A method for preparing exosomes, exosomes prepared thereby, and applications thereof

Abstract

The present invention provides a method for preparing exosomes, and the exosomes prepared thereby and their application. The preparation method comprises the following steps: (1) digesting autologous adipose tissue with a trypsin digestion solution, washing, and obtaining fat cells; (2) culturing the fat cells obtained in step (1) in a culture medium to obtain a cell suspension; (3) mixing the cell suspension obtained in step (2) with tranexamic acid, culturing, and collecting to obtain exosomes produced by autologous mesenchymal adipose stem cells modified with tranexamic acid. The active ingredients of the exosome preparation include exosomes produced by autologous mesenchymal adipose stem cells and tranexamic acid modification, and the exosome preparation for burn wound repair needs to be able to promote wound healing, reduce melanin deposition on the wound, and reduce the marks after wound healing.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to a method for preparing exosomes, and the exosomes prepared thereby and applications thereof. Background Art

[0002] Skin burns are open pathological injuries that can damage the structure and function of the skin. The healing of the skin after a burn is a complex physiological process that goes through hemostasis, inflammatory response, hyperplasia, tissue remodeling, and scarring. This process requires the participation of multiple tissue cells, such as epithelial cells, endothelial cells, immune cells, and fibroblasts. Because burn wounds contain a large amount of burn toxins and are exposed to a complex microbial environment, the appropriate participation of inflammatory cells can reduce the risk of infection, but excessive inflammatory reactions affect the healing of burn wounds.

[0003] For patients with large-area burns, autologous skin transplantation is limited due to the lack of autologous skin sources; allogeneic skin is not only limited, but also has problems such as immune rejection. New wound repair technologies are urgently needed for the rescue of burn patients, skin function recovery, and scar fading. Adipose-derived mesenchymal stem cells are a type of pluripotent stem cells that are widely distributed, easy to obtain, and have certain self-renewal and differentiation potentials. They can secrete a variety of cytokines that promote tissue repair, regulate tissue microenvironment, and are anti-inflammatory and anti-fibrotic. Although adipose-derived mesenchymal stem cells are relatively safe, stem cells with intact structures still have a certain risk of tumorigenicity. Exosomes derived from adipose-derived mesenchymal stem cells take into account both safety and effectiveness. Exosomes derived from adipose-derived mesenchymal stem cells can transport stem cell nucleic acids, proteins, lipids, and signaling molecules to target cells, playing a similar role to stem cells in burn wound repair.

[0004] CN113018501A discloses an endothelial progenitor cell exosome medical dressing, a preparation method and its application, comprising the following steps: obtaining endothelial progenitor cells by three-dimensional culture, preparing exosomes using endothelial progenitor cells, and obtaining exosome medical dressings through exosomes. The endothelial progenitor cell exosome medical dressing described in the invention uses healthy umbilical cord blood endothelial progenitor cells as raw materials, contains highly active exosomes, and nano-scale phospholipid vesicles encapsulate hundreds of proteins and bioactive molecules such as microRNA that are closely related to cell growth and repair functions. It promotes wound tissue repair of diseases such as burns and scalds by activating the patient's endogenous tissue-specific stem cells and establishing a regenerative microenvironment for tissue damage repair.

[0005] CN111671772A discloses the use of exosomes in the preparation of medicines or cosmetics for repairing skin damage, belonging to the field of skin treatment technology. The invention separates and obtains mesenchymal stem cells from primary antler tissue; after in vitro expansion culture, the cell culture fluid is collected, and exosomes derived from antler mesenchymal stem cells are obtained by separation and extraction. It has been confirmed that the inflammatory response of HaCat cells induced by LPS can be inhibited through the NLRP3 inflammasome signaling pathway, and the wound repair and inflammatory response of the skin injury mouse model can be accelerated. The present invention can be used to prepare medicines or cosmetics for treating skin damage and repairing.

[0006] CN110693912A discloses the use of cell exosomes in the preparation of products that promote wound healing. The stem cell exosomes of the invention are non-immunogenic and non-tumorigenic, and the extraction method is mature and easy to store. When attached to the skin of pressure sores, it can reduce the content of myocollagen fibers, type I collagen and / or type III collagen and / or HMGB1 and / or TGF-β expression in the wound tissue, thereby promoting wound healing. The invention uses exosomes to replace stem cells that are strictly used in clinical practice due to immunogenicity and tumorigenicity, eliminates the limitations of stem cell therapy, and can achieve corresponding therapeutic effects.

[0007] Therefore, providing a method for preparing and modifying exosomes of autologous adipose-derived mesenchymal stem cells, which can be used to promote wound repair, reduce inflammation, and reduce scars in patients with skin burns, is a research focus in this field. Summary of the invention

[0008] In view of the deficiencies in the prior art, the object of the present invention is to provide a method for preparing exosomes, the exosomes prepared thereby, and applications thereof, and in particular to a method for preparing exosomes produced by autologous mesenchymal adipose stem cells modified with tranexamic acid, the exosomes prepared thereby, and applications thereof.

[0009] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0010] In a first aspect, the present invention provides a method for preparing exosomes, the method for preparing exosomes comprising the following steps:

[0011] (1) digesting autologous adipose tissue with trypsin digestion solution, washing it, and obtaining adipocytes;

[0012] (2) culturing the adipocytes obtained in step (1) in a culture medium to obtain a cell suspension;

[0013] (3) The cell suspension obtained in step (2) is mixed with tranexamic acid, cultured, and collected to obtain exosomes produced by autologous mesenchymal adipose stem cells modified with tranexamic acid.

[0014] In the present invention, in the preparation of the exosomes, the source of the exosomes is autologous adipose-derived mesenchymal stem cells, which can transport nucleic acids, proteins, lipids and signal molecules of stem cells to target cells, and play a function similar to that of stem cells in burn wound repair.

[0015] Preferably, in step (1), the volume ratio of the trypsin digestion solution to the autologous adipose tissue is (15-25):(25-50);

[0016] Among them, "15-25" can be, for example, 15, 17, 19, 21, 23, 25, etc.;

[0017] Here, “25-50” can be, for example, 25, 30, 35, 40, 45, 50, etc.

[0018] Preferably, in step (1), the trypsin digestion solution comprises: trypsin and EDTA solution.

[0019] Preferably, the mass percentage of trypsin in the trypsin digestion solution is 0.05-0.2%, for example, it can be 0.05%, 0.1%, 0.15%, 0.2%, etc.

