Preparation and preservation method of stem cell source exosome

Abstract

The invention belongs to the technical field of cell sorting, and particularly relates to a preparation and preservation method of a stem cell source exosome. The preparation method comprises the following steps: cutting umbilical cord tissues into small blocks, adding digestive juice containing collagenase I and hyaluronidase for digestion, and collecting tissue precipitates for culture to obtain cell culture fluid; centrifuging the cell culture solution, collecting cell precipitates, resuspending, adding fluorophore coupled positive antibodies and / or negative antibodies to mark the human umbilical cord mesenchymal stem cells, and sorting the human umbilical cord mesenchymal stem cells; the method comprises the following steps: culturing human umbilical cord mesenchymal stem cells, collecting a stem cell culture solution, and extracting exosomes in the stem cell culture solution through a differential centrifugation method. The invention provides a large-scale and reliable exosome preparation scheme for clinical application, provides powerful guarantee for long-term storage and transportation of the exosome and wide application in clinical application and scientific research, and has important scientific significance and wide market prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of exosome preparation, and particularly relates to a method for preparing and preserving exosomes derived from stem cells. Background Art

[0002] Ovarian aging is one of the core pathological features of the decline in female reproductive system function, manifested as reduced follicle reserve, disordered ovarian endocrine function, and loss of reproductive ability. With the intensification of the global population aging trend, ovarian aging-related problems (such as menopause, premature ovarian failure, and the resulting metabolic syndrome, etc.) have an increasingly significant impact on women's health. Currently, the main treatment methods for ovarian aging mainly include hormone replacement therapy (HRT) and drug intervention, but these methods often have side effects or limited effects. Therefore, exploring new intervention strategies, especially regenerative medicine-based treatment means, has become a key direction in ovarian aging research.

[0003] In recent years, exosomes, as key mediators of intercellular information transmission, have gradually become a hot topic in regenerative medicine and disease treatment research. Exosomes are cell-secreted vesicles with a diameter of 30 - 150 nm, carrying biomolecules such as proteins, RNAs, and lipids, and play important roles in cell repair, immune regulation, anti-inflammation, and tissue regeneration by regulating the signal pathways of recipient cells. Among exosomes from various sources, exosomes derived from human mesenchymal stem cells (hMSCs) have attracted much attention due to their extensive tissue repair and immunomodulatory potential, and have become a powerful candidate tool for ovarian aging treatment. Existing studies have shown that hMSCs exosomes can regulate cell functions in ovarian tissue by carrying reparative factors (such as miRNAs, proteins, etc.), inhibit ovarian inflammation, and promote follicle formation, thereby delaying the process of ovarian aging. Specifically, hMSCs exosomes have shown significant application potential in the following aspects: (1) Anti-inflammatory and antioxidant effects: Ovarian aging is often accompanied by oxidative stress and chronic inflammatory responses. hMSCs exosomes can relieve ovarian tissue damage and delay its functional degradation by inhibiting the expression of oxidative stress markers and inflammatory factors; (2) Promote ovarian cell proliferation and apoptosis regulation: hMSCs exosomes can regulate the proliferation and apoptosis of ovarian granulosa cells and oocytes, improve the ovarian microenvironment, promote the development and maturation of follicles, and thus enhance the reserve function of the ovary; (3) Regulate intercellular communication: The biomolecules transmitted through exosomes can effectively regulate signal transmission between ovarian cells and promote tissue repair and regeneration.

