Extracellular vesicles / exosomes storage solutions and their mixtures

Abstract

To provide a solution for preserving extracellular vesicles / exosomes, and a mixed solution thereof, which overcome the challenges of difficulty in preservation and susceptibility to degradation of the extracellular vesicles / exosomes after purification.SOLUTION: A solution for preserving extracellular vesicles / exosomes includes: a carrier solvent selected from the group consisting of polysorbate 80, sucrose, and Polyethylene Glycol 3350 / 4000 (PEG-3350 / 4000); and a stabilizer selected from the group consisting of salts, amino acids, and amino acid salts, wherein the pH of the preservation solution ranges from 5 to 7.4.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a preservation solution, particularly a formulation of a preservation solution that allows extracellular vesicles / exosomes to be preserved for a long period of time in a frozen environment, and a preservation method thereof. [Background technology]

[0002] Because extracellular vesicles / exosomes are difficult to store after purification and are prone to degradation, they can often only be purified in the amount required for each use. Furthermore, when it comes to applications such as modification or drug binding, the overall time required is lengthened, making industrial development difficult. Summary of the Invention [Problem to be solved by the invention]

[0003] We provide extracellular vesicles / exosomes preservation solutions and mixture solutions thereof that overcome the problems of extracellular vesicles / exosomes being difficult to store after purification and being prone to degradation. [Means for solving the problem]

[0004] To overcome the problems that extracellular vesicles / exosomes are difficult to store and prone to degradation after purification, the present invention provides a storage solution for extracellular vesicles / exosomes, which comprises a carrier solvent selected from the group consisting of polysorbate 80 (Tween 80), sucrose, and polyethylene glycol 3350 / 4000 (PEG-3350 / 4000), and a stabilizer selected from the group consisting of salts, amino acids, and amino acid salts, and the pH value of the storage mixed solution is 5 to 7.4.

[0005] In the preservation solution, the salt is sodium acetate or sodium chloride, and the weight percent concentration of the salt is 0.1 to 2 of the total volume of the preservation solution.

[0006] In the preservation solution, the amino acid is selected from the group consisting of histidine, arginine, and glycine, and the weight percent concentration of the amino acid is 0.5 to 3 of the total volume of the preservation solution.

[0007] In the preservation solution, the amino acid salt is selected from the group consisting of histidine hydrochloride, arginine hydrochloride, and glycine hydrochloride, and the weight percent concentration of the amino acid salt is 0.05 to 0.25 of the total volume of the preservation solution.

[0008] In the preservative solution, the weight percent concentration of the polysorbate 80 (Polysorbate 80, Tween 80) is 0.01 to 0.03 of the total volume of the preservative solution, the weight percent concentration of the sucrose is 5 to 10 of the total volume of the preservative solution, and the weight percent concentration of the polyethylene glycol 3350 / 4000 (PEG-3350 / 4000) is 1 to 5 of the total volume of the preservative solution.

[0009] The present invention further comprises: Adding extracellular vesicles / exosomes to a storage solution to form a storage mixture; Gradually lowering the temperature of the storage mixture solution and storing it in a frozen storage environment; The present invention provides a method for preserving extracellular vesicles / exosomes, comprising:

[0010] In the above-described preservation method, the temperature of the frozen storage environment is -20°C or -80°C.

[0011] In the preservation method, after the preservation mixed solution is prepared, the solution is first drained and dried to form a drained and dried preservation sample, and the drained and dried preservation sample is then stored in the frozen storage environment.

[0012] The present invention further provides a mixed solution comprising a preservation solution and extracellular vesicles / exosomes.

[0013] In the mixed solution, the extracellular vesicles / exosomes were present at a concentration of 10 per milliliter in the storage mixed solution. 11 ~10 13 is the number of cells.

[0014] In the mixed solution, the extracellular vesicles / exosomes are modified extracellular vesicles modified with specific proteins.

[0015] In the mixed solution, the extracellular vesicles / exosomes are loaded with a drug. [Effects of the Invention]

[0016] The preservation solution provided by the present invention can be stored for a long period of time under the refrigerated environment conditions by selecting the carrier solvent and stabilizer to set storage conditions and pH suitable for different types of extracellular vesicles / exosomes.