[0020] Preferably, the concentration of the EDTA solution is 0.05-0.15 mM, for example, it can be 0.05 mM, 0.06 mM, 0.1 mM, 0.12 mM, 0.15 mM, etc.

[0021] Preferably, the pH of the EDTA solution is 7.5-8.5, for example, it can be 7.5, 7.6, 7.8, 8.0, 8.2, 8.5, etc.

[0022] Preferably, in step (1), the autologous adipose tissue is autologous abdominal adipose tissue.

[0023] Preferably, in step (1), the digestion specifically comprises the following steps: mixing the autologous adipose tissue with part of the trypsin digestion solution, performing a primary digestion, and collecting the partially digested cell suspension; then mixing the remaining undigested autologous adipose tissue with the remaining trypsin digestion solution, performing a secondary digestion, and combining and collecting to obtain a digested cell suspension.

[0024] Preferably, the volume ratio of the autologous adipose tissue, part of the trypsin digestion solution, and the remaining trypsin digestion solution is (15-25):(15-30):(10-20);

[0025] Among them, "15-25" can be, for example, 15, 17, 19, 21, 23, 25, etc.;

[0026] Here, "15-30" can be, for example, 15, 20, 25, 30, etc.;

[0027] Here, “10-20” can be, for example, 10, 12, 14, 16, 18, 20, etc.

[0028] Preferably, the temperature of the primary digestion is 36.5-38°C, for example, 36.5°C, 37°C, 37.5°C, 38°C, etc., and the time of the primary digestion is 8-12min, for example, 8min, 9min, 10min, 11min, 12min, etc.

[0029] Preferably, the temperature of the secondary digestion is 36.5-38°C, for example, 36.5°C, 37°C, 37.5°C, 38°C, etc., and the time of the secondary digestion is 4-10min, for example, 4min, 5min, 6min, 7min, 8min, 9min, 10min, etc.

[0030] Preferably, the washing specifically comprises the following steps: after centrifuging the digested cell suspension, discarding the supernatant, adding PBS buffer, shaking and washing, and obtaining fat cells.

[0031] Preferably, the centrifugal rotation speed is 800-1500rpm, for example, it can be 800rpm, 900rpm, 1000rpm, 1100rpm, 1200rpm, 1300rpm, 1400rpm, 1500rpm, etc., and the centrifugal time is 2-5min, for example, it can be 2min, 3min, 4min, 5min, etc.

[0032] Preferably, the shaking cleaning is performed at least twice, for example, 2 times, 3 times, 4 times, 5 times, etc.

[0033] Preferably, in step (2), the culture medium is a serum-free culture medium.

[0034] Preferably, in step (2), the inoculation amount of the fat cells is 15-25 cm 3 , for example, it can be 15cm 3 、16cm 3 、17cm 3 、18cm 3 、19cm 3 , 20cm 3 、21cm 3 、22cm 3 、23cm 3 、24cm 3 、25cm 3 wait.

[0035] Preferably, in step (2), the culture temperature is 36.5-38°C, for example, it can be 36.5°C, 37°C, 37.5°C, 38°C, etc., the culture gas is 5% carbon dioxide, and the culture time is 22-28h, for example, it can be 22h, 23h, 24h, 25h, 26h, 27h, 28h, etc.

[0036] Preferably, in step (3), the volume to mass ratio of the cell suspension to tranexamic acid is (20-30) mL: (4-15) mg;

[0037] Among them, "20-30" can be, for example, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, etc.; "4-15" can be, for example, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, etc.

[0038] Preferably, in step (3), the culture temperature is 36.5-38°C, for example, it can be 36.5°C, 37°C, 37.5°C, 38°C, etc., the culture gas is 5% carbon dioxide, and the culture time is 1-3h, for example, it can be 1h, 1.5h, 2h, 2.5h, 3h, etc.

[0039] Preferably, in step (3), the collection specifically comprises the following steps: harvesting the cultured exosomes, performing a centrifugation, and collecting the supernatant; performing a second centrifugation, and collecting the supernatant; and finally performing a third centrifugation, discarding the supernatant, and collecting the precipitate.

[0040] Preferably, the temperature of the first centrifugation, the second centrifugation, and the third centrifugation is independently 0-8°C, for example, 0°C, 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, etc.

[0041] Preferably, the rotation speed of the centrifugation is 1000-3000rpm, for example, it can be 1000rpm, 1500rpm, 2000rpm, 2500rpm, 3000rpm, etc., and the time of the centrifugation is 10-15min, for example, it can be 10min, 11min, 12min, 13min, 14min, 15min, etc.

[0042] Preferably, the rotation speed of the secondary centrifugation is 1000-3000rpm, for example, it can be 1000rpm, 1500rpm, 2000rpm, 2500rpm, 3000rpm, etc., and the time of the secondary centrifugation is 30-40min, for example, it can be 30min, 32min, 34min, 36min, 38min, 40min, etc.

[0043] Preferably, the rotation speed of the three centrifugations is 8000-12000rpm, for example, it can be 8000rpm, 9000rpm, 10000rpm, 11000rpm, 12000rpm, etc., and the time of the three centrifugations is 50-60min, for example, it can be 50min, 52min, 54min, 56min, 58min, 60min, etc.

[0044] Preferably, the precipitate is further preliminarily suspended in 7-10 mL PBS buffer (for example, 7 mL, 7.5 mL, 8 mL, 8.5 mL, 9 mL, 9.5 mL, 10 mL, etc.), and the total RNA concentration is measured after the suspension.

[0045] Preferably, according to the total RNA concentration of the preliminary exosome PBS suspension, the PBS buffer is used for further dilution to dilute the final exosome total RNA concentration to 15-30 pg / mL. For example, the total RNA concentration can be 15 pg / mL, 18 pg / mL, 21 pg / mL, 24 pg / mL, 27 pg / mL, 30 pg / mL, etc.

[0046] In a second aspect, the present invention provides an exosome, wherein the exosome is prepared by the method for preparing the exosome described in the first aspect.

[0047] In a third aspect, the present invention provides an exosome gel, comprising the exosomes described in the second aspect, sodium alginate, calcium chloride and PBS buffer.

[0048] Preferably, in the exosome gel, the concentration of total exosome RNA is 15-30 pg / mL, the mass concentration of sodium alginate is 2.8-3.4%, the mass concentration of calcium chloride is 0.4-0.6%, and the solvent is PBS buffer.

