Stem cell exosome high-activity freeze-drying formula and freeze-drying process thereof

Abstract

The invention discloses a stem cell exosome high-activity freeze-drying formula and a freeze-drying process thereof, and relates to the technical field of biology, the stem cell exosome high-activity freeze-drying formula is prepared from the following components: 90-100 parts of stem cell exosome, 1-5 parts of sodium alginate, 3-6 parts of a sericin protein mixture, 5-15 parts of mannitol, 0.3-0.6 part of a tartary buckwheat extract, and beta-mercaptoethanol; the stem cell exosome freeze-drying formula prepared by the invention has higher activity, and can maintain higher recovery rate after hydration, so that the biological stability of the stem cell exosome freeze-drying formula is greatly improved. According to the method, moisture is directly sublimated from a solid state to a gaseous state under a vacuum condition, so that the damage to an exosome structure caused by ice crystal formation is avoided, the low-temperature and low-pressure environment can effectively reduce the oxidation and degradation reaction of the exosome, and the biological activity of the obtained stem cell exosome freeze-drying formula is kept.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular to a high-activity freeze-dried formula of stem cell exosomes. Background Art

[0002] With the rapid development of biotechnology, stem cell research has been continuously developed. In view of the development of stem cells, freeze-dried stem cell exosomes have been further developed due to their unique biological properties and broad application prospects.

[0003] Stem cell exosomes are tiny vesicles secreted by cells under specific conditions, with a diameter of about 30-150 nanometers. They are rich in a variety of bioactive substances, such as proteins, lipids, nucleic acids, etc., which play an important role in information transmission and material exchange between cells.

[0004] In order to facilitate the storage and transportation of stem cell exosomes, the existing technology uses freeze-drying technology to make a powdered product. This freeze-dried powder retains the complete structure and biological activity of the exosomes.

[0005] After freeze-drying, stem cell exosomes can be used in the medical field. After freeze-drying, stem cell exosomes can be used for tissue repair and treatment of different disease models.

[0006] However, the exosome recovery rate of the freeze-dried stem cell exosome formula prepared by the prior art is relatively low, resulting in a significant decrease in the activity of the exosomes, causing a significant decrease in its effect on tissue repair and treatment.

[0007] Based on this, a high-activity freeze-dried formula of stem cell exosomes is provided to solve existing technical problems. Summary of the invention

[0008] The purpose of the present invention is to provide a high-activity freeze-dried formula of stem cell exosomes and a freeze-drying process thereof to solve the deficiencies in the prior art.

[0009] The technical solution adopted by the present invention is as follows:

[0010] A high-activity freeze-dried formula of stem cell exosomes comprises the following ingredients by weight: 90-100 parts of stem cell exosomes, 1-5 parts of sodium alginate, 3-6 parts of sericin mixture, 5-15 parts of mannitol, 0.3-0.6 parts of tartary buckwheat extract, and 0.3-0.6 parts of β-mercaptoethanol.

[0011] As a further technical solution, the weight ratio of the sericin mixture: buckwheat extract: β-mercaptoethanol is 10:1:1.

[0012] As a further technical solution, the method for preparing the tartary buckwheat extract is:

[0013] The dried tartary buckwheat is added into a grinder, pulverized, and passed through an 80-mesh sieve to obtain tartary buckwheat flour;

[0014] Then, the tartary buckwheat flour is defatted by using petroleum ether to obtain defatted tartary buckwheat flour;

[0015] The defatted buckwheat flour is mixed with an ethanol solution having a mass fraction of 80%, and then refluxed for 2 hours, filtered, and the filtrate is removed to obtain a filter residue, which is dried to a constant weight to obtain a preliminary treated product;

[0016] The purified water was subjected to plasma treatment for 10 minutes to obtain pre-treated purified water;

[0017] The obtained pre-treated pure water is mixed with the preliminary treated product, subjected to ultrasonic dispersion for 10 minutes, and then subjected to heating reflux extraction for 4-5 hours, and finally subjected to a centrifugal treatment to obtain the supernatant;

[0018] The supernatant was concentrated to 40% of the original volume;

[0019] Add 3 times the volume of anhydrous ethanol to the concentrated supernatant, place it at 4°C for alcohol precipitation for 24 hours, and then perform a second centrifugation treatment, collect the precipitate, and obtain it after drying.

[0020] As a further technical solution, petroleum ether is used to degrease the tartary buckwheat flour as follows:

[0021] The tartary buckwheat flour and petroleum ether were mixed, stirred at a speed of 500 r / min for 2 hours, allowed to stand for 1 hour, and then dried by rotary evaporation;

[0022] The mixing ratio of tartary buckwheat flour and petroleum ether is 12-18 g: 150 mL;

[0023] The ethanol solution with a mass fraction of 80% and the defatted buckwheat flour are mixed in a mass ratio of 1:5.

[0024] As a further technical solution: the method for preparing pretreated pure water is:

[0025] The purified water is subjected to plasma treatment;

[0026] Among them, the plasma treatment time is 30min and the power is 180W;

[0027] The plasma processing gas is helium.

[0028] As a further technical solution, the mixing ratio of the pre-treated pure water to the primary treated product is 1:12-15;

[0029] The rotation speed of the centrifugal treatment is 7500r / min;

[0030] The rotation speed of the secondary centrifugal treatment is 6000r / min.

[0031] As a further technical solution, the method for preparing the sericin mixture is:

[0032] First, the cocoons were washed with distilled water for 10 minutes and then dried to constant weight at 60°C for 10 hours.

[0033] Cutting the cleaned and dried silkworm cocoons into pieces to obtain silkworm cocoon scraps;

[0034] Adding the silkworm cocoon fragments to distilled water, stirring and mixing, to obtain a mixed solution;

[0035] Adjust the pH of the mixed solution to 6.5;

[0036] Papain is added to the mixed solution, and the mixture is stirred for 2-3 hours. The mixture is then inactivated and centrifuged. The supernatant is taken and freeze-dried to obtain a sericin mixture.

[0037] As a further technical solution: the cocoon fragments and distilled water are mixed in a mass ratio of 1:40-50;

[0038] The mixing ratio of the mixed solution to papain is 1kg:3g;

[0039] After the papain is added, stirring and mixing is performed at 55° C.;

[0040] Among them, inactivation was treated at 95°C for 5 min;

[0041] The centrifugal speed is 4500r / min.

[0042] A freeze-drying process for a high-activity freeze-dried formula of stem cell exosomes comprises the following steps:

[0043] S1. First, add the tartary buckwheat extract and the sericin mixture to sterile water in sequence, and stir to mix evenly to obtain a first mixed solution;

[0044] S2 adds the stem cell exosomes to the first mixed solution, mixes them evenly, and cultures them at 37° C. for 2 hours to obtain a culture solution;

[0045] S3: adding sodium alginate, mannitol, and β-mercaptoethanol to the culture solution in sequence, mixing them evenly, and culturing them at 37° C. for 30 minutes to obtain a second mixed solution;

[0046] S4 pre-freezes the second mixed solution for 24 hours;

[0047] S5 is pre-frozen and then frozen for 24 hours, and then stored at 4°C.

[0048] As a further technical solution: the concentration of the tartary buckwheat extract in the first mixed solution is 1 mol / L;

[0049] The pre-freezing temperature is -80℃;

[0050] The freeze-drying temperature was -55°C and the vacuum pressure was 12Pa.

[0051] Beneficial effects:

[0052] The stem cell exosome freeze-dried formula prepared by the present invention has high activity and can maintain a high recovery rate after hydration, thereby greatly improving its biological stability.

[0053] The present invention mainly avoids the destruction of the exosome structure by ice crystal formation by directly sublimating water from solid to gas under vacuum conditions. This low temperature and low pressure environment can effectively reduce the oxidation and degradation reactions of exosomes, so that the obtained stem cell exosome freeze-dried formula maintains its biological activity.

[0054] In the process of the present invention, a pre-freezing treatment is performed, mainly placing the solution in a low-temperature environment so that the water therein freezes to form a stable crystal structure, paving the way for subsequent freeze-drying. In the subsequent freeze-drying process, the ice crystals are sublimated to finally obtain dry exosome powder.

[0055] The present invention introduces a sericin mixture, which helps to maintain the structural and functional integrity of biomacromolecules such as proteins and nucleic acids in exosomes, and can reduce oxidation and degradation during the freeze-drying process.

[0056] The introduced bitter buckwheat extract can protect the cell membrane structure during the freeze-drying process, and the cell membrane structure of the liver cell exosomes can be protected, which has an extremely important impact on ensuring the biological activity of the exosomes. A small amount of water will remain in the freeze-dried powder in an amorphous state, and with the effect of β-mercaptoethanol, it can further protect the cell membrane from mechanical damage. The introduction of sodium alginate and mannitol can further improve the stability of the exosome freeze-dried powder, and it can be stored for a long time at low temperature without affecting its biological activity, and has a high recovery rate.

[0057] The highly active freeze-dried exosome formula product prepared by the present invention can be significantly applied in the fields of regenerative medicine, disease diagnosis and treatment, and medical cosmetology. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 This is a picture of the product of the highly active freeze-dried formula of exosomes in Example 1;

[0059] Figure 2 This is a picture of the sericin mixture product in Example 1. DETAILED DESCRIPTION

[0060] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0061] Example 1

[0062] A highly active freeze-dried formula of stem cell exosomes:

[0063] Components Weight Stem Cell Exosomes 90 servings Sodium alginate 1 serving Sericin Blend 3 servings Mannitol 5 servings Buckwheat Extract 0.3 parts β-Mercaptoethanol 0.3 parts

[0064] The weight ratio of sericin mixture: tartary buckwheat extract: β-mercaptoethanol is 10:1:1.

[0065] The preparation method of tartary buckwheat extract is as follows:

[0066] The dried tartary buckwheat is added into a grinder, pulverized, and passed through an 80-mesh sieve to obtain tartary buckwheat flour;

[0067] Then, the tartary buckwheat flour is defatted by using petroleum ether to obtain defatted tartary buckwheat flour;

[0068] The defatted buckwheat flour is mixed with an ethanol solution having a mass fraction of 80%, and then refluxed for 2 hours, filtered, and the filtrate is removed to obtain a filter residue, which is dried to a constant weight to obtain a preliminary treated product;

[0069] The purified water was subjected to plasma treatment for 10 minutes to obtain pre-treated purified water;

[0070] The obtained pre-treated pure water is mixed with the preliminary treated product, subjected to ultrasonic dispersion for 10 minutes, and then subjected to heating reflux extraction for 4-5 hours, and finally subjected to a centrifugal treatment to obtain a supernatant; the rotation speed of the centrifugal treatment is 7500r / min;

[0071] The supernatant was concentrated to 40% of the original volume;

[0072] Add 3 times the volume of anhydrous ethanol to the concentrated supernatant, place it at 4°C for alcohol precipitation for 24 hours, and then centrifuge it for the second time, collect the precipitate, and dry it to obtain;

[0073] The rotation speed of the secondary centrifugal treatment is 6000r / min.

[0074] The degreasing treatment of tartary buckwheat flour with petroleum ether is as follows:

[0075] The tartary buckwheat flour and petroleum ether were mixed, stirred at a speed of 500 r / min for 2 hours, allowed to stand for 1 hour, and then dried by rotary evaporation;

[0076] The mixing ratio of tartary buckwheat flour and petroleum ether is 12 g: 150 mL;

[0077] The ethanol solution with a mass fraction of 80% and the defatted buckwheat flour are mixed in a mass ratio of 1:5.

[0078] The method for preparing pretreated pure water is:

[0079] The purified water is subjected to plasma treatment;

[0080] Among them, the plasma treatment time is 30min and the power is 180W;

[0081] The plasma processing gas is helium.

[0082] The mixing ratio of pre-treated purified water and primary treated material is 1:12;

[0083] The sericin mixture is prepared as follows:

[0084] First, the silk cocoons are washed with distilled water for 10 minutes, and then dried to constant weight, wherein the drying temperature is 60°C and the time is 10 hours; the washed and dried silk cocoons are cut into pieces to obtain silk cocoon scraps; the silk cocoon scraps and distilled water are mixed in a mass ratio of 1:40; the silk cocoon scraps are added to the distilled water, stirred and mixed to obtain a mixed solution; the pH of the mixed solution is adjusted to 6.5; papain is added to the mixed solution, stirred and mixed at 55°C for 2 hours, and then inactivated, and then centrifuged at a centrifugal speed of 4500r / min, the supernatant is taken, and freeze-dried to obtain a sericin mixture; the mixing ratio of the mixed solution to papain is 1kg:3g; wherein the inactivation is treated at 95°C for 5 minutes;

[0085] A freeze-drying process for a high-activity freeze-dried formula of stem cell exosomes comprises the following steps:

[0086] S1. First, add the tartary buckwheat extract and the sericin mixture to sterile water in sequence, and stir to mix evenly to obtain a first mixed solution;

[0087] S2 adds the stem cell exosomes to the first mixed solution, mixes them evenly, and cultures them at 37° C. for 2 hours to obtain a culture solution;

[0088] S3: adding sodium alginate, mannitol, and β-mercaptoethanol to the culture solution in sequence, mixing them evenly, and culturing them at 37° C. for 30 minutes to obtain a second mixed solution;

[0089] S4 pre-freezes the second mixed solution for 24 hours;

[0090] S5 is pre-frozen and then frozen for 24 hours, and then stored at 4°C.

[0091] The concentration of the tartary buckwheat extract in the first mixed solution is 1 mol / L;

[0092] The pre-freezing temperature is -80℃;

[0093] The freeze-drying temperature was -55°C and the vacuum pressure was 12Pa.

[0094] Example 2

[0095] A highly active freeze-dried formula of stem cell exosomes:

[0096] Components Weight Stem Cell Exosomes 92 copies Sodium alginate 2 servings Sericin Blend 4 servings Mannitol 7 servings Buckwheat Extract 0.4 parts β-Mercaptoethanol 0.4 parts

[0097] The weight ratio of sericin mixture: tartary buckwheat extract: β-mercaptoethanol is 10:1:1.

[0098] The preparation method of tartary buckwheat extract is as follows:

[0099] The dried tartary buckwheat is added into a grinder, pulverized, and passed through an 80-mesh sieve to obtain tartary buckwheat flour;

[0100] Then, the tartary buckwheat flour is defatted by using petroleum ether to obtain defatted tartary buckwheat flour;

[0101] The defatted buckwheat flour is mixed with an ethanol solution having a mass fraction of 80%, and then refluxed for 2 hours, filtered, and the filtrate is removed to obtain a filter residue, which is dried to a constant weight to obtain a preliminary treated product;

[0102] The purified water was subjected to plasma treatment for 10 minutes to obtain pre-treated purified water;

[0103] The obtained pre-treated pure water was mixed with the preliminary treated product, subjected to ultrasonic dispersion for 10 minutes, and then subjected to heating reflux extraction for 5 hours, and finally subjected to a centrifugal treatment to obtain a supernatant; the rotation speed of the centrifugal treatment was 7500 r / min;

[0104] The supernatant was concentrated to 40% of the original volume;

[0105] Add 3 times the volume of anhydrous ethanol to the concentrated supernatant, place it at 4°C for alcohol precipitation for 24 hours, and then centrifuge it for the second time, collect the precipitate, and dry it to obtain;

[0106] The rotation speed of the secondary centrifugal treatment is 6000r / min.

[0107] The degreasing treatment of tartary buckwheat flour with petroleum ether is as follows:

[0108] The tartary buckwheat flour and petroleum ether were mixed, stirred at a speed of 500 r / min for 2 hours, allowed to stand for 1 hour, and then dried by rotary evaporation;

[0109] The mixing ratio of tartary buckwheat flour and petroleum ether is 13 g:150 mL;

[0110] The ethanol solution with a mass fraction of 80% and the defatted buckwheat flour are mixed in a mass ratio of 1:5.

[0111] The method for preparing pretreated pure water is:

[0112] The purified water is subjected to plasma treatment;

[0113] Among them, the plasma treatment time is 30min and the power is 180W;

[0114] The plasma processing gas is helium.

[0115] The mixing ratio of pre-treated purified water and primary treated material is 1:14;

[0116] The sericin mixture is prepared as follows:

[0117] First, the silk cocoons are washed with distilled water for 10 minutes, and then dried to constant weight, wherein the drying temperature is 60°C and the time is 10 hours; the washed and dried silk cocoons are cut into pieces to obtain silk cocoon scraps; the mass ratio of the silk cocoon scraps to distilled water is 1:45; the silk cocoon scraps are added to the distilled water, stirred and mixed to obtain a mixed solution; the pH of the mixed solution is adjusted to 6.5; papain is added to the mixed solution, stirred and mixed at 55°C for 2-3 hours, and then inactivated, and then centrifuged at a centrifugal speed of 4500r / min, the supernatant is taken, and freeze-dried to obtain a sericin mixture; the mixing ratio of the mixed solution to papain is 1kg:3g; wherein the inactivation is treated at 95°C for 5 minutes;

[0118] A freeze-drying process for a high-activity freeze-dried formula of stem cell exosomes comprises the following steps:

[0119] S1. First, add the tartary buckwheat extract and the sericin mixture to sterile water in sequence, and stir to mix evenly to obtain a first mixed solution;

[0120] S2 adds the stem cell exosomes to the first mixed solution, mixes them evenly, and cultures them at 37° C. for 2 hours to obtain a culture solution;

[0121] S3: adding sodium alginate, mannitol, and β-mercaptoethanol to the culture solution in sequence, mixing them evenly, and culturing them at 37° C. for 30 minutes to obtain a second mixed solution;

[0122] S4 pre-freezes the second mixed solution for 24 hours;

[0123] S5 is pre-frozen and then frozen for 24 hours, and then stored at 4°C.

[0124] The concentration of the tartary buckwheat extract in the first mixed solution is 1 mol / L;

[0125] The pre-freezing temperature is -80℃;

[0126] The freeze-drying temperature was -55°C and the vacuum pressure was 12Pa.

[0127] Example 3

[0128] A highly active freeze-dried formula of stem cell exosomes:

[0129] Components Weight Stem Cell Exosomes 98 copies Sodium alginate 3 servings Sericin Blend 5 servings Mannitol 8 servings Buckwheat Extract 0.5 serving β-Mercaptoethanol 0.5 serving

[0130] The weight ratio of sericin mixture: tartary buckwheat extract: β-mercaptoethanol is 10:1:1.

[0131] The preparation method of tartary buckwheat extract is as follows:

[0132] The dried tartary buckwheat is added into a grinder, pulverized, and passed through an 80-mesh sieve to obtain tartary buckwheat flour;

[0133] Then, the tartary buckwheat flour is defatted by using petroleum ether to obtain defatted tartary buckwheat flour;

[0134] The defatted buckwheat flour is mixed with an ethanol solution having a mass fraction of 80%, and then refluxed for 2 hours, filtered, and the filtrate is removed to obtain a filter residue, which is dried to a constant weight to obtain a preliminary treated product;

[0135] The purified water was subjected to plasma treatment for 10 minutes to obtain pre-treated purified water;

[0136] The obtained pre-treated pure water was mixed with the preliminary treated product, subjected to ultrasonic dispersion for 10 minutes, and then subjected to heating reflux extraction for 4.5 hours, and finally subjected to a centrifugal treatment to obtain a supernatant; the rotation speed of the centrifugal treatment was 7500 r / min;

[0137] The supernatant was concentrated to 40% of the original volume;

[0138] Add 3 times the volume of anhydrous ethanol to the concentrated supernatant, place it at 4°C for alcohol precipitation for 24 hours, and then centrifuge it for the second time, collect the precipitate, and dry it to obtain;

[0139] The rotation speed of the secondary centrifugal treatment is 6000r / min.

[0140] The degreasing treatment of tartary buckwheat flour with petroleum ether is as follows:

[0141] The tartary buckwheat flour and petroleum ether were mixed, stirred at a speed of 500 r / min for 2 hours, allowed to stand for 1 hour, and then dried by rotary evaporation;

[0142] The mixing ratio of tartary buckwheat flour and petroleum ether is 15 g:150 mL;

[0143] The ethanol solution with a mass fraction of 80% and the defatted buckwheat flour are mixed in a mass ratio of 1:5.

[0144] The method for preparing pretreated pure water is:

[0145] The purified water is subjected to plasma treatment;

[0146] Among them, the plasma treatment time is 30min and the power is 180W;

[0147] The plasma processing gas is helium.

[0148] The mixing ratio of pre-treated purified water and primary treated material is 1:12;

[0149] The sericin mixture is prepared as follows:

[0150] First, the silk cocoons are washed with distilled water for 10 minutes, and then dried to constant weight, wherein the drying temperature is 60°C and the time is 10 hours; the washed and dried silk cocoons are cut into pieces to obtain silk cocoon scraps; the silk cocoon scraps and distilled water are mixed in a mass ratio of 1:42; the silk cocoon scraps are added to the distilled water, stirred and mixed to obtain a mixed solution; the pH of the mixed solution is adjusted to 6.5; papain is added to the mixed solution, stirred and mixed at 55°C for 2-3 hours, and then inactivated, and then centrifuged at a centrifugal speed of 4500r / min, the supernatant is taken, and freeze-dried to obtain a sericin mixture; the mixing ratio of the mixed solution and papain is 1kg:3g; wherein the inactivation is treated at 95°C for 5 minutes;

[0151] A freeze-drying process for a high-activity freeze-dried formula of stem cell exosomes comprises the following steps:

[0152] S1. First, add the tartary buckwheat extract and the sericin mixture to sterile water in sequence, and stir to mix evenly to obtain a first mixed solution;

[0153] S2 adds the stem cell exosomes to the first mixed solution, mixes them evenly, and cultures them at 37° C. for 2 hours to obtain a culture solution;

[0154] S3: adding sodium alginate, mannitol, and β-mercaptoethanol to the culture solution in sequence, mixing them evenly, and culturing them at 37° C. for 30 minutes to obtain a second mixed solution;

[0155] S4 pre-freezes the second mixed solution for 24 hours;

[0156] S5 is pre-frozen and then frozen for 24 hours, and then stored at 4°C.

[0157] The concentration of the tartary buckwheat extract in the first mixed solution is 1 mol / L;

[0158] The pre-freezing temperature is -80℃;

[0159] The freeze-drying temperature was -55°C and the vacuum pressure was 12Pa.

[0160] Example 4

[0161] A highly active freeze-dried formula of stem cell exosomes:

[0162] Components Weight Stem Cell Exosomes 95 copies Sodium alginate 4.5 servings Sericin Blend 5 servings Mannitol 10 servings Buckwheat Extract 0.45 β-Mercaptoethanol 0.45

[0163] The weight ratio of sericin mixture: tartary buckwheat extract: β-mercaptoethanol is 10:1:1.

[0164] The preparation method of tartary buckwheat extract is as follows:

[0165] The dried tartary buckwheat is added into a grinder, pulverized, and passed through an 80-mesh sieve to obtain tartary buckwheat flour;

[0166] Then, the tartary buckwheat flour is defatted by using petroleum ether to obtain defatted tartary buckwheat flour;

[0167] The defatted buckwheat flour is mixed with an ethanol solution having a mass fraction of 80%, and then refluxed for 2 hours, filtered, and the filtrate is removed to obtain a filter residue, which is dried to a constant weight to obtain a preliminary treated product;

[0168] The purified water was subjected to plasma treatment for 10 minutes to obtain pre-treated purified water;

[0169] The obtained pre-treated pure water was mixed with the preliminary treated product, subjected to ultrasonic dispersion for 10 minutes, and then subjected to heating reflux extraction for 4.5 hours, and finally subjected to a centrifugal treatment to obtain a supernatant; the rotation speed of the centrifugal treatment was 7500 r / min;

[0170] The supernatant was concentrated to 40% of the original volume;

[0171] Add 3 times the volume of anhydrous ethanol to the concentrated supernatant, place it at 4°C for alcohol precipitation for 24 hours, and then centrifuge it for the second time, collect the precipitate, and dry it to obtain;

[0172] The rotation speed of the secondary centrifugal treatment is 6000r / min.

[0173] The degreasing treatment of tartary buckwheat flour with petroleum ether is as follows:

[0174] The tartary buckwheat flour and petroleum ether were mixed, stirred at a speed of 500 r / min for 2 hours, allowed to stand for 1 hour, and then dried by rotary evaporation;

[0175] The mixing ratio of tartary buckwheat flour and petroleum ether is 15 g:150 mL;

[0176] The ethanol solution with a mass fraction of 80% and the defatted buckwheat flour are mixed in a mass ratio of 1:5.

[0177] The method for preparing pretreated pure water is:

[0178] The purified water is subjected to plasma treatment;

[0179] Among them, the plasma treatment time is 30min and the power is 180W;

[0180] The plasma processing gas is helium.

[0181] The mixing ratio of pre-treated purified water and primary treated material is 1:13;

[0182] The sericin mixture is prepared as follows:

[0183] First, the silk cocoons are washed with distilled water for 10 minutes, and then dried to constant weight, wherein the drying temperature is 60°C and the time is 10 hours; the washed and dried silk cocoons are cut into pieces to obtain silk cocoon scraps; the silk cocoon scraps and distilled water are mixed in a mass ratio of 1:45; the silk cocoon scraps are added to the distilled water, stirred and mixed to obtain a mixed solution; the pH of the mixed solution is adjusted to 6.5; papain is added to the mixed solution, stirred and mixed at 55°C for 2 hours, and then inactivated, and then centrifuged at a centrifugal speed of 4500r / min, the supernatant is taken, and freeze-dried to obtain a sericin mixture; the mixing ratio of the mixed solution and papain is 1kg:3g; wherein the inactivation is treated at 95°C for 5 minutes;

[0184] A freeze-drying process for a high-activity freeze-dried formula of stem cell exosomes comprises the following steps:

[0185] S1. First, add the tartary buckwheat extract and the sericin mixture to sterile water in sequence, and stir to mix evenly to obtain a first mixed solution;

[0186] S2 adds the stem cell exosomes to the first mixed solution, mixes them evenly, and cultures them at 37° C. for 2 hours to obtain a culture solution;

[0187] S3: adding sodium alginate, mannitol, and β-mercaptoethanol to the culture solution in sequence, mixing them evenly, and culturing them at 37° C. for 30 minutes to obtain a second mixed solution;

[0188] S4 pre-freezes the second mixed solution for 24 hours;

[0189] S5 is pre-frozen and then frozen for 24 hours, and then stored at 4°C.

[0190] The concentration of the tartary buckwheat extract in the first mixed solution is 1 mol / L;

[0191] The pre-freezing temperature is -80℃;

[0192] The freeze-drying temperature was -55°C and the vacuum pressure was 12Pa.

[0193] Example 5

[0194] A highly active freeze-dried formula of stem cell exosomes:

[0195] Components Weight Stem Cell Exosomes 100 copies Sodium alginate 5 servings Sericin Blend 6 servings Mannitol 15 servings Buckwheat Extract 0.6 parts β-Mercaptoethanol 0.6 parts

[0196] The weight ratio of sericin mixture: tartary buckwheat extract: β-mercaptoethanol is 10:1:1.

[0197] The preparation method of tartary buckwheat extract is as follows:

[0198] The dried tartary buckwheat is added into a grinder, pulverized, and passed through an 80-mesh sieve to obtain tartary buckwheat flour;

[0199] Then, the tartary buckwheat flour is defatted by using petroleum ether to obtain defatted tartary buckwheat flour;

[0200] The defatted buckwheat flour is mixed with an ethanol solution having a mass fraction of 80%, and then refluxed for 2 hours, filtered, and the filtrate is removed to obtain a filter residue, which is dried to a constant weight to obtain a preliminary treated product;

[0201] The purified water was subjected to plasma treatment for 10 minutes to obtain pre-treated purified water;

[0202] The obtained pre-treated pure water was mixed with the preliminary treated product, subjected to ultrasonic dispersion for 10 minutes, and then subjected to heating reflux extraction for 5 hours, and finally subjected to a centrifugal treatment to obtain a supernatant; the rotation speed of the centrifugal treatment was 7500 r / min;

[0203] The supernatant was concentrated to 40% of the original volume;

[0204] Add 3 times the volume of anhydrous ethanol to the concentrated supernatant, place it at 4°C for alcohol precipitation for 24 hours, and then centrifuge it for the second time, collect the precipitate, and dry it to obtain;

[0205] The rotation speed of the secondary centrifugal treatment is 6000r / min.

[0206] The degreasing treatment of tartary buckwheat flour with petroleum ether is as follows:

[0207] The tartary buckwheat flour and petroleum ether were mixed, stirred at a speed of 500 r / min for 2 hours, allowed to stand for 1 hour, and then dried by rotary evaporation;

[0208] The mixing ratio of tartary buckwheat flour and petroleum ether is 18 g:150 mL;

[0209] The ethanol solution with a mass fraction of 80% and the defatted buckwheat flour are mixed in a mass ratio of 1:5.

[0210] The method for preparing pretreated pure water is:

[0211] The purified water is subjected to plasma treatment;

[0212] Among them, the plasma treatment time is 30min and the power is 180W;

[0213] The plasma processing gas is helium.

[0214] The mixing ratio of pre-treated purified water and primary treated material is 1:15;

[0215] The sericin mixture is prepared as follows:

[0216] First, the silk cocoons are washed with distilled water for 10 minutes, and then dried to constant weight, wherein the drying temperature is 60°C and the time is 10 hours; the washed and dried silk cocoons are cut into pieces to obtain silk cocoon scraps; the mass ratio of the silk cocoon scraps to distilled water is 1:50; the silk cocoon scraps are added to the distilled water, stirred and mixed to obtain a mixed solution; the pH of the mixed solution is adjusted to 6.5; papain is added to the mixed solution, stirred and mixed at 55°C for 3 hours, and then inactivated, and then centrifuged at a centrifugal speed of 4500r / min, the supernatant is taken, and freeze-dried to obtain a sericin mixture; the mixing ratio of the mixed solution to papain is 1kg:3g; wherein the inactivation is treated at 95°C for 5 minutes;

[0217] A freeze-drying process for a high-activity freeze-dried formula of stem cell exosomes comprises the following steps:

[0218] S1. First, add the tartary buckwheat extract and the sericin mixture to sterile water in sequence, and stir to mix evenly to obtain a first mixed solution;

[0219] S2 adds the stem cell exosomes to the first mixed solution, mixes them evenly, and cultures them at 37° C. for 2 hours to obtain a culture solution;

[0220] S3: adding sodium alginate, mannitol, and β-mercaptoethanol to the culture solution in sequence, mixing them evenly, and culturing them at 37° C. for 30 minutes to obtain a second mixed solution;

[0221] S4 pre-freezes the second mixed solution for 24 hours;

[0222] S5 is pre-frozen and then frozen for 24 hours, and then stored at 4°C.

[0223] As a further technical solution: the concentration of the tartary buckwheat extract in the first mixed solution is 1 mol / L;

[0224] The pre-freezing temperature is -80℃;

[0225] The freeze-drying temperature was -55°C and the vacuum pressure was 12Pa.

[0226] Comparative Example 1:

[0227] Based on Example 1, no tartary buckwheat extract was added, and the rest of the technical solutions remained unchanged.

[0228] Comparative Example 2:

[0229] Based on Example 1, no sericin mixture is added, and the rest of the technical solutions remain unchanged.

[0230] test:

[0231] Adipose stem cell exosomes were used as samples and freeze-dried in the manners of the embodiment and the comparative example respectively;

[0232] Preparation of hydration solution: Under sterile conditions, mix 10 ml of fetal bovine serum, 0.8 ml of penicillin-streptomycin, and 40 ml of DMEM / low-glucose medium to obtain hydration solution;

[0233] The lyophilized formula obtained in the embodiment and the comparative example was mixed with the hydration solution at a mass ratio of 1:5, and then centrifuged at 4°C and 1050×g for 5 min, the supernatant was discarded, and the precipitate was resuspended in an equal volume of PBS, and cultured in a 5% CO2 cell culture incubator for 12 h. The active nucleated cells (hUMSCs) in the hydration solution were detected by flow cytometry, and the ratio was calculated with the active nucleated cells in the exosome raw material to obtain the recovery rate;

[0234] Table 1

[0235] Recovery rate% Example 1 93.3 Example 2 95.1 Example 3 95.8 Example 4 94.6 Example 5 94.1 Comparative Example 1 70.7 Comparative Example 2 88.5

[0236] It can be seen from Table 1 that the freeze-dried exosomes prepared in the present invention have a high recovery rate.

[0237] Taking Example 1 as the basic sample, the effects of different weight portions of tartary buckwheat extract on the recovery rate of the freeze-dried formula were compared:

[0238] Table 2

[0239] Weight Recovery rate% 0 70.7 0.1 82.6 0.2 88.5 0.3 93.3 0.4 93.6 0.5 93.8 0.6 94.0 0.7 93.8 0.8 93.9

[0240] It can be seen from Table 2 that with the increase of tartary buckwheat extract, the exosome recovery rate of the freeze-dried formula continued to increase and could not continue to increase beyond a certain limit.

[0241] Safety Testing

[0242] 50 male rabbits weighing 1.5±0.3 kg were randomly divided into 5 groups, with 10 rabbits in each group. 75 μg of the lyophilized exosome formula of the embodiment was hydrated and then diluted to 8 mL with normal saline;

[0243] 8 mL of the drug was dripped into the right ear vein of the rabbit once a day for three consecutive days, and the area around the injection site was observed for allergic symptoms:

[0244] The allergy evaluation criteria are:

[0245] Grade 0, no obvious reaction at the administration site;

[0246] Grade 1: mild bruising and redness and swelling of the veins at the administration site;

[0247] Grade 2, moderate bruising and redness and swelling of the venous vessels at the administration site;

[0248] Grade 3: severe venous congestion at the drug administration site and severe redness and swelling of the surrounding tissues;

[0249] Table 3

[0250] Allergy evaluation standard / grade Example 1 0 Example 2 0 Example 3 0 Example 4 0 Example 5 0

[0251] It can be seen from Table 3 that the freeze-dried exosome formula prepared by the present invention is safe and reliable.

[0252] The above description is only a preferred embodiment of the present invention, but the present invention is not limited to the scope of implementation shown. Any changes made in accordance with the concept of the present invention, or modifications to equivalent embodiments with equivalent changes, which still do not exceed the spirit covered by the specification, should be within the protection scope of the present invention.

Claims

1. A high-activity freeze-dried formula of stem cell exosomes, characterized in that: The invention comprises the following ingredients by weight: 90-100 parts of stem cell exosomes, 1-5 parts of sodium alginate, 3-6 parts of sericin mixture, 5-15 parts of mannitol, 0.3-0.6 parts of tartary buckwheat extract and 0.3-0.6 parts of beta-mercaptoethanol.

2. A highly active freeze-dried formula of stem cell exosomes according to claim 1, characterized in that: The weight ratio of the sericin mixture: the tartary buckwheat extract: the β-mercaptoethanol is 10:1:

1.

3. A highly active freeze-dried formula of stem cell exosomes according to claim 2, characterized in that: The preparation method of the tartary buckwheat extract is as follows: The dried tartary buckwheat is added into a grinder, pulverized, and passed through an 80-mesh sieve to obtain tartary buckwheat flour; Then, the tartary buckwheat flour is defatted by using petroleum ether to obtain defatted tartary buckwheat flour; The defatted buckwheat flour is mixed with an ethanol solution having a mass fraction of 80%, and then refluxed for 2 hours, filtered, and the filtrate is removed to obtain a filter residue, which is dried to a constant weight to obtain a preliminary treated product; The purified water was subjected to plasma treatment for 10 minutes to obtain pre-treated purified water; The obtained pre-treated pure water is mixed with the preliminary treated product, subjected to ultrasonic dispersion for 10 minutes, and then subjected to heating reflux extraction for 4-5 hours, and finally subjected to a centrifugal treatment to obtain the supernatant; The supernatant was concentrated to 40% of the original volume; Add 3 times the volume of anhydrous ethanol to the concentrated supernatant, place it at 4°C for alcohol precipitation for 24 hours, and then perform a second centrifugation treatment, collect the precipitate, and obtain it after drying.

4. A highly active freeze-dried formula of stem cell exosomes according to claim 3, characterized in that: The degreasing treatment of tartary buckwheat flour with petroleum ether is as follows: The tartary buckwheat flour and petroleum ether were mixed, stirred at a speed of 500 r / min for 2 hours, allowed to stand for 1 hour, and then dried by rotary evaporation; The mixing ratio of tartary buckwheat flour and petroleum ether is 12-18 g: 150 mL; The ethanol solution with a mass fraction of 80% and the defatted buckwheat flour are mixed in a mass ratio of 1:

5.

5. The high-activity freeze-dried formula of stem cell exosomes according to claim 1, characterized in that: The method for preparing pretreated pure water is as follows: The purified water is subjected to plasma treatment; Among them, the plasma treatment time is 30min and the power is 180W; The plasma processing gas is helium.

6. A highly active freeze-dried formula of stem cell exosomes according to claim 5, characterized in that: The mixing ratio of the pre-treated pure water to the primary treated product is 1:12-15; The rotation speed of the centrifugal treatment is 7500r / min; The rotation speed of the secondary centrifugal treatment is 6000r / min.

7. A highly active freeze-dried formula of stem cell exosomes according to claim 1, characterized in that: The preparation method of the sericin mixture is as follows: First, the cocoons were washed with distilled water for 10 minutes and then dried to constant weight at 60°C for 10 hours. Cutting the cleaned and dried silkworm cocoons into pieces to obtain silkworm cocoon scraps; Adding the silkworm cocoon fragments to distilled water, stirring and mixing, to obtain a mixed solution; Adjust the pH of the mixed solution to 6.5; Papain is added to the mixed solution, and the mixture is stirred for 2-3 hours. The mixture is then inactivated and centrifuged. The supernatant is taken and freeze-dried to obtain a sericin mixture.

8. A highly active freeze-dried formula of stem cell exosomes according to claim 7, characterized in that: The mixing mass ratio of the silkworm cocoon fragments to distilled water is 1:40-50; The mixing ratio of the mixed solution to papain is 1kg:3g; After the papain is added, stirring and mixing is performed at 55° C.; Among them, inactivation was treated at 95°C for 5 min; The centrifugal speed is 4500r / min.

9. The freeze-drying process of the high-activity freeze-dried formula of stem cell exosomes according to claim 1, characterized in that: The following steps are involved: S1. First, add the tartary buckwheat extract and the sericin mixture to sterile water in sequence, and stir to mix evenly to obtain a first mixed solution; S2 adds the stem cell exosomes to the first mixed solution, mixes them evenly, and cultures them at 37° C. for 2 hours to obtain a culture solution; S3: adding sodium alginate, mannitol, and β-mercaptoethanol to the culture solution in sequence, mixing them evenly, and culturing them at 37° C. for 30 minutes to obtain a second mixed solution; S4 pre-freezes the second mixed solution for 24 hours; S5 is pre-frozen and then frozen for 24 hours, and then stored at 4°C.

10. The freeze-drying process of the high-activity freeze-dried formula of stem cell exosomes according to claim 9, characterized in that: The concentration of the tartary buckwheat extract in the first mixed solution is 1 mol / L; The pre-freezing temperature is -80℃; The freeze-drying temperature was -55°C and the vacuum pressure was 12Pa.

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