Method for preparing exosomes from human platelet

Abstract

To provide a method for preparing exosomes from a human platelet.SOLUTION: A method for producing exosomes using a human platelet according to the present invention includes: a step of preparing a human platelet; a step of treating the human platelet with a buffer to obtain a platelet solution; and a step of collecting supernatant of the platelet solution to obtain an exosome solution, wherein the buffer is one selected from the group consisting of a calcium ion buffer, phosphate buffered saline (PBS), tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl, pH 7.0 to 8.0) buffer, and hydroxyethylpiperazine ethane sulfonic acid buffer (HEPES buffer).SELECTED DRAWING: Figure 1

Description

Technical Field

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

Background Art

[0002] Exosomes are small endosomal-derived membrane microvesicles, which are a type of extracellular vesicles. The lipid bilayer vesicles of exosomes contain various signal factors such as nucleic acids, proteins, carbohydrates, and lipids. As carriers of these functional substances and as mediators of intercellular signal transduction, they can efficiently regulate gene expression and protein function expression in cells. Moreover, the functions of exosomes are determined by the type of cells from which they are derived, have various application aspects, and are involved in various clinical studies.

Summary of the Invention

Problems to be Solved by the Invention

[0003] An object of the present invention is to provide a method for producing exosomes from human platelets.

Means for Solving the Problems

[0004] In some embodiments, the method for producing exosomes includes the steps of 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, phosphate buffered saline (PBS), tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl, pH 7.0 - 8.0) buffer solution, and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer solution (HEPES Buffer).

[0005] In some embodiments, the calcium ion buffer is phosphate buffered saline containing at least one of calcium chloride, calcium carbonate, and calcium gluconate.

[0006] In some embodiments, the concentration of the calcium chloride is 2 mM to 20 mM.

[0007] In some embodiments, the average particle size range of exosomes in the exosome solution is 118.8 nm ± 1.0 nm to 156.3 nm ± 0.6 nm.

[0008] In some embodiments, 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 obtained by the above preparation method is 75.4 nm ± 1.5 nm to 171.8 nm ± 3.1 nm.

[0009] In some embodiments, 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 obtained by the preparation method is 95.3 nm ± 2.5 nm to 219.5 nm ± 5.9 nm.

[0010] In some embodiments, the buffer is 0.1× to 2× phosphate buffered saline.

[0011] In some embodiments, the buffer is phosphate buffered saline, and the average particle size range of exosomes in the exosome solution obtained by the preparation method is 167.9 nm ± 3.6 nm.

[0012] In some embodiments, the buffer is the phosphate buffered saline, and the particle size distribution (D10 - D90) of 10% to 90% of the exosomes in the exosome solution obtained by the preparation method is 103.0 nm ± 4.0 nm to 252.7 nm ± 8.0 nm.

[0013] In some embodiments, the tris(hydroxymethyl)aminomethane hydrochloride buffer solution contains 0.1 M to 1 M of tris(hydroxymethyl)aminomethane hydrochloride.

[0014] In some embodiments, the buffer solution is a tris(hydroxymethyl)aminomethane hydrochloride buffer solution, and the average particle size of the exosomes in the exosome solution obtained by the preparation method is 182.0 nm ± 1.4 nm.

[0015] In some embodiments, the 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 preparation method is 119.6 nm ± 1.5 nm to 272.5 nm ± 8.1 nm.

[0016] In some embodiments, the 2-(4-(2-hydroxyethyl)piperazin-1-yl)ethanesulfonic acid buffer solution contains 0.1 M to 1 M of 2-(4-(2-hydroxyethyl)piperazin-1-yl)ethanesulfonic acid.

[0017] In some embodiments, the buffer solution is a 2-(4-(2-hydroxyethyl)piperazin-1-yl)ethanesulfonic acid buffer solution, and the average particle size of most of the exosomes in the exosome solution obtained by the preparation method is 176.8 nm ± 2.8 nm.

[0018] In some embodiments, the buffer solution is a 2-(4-(2-hydroxyethyl)piperazin-1-yl)ethanesulfonic acid buffer solution, and the particle size distribution (D10 - D90) of 10% to 90% of the exosomes in the exosome solution obtained by the preparation method is 117.2 nm ± 2.9 nm to 266.3 nm ± 5.3 nm.

[0019] In some embodiments, the exosome solution further contains epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), and glial cell line-derived neurotrophic factor (GDNF).

Advantages of the Invention

[0020] In summary, the exosome preparation methods of all embodiments can provide an exosome solution containing the required exosomes in a relatively simple environment. In some embodiments, the obtained exosome solution has an exosome composition with different concentrations, average particle sizes, particle size distributions, or any combination thereof, based on the use of different types and / or different concentrations of buffer solutions. In some embodiments, the obtained exosome solution further contains growth factors such as epidermal growth factor, vascular endothelial growth factor, and glial cell line-derived neurotrophic factor, and an exosome solution containing growth factors with different concentration compositions can be obtained based on different types and / or different concentrations of buffer solutions in the exosome solution.

