Exosome-rich umbilical plasma concentrated preparation as well as preparation method and application thereof

Abstract

The invention discloses an exosome-rich umbilical plasma concentrated preparation as well as a preparation method and application thereof. The method comprises the following steps: carrying out primary centrifugation and secondary centrifugation on umbilical cord blood to obtain an umbilical cord blood supernatant, carrying out primary centrifugation and secondary centrifugation on the umbilical cord blood supernatant to obtain umbilical cord blood exosomes, and remelting the umbilical cord blood exosomes by using O-type umbilical cord blood plasma to obtain the exosome-rich umbilical cord blood plasma concentrated preparation. The method is safe and high in repeatability, potential hazards of external additives to the human body can be reduced, and the prepared exosome-rich umbilical plasma concentrated preparation can be used for preparing products for accelerating wound healing and improving non-scar alopecia and acne.

Description

Technical Field

[0001] The present invention belongs to the technical field of umbilical cord blood exosome treatment, and relates to a concentrated preparation of umbilical cord plasma rich in exosomes, a preparation method thereof, and an application thereof. Background Art

[0002] In the fields of biotechnology, blood product preparation technology, and cell engineering, the research and application of umbilical cord blood have gradually become a hot topic. Umbilical cord blood is a precious biological resource, containing abundant hematopoietic stem cells and immune cells, and is widely used in clinical treatment, such as treating diseases such as leukemia, anemia, and immune deficiency. Stem cell therapy has shown encouraging effects in regenerative medicine. Generally speaking, stem cells have the ability to differentiate into various cell types and can replace damaged cells at the injured site. In addition, stem cells also secrete biofactors that affect surrounding tissues. Although stem cell therapy brings hope for degenerative diseases, there are still some challenges, including host cell rejection, phenotypic stability, and tumor risk. This requires the development of safe and effective cell-free therapies. In recent years, researchers have found that exosomes in umbilical cord blood also have important biological functions, such as promoting cell proliferation, differentiation, migration, and anti-apoptosis. Exosomes are membranous vesicles with a diameter between 30 and 150 nanometers, containing bioactive substances such as proteins, lipids, and nucleic acids, and can regulate various physiological and pathological processes through cell-to-cell communication. One of the focuses of recent research is exosomes as a therapeutic agent. Exosomes can be produced by various cells and are also present in various body fluids, including blood, urine, plasma, amniotic fluid, and semen. Research shows that exosomes of stem cells are responsible for cell-to-cell communication and signal transduction and promote the repair of damaged tissues. Umbilical cord blood contains various stem cells such as mesenchymal stem cells and hematopoietic stem cells, and the proliferation and functions of these stem cells are affected by various cytokines, growth factors, and immunomodulators. Umbilical cord plasma exosomes have the advantages of low immunogenicity, few side effects, and high safety, and have broad application prospects.

[0003] Although there are reports on methods for preparing, cryopreserving, and resuscitating various umbilical cord blood exosomes at home and abroad, due to the strict requirements for clinical application safety, there is still an urgent need for a more original and safer method for preparing a concentrated preparation of umbilical cord plasma rich in exosomes and developing more applications thereof. Summary of the Invention

[0004] In view of the deficiencies of the prior art and the actual needs, the present invention provides a preparation method and application of an exosome-rich umbilical cord plasma concentrate. The method of the present invention is safe and highly reproducible, and can reduce the potential harm of external additives to the human body. The exosome-rich umbilical cord plasma concentrate prepared by the method of the present invention is rich in immunoglobulin-related proteins compared with the plasma of normal people, and has lower heat shock proteins and stress response-related proteins, meeting the characteristics of the primitiveness, low differentiation degree and immunomodulatory function of umbilical cord blood mononuclear cells.

[0005] To achieve the object of the present invention, the following technical solutions are adopted:

[0006] In the first aspect, the present invention provides a method for preparing an exosome-rich umbilical cord plasma concentrate, the method comprising subjecting umbilical cord blood to primary centrifugation and secondary centrifugation to obtain umbilical cord blood supernatant, subjecting the umbilical cord blood supernatant to primary centrifugation and secondary centrifugation to obtain umbilical cord blood exosomes, and thawing the umbilical cord blood exosomes with O-type umbilical cord plasma to obtain the exosome-rich umbilical cord plasma concentrate.

[0007] The method in the present invention is safe and highly reproducible. The exosome-rich umbilical cord plasma concentrate prepared has low immunogenicity, is not likely to cause coagulation reactions of plasma with different blood types, can reduce the potential harm of external additives to the human body, and the exosome-rich umbilical cord plasma concentrate prepared can be used in the preparation of products for accelerating wound healing, improving non-scarring alopecia and improving acne.

[0008] As a preferred technical solution, the method for preparing the exosome-rich umbilical cord plasma concentrate in the present invention comprises the following steps:

[0009] (1) Collect umbilical cord blood, preserve it, and transport it to the umbilical cord blood hematopoietic stem cell bank;

[0010] (2) Conduct virus detection on the umbilical cord blood;

[0011] (3) Under aseptic conditions, subject the umbilical cord blood to primary centrifugation and secondary centrifugation to obtain umbilical cord blood supernatant, and subject the umbilical cord blood supernatant to primary centrifugation and secondary centrifugation to obtain a precipitate, which is umbilical cord blood exosomes;

[0012] (4) Thaw the umbilical cord blood exosomes with O-type umbilical cord plasma to obtain the exosome-rich umbilical cord plasma concentrate.

[0013] In the present invention, primary centrifugation of umbilical cord blood can effectively remove cell debris, secondary centrifugation of umbilical cord blood can obtain relatively pure umbilical cord blood supernatant, primary centrifugation of umbilical cord blood supernatant can effectively remove subcellular components, and secondary centrifugation of umbilical cord blood supernatant can effectively obtain umbilical cord blood exosomes.