[0004] However, despite the great potential of hMSCs exosomes in ovarian aging research, their application and promotion still face a series of challenges, especially in the purification, preparation, and preservation of exosomes. When exosomes derived from HUMSCs (human umbilical cord mesenchymal stem cells) are stored in liquid nitrogen, although the low-temperature environment can greatly slow down the degradation rate of exosomes, it may still cause some microscopic structural changes. First of all, although liquid nitrogen storage can avoid the formation of ice crystals, long-term exposure to ultra-low temperatures may still lead to the rearrangement of exosomal membrane lipids, thereby affecting the fluidity and integrity of its membrane. This structural change of the membrane will directly affect the binding ability of exosomes to target cells after resuscitation. In addition, certain protein structures may undergo irreversible conformational changes at ultra-low temperatures, thus affecting their activity as signaling molecules. Similarly, nucleic acids inside exosomes, such as miRNAs and mRNAs, may undergo partial degradation or chemical modification during long-term storage, thereby reducing the efficiency of their functional transmission. To address these potential problems brought about by storage in liquid nitrogen, researchers are developing some new preservation methods, mainly including: (1) Lyophilization: Lyophilization removes water at ultra-low temperatures to maintain the structure and function of exosomes. This method can not only avoid the physical damage of exosomes by ice crystals during the freezing process but also enable long-term storage without ultra-low temperature conditions. However, the main challenge of this technology lies in how to select appropriate lyoprotectants to avoid damage to the exosomal membrane during lyophilization and rehydration, preventing membrane rupture and content leakage. (2) Vitrification: Different from traditional freezing methods, vitrification avoids the formation of ice crystals by rapid cooling, thus protecting the membrane structure and internal components of exosomes to the greatest extent. This method is particularly suitable for long-term storage of exosomes, but the preservation medium still needs to be optimized to improve its activity after resuscitation. (3) Nanopackaging technology: Encapsulating exosomes in biocompatible nanoparticles to improve their stability. This method protects exosomes from environmental stresses, such as oxidation and enzymatic degradation, thereby extending their effective shelf life. In addition, this nanopackaging may also improve the targeted delivery efficiency of exosomes in vivo. These new preservation strategies help to improve the long-term stability and functional maintenance of exosome preservation, but there are still many problems.

[0005] In view of this, developing new methods for large-scale preparation and preservation of exosomes and improving the maintenance of functional activity during long-term exosome preservation are beneficial for providing innovative technical means and scientific bases for the intervention and treatment of ovarian aging. Summary of the Invention

[0006] In view of the problems existing in the prior art, the present invention provides a method for preparing and preserving stem cell-derived exosomes, which provides a large-scale and reliable exosome preparation scheme for clinical applications, and provides strong guarantees for the long-term storage, transportation of exosomes, and their extensive use in clinical applications and scientific research, having important scientific significance and broad market prospects.

[0007] To achieve the above object, the present invention is specifically realized through the following technical solutions:

[0008] In the first aspect of the present invention, a method for preparing stem cell-derived exosomes is provided, including the following steps:

[0009] S1. Cut umbilical cord tissue into small pieces, add a digestive solution containing collagenase I and hyaluronidase for digestion, centrifuge the digested suspension, collect the tissue precipitate, inoculate it in α-MEM culture medium for culture, and obtain a cell culture solution containing human umbilical cord mesenchymal stem cells;

[0010] S2. Centrifuge the cell culture solution in step S1, collect the cell precipitate and resuspend it, then add fluorescent group-conjugated positive antibodies and negative antibodies to label the human umbilical cord mesenchymal stem cells. The positive antibodies include anti-CD73 antibody, anti-CD90 antibody, anti-CD105 antibody and anti-CD44 antibody, and the negative antibodies include anti-HLA-DR antibody, anti-CD34 antibody and anti-CD45 antibody. Sort out the cell population that is positive for CD73, CD90, CD105, CD44 and negative for HLA-DR, CD34, CD45 to obtain the human umbilical cord mesenchymal stem cells;

[0011] S3. Culture the human umbilical cord mesenchymal stem cells, collect the stem cell culture solution, and extract exosomes from the stem cell culture solution by differential centrifugation.

[0012] Further, in step S1, the digestive solution includes 1 mg / mL of collagenase I and 1 U / mL of hyaluronidase.

[0013] Further, in step S1, digest at 37 °C for 1-2 hours.

[0014] In step S2, the method for labeling the human umbilical cord mesenchymal stem cells includes: centrifuge the cell culture solution in step S1, collect the cell precipitate and resuspend it with PBS, adjust the cell density to 1×10 6 cells / mL, add fluorescent group-conjugated positive antibodies and negative antibodies, mix well and incubate in the dark at 4 °C for 30 minutes; then centrifuge at 300×g for 5 minutes, discard the supernatant, and resuspend with PBS.

[0015] Further, in step S2, the fluorescent group is selected from at least one of PE, Cy7, APC, and FITC, or a combination of multiple ones of PE, Cy7, APC, and FITC.

