Method for preparing exosome from human platelets

Abstract

A method for preparing exosomes from human platelets includes preparing the human platelets, treating the human platelets with a buffer solution to obtain a platelet solution, and collecting a supernatant of the platelet solution to obtain an exosome solution. Wherein the buffer solution is any one of a calcium ion buffer solution, a phosphate buffer solution (PBS), a tris (hydroxymethyl) aminomethane hydrochloride (Tris-HCl, the pH value is 7.0-8.0) buffer solution and a hydroxyethyl piperazine ethanesulfonic acid buffer solution (HEPES Buffer).

Description

Technical Field

[0001] The present invention relates to a method for preparing exosomes, and particularly to a method for preparing exosomes by treating platelets with a specific buffer solution. Background Art

[0002] Exosomes are a type of small endosomal-derived membrane microvesicles and are a kind of extracellular vesicles. The lipid bilayer membrane vesicles of exosomes carry various signaling factors such as nucleic acids, proteins, saccharides, and lipids. They are carriers of these functional substances, mediators of cell-to-cell signal transduction, and can efficiently control cell gene expression and protein function expression. Moreover, the functions of exosomes depend on the cell types from which they are derived. They have multiple applications and are related to various clinical studies. Summary of the Invention

[0003] In some embodiments, a method for preparing exosomes includes preparing human platelets, treating the human platelets with a buffer solution to obtain a platelet solution, and collecting the supernatant of the platelet solution to obtain an exosome solution. Among them, the buffer solution is any one of a calcium ion buffer solution, a phosphate buffer solution (PBS), a tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl, pH 7.0 - 8.0) buffer solution, and a 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer solution (HEPES Buffer).

[0004] In some embodiments, the calcium ion buffer solution is a phosphate buffer solution containing at least one of calcium chloride, calcium carbonate, and calcium gluconate.

[0005] In some embodiments, the concentration of the aforementioned calcium chloride is 2 mM to 20 mM.

[0006] In some embodiments, the average particle size range of exosomes in the exosome solution is from 118.8 nm ± 1.0 nm to 156.3 nm ± 0.6 nm.

[0007] In some embodiments, the concentration of the aforementioned calcium chloride is 5 mM, and the particle size distribution (D10 - D90) of 10% to 90% of the exosomes in the exosome solution obtained by the aforementioned preparation method is from 75.4 nm ± 1.5 nm to 171.8 nm ± 3.1 nm.

[0008] In some embodiments, the concentration of the aforementioned calcium chloride is 7.5 mM, and the particle size distribution (D10 - D90) of 10% to 90% of the exosomes in the exosome solution obtained by the aforementioned preparation method is from 95.3 nm ± 2.5 nm to 219.5 nm ± 5.9 nm.

[0009] In some embodiments, the aforementioned buffer solution is a phosphate buffer solution with a concentration of 0.1× to 2×.

[0010] In some embodiments, the aforementioned buffer solution is a phosphate buffer solution, and the average particle size range of exosomes in the exosome solution obtained by the aforementioned preparation method is 167.9 nm ± 3.6 nm.

[0011] In some embodiments, the aforementioned buffer solution is the phosphate buffer solution, and the particle size distribution (D10 - D90) of 10% to 90% of the exosomes in the exosome solution obtained by the aforementioned preparation method is 103.0 nm ± 4.0 nm to 252.7 nm ± 8.0 nm.

[0012] In some embodiments, the aforementioned tris(hydroxymethyl)aminomethane hydrochloride buffer solution contains 0.1 M to 1 M tris(hydroxymethyl)aminomethane hydrochloride.

[0013] In some embodiments, the aforementioned buffer solution is a tris(hydroxymethyl)aminomethane hydrochloride buffer solution, and the average particle size of exosomes in the exosome solution obtained by the aforementioned preparation method is 182.0 nm ± 1.4 nm.

[0014] In some embodiments, the aforementioned buffer solution is a tris(hydroxymethyl)aminomethane hydrochloride buffer solution, and the particle size distribution (D10 - D90) of 10% to 90% of the exosomes in the exosome solution obtained by the aforementioned preparation method is 119.6 nm ± 1.5 nm to 272.5 nm ± 8.1 nm.

[0015] In some embodiments, the aforementioned 2-(4-(2-hydroxyethyl)piperazin-1-yl)ethanesulfonic acid buffer contains 0.1 M to 1 M 2-(4-(2-hydroxyethyl)piperazin-1-yl)ethanesulfonic acid.

[0016] In some embodiments, the aforementioned buffer solution is a 2-(4-(2-hydroxyethyl)piperazin-1-yl)ethanesulfonic acid buffer, and the average particle size of most of the exosomes in the exosome solution obtained by the aforementioned preparation method is 176.8 nm ± 2.8 nm.

[0017] In some embodiments, the aforementioned buffer solution is the 2-(4-(2-hydroxyethyl)piperazin-1-yl)ethanesulfonic acid buffer, and the particle size distribution (D10 - D90) of 10% to 90% of the exosomes in the exosome solution obtained by the aforementioned preparation method is 117.2 nm ± 2.9 nm to 266.3 nm ± 5.3 nm.

[0018] In some embodiments, the aforementioned exosome solution further comprises epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), and glial cell line-derived neurotrophic factor (GDNF).