[0017] The preservation solution can also preserve the extracellular vesicles / exosomes in different storage environments, which can aid in the commercial development of the product.

[0018] The present invention allows extracellular vesicles / exosomes to be frozen and stored in a specific liquid or dry form as needed, depending on the composition of the preservation solution. This allows them to be preserved for a long period of time, and maintains a certain reaction effect when thawed and reused, thereby facilitating research, development, and application in the medical industry, and improving time costs and commercial value. [Brief explanation of the drawings]

[0019] [Figure 1] FIG. 1 is a block diagram of the steps of a first preferred embodiment of the preparation method provided by the present invention. [Figure 2] FIG. 1 is a concentration test diagram of the extracellular vesicles of the first preferred embodiment provided by the present invention. [Figure 3] This is an expression diagram of marker proteins in extracellular vesicles in the first preferred embodiment provided by the present invention. [Figure 4] FIG. 2 is a block diagram of the steps of a second preferred embodiment of the preparation method provided by the present invention. [Figure 5] FIG. 10 is a concentration test diagram of the extracellular vesicles of the second preferred embodiment provided by the present invention. [Figure 6] This is an expression diagram of marker proteins in extracellular vesicles in the second preferred embodiment provided by the present invention. [Figure 7] FIG. 1 is a size analysis diagram of extracellular vesicles according to a second preferred embodiment of the present invention. [Figure 8] This is a drug loading analysis diagram of extracellular vesicles of a preferred embodiment provided by the present invention. [Figure 9] FIG. 1 is a first test diagram of the cytotoxicity effect of the extracellular vesicles of a preferred embodiment provided by the present invention. [Figure 10] FIG. 2 is a second test diagram of the cytotoxic effect of the extracellular vesicles of a preferred embodiment provided by the present invention. [Figure 11] This is a concentration test diagram for long-term frozen storage of extracellular vesicles of a preferred embodiment provided by the present invention. [Figure 12] FIG. 1 is the first test diagram of the cytotoxicity effect of long-term cryopreservation of extracellular vesicles of a preferred embodiment provided by the present invention. [Figure 13]FIG. 2 is a second test diagram showing the cytotoxic effect of long-term cryopreservation of extracellular vesicles of a preferred embodiment provided by the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0020] The present invention provides a storage solution for storing extracellular vesicles / exosomes, comprising water, a carrier solvent, and a stabilizer, the pH value of which is 5 to 7.4. The carrier solvent is selected from the group consisting of polysorbate 80 (Tween 80), sucrose, and polyethylene glycol 3350 / 4000 (PEG-3350 / 4000). The stabilizer stabilizes the biological activity of the extracellular vesicles / exosomes when mixed with the storage solution and allows them to adapt to different storage environments.

[0021] Preferably, the stabilizer comprises an amino acid or a salt of an amino acid, and more preferably, the stabilizer is selected from the group consisting of histidine, arginine, glycine, histidine hydrochloride, arginine hydrochloride, and glycine hydrochloride.

[0022] Preferably, the stabilizing agent comprises a salt for stabilizing the pH environment of the storage solution, and preferably, the salt is sodium acetate or sodium chloride.

[0023] Here, the weight percent concentration of the polysorbate 80 (Tween 80) is 0.01 to 0.03 of the total volume of the preservative solution, and the weight percent concentration of the sucrose is 5 to 10 of the total volume of the preservative solution.

[0024] The weight percent concentration of the amino acid is 0.5 to 3 based on the total volume of the preservation solution, and the weight percent concentration of the amino acid salt is 0.05 to 0.25 based on the total volume of the preservation solution.

[0025] The amount of the salts added to the preservation solution is adjusted according to the required pH value of the preservation solution.

[0026] Furthermore, the present invention provides Examples 1, 2, and 3, in which the extracellular vesicles / exosomes are preserved by cryopreservation. The cryopreservation may be liquid or dry. Here, phosphate buffered saline (PBS), a commonly used buffer solution, and dimethyl sulfoxide (DMSO) solution, a commonly used solution for preserving the extracellular vesicles / exosomes, are referred to as Comparative Examples 1 and 2, respectively.

