), LPS control group well, platelet-rich plasma experimental group well, and pure platelet experimental group well to microcentrifuge tubes respectively. The concentration of human interleukin (Human IL-6) in the cell culture medium is interleukin (IL-6, inflammatory factor) Measure using an 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.

[0151] From the experimental results, it can be seen that a high-purity platelet exosome composition (that is, the one from which exosomes from other cells other than platelets have been removed) can effectively alleviate the inflammatory response.

[0152] Experiment 5-3: The effect of exosomes in different biological solutions on the cell migration of human dermal fibroblasts Using an isotonic sodium chloride solution injection, adjust the concentration of the pure platelet group exosome solution prepared in Experiment 5-1, adjust the concentration of the platelet plasma group exosome solution prepared in Experiment 5-1, so that the exosome concentrations of both are the same (the total number of exosomes per unit volume is the same).

[0153] The experiment was conducted using the same human dermal fibroblasts (HDF) as in Experiment 4-1. 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 a new medium.

[0154] A 96-well plate containing a cell culture insert was divided into untreated wells, platelet-rich plasma experimental wells, and pure platelet experimental wells, and three replicates were created for each well. Human dermal fibroblasts (HDF) were added to the untreated wells, platelet-rich plasma experimental wells, and pure platelet experimental wells such 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. The old medium and the cell culture insert of each group were removed, and photographs and records were taken with a microscope. In the wells of each group, it was confirmed that the area occupied by the cell culture insert was not covered with cells. Next, the following steps were performed for each group.

[0155] Untreated wells: Continue culturing in a DMEM medium (containing 2% FBS) in an incubator at 37 °C and 5% carbon dioxide (CO2) for 6 hours.

[0156] Platelet-rich plasma experimental wells: Mix the platelet-rich plasma group exosome solution with a DMEM medium (containing 2% FBS) (the volume ratio of the platelet-rich plasma group exosome solution to the DMEM medium is 1:19) to produce a platelet-rich plasma experimental culture medium. Remove the old medium. Suspend the cells in the platelet-rich plasma experimental wells in the platelet-rich plasma experimental culture medium. Continue culturing in an incubator at 37 °C and 5% carbon dioxide (CO2) for 6 hours.

[0157] Pure platelet experimental wells: Mix the pure platelet group exosome solution with a DMEM medium (containing 2% FBS) (the volume ratio of the pure platelet group exosome solution to the DMEM medium is 1:19) to produce a pure platelet experimental culture medium. Remove the old medium. Suspend the cells in the pure platelet experimental group wells in the pure platelet experimental culture medium. Continue culturing in an incubator at 37 °C and 5% carbon dioxide (CO2) for 6 hours.

[0158] 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 untreated group (p < 0.05).

[0159] The experimental results are shown in Fig. 5A. Fig. 5A is a bar graph showing the effect of exosomes in different biological solutions on the cell migration of human skin fibroblasts.

[0160] The results in Fig. 5A showed that the average cell migration area of the untreated group was 0.5 square millimeters; the average cell migration area of the platelet-rich plasma experimental group was 0.56 square millimeters; the average cell migration area of the pure platelet experimental group was 0.81 square millimeters, indicating that there was a significant difference in the cell migration area between the pure platelet experimental group and the untreated group (p < 0.05). Therefore, it was found that a high-purity platelet exosome composition (i.e., one from which exosomes from other cells other than platelets have been removed) can promote the migration reaction of human skin fibroblasts, and thus promote tissue repair, obtaining unexpected effects.

[0161] Experiment 5-4: The effect of exosomes in different biological solutions on the cell proliferation of human skin fibroblasts Using an isotonic sodium chloride solution injection, adjust the concentration of the pure platelet group exosome solution prepared in Experiment 5-1, and also adjust the concentration of the platelet-rich plasma group exosome solution prepared in Experiment 5-1 so that the exosome concentrations of both are the same (the total number of exosomes per unit volume is the same).

[0162] This experiment is carried out using the same human dermal fibroblasts (HDF) as in Experiment 4-1. 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 is replaced with fresh medium.

[0163] A 96-well plate is divided into untreated wells, platelet-rich plasma experimental wells, and pure platelet experimental wells, and two replicates are created for each well. Human dermal fibroblasts (HDF) are added to the untreated wells, platelet-rich plasma experimental wells, and pure platelet experimental wells such 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.

[0164] Untreated wells: Remove the old culture medium. Continue culturing in DMEM culture medium (containing 2% FBS) in an incubator at 37 °C and 5% carbon dioxide (CO2) for 6 hours.