Brief Description of the Drawings

[0021]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Modes for Carrying Out the Invention

[0022] Refer to FIG. 1. First, human platelets are prepared (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 to obtain a precipitate, thereby removing the plasma. Subsequently, phosphate buffered saline filtered through a 0.02 μm filter is added to redissolve the precipitate, and after centrifuging at 3000×g for 30 minutes, the supernatant is removed to obtain a precipitate, thereby removing residual plasma and obtaining human platelets from which plasma has been removed.

[0023] After step S100, the human platelets are treated with a buffer to obtain a platelet solution (i.e., step S200). Here, the platelet solution already contains exosomes secreted by human platelets upon stimulation by the buffer.

[0024] In some embodiments of step S200, after adding a buffer to the human platelets, the human platelets are suspended in the buffer to obtain a platelet solution. In another part of the embodiments of step S200, after adding a buffer to the human platelets, the human platelets are uniformly mixed in the buffer by methods such as stirring or shaking to obtain a platelet solution. For example, after the human platelets come into contact with the buffer, the buffer activates the human platelets or reacts with the human platelets, thereby forming a platelet solution.

[0025] In some embodiments of step S200, the human platelets are treated with a buffer at an effective temperature to obtain a platelet solution. For example, the effective temperature may be 37°C. In one embodiment, the human platelets are treated with a buffer at 37°C to obtain a platelet solution.

[0026] In some embodiments of step S200, human platelets are treated with a buffer solution for an effective time. For example, after suspending human platelets in a buffer solution to form a platelet suspension, the platelet suspension is allowed to stand for an effective time to obtain a platelet solution. In some embodiments, the effective time may be from 1 hour to 3 hours. For example, the effective time may be 60 minutes, 90 minutes, 120 minutes, 150 minutes, or 180 minutes. In one embodiment, human platelets are cultured in a buffer solution at 37 °C for 1 hour.

[0027] Among them, the buffer solution is any one of a calcium ion buffer solution, phosphate buffered saline (PBS; abbreviated as PBS buffer solution), tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl, pH 7.5) buffer solution (abbreviated as Tris-HCl buffer solution (pH 7.5)), and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer solution (HEPES Buffer; abbreviated as HEPES buffer solution).

[0028] 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 from 2 mM to 20 mM. For example, the calcium ion buffer solution is prepared by mixing phosphate buffered saline and calcium chloride, and the concentration of calcium chloride is from 2 mM to 20 mM. In some embodiments, the concentration of calcium ions or calcium chloride is any one of 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, and 20 mM.

[0029] In some embodiments, phosphate buffered saline can be obtained by dissolving commercially available PBS tablets (e.g., Sigma tablets) in deionized water and then adding sodium chloride (NaCl) and potassium chloride (KCl). For example, commercially available PBS tablets can be dissolved in 200 milliliters of deionized water to obtain 1× phosphate buffered saline, which contains 137 mM of sodium chloride, 2.7 mM of potassium chloride, and 10 mM of phosphate buffer. In some embodiments, if necessary, phosphate buffered saline with a concentration ranging from 0.1-fold (0.1×) to 2-fold (2×) can be obtained by adjusting the amount of deionized water added (concentrations such as 0.1×, 0.5×, 1×, 1.5×, 2×, etc.). In some embodiments, the buffer is 1× phosphate buffered saline.

[0030] In some embodiments, Tris-HCl buffer (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 used is a 0.1 M to 1 M Tris-HCl buffer (pH 7.0 - 8.0). For example, the concentration of the Tris-HCl buffer (pH 7.0 - 8.0) can be 0.1 M, 0.2 M, 0.3 M, 0.4 M, 0.5 M, 0.6 M, 0.7 M, 0.8 M, 0.9 M, or 1.0 M. In some embodiments, the buffer is 0.1 M Tris-HCl buffer (pH 7.5).

[0031] In some embodiments, 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 used is a 0.1 M to 1 M HEPES buffer. For example, the concentration of the HEPES buffer can be 0.1 M, 0.2 M, 0.3 M, 0.4 M, 0.5 M, 0.6 M, 0.7 M, 0.8 M, 0.9 M, or 1.0 M. In some embodiments, the buffer is 0.1 M HEPES buffer and its pH value is 7.5.

[0032] After step S200, the supernatant of the platelet solution is collected to obtain an 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. When the supernatant of the platelet solution after centrifugation is obtained, an exosome solution is obtained.

[0033] Moreover, when different buffers are used to treat platelets for different times, both the concentration and particle size of the obtained exosomes are different.

[0034] In some embodiments, the exosome solution obtained by treating with calcium ion buffers containing calcium ions at different concentrations (e.g., 5 mM, 7.5 mM, 15 mM, or 20 mM, etc.) has an average exosome concentration of the contained exosomes of 6200 ± 403×10 7 particles / ml to 9740 ± 252×10 7 particles / ml, and the average particle size range of these exosomes is from 118.8 nm ± 1.0 nm to 156.3 nm ± 0.6 nm.