[0027] Based on the concentration of extracellular vesicles added to each Comparative Example and Example, the number of extracellular vesicles in each Comparative Example and Example was compared using a nanoparticle tracking analysis, and the remaining concentration percentage of the extracellular vesicles in each Comparative Example and Example was calculated. Furthermore, the remaining concentration percentage of the extracellular vesicles in each Comparative Example and Example after the cryopreservation was compared to evaluate the decomposition rate of the extracellular vesicles in each Comparative Example and Example, and the preservation effect of the preservation solution during the cryopreservation was verified.

[0028] In Examples 1, 2, and 3, the carrier solvent and the stabilizer were added, and after confirming that the pH value was within an appropriate range, water was added to the preservative solution to the final required volume. The formulations of Examples 1, 2, and 3 are as shown in Tables 1 to 3, respectively.

[0029] [Table 1]

[0030] [Table 2]

[0031] [Table 3]

[0032] In Experiment 1, referring to Figures 1 to 3, the cryopreservation method was carried out on extracellular vesicles / exosomes by liquid preservation using the preservation solution, and the steps included the following:

[0033] S1: Prepare the preservative solution. The present invention uses the above Tables 1 to 3 as examples hereinafter, and the components in Tables 1 to 3 are mixed to form Example 1, Example 2, and Example 3, respectively.

[0034] S2. Form a storage mixture solution. The extracellular vesicles / exosomes are added to the storage solution to form a storage mixture solution.

[0035] Here, the concentration of the extracellular vesicles / exosomes in the storage mixture, PBS, and DMSO is 10 per milliliter. 11 ~10 13 It is preferable that the number of cells is .

[0036] In this embodiment, the extracellular vesicles are obtained by a common extraction technique. Here, the extracellular vesicles / exosomes may be modified extracellular vesicles modified with specific proteins produced by a conventional cellular process. The extracellular vesicles / exosomes or modified extracellular vesicles may be coated / loaded with a drug after undergoing a conventional cellular process.

[0037] S3: Stepwise temperature reduction: The temperature of the preservation mixture solution is gradually reduced by stepwise temperature reduction, and the preservation mixture solution is stored in a frozen storage environment. Here, the temperature reduction rate of the stepwise temperature reduction is 0.5 to 2°C per minute.

[0038] Here, the temperature of the frozen storage environment may be -20°C or -80°C.

[0039] Here, the temperature of the frozen storage environment is -80°C, for example, and the stepwise temperature reduction involves preparing the storage mixture at room temperature, then leaving it at -4°C and -20°C, respectively, and finally storing it in the storage environment at -80°C.

[0040] Next, referring to Figure 2, after the extracellular vesicles were mixed with Comparative Example 1, Comparative Example 2, Example 1, Example 2, and Example 3, the concentrations of the extracellular vesicles were tested before being placed in the frozen storage environment (D0) and after being stored in the frozen storage environment for 1 week (W1), 2 weeks (W2), 3 weeks (W3), and 4 weeks (W4). The residual concentrations of the extracellular vesicles in the frozen environment for each Comparative Example and each Example were compared to evaluate the effect of storing the extracellular vesicles in the frozen environment for each Example.

[0041] 2, it can be seen that the extracellular vesicles in Comparative Examples 1 and 2 were already significantly decomposed before cryopreservation, but that the extracellular vesicles in Examples 1, 2, and 3 all exhibited excellent cryopreservation effects. In particular, after 4 weeks (W4), the concentration of the extracellular vesicles in Example 1 was still maintained at 85% or more of the original concentration, while the concentrations of the extracellular vesicles in Examples 2 and 3 were maintained at 80-85%.

[0042] Next, referring to Figure 3, the conventional marker proteins CD9 and CD81 of the extracellular vesicles were marked with prominent marks, respectively, and the extracellular vesicles surviving after 4 weeks of cryopreservation in Comparative Example 2 and each Example were examined for biological activity, thereby verifying the applicability of the cryopreserved extracellular vesicles in Examples 1, 2, and 3. As is clear from the detection results of the marker protein CD9 in Examples 1, 2, and 3, the cryopreservation of the extracellular vesicles in Examples 1, 2, and 3 did not affect the biological activity of the extracellular vesicles, demonstrating their advantageous use in subsequent applications.

[0043] In Experiment 2, referring to Figures 4 to 6, the preservation solution was used to dry and preserve extracellular vesicles / exosomes, and the cryopreservation method was carried out. The difference from Experiment 1 is that the preservation mixture solution was formed, and after a stepwise temperature drop, step S4 was carried out, followed by draining, drying, and cryopreservation. The preservation mixture solution was drained and dried to form a drained and dried preservation sample, which was then stored in a -80°C environment.

[0044] Next, referring to Figure 5, the present invention compared the residual concentrations of the extracellular vesicles after storing the preservation mixed solution and the drained and dried preservation samples of Comparative Example 2 and each Example in the frozen environment at -80°C for 4 weeks, and compared the effects of the frozen preservation methods on the preservation of the extracellular vesicles during liquid preservation and dry preservation in each Example.

[0045] In Comparative Example 2, the extracellular vesicles after dry storage using DMSO were significantly more decomposed than those stored in liquid form, but excellent preservation effects were observed for all extracellular vesicles when cryopreserved using the preservation solutions provided in Examples 1 to 3. Furthermore, there was no significant difference in the preservation effects of the extracellular vesicles between the liquid storage method and the dry storage method.

[0046] Similarly, Figure 6 also shows that the marker proteins CD9 and CD81 of the extracellular vesicles are marked with prominent marks, respectively. The biological activity of the surviving extracellular vesicles after 4 weeks of dry storage in Comparative Example 2 and each Example was examined, and the applicability of the extracellular vesicles after cryopreservation in Examples 1, 2, and 3 was verified. As is clear from the detection results of the marker protein CD9 in Examples 1, 2, and 3, the biological activity of the extracellular vesicles of Examples 1, 2, and 3 is not affected after cryopreservation, which is consistent with the above-mentioned experimental results.

[0047] Furthermore, in Figure 7, the size of each extracellular vesicle after freeze-drying storage in Comparative Example 2 and Examples 1, 2, and 3 was compared and analyzed, confirming that the physiological form of the extracellular vesicles was not affected by the preservation solution and the cryopreservation method. As can be seen from the figure, there was no difference in the size of each extracellular vesicle after freeze-drying storage in Comparative Example 2 and each Example, but it can be seen that the concentration of extracellular vesicles after freeze-drying storage in Comparative Example 2 was significantly reduced.

[0048] As shown in Experiment 3, Figure 8, the present invention performs a drug loading process after thawing the extracellular vesicles, and drug testing confirms that the extracellular vesicles still have the ability to incorporate the drug after being cryopreserved in the preservation solution.

[0049] In this embodiment, doxorubicin (DOX) is used as the drug, and the reflected wavelength of the drug is detected to confirm whether the drug has been incorporated into the extracellular vesicles. The better the effect of loading the drug into the extracellular vesicles, the greater the rate at which the drug is incorporated, and the more prominent the reflected wavelength of the extracellular vesicles between 400 and 550, indicating that the extracellular vesicles are effective in transporting the drug.

[0050] The drug loading process provided by the present invention first 11The extracellular vesicles of the number of cells were added to 2 mg / mL of doxorubicin (DOX) and allowed to react, allowing the drug to be incorporated into the extracellular vesicles. After centrifugation, the uncoated DOX was removed to clear the extracellular vesicles, which could then be used for subsequent testing.

[0051] In this experiment, the group containing only the drug was designated as Control Group 1; the group containing the extracellular vesicles without drug loading was designated as Control Group 2; the group containing the extracellular vesicles after drug loading was designated as Control Group 3; and the group containing the extracellular vesicles after drug loading, frozen and stored in a dry storage form, and then redissolved was designated as Experimental Group 1. Here, Control Group 3 used the formulation provided in Example 1 as the medium for the drug loading process on the extracellular vesicles. Experimental Group 1 also used the formulation provided in Example 1 as the storage solution for the drug loading process and then frozen and stored. The results show that the drug content of the extracellular vesicles carrying the drug was relatively low after dry storage.

[0052] To prove that experimental group 1 still retains efficacy even after re-dissolving after undergoing the drug loading process and cryopreservation, the present invention further tests the therapeutic efficacy of the extracellular vesicles incorporating drugs through cryopreservation via the preservation solution.

[0053] Experiment 4: In this experiment, the extracellular vesicles were first loaded with the drug and then frozen in the storage solution (dry storage format). After thawing the extracellular vesicles containing the drug (doxorubicin), a cytotoxicity test was conducted on human breast cancer cells to confirm the cytotoxic effect of the extracellular vesicles containing the drug (doxorubicin).

[0054] Referring to Figure 9, the drug-containing extracellular vesicles (control group 3) and the freeze-dried drug-containing extracellular vesicles (experimental group 1) were added to the culture medium of the human breast cancer cells and incubated. After 12, 24, and 48 hours, the cell viability (%) of the human cancer cells in each group was compared with that before incubation, and the cytotoxic effects of control group 3 and experimental group 1 were compared. The results show that the drug-containing extracellular vesicles still have therapeutic efficacy for drug uptake, although the cytotoxic effect after cryopreservation was lower than that of the group that had not been cryopreserved.

[0055] Experiment 5: In this experiment, the extracellular vesicles were extracted after frozen storage (dried storage form), and then thawed and subjected to the drug loading process. A cytotoxicity test was also conducted on the human breast cancer cells to confirm the cytotoxic effect of the extracellular vesicles carrying the drug (doxorubicin).

[0056] 10, the present invention conducted a cytotoxicity test on human breast cancer cells using a group in which the extracellular vesicles were frozen in a dry state and then mixed with the drug (DOX) (experimental group 4), and a group in which the extracellular vesicles containing the drug were not frozen (control group 4). The results show that the cytotoxic effect of the cryopreserved extracellular vesicles thawed and combined with the drug (experimental group 4) on human breast cancer cells was not significantly different from that of control group 4, and both groups showed excellent cytotoxic effects.

[0057] Furthermore, compared with the results of Figure 9, a better procedure for preserving the extracellular vesicles and loading the drug can be established using the preservation solution provided by the present invention.

[0058] Experiment 6 (Figure 11) further compares the preservation effects of the extracellular vesicles / exosomes in different storage environments using the preservation solution provided by the present invention, and verifies the appropriateness of storage. The extracellular vesicles were placed in the preservation solution and stored at -80°C, -20°C, 4°C, room temperature, and 40°C. The concentration of the extracellular vesicles before storage was used as the standard, and the remaining concentration (%) of the extracellular vesicles in each group was measured after 1 week, 2 weeks, 4 weeks, 8 weeks, 16 weeks, and 26 weeks of storage.

[0059] The results show that under the conditions of the preservation solution provided by the present invention, the extracellular vesicles have a sufficiently excellent preservation effect in the frozen environment (-80°C and -20°C), and the preservation effect can be extended to more than six months (26 weeks), with the remaining concentration of the extracellular membrane reaching 80%. As the temperature of the preservation environment increases, the preservation effect of the extracellular vesicles decreases with the length of storage time.

[0060] Figures 11 and 12 further show that the drug (doxorubicin) loading process was carried out on the extracellular vesicles after they had been stored in the frozen environment for 12 and 14 weeks using the preservation solution provided by the present invention, and a cytotoxicity test was then carried out on the human breast cancer cells to confirm the drug uptake efficiency of the extracellular vesicles after long-term storage and the cytotoxic effect of the extracellular vesicles.

[0061] Here, the group of human breast cancer cells treated with the drug was designated as control group 5, the group of human breast cancer cells treated with the extracellular vesicles not loaded with the drug was designated as control group 6, and the group of extracellular vesicles loaded with the drug that had not been cryopreserved was designated as control group 7. The group of extracellular vesicles loaded with the drug after cryopreservation at -20°C was designated as experimental group 5, and the group of extracellular vesicles loaded with the drug after cryopreservation at -80°C was designated as experimental group 6.

[0062] The human breast cancer cells were co-cultured with each group and the cytotoxicity test was performed. The number of human breast cancer cells in each group was calculated on the first, second, and third days, and the total number of human breast cancer cells not subjected to the cytotoxicity test was compared to calculate the cytotoxicity death rate (%) of the human breast cancer cells.

[0063] As can be seen from the results, experimental groups 5 and 6 showed significant cytotoxicity on day 1. As the co-culture time increased, the toxic death rates (%) of the human breast cancer cells in experimental groups 5 and 6 continued to increase, and on days 2 and 3, experimental group 6 showed similar cytotoxicity results to control group 7. It has been proven that the preservation solution provided by the present invention can not only preserve the extracellular vesicles / exosomes for a long period of time in the refrigerated environment, but also maintain the biological activity of the extracellular vesicles / exosomes.

[0064] The preservation solution provided by the present invention can be stored for a long period of time under the refrigerated environment conditions by selecting the carrier solvent and stabilizer to set storage conditions and pH suitable for different types of extracellular vesicles / exosomes.

[0065] The preservation solution can also preserve the extracellular vesicles / exosomes in different storage environments, which can aid in the commercial development of the product.

[0066] The present invention allows extracellular vesicles / exosomes to be frozen and stored in a specific liquid or dry form as needed, depending on the composition of the preservation solution. This allows them to be preserved for a long period of time, and maintains a certain reaction effect when thawed and reused, thereby facilitating research, development, and application in the medical industry, and improving time costs and commercial value. [Explanation of symbols]

[0067] S1 Step S2 Step S3 Step S4 Step

Claims

1. A storage solution for storing extracellular vesicles / exosomes, comprising: a carrier solvent containing sucrose and polyethylene glycol 3350 / 4000 (PEG-3350 / 4000); a stabilizer selected from the group consisting of salts, amino acids, and amino acid salts; Including, The weight percent concentration of the sucrose is 5% to 10% of the total volume of the preservative solution, and the weight percent concentration of the polyethylene glycol 3350 / 4000 (PEG-3350 / 4000) is 1% of the total volume of the preservative solution; the pH value of the preservative solution is 5 to 7.4; The preservation solution maintains the concentration of extracellular vesicles / exosomes at least 80% of the concentration before storage when the extracellular vesicles / exosomes are stored in a frozen storage environment for at least 4 weeks.

2. 2. The preservative solution of claim 1, wherein the salt is sodium acetate or sodium chloride.

3. 2. The preservation solution according to claim 1, wherein the amino acid is selected from the group consisting of histidine, arginine, and glycine, and the weight percent concentration of the amino acid is 0.5 to 3 of the total volume of the preservation solution.

4. 2. The preservation solution according to claim 1, wherein the amino acid salt is selected from the group consisting of histidine hydrochloride, arginine hydrochloride, and glycine hydrochloride, and the weight percent concentration of the amino acid salt is 0.05 to 0.25 of the total volume of the preservation solution.

5. 10. A method for treating a skin ulcer comprising the preservative solution of claim 1 , Adding extracellular vesicles / exosomes to a storage solution to form a storage mixture; Gradually lowering the temperature of the storage mixture solution and storing it in a frozen storage environment; A preservation method for preserving extracellular vesicles / exosomes, comprising:

6. The method for preserving food according to claim 5, wherein the temperature of the frozen storage environment is -20°C or -80°C.

7. The preservation method according to claim 5, wherein the preservation mixed solution is first drained and dried to form a drained and dried preservation sample, and the drained and dried preservation sample is then stored in the frozen storage environment.

8. A mixed solution comprising the preservation solution according to claim 1 and extracellular vesicles / exosomes.

9. The extracellular vesicles / exosomes were added to the mixed solution at a concentration of 10 per milliliter. １１ ~10 １３ The mixed solution according to claim 8, wherein the number of cells is

10. The mixed solution according to claim 8, wherein the extracellular vesicles / exosomes are modified extracellular vesicles modified with a specific protein.

11. The mixed solution according to claim 8, wherein the extracellular vesicles / exosomes are loaded with a drug.

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