[0019] In summary, the method for preparing exosomes according to any one of the embodiments can provide an exosome solution containing the required exosomes in a relatively simple environment. In some embodiments, the obtained exosome solution will have different concentrations, average particle sizes, particle size distributions, or any combination thereof of exosome compositions depending on the types and / or concentrations of buffer solutions used. In some embodiments, the obtained exosome solution further comprises growth factors such as epidermal growth factor, vascular endothelial growth factor, and glial cell line-derived neurotrophic factor, and the exosome solution will obtain an exosome solution containing different concentration compositions of growth factors depending on different types and / or different concentrations of buffer solutions. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 is the preparation steps of exosomes according to any one of the embodiments;

[0021] Figure 2 is the experimental result graph of the exosome concentration obtained using calcium ion buffer solutions with different concentrations;

[0022] Figure 3 is the experimental result graph of the exosome concentration obtained using different buffer solutions;

[0023] Figure 4 is the experimental result graph of the cell migration experiment of exosomes obtained using calcium ion buffer solutions with different concentrations; and

[0024] Figure 5 is the experimental result graph of the cell migration experiment of exosomes obtained using different buffer solutions.

[0025] Wherein, reference numerals:

[0026] S100 - S300: Steps DETAILED DESCRIPTION OF THE EMBODIMENTS

[0027] Please refer to Figure 1. First, prepare human platelets (i.e., step S100). In some embodiments of step S100, after obtaining a fresh human platelet concentrate unit, the plasma in the human platelet concentrate unit is removed to obtain human platelets. For example, after obtaining a fresh human platelet concentrate unit from a registered blood bank, the obtained human platelet concentrate unit is centrifuged at 3000×g for 30 minutes, and then the supernatant is removed and the precipitate is obtained to remove the plasma. Then, a phosphate buffer solution filtered through a 0.02μm filter is added to redissolve the precipitate, and the mixture is centrifuged at 3000×g for 30 minutes, the supernatant is removed and the precipitate is obtained to remove the residual plasma, that is, human platelets with plasma removed are obtained.

[0028] After step S100, the human platelets are treated with a buffer solution to obtain a platelet solution (i.e., step S200). Here, the platelet solution already contains exosomes secreted by the human platelets stimulated by the buffer solution.

[0029] In some embodiments of step S200, after the human platelets are added to the buffer solution, the human platelets are suspended in the buffer solution to obtain a platelet solution. In other embodiments of step S200, after the human platelets are added to the buffer solution, the human platelets are mixed in the buffer solution by stirring or shaking evenly to obtain a platelet solution. For example, when the human platelets come into contact with the buffer solution, the buffer solution will activate the human platelets or react with the human platelets to form a platelet solution.

[0030] In some embodiments of step S200, the human platelets are treated with a buffer solution at an effective temperature to obtain a platelet solution. For example, the effective temperature can be 37°C. In a demonstration example, the human platelets are treated with a buffer solution at 37°C to obtain a platelet solution.

[0031] In some embodiments of step S200, the human platelets are treated with a buffer solution for an effective time. For example, the human platelets are suspended in the buffer solution to form a platelet suspension, and then the platelet suspension is allowed to stand for an effective time to obtain a platelet solution. In some embodiments, the effective time can be from 1 hour to 3 hours. For example, the effective time can be 60 minutes, 90 minutes, 120 minutes, 150 minutes or 180 minutes. In a demonstration example, the human platelets are cultured in the buffer solution at 37°C for 1 hour.

[0032] Among them, the aforementioned buffer solution is any one of a calcium ion buffer solution, a phosphate buffer solution (PBS; hereinafter referred to as PBS buffer solution), a tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl, pH 7.5) buffer solution (hereinafter referred to as Tris-HCl buffer solution (pH 7.5)), and a 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer solution (HEPES Buffer; hereinafter referred to as HEPES buffer solution).

[0033] In some embodiments, the calcium ion buffer solution is phosphate buffered saline containing at least one of calcium chloride (CaCl2), calcium carbonate (CaCO3), and calcium gluconate (C 12 H 22 CaO 14 ). In some embodiments, the calcium ion concentration of the calcium ion buffer solution is 2 mM to 20 mM. For example, the calcium ion buffer solution is prepared from a phosphate buffer solution and calcium chloride, and the concentration of calcium chloride is 2 mM to 20 mM. In some exemplary cases, the concentration of calcium ion or calcium chloride can be 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 7.5 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, or 20 mM.

[0034] In some embodiments, the PBS buffer solution is a phosphate buffer solution containing sodium chloride (NaCl) and potassium chloride (KCl) obtained by dissolving a commercially available PBS tablet (such as a Sigma tablet) in deionized water. For example, dissolving a commercially available PBS tablet in 200 ml of deionized water can obtain a 1× PBS buffer solution, which contains 137 mM of sodium chloride, 2.7 mM of potassium chloride, and 10 mM of phosphate buffer. In some embodiments, according to the need, PBS buffer solutions with concentrations of 0.1 times (0.1×) to 2 times (2×), such as 0.1×, 0.5×, 1×, 1.5×, 2×, etc., can be obtained by adjusting the amount of deionized water added. In some exemplary cases, the buffer solution is a 1× PBS buffer solution.

[0035] In some embodiments, the Tris-HCl buffer solution (pH 7.0 - 8.0) is prepared by dissolving tris(hydroxymethyl)aminomethane (Tris) in deionized water and adjusting the pH value to 7.0 - 8.0 with hydrochloric acid (HCl). In some embodiments, the buffer solution used is a 0.1M to 1M Tris-HCl buffer solution (pH 7.0 - 8.0). For example, the concentration of the Tris-HCl buffer solution (pH 7.0 - 8.0) can be 0.1M, 0.2M, 0.3M, 0.4M, 0.5M, 0.6M, 0.7M, 0.8M, 0.9M, 1.0M. In some exemplary embodiments, the buffer solution is a 0.1M Tris-HCl buffer solution (pH 7.5).

