Preparation method and application of function-enhanced mesenchymal stem cell exosome

Abstract

The invention relates to the technical field of biological medicine, in particular to a preparation method and application of a function-enhanced mesenchymal stem cell exosome, the preparation method comprises the following steps: S1, preparing an NK cell exosome, collecting supernate in an NK cell culture medium, and treating the supernate by using an optimized exosome separation and purification process to obtain an NK cell exosome; finishing preparation of a stock solution; s2, culturing the mesenchymal stem cells, culturing the cells in a 3D suspension culture mode, treating and controlling low oxygen concentration by combining with NK cell exosomes, and simulating an in-vivo inflammation microenvironment in a multi-azimuth manner, so that the exosomes secreted by the mesenchymal stem cells have a stronger immune regulation function and an anti-inflammatory effect; s3, preparation of mesenchymal stem cell exosomes: preparing a stock solution through an exosome separation and purification process combining a tangential flow ultrafiltration technology and a PS affinity chromatography technology; the exosome stock solution obtained by the method is high in purity and high in yield, and has more excellent biological activity and excellent treatment effect.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and specifically to a method for preparing function-enhanced mesenchymal stem cell exosomes and applications thereof. Background Art

[0002] Anti-allergic drugs are often used in domestic clinics to treat skin allergic diseases, but long-term or frequent use can cause adverse reactions in the respiratory tract, digestive tract, etc. Glucocorticoid drugs can be used for severe cases, but the side effects are strong and seriously affect the health of the body. Mesenchymal stem cells (MSCs) mainly exert their immunoregulatory function through paracrine effects. Exosomes are tiny vesicles with a lipid bilayer membrane structure. They are nanoscale substances secreted by cells to achieve intercellular information exchange. They can transport proteins, fats, metabolites, nucleic acids and other substances to receptor cells. Mesenchymal stem cell exosomes (MSC-EXO) carry the bioactive substances of MSCs and inherit the immunoregulatory function of MSCs. Using exosome therapy instead of stem cell therapy can avoid most of the problems associated with cell therapy, reproduce the therapeutic effects of stem cells, and avoid the side effects of traditional drug therapy on the human body.

[0003] Under the stimulation of the inflammatory microenvironment in vivo, MSCs will produce a large number of immunomodulatory factors, cell activation factors and growth factors, which can regulate the immune microenvironment of tissues, downregulate inflammatory factors and promote tissue repair. Traditional technology usually uses 2D adherent culture plus cytokine stimulation to culture MSCs. 2D adherent culture is to attach cells to the surface of a planar material for static culture, and the cells are usually placed in a normoxic environment during culture. This culture method cannot well simulate the natural three-dimensional, hypoxic growth microenvironment of cells in the human body, affecting the physiological function of cells. Cytokine stimulation generally uses pro-inflammatory factors such as TNF-α, INF-γ and IL-1β to simulate inflammatory signals in vivo to activate the expression of related genes of MSCs. However, cytokines have a single effect, and the use of cytokine stimulation cannot achieve the best activation effect. Therefore, the MSC-EXO obtained by the prior art cannot exert satisfactory clinical efficacy. Therefore, a method for preparing mesenchymal stem cell exosomes with enhanced function and its application are proposed. Summary of the invention

[0004] The object of the present invention is to provide a method for preparing mesenchymal stem cell exosomes with enhanced function and its application, so as to solve the problems raised in the above background technology.

[0005] The purpose of the present invention can be achieved through the following technical solutions:

[0006] A method for preparing mesenchymal stem cell exosomes with enhanced function, the preparation method comprising the following steps:

[0007] S1. Preparation of NK cell exosomes

[0008] The supernatant in the NK cell culture medium was collected, and the supernatant was treated using an optimized exosome separation and purification process to enrich the NK cell exosomes;

[0009] S2. Mesenchymal stem cell culture

[0010] Cultivating mesenchymal stem cells using a 3D suspension culture method, maintaining a low level of dissolved oxygen concentration during the culture process, and using the NK cell exosomes obtained in step S1 to treat the mesenchymal stem cells, activate the mesenchymal stem cells to express immune regulation-related proteins, and release the exosomes into the culture medium supernatant;

[0011] S3. Preparation of mesenchymal stem cell exosomes

[0012] The culture medium supernatant obtained in step S2 is collected, and the mesenchymal stem cell exosomes are enriched using the optimized exosome separation and purification process.