[0049] In the exosome gel, the concentration of total exosome RNA is 15-30 pg / mL, for example, 15 pg / mL, 18 pg / mL, 21 pg / mL, 24 pg / mL, 27 pg / mL, 30 pg / mL, etc.

[0050] In the exosome gel, the mass concentration of sodium alginate is 2.8-3.4%, for example, it can be 2.8%, 2.9%, 3.0%, 3.1%, 3.2%, 3.3%, 3.4%, etc.

[0051] In the exosome gel, the mass concentration of calcium chloride is 0.4-0.6%, for example, it can be 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, etc.

[0052] In a fourth aspect, the present invention provides a method for preparing the exosome gel according to the third aspect, the method for preparing the exosome gel comprising the following steps: mixing exosomes, sodium alginate and PBS buffer, and then mixing with calcium chloride to obtain the exosome gel.

[0053] Preferably, the temperature of the mixture of the exosomes, sodium alginate and PBS buffer is 4-25°C (for example, 4°C, 5°C, 8°C, 10°C, 15°C, 20°C, 25°C, etc.), and the mixture is vortexed at a speed of 1500-2000rpm (for example, 1500rpm, 1600rpm, 1800rpm, 2000rpm, etc.) for 3-10s (for example, 3s, 4s, 5s, 6s, 7s, 8s, 9s, 10s, etc.).

[0054] Preferably, the temperature of mixing with calcium chloride is 4-25°C (for example, it can be 4°C, 5°C, 8°C, 10°C, 15°C, 20°C, 25°C, etc.), and a vortex oscillator is used to oscillate at a speed of 1500-2000rpm (for example, it can be 1500rpm, 1600rpm, 1800rpm, 2000rpm, etc.) for 6-15s (for example, it can be 6s, 7s, 8s, 9s, 10s, 11s, 12s, 13s, 14s, 15s, etc.).

[0055] In a fifth aspect, the present invention provides a use of the exosomes as described in the second aspect, or the exosome gel as described in the third aspect, in the preparation of a preparation for repairing burn wounds.

[0056] Compared with the prior art, the present invention has the following beneficial effects:

[0057] (1) The active ingredients of the exosomes of the present invention include exosomes produced by autologous mesenchymal adipose stem cells and modified with tranexamic acid;

[0058] (2) The exosome gel of the present invention can promote wound healing, reduce melanin deposition on the wound, and reduce the traces of wound healing;

[0059] (3) The concentration of tranexamic acid in the exosome gel of the present invention should be no less than 25 μg / mL, and the total amount of RNA should be no less than 15 pg / mL. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 This is a curve chart showing the change of tranexamic acid concentration over time. DETAILED DESCRIPTION

[0061] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0062] The sources of the components in the following examples and comparative examples are as follows:

[0063]

[0064] Example 1

[0065] This embodiment provides an exosome gel, which is prepared by the following preparation method:

[0066] (1) Take 20.01 mL of autologous abdominal adipose tissue (sample number S01025) and digest it with 20 mL of trypsin digestion solution at 37°C for 10 min. Gently shake the digested cell suspension and transfer it to a centrifuge tube. Then, digest the remaining tissue again with 15 mL of trypsin digestion solution at 37°C for 5 min. Gently shake the digested suspension and transfer it to a centrifuge tube. Combine and collect the digested cell suspension.

[0067] Wherein, the trypsin digestion solution comprises, by mass percentage: 0.1% trypsin, a solvent of 0.1 mM EDTA solution with a pH of 8.0;

[0068] The collected digested cell suspension was centrifuged at 1000 rpm for 3 min, and the supernatant was discarded; 10 mL of PBS buffer was added, and after shaking and washing, it was centrifuged at 1000 rpm for 3 min, and the supernatant was discarded; the washing step was repeated once to obtain adipocytes;

[0069] (2) The digested and washed adipocytes were cultured in 25 mL of serum-free medium (cultured at 37° C., 5% carbon dioxide concentration for 24 h to obtain a cell suspension;

[0070] (3) Add 6 mg of tranexamic acid to the cell suspension cultured for 24 h and continue culturing for 2 h. Harvest exosomes. Centrifuge the culture solution at 2000 rpm for 12 min at 4 °C using a low-temperature centrifuge to obtain the supernatant. Centrifuge the supernatant at 2000 rpm for 35 min at 4 °C to obtain the supernatant. Centrifuge the supernatant at 10000 rpm for 55 min at 4 °C, discard the supernatant, and collect the precipitate.

[0071] (4) Add 12 mL of PBS buffer to the precipitate to preliminarily obtain a concentrated suspension of adipose mesenchymal exosomes modified with tranexamic acid;

[0072] (5) Take 1 mL of concentrated suspension sample to measure total RNA, the concentration is 50 pg / mL, take 10 mL of concentrated suspension sample and add 10 mL of PBS buffer to obtain 20 mL of exosome suspension;

[0073] (6) Add 620 mg of sodium alginate to 20 mL of the obtained PBS suspension of exosomes, dissolve and shake evenly, then add 0.2 mL of double-distilled calcium chloride solution containing 50 mg, and shake quickly to obtain 20 mL of exosome gel preparation.

[0074] Example 2

[0075] This embodiment provides an exosome gel, which is prepared by the following preparation method:

[0076] (1) 21.43 mL of autologous abdominal adipose tissue (sample number S01026) was digested with 20 mL of trypsin digestion solution at 37°C for 10 min, the digested cell suspension was gently shaken and transferred to a centrifuge tube; the remaining tissue was digested again with 15 mL of trypsin digestion solution at 37°C for 5 min; the digested suspension was gently shaken and transferred to a centrifuge tube, and the digested cell suspension was combined and collected;

[0077] Wherein, the trypsin digestion solution comprises, by mass percentage: 0.1% trypsin, a solvent of 0.1 mM EDTA solution with a pH of 8.0;

[0078] The collected digested cell suspension was centrifuged at 1000 rpm for 3 min, and the supernatant was discarded; 10 mL of PBS buffer was added, and after shaking and washing, it was centrifuged at 1000 rpm for 3 min, and the supernatant was discarded; the washing step was repeated once to obtain adipocytes;

[0079] (2) The digested and washed adipocytes were cultured in 25 mL of serum-free medium (cultured at 37° C., 5% carbon dioxide concentration for 24 h to obtain a cell suspension;

[0080] (3) Add 7 mg of tranexamic acid to the cell suspension cultured for 24 h and continue culturing for 2 h. Harvest exosomes. Centrifuge the culture solution at 2000 rpm for 12 min at 4 °C using a low-temperature centrifuge to obtain the supernatant. Centrifuge the supernatant at 2000 rpm for 35 min at 4 °C to obtain the supernatant. Centrifuge the supernatant at 10000 rpm for 55 min at 4 °C, discard the supernatant, and collect the precipitate.