[0016] Further, in step S2, the positive rate of the positive antibody is greater than 99%, and the positive rate of the negative antibody is less than 1%.

[0017] Further, in step S2, it further includes the step of inducing osteogenic and adipogenic differentiation of the human umbilical cord mesenchymal stem cells.

[0018] Furthermore, the osteogenic and adipogenic induction differentiation includes the following steps: subculturing the human umbilical cord mesenchymal stem cells to a confluence of 80%-90%, adding osteogenic induction medium, and continuously culturing for 21 days, during which the medium is changed every 3 days, and the osteogenic differentiation is detected by alizarin red staining; then replacing the osteogenic induction medium with adipogenic induction medium, continuously culturing for 14 days, during which the medium is changed every 2 days, and the formation of lipid droplets is detected by oil red O staining; selecting the human umbilical cord mesenchymal stem cells with osteogenic and adipogenic differentiation ability for subsequent culture.

[0019] Further, in step S3, extracting exosomes from the stem cell culture solution by differential centrifugation includes the following steps: subculturing the human umbilical cord mesenchymal stem cells to a confluence of 80%-90%, collecting the stem cell culture solution, centrifuging the stem cell culture solution at 300×g for 10 minutes, then at 2000×g for 20 minutes, and then at 10000×g for 30 minutes, collecting the supernatant and filtering it through a 0.22μm filter membrane, centrifuging the obtained filtrate at 100000×g for 70 minutes, resuspending the exosome precipitate with PBS, and then centrifuging at 100000×g for 70 minutes, discarding the supernatant, and collecting the exosome precipitate.

[0020] The second aspect of the present invention provides a preservation method for stem cell-derived exosomes, including the following steps:

[0021] Adding the exosome precipitate prepared by the preparation method of stem cell-derived exosomes as described above to a PBS buffer solution containing human serum albumin, trehalose, and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid for preservation.

[0022] Further, in the PBS buffer solution, the mass concentration of human serum albumin is 0.2%, the molar concentration of trehalose is 25 mM, and the molar concentration of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid is 25 mM.

[0023] Further, the preservation conditions include normoxia and / or hypoxia conditions, and the hypoxia condition is that the oxygen content is greater than or equal to 1% and less than 21%.

[0024] The advantages and positive effects of the present invention are as follows:

[0025] 1. The present invention uses collagenase I and hyaluronidase to digest and extract human umbilical cord tissue, combines fluorescent group-coupled antibodies to identify specific cell surface antigens, and sorts out human umbilical cord mesenchymal stem cells with high abundance, purity and activity with stem cell characteristics. Subsequently, exosomes are extracted from the culture medium of human umbilical cord mesenchymal stem cells by differential centrifugation method. The obtained exosomes have a typical bilayer membrane vesicle morphology and a complete membrane structure, greatly improving the extraction concentration of exosomes and enhancing the biological activity of exosomes, providing a large-scale and reliable exosome preparation scheme for clinical application, and having important scientific significance and broad market prospects.

[0026] 2. The present invention introduces three components, namely human serum albumin, trehalose and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, into PBS buffer solution. Preserving exosomes with this is beneficial to increasing their concentration and maintaining their typical vesicle structure, effectively preventing the degradation and morphological changes of exosomes during long-term preservation, significantly improving the structural stability of exosomes, and prolonging the preservation time of exosomes under both normoxic and hypoxic conditions. This provides a strong guarantee for the long-term storage, transportation and wide use of exosomes in clinical application and scientific research, and provides new impetus for the research and application of exosomes in the field of biomedicine. Description of the Drawings

[0027] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0028] Figure 1 It is a fluorescence signal statistical chart of the surface markers of human umbilical cord mesenchymal stem cells sorted by flow cytometry in the embodiment of the present invention;

[0029] Figure 2 It is a staining diagram of the 4th generation of human umbilical cord mesenchymal stem cells induced to differentiate into adipocytes and osteoblasts in the embodiment of the present invention;

[0030] Figure 3 It is a nanoparticle tracking analysis result diagram of exosomes derived from human umbilical cord mesenchymal stem cells in the embodiment of the present invention;

[0031] Figure 4 It is a transmission electron microscope diagram of exosomes derived from human umbilical cord mesenchymal stem cells in the embodiment of the present invention;