[0035] In some embodiments, the exosome solution obtained by treating with a calcium ion buffer of 5 mM calcium ions has a particle size distribution (D10 - D90) of 10% to 90% of the contained exosomes between 75.4 nm ± 1.5 nm and 171.8 nm ± 3.1 nm.

[0036] In some embodiments, the exosome solution obtained by treating with a calcium ion buffer of 7.5 mM calcium ions has a particle size distribution (D10 - D90) of 10% to 90% of the contained exosomes between 95.3 nm ± 2.5 nm and 219.5 nm ± 5.9 nm.

[0037] In some embodiments, the exosome solution obtained by treating with phosphate buffered saline has an average exosome concentration of the contained exosomes of 1890 ± 34.6×10 7is in the unit of particles per milliliter, and the average particle size range of these exosomes is 167.9 nm ± 3.6 nm.

[0038] In some embodiments, the exosome solution obtained by treatment with phosphate buffered saline has a particle size distribution (D10 - D90) of 10% to 90% of the exosomes contained therein between 103.0 nm ± 4.0 nm and 252.7 nm ± 8.0 nm.

[0039] In some embodiments, the exosome solution obtained by treatment with 0.1 M tris(hydroxymethyl)aminomethane hydrochloride buffer has an average exosome concentration of 4230 ± 322×10 7 particles per milliliter, and the average particle size range of these exosomes is 182.0 nm ± 1.4 nm.

[0040] In some embodiments, the exosome solution obtained by treatment with 0.1 M tris(hydroxymethyl)aminomethane hydrochloride buffer has a particle size distribution (D10 - D90) of 10% to 90% of the exosomes contained therein between 119.6 nm ± 1.5 nm and 272.5 nm ± 8.1 nm.

[0041] In some embodiments, the exosome solution obtained by treatment with 0.1 M hydroxyethylpiperazineethanesulfonic acid buffer has an average exosome concentration of 2460 ± 178×10 7 particles per milliliter, and the average particle size range of these exosomes is 176.8 nm ± 2.8 nm.

[0042] In some embodiments, the exosome solution obtained by treatment with 0.1 M hydroxyethylpiperazineethanesulfonic acid buffer has a particle size distribution (D10 - D90) of 10% to 90% of the exosomes contained therein between 117.2 nm ± 2.9 nm and 266.3 nm ± 5.3 nm.

[0043] In some embodiments, the exosome solution can promote cell migration and has potential in applications for wound healing. In some embodiments, the aforementioned exosome solution can promote the cell migration of epidermal cells and further promote skin wound healing and / or repair. Related technologies and research can refer to the literature of scholars such as Ayman et al. (Ayman Grada et al. (February 2017) J Invest Dermatol., 137(2):e11-e16), Luis et al. (Luis G Rodriguez et al. (2005) Methods Mol Biol., 294:23-9), Bereiter-Hahn et al. (Bereiter-Hahn, J (1984) Biology of the Integument (Berlin and HeidelbergtenSpringer-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), Marcia et al. (Marcia L. Usui (2008) Journal of Histochemistry and Cytochemistry, 56(7):687-696).

[0044] In some embodiments, the exosome solution further contains 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 the exosomes or are in the buffer outside the exosomes.

[0045] In some embodiments, the exosome solution containing EGF can promote the growth of epidermal cells to cover wounds, and can not only be applied clinically for wound treatment and use as a drug, but also has potential in the treatment of chronic diabetic foot ulcers. In some embodiments, the exosome solution containing VEGF can stimulate angiogenesis of granulation tissue and indirect angiogenesis in the periphery, and can be applied to research related to wound healing and diabetic foot ulcers in clinical practice. In some embodiments, the exosome solution containing GDNF can promote wound healing.

[0046] Related technologies and research can refer to the literature of scholars such as Kanchan (Kanchan Shakhakarmi et al. (2023) Archives of Pharmacal Research, 46:299 - 322), Hardwick (J Hardwicke et al. (June 2008) Surgeon, 6(3):172 - 7), Mert (Mert Dumantepe et al. (April 2015) Growth Factors. 33(2):128 - 32), Tiaka (Tiaka EK, Papanas N et al. (March 2012) Perspect Vasc Surg Endovasc Ther.;24(1):37 - 44), Jorge (Jorge Berlanga - Acosta et al. (July 2020) MEDICC Rev;22(3):24 - 31), Stephan (Stephan Barrientos et al. (2014) Wound Repair Regen;22(5):569 - 578), David (David O Bates et al. (June 2003) Int J Low Extrem Wounds;2(2):107 - 20), Aakansha (Aakansha Giri Goswami et al. (August 2022) Growth Factors.;40(3 - 4):73 - 88), Mohammad (Mohammad Zubair & Jamal Ahmad (2019) Reviews in Endocrine and Metabolic isorders 20:p207 - 217), Neda (Neda Vishlaghi et al. (April 2022) Exp Dermatol.;31(4):577 - 581), Thomas (Thomas S Lisse et al. (June 12, 2020) NPJ Regen Med.;5:13), Simon (Simon Mwangi et al. (March 2008) Gastroenterology;134(3):727 - 37).