[0081] (4) Add 12 mL of PBS buffer to the precipitate to obtain a concentrated suspension of adipose mesenchymal exosomes modified with tranexamic acid;

[0082] (5) Take 1 mL of concentrated suspension sample to measure total RNA, the concentration is 42 pg / mL, take 10 mL of concentrated suspension sample and add 10 mL of PBS buffer to obtain 20 mL of exosome suspension;

[0083] (6) Add 620 mg of sodium alginate to 20 mL of the obtained PBS suspension of exosomes, dissolve and shake evenly, then add 0.2 mL of double-distilled calcium chloride solution containing 50 mg, and shake quickly to obtain 20 mL of exosome gel preparation.

[0084] Example 3

[0085] This embodiment provides an exosome gel, which is prepared by the following preparation method:

[0086] (1) 16.23 mL of autologous abdominal adipose tissue (sample number S01028) was digested with 15 mL of trypsin digestion solution at 37°C for 10 min, the digested cell suspension was gently shaken and transferred to a centrifuge tube; the remaining tissue was digested again with 15 mL of trypsin digestion solution at 37°C for 5 min; the digested suspension was gently shaken and transferred to a centrifuge tube, and the digested cell suspension was combined and collected;

[0087] Wherein, the trypsin digestion solution comprises, by mass percentage: 0.1% trypsin, a solvent of 0.1 mM EDTA solution with a pH of 8.0;

[0088] The collected digested cell suspension was centrifuged at 1000 rpm for 3 min, and the supernatant was discarded; 10 mL of PBS buffer was added, and after shaking and washing, it was centrifuged at 1000 rpm for 3 min, and the supernatant was discarded; the washing step was repeated once to obtain adipocytes;

[0089] (2) The digested and washed adipocytes were cultured in 25 mL of serum-free medium (cultured at 37° C., 5% carbon dioxide concentration for 24 h to obtain a cell suspension;

[0090] (3) Add 6 mg of tranexamic acid to the cell suspension cultured for 24 h and continue culturing for 2 h. Harvest exosomes. Centrifuge the culture solution at 2000 rpm for 15 min at 4°C using a low-temperature centrifuge to obtain the supernatant. Centrifuge the supernatant at 2000 rpm for 40 min at 4°C to obtain the supernatant. Centrifuge the supernatant at 10000 rpm for 60 min at 4°C, discard the supernatant, and collect the precipitate.

[0091] (4) Add 11 mL of PBS buffer to the precipitate to obtain a concentrated suspension of adipose mesenchymal exosomes modified with tranexamic acid;

[0092] (5) Take 1 mL of concentrated suspension sample to measure total RNA, the concentration is 24 pg / mL, take 10 mL of concentrated suspension sample and add 15 mL of PBS buffer to obtain 15 mL of exosome suspension;

[0093] (6) Add 465 mg of sodium alginate to 15 mL of the obtained exosome PBS suspension, dissolve and shake evenly, then add 0.15 mL of double-distilled calcium chloride solution containing 50 mg, and shake quickly to obtain 15 mL of exosome gel preparation.

[0094] Example 4

[0095] This embodiment provides an exosome gel, which is prepared by the following preparation method:

[0096] (1) 11.92 mL of autologous abdominal adipose tissue (sample number S01029) was digested with 15 mL of trypsin digestion solution at 37°C for 10 min, the digested cell suspension was gently shaken and transferred to a centrifuge tube; the remaining tissue was digested again with 10 mL of trypsin digestion solution at 37°C for 5 min; the digested suspension was gently shaken and transferred to a centrifuge tube, and the digested cell suspension was combined and collected;

[0097] Wherein, the trypsin digestion solution comprises, by mass percentage: 0.1% trypsin, a solvent of 0.1 mM EDTA solution with a pH of 8.0;

[0098] The collected digested cell suspension was centrifuged at 1000 rpm for 3 min, and the supernatant was discarded; 10 mL of PBS buffer was added, and after shaking and washing, it was centrifuged at 1000 rpm for 3 min, and the supernatant was discarded; the washing step was repeated once to obtain adipocytes;

[0099] (2) The digested and washed adipocytes were cultured in 25 mL of serum-free medium (cultured at 37° C., 5% carbon dioxide concentration for 24 h to obtain a cell suspension;

[0100] (3) Add 4 mg of tranexamic acid to the cell suspension cultured for 24 h and continue culturing for 2 h. Harvest exosomes. Centrifuge the culture solution at 2000 rpm for 15 min at 4°C using a low-temperature centrifuge to obtain the supernatant. Centrifuge the supernatant at 2000 rpm for 40 min at 4°C to obtain the supernatant. Centrifuge the supernatant at 10000 rpm for 60 min at 4°C, discard the supernatant, and collect the precipitate.

[0101] (4) Add 9 mL of PBS buffer to the precipitate to obtain a concentrated suspension of adipose mesenchymal exosomes modified with tranexamic acid;

[0102] (5) Take 1 mL of concentrated suspension sample to measure total RNA, the concentration is 17 pg / mL, take 8 mL of concentrated suspension sample and add 7 mL of PBS buffer to obtain 15 mL of exosome suspension;

[0103] (6) Add 465 mg of sodium alginate to 15 mL of the obtained exosome PBS suspension, dissolve and shake evenly, then add 0.15 mL of double-distilled calcium chloride solution containing 50 mg, and shake quickly to obtain 15 mL of exosome gel preparation.

[0104] Results: Due to insufficient adipose tissue sampling, the total RNA content of the final preparation was only 9 pg / mL, which did not meet the preparation requirements.