[0036] In some embodiments, the HEPES buffer is prepared by dissolving 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) in double-distilled water (ddH2O) and adjusting the pH value to 7.0 to 8.0 with sodium hydroxide (NaOH). In some embodiments, the buffer solution used is a 0.1M to 1M HEPES buffer solution. For example, the concentration of the HEPES buffer solution can be 0.1M, 0.2M, 0.3M, 0.4M, 0.5M, 0.6M, 0.7M, 0.8M, 0.9M, 1.0M. In some exemplary embodiments, the buffer solution is a 0.1M HEPES buffer solution and its pH value is 7.5.

[0037] After step S200, the supernatant of the platelet solution is collected to obtain the exosome solution (i.e., step S300). For example, after collecting the platelet solution obtained in step S200, it is centrifuged at 3000×g for 30 minutes to separate the platelet solution into a supernatant and a precipitate. The supernatant of the centrifuged platelet solution is obtained to get the exosome solution.

[0038] Moreover, when platelets are treated with different buffer solutions at different times, the concentration and particle size of the obtained exosomes are different.

[0039] In some embodiments, the obtained exosome solution is treated with a calcium ion buffer solution containing calcium ions at different concentrations (such as 5 mM, 7.5 mM, 15 mM, or 20 mM, etc.), and the average exosome concentration of the exosomes contained therein is 6200 ± 403×10 7 particles / ml to 9740 ± 252×10 7 particles / ml, and the average particle size range of these exosomes is 118.8 nm ± 1.0 nm to 156.3 nm ± 0.6 nm.

[0040] In some embodiments, the obtained exosome solution is treated with a calcium ion buffer solution containing 5 mM calcium ions, and 10% to 90% of the exosomes contained therein have a particle size distribution (D10-D90) falling between 75.4 nm ± 1.5 nm and 171.8 nm ± 3.1 nm.

[0041] In some embodiments, the obtained exosome solution is treated with a calcium ion buffer solution containing 7.5 mM calcium ions, and 10% to 90% of the exosomes contained therein have a particle size distribution (D10-D90) falling between 95.3 nm ± 2.5 nm and 219.5 nm ± 5.9 nm.

[0042] In some embodiments, the obtained exosome solution is treated with a phosphate buffer solution, and the average exosome concentration of the exosomes contained therein is 1890 ± 34.6×10 7 particles / ml, and the average particle size range of these exosomes is 167.9 nm ± 3.6 nm.

[0043] In some embodiments, the obtained exosome solution is treated with a phosphate buffer solution, and 10% to 90% of the exosomes contained therein have a particle size distribution (D10-D90) falling between 103.0 nm ± 4.0 nm and 252.7 nm ± 8.0 nm.

[0044] In some embodiments, the obtained exosome solution is treated with a 0.1 M tris(hydroxymethyl)aminomethane hydrochloride buffer solution, and the average exosome concentration of the exosomes contained therein is 4230 ± 322×10 7 particles / ml, and the average particle size range of these exosomes is 182.0 nm ± 1.4 nm.

[0045] In some embodiments, the obtained exosome solution is treated with a 0.1 M tris(hydroxymethyl)aminomethane hydrochloride buffer solution, and 10% to 90% of the exosomes contained therein have a particle size distribution (D10-D90) falling between 119.6 nm ± 1.5 nm and 272.5 nm ± 8.1 nm.

[0046] In some embodiments, the obtained exosome solution is treated with a 0.1 M N-(2-hydroxyethyl)piperazine-N'-ethanesulfonic acid buffer solution, and the average exosome concentration of the exosomes contained therein is 2460 ± 178×10 7 particles / ml, and the average particle size range of these exosomes is 176.8 nm ± 2.8 nm.

[0047] In some embodiments, the exosome solution obtained by treating with 0.1M N-(2-Hydroxyethyl)piperazine-N'-ethanesulfonic acid buffer contains 10% to 90% of exosomes with a particle size distribution (D10-D90) ranging from 117.2nm ± 2.9nm to 266.3nm ± 5.3nm.

[0048] In some embodiments, the aforementioned exosome solution can promote cell migration and has the potential to be applied to wound healing. In some embodiments, the aforementioned exosome solution can promote the cell migration of epidermal cells, thereby promoting the wound healing and / or repair of the skin. For related technologies and research, reference can be made to the articles of scholars such as Ayman et al. (Ayman Grada et al. (2017, Feb) J Invest Dermatol., 137(2):e11-e16), Luis et al. (Luis G Rodriguez etal. (2005) Methods Mol Biol., 294:23-9), Bereiter-Hahn et al. (Bereiter-Hahn, J(1984) Biology of the Integument (Berlin and Heidelberg: Springer-Verlag), 443–471), Roberta et al. (Roberta Addis et al. (2020) Int J Med Sci, 17(8):1030-1042), Yuan et al. (Yuan Hu Xuan et al. (2014) PLoS ONE:9(9):e108182), Satish et al. (Satish Patel et al. (2019) Biomedicine&Pharmacotherapy 112:1086), Madhyastha et al. (R Madhyastha et al(2012) Int Wound J, 9:355–361), and Marcia et al. (Marcia L. Usui(2008) Journal of Histochemistry andCytochemistry, 56(7):687-696).

[0049] In some embodiments, the exosome solution further comprises growth factors such as epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), and glial cell line-derived neurotrophic factor (GDNF). In some embodiments, growth factors such as EGF, VEGF, and GDNF are encapsulated in exosomes or located in the buffer solution outside the exosomes.

[0050] In some embodiments, the exosome solution containing EGF can promote the growth of epidermal cells to cover the wound, so it can be clinically applied to wound treatment and used as a drug, and also has the potential to treat chronic diabetic foot ulcers. In some embodiments, the exosome solution containing VEGF can stimulate angiogenesis of granulation tissue and stimulate peripheral collateral angiogenesis, so it can be clinically applied to wound healing and related research on diabetic foot ulcers. In some embodiments, the exosome solution containing GDNF can promote wound healing.