[0165] Platelet-rich plasma experimental wells: Remove the old culture medium. Suspend the cells in the platelet-rich plasma experimental wells with the platelet-rich plasma experimental culture medium described in Experiment 5-3, and continue culturing in an incubator at 37 °C and 5% carbon dioxide (CO2) for 6 hours.

[0166] Pure platelet experimental wells: Remove the old culture medium. Suspend the cells in the pure platelet experimental wells with the pure platelet experimental group culture medium described in Experiment 5-3, and continue culturing in an incubator at 37 °C and 5% carbon dioxide (CO2) for 6 hours.

[0167] 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 to the number of viable cells.

[0168] The experimental results are shown in Figure 5B. Figure 5B is a bar graph of the effect of exosomes in different biological solutions on the cell proliferation of human skin fibroblasts.

[0169] The results in Figure 5B showed that the average number of cells in the untreated group was 13,947 cells; the average number of cells in the platelet-rich plasma experimental group was 15,357 cells; the average number of cells in the pure platelet experimental group increased to 21,797 cells, indicating that there was a significant difference in the number of cells (p < 0.05) between the pure platelet experimental group and the untreated group. It was found that high-purity platelet exosomes (that is, exosomes removed from cells other than platelets) can promote the proliferation reaction of human skin fibroblasts, and thus promote tissue repair, obtaining unexpected effects.

[0170] Experiment 6: The effect of the freeze-drying process on the anti-inflammatory effect of the product.

[0171] According to steps (S11’) to (S13’) of Experiment 1, a purified platelet exosome solution was prepared.

[0172] Prepare human dermal fibroblasts required for this experiment according to the experimental procedure of Experiment 4-1. The 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 well contained the same number of cells, and cultured in an incubator at 37 °C and 5% carbon dioxide (CO2) for 24 hours.

[0173] 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. Culture in an incubator at 37 °C and 5% carbon dioxide (CO2) for 1 hour. Next, perform the following steps for each group.

[0174] 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.

[0175] 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.

[0176] Non-lyophilized group wells: Remove the old culture medium (used medium). Collect 1 mL of the purified platelet exosome solution and 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 for 24 hours in an incubator at 37°C and 5% carbon dioxide (CO2).

[0177] 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 and 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 for 24 hours in an incubator at 37°C and 5% carbon dioxide (CO2).

[0178] Transfer the cell culture medium from the untreated well, LPS control group well, non-lyophilized group well, and lyophilized group well to a microcentrifuge tube 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.

[0179] Data are presented as mean ± SD, and statistical analysis is performed using a t test. Groups marked with "**" show statistical differences from the non-lyophilized group (p < 0.01).

[0180] Refer to Table 3 and Figure 6 for the experimental results. Figure 6 is a bar graph showing the effect of the freeze-drying process on the anti-inflammatory effect of the product.

[0181]

Table 3

[0182] The experimental results in Figure 6 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 382.9 pg / mL; and the average inflammatory factor release in the lyophilized group was 79.3 pg / mL. Compared with the non-lyophilized group, the average inflammatory factor release in the lyophilized group was significantly decreased (p<0.01). On the other hand, compared with the inflammatory factor release in the non-lyophilized group (382.9 pg / mL), the inflammatory factor release in the lyophilized group was significantly decreased to 79.3 pg / mL (decreased from 100% to 21%; calculation formula: 79.3÷382.9×100% = 21%) (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.

[0183] After the inventors induced the inflammatory reaction of other human tissues with a stimulant (such as LPS), they predicted that the platelet exosome dry powder obtained from the lyophilization step of the present invention could also suppress the induced inflammatory reaction, thereby alleviating the inflammation of other human tissues.

[0184] 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.

[0185] References

[0186]

Table 4

Claims

Claim 1 A method for manufacturing a purified platelet-derived exosome dry powder, the manufacturing process comprising: (a) Taking one unit of a blood-derived solution containing platelets and performing a process for 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 a 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 co-treatment process of collagen and thrombin; (c) Performing a purification process to obtain a purified platelet-derived exosome solution; and A method comprising a step of performing a drying process on a purified platelet-derived exosome solution to obtain a purified platelet-derived exosome dry powder containing a plurality of platelet exosomes. **Claim 2** The method according to claim 1, wherein the drying process is a freeze-drying process. **Claim 3** The step (a3) is the pure platelet solution per milliliter (mL) The method according to claim 1, further comprising a process of removing leukocytes so that the number of leukocytes in the 6 solution) is less than 10. **Claim 4** The 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 the 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 the step (a5) further comprises 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. **Claim 5** The solution containing blood-derived platelets 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. **Claim 6** The activation process includes a step of mixing collagen and thrombin with the pure platelet solution, and a step of vortexing at room temperature for 30 minutes. The method according to claim 1. **Claim 7** The activation process is (b1) a step of mixing collagen and thrombin with the pure platelet solution to obtain a mixed solution, wherein the collagen concentration in the mixed solution is 5-20 μg / mL and the thrombin concentration in the mixed solution is 0.1-2 U / mL; and (b2) a step of vortexing at room temperature for 30 minutes. The method according to claim 1. **Claim 8** The activation process is (b1') a step of mixing collagen and thrombin with the pure platelet solution to obtain a mixed solution, wherein the collagen concentration in the mixed solution is 7-12 μg / mL and the thrombin concentration in the mixed solution is 0.5-1.5 U / mL; and (b2') a step of shaking at room temperature for 30 minutes, the method according to claim 1.