[0014] Preferably, the temperature for storage in step (1) is -2°C to 8°C, such as -2°C, -1°C, 0°C, 1°C, 2°C, 4°C or 8°C, etc.; the temperature for transportation is 4°C to 15°C, such as 4°C, 5°C, 6°C, 10°C, 12°C, 14°C or 15°C, etc.

[0015] Preferably, the method for virus detection in step (2) includes the serological antigen-antibody reaction method.

[0016] Preferably, the virus detection in step (2) includes umbilical cord blood hepatitis B surface antigen detection, hepatitis C virus antibody detection, human immunodeficiency virus antibody detection, cytomegalovirus antibody detection and syphilis spirochete antibody detection.

[0017] Preferably, the umbilical cord blood in step (2) includes umbilical cord blood whole blood samples.

[0018] Preferably, in the first centrifugation and the second centrifugation of the umbilical cord blood in step (3), the speed of the first centrifugation is 250×g to 350×g (such as 250×g, 260×g, 280×g, 300×g or 350×g, etc.), the time is 8 to 15 minutes (such as 8 minutes, 9 minutes, 10 minutes, 12 minutes or 15 minutes, etc.), the temperature is 2°C to 8°C (such as 2°C, 3°C, 4°C, 5°C, 6°C, 7°C or 8°C, etc.); the speed of the second centrifugation is 1800×g to 2500×g (such as 1800×g, 1900×g, 2000×g, 2400×g or 2500×g, etc.), the time is 8 to 15 minutes (such as 8 minutes, 9 minutes, 10 minutes, 12 minutes or 15 minutes, etc.), the temperature is 2°C to 8°C (such as 2°C, 3°C, 4°C, 5°C, 6°C, 7°C or 8°C, etc.).

[0019] Preferably, in the first centrifugation and the second centrifugation of the umbilical cord blood supernatant in step (3), the speed of the first centrifugation is 12000×g to 16000×g (such as 12000×g, 13000×g, 14000×g, 15000×g or 16000×g, etc.), the time is 20 to 30 minutes (such as 20 minutes, 22 minutes, 24 minutes, 26 minutes or 30 minutes, etc.), the temperature is 2°C to 8°C (such as 2°C, 3°C, 4°C, 5°C, 6°C, 7°C or 8°C, etc.); the speed of the second centrifugation is 80000×g to 150000×g (such as 80000×g, 90000×g, 100000×g, 120000×g or 150000×g, etc.), the time is 60 to 80 minutes (such as 60 minutes, 62 minutes, 70 minutes, 75 minutes or 80 minutes, etc.), the temperature is 2°C to 8°C (such as 2°C, 3°C, 4°C, 5°C, 6°C, 7°C or 8°C, etc.).

[0020] Preferably, the method for preparing the O-type umbilical cord plasma in step (4) includes: subjecting the virus-tested O-type umbilical cord blood to primary centrifugation and secondary centrifugation to obtain the supernatant of the O-type umbilical cord blood, and subjecting the supernatant of the O-type umbilical cord blood to primary centrifugation and secondary centrifugation under sterile conditions, and the supernatant is the O-type umbilical cord plasma.

[0021] Preferably, the preparation method further includes collecting O-type umbilical cord blood, storing it, and transporting it to the umbilical cord blood hematopoietic stem cell bank.

[0022] Preferably, the storage temperature is -2°C to 8°C, such as -2°C, -1°C, 0°C, 1°C, 2°C, 4°C, or 8°C, etc.; the transportation temperature is 4°C to 15°C, such as 4°C, 5°C, 6°C, 10°C, 12°C, 14°C, or 15°C, etc.

[0023] Preferably, the method for virus detection includes the serological antigen-antibody reaction method.

[0024] Preferably, the virus detection includes the detection of hepatitis B surface antigen in umbilical cord blood, the detection of hepatitis C virus antibody, the detection of human immunodeficiency virus antibody, the detection of cytomegalovirus antibody, and the detection of syphilis spirochete antibody.

[0025] Preferably, the O-type umbilical cord blood includes the whole blood sample of O-type umbilical cord blood.

[0026] Preferably, in the primary centrifugation and secondary centrifugation of the O-type umbilical cord blood, the speed of the primary centrifugation is 250×g to 350×g (such as 250×g, 260×g, 280×g, 300×g, or 350×g, etc.), the time is 8 to 15 minutes (such as 8 minutes, 9 minutes, 10 minutes, 12 minutes, or 15 minutes, etc.), the temperature is 2°C to 8°C (such as 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, or 8°C, etc.), the speed of the secondary centrifugation is 1800×g to 2500×g (such as 1800×g, 1900×g, 2000×g, 2400×g, or 2500×g, etc.), the time is 8 to 15 minutes (such as 8 minutes, 9 minutes, 10 minutes, 12 minutes, or 15 minutes, etc.), and the temperature is 2°C to 8°C (such as 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, or 8°C, etc.).

[0027] Preferably, the speed of the first centrifugation in the primary centrifugation and the secondary centrifugation of the O-type cord blood supernatant is 12,000×g to 16,000×g (such as 12,000×g, 13,000×g, 14,000×g, 15,000×g or 16,000×g, etc.), the time is 20 to 30 minutes (such as 20 minutes, 22 minutes, 24 minutes, 26 minutes or 30 minutes, etc.), and the temperature is 2°C to 8°C (such as 2°C, 3°C, 4°C, 5°C, 6°C, 7°C or 8°C, etc.); the speed of the secondary centrifugation is 80,000×g to 150,000×g (such as 80,000×g, 90,000×g, 100,000×g, 120,000×g or 150,000×g, etc.), the time is 60 to 80 minutes (such as 60 minutes, 62 minutes, 70 minutes, 75 minutes or 80 minutes, etc.), and the temperature is 2°C to 8°C (such as 2°C, 3°C, 4°C, 5°C, 6°C, 7°C or 8°C, etc.).