[0032] Figure 5This is a diagram showing the results of immunoblotting detection of specific markers of exosomes derived from human umbilical cord mesenchymal stem cells in an embodiment of the present invention;

[0033] Figure 6 This is a graph showing the concentration detection results of exosomes derived from human umbilical cord mesenchymal stem cells resuspended in different buffers according to an embodiment of the present invention;

[0034] Figure 7 This is a graph showing the concentration detection results of exosomes derived from human umbilical cord mesenchymal stem cells stored under normoxic and hypoxic conditions after being resuspended in different buffers according to an embodiment of the present invention. DETAILED DESCRIPTION

[0035] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with embodiments. The embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0036] Based on the information contained in the present invention, it is easy for those skilled in the art to make various changes to the precise description of the present invention without departing from the spirit and scope of the appended claims. It should be understood that the scope of the present invention is not limited to the defined processes, properties or components, because these embodiments and other descriptions are only for the purpose of illustrating specific aspects of the present invention. In fact, various changes that a person skilled in the art or related fields can obviously make to the embodiments of the present invention are covered within the scope of the appended claims.

[0037] In order to better understand the present invention rather than limit the scope of the present invention, all the numerals and other numerical values ​​used in the present invention indicating dosage, percentage, etc. should be understood as modified by the word "about" in all cases. Therefore, unless otherwise specified, the numerical parameters listed in the specification and the appended claims are approximate values, which may be changed according to the different ideal properties attempted to be obtained. Each numerical parameter should at least be regarded as being obtained based on the reported significant figures and by conventional rounding methods. In addition, the meaning of the terms "include", "comprise", "contain", "have" and the like is non-restrictive, that is, other steps and other ingredients that do not affect the results can be added.

[0038] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, specific embodiments of the present invention are described in detail below with reference to the accompanying drawings.

[0039] The present invention provides a method for preparing stem cell-derived exosomes, comprising the following steps:

[0040] S1. Cut the umbilical cord tissue into small pieces, add a digestive solution containing collagenase I and hyaluronidase for digestion, centrifuge the digested suspension, collect the tissue precipitate, inoculate it in α-MEM culture medium for culture, and obtain a cell culture solution containing human umbilical cord mesenchymal stem cells;

[0041] S2. Centrifuge the cell culture solution in step S1, collect the cell precipitate and resuspend it, then add a fluorescent group-conjugated positive antibody and / or negative antibody to label the human umbilical cord mesenchymal stem cells. The positive antibodies include anti-CD73 antibody, anti-CD90 antibody, anti-CD105 antibody and anti-CD44 antibody, and the negative antibodies include anti-HLA-DR antibody, anti-CD34 antibody and anti-CD45 antibody. Sort out a cell population that is positive for CD73, CD90, CD105, CD44 and negative for HLA-DR, CD34, CD45 to obtain the human umbilical cord mesenchymal stem cells;

[0042] S3. Culture the human umbilical cord mesenchymal stem cells, collect the stem cell culture solution, and extract the exosomes in the stem cell culture solution by differential centrifugation.

[0043] The present invention uses an innovative enzyme digestion method to extract human umbilical cord mesenchymal stem cells (HUMSCs), which can obtain a high-purity cell population in a short time. Combining fluorescent group-conjugated antibodies to identify and sort the surface specific antigens of HUMSCs provides reliable cell characteristic data for subsequent preparation of stem cell-derived exosomes, and improves the abundance, purity and activity of target cells. Subsequently, exosomes are extracted from the HUMSCs culture solution by differential centrifugation, which ensures the typical bilayer membrane vesicle morphology and intact membrane structure of exosomes, and greatly improves the extraction concentration of exosomes and the biological activity of exosomes, providing a large-scale and reliable exosome preparation scheme for clinical applications, with important scientific significance and broad market prospects.

[0044] Optionally, in step S1, the digestive solution includes 1 mg / mL of collagenase I and 1 U / mL of hyaluronidase; place the small pieces of umbilical cord tissue in the enzyme digestive solution and perform enzyme digestion at 37 °C for 1-2 hours to disperse the cells in the tissue.