[0051] For relevant technologies and research, refer to the articles of scholars such as Kanchan et al. (Kanchan Shakhakarmi et al. (2023) Archives of Pharmacal Research, 46:299–322), Hardwick et al. (J Hardwicke et al. (2008, Jun) Surgeon, 6(3):172-7), Mert et al. (Mert Dumantepe et al. (2015, Apr) Growth Factors. 33(2):128-32), Tiaka et al. (Tiaka EK, Papanas N et al. (2012, Mar) Perspect Vasc Surg Endovasc Ther.; 24(1):37-44), Jorge et al. (Jorge Berlanga-Acosta et al. (2020, Jul) MEDICC Rev; 22(3):24-31), Stephan et al. (Stephan Barrientos et al. (2014) Wound Repair Regen; 22(5):569–578), David et al. (David O Bates et al. (2003, Jun) Int J Low Extrem Wounds; 2(2):107-20), Aakansha et al. (Aakansha Giri Goswami et al. (2022, Aug) Growth Factors.; 40(3-4):73-88), Mohammad et al. (Mohammad Zubair & Jamal Ahmad (2019) Reviews in Endocrine and Metabolic isorders 20:p207–217), Neda et al. (Neda Vishlaghi et al. (2022, Apr) Exp Dermatol.; 31(4):577-581), Thomas et al. (Thomas S Lisse et al. (2020, Jun 12) NPJ Regen Med.; 5:13), and Simon et al. (Simon Mwangi et al. (2008, Mar) Gastroenterology; 134(3):727-37).

[0052] In some embodiments, the concentration range of EGF contained in the exosome solution is between 20.95 ng / mL and 128 ng / mL. In some embodiments, the concentration range of VEGF contained in the exosome solution is between 417.88 ng / mL and 1332 ng / mL. In some embodiments, the concentration range of GDNF contained in the exosome solution is between 18.722 ng / mL and 184 ng / mL.

[0053] The following experimental data are expressed as mean ± standard deviation (SD), and the differences between the two groups are analyzed by the student’s t-test. In the figures, "*" represents a p-value less than 0.05, "**" represents a p-value less than 0.01, and "***" represents a p-value less than 0.001. The more "*", the more significant the statistical difference.

[0054] Example 1: Preparation of Exosome Solution - Calcium Ion Buffer Solution

[0055] First, a fresh human platelet concentrate unit obtained from a registered blood bank (source: Taipei Blood Donation Center, China) was centrifuged at 3000×g for 30 minutes, and then the supernatant was removed and the precipitate was obtained to remove plasma. Then, 1×PBS buffer solution (purchased from Gibco) filtered through a 0.02 μm filter was added to wash the precipitate, and then centrifuged at 3000×g for 30 minutes to obtain the precipitate to remove residual plasma, that is, human platelets with plasma removed were obtained.

[0056] The human platelets with plasma removed were divided into five groups, namely the control group, experimental group (A), experimental group (B), experimental group (C), and experimental group (D). Among them, the buffer solution used in the control group was 1×PBS buffer solution (purchased from Sigma; prepared with Sigma tablets), the buffer solution used in experimental group (A) was a 5 mM calcium ion buffer solution prepared from 1×PBS buffer solution and calcium chloride (purchased from Spectrum), the buffer solution used in experimental group (B) was a 7.5 mM calcium ion buffer solution prepared from 1×PBS buffer solution and calcium chloride, the buffer solution used in experimental group (C) was a 15 mM calcium ion buffer solution prepared from 1×PBS buffer solution and calcium chloride, and the buffer solution used in experimental group (D) was a 20 mM calcium ion buffer solution prepared from 1×PBS buffer solution and calcium chloride.

[0057] Next, the plasma-depleted human platelets in each group were treated with five different buffer solutions at 37°C for 1 hour to obtain platelet solutions in each group. The platelet solutions in each group were centrifuged at 3000×g for 30 minutes, and the supernatants of the platelet solutions in each group were collected to obtain exosome solutions in each group.

[0058] Example 2: Preparation of exosome solution - Different types of buffer solutions

[0059] First, a fresh human platelet concentrate unit obtained from a registered blood bank (source: Taipei Blood Donation Center, China) was centrifuged at 3000×g for 30 minutes. Then, the supernatant was removed and the precipitate was obtained to remove plasma. Next, the precipitate was washed with 1×PBS buffer solution filtered through a 0.02μm filter, and then centrifuged at 3000×g for 30 minutes to obtain the precipitate, thereby removing residual plasma, and plasma-depleted human platelets were obtained.

[0060] The plasma-depleted human platelets were divided into 4 groups, namely the control group, experimental group (1), experimental group (2), and experimental group (3). Among them, the buffer solution used in the control group was saline (purchased from Sigma), the buffer solution used in experimental group (1) was 1×PBS buffer solution (purchased from Sigma; prepared with Sigma tablets), the buffer solution used in experimental group (2) was 0.1M Tris-HCl buffer solution (pH 7.5; purchased from Sigma), and the buffer solution used in experimental group (3) was 0.1M HEPES buffer solution (purchased from Sigma).

[0061] Next, the plasma-depleted human platelets in each group were treated with four different buffer solutions at 37°C for 1 hour to obtain platelet solutions in each group. The platelet solutions in each group were centrifuged at 3000×g for 30 minutes, and the supernatants of the platelet solutions in each group were collected to obtain exosome solutions in each group.

[0062] Example 3: Detection of exosome concentration in each group of exosome solutions

[0063] The 5 groups of exosome solutions prepared in Example 1 and the 4 groups of exosome solutions obtained in Example 2 were subjected to nanoparticle tracking analysis (NTA) using a Nanosight instrument to obtain the exosome concentration of the exosome solutions in each group.

[0064] The detection results of the exosome concentration of the 5 groups of exosome solutions prepared in Example 1 are shown in Table 1 and Figure 2 as follows.

[0065] Table 1

[0066]

[0067]

[0068] Herein, the control group of Example 1 is labeled as "Control Group [1]" to distinguish it from the control group of Example 2.

[0069] From Table 1 and Figure 2 it can be seen that the buffer solution used in Control Group [1] is 1×PBS buffer solution. Therefore, when human platelets are treated with 1×PBS buffer solution, the concentration of exosomes secreted by the stimulated human platelets is 1890×10 7 per mL. The buffer solution used in Experimental Group (A) is 5 mM calcium ion buffer solution. Therefore, when human platelets are treated with 5 mM calcium ion buffer solution, the concentration of exosomes secreted by the stimulated human platelets is 9060×10 7 per mL. The buffer solution used in Experimental Group (B) is 7.5 mM calcium ion buffer solution. Therefore, when human platelets are treated with 7.5 mM calcium ion buffer solution, the concentration of exosomes secreted by the stimulated human platelets is 9740×10 7 per mL. The buffer solution used in Experimental Group (C) is 15 mM calcium ion buffer solution. Therefore, when human platelets are treated with 15 mM calcium ion buffer solution, the concentration of exosomes secreted by the stimulated human platelets is 7510×10 7 per mL. The buffer solution used in Experimental Group (D) is 20 mM calcium ion buffer solution. Therefore, when human platelets are treated with 20 mM calcium ion buffer solution, the concentration of exosomes secreted by the stimulated human platelets is 6200×10 7 per mL.

[0070] It can be seen from this that the addition of calcium ions can promote the secretion of exosomes by human platelets, and when human platelets are treated with 5 mM calcium ion buffer solution or 7.5 mM calcium ion buffer solution, the concentration of exosomes in the obtained exosome solution is at least 4.5 times higher than that treated with 1×PBS buffer solution.

[0071] The detection results of the exosome concentration of the 4 groups of exosome solutions prepared in Example 2 are shown in Table 3:

[0072] Table 2

[0073]

[0074] Herein, the control group of Example 2 is labeled as "Control Group [2]" to distinguish it from the control group of Example 1

[0075] As can be seen from Table 2 and Figure 3 it is known that the buffer solution used in the control group [2] is physiological saline. Therefore, when human platelets are treated with physiological saline, the concentration of exosomes secreted by the stimulated human platelets is 1820×10 7 per mL. The buffer solution used in experimental group (1) is 1×PBS buffer solution. Therefore, when human platelets are treated with 1×PBS buffer solution, the concentration of exosomes secreted by the stimulated human platelets is 1890×10 7 per mL. The buffer solution used in experimental group (2) is 0.1M Tris-HCl buffer solution (pH 7.5). Therefore, when human platelets are treated with Tris-HCl buffer solution (pH 7.5), the concentration of exosomes secreted by the stimulated human platelets is 4230×10 7 per mL. The buffer solution used in experimental group (3) is 0.1M HEPES buffer solution. Therefore, when human platelets are treated with 0.1M HEPES buffer solution, the concentration of exosomes secreted by the stimulated human platelets is 2460×10 7 per mL.

[0076] It can be seen from this that different types of buffer solutions can promote the secretion of exosomes by human platelets compared to physiological saline. Based on this, users can select different buffer solutions to prepare exosome solutions according to the required concentration of exosomes.

[0077] Example 4: Exosome particle size information of each group of exosome solutions

[0078] The 4 groups of exosome solutions prepared in Example 1 and the 4 groups of exosome solutions obtained in Example 2 were subjected to nanoparticle tracking analysis (NTA) using a Nanosight instrument to obtain particle size information such as the average particle size and particle size distribution of each group of exosome solutions.

[0079] The detection results of the particle size information of the 4 groups of exosome solutions prepared in Example 1 are shown in Table 3.

[0080] Table 3

[0081]

[0082] In Table 3, D10-D90 represents the particle size distribution from 10% to 90%.

[0083] As can be seen from Table 3, the buffer solution used in experimental group (A) was a 5 mM calcium ion buffer solution. Therefore, when human platelets were treated with a 5 mM calcium ion buffer solution, the average particle size of the exosomes secreted by the stimulated human platelets was 118.8 ± 1.0 nm, the maximum particle size was 91.2 ± 6.2 nm, and the particle size distribution of 10% to 90% of the exosomes was 75.4 ± 1.5 nm to 171.8 ± 3.1 nm. The buffer solution used in experimental group (B) was a 7.5 mM calcium ion buffer solution. Therefore, when human platelets were treated with a 7.5 mM calcium ion buffer solution, the average particle size of the exosomes secreted by the stimulated human platelets was 156.3 ± 0.6 nm, the maximum particle size was 128.6 ± 7.5 nm, and the particle size distribution of 10% to 90% of the exosomes was 95.3 ± 2.5 nm to 219.5 ± 5.9 nm. The buffer solution used in experimental group (C) was a 15 mM calcium ion buffer solution. Therefore, when human platelets were treated with a 15 mM calcium ion buffer solution, the average particle size of the exosomes secreted by the stimulated human platelets was 149.1 ± 4.8 nm, the maximum particle size was 148.1 ± 6.5 nm, and the particle size distribution of 10% to 90% of the exosomes was 79.2 ± 8.4 nm to 217.9 ± 0.9 nm. The buffer solution used in experimental group (D) was a 20 mM calcium ion buffer solution. Therefore, when human platelets were treated with a 20 mM calcium ion buffer solution, the average particle size of the exosomes secreted by the stimulated human platelets was 152.9 ± 3.9 nm, the maximum particle size was 155.0 ± 9.1 nm, and the particle size distribution of 10% to 90% of the exosomes was 59.5 ± 7.2 nm to 221.5 ± 21.5 nm.

[0084] That is to say, when the calcium ion concentration is adjusted, exosomes with different particle sizes can be obtained. Therefore, users can select different calcium ion buffer solutions according to the required particle size of the exosomes to obtain an exosome solution containing exosomes with the corresponding particle size.

[0085] The detection results of the particle size information of the 4 groups of exosome solutions prepared in Example 2 are shown in Table 4.

[0086] Table 4

[0087]

[0088]

[0089] In Table 4, D10 - D90 represents the particle size distribution from 10% to 90%.

[0090] The buffer solution used in the control group was physiological saline. Therefore, when human platelets were treated with physiological saline, the average particle size of the exosomes secreted by the stimulated human platelets was 167.2 ± 1.1 nm, the maximum particle size was 143.8 ± 5.4 nm, and the particle size distribution of 10% to 90% of the exosomes was 108.9 ± 3.0 nm to 242.5 ± 6.4 nm. The buffer solution used in experimental group (1) was 1×PBS buffer solution. Therefore, when human platelets were treated with 1×PBS buffer solution, the average particle size of the exosomes secreted by the stimulated human platelets was 167.9 ± 3.6 nm, the maximum particle size was 142.8 ± 2.0 nm, and the particle size distribution of 10% to 90% of the exosomes was 103.0 ± 4.0 nm to 252.7 ± 8.0 nm. The buffer solution used in experimental group (2) was 0.1 M Tris-HCl buffer solution (pH 7.5). Therefore, when human platelets were treated with Tris-HCl buffer solution (pH 7.5), the average particle size of the exosomes secreted by the stimulated human platelets was 182.0 ± 1.4 nm, the maximum particle size was 156.5 ± 4.2 nm, and the particle size distribution of 10% to 90% of the exosomes was 119.6 ± 1.5 nm to 272.5 ± 8.1 nm. The buffer solution used in experimental group (3) was 0.1 M HEPES buffer solution. Therefore, when human platelets were treated with 0.1 M HEPES buffer solution, the average particle size of the exosomes secreted by the stimulated human platelets was 176.8 ± 2.8 nm, the maximum particle size was 134.4 ± 2.4 nm, and the particle size distribution of 10% to 90% of the exosomes was 117.2 ± 2.9 nm to 266.3 ± 5.3 nm.

[0091] That is to say, different types of buffer solutions can be used to obtain exosomes with different particle sizes. Therefore, users can select different types of buffer solutions according to the required particle size of the exosomes to obtain an exosome solution containing exosomes with the corresponding particle size.

[0092] Example 5: Cell migration experiment - calcium ion buffer solution

[0093] Injury will cause the loss of some cells and tissues in the body, and the body needs to repair the injury. There are two different forms of repair: "regeneration" and "fibrous repair", and both processes involve cell migration. Therefore, the design of this experiment is to observe whether the sample has the ability to promote cell migration, so as to judge whether it has the potential to repair wounds / wound healing.

[0094] Herein, the cells used were normal human dermal fibroblasts (purchased from Lonza). The cell culture medium used was Fibroblast Growth Medium-2 (FGM2; purchased from Lonza). The working medium used was 5×DMEM medium containing 0.1% fetal bovine serum (FBS) (purchased from Gibco). The samples used were exosome solutions prepared by treating Example 1 with different calcium ion buffer solutions, a total of 5 groups, namely the control group, experimental groups (A) to (D). The experimental instrument used was a 96-well system (96-well system) device, which included a lid, a filter membrane, and a microplate. The analytical instrument used was an automated cell imaging system (ImageXpress Micro XLS).

[0095] First, the normal human dermal fibroblasts were activated for standby. And, the 5 groups of exosome solutions were diluted to a concentration of 1.75×10 10 per mL with DPBS (source: Gibco), and then diluted to a concentration of 1.4×10 10 per mL with the working medium to be used as the test samples for subsequent experiments.

[0096] Each group of test samples was respectively injected into a 96-well microplate, the filter membrane was placed on the 96-well microplate, and the treated normal human dermal fibroblasts were added to the corresponding filter membrane at 1500 cells per well. Through the chemotaxis of normal human dermal fibroblasts to each group of test samples, the normal human dermal fibroblasts would be attracted and pass through the 10-μm filter membrane and move to the lower part of the filter membrane. Then, it was cultured in a CO2 incubator for 5 hours. The ones that did not penetrate the filter membrane above each group were wiped off and fixed with methanol (source: Merck), and the normal human dermal fibroblasts were stained with a DAPI dye (4’,6-diamidino-2-phenylindole; source: Calbiochem). Finally, each group was analyzed with an ImageXpress Micro XLS instrument, and the results were as Figure 4 shown in Table 5. In Figure 4 , the p-value was compared with the control group.

[0097] Table 5

[0098]

[0099] Please refer to Figure 4 and Table 5. The average number of cell migrations in the control group was 41.75 ± 21.2855, while the average number of cell migrations in experimental group (A) was 111.75 ± 43.7844, in experimental group (B) was 117.875 ± 35.5063, in experimental group (C) was 74.875 ± 42.0695, and in experimental group (D) was 51 ± 20.9626. From the above results, it can be seen that when the buffer solution contains calcium ions, the prepared exosome solution can increase the number of migrating cells. Among them, when the calcium ion concentration is 5 mM or 7.5 mM, the number of migrating cells can be increased by at least 2 times compared with the control group. And when the calcium ion concentration is 15 mM, the number of migrating cells can be increased by at least 1.5 times compared with the control group.

[0100] Therefore, preparing an exosome solution with a calcium ion buffer solution can promote cell migration and has the potential to promote wound healing.

[0101] Example 6: Cell migration experiment - different buffer solutions

[0102] Similar to Example 5, the design of this experiment is to observe whether the sample has the ability to promote cell migration, so as to judge whether it has the potential to repair wounds / wound healing.

[0103] Herein, the cells used were normal human dermal fibroblasts (purchased from Lonza). The cell culture medium used was Fibroblast Growth Medium-2 (FGM2; purchased from Lonza). The working medium used was 5×DMEM medium containing 0.1% fetal bovine serum (FBS) (purchased from Gibco). The samples used were exosome solutions prepared by treating with different calcium ion buffer solutions in Example 1, with a total of 4 groups, namely the control group and experimental groups (one) to (three). The experimental instrument used was a 96-well system (96-well system) device, which included a lid, a filter membrane, and a microplate. The analytical instrument used was an automated cell imaging system (ImageXpress Micro XLS).

[0104] First, activate the normal human dermal fibroblasts for standby. And dilute the concentration of the exosome in 5 groups of exosome solutions to 1.75×10 10 cells / mL with DPBS (source: Gibco), and then dilute the concentration of the exosome to 1.4×1010 cells / mL as the test samples for subsequent experiments.

[0105] Inject the test samples of each group into a 96-well microplate respectively. Place the filter membrane on the 96-well microplate, and add 5000 cells per well of the treated normal human skin fibroblasts onto the corresponding filter membranes of each group. Through the chemotaxis of normal human skin fibroblasts to the test samples of each group, the normal human skin fibroblasts will be attracted and pass through the 10-μm filter membrane and move to the lower part of the filter membrane. Then, culture in a CO₂ incubator for 5 hours. Wipe off the part above the filter membrane of each group that has not penetrated, and then fix it with methanol (source: Merck), and stain the normal human skin fibroblasts with DAPI dye (4’,6-diamidino-2-phenylindole; source: Calbiochem). Finally, analyze each group with an ImageXpress Micro XLS instrument, and the results are as Figure 5 and Table 6 shown. In Figure 5 , the p-value is compared with the control group.

[0106] Table 6

[0107]

[0108] Please refer to Figure 5 and Table 6. The average number of cell migrations in the control group is 201 ± 47.91063, while the average number of cell migrations in experimental group (I) is 268.5 ± 43.52011, the average number of cell migrations in experimental group (II) is 281.625 ± 50.93957, and the average number of cell migrations in experimental group (III) is 346.375 ± 92.24956. From the above results, it can be seen that when preparing exosome solutions with different buffer solutions, it has the ability to increase the number of migrating cells. Among them, when the buffer solution is 0.1 M HEPES buffer solution, the number of migrating cells can be increased by at least 1.5 times compared with the control group. Based on this, the exosome solutions prepared with different buffer solutions can promote cell migration and have the potential to promote wound healing.

[0109] Example 7: Growth factor analysis

[0110] Here, the exosome solutions of 3 groups including the control group, experimental group (A), and experimental group B in Example 1, and the exosome solutions of 4 groups including the control group, experimental group (I), experimental group (II), and experimental group (III) in Example 2 are entrusted to Cenxiang Co., Ltd. (Raybiotech) to analyze the growth factors contained in the solutions for the detection item of human growth factor array Q1 (#QAH-GF-1-SERV). The analysis results are shown in Table 7 and Table 8.

[0111] The aforementioned test item website: https: / / www.raybiotech.com / human-growth-factor-array-q1-qah-gf-1.

[0112] The growth factor analysis results of the exosome solutions of the control group, experimental group (A), and experimental group B in Example 1 are shown in Table 7:

[0113] Table 7

[0114]

[0115] Herein, the control group of Example 1 is labeled as "Control Group [1]" to distinguish it from the control group of Example 2.

[0116] As can be seen from Table 7, the growth factors detected in the control group [1], experimental group (A), and experimental group (B) all include EGF, VEGF, and GDNF. The detected concentration of EGF in the control group [1] is 20.95 pg / ml, the detected concentration of VEGF is 974.33 pg / ml, and the detected concentration of GDNF is 18.72 pg / ml. The detected concentration of EGF in experimental group (A) is 128 pg / ml, the detected concentration of VEGF is 816 pg / ml, and the detected concentration of GDNF is 102 pg / ml. The detected concentration of EGF in experimental group (B) is 74 pg / ml, the detected concentration of VEGF is 1332 pg / ml, and the detected concentration of GDNF is 184 pg / ml. It can be seen therefrom that the concentrations of EGF and GDNF contained in the exosome solution prepared with a calcium ion buffer solution of 5 mM or 7.5 mM are both higher than the concentrations measured in the control group [1], and the concentration of VEGF contained in the exosome solution prepared with a calcium ion buffer solution of 7.5 mM is also higher than that in the control group [1]. In other words, the exosome solutions prepared with calcium ion buffer solutions of different concentrations contain growth factors with different concentration compositions. Moreover, EGF, VEGF, and GDNF are all growth factors beneficial to wound healing, so the exosome solutions prepared with calcium ion buffer solutions of different concentrations have the potential to be applied to wound healing.

[0117] The growth factor analysis results of the exosome solutions of the control group, experimental group (1), experimental group (2), and experimental group (3) in Example 2 are shown in Table 8:

[0118] Table 8

[0119]

[0120] Herein, the control group of Example 2 is labeled as "Control Group [2]" to distinguish it from the control group of Example 1.

[0121] As can be seen from Table 8, the growth factors detected in the control group [2], experimental group (I), experimental group (II), and experimental group (III) all include EGF, VEGF, and GDNF. The detected concentration of EGF in the control group [2] was 12.17 pg / ml, the detected concentration of VEGF was 108.61 pg / ml, and the concentration of GDNF was not detected. The detected concentration of EGF in experimental group (I) was 20.95 pg / ml, the detected concentration of VEGF was 974.33 pg / ml, and the detected concentration of GDNF was 18.72 pg / ml. The detected concentration of EGF in experimental group (II) was 21.47 pg / ml, the detected concentration of VEGF was 794.75 pg / ml, and the detected concentration of GDNF was 81.7 pg / ml. The detected concentration of EGF in experimental group (III) was 29.84 pg / ml, the detected concentration of VEGF was 417.88 pg / ml, and the detected concentration of GDNF was 72.55 pg / ml. It can be seen from this that the concentrations of EGF and GDNF contained in experimental groups (I) to (III) are higher than those in the control group, and the VEGF concentration in experimental group (I) is higher than that in the other three groups, the GNDF concentration in experimental group (II) is higher than that in the other three groups, and the EGF concentration in experimental group (III) is higher than that in the other three groups. In other words, the highest-concentration growth factors in different groups are different, so the compositions of the growth factors contained are also different. Moreover, the exosome solutions of experimental groups (I) to (III) all have the potential to be applied to wound healing.

[0122] In summary, according to the method for preparing exosomes of any embodiment of the present invention, an exosome solution containing the required exosomes can be provided in a relatively simple environment, so as to avoid the variables caused by unwanted components (such as growth factors from the culture solution, etc.) when the obtained exosomes are used in other tests or applications. In some embodiments, the obtained exosome solution will have exosome compositions in different states such as different concentrations, average particle sizes, particle size distributions, or any combination thereof according to the different types and / or concentrations of buffer solutions used.

[0123] Certainly, the present invention can also have many other embodiments. Without departing from the spirit and essence of the present invention, those skilled in the art can make various corresponding changes and deformations according to the present invention, but these corresponding changes and deformations should all fall within the protection scope of the claims of the present invention.

Claims

1. A method for preparing exosomes from human platelets, characterized in that, Comprising: Preparing human platelets; Treating the human platelets with a buffer solution to obtain a platelet solution, wherein the buffer solution is any one of a calcium ion buffer solution, a phosphate buffer solution (PBS), a tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl, pH 7.0 - 8.0) buffer solution, and a 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer solution (HEPES Buffer); and Collecting the supernatant of the platelet solution to obtain an exosome solution.

2. The method according to claim 1, wherein The calcium ion buffer solution is a phosphate buffer solution containing at least one of calcium chloride, calcium carbonate, and calcium gluconate.

3. The method according to claim 2, wherein The concentration of the calcium chloride is 2 mM to 20 mM.

4. The method according to claim 3, wherein The average particle size range of the exosomes in the exosome solution is 118.8 nm ± 1.0 nm to 156.3 nm ± 0.6 nm.

5. The method according to claim 2, characterized in that The concentration of the calcium chloride is 5 mM, and the particle size distribution (D10 - D90) of 10% to 90% of the exosomes in the exosome solution is 75.4 nm ± 1.5 nm to 171.8 nm ± 3.1 nm.

6. The method according to claim 2, wherein The concentration of the calcium chloride is 7.5 mM, and the particle size distribution (D10 - D90) of 10% to 90% of the exosomes in the exosome solution is 95.3 nm ± 2.5 nm to 219.5 nm ± 5.9 nm.

7. The method according to claim 1, wherein The buffer solution is 0.1× to 2× of the phosphate buffer solution.

8. The method according to claim 1, wherein The buffer solution is the phosphate buffer solution, and the average particle size range of the exosomes in the exosome solution is 167.9 nm ± 3.6 nm.

9. The method according to claim 1, wherein The buffer solution is the phosphate buffer solution, and the particle size distribution (D10 - D90) of 10% to 90% of the exosomes in the exosome solution is 103.0 nm ± 4.0 nm to 252.7 nm ± 8.0 nm.

10. The method according to claim 1, characterized in that, The tris(hydroxymethyl)aminomethane hydrochloride buffer solution contains 0.1 M to 1 M of tris(hydroxymethyl)aminomethane hydrochloride.

11. The method according to claim 1, characterized in that, The buffer solution is the tris(hydroxymethyl)aminomethane hydrochloride buffer solution, and the average particle size of the exosomes in the exosome solution is 182.0 nm ± 1.4 nm.

12. The method according to claim 1, wherein The buffer solution is the tris(hydroxymethyl)aminomethane hydrochloride buffer solution, and the particle size distribution (D10 - D90) of 10% to 90% of the exosomes in the exosome solution is 119.6 nm ± 1.5 nm to 272.5 nm ± 8.1 nm.

13. The method according to claim 1, characterized in that, The 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer solution contains 0.1 M to 1 M of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid.

14. The method according to claim 1, wherein The buffer solution is the 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer solution, and the average particle size of most of the exosomes in the exosome solution is 176.8 nm ± 2.8 nm.

15. The method according to claim 1, wherein The buffer solution is the 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer solution, and the particle size distribution (D10 - D90) of 10% to 90% of the exosomes in the exosome solution is 117.2 nm ± 2.9 nm to 266.3 nm ± 5.3 nm.

16. The method according to claim 1, characterized in that, This exosome solution further includes epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), and glial cell line-derived neurotrophic factor (GDNF).

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