[0105] Example 5

[0106] This embodiment provides an exosome gel, which is prepared by the following preparation method:

[0107] (1) 13.46 mL of autologous abdominal adipose tissue (sample number S01030) was digested with 15 mL of trypsin digestion solution at 37°C for 10 min, the digested cell suspension was gently shaken and transferred to a centrifuge tube; the remaining tissue was digested again with 10 mL of trypsin digestion solution at 37°C for 5 min; the digested suspension was gently shaken and transferred to a centrifuge tube, and the digested cell suspension was combined and collected;

[0108] Wherein, the trypsin digestion solution comprises, by mass percentage: 0.1% trypsin, a solvent of 0.1 mM EDTA solution with a pH of 8.0;

[0109] The collected digested cell suspension was centrifuged at 1000 rpm for 3 min, and the supernatant was discarded; 10 mL of PBS buffer was added, and after shaking and washing, it was centrifuged at 1000 rpm for 3 min, and the supernatant was discarded; the washing step was repeated once to obtain adipocytes;

[0110] (2) The digested and washed adipocytes were cultured in 25 mL of serum-free medium (cultured at 37° C., 5% carbon dioxide concentration for 24 h to obtain a cell suspension;

[0111] (3) Add 5 mg of tranexamic acid to the cell suspension cultured for 24 h and continue culturing for 2 h. Harvest exosomes. Centrifuge the culture solution at 2000 rpm for 15 min at 4°C using a low-temperature centrifuge to obtain the supernatant. Centrifuge the supernatant at 2000 rpm for 40 min at 4°C to obtain the supernatant. Centrifuge the supernatant at 10000 rpm for 60 min at 4°C, discard the supernatant, and collect the precipitate.

[0112] (4) Add 10 mL of PBS buffer to the precipitate to obtain a concentrated suspension of adipose mesenchymal exosomes modified with tranexamic acid;

[0113] (5) Take 1 mL of concentrated suspension sample to measure total RNA, the concentration is 15 pg / mL, take 9 mL of concentrated suspension sample and add 6 mL of PBS buffer to obtain 15 mL of exosome suspension;

[0114] (6) Add 465 mg of sodium alginate to 15 mL of the obtained exosome PBS suspension, dissolve and shake evenly, then add 0.15 mL of double-distilled calcium chloride solution containing 50 mg, and shake quickly to obtain 15 mL of exosome gel preparation.

[0115] Results: Due to insufficient adipose tissue sampling, the total RNA content of the final preparation was only 9 pg / mL, which did not meet the preparation requirements.

[0116] Example 6

[0117] This embodiment provides an exosome gel, which is prepared by the following preparation method:

[0118] (1) Take 17.00 mL of autologous abdominal adipose tissue (sample number S01033) and digest it with 20 mL of trypsin digestion solution at 37°C for 10 min. Gently shake the digested cell suspension and transfer it to a centrifuge tube. Then, digest the remaining tissue again with 15 mL of trypsin digestion solution at 37°C for 5 min. Gently shake the digested suspension and transfer it to a centrifuge tube. Combine and collect the digested cell suspension.

[0119] Wherein, the trypsin digestion solution comprises, by mass percentage: 0.1% trypsin, a solvent of 0.1 mM EDTA solution with a pH of 8.0;

[0120] The collected digested cell suspension was centrifuged at 1000 rpm for 3 min, and the supernatant was discarded; 10 mL of PBS buffer was added, and after shaking and washing, it was centrifuged at 1000 rpm for 3 min, and the supernatant was discarded; the washing step was repeated once to obtain adipocytes;

[0121] (2) The digested and washed adipocytes were cultured in 25 mL of serum-free medium (cultured at 37° C., 5% carbon dioxide concentration for 24 h to obtain a cell suspension;

[0122] (3) Add 7 mg of tranexamic acid to the cell suspension cultured for 24 h and continue culturing for 2 h. Harvest exosomes. Centrifuge the culture solution at 2000 rpm for 12 min at 4 °C using a low-temperature centrifuge to obtain the supernatant. Centrifuge the supernatant at 2000 rpm for 35 min at 4 °C to obtain the supernatant. Centrifuge the supernatant at 10000 rpm for 55 min at 4 °C, discard the supernatant, and collect the precipitate.

[0123] (4) Add 9 mL of PBS buffer to the precipitate to obtain a concentrated suspension of adipose mesenchymal exosomes modified with tranexamic acid;

[0124] (5) Take 1 mL of concentrated suspension sample to measure total RNA, the concentration is 36 pg / mL, take 8 mL of concentrated suspension sample and add 7 mL of PBS buffer to obtain 15 mL of exosome suspension;

[0125] (6) Add 465 mg of sodium alginate to 15 mL of the obtained exosome PBS suspension, dissolve and shake evenly, then add 0.15 mL of double-distilled calcium chloride solution containing 50 mg, and shake quickly to obtain 15 mL of exosome gel preparation.

[0126] Example 7

[0127] This embodiment provides an exosome gel, which is prepared by the following preparation method:

[0128] (1) 19.01 mL of autologous abdominal adipose tissue (sample number S01034) was digested with 20 mL of trypsin digestion solution at 37°C for 10 min, the digested cell suspension was gently shaken and transferred to a centrifuge tube; the remaining tissue was digested again with 15 mL of trypsin digestion solution at 37°C for 5 min; the digested suspension was gently shaken and transferred to a centrifuge tube, and the digested cell suspension was combined and collected;

[0129] Wherein, the trypsin digestion solution comprises, by mass percentage: 0.1% trypsin, a solvent of 0.1 mM EDTA solution with a pH of 8.0;

[0130] The collected digested cell suspension was centrifuged at 1000 rpm for 3 min, and the supernatant was discarded; 10 mL of PBS buffer was added, and after shaking and washing, it was centrifuged at 1000 rpm for 3 min, and the supernatant was discarded; the washing step was repeated once to obtain adipocytes;

[0131] (2) The digested and washed adipocytes were cultured in 25 mL of serum-free medium (cultured at 37° C., 5% carbon dioxide concentration for 24 h to obtain a cell suspension;

[0132] (3) Add 7 mg of tranexamic acid to the cell suspension cultured for 24 h and continue culturing for 2 h. Harvest exosomes. Centrifuge the culture solution at 2000 rpm for 12 min at 4 °C using a low-temperature centrifuge to obtain the supernatant. Centrifuge the supernatant at 2000 rpm for 35 min at 4 °C to obtain the supernatant. Centrifuge the supernatant at 10000 rpm for 55 min at 4 °C, discard the supernatant, and collect the precipitate.

[0133] (4) Add 12 mL of PBS buffer to the precipitate to obtain a concentrated suspension of adipose mesenchymal exosomes modified with tranexamic acid;

[0134] (5) Take 1 mL of concentrated suspension sample to measure total RNA, the concentration is 34 pg / mL, take 10 mL of concentrated suspension sample and add 10 mL of PBS buffer to obtain 20 mL of exosome suspension;

[0135] (6) Take 20 mL of the obtained PBS suspension of exosomes, add 620 mg of sodium alginate, dissolve and shake evenly, then add 0.2 mL of double-distilled calcium chloride solution containing 50 mg, and shake quickly to obtain 20 mL of exosome gel preparation.

[0136] Example 8

[0137] This embodiment provides an exosome gel, which is prepared by the following preparation method:

[0138] (1) Take 26.20 mL of autologous abdominal adipose tissue (sample number S01040) and digest it with 25 mL of trypsin digestion solution at 37°C for 10 min. Gently shake the digested cell suspension and transfer it to a centrifuge tube. Then, digest the remaining tissue again with 15 mL of trypsin digestion solution at 37°C for 5 min. Gently shake the digested suspension and transfer it to a centrifuge tube. Combine and collect the digested cell suspension.

[0139] Wherein, the trypsin digestion solution comprises, by mass percentage: 0.1% trypsin, a solvent of 0.1 mM EDTA solution with a pH of 8.0;

[0140] The collected digested cell suspension was centrifuged at 1000 rpm for 3 min, and the supernatant was discarded; 10 mL of PBS buffer was added, and after shaking and washing, it was centrifuged at 1000 rpm for 3 min, and the supernatant was discarded; the washing step was repeated once to obtain adipocytes;

[0141] (2) The digested and washed adipocytes were cultured in 25 mL of serum-free medium (cultured at 37° C., 5% carbon dioxide concentration for 24 h to obtain a cell suspension;

[0142] (3) Add 9 mg of tranexamic acid to the cell suspension cultured for 24 h and continue culturing for 2 h. Harvest exosomes. Centrifuge the culture solution at 2000 rpm for 10 min at 4 °C using a low-temperature centrifuge to obtain the supernatant. Centrifuge the supernatant at 2000 rpm for 35 min at 4 °C to obtain the supernatant. Centrifuge the supernatant at 10000 rpm for 50 min at 4 °C, discard the supernatant, and collect the precipitate.

[0143] (4) Add 15 mL of PBS buffer to the precipitate to obtain a concentrated suspension of adipose mesenchymal exosomes modified with tranexamic acid;

[0144] (5) Take 1 mL of concentrated suspension sample to measure total RNA, the concentration is 40 pg / mL, take 10 mL of concentrated suspension sample and add 10 mL of PBS buffer to obtain 20 mL of exosome suspension;

[0145] (5) Take 20 mL of the obtained PBS suspension of exosomes, add 620 mg of sodium alginate, dissolve and shake evenly, then add 0.2 mL of double-distilled calcium chloride solution containing 50 mg, and shake quickly to obtain 20 mL of exosome gel preparation.

[0146] Example 9

[0147] This embodiment provides an exosome gel, which is prepared by the following preparation method:

[0148] (1) 18.35 mL of autologous abdominal adipose tissue (sample number S01041) was digested with 20 mL of trypsin digestion solution at 37°C for 10 min, the digested cell suspension was gently shaken and transferred to a centrifuge tube; the remaining tissue was digested again with 15 mL of trypsin digestion solution at 37°C for 5 min; the digested suspension was gently shaken and transferred to a centrifuge tube, and the digested cell suspension was combined and collected;

[0149] Wherein, the trypsin digestion solution comprises, by mass percentage: 0.1% trypsin, a solvent of 0.1 mM EDTA solution with a pH of 8.0;

[0150] The collected digested cell suspension was centrifuged at 1000 rpm for 3 min, and the supernatant was discarded; 10 mL of PBS buffer was added, and after shaking and washing, it was centrifuged at 1000 rpm for 3 min, and the supernatant was discarded; the washing step was repeated once to obtain adipocytes;

[0151] (2) The digested and washed adipocytes were cultured in 25 mL of serum-free medium (cultured at 37° C., 5% carbon dioxide concentration for 24 h to obtain a cell suspension;

[0152] (3) Add 7 mg of tranexamic acid to the cell suspension cultured for 24 h and continue culturing for 2 h. Harvest exosomes. Centrifuge the culture solution at 2000 rpm for 15 min at 4°C using a low-temperature centrifuge to obtain the supernatant. Centrifuge the supernatant at 2000 rpm for 40 min at 4°C to obtain the supernatant. Centrifuge the supernatant at 10000 rpm for 55 min at 4°C, discard the supernatant, and collect the precipitate.

[0153] (4) Add 12 mL of PBS buffer to the precipitate to obtain a concentrated suspension of adipose mesenchymal exosomes modified with tranexamic acid;

[0154] (5) Take 1 mL of concentrated suspension sample to measure total RNA, the concentration is 35 pg / mL, take 10 mL of concentrated suspension sample and add 5 mL of PBS buffer to obtain 15 mL of exosome suspension;

[0155] (6) Add 465 mg of sodium alginate to 15 mL of the obtained exosome PBS suspension, dissolve and shake evenly, then add 0.15 mL of double-distilled calcium chloride solution containing 50 mg, and shake quickly to obtain 15 mL of exosome gel preparation.

[0156] Example 10

[0157] This embodiment provides an exosome gel, which is prepared by the following preparation method:

[0158] (1) 29.09 mL of autologous abdominal adipose tissue (sample number S01048) was digested with 25 mL of trypsin digestion solution at 37°C for 10 min, the digested cell suspension was gently shaken and transferred to a centrifuge tube; the remaining tissue was digested again with 20 mL of trypsin digestion solution at 37°C for 5 min; the digested suspension was gently shaken and transferred to a centrifuge tube, and the digested cell suspension was combined and collected;

[0159] Wherein, the trypsin digestion solution comprises, by mass percentage: 0.1% trypsin, a solvent of 0.1 mM EDTA solution with a pH of 8.0;

[0160] The collected digested cell suspension was centrifuged at 1000 rpm for 3 min, and the supernatant was discarded; 10 mL of PBS buffer was added, and after shaking and washing, it was centrifuged at 1000 rpm for 3 min, and the supernatant was discarded; the washing step was repeated once to obtain adipocytes;

[0161] (2) The digested and washed adipocytes were cultured in 25 mL of serum-free medium (cultured at 37° C., 5% carbon dioxide concentration for 24 h to obtain a cell suspension;

[0162] (3) Add 10 mg of tranexamic acid to the cell suspension cultured for 24 h and continue culturing for 2 h. Harvest exosomes. Centrifuge the culture solution at 2000 rpm for 10 min at 4 °C using a low-temperature centrifuge to obtain the supernatant. Centrifuge the supernatant at 2000 rpm for 35 min at 4 °C to obtain the supernatant. Centrifuge the supernatant at 10000 rpm for 50 min at 4 °C, discard the supernatant, and collect the precipitate.

[0163] (4) Add 12 mL of PBS buffer to the precipitate to obtain adipose mesenchymal exosomes modified with tranexamic acid;

[0164] (5) Take 1 mL of concentrated suspension sample to measure total RNA, the concentration is 44 pg / mL, take 10 mL of concentrated suspension sample and add 10 mL of PBS buffer to obtain 20 mL of exosome suspension;

[0165] (6) Take 20 mL of the obtained PBS suspension of exosomes, add 620 mg of sodium alginate, dissolve and shake evenly, then add 0.2 mL of double-distilled calcium chloride solution containing 50 mg, and shake quickly to obtain 20 mL of exosome gel preparation.

[0166] Test Example 1

[0167] Tranexamic acid content test

[0168] The content of tranexamic acid is detected by derivative ultra-high performance liquid chromatography (ULPC), and the specific detection method is as follows:

[0169] (1) Preparation of derivatization reagent: Dissolve 30 mg of o-phthalaldehyde in 1 mL of methanol; dissolve 0.26 g of sodium sulfite in 1 mL of double distilled water; mix the o-phthalaldehyde and sodium sulfite solutions, add 0.1 mol / L pH 10 borax buffer to make up to 200 mL, and use only for 24 hours;

[0170] (2) Standard curve samples: PBS solutions of tranexamic acid with concentrations of 0, 10, 20, 30, 40, and 50 μg / mL were prepared using tranexamic acid;

[0171] (3) Take 1 mL of exosome PBS suspension, add 1 mL of derivatization reagent, mix well and react for 15 min;

[0172] Wherein, the exosome PBS suspension is the final product obtained in step (4) of Examples 1-10 and Comparative Example 1 respectively;

[0173] (4) Take 1 μL of tranexamic acid derivatized sample and use Waters Acquity ULPC BEH C18 column, detection temperature 37°C, detection voltage controlled at 500 mV, flow rate 0.55 mL / min mobile phase (acetonitrile: 28 mmol / L ammonium acetate solution = 2:8, pH 4.1) isocratic elution, and the expected chromatographic peak time is 2 min 05s to 2 min 15s; the specific test results are shown in Table 1 below:

[0174] Table 1

[0175]

[0176] From the test data in Table 1, it can be seen that the concentration of tranexamic acid in the exosome preparation of the present invention should not be less than 25 μg / mL to meet the standard. Figure 1 is the change of tranexamic acid concentration over time, such as Figure 1 As shown, adipose tissue and serum-free medium were cultured for 22 hours, and 50 mg of tranexamic acid was co-cultured for 2 hours, and the volume of the prepared exosome PBS suspension was 20 mL. The study found that after 90 minutes of co-culture with tranexamic acid, the concentration of tranexamic acid in the exosome preparation was close to the peak.

[0177] Test Example 2

[0178] Total RNA content test

[0179] The total RNA content was used to evaluate the harvest quality of exosomes. The concentration of RNA was detected by spectrophotometry. The extraction kit was Invitrogen PureLink RNA Micro Extraction Kit (Cat. No. 12183016). The specific detection method is as follows:

[0180] (1) Pipette 1 mL of exosome preparation and centrifuge at 15000 rpm for 60 min at 0-8°C, and discard the supernatant;

[0181] (2) Add 100 μL of lysis buffer to resuspend the exosome precipitate and let stand for 5 min;

[0182] (3) Add 50 μL of ethanol to 100 μL of lysate and mix thoroughly. Add the mixed lysate-ethanol solution to the micro filter cartridge assembly and cover it. Centrifuge at 13,000 rpm for 10 seconds and discard the flow-through.

[0183] (4) Add 180 μL of Wash Solution 1 to the micro filter cartridge, centrifuge at 13,000 rpm for 10 s, and discard the flow-through;

[0184] (5) Add 180 μL of Wash Solution 2 / 3 to the micro filter cartridge, centrifuge at 13,000 rpm for 10 s, and discard the flow-through;

[0185] (6) Repeat step 5;

[0186] (7) Place the micro filter cartridge in a clean collection tube, centrifuge at 13,000 rpm for 60 seconds, discard the residual liquid, and dry the micro filter cartridge for 60 seconds;

[0187] (8) Place the micro filter cartridge into a clean elution tube, add 10 μL of preheated elution buffer, and incubate at room temperature for 60 seconds;

[0188] (9) Centrifuge at 13000 rpm for 30 seconds, add 10 μL of preheated elution buffer to the micro filter cartridge again, and incubate at room temperature for 60 seconds;

[0189] (10) Determination of the total amount of RNA: aspirate the entire RNA solution (about 20 μL) and measure the absorbance (OD) at 260 nm without dilution. The RNA content is calculated based on an OD of about 40 ng / μL.

[0190] The specific test results are shown in Table 2 below:

[0191] Table 2

[0192]

[0193] From the test data in Table 2, it can be seen that the RNA concentration in the exosome preparation of the present invention should not be less than 15pg / mL. This fully illustrates that the active ingredients of the exosome preparation of the present invention include exosomes produced by autologous mesenchymal adipose stem cells and modified with tranexamic acid, and the exosome preparation for burn wound repair needs to be able to promote wound healing, reduce melanin deposition on the wound, and reduce the traces after wound healing.

[0194] Test Example 3

[0195] Mouse wound healing rate test

[0196] Eight 6-week-old female SPF BALB / 6J mice were selected and anesthetized with 3% sodium pentobarbital at a dose of 30 mg / kg. Depilatory cream containing mercaptoethanol was used to depilate both sides of the back of the mice. Each depilatory area was >1 cm 2 , use 90℃ constant temperature and constant pressure electric heat source to burn for 15s to cause deep second degree burns, the diameter of the burn is 0.4cm round (area 0.5cm 2 ).