[0013] Preferably, the mesenchymal stem cells in step S2 are derived from human umbilical cord or human fat;

[0014] The 3D suspension culture method in step S2 is to culture mesenchymal stem cells on microcarriers at a rate of 1-3×10 5 The density of cells / ml was inoculated in a bioreactor for suspension culture. The ratio of the number of mesenchymal stem cells to the effective attachment area of ​​the microcarrier was 6000-8000 cells / cm 2 ;

[0015] The 3D suspension culture conditions in step S2 are as follows: pH value is controlled at 6.5-7.5, temperature is controlled at 36-38°C, stirring speed is controlled at 20-40 rpm on the first day after inoculation, stirring speed is controlled at 60-100 rpm from the second day onwards, and cell culture fluid is harvested on the fifth day.

[0016] Preferably, the low level of dissolved oxygen concentration in step S2 is achieved by controlling the volume fraction of oxygen introduced into the reactor to be 2%-8%;

[0017] The concentration of NK cell exosomes in step S2 is 100-200 particles / cell, and the particle-protein ratio of NK cell exosomes is ≥1×10 7 Particles / μg, NK cell exosomes were added on the second day after mesenchymal stem cells were inoculated.

[0018] Preferably, the optimized exosome separation and purification process in step S1 and step S3 comprises the following steps:

[0019] P1. Clarification and Filtration

[0020] The collected cell culture medium supernatant is centrifuged and then filtered through a filter membrane to obtain a clarified filtered harvest solution;

[0021] P2, ultrafiltration concentration

[0022] The clarified filtered harvest liquid obtained in step P1 is treated by a tangential flow concentration process to obtain a concentrated harvest liquid;

[0023] P3, affinity chromatography

[0024] The concentrated harvest liquid obtained in step P2 is treated by PS affinity chromatography to obtain a purified harvest liquid;

[0025] P4. Preparation of stock solution

[0026] The stock buffer was replaced with the solvent of the purified harvest solution obtained in step P3 by the tangential flow replacement process, and the concentration of exosome particles was adjusted to 1-3×10 9 particles / ml, and the stock solution was sterilized and filtered to obtain a sterile exosome stock solution.

[0027] Preferably, the centrifugal conditions in step P1 are: centrifugal force of 1000-3000×g, centrifugal time of 5-10 min, and centrifugal temperature of 16-25°C;

[0028] The material of the filter membrane in step P1 is at least one of cellulose acetate, polyethersulfone or polyvinylidene fluoride, and the retention pore size of the filter membrane is 0.20-0.45 μm.

[0029] Preferably, the tangential flow concentration process in step P2 is: using a tangential flow system to apply back pressure at the reflux end of the ultrafiltration membrane structure, so that the filtrate is filtered out from the permeate end of the ultrafiltration membrane structure, the volume of the reflux liquid is reduced, and the clarified filtration harvest liquid is continuously concentrated during the circulation process;

[0030] The ultrafiltration membrane structure is at least one of a cassette membrane package and a hollow fiber column, the material of the ultrafiltration membrane is one of regenerated cellulose and polyethersulfone, and the molecular weight cutoff of the ultrafiltration membrane is 300-500KD;

[0031] In step P2, the transmembrane pressure is controlled to be 2-4 psi, the inlet flow rate is adjusted to 60-120 LMH, and the inlet shear flow rate is controlled to be less than 4000 s -1 The concentration multiple of the prepared concentrated harvest solution is controlled to be 10-50 times.