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

10. The blood-derived solution containing platelets is whole blood, apheresis platelets, leukocytes-reduced platelets apheresis, Platelet-Rich Plasma (PRP), or any combination thereof; or the blood-derived solution 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. In the 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 the 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.

12. The purification process includes (c1) performing a centrifugation process to remove most of the platelet-associated structures; and (c2) performing a membrane filtration process to completely remove the platelet-associated structures. The membrane filtration process in step (c2) utilizes 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. Purified platelet-derived exosome dry powder, manufactured by a manufacturing method, the manufacturing method is (a) Taking 1 unit of a blood-derived solution containing platelets and performing a process for purifying platelets to obtain a pure platelet solution; wherein step (a) comprises (a1) Taking 1 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 the 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, release exosomes, and obtain an activated pure platelet solution; wherein the activation process is a co-treatment process of collagen and thrombin; (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 activation process is (b1') A step of mixing collagen and thrombin with the pure platelet solution to obtain a mixed solution, wherein the collagen concentration in the mixed solution is 5 to 20 μg / mL and the thrombin concentration in the mixed solution is 0.1 to 2 U / mL; and (b2') The purified platelet exosome dry powder according to claim 13, comprising a step of vortexing at room temperature for 30 minutes.

16. In the purified platelet 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 (1) The purified platelet exosome dry powder per 1 gram (g) contains at least 1×10 10 exosomes; or, (2) Per 1 gram (g) of purified platelet exosome dry powder, containing 1×10 11 to 1×10 15 platelet exosomes The purified platelet exosome dry powder according to any one of claims 13 to 15.

17. (1) The purified platelet exosome dry powder per gram (g) contains at least 50 μg of microRNA; or, (2) The content of microRNA in the purified platelet exosome dry powder per gram (g) is 100 μg to 2,500 μg. The purified platelet exosome 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 powder according to any one of claims 13 to 15. **Claim 19** (1) In the purified platelet-derived exosome 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 a leukocyte marker; the leukocyte marker is CD45. The purified platelet-derived exosome powder according to any one of claims 13 to 15. **Claim 20** (1) In the purified platelet-derived exosome 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 an erythrocyte marker; the erythrocyte marker is at least one of CD235ar and Annexin V, or a combination thereof. The purified platelet-derived exosome powder according to any one of claims 13 to 15. **Claim 21** (1) In the purified platelet-derived exosome powder per gram (g), other 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. **Claim 22** Use of the purified platelet-derived exosome powder according to any one of claims 13 to 15 for manufacturing 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. **Claim 23** Use according to claim 22 for manufacturing 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. **Claim 24** Use of the purified platelet-derived exosome powder according to any one of claims 13 to 15 for manufacturing a pharmaceutical composition or a health composition for enhancing the migratory ability of skin fibroblasts to the skin site. **Claim 25** Use according to claim 24, wherein the skin site is a skin lesion site.

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

27. A method for producing a purified platelet-derived exosome dry powder, the production process comprising: (a) Taking 1 unit of a blood-derived solution containing platelets and performing a process for purifying platelets to obtain a pure platelet solution; wherein step (a) comprises: (a1) Taking 1 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 the platelets to 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 to release exosomes and obtain an activated pure platelet solution; wherein the activation process is a co-treatment process with collagen and adenosine diphosphate (ADP); (c) Performing a purification process to obtain a purified platelet-derived exosome solution; and (d) A method comprising a step of performing a drying process on a purified platelet-derived exosome solution to obtain a purified platelet-derived exosome dry powder containing a plurality of platelet-derived exosomes. **Claim 28** The activation process is (b1') A step of mixing collagen and adenosine diphosphate with the pure platelet solution to obtain a mixed solution, wherein the collagen concentration in the mixed solution is 5 to 20 μg / mL and the adenosine diphosphate concentration in the mixed solution is 40 to 80 μM; and (b2') A step of vortexing at room temperature for 30 minutes, The method according to claim 27.

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