[0197] S01034 and S01048 samples were used for the mouse burn healing test. The exosome gel preparation samples were used for the burn wound on the left side of the mouse, and PBS gel was used for the burn wound on the right side. The amount used each time was 0.2 mL, which was applied to the burn site once every 12 hours. The wound healing area was observed on the 0th, 7th, 14th, and 21st days. The wound healing rate was calculated as follows: Wound healing rate (%) = (initial wound area - wound area during measurement) / initial wound area × 100%.

[0198] The test results are shown in Table 3:

[0199] Table 3

[0200]

[0201]

[0202] From the test data in Table 3, it can be seen that the healing rate of all mice on the 21st day was above 90%, and the healing rate of some mice could reach 100%, which shows that the source of the exosomes of the present invention is autologous adipose mesenchymal stem cells, which can transport the nucleic acids, proteins, lipids and signal molecules of stem cells to target cells, and play a function similar to that of stem cells in burn wound repair, and the exosome gel can promote wound healing, reduce melanin deposition on the wound, and reduce the traces after wound healing.

[0203] The applicant declares that the present invention illustrates the preparation method of exosomes of the present invention and the exosomes prepared thereby and their applications through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned process steps, that is, it does not mean that the present invention must rely on the above-mentioned process steps to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of the raw materials selected by the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the scope of protection and disclosure of the present invention.

Claims

1. A method for preparing exosomes, characterized in that: The preparation method comprises the following steps: (1) digesting autologous adipose tissue with trypsin digestion solution, washing it, and obtaining adipocytes; The trypsin digestion solution comprises: trypsin and EDTA solution; the mass percentage of trypsin in the trypsin digestion solution is 0.05-0.2%; The digestion specifically comprises the following steps: mixing the autologous fat tissue with the trypsin digestion solution used for the first digestion, performing the first digestion, and collecting a partially digested cell suspension; then mixing the remaining undigested autologous fat tissue with the trypsin digestion solution used for the second digestion, performing the second digestion, combining and collecting, and obtaining a digested cell suspension; The volume ratio of the autologous adipose tissue, the trypsin digestion solution used for the first digestion, and the trypsin digestion solution used for the second digestion is (15-25):(15-20):(10-20); (2) culturing the adipocytes obtained in step (1) in a culture medium to obtain a cell suspension; (3) mixing the cell suspension obtained in step (2) with tranexamic acid, culturing, and collecting to obtain exosomes produced by autologous mesenchymal adipose stem cells modified with tranexamic acid; In step (3), the volume-to-mass ratio of the cell suspension to tranexamic acid is (20-30) mL: (4-15) mg; the culture temperature is 36.5-38° C., the culture gas is 5% carbon dioxide, and the culture time is 1-3 h; the collection specifically comprises the following steps: harvesting the cultured exosomes, performing a first centrifugation, and collecting the supernatant; performing a second centrifugation, and collecting the supernatant; and finally performing a third centrifugation, discarding the supernatant, and collecting the precipitate; The temperatures of the first centrifugation, the second centrifugation and the third centrifugation are independently 0-8°C; the speed of the first centrifugation is 1000-3000rpm, and the time of the first centrifugation is 10-15min; the speed of the second centrifugation is 1000-3000rpm, and the time of the second centrifugation is 30-40min; the speed of the third centrifugation is 8000-12000rpm, and the time of the third centrifugation is 50-60min; The precipitate also needs to be initially suspended with 7-10 mL of PBS buffer, and the total RNA concentration is measured after suspension; based on the total RNA concentration of the preliminary exosome PBS suspension, the PBS buffer is used for further dilution to dilute the final exosome total RNA concentration to 15-30 pg / mL.

2. The method for preparing exosomes according to claim 1, characterized in that: In step (1), the concentration of the EDTA solution is 0.05-0.15 mM.

3. The method for preparing exosomes according to claim 1, characterized in that: In step (1), the pH of the EDTA solution is 7.5-8.

5.

4. The method for preparing exosomes according to claim 1, characterized in that: In step (1), the autologous adipose tissue is autologous abdominal adipose tissue.

5. The method for preparing exosomes according to claim 1, characterized in that: The temperature of the first digestion is 36.5-38°C, and the time of the first digestion is 8-12 minutes.

6. The method for preparing exosomes according to claim 1, characterized in that: The temperature of the second digestion is 36.5-38°C, and the time of the second digestion is 4-10 minutes.

7. The method for preparing exosomes according to claim 1, characterized in that: In step (1), the washing specifically includes the following steps: after the digested cell suspension is centrifuged, the supernatant is discarded, PBS buffer is added, and the suspension is shaken and washed to obtain adipocytes.

8. The method for preparing exosomes according to claim 7, characterized in that: The shaking cleaning is performed at least twice.

9. The method for preparing exosomes according to claim 1, characterized in that: In step (2), the culture medium is a serum-free culture medium.

10. The method for preparing exosomes according to claim 1, characterized in that: In step (2), the inoculation volume of the fat cells is 15-25 cm 3 .

11. The method for preparing exosomes according to claim 1, characterized in that: In step (2), the culture temperature is 36.5-38°C, the culture gas is 5% carbon dioxide, and the culture time is 20-28 hours.

12. An exosome gel, characterized in that: The exosome gel comprises exosomes prepared by the method for preparing exosomes according to any one of claims 1 to 11, sodium alginate, calcium chloride and PBS buffer; In the exosome gel, the concentration of total RNA is 15-30 pg / mL, the mass concentration of sodium alginate is 2.8-3.4%, the mass concentration of calcium chloride is 0.4-0.6%, and the solvent is PBS buffer.

13. The method for preparing the exosome gel according to claim 12, characterized in that: The method for preparing the exosome gel comprises the following steps: mixing the exosomes prepared by the method for preparing exosomes according to any one of claims 1 to 11, sodium alginate and PBS buffer, and then mixing with calcium chloride to obtain the exosome gel; The mass concentration of the sodium alginate is 2.8-3.4%, and the mass concentration of the calcium chloride is 0.4-0.6%; the temperature of the mixture of the exosomes, sodium alginate and PBS buffer is 4-25° C., and the mixture is vortexed at a speed of 1500-2000 rpm for 3-10 seconds; The temperature of the mixture with calcium chloride is 4-25° C., and a vortex shaker is used at a speed of 1500-2000 rpm for 6-15 seconds.

14. Use of the exosome gel according to claim 12 in preparing a preparation for repairing burn wounds.

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