[0032] Preferably, the PS affinity chromatography process in step P3 is: utilizing the property of phosphatidylserine on the surface of the exosome membrane to specifically bind to Tim4, when calcium ions are present, Tim4 fixed on the carrier captures the exosomes in the concentrated harvest solution, after the capture is completed, the carrier is cleaned to remove impurities, and then a calcium ion chelator is added thereto to elute the exosomes on Tim4 to obtain a purified harvest solution;

[0033] The material of the carrier is resin, and the specific surface area of ​​the carrier is 120m 2 / g, the density of the carrier is 0.95g / cm 3 , the particle size of the carrier is 45 μm, Tim4 is fixed on the surface of the carrier, and loaded into the separation column;

[0034] The PS affinity chromatography process in step P3 is characterized by comprising the following steps:

[0035] a. Adding a buffer and the concentrated harvest solution obtained in step P2 to the separation column to allow the exosomes to bind to Tim4;

[0036] b. Adding buffer to the separation column to wash the carrier and remove impurities;

[0037] c. adding an eluent to the separation column to recover the exosomes;

[0038] The eluent in step c contains a calcium ion chelating agent;

[0039] The stock solution buffer in step P4 contains 0.15 mol / L NaCl and 0.1 M sucrose, and the pH value of the stock solution buffer is 7.0-7.4;

[0040] The tangential flow displacement process in step P4 is: the tangential flow system in step P2 is used to continuously perform solvent displacement multiple times with the same volume, thereby removing exogenous pollutants, and the displacement volume multiple is 6-10 times;

[0041] In step P4, a sterilizing filter is used for sterilizing filtration, the filter membrane of the sterilizing filter is made of polyethersulfone, and the filter membrane retention pore size is 0.22 μm.

[0042] The invention discloses an application of function-enhanced mesenchymal stem cell exosomes. The function-enhanced mesenchymal stem cell exosomes prepared by a method for preparing function-enhanced mesenchymal stem cell exosomes can downregulate cellular inflammatory factors and are used to prepare drugs for treating skin allergic diseases. The drugs can slow down or eliminate skin inflammation and allergic reactions and promote skin cell repair.

[0043] Beneficial effects of the present invention:

[0044] 1. The present invention treats mesenchymal stem cells (MSCs) with NK cell exosomes (NK-EXO), which can better activate the immune regulatory function of MSCs, stimulate MSCs to express and secrete immune regulatory factors, and promote MSCs to secrete mesenchymal stem cell exosomes (MSC-EXO) with enhanced immune regulatory function. MSC-EXO has stronger anti-inflammatory and anti-allergic effects.

[0045] 2. The present invention adopts 3D suspension culture combined with low oxygen content culture, which is closer to the natural MSCs growth microenvironment in the body, has better repeatability and stability, and is easier to achieve large-scale production of high-concentration exosome raw materials.

[0046] 3. The present invention completes the preparation of MSC-EXO stock solution through the optimized exosome separation and purification process, combined with tangential flow ultrafiltration technology and PS affinity chromatography technology. Compared with the previous exosome preparation process, the MSC-EXO obtained in the present invention has high purity, large yield, better biological activity and excellent therapeutic effect, and has broad application prospects in the clinical treatment of skin allergic diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative work.

[0048] Figure 1 is a particle size distribution diagram of NK cell exosomes in the embodiment provided by the present invention;

[0049] Figure 2 is a transmission electron microscopy image of NK cell exosomes in an embodiment provided by the present invention;

[0050] Figure 3 The cell morphology of mesenchymal stem cells (MSCs) after culture in the examples and comparative examples provided by the present invention: Example (A), Comparative Example 1 (B), Comparative Example 2 (C);

[0051] Figure 4 It is a comparison chart of the cell proliferation multiples of MSCs after culture in the examples and comparative examples provided by the present invention (*P<0.05, **P<0.01);

[0052] Figure 5 It is a comparison chart of the content of the anti-inflammatory factor TGF-β in the supernatant of MSCs after culture in the examples provided by the present invention and the comparative examples (*P<0.05, **P<0.01);

[0053] Figure 6It is a comparison chart of the content of the anti-inflammatory factor IL-10 in the supernatant of MSCs after culture in the embodiment provided by the present invention and the comparative example (*P<0.05, **P<0.01);

[0054] Figure 7 1 is a particle size distribution diagram of mesenchymal stem cell exosomes (MSC-EXO) prepared in the examples and comparative examples provided by the present invention: Example (A), Comparative Example 1 (B), Comparative Example 2 (C);

[0055] Figure 8 TEM images of MSC-EXO prepared in the embodiments and comparative examples provided by the present invention: embodiment (A), comparative example 1 (B), comparative example 2 (C);

[0056] Fig. 9 It is a comparison diagram of the proportion of Treg cells in activated human cord blood mononuclear cells (PBMC) after MSC-EXO prepared in the embodiment and the comparative example provided by the present invention were added to PBMC for culture (*P<0.05, **P<0.01); DETAILED DESCRIPTION

[0057] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0058] A method for preparing mesenchymal stem cell exosomes with enhanced function, the preparation method comprising the following steps:

[0059] S1. Preparation of NK cell exosomes

[0060] The supernatant in the NK cell culture medium was collected, and the supernatant was treated using an optimized exosome separation and purification process to enrich the NK cell exosomes;

[0061] S2. Mesenchymal stem cell culture

[0062] Cultivating mesenchymal stem cells using a 3D suspension culture method, maintaining a low level of dissolved oxygen concentration during the culture process, and using the NK cell exosomes obtained in step S1 to treat the mesenchymal stem cells, activate the mesenchymal stem cells to express immune regulation-related proteins, and release the exosomes into the culture medium supernatant;

[0063] S3. Preparation of mesenchymal stem cell exosomes

[0064] The culture medium supernatant obtained in step S2 is collected, and the mesenchymal stem cell exosomes are enriched using the optimized exosome separation and purification process.

[0065] NK cell exosomes (NK-EXO) contain a variety of cytokines (TNF-α, IL-10, IFN-γ, etc.), chemokines (CCL3, CCL4, CCL5, etc.) and growth factors (GM-CSF, etc.). Compared with a single cytokine, using NK-EXO to treat mesenchymal stem cells (MSCs) can better promote MSCs to express anti-inflammatory factors (IL-4, IL-10 and COX2).

[0066] Example

[0067] (I) Preparation and identification of NK cell exosomes (NK-EXO):

[0068] 1. When NK cells are cultured for 16-18 days, extract the supernatant of the cell culture medium and centrifuge at a centrifugal force of 2000×g, a centrifugal time of 5 minutes, and a centrifugal temperature of 20°C.

[0069] 2. Collect the supernatant after centrifugation and transfer it to a disposable sterile vacuum filtration cup. Use a polyethersulfone filter membrane with a retention pore size of 0.22 μm to filter and remove impurities such as residual cell debris and macromolecules in the supernatant under a negative pressure of 0.08 MPa to obtain a clarified filtered harvest liquid.

[0070] 3. Use a tangential flow system to concentrate, clarify and filter the harvested liquid. The tangential flow system includes a hollow fiber column with a molecular weight cutoff of 500 KD.

[0071] Before loading, flush the pipeline and hollow fiber column with PBS and balance the system until the pH value of the effluent at the reflux end and the permeate end is neutral; after loading, the whole process is controlled at a transmembrane pressure of 3.5psi, an inlet flow rate of 80LMH, and an inlet shear flow rate of 3000s -1 , so that the clarified filtered harvest liquid is concentrated by 20 times in volume to obtain a concentrated harvest liquid.

[0072] 4. Take a PS affinity chromatography separation column, add a buffer balance system, and then slowly add the concentrated harvest liquid to allow the exosomes to bind to Tim4 on the carrier. After the liquid in the separation column has flowed out, add buffer to wash the carrier to remove impurities, and then add eluent to the separation column to obtain a purified harvest liquid.

[0073] 5. Use the tangential flow system to replace the purified harvest solution, add an equal volume of stock buffer to the purified harvest solution, and use the tangential flow system to concentrate the harvest solution to half the volume. Repeat this process 10 times.

[0074] The stock buffer solution contained 0.15 mol / L NaCl, 0.1 M sucrose, the pH value was controlled between 7.0 and 7.4, the molecular weight cutoff of the hollow fiber column was 500 KD, the transmembrane pressure of the whole process was controlled at 3.5 psi, the inlet flow rate was controlled at 80 LMH, and the inlet shear flow rate was controlled at 3000 s -1 .

[0075] 6. The concentration of exosome particles after replacement was determined by nanoparticle tracking analysis (NTA). NTA used the German ZetaView nanoparticle tracking analyzer, model ZetaViewPMX110. The samples were tested three times, and the final concentration and particle size distribution are shown in Table 1 and Figure 1 As shown;

[0076] The morphology of exosomes was observed using transmission electron microscopy. Figure 2 As shown, the typical exosome morphology, i.e., a saucer or cup-shaped structure, was confirmed;

[0077] The protein concentration was determined using the BCA method, and the particle-protein ratio of the exosomes was calculated to control the purity of the exosomes. The results are shown in Table 1:

[0078] Table 1

[0079]

[0080] 7. Based on the above particle concentration, dilute the exosome particle concentration to 1×10 9 particles / ml, which is convenient for subsequent addition or use. The diluted exosomes are sterilized and filtered using a polyethersulfone filter membrane with a pore size of 0.22 μm to obtain the NK-EXO stock solution.

[0081] (II) Mesenchymal stem cells (MSCs) culture:

[0082] 1. After the seed cells of MSCs working cell bank were recovered, 1×10 5 The density of cells / ml was inoculated in the bioreactor, and the ratio of MSCs number to the effective attachment area of ​​microcarriers was 7500 cells / cm 2 , weigh the microcarriers in advance, soak them in PBS overnight, sterilize them with wet heat, replace PBS with culture medium, and transfer the microcarrier suspension to the bioreactor.

[0083] 2. On the first day after inoculation, the stirring speed of the bioreactor was controlled to 30 rpm, and the speed was adjusted to 80 rpm from the second day onwards. The pH value of the whole process was controlled to 7.1-7.3, the temperature was controlled to 36.5-37.5°C, and the volume fraction of oxygen was controlled to 4.5-5.5%.

[0084] 3. On the second day after inoculation, NK-EXO was added to the bioreactor at a concentration of 150 particles / cell.

[0085] 4. On the 5th day after inoculation, collect the cell culture supernatant.

[0086] (III) Preparation of mesenchymal stem cell exosomes (MSC-EXO):

[0087] 1. Centrifuge the MSCs culture supernatant and control the centrifugal force to 2000×g, the centrifugal time to 10 min, and the centrifugal temperature to 20°C.

[0088] 2. Collect the supernatant after centrifugation and transfer it to a disposable sterile vacuum filtration cup. Use a polyethersulfone filter membrane with a retention pore size of 0.22 μm to filter and remove impurities such as residual cell debris and macromolecules in the supernatant under a negative pressure of 0.08 MPa to obtain a clarified filtered harvest liquid.

[0089] 3. Use a tangential flow system to concentrate, clarify and filter the harvested liquid. The tangential flow system includes a hollow fiber column with a molecular weight cutoff of 500 KD.

[0090] Before loading, flush the pipeline and hollow fiber column with PBS and balance the system until the pH value of the effluent at the reflux end and the permeate end is neutral; after loading, the whole process is controlled at a transmembrane pressure of 3.5psi, an inlet flow rate of 80LMH, and an inlet shear flow rate of 3000s -1 , so that the clarified filtered harvest liquid is concentrated by 20 times in volume to obtain a concentrated harvest liquid.

[0091] 4. Take a PS affinity chromatography separation column, add a buffer balance system, and then slowly add the concentrated harvest liquid to allow the exosomes to bind to Tim4 on the carrier. After the liquid in the separation column has flowed out, add buffer to wash the carrier to remove impurities, and then add eluent to the separation column to obtain a purified harvest liquid.

[0092] 5. Use the tangential flow system to replace the purified harvest solution, add an equal volume of stock buffer to the purified harvest solution, and use the tangential flow system to concentrate the harvest solution to half the volume. Repeat this process 10 times.

[0093] The stock buffer solution contained 0.15 mol / L NaCl, 0.1 M sucrose, the pH value was controlled between 7.0 and 7.4, the molecular weight cutoff of the hollow fiber column was 500 KD, the transmembrane pressure of the whole process was controlled at 3.5 psi, the inlet flow rate was controlled at 80 LMH, and the inlet shear flow rate was controlled at 3000 s -1 .

[0094] 6. According to the exosome particle concentration determined by NTA, dilute the exosome particle concentration with stock solution buffer to a final concentration of 1×109 particles / ml, which is convenient for subsequent use. The diluted exosomes were sterilized and filtered using a polyethersulfone filter membrane with a pore size of 0.22 μm to obtain the MSC-EXO stock solution.

[0095] Comparative Example 1

[0096] MSCs were cultured using the traditional 2D adherent culture method. The revived MSCs were cultured at 7500 cells / cm 2 The cells were inoculated into a cell culture flask at a density of 1000 cells / mL, and an appropriate amount of cell culture fluid was added to the culture flask. The flask was cultured in a normoxic incubator at a temperature of 36-38° C. and containing 5% carbon dioxide. NK-EXO was not added to the culture fluid during the MSCs culture process. The cell culture supernatant was collected on the 5th day after inoculation. The subsequent exosome preparation steps were the same as those in the embodiment.

[0097] Comparative Example 2

[0098] The same method as in Example 1 was used, except that NK-EXO was not used to treat MSCs during the MSCs culture process.

[0099] Effect verification

[0100] (I) Effects of different culture processes on MSCs

[0101] 1. When collecting MSCs cell culture supernatant in Example, Comparative Example 1 and Comparative Example 2, cells were sampled and observed under a microscope.

[0102] like Figure 3 As shown in (B), it can be seen that in Comparative Example 1, MSCs grow attached to the bottom surface of the culture container, which is a 2D static culture;

[0103] like Figure 3 (A) and Figure 3 As shown in (C), in Example 2 and Comparative Example 2, MSCs grew on the surface of microcarriers, and 3D suspension culture was achieved by stirring the culture medium. After adding NK-EXO, MSCs still maintained good adhesion and the cell morphology remained unchanged. Compared with the 2D culture method, 3D suspension culture enhanced cell-cell interaction or cell-ECM interaction.

[0104] 2. When collecting the MSCs cell culture supernatant in Example 1 and Comparative Example 2, the cells were digested and separated and then counted to calculate the cell proliferation multiples. The results are as follows: Figure 4 The collected supernatants were sampled and the contents of anti-inflammatory factors TGF-β and IL-10 were detected by Elisa kit. The results are shown in Figure 5 and Figure 6Compared with the 2D culture method, the 3D suspension culture combined with the low oxygen content culture method promoted the proliferation of MSCs and partially activated the immune regulation function of MSCs. After treatment with NK-EXO, the biological activity of MSCs was further enhanced, the proliferation ability was improved, and the expression of anti-inflammatory factors was significantly increased.

[0105] (II) Effects of different culture processes on MSC-EXO

[0106] 1. The particle concentration and particle size distribution of MSC-EXO before adjustment to the final concentration in Example, Comparative Example 1 and Comparative Example 2 were determined by NTA, the protein concentration was determined by BCA method, and the particle-protein ratio was calculated. The results are shown in Table 2 and Figure 7 Compared with the comparative example, the MSC-EXO separated and purified from the MSCs cell culture supernatant in the embodiment has a higher particle concentration and a larger yield. The particle protein ratio values ​​in the three groups of data are close, indicating that the optimized exosome separation and purification process is stable and reproducible, and the obtained MSC-EXO has a high purity.

[0107] The morphology of MSC-EXO was observed by transmission electron microscopy. Typical exosome morphology, i.e., a saucer or cup-shaped structure, was observed in Example 1, Comparative Example 2. Figure 8 shown.

[0108] Table 2

[0109]

[0110] 2. Add the MSC-EXO finally obtained in Example, Comparative Example 1, and Comparative Example 2 to activated human umbilical cord blood mononuclear cells (PBMC) for co-culture, and detect the effect of MSC-EXO on the proportion of regulatory T cells (CD4+CD25+Treg) in PBMC. Regulatory T cells are a type of T cell subset with immunoregulatory function and have immunosuppressive effects. The umbilical cord blood of healthy volunteers (signed informed consent) was diluted with PBS at a ratio of 1:1, and then slowly added to the upper layer of Ficoll lymphocyte separation solution at a ratio of 1:1. After centrifugation at 700×g, the white film layer was taken, and PBMC was obtained after washing and resuspending with PBS. PBMC was diluted to 5×10 5 / ml were inoculated in RPMI 1640 induction medium containing 1μg / ml anti-CD3 and 1μg / ml anti-CD28, and then divided into groups and added with a final particle concentration of 1×10 8 MSC-EXO prepared by Example, Comparative Example 1 and Comparative Example 2 were added with an equal volume of PBS as a blank control. After 5 days of co-culture, the proportion of CD4+CD25+Treg cells was detected by flow cytometry. The results are as follows Fig. 9 Compared with the control group, after adding MSC-EXO and co-culturing with PBMC, the cell ratio of CD4+CD25+Treg in PBMC increased, among which, the MSC-EXO prepared by the embodiment can significantly upregulate CD4+CD25+Treg with immunosuppressive effect.

[0111] The invention discloses an application of function-enhanced mesenchymal stem cell exosomes. The function-enhanced mesenchymal stem cell exosomes prepared by a method for preparing function-enhanced mesenchymal stem cell exosomes can downregulate cellular inflammatory factors and are used to prepare drugs for treating skin allergic diseases. The drugs can slow down or eliminate skin inflammation and allergic reactions and promote skin cell repair.

[0112] A trial of MSC-EXO in the treatment of allergic skin inflammation

[0113] 1. Subjects: 6 people, all female, aged 25-48 years old, average age 35 years old, all with skin inflammation, 1-3 years of disease course, no acute or chronic diseases, no strong sunlight or ultraviolet rays during treatment, no medication for nearly 1 month. One injection every 3 weeks is a treatment cycle, the injection method is water light needle, each injection of MSC-EXO stock solution is controlled at 3-5ml, and the whole course of treatment is 4-6 treatment cycles depending on the severity of symptoms.

[0114] 2. Before treatment, all subjects had obvious symptoms of skin inflammation, which were significantly alleviated after treatment, including reduced facial redness and swelling, disappearance of acne, elimination of inflammation, and significant improvement in skin condition. At the same time, no side effects such as allergies and pigmentation occurred during the treatment.

[0115] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, and these changes and improvements all fall within the scope of the present invention to be protected.

Claims

1. A method for preparing function-enhanced mesenchymal stem cell exosomes, characterized in that: The preparation method comprises the following steps: S1. Preparation of NK cell exosomes The supernatant in the NK cell culture medium was collected, and the supernatant was treated using an optimized exosome separation and purification process to enrich the NK cell exosomes; S2. Mesenchymal stem cell culture Cultivating mesenchymal stem cells using a 3D suspension culture method, maintaining a low level of dissolved oxygen concentration during the culture process, and using the NK cell exosomes obtained in step S1 to treat the mesenchymal stem cells, activate the mesenchymal stem cells to express immune regulation-related proteins, and release the exosomes into the culture medium supernatant; S3. Preparation of mesenchymal stem cell exosomes The culture medium supernatant obtained in step S2 is collected, and the mesenchymal stem cell exosomes are enriched using the optimized exosome separation and purification process.

2. The method for preparing function-enhanced mesenchymal stem cell exosomes according to claim 1, characterized in that: The mesenchymal stem cells in step S2 are derived from human umbilical cord or human fat; The 3D suspension culture method in step S2 is to culture mesenchymal stem cells on microcarriers at a rate of 1-3×10 5 The density of cells / ml was inoculated in a bioreactor for suspension culture. The ratio of the number of mesenchymal stem cells to the effective attachment area of ​​the microcarrier was 6000-8000 cells / cm 2 ; The 3D suspension culture conditions in step S2 are as follows: pH value is controlled at 6.5-7.5, temperature is controlled at 36-38°C, stirring speed is controlled at 20-40 rpm on the first day after inoculation, stirring speed is controlled at 60-100 rpm from the second day onwards, and cell culture fluid is harvested on the fifth day.

3. The method for preparing function-enhanced mesenchymal stem cell exosomes according to claim 1, characterized in that: The low level of dissolved oxygen concentration in step S2 is achieved by controlling the volume fraction of oxygen introduced into the reactor to be 2%-8%; The concentration of NK cell exosomes in step S2 is 100-200 particles / cell, and the particle-protein ratio of NK cell exosomes is ≥1×10 7 Particles / μg, NK cell exosomes were added on the second day after mesenchymal stem cells were inoculated.

4. The method for preparing function-enhanced mesenchymal stem cell exosomes according to claim 1, characterized in that: The optimized exosome separation and purification process in step S1 and step S3 comprises the following steps: P1. Clarification and Filtration The collected cell culture medium supernatant is centrifuged and then filtered through a filter membrane to obtain a clarified filtered harvest solution; P2, ultrafiltration concentration The clarified filtered harvest liquid obtained in step P1 is treated by a tangential flow concentration process to obtain a concentrated harvest liquid; P3, affinity chromatography The concentrated harvest liquid obtained in step P2 is treated by PS affinity chromatography to obtain a purified harvest liquid; P4. Preparation of stock solution The stock buffer was replaced with the solvent of the purified harvest solution obtained in step P3 by the tangential flow replacement process, and the concentration of exosome particles was adjusted to 1-3×10 9 particles / ml, and the stock solution was sterilized and filtered to prepare a sterile exosome stock solution.

5. The method for preparing function-enhanced mesenchymal stem cell exosomes according to claim 4, characterized in that: The centrifugal conditions in step P1 are: centrifugal force of 1000-3000×g, centrifugal time of 5-10 min, and centrifugal temperature of 16-25°C; The material of the filter membrane in step P1 is at least one of cellulose acetate, polyethersulfone or polyvinylidene fluoride, and the retention pore size of the filter membrane is 0.20-0.45 μm.

6. The method for preparing function-enhanced mesenchymal stem cell exosomes according to claim 4, characterized in that: The tangential flow concentration process in step P2 is: using a tangential flow system to apply back pressure at the reflux end of the ultrafiltration membrane structure, so that the filtrate is filtered out from the permeate end of the ultrafiltration membrane structure, the volume of the reflux liquid is reduced, and the clarified filtration harvest liquid is continuously concentrated during the circulation process; The ultrafiltration membrane structure is at least one of a cassette membrane package and a hollow fiber column, the material of the ultrafiltration membrane is one of regenerated cellulose and polyethersulfone, and the molecular weight cutoff of the ultrafiltration membrane is 300-500KD; In step P2, the transmembrane pressure is controlled to be 2-4 psi, the inlet flow rate is adjusted to 60-120 LMH, and the inlet shear flow rate is controlled to be less than 4000 s -1 The concentration multiple of the prepared concentrated harvest solution is controlled to be 10-50 times.

7. The method for preparing function-enhanced mesenchymal stem cell exosomes according to claim 4, characterized in that: The PS affinity chromatography process in step P3 is as follows: utilizing the property of phosphatidylserine on the surface of the exosome membrane to specifically bind to Tim4, when calcium ions are present, Tim4 fixed on the carrier captures the exosomes in the concentrated harvest solution, after the capture is completed, the carrier is cleaned to remove impurities, and then a calcium ion chelator is added thereto to elute the exosomes on Tim4 to obtain a purified harvest solution; The material of the carrier is resin, and the specific surface area of ​​the carrier is 120m 2 / g, the density of the carrier is 0.95g / cm 3 , the particle size of the carrier is 45 μm, Tim4 is fixed on the surface of the carrier, and loaded into the separation column; The PS affinity chromatography process in step P3 is characterized by comprising the following steps: a. Adding a buffer and the concentrated harvest solution obtained in step P2 to the separation column to allow the exosomes to bind to Tim4; b. Adding buffer to the separation column to wash the carrier and remove impurities; c. adding an eluent to the separation column to recover the exosomes; The eluent in step c contains a calcium ion chelating agent; The stock solution buffer in step P4 contains 0.15 mol / L NaCl and 0.1 M sucrose, and the pH value of the stock solution buffer is 7.0-7.4; The tangential flow displacement process in step P4 is: the tangential flow system in step P2 is used to continuously perform solvent displacement multiple times with the same volume, thereby removing exogenous pollutants, and the displacement volume multiple is 6-10 times; In step P4, a sterilizing filter is used for sterilizing filtration, the filter membrane of the sterilizing filter is made of polyethersulfone, and the filter membrane retention pore size is 0.22 μm.

8. The use of a function-enhanced mesenchymal stem cell exosome according to claims 1-7, characterized in that: The function-enhanced mesenchymal stem cell exosomes prepared by a method for preparing function-enhanced mesenchymal stem cell exosomes can downregulate cellular inflammatory factors and are used to prepare drugs for treating skin allergic diseases. They can slow down or eliminate skin inflammation and allergic reactions and promote skin cell repair.

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