[0045] In step S2, the method for labeling the human umbilical cord mesenchymal stem cells includes: centrifuge the cell culture solution in step S1, collect the cell precipitate and resuspend it with PBS, adjust the cell density to 1×10 6 cells / mL, add a fluorescent group-conjugated positive antibody and / or negative antibody, mix well and incubate in the dark at 4 °C for 30 minutes; then centrifuge at 300×g for 5 minutes, discard the supernatant, and resuspend with PBS to obtain the antibody-labeled human umbilical cord mesenchymal stem cells.

[0046] In the above step S2, the sorting method usually adopts flow cytometry; when flow cytometry is used, the antibodies used in each step are conjugated with fluorescent groups, and thus after antibody labeling, cells are sorted based on the fluorescence signal. Of course, those skilled in the art can also adopt other sorting methods, such as immunomagnetic cell sorting; when immunomagnetic cell sorting is used, the antibodies used in each step bind to magnetic beads, and thus after antibody labeling, magnetic cells are adsorbed and sorted. This is conventional technology in this field and will not be elaborated here.

[0047] It should be noted that the fluorescence or magnetic beads carried by the antibodies in each step can be the same or different, and are adaptively adjusted according to specific operations. In some embodiments, when the antibodies in step S2, such as anti-CD73 antibody and anti-CD90 antibody, are labeled and sorted simultaneously (sorted in one reaction system), the fluorescence or magnetic beads used for the two need to be different to be distinguished by different signals; however, it should be noted that when multiple negative antibodies are labeled and sorted in the same reaction system, their fluorescent groups can be the same. When labeled and sorted separately, for example, first label with a fluorescent group-conjugated anti-CD73 antibody, then sort out CD73-positive cells, wash away the anti-CD73 antibody, and then further through the steps of labeling and sorting with a fluorescent group-conjugated CD90 antibody, the fluorescence or magnetic beads used for the two can be the same or different at this time.

[0048] Optionally, the fluorescent groups conjugated to the antibodies include but are not limited to: PE, Cy7, APC, and FITC, or combinations selected from at least two of the aforementioned fluorescent groups, such as PE-Cy7 and APC-Cy7.

[0049] Optionally, in step S2, the positive rate during positive antibody sorting is greater than 99%, and the positive rate during negative antibody sorting is less than 1%. Screening positive and negative labeled cell populations based on this standard can obtain high-purity human umbilical cord mesenchymal stem cells.

[0050] Optionally, in step S2, it further includes the step of inducing osteogenic and adipogenic differentiation of the human umbilical cord mesenchymal stem cells. Through osteogenic and adipogenic induction culture, the multi-directional differentiation potential of HUMSCs is clarified, the differentiation ability of the isolated human umbilical cord mesenchymal stem cells is ensured, a reliable cell source is provided for the preparation of regenerative medicine exosomes, and the credibility of the exosome source is enhanced.

[0051] Specifically, osteogenic and adipogenic induction differentiation includes the following steps: subculturing the human umbilical cord mesenchymal stem cells to 80%-90% confluence, adding osteogenic induction medium, and continuously culturing for 21 days, during which the medium is changed every 3 days, and the osteogenic differentiation is detected by alizarin red staining; then replacing the osteogenic induction medium with adipogenic induction medium and continuously culturing for 14 days, during which the medium is changed every 2 days, and the lipid droplet formation is detected by oil red O staining; selecting the human umbilical cord mesenchymal stem cells with osteogenic and adipogenic differentiation abilities for subsequent culture to prepare exosomes.

[0052] The osteogenic induction medium is used to induce the differentiation of mesenchymal stem cells into osteoid cells, and its medium components include: basal medium (α-MEM medium), 10 mM β-glycerophosphate sodium (β-Glycerophosphate Sodium, used to provide the phosphate source of bone matrix), 100 μg / mL vitamin C (L-ascorbic acid, which helps collagen synthesis and mineralization), and 100 nM dexamethasone (Dexamethasone, as a glucocorticoid, can promote bone differentiation).

[0053] The adipogenic induction medium is used to induce the differentiation of mesenchymal stem cells into adipocytes (adipocyte-like cells), and its medium components include: basal medium (α-MEM medium), 10 μg / mL insulin (Insulin, which helps to promote the differentiation and lipid accumulation of adipocytes), 1 μM dexamethasone (Dexamethasone, as a glucocorticoid, helps to induce adipogenic differentiation), 1 mM isobutylmethylxanthine (3-isobutyl-1-methylxanthine, IBMX, used to activate the adipogenic differentiation process), and 10 μM indomethacin (Indomethacin, which has the effect of promoting adipogenic differentiation).