[0047] 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.

[0048] The following experimental data are presented as mean ± standard deviation (SD) and analyzed for differences between two groups using 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.

Example

[0049] Preparation of exosome solution - Calcium ion buffer First, obtain a fresh human platelet concentrate unit from a registered blood bank (provider: Taipei Blood Center). After centrifuging the obtained human platelet concentrate unit at 3000 × g for 30 minutes, remove the supernatant to obtain a precipitate, thereby removing plasma. Subsequently, add 1× phosphate buffered saline (purchased from Gibco) filtered through a 0.02 μm filter to wash the precipitate, and centrifuge again at 3000 × g for 30 minutes to obtain a precipitate, thereby removing residual plasma and obtaining human platelets with plasma removed.

[0050] The human platelets with plasma removed are divided into five groups: a 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 is 1× phosphate buffered saline (purchased from Sigma; prepared with Sigma tablets), the buffer solution used in experimental group (A) is a 5 mM calcium ion buffer solution prepared with 1× phosphate buffered saline and calcium chloride (purchased from Spectrum), the buffer solution used in experimental group (B) is a 7.5 mM calcium ion buffer solution prepared with 1× phosphate buffered saline and calcium chloride, the buffer solution used in experimental group (C) is a 15 mM calcium ion buffer solution prepared with 1× phosphate buffered saline and calcium chloride, and the buffer solution used in experimental group (D) is a 20 mM calcium ion buffer solution prepared with 1× phosphate buffered saline and calcium chloride.

[0051] Subsequently, the human platelets with plasma removed in each group are treated with five different buffer solutions at 37°C for 1 hour respectively to obtain the platelet solutions of each group. The platelet solutions of each group are centrifuged at 3000×g for 30 minutes, and the supernatant of the platelet solution of each group is collected to obtain the exosome solution of each group.

Example

[0052] Preparation of exosome solution - Different types of buffer solutions First, a fresh human platelet concentrate unit is obtained from a registered blood bank (provider: Taipei Blood Center). After centrifuging the obtained human platelet concentrate unit at 3000×g for 30 minutes, the supernatant is removed to obtain a precipitate, thereby removing the plasma. Subsequently, 1× phosphate buffered saline filtered through a 0.02 μm filter is added to wash the precipitate, and the precipitate is obtained again by centrifuging at 3000×g for 30 minutes, thereby removing the residual plasma and obtaining human platelets with plasma removed.

[0053] Human platelets from which plasma has been removed are divided into four groups: a control group, experimental group (1), experimental group (2), and experimental group (3). Among them, the buffer solution used in the control group is physiological saline (Saline; purchased from Sigma), the buffer solution used in experimental group (1) is 1× phosphate buffered saline (purchased from Sigma; prepared with Sigma tablets), the buffer solution used in experimental group (2) is 0.1 M Tris-HCl buffer (pH 7.5; purchased from Sigma), and the buffer solution used in experimental group (3) is 0.1 M HEPES buffer (purchased from Sigma).

[0054] Subsequently, human platelets from which plasma has been removed in each group are treated with four different buffer solutions at 37°C for 1 hour each to obtain platelet solutions for each group. The platelet solutions for each group are centrifuged at 3000×g for 30 minutes, and the supernatant of the platelet solution for each group is collected to obtain exosome solutions for each group.

Example

[0055] Measurement of exosome concentration in the exosome solution of each group Using a Nanosight instrument, nanoparticle tracking analysis (NTA) is performed on the five groups of exosome solutions obtained in Example 1 and the four groups of exosome solutions obtained in Example 2 to obtain the exosome concentration of the exosome solution for each group.

[0056] The measurement results of the exosome concentration of the five groups of exosome solutions obtained in Example 1 are shown in Table 1 and Figure 2.

[0057]

Table 1

[0058] Here, the control group of Example 1 is labeled as "Control Group [1]" to distinguish it from the control group of Example 2.

[0059] As can be seen from Table 1 and Figure 2, since the buffer used for the control group [1] is 1× phosphate buffered saline, after treating human platelets with 1× phosphate buffered saline, the exosome concentration obtained by the secretion due to the stimulation of human platelets is 1890×10 7 per mL. Since the buffer used for experimental group (A) is 5 mM calcium ion buffer, after treating human platelets with 5 mM calcium ion buffer, the exosome concentration obtained by the secretion due to the stimulation of human platelets is 9060×10 7 per mL. Since the buffer used for experimental group (B) is 7.5 mM calcium ion buffer, after treating human platelets with 7.5 mM calcium ion buffer, the exosome concentration obtained by the secretion due to the stimulation of human platelets is 9740×10 7 per mL. Since the buffer used for experimental group (C) is 15 mM calcium ion buffer, after treating human platelets with 15 mM calcium ion buffer, the exosome concentration obtained by the secretion due to the stimulation of human platelets is 7510×10 7 per mL. Since the buffer used for experimental group (D) is 20 mM calcium ion buffer, after treating human platelets with 20 mM calcium ion buffer, the exosome concentration obtained by the secretion due to the stimulation of human platelets is 6200×10 7 per mL.

[0060] As can be seen from this, the addition of calcium ions can promote the exosome secretion of human platelets, and the exosome concentration in the exosome solution obtained by treating human platelets with 5 mM calcium ion buffer or 7.5 mM calcium ion buffer is at least 4.5 times higher than that of the treatment using 1× phosphate buffered saline.

[0061] Table 3 shows the measurement results of the exosome concentrations of the four groups of exosome solutions obtained in Example 2.

[0062]

Table 2

[0063] Here, the control group of Example 2 is labeled as "Control Group [2]" to distinguish it from the control group of Example 1.

[0064] As can be seen from Table 2 and Figure 3, since the buffer used for Control Group [2] is physiological saline, after treating human platelets with physiological saline, the exosome concentration obtained by the secretion due to the stimulation of human platelets was 1820×10 7 per mL. Since the buffer used for Experimental Group (I) is 1× phosphate buffered saline, after treating human platelets with 1× phosphate buffered saline, the exosome concentration obtained by the secretion due to the stimulation of human platelets was 1890×10 7 per mL. Since the buffer used for Experimental Group (II) is 0.1 M Tris-HCl buffer (pH 7.5), after treating human platelets with Tris-HCl buffer (pH 7.5), the exosome concentration obtained by the secretion due to the stimulation of human platelets was 4230×10 7 per mL. Since the buffer used for Experimental Group (III) is 0.1 M HEPES buffer, after treating human platelets with 0.1 M HEPES buffer, the exosome concentration obtained by the secretion due to the stimulation of human platelets was 2460×10 7 per mL.

[0065] From this, it can be seen that different types of buffers can promote the exosome secretion of human platelets compared to physiological saline. Based on this, users can select and use different buffers according to the required exosome concentration to prepare exosome solutions.

Example

[0066] Information regarding the exosome particle size of the exosome solutions of each group Nanoparticle Tracking Analysis (NTA) was performed on the four groups of exosome solutions obtained in Example 1 and the four groups of exosome solutions obtained in Example 2 using a Nanosight instrument to obtain information on the particle size, such as the average particle size and particle size distribution, of each group of exosome solutions.

[0067] Table 3 shows the measurement results of the particle size information of the four groups of exosome solutions obtained in Example 1.

[0068]

Table 3

[0069] In Table 3, D10 to D90 represent the particle size distribution from 10% to 90%.

[0070] As can be seen from Table 3, since the buffer solution used in experimental group (A) is a 5 mM calcium ion buffer solution, after treating human platelets with a 5 mM calcium ion buffer solution, the average particle size of the exosomes obtained by secretion due to stimulation of human platelets is 118.8 ± 1.0 nm, the mode diameter is 91.2 ± 6.2 nm, and the particle size distribution of 10% to 90% of the exosomes is 75.4 ± 1.5 nm to 171.8 ± 3.1 nm. Since the buffer solution used in experimental group (B) is a 7.5 mM calcium ion buffer solution, after treating human platelets with a 7.5 mM calcium ion buffer solution, the average particle size of the exosomes obtained by secretion due to stimulation of human platelets is 156.3 ± 0.6 nm, the mode diameter is 128.6 ± 7.5 nm, and the particle size distribution of 10% to 90% of the exosomes is 95.3 ± 2.5 nm to 219.5 ± 5.9 nm. Since the buffer solution used in experimental group (C) is a 15 mM calcium ion buffer solution, after treating human platelets with a 15 mM calcium ion buffer solution, the average particle size of the exosomes obtained by secretion due to stimulation of human platelets is 149.1 ± 4.8 nm, the mode diameter is 148.1 ± 6.5 nm, and the particle size distribution of 10% to 90% of the exosomes is 79.2 ± 8.4 nm to 217.9 ± 0.9 nm. Since the buffer solution used in experimental group (D) is a 20 mM calcium ion buffer solution, after treating human platelets with a 20 mM calcium ion buffer solution, the average particle size of the exosomes obtained by secretion due to stimulation of human platelets is 152.9 ± 3.9 nm, the mode diameter is 155.0 ± 9.1 nm, and the particle size distribution of 10% to 90% of the exosomes is 59.5 ± 7.2 nm to 221.5 ± 21.5 nm.

[0071] That is, after adjusting the calcium ion concentration, exosomes with different particle size sizes can be obtained. Therefore, the user can select different calcium ion buffer solutions for exosomes with the required particle size sizes, and obtain an exosome solution containing exosomes with the corresponding particle size sizes. Table 4 shows the measurement results of the particle size information of the four groups of exosome solutions obtained in Example 2.

[0072]

Table 4

[0073] In Table 4, D10 to D90 represent the particle size distribution from 10% to 90%.

[0074] Since the buffer solution used for the control group is physiological saline, after treating human platelets with physiological saline, the average particle size of exosomes obtained by secretion due to stimulation of human platelets is 167.2 ± 1.1 nm, the mode diameter is 143.8 ± 5.4 nm, and the particle size distribution of 10% to 90% of exosomes is from 108.9 ± 3.0 nm to 242.5 ± 6.4 nm. Since the buffer solution used for experimental group (one) is 1× phosphate buffered saline, after treating human platelets with 1× phosphate buffered saline, the average particle size of exosomes obtained by secretion due to stimulation of human platelets is 167.9 ± 3.6 nm, the mode diameter is 142.8 ± 2.0 nm, and the particle size distribution of 10% to 90% of exosomes is from 103.0 ± 4.0 nm to 252.7 ± 8.0 nm. Since the buffer solution used for experimental group (two) is 0.1 M Tris-HCl buffer (pH 7.5), after treating human platelets with Tris-HCl buffer (pH 7.5), the average particle size of exosomes obtained by secretion due to stimulation of human platelets is 182.0 ± 1.4 nm, the mode diameter is 156.5 ± 4.2 nm, and the particle size distribution of 10% to 90% of exosomes is from 119.6 ± 1.5 nm to 272.5 ± 8.1 nm. Since the buffer solution used for experimental group (three) is 0.1 M HEPES buffer, after treating human platelets with 0.1 M HEPES buffer, the average particle size of exosomes obtained by secretion due to stimulation of human platelets is 176.8 ± 2.8 nm, the mode diameter is 134.4 ± 2.4 nm, and the particle size distribution of 10% to 90% of exosomes is from 117.2 ± 2.9 nm to 266.3 ± 5.3 nm.

[0075] That is, by using different types of buffers, exosomes with different particle size distributions can be obtained. Therefore, the user can select different types of buffers for exosomes with the required particle size distribution, and obtain an exosome solution containing exosomes with the corresponding particle size distribution.

Example

[0076] Cell migration experiment - calcium ion buffer When a part of the body's cells and tissues is lost due to injury, the body needs to repair the injury. There are two different forms of repair: "regeneration" and "fibrous repair", and cell migration is involved in both of these two processes. Therefore, this experiment is designed to observe whether the sample has the ability to promote cell migration and to determine whether it has the potential ability to repair / heal wounds.

[0077] The cells used here are normal human dermal fibroblasts (purchased from Lonza). The cell culture medium used is Fibroblast Growth Medium-2 (FGM2; purchased from Lonza). The working medium used is 5×DMEM medium (purchased from Gibco) containing 0.1% fetal bovine serum (FBS). The samples used are exosome solutions obtained after treatment with different calcium ion buffers in Example 1, and are divided into five groups: a control group, and experimental groups (A) to (D). The experimental apparatus used is a 96-well ChemoTx (R) system (96-well ChemoTx (R) system), which includes an upper lid, a filter membrane, and a microplate. The analysis apparatus used is an automatic cell imaging system (ImageXpress Micro XLS).

[0078] First, normal human dermal fibroblasts are activated. And five groups of exosome solutions are diluted with DPBS (supplier: Gibco) to a concentration of 1.75×10 10 particles / mL, and then further diluted with the working medium to a concentration of 1.4×10 10 particles / mL to obtain the samples to be measured for subsequent experiments.

[0079] The samples to be measured for each group are respectively injected into a 96-well microplate. A filter membrane is placed on the 96-well microplate, and the treated normal human dermal fibroblasts are added onto the corresponding filter membranes of each well at a cell number of 1500 cells per well. Due to the chemotaxis of normal human dermal fibroblasts towards the samples to be measured for each group, the normal human dermal fibroblasts are attracted to pass through the 10-μm filter membrane and move below the filter membrane. Subsequently, they are cultured in a CO2 incubator for 5 hours. The ones that have not passed through the filter membrane above each group are wiped off and removed, fixed with methanol (supplier: Merck), and stained with DAPI staining solution (4’,6-diamidino-2-phenylindole; supplier: Calbiochem) for normal human dermal fibroblasts. Finally, analysis is performed for each group with an ImageXpress Micro XLS device, and the results are shown in Figure 4 and Table 5. In Figure 4, the p-value is a comparison with the control group.

[0080]

Table 5

[0081] Refer to Figure 4 and Table 5. The average number of cell migrations in the control group was 41.75 ± 21.2855, 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 calcium ions are contained in the buffer solution, the obtained 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 improved by at least twice compared with the control group. And when the calcium ion concentration is 15 mM, the number of migrating cells can be improved by at least 1.5 times compared with the control group.

[0082] From this, it can be seen that when preparing an exosome solution with a calcium ion buffer solution, cell migration can be promoted and it has the potential for wound healing.

Example

[0083] Cell Migration Experiment - Different Buffer Solutions Similar to Example 5, this experiment is designed to observe whether the sample has the ability to promote cell migration and to determine whether it has the potential ability to repair / heal wounds.

[0084] The cells used here are normal human dermal fibroblasts (purchased from Lonza). The cell culture medium used is Fibroblast Growth Medium - 2 (FGM2; purchased from Lonza). The working medium used is 5×DMEM medium (purchased from Gibco) containing 0.1% fetal bovine serum (FBS). The samples used are exosome solutions obtained after treatment with different calcium ion buffer solutions in Example 1, and are divided into four groups: a control group and experimental groups (one) to (three). The experimental device used is a 96-well ChemoTx (R) System (96-well ChemoTx (R)a system, including an upper lid, a filter membrane, and a microplate. The analysis device used is an automated cell imaging system (ImageXpress Micro XLS).

[0085] First, normal human dermal fibroblasts are activated. And five groups of exosome solutions are diluted with DPBS (supplier: Gibco) to a concentration of 1.75×10 10 particles / mL, and then further diluted with the working medium to a concentration of 1.4×10 10 particles / mL to obtain the samples to be measured for subsequent experiments.

[0086] Each group of samples to be measured is respectively injected into a 96-well microplate. A filter membrane is placed on the 96-well microplate, and the treated normal human dermal fibroblasts are added onto the corresponding filter membrane of each well at a cell count of 5000 cells per well. Due to the chemotaxis of normal human dermal fibroblasts towards each group of samples to be measured, the normal human dermal fibroblasts are attracted to pass through the 10-μm filter membrane and move below the filter membrane. Subsequently, they are cultured in a CO2 incubator for 5 hours. The ones that have not passed through the filter membrane above each group are wiped off and removed, fixed with methanol (supplier: Merck), and stained with DAPI staining solution (4’,6-diamidino-2-phenylindole; supplier: Calbiochem) for the normal human dermal fibroblasts. Finally, analysis is performed on each group using the ImageXpress Micro XLS device, and the results are shown in Figure 5 and Table 6. In Figure 5, the p-value is a comparison with the control group.

[0087]

Table 6

[0088] Refer to FIG. 5 and Table 6. The average number of cell migrations in the control group is 201 ± 47.91063, 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 using exosome solutions prepared with different buffers, they have the ability to increase the number of migrating cells. Among them, when the buffer is 0.1 M HEPES buffer, the number of migrating cells can be improved by at least 1.5 times compared with the control group. From this, it can be seen that exosome solutions prepared with different buffers can promote cell migration and have the potential for wound healing.

Example

[0089] Analysis of Growth Factors Here, a total of 3 groups of exosome solutions, namely the control group, experimental group (A), and experimental group B in Example 1, and a total of 4 groups of exosome solutions, namely the control group, experimental group (I), experimental group (II), and experimental group (III) in Example 2, were entrusted to Cenxianggu Co., Ltd. (Raybiotech) to analyze the growth factors contained in the solutions for the measurement items of the human growth factor array Q1 (#QAH-GF-1-SERV). The analysis results are shown in Table 7 and Table 8.

[0090] Regarding the above-mentioned measurement items, refer to the website: https: / / www.raybiotech.com / human-growth-factor-array-q1-qah-gf-1.

[0091] The growth factor analysis results of a total of 3 groups of exosome solutions, namely the control group, experimental group (A), and experimental group B in Example 1, are shown in Table 7.

[0092]

Table 7

[0093] Here, the control group in Example 1 is labeled as "Control Group [1]" to distinguish it from the control group in Example 2.

[0094] As can be seen from Table 7, the growth factors of the control group [1], experimental group (A), and experimental group (B) were measured, and all of them contained EGF, VEGF, and GDNF. The measured concentration of EGF in the control group [1] was 20.95 pg / ml, the measured concentration of VEGF was 974.33 pg / ml, and the measured concentration of GDNF was 18.72 pg / ml. The measured concentration of EGF in the experimental group (A) was 128 pg / ml, the measured concentration of VEGF was 816 pg / ml, and the measured concentration of GDNF was 102 pg / ml. The measured concentration of EGF in the experimental group (B) was 74 pg / ml, the measured concentration of VEGF was 1332 pg / ml, and the measured concentration of GDNF was 184 pg / ml. From this, it can be seen that the concentrations of EGF and GDNF contained in the exosome solution obtained with 5 mM or 7.5 mM calcium ion buffer were both higher than the concentrations measured in the control group [1], and the concentration of VEGF contained in the exosome solution obtained with 7.5 mM calcium ion buffer was also higher than that of the control group [1]. That is, the exosome solutions obtained with calcium ion buffers of different concentrations contain growth factors with different concentration compositions. And since EGF, VEGF, and GDNF are all growth factors beneficial to wound healing, the exosome solutions obtained with calcium ion buffers of different concentrations have potential in the application to wound healing.