[0028] In a second aspect, the present invention provides a concentrated preparation of umbilical cord plasma rich in exosomes, and the concentrated preparation of umbilical cord plasma rich in exosomes is prepared by the method described in the first aspect.

[0029] In a third aspect, the present invention provides the use of the concentrated preparation of umbilical cord plasma rich in exosomes described in the second aspect in the preparation of products for accelerating wound healing, improving non-scarring alopecia and improving acne.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] (1) The concentrated preparation of umbilical cord plasma rich in exosomes prepared by the method of the present invention has low immunogenicity and is not likely to cause coagulation reactions of plasma of different blood types;

[0032] (2) The conditions of the method of the present invention are mild, and the concentrated preparation of umbilical cord plasma rich in exosomes prepared does not affect the biological functions of exosomes and does not limit their application in clinics. In addition, umbilical cord plasma is used for reconstitution in the final resuspension stage, reducing the potential harm to the human body caused by external additives such as culture medium, PBS buffer, etc.;

[0033] (3) The concentrated preparation of umbilical cord plasma rich in exosomes prepared by the method of the present invention is rich in immunoglobulin-related proteins compared with the plasma of normal people, and has lower heat shock proteins and stress response-related proteins, meeting the characteristics of the originality of umbilical cord blood mononuclear cells, low differentiation degree and immunomodulatory function. Description of the Drawings

[0034] Figure 1 It is a graph of the particle size and concentration results of exosomes in Example 1;

[0035] Figure 2 It is a graph of the particle size and concentration results of exosomes in Comparative Example 1;

[0036] Figure 3 Comparison chart before and after the treatment of acne with the exosome-rich umbilical cord plasma concentrate preparation obtained by using the method of Example 1;

[0037] Figure 4 Chart of the differential data results of the protein mass spectrometry part of Experimental Example 1 and Comparative Example 1. Detailed implementation manners

[0038] To further elaborate on the technical means and effects adopted by the present invention, the present invention will be further described below in conjunction with examples and drawings. It can be understood that the specific implementation manners described herein are only used to explain the present invention, rather than limiting the present invention.

[0039] For those not specifying specific techniques or conditions in the examples, they shall be in accordance with the techniques or conditions described in the literature in this field or in accordance with the product specifications. For the reagents or instruments not specifying the manufacturer, they are all conventional products that can be obtained through regular commercial channels.

[0040] Example 1

[0041] This example provides a method for preparing an exosome-rich umbilical cord plasma concentrate preparation.

[0042] (1) After collection, the umbilical cord blood is stored and transported to the umbilical cord blood hematopoietic stem cell bank

[0043] Using a triple blood bag containing citrate-phosphate-dextrose anticoagulant, the umbilical cord blood is collected immediately after the birth of the newborn. The umbilical cord blood (from Guangdong Province) is shaken well after collection in the operating room and immediately stored in a 4°C refrigerator. Then, it is transported to the umbilical cord blood hematopoietic stem cell bank by the staff in a 15°C blood transport box. The information of each umbilical cord blood is reviewed by two people. After heat-sealing to remove the blood collection needle and the redundant pipeline, the blood bag is weighed and the umbilical cord blood volume is calculated. Each umbilical cord blood is encoded with a unique number, and then the surface of the blood bag is disinfected.

[0044] (2) Detection of five virus indicators of umbilical cord blood

[0045] About 5 mL of umbilical cord blood was collected, and plasma was separated. Enzyme-linked immunosorbent assay was used to detect HBsAg, HCV-Ab, HIV-Ab, TP-Ab, and CMV-IgM according to the operating procedures of the diagnostic kits for hepatitis B virus surface antigen (HBsAg) (purchased from Beijing Wantai Biological Pharmacy Co., Ltd.), human hepatitis C antibody (HCV-Ab) ELISA kit (purchased from Beijing Wantai Biological Pharmacy Co., Ltd.), human immunodeficiency virus antigen and antibody HIV-Ab ELISA kit (purchased from Beijing Wantai Biological Pharmacy Co., Ltd.), treponema pallidum antibody (TP-Ab) ELISA detection kit (purchased from Beijing Wantai Biological Pharmacy Co., Ltd.), and human cytomegalovirus antibody IgM (CMV-IgM) ELISA detection kit (purchased from Beijing Baier Biotechnology Co., Ltd.).

[0046] (3) Concentrate umbilical cord plasma exosomes

[0047] Two bags of umbilical cord blood (one bag is type O blood, about 100 mL in total) were taken out of the blood bag and transferred into two centrifuge tubes, which were marked, especially the centrifuge tube containing type O blood. After the centrifuge was balanced, low-speed centrifugation (300×g, 10 min, 4°C) was used to remove cell debris. The supernatant was taken into a new centrifuge tube and centrifuged again (2,000×g, 10 min, 4°C). The supernatant was taken into a new centrifuge tube. The supernatant was centrifuged at 4°C at a speed of 15,000×g for 30 min. The supernatant was aspirated into a new centrifuge tube, and the precipitate was discarded to remove subcellular components. The supernatant was ultracentrifuged at 4°C at a speed of 100,000×g for 70 min. Pay attention to distinguishing the centrifuge tubes of type O blood. Aspirate the supernatant of the non-type O blood centrifuge tube, retain 10 mL of the supernatant of the type O blood centrifuge tube, and discard the rest of the supernatant. Resuspend the precipitate in each tube with 30 mL of PBS solution, and then centrifuge at 4°C, 100,000×g for 70 min, and discard the supernatant. Resuspend the exosomes precipitated in the two centrifuge tubes with the 10 mL of the supernatant of the type O blood centrifuge tube reserved and combine them into one centrifuge tube.