[0054] Optionally, in step S3, extracting exosomes from the stem cell culture medium by differential centrifugation includes the following steps: subculturing the human umbilical cord mesenchymal stem cells to 80%-90% confluence, collecting the stem cell culture medium, centrifuging the stem cell culture medium at 300×g for 10 minutes, then at 2000×g for 20 minutes, and then at 10000×g for 30 minutes, collecting the supernatant and filtering it through a 0.22 μm filter membrane, centrifuging the obtained filtrate at 100000×g for 70 minutes, resuspending the exosome precipitate with PBS, and then centrifuging at 100000×g for 70 minutes, discarding the supernatant, and collecting the exosome precipitate.

[0055] Another embodiment of the present invention provides a method for preserving stem cell-derived exosomes, including the following steps:

[0056] The exosome precipitate prepared by the method for preparing stem cell-derived exosomes as described above is added to a PBS buffer containing human serum albumin (HSA), trehalose, and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) for preservation.

[0057] In the present invention, three components, namely HSA, trehalose, and HEPES, are introduced into the PBS buffer. As a protein stabilizer, HSA has a strong ability to protect the exosome structure, preventing exosomes from aggregating and degrading during storage and transportation; trehalose, a disaccharide, can effectively maintain the morphology of exosomes, prevent them from dehydrating and shrinking, and provide protection inside cells to avoid damage to exosome functions due to environmental changes such as oxidative stress; HEPES, as a buffering component, can maintain the pH stability of the solution, ensuring the activity and integrity of the membrane structure of exosomes during storage. The exosomes resuspended in the buffer of the present invention have a higher concentration and typical vesicle structure, can effectively prevent exosome degradation and morphological changes during long-term storage, significantly improve the structural stability of exosomes, and can extend the storage time of exosomes under both normoxic and hypoxic conditions, providing strong guarantees for the long-term storage, transportation, and wide use of exosomes in clinical applications and scientific research, and providing new impetus for the research and application of exosomes in the biomedical field.

[0058] Optionally, in the PBS buffer, the mass concentration of human serum albumin is 0.2 (wt)%, the molar concentration of trehalose is 25 mM, and the molar concentration of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) is 25 mM.

[0059] The above preservation conditions include normoxic and / or hypoxic conditions, and the hypoxic condition is that the oxygen content is greater than or equal to 1% and less than 21%.

[0060] There is no special limitation on the addition amount of the buffer of the present invention, which is subject to being able to resuspend exosomes and is adaptively adjusted according to the preservation concentration requirements of exosomes. For example, when a higher exosome concentration is required, the amount of buffer can be appropriately reduced, and when a lower exosome concentration is required, the amount of buffer can be appropriately increased.

[0061] The present invention will be further illustrated below in conjunction with specific examples. The experimental methods without specific conditions noted in the following examples are usually carried out under conventional conditions, such as the conditions described in "Molecular Cloning: A Laboratory Manual (Fourth Edition)" published by Cold Spring Harbor Laboratory, or usually according to the conditions recommended by the manufacturer.

[0062] In the following examples, the antibodies used for labeling human umbilical cord mesenchymal stem cells include: Anti-Human CD73PE labeled with PE, purchased from eBioscience, catalog number 12-0739-42; Anti-Human CD90PE-Cy7 labeled with PE-Cy7, purchased from eBioscience, catalog number 12-0909-42; Anti-Human CD105APC labeled with APC, purchased from eBioscience, catalog number 14-1057-82; Anti-Human CD44FITC labeled with FITC, purchased from eBioscience, catalog number 14-0441-82; Anti-Human HLA-DR PE labeled with PE, purchased from eBioscience, catalog number 14-9956-82; Anti-Human CD34APC-Cy7 labeled with APC-Cy7, purchased from eBioscience, catalog number 14-0349-82; Anti-Human CD45FITC labeled with FITC, purchased from eBioscience, catalog number 14-0459-82. The antibodies used for detecting exosome-specific markers include: Anti-Calnexin antibody, purchased from Abcam, catalog number ab22595; Anti-TSG101 antibody, purchased from Abcam, catalog number ab125011.