[0095] Table 8 shows the results of the growth factor analysis of the exosome solutions of a total of 4 groups, namely the control group, experimental group (1), experimental group (2), and experimental group (3) in Example 2.

[0096]

Table 8

[0097] Here, the control group in Example 2 is denoted as "control group [2]" to distinguish it from the control group in Example 1.

[0098] As can be seen from Table 8, the growth factors of the control group [2], experimental group (one), experimental group (two), and experimental group (three) were measured, and all of them contained EGF, VEGF, and GDNF in the growth factors. The measured concentration of EGF in the control group [2] was 12.17 pg / ml, the measured concentration of VEGF was 108.61 pg / ml, and the GDNF concentration was not measured. The measured concentration of EGF in the experimental group (one) was 20.95 pg / ml, the measured concentration of VEGF was 974.33 pg / ml, and the measured concentration of GDNF was 18.72 pg / ml. The measured concentration of EGF in the experimental group (two) was 21.47 pg / ml, the measured concentration of VEGF was 794.75 pg / ml, and the measured concentration of GDNF was 81.7 pg / ml. The measured concentration of EGF in the experimental group (three) was 29.84 pg / ml, the measured concentration of VEGF was 417.88 pg / ml, and the measured concentration of GDNF was 72.55 pg / ml. As can be seen from this, the concentrations of EGF and GDNF contained in the experimental groups (one) to (three) were all higher than those of the control group, and the VEGF concentration in the experimental group (one) was higher than that of the other three groups, the GNDF concentration in the experimental group (two) was higher than that of the other three groups, and the EGF concentration in the experimental group (three) was higher than that of the other three groups. That is, since the highest-concentration growth factors in different groups are all different, the composition of the contained growth factors is also different. Moreover, the exosome solutions of the experimental groups (one) to (three) all have potential in the application to wound healing.

[0099] Summarizing the above, the method for preparing exosomes based on any embodiment of the present invention can provide an exosome solution containing the required exosomes in a relatively simple environment, and when using the obtained exosomes in other tests and applications, variables caused by unnecessary components (such as growth factors derived from the culture solution) can be avoided. In some embodiments, the obtained exosome solution has an exosome composition in a state such as different concentrations, average particle sizes, particle size distributions, or any combination thereof based on the use of different types and / or different concentrations of buffer solutions.

[0100] Although the technical content of the present invention has already been disclosed through the above-described embodiments, the above description is not used to limit the present invention. Those skilled in the art can make several changes and modifications without departing from the gist of the present invention, and all of them are included in the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the definition of the scope of claims attached to the specification.

Explanation of Reference Numerals

[0101] Steps S100 to S300

Claims

1. A method for producing exosomes from human platelets, comprising: a step of preparing human platelets; a step of 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, phosphate buffered saline (PBS), tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl, pH 7.0 to 8.0) buffer solution, and hydroxyethylpiperazineethanesulfonic acid buffer solution (HEPES Buffer); a step of collecting the supernatant of the platelet solution to obtain an exosome solution; A method for producing exosomes from human platelets, characterized by comprising the above steps.

2. The method for producing exosomes from human platelets according to claim 1, wherein the calcium ion buffer solution is phosphate buffered saline containing at least one of calcium chloride, calcium carbonate, and calcium gluconate.

3. The method for producing exosomes from human platelets according to claim 2, wherein the concentration of the calcium chloride is from 2 mM to 20 mM.

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

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

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

7. The method for producing exosomes from human platelets according to claim 1, wherein the buffer solution is the phosphate buffered saline from 0.1× to 2×.

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

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

10. The method for producing exosomes from human platelets according to claim 1, wherein the tris(hydroxymethyl)aminomethane hydrochloride buffer solution contains tris(hydroxymethyl)aminomethane hydrochloride from 0.1 M to 1 M.

11. The method for producing exosomes from human platelets according to claim 1, wherein the buffer solution is the tris(hydroxymethyl)aminomethane hydrochloride buffer solution, and the average particle size of exosomes in the exosome solution is 182.0 nm ± 1.4 nm.

12. The method for producing exosomes from human platelets 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 from 119.6 nm ± 1.5 nm to 272.5 nm ± 8.1 nm.

13. The method for producing exosomes from human platelets according to claim 1, wherein the hydroxyethylpiperazineethanesulfonic acid buffer solution contains hydroxyethylpiperazineethanesulfonic acid from 0.1 M to 1 M.

14. The method for producing exosomes from human platelets according to claim 1, wherein the buffer solution is the hydroxyethylpiperazineethanesulfonic 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 buffer solution is the hydroxyethylpiperazineethanesulfonic acid buffer solution, and 10% to 90% of the particle size distribution (D10 to D90) of the exosomes in the exosome solution is from 117.2 nm ± 2.9 nm to 266.3 nm ± 5.3 nm. A method for producing exosomes from human platelets according to claim 1, characterized in that.

16. The exosome solution further contains epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), and glial cell line-derived neurotrophic factor (GDNF). A method for producing exosomes from human platelets according to claim 1, characterized in that.

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