[0048] (5) Filtration, storage and detection

[0049] The exosome-rich umbilical cord plasma concentrate suspension was filtered through a 0.22 μm filter and stored in a -80°C refrigerator for future use. At the same time, the dynamic light scattering (DLS) of the Nanosizer TM instrument was used to measure the size distribution and quantity of UCB-Exos for quality control and clinical applications.

[0050] (6) Test results

[0051] The average particle size of the exosome-rich umbilical cord plasma concentrate is 135.6 nm, and the exosomes are concentrated to 8.9×10 10cells / mL, and the final volume of concentration is 10 mL.

[0052] (7) Protein mass spectrometry detection

[0053] Sample treatment: Add lysis buffer (1.5% SDS / 100 mM Tris-CI) to the sample, mix well, perform tissue homogenization and centrifugation to obtain the supernatant. Precipitate the protein in the solution by the acetone precipitation method. After resuspending the obtained protein precipitate with resuspension solution (8 M Urea / 100 mM Tris-Cl), add dithiothreitol (DTT) and incubate at 37 °C for 1 h; subsequently, add iodoacetamide (IAA) and perform alkylation reaction at room temperature in the dark to block sulfhydryl groups. Determine the protein concentration by the Bradford method. Add 100 mM Tris-HCl solution to the reduced and alkylated sample to dilute the Urea concentration to below 2 M, add trypsin according to the mass ratio of enzyme to protein of 1:50, and incubate and shake overnight at 37 °C for digestion. Add TFA the next day to terminate the digestion, and take the supernatant for Sep-Pak C18 desalting. After drying, store at -20 °C for later use.

[0054] Mass spectrometry detection: Appropriate amounts of peptide segments were taken from each sample, and chromatographic separation was performed using a Vanquish Neo UHPLC system, operated with a Neo UHPLC chromatographic system (Thermo Scientific). Buffer: Solution A was an aqueous solution of 0.1% formic acid, and solution B was an aqueous solution of 0.1% formic acid in acetonitrile (acetonitrile was 80%). The chromatographic column was equilibrated with 96% of solution A. After the sample was injected into the Trap Column (PepMap Neo 5μm C18 300μm X 5mm, Thermo Scientific), gradient separation was performed through the analytical chromatographic column (uPAC Neo High Throughput column, Thermo Scientific). The liquid phase gradient was set as follows: from 0 min to 0.1 min, the linear gradient of solution B was from 4% to 6%; from 0.1 min to 1.1 min, the linear gradient of solution B was from 6% to 12%; from 1.1 min to 4.3 min, the linear gradient of solution B was from 12% to 22.5%; from 4.3 min to 6.1 min, the linear gradient of solution B was from 22.5% to 45%; from 6.1 min to 8 min, solution B was maintained at 99%. After peptide separation, DIA (data-independent acquisition) mass spectrometry analysis was performed using an Orbitrap Astral mass spectrometer (Thermo Scientific). The analysis duration was 8 min, the electrospray voltage was 2.2 kV, the detection mode was positive ion, the precursor ion scan range was 380 - 980 m / z, the resolution of the first-stage mass spectrometry was 240,000, the AGC target was 500%, and the first-stage Maximum IT was 3 ms. The resolution of the second-stage mass spectrometry was 80,000, the AGC target was 500%, the second-stage Maximum IT was 3 ms, the RF-lens was 40%, the MS2 Activation Type was HCD, the Isolation window was 2 Th, the Normalized collision energy was 25%, and the cycle time was 0.6.

[0055] (8) One patient with acne was recruited. After washing the face morning and evening every day, the exosome-rich umbilical plasma concentrate preparation was applied to the acne skin specimens. After 15 days of use, the acne and nodules had significantly regressed compared to 0 day, 3 days, and 9 days of use, and the number of newly developed suppurative acne decreased by 70%. See Figure 3 .

[0056] Example 2

[0057] This example provides a method for preparing an exosome-rich umbilical plasma concentrate preparation.

[0058] (1) After the umbilical cord blood was collected, it was stored and transported to the umbilical cord blood hematopoietic stem cell bank

[0059] Using a triple blood bag containing citrate-phosphate-dextrose anticoagulant, cord blood was collected immediately after the birth of the neonate. The cord blood (from Guangdong Province) was shaken well after collection in the operating room and immediately stored in a 4°C refrigerator. It was then transported to the cord blood hematopoietic stem cell bank by staff in a 15°C blood transport box after collection. The information of each cord blood was double-checked by two people. After heat-sealing to remove the blood collection needle and excess tubing, the blood bag was weighed and the cord blood volume was calculated. Each cord blood was coded with a unique number, and then the surface of the blood bag was disinfected.

[0060] (2) Detection of five viral indicators in cord blood

[0061] About 5 mL of cord blood was taken, and plasma was separated. Enzyme-linked immunosorbent assay was used. According to the operating steps of the hepatitis B surface antigen (HBsAg) diagnostic kit (purchased from Beijing Wantai Biological Pharmacy Co., Ltd.), human hepatitis C antibody (HCV-Ab) ELISA kit (purchased from Beijing Wantai Biological Pharmacy Co., Ltd.), human immunodeficiency virus antigen and antibody HIV-Ab ELISA kit (purchased from Beijing Wantai Biological Pharmacy Co., Ltd.), syphilis treponema antibody (TP-Ab) ELISA detection kit (purchased from Beijing Wantai Biological Pharmacy Co., Ltd.) and human cytomegalovirus antibody IgM (CMV-IgM) ELISA detection kit (purchased from Beijing Bell Biological Engineering Co., Ltd.) instruction manuals, HBsAg, HCV-Ab, HIV-Ab, TP-Ab, and CMV-IgM were detected.

[0062] (3) Concentrated umbilical plasma exosomes

[0063] Two bags of cord blood (one bag was type O blood, about 100 mL in total) were taken out from the blood bag and transferred into two centrifuge tubes, which were marked well, especially the centrifuge tube containing type O blood. After the centrifuge was balanced, low-speed centrifugation (250×g, 8 min, 2°C) was used to remove cell debris. The supernatant was taken into a new centrifuge tube and centrifuged again (1800×g, 8 min, 2°C). The supernatant was taken into a new centrifuge tube. The supernatant was centrifuged at a speed of 12,000×g at 2°C for 20 min. The supernatant was aspirated into a new centrifuge tube, and the precipitate was discarded to remove subcellular components. The supernatant was ultracentrifuged at a speed of 80,000×g at 2°C for 60 min. Pay attention to distinguishing the centrifuge tube of type O blood. Aspirate the supernatant of the non-type O blood centrifuge tube, and retain 10 mL of the supernatant of the type O blood centrifuge tube, and discard the rest of the supernatant. The precipitate in each tube was resuspended with 30 mL of PBS solution and then centrifuged at 80,000×g at 2°C for 60 min, and the supernatant was discarded. The precipitate of exosomes in the two centrifuge tubes was resuspended with the retained 10 mL of the supernatant of the type O blood centrifuge tube and combined into one centrifuge tube.

[0064] (5) Filtration, storage and detection

[0065] After the exosome-rich umbilical cord plasma concentrate suspension was filtered through a 0.22-μm filter, it was stored in a -80 °C refrigerator for future use. Meanwhile, the dynamic light scattering (DLS) of the Nanosizer TM instrument was used to measure the size distribution and quantity of UCB-Exos for quality control and clinical applications.

[0066] (6) Test results

[0067] The average particle size of the exosome-rich umbilical cord plasma concentrate was 182.6 nm, and the exosomes were concentrated to 5.6×10 10 per mL, and the final concentrated volume was 10 mL.

[0068] (7) Protein mass spectrometry detection

[0069] Sample treatment: Lysis buffer (1.5% SDS / 100 mM Tris-Cl) was added to the sample and mixed thoroughly. Tissue homogenate was centrifuged to obtain the supernatant. The proteins in the solution were precipitated by the acetone precipitation method. After the obtained protein precipitate was redissolved with a reconstitution solution (8 M Urea / 100 mM Tris-Cl), dithiothreitol (DTT) was added and incubated at 37 °C for 1 h. Subsequently, iodoacetamide (IAA) was added for alkylation reaction at room temperature in the dark to block sulfhydryl groups. The protein concentration was determined by the Bradford method. A 100 mM Tris-HCl solution was added to the reduced and alkylated sample to dilute the Urea concentration below 2 M. Trypsin was added at a mass ratio of enzyme to protein of 1:50 and incubated with shaking overnight at 37 °C for digestion. The next day, TFA was added to terminate the digestion, and the supernatant was taken for Sep-Pak C18 desalting. After drying, it was stored at -20 °C for future use.

[0070] Mass spectrometry detection: An appropriate amount of peptide segments was taken from each sample, and chromatographic separation was performed using a Vanquish Neo UHPLC system, operated by the Neo UHPLC chromatographic system (Thermo Scientific). Buffer: Solution A was an aqueous solution of 0.1% formic acid, and solution B was an aqueous solution of 0.1% formic acid in acetonitrile (acetonitrile was 80%). The chromatographic column was equilibrated with 96% of solution A. After the sample was injected into the Trap Column (PepMap Neo 5μm C18 300μm X 5mm, Thermo Scientific), gradient separation was carried out through the analytical chromatographic column (uPAC Neo High Throughput column, Thermo Scientific). The liquid phase gradient was set as follows: from 0 min to 0.1 min, the linear gradient of solution B was from 4% to 6%; from 0.1 min to 1.1 min, the linear gradient of solution B was from 6% to 12%; from 1.1 min to 4.3 min, the linear gradient of solution B was from 12% to 22.5%; from 4.3 min to 6.1 min, the linear gradient of solution B was from 22.5% to 45%; from 6.1 min to 8 min, solution B was maintained at 99%. After the peptide segments were separated, DIA (data-independent acquisition) mass spectrometry analysis was performed using an Orbitrap Astral mass spectrometer (Thermo Scientific). The analysis duration was 8 min, the electrospray voltage was 2.2 kV, the detection mode: positive ion, the parent ion scanning range: 380 - 980 m / z, the resolution of the first-stage mass spectrometry: 240000, AGC target: 500%, the first-stage Maximum IT: 3 ms. The resolution of the second-stage mass spectrometry: 80000, AGC target: 500%, the second-stage Maximum IT: 3 ms, RF-lens: 40%, MS2 Activation Type: HCD, Isolation window: 2 Th, Normalized collision energy: 25%, cycle time: 0.6.

[0071] (8) One patient with acne was recruited. After washing the face in the morning and evening every day, the patient used the exosome-rich umbilical plasma concentrate preparation to apply on the acne skin specimens. After 15 days of use, the acne and nodules significantly regressed compared with those at 0 day, 3 days, and 9 days of use, and the number of newly developed suppurative acne decreased by 70%.

[0072] Example 3

[0073] This example provides a preparation method for an exosome-rich umbilical plasma concentrate preparation.

[0074] (1) After the umbilical cord blood was collected, it was stored and transported to the umbilical cord blood hematopoietic stem cell bank

[0075] Using a triple blood bag containing citrate-phosphate-dextrose anticoagulant, cord blood was collected immediately after the birth of the neonate. The cord blood (from Guangdong Province) was shaken well after collection in the operating room and immediately stored in a -2°C refrigerator. It was then transported to the cord blood hematopoietic stem cell bank by staff in a 4°C blood transport box after collection. The information of each cord blood was double-checked by two people. After heat-sealing to remove the blood collection needle and excess tubing, the blood bag was weighed and the cord blood volume was calculated. Each cord blood was coded with a unique number, and then the surface of the blood bag was disinfected.

[0076] (2) Detection of five viral indicators in cord blood

[0077] About 5 mL of cord blood was collected, and plasma was separated. Enzyme-linked immunosorbent assay was used. According to the operating steps of the hepatitis B surface antigen (HBsAg) diagnostic kit (purchased from Beijing Wantai Biological Pharmacy Co., Ltd.), human hepatitis C antibody (HCV-Ab) ELISA kit (purchased from Beijing Wantai Biological Pharmacy Co., Ltd.), human immunodeficiency virus antigen and antibody HIV-Ab ELISA kit (purchased from Beijing Wantai Biological Pharmacy Co., Ltd.), treponema pallidum antibody (TP-Ab) ELISA detection kit (purchased from Beijing Wantai Biological Pharmacy Co., Ltd.) and human cytomegalovirus antibody IgM (CMV-IgM) ELISA detection kit (purchased from Beijing Bell Biological Engineering Co., Ltd.) instructions, HBsAg, HCV-Ab, HIV-Ab, TP-Ab, and CMV-IgM were detected.

[0078] (3) Concentrated umbilical plasma exosomes

[0079] Two bags of cord blood (one bag was type O blood, about 100 mL in total) were taken out from the blood bag and transferred to two centrifuge tubes. The centrifuge tubes were marked, especially the centrifuge tube containing type O blood. After the centrifuge was balanced, low-speed centrifugation (350×g, 15 min, 8°C) was used to remove cell debris. The supernatant was taken to a new centrifuge tube and centrifuged again (2500×g, 15 min, 8°C). The supernatant was taken to a new centrifuge tube. The supernatant was centrifuged at 8°C, 16,000×g for 30 min. The supernatant was aspirated into a new centrifuge tube, and the precipitate was discarded to remove subcellular components. The supernatant was ultracentrifuged at 8°C, 150,000×g for 80 min. Pay attention to distinguish the centrifuge tube of type O blood. Aspirate the supernatant of the non-type O blood centrifuge tube, and retain 10 mL of the supernatant of the type O blood centrifuge tube. The rest of the supernatant was discarded. The precipitate in each tube was resuspended with 30 mL of PBS solution and then centrifuged at 8°C, 150,000×g for 80 min, and the supernatant was discarded. The precipitate of exosomes in the two centrifuge tubes was resuspended with the 10 mL of the supernatant of the type O blood centrifuge tube reserved and combined into one centrifuge tube.

[0080] (5) Filtration, storage and detection

[0081] The exosome-rich umbilical cord plasma concentrate suspension was filtered through a 0.22 μm filter and stored in a -80 °C refrigerator for later use. Meanwhile, the dynamic light scattering (DLS) of the Nanosizer TM instrument was used to measure the size distribution and quantity of UCB-Exos for quality control and clinical applications.

[0082] (6) Test results

[0083] The average particle size of the exosome-rich umbilical cord plasma concentrate was 125.3 nm, and the exosomes were concentrated to 6.4×10 10 per mL, and the final concentrated volume was 10 mL.

[0084] (7) Protein mass spectrometry detection

[0085] Sample treatment: Lysis buffer (1.5% SDS / 100 mM Tris-Cl) was added to the sample, mixed thoroughly, and tissue homogenate was centrifuged to obtain the supernatant. The proteins in the solution were precipitated by the acetone precipitation method. After the obtained protein precipitate was redissolved with a reconstitution solution (8 M Urea / 100 mM Tris-Cl), dithiothreitol (DTT) was added and incubated at 37 °C for 1 h; subsequently, iodoacetamide (IAA) was added for alkylation reaction at room temperature in the dark to block sulfhydryl groups. The protein concentration was determined by the Bradford method. A 100 mM Tris-HCl solution was added to the reduced and alkylated sample to dilute the Urea concentration to below 2 M, and trypsin was added at a mass ratio of enzyme to protein of 1:50 and incubated with shaking at 37 °C overnight for digestion. The next day, TFA was added to terminate the digestion, and the supernatant was taken for Sep-Pak C18 desalting. After drying, it was stored at -20 °C for later use.

[0086] Mass spectrometry detection: An appropriate amount of peptide segments was taken from each sample, and chromatographic separation was performed using a Vanquish Neo UHPLC system, operated by the Neo UHPLC chromatographic system (Thermo Scientific). Buffer: Solution A was an aqueous solution of 0.1% formic acid, and solution B was an aqueous solution of 0.1% formic acid in acetonitrile (acetonitrile was 80%). The chromatographic column was equilibrated with 96% of solution A. After the sample was injected into the Trap Column (PepMap Neo 5μm C18 300μm X 5mm, Thermo Scientific), gradient separation was carried out through the analytical chromatographic column (uPAC Neo High Throughput column, Thermo Scientific). The liquid phase gradient was set as follows: from 0 min to 0.1 min, the linear gradient of solution B was from 4% to 6%; from 0.1 min to 1.1 min, the linear gradient of solution B was from 6% to 12%; from 1.1 min to 4.3 min, the linear gradient of solution B was from 12% to 22.5%; from 4.3 min to 6.1 min, the linear gradient of solution B was from 22.5% to 45%; from 6.1 min to 8 min, solution B was maintained at 99%. After the peptide segments were separated, DIA (data-independent acquisition) mass spectrometry analysis was performed using an Orbitrap Astral mass spectrometer (Thermo Scientific). The analysis duration was 8 min, the electrospray voltage was 2.2 kV, the detection mode: positive ion, the precursor ion scan range: 380 - 980 m / z, the resolution of the first-stage mass spectrometry: 240000, AGC target: 500%, the first-stage Maximum IT: 3 ms. The resolution of the second-stage mass spectrometry: 80000, AGC target: 500%, the second-stage Maximum IT: 3 ms, RF-lens: 40%, MS2 Activation Type: HCD, Isolation window: 2 Th, Normalized collision energy: 25%, cycle time: 0.6.

[0087] (8) One patient with acne was recruited. After washing the face in the morning and evening every day, the patient used the exosome-rich umbilical cord plasma concentrate preparation to smear on the acne approval specimens. After 15 days of use, the acne and nodules had significantly regressed compared with 0 day, 3 days, and 9 days of use, and the number of newly developed suppurative acne decreased by 70%.

[0088] Comparative example

[0089] This comparative example provides a preparation method of an exosome-rich umbilical cord plasma concentrate preparation.

[0090] (1) After the umbilical cord blood was collected, it was stored and transported to the umbilical cord blood hematopoietic stem cell bank

[0091] Refer to Example 1.

[0092] (2) Detection of Five Virus Markers in Umbilical Cord Blood

[0093] Refer to Example 1.

[0094] (3) Concentrated Umbilical Cord Plasma Exosomes

[0095] Take out 2 bags of umbilical cord blood (both non-O blood type) from the blood bag and transfer them into 2 centrifuge tubes, which are marked well. After the centrifuge is balanced, use low-speed centrifugation (300×g, 10 min, 4°C) to remove cell debris. Take the supernatant into a new centrifuge tube and centrifuge again (2,000×g, 10 min, 4°C), then take the supernatant into a new centrifuge tube. The supernatant is centrifuged at a speed of 15,000×g at 4°C for 30 min. Aspirate the supernatant into a new centrifuge tube, discard the precipitate, and remove subcellular components; the supernatant is ultracentrifuged at a speed of 100,000×g at 4°C for 70 min, and the supernatant is discarded. Keep the plasma in 1 tube. Resuspend the exosomes precipitated in 2 centrifuge tubes with the supernatant of the centrifuge tube and combine them into one centrifuge tube.

[0096] (5) Filtration, Storage and Detection

[0097] The suspension of the exosome-rich umbilical cord plasma concentrate is filtered through a 0.22μm filter and stored in a -80°C refrigerator for future use. At the same time, use a Nanosizer TM instrument's dynamic light scattering (DLS) to measure the size distribution and quantity of UCB-Exos for quality control and clinical applications.

[0098] (6) Detection Results

[0099] Visible agglomerated substances appeared in the exosome-rich umbilical cord plasma after preparation, which were removed by filtration through a 0.22μm filter, but there is a risk. Finally, the average particle size of the exosome-rich umbilical cord plasma concentrate is 142.2nm, but the peak width is significantly wider than that in Example 1. The exosomes are concentrated to 7.5×10 10 per mL, and the final concentrated volume is 10 mL.

[0100] (7) Protein Mass Spectrometry Detection

[0101] Sample treatment: Add lysis buffer (1.5% SDS / 100 mM Tris-CI) to the sample, mix well, perform tissue homogenization and centrifugation to obtain the supernatant. Precipitate the protein in the solution using the acetone precipitation method. After resuspending the obtained protein precipitate with the resuspension solution (8 M Urea / 100 mM Tris-Cl), add dithiothreitol (DTT) and incubate at 37 °C for 1 h; subsequently, add iodoacetamide (IAA) and perform alkylation reaction at room temperature in the dark to block sulfhydryl groups. Determine the protein concentration using the Bradford method. Add 100 mM Tris-HCl solution to the reduced and alkylated sample to dilute the Urea concentration below 2 M, add trypsin according to the mass ratio of enzyme to protein of 1:50, and incubate and shake overnight at 37 °C for digestion. Add TFA the next day to terminate the digestion, and take the supernatant for Sep-Pak C18 desalting. After drying by evaporation, store at -20 °C for later use.

[0102] Mass spectrometry detection: An appropriate amount of peptide segments was taken from each sample, and chromatographic separation was performed using a Vanquish Neo UHPLC system, operated by the Neo UHPLC chromatographic system (Thermo Scientific). Buffer: Solution A was 0.1% formic acid aqueous solution, and solution B was 0.1% formic acid acetonitrile aqueous solution (acetonitrile was 80%). The chromatographic column was equilibrated with 96% of solution A. After the sample was injected into the Trap Column (PepMap Neo 5μm C18 300μm X 5mm, Thermo Scientific), gradient separation was carried out through the analytical chromatographic column (uPAC Neo High Throughput column, Thermo Scientific). The liquid phase gradient was set as follows: from 0 min to 0.1 min, the linear gradient of solution B was from 4% to 6%; from 0.1 min to 1.1 min, the linear gradient of solution B was from 6% to 12%; from 1.1 min to 4.3 min, the linear gradient of solution B was from 12% to 22.5%; from 4.3 min to 6.1 min, the linear gradient of solution B was from 22.5% to 45%; from 6.1 min to 8 min, solution B was maintained at 99%. After the peptide segments were separated, DIA (data-independent acquisition) mass spectrometry analysis was performed using an Orbitrap Astral mass spectrometer (Thermo Scientific). The analysis duration was 8 min, the electrospray voltage was 2.2 kV, the detection mode was positive ion, the precursor ion scan range was 380 - 980 m / z, the resolution of the first-stage mass spectrometry was 240,000, AGC target: 500%, the first-stage Maximum IT: 3 ms. The resolution of the second-stage mass spectrometry was 80,000, AGC target: 500%, the second-stage Maximum IT: 3 ms, RF-lens: 40%, MS2 Activation Type: HCD, Isolation window: 2 Th, Normalized collision energy: 25%, cycle time: 0.6.

[0103] The differential data of the protein mass spectrometry part of Example 1 and the comparative example are shown in Figure 4 ... The detection results showed that immunoglobulin-related proteins were generally higher in the exosome-rich umbilical plasma concentrate preparation compared to the blood content of normal people, and specific immunoglobulins were significantly up-regulated; the contents of heat shock proteins and stress response-related proteins were significantly lower, demonstrating the originality of the exosome-rich umbilical plasma concentrate preparation.

[0104] In summary, the method of the present invention is safe and highly reproducible, can reduce the potential harm of external additives to the human body, and the prepared exosome-rich umbilical plasma concentrate preparation can be used in the preparation of products for accelerating wound healing, improving non-scarring alopecia, and improving acne.

[0105] The applicant declares that the present invention illustrates the detailed method of the present invention through the above embodiments, but the present invention is not limited to the above detailed method, that is, it does not mean that the present invention must rely on the above detailed method to be implemented. Those skilled in the art should understand that any improvement to the present invention, the equivalent substitution of each raw material of the present invention's product, the addition of auxiliary components, the selection of specific methods, etc., all fall within the protection scope and the disclosure scope of the present invention.

Claims

1. A method for preparing an exosome-rich umbilical plasma concentrate preparation, characterized in that, The method includes subjecting umbilical cord blood to primary centrifugation and secondary centrifugation to obtain umbilical cord blood supernatant, subjecting the umbilical cord blood supernatant to primary centrifugation and secondary centrifugation to obtain umbilical cord blood exosomes, and using O-type umbilical cord plasma to reconstitute the umbilical cord blood exosomes, thereby obtaining the exosome-rich umbilical cord plasma concentrate preparation.

2. The method for preparing an exosome-rich umbilical plasma concentrate preparation according to claim 1, characterized in that, The method includes the following steps: (1) Collect umbilical cord blood, store it, and transport it to the umbilical cord blood hematopoietic stem cell bank; (2) Conduct virus detection on the umbilical cord blood; (3) Under sterile conditions, subject the umbilical cord blood to primary centrifugation and secondary centrifugation to obtain umbilical cord blood supernatant, and subject the umbilical cord blood supernatant to primary centrifugation and secondary centrifugation to obtain a precipitate, which is the umbilical cord blood exosomes; (4) Use O-type umbilical cord plasma to reconstitute the umbilical cord blood exosomes to obtain the exosome-rich umbilical cord plasma concentrate preparation.

3. The method for preparing an exosome-rich umbilical plasma concentrate preparation according to claim 2, wherein In step (1), the storage temperature is -2°C to 8°C; the transportation temperature is 4°C to 15°C.

4. The method for preparing an exosome-rich umbilical plasma concentrate preparation according to claim 2 or 3, characterized in that, In step (2), the method for virus detection includes the serological antigen-antibody reaction method; Preferably, in step (2), the virus detection includes detection of hepatitis B surface antigen in umbilical cord blood, detection of hepatitis C virus antibody, detection of human immunodeficiency virus antibody, detection of cytomegalovirus antibody, and detection of Treponema pallidum antibody.

5. The method for preparing an exosome-rich umbilical plasma concentrate preparation according to any one of claims 2-4, characterized in that, In step (2), the umbilical cord blood includes the whole blood sample of umbilical cord blood.

6. The method for preparing an exosome-rich umbilical plasma concentrate preparation according to any one of claims 2-5, characterized in that, In step (3), for the primary centrifugation and secondary centrifugation of umbilical cord blood, the speed of the primary centrifugation is 250×g to 350×g, the time is 8 to 15 minutes, the temperature is 2°C to 8°C, the speed of the secondary centrifugation is 1800×g to 2500×g, the time is 8 to 15 minutes, and the temperature is 2°C to 8°C.

7. The method for preparing an exosome-rich umbilical plasma concentrate preparation according to any one of claims 2-6, characterized in that, In step (3), for the primary centrifugation and secondary centrifugation of the umbilical cord blood supernatant, the speed of the primary centrifugation is 12000×g to 16000×g, the time is 20 to 30 minutes, the temperature is 2°C to 8°C; the speed of the secondary centrifugation is 80000×g to 150000×g, the time is 60 to 80 minutes, and the temperature is 2°C to 8°C.

8. The method for preparing an exosome-rich umbilical plasma concentrate preparation according to any one of claims 2-7, characterized in that, In step (4), the preparation method of the O-type umbilical cord plasma includes: subjecting the O-type umbilical cord blood after virus detection to primary centrifugation and secondary centrifugation to obtain O-type umbilical cord blood supernatant, and under sterile conditions, subjecting the O-type umbilical cord blood supernatant to primary centrifugation and secondary centrifugation, and the supernatant is the O-type umbilical cord plasma.

9. An exosome-rich umbilical plasma concentrate preparation, characterized in that, The exosome-rich umbilical cord plasma concentrate preparation is prepared by the method for preparing the exosome-rich umbilical cord plasma concentrate preparation according to any one of claims 1-8.

10. Use of the exosome-rich umbilical cord plasma concentrate preparation according to claim 9 in the preparation of products for accelerating wound healing, improving non-scarring alopecia, and improving acne.

Images