[0063] Example

[0064] 1. Extraction of human umbilical cord mesenchymal stem cells

[0065] The human umbilical cord used for isolating human umbilical cord mesenchymal stem cells (HUMSCs) was provided by Wuhan Optics Valley Zhongyuan Xiehe Cell Gene Technology Co., Ltd., and materials such as the "Informed Consent Form for Umbilical Cord Collection" and the "Informed Confirmation Form for Donation Matters" were signed with the sample provider. The operations for isolating the HUMSCs cell population from the umbilical cord include:

[0066] Take a fresh human umbilical cord, remove the blood vessels and amnion under sterile conditions, and then cut the umbilical cord tissue into small pieces of 1-2 mm 3 and place them in an enzyme digestion solution containing 1 mg / mL collagenase I and 1 U / mL hyaluronidase, and perform enzyme digestion at 37 °C for 1-2 hours. After digestion is completed, add Gibco α-MEM culture medium (catalog number C12571500BT) to terminate the enzyme digestion effect, filter the suspension through a 70 μm filter, and collect the filtered cell suspension. Then centrifuge the cell suspension at 1000 rpm for 5-10 minutes, discard the supernatant, collect the cell precipitate, resuspend it in the medium, and place it at 37 °C, 5% CO2 Cultivate under the following conditions, regularly change the culture medium. Wait for the cells to adhere and proliferate to form colonies, then perform subculture to finally obtain human umbilical cord mesenchymal stem cells (HUMSCs).

[0067] 2. Isolation of human umbilical cord mesenchymal stem cells

[0068] To improve the cell purity of HUMSCs, label HUMSCs with fluorescently labeled positive marker (CD73, CD90, CD105, CD44) antibodies and negative marker (HLA-DR, CD34, CD45) antibodies. Then, through flow cytometry, obtain a cell population that is positive for CD73, CD90, CD105, CD44 and negative for HLA-DR, CD34, CD45, which is the target HUMSCs population, named CD73 + CD90 + CD105 + CD44 + CD34 - CD45 - HLA-DR - . The specific operation is as follows:

[0069] Cultivate HUMSCs to the logarithmic growth phase, collect the cell suspension, wash it twice with PBS and then centrifuge to discard the supernatant. Resuspend the cells with an appropriate amount of PBS and adjust the density to 1×10 6 cells / mL. Take 100 μL of the cell suspension to be tested, add 5 μL of the fluorescent group positive antibody Anti-Human CD73PE, gently mix and incubate at 4 °C in the dark for 30 minutes. After the incubation, centrifuge at 300×g for 5 minutes, discard the supernatant, resuspend the cells with 1 mL of PBS, repeat the above steps twice to wash the cells and remove the unbound antibodies, and finally resuspend the cells with 500 μL of PBS. Load the stained cell suspension onto a flow cytometer for detection, collect at least 10,000 cell events, and record the percentage of cells expressing the positive antigen marker; sort to obtain the CD73 + cell population. Then, successively add the fluorescent group-conjugated positive antibodies (Anti-Human CD90PE-Cy7, Anti-HumanCD105APC, and Anti-Human CD44FITC) and negative antibodies (Anti-Human HLA-DR PE, Anti-HumanCD34APC-Cy7, and Anti-Human CD45FITC) according to the above operation, respectively record the percentage of cells expressing the positive and negative antigen markers, verify the antigen characteristics of the isolated HUMSCs cells, and sort to obtain CD73 + CD90 + CD105 +CD44 + CD34 - CD45 - HLA-DR - Cell population.

[0070] Figure 1 The fluorescence signal statistical chart of representative HUMSCs surface markers measured by flow cytometry is shown. The positive rate of cell surface positive marker antigen detected by flow cytometry should be greater than 99%, and the positive rate of negative marker antigen should be less than 1%. It can be seen from the figure that the purity of the HUMSCs cell population obtained in this example is higher and can be used for the preparation of stem cell exosomes in subsequent experiments.

[0071] 3. Identification of the differentiation ability of human umbilical cord mesenchymal stem cells

[0072] For the above sorted CD73 + CD90 + CD105 + CD44 + CD34 - CD45 - HLA-DR - cell population, perform osteogenic and adipogenic induction experiments to verify the differentiation ability of HUMSCs, and further identify that the cells prepared by the present invention are stem cells through their differentiation ability. The specific operations are as follows:

[0073] Adjust the density of CD73 + CD90 + CD105 + CD44 + CD34 - CD45 - HLA-DR - The cell density is 5×104 cells / cm 2 , inoculate into the basal medium, and subculture in an environment of 37°C and 5% CO 2 until the confluence reaches 80%-90%. Subsequently, replace the basal medium with the osteogenic induction medium, continuously culture and change the medium every 3 days, culture for about 21 days, and detect the osteogenic differentiation by alizarin red staining. Then replace the osteogenic induction medium with the adipogenic induction medium, continuously culture and change the medium every 2 days, culture for about 14 days, and determine the formation of lipid droplets by oil red O staining. After the culture is completed, wash the cells 2-3 times with PBS.

[0074] The components of the osteogenic induction medium include: basal medium (α-MEM medium), 10 mM β-glycerophosphate sodium, 100 μg / mL vitamin C (L-ascorbic acid), and 100 nM dexamethasone.

[0075] The components of the adipogenic induction medium include: basal medium (α-MEM medium), 10 μg / mL insulin, 1 μM dexamethasone, 1 mM 3-isobutyl-1-methylxanthine (IBMX), and 10 μM indomethacin.

[0076] Figure 2 The staining images of the 4th generation of HUMSCs after adipogenic and osteogenic induction and differentiation are shown. From left to right, they are the bright-field image and alizarin red staining image after osteogenic induction of HUMSCs, and the bright-field image and oil red O staining image after adipogenic induction of HUMSCs. The figure shows that HUMSCs have typical adipogenic and osteogenic differentiation conditions, indicating that the extracted CD73 + CD90 + CD105 + CD44 + CD34 - CD45 - HLA-DR - cells have typical stem cell differentiation potential and can be used for exosome extraction.

[0077] 4. Purification of exosomes in human umbilical cord mesenchymal stem cell culture medium

[0078] Exosomes in human umbilical cord mesenchymal stem cell culture medium were obtained by differential centrifugation. Human umbilical cord mesenchymal stem cells (CD73 + CD90 + CD105 + CD44 + CD34 - CD45 - HLA-DR - cells) were inoculated into the basal medium and cultured at 37 °C and 5% CO 2Cultured in an environment until 80%-90% confluence, then collect the cell culture medium. First, centrifuge at 300×g for 10 minutes to remove cell impurities, then centrifuge at 2000×g for 20 minutes to remove cell debris and large particulate matter, and then centrifuge at 10000×g for 30 minutes to remove smaller cell debris and fine particles. The obtained supernatant is filtered through a 0.22μm filter membrane; then, the filtered supernatant is centrifuged at 100000×g for 70 minutes to remove ultrafine particles and obtain a precipitate. Finally, resuspend the exosome precipitate with PBS, and further centrifuge at 100000×g for 70 minutes. Discard the supernatant and collect the final exosome precipitate, and resuspend it with 1×PBS. 1×PBS adopts the standard concentration, including 137mM NaCl, 2.7mM KCl, 10mM Na 2 HPO 4 and 1.8mM KH 2 PO 4 , with a pH of 7.2-7.4, consistent with physiological conditions.

[0079] 5. Identification of Exosomes Derived from Human Umbilical Cord Mesenchymal Stem Cells

[0080] The above-mentioned isolated and purified exosomes were identified by methods such as nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and western blot (WB). First, use NTA to measure the particle size and concentration of the extracted exosome samples, and record the particle size distribution and concentration information. Then, observe the exosome samples through transmission electron microscopy to obtain the morphological and structural characteristics of the exosomes. Finally, perform western blot analysis, and use antibodies against exosome-specific markers TSG101 and Calnexin to detect the samples by immunoblotting (Western blot, WB) to verify their exosome identity; TSG101 plays a key role in exosome formation and has a high content in exosomes. Calnexin is an endoplasmic reticulum marker and is usually absent in exosomes.

[0081] Figure 3 Shows the results of exosome NTA detection, indicating that the particle size range of exosomes is distributed within the normal range of 30-150nm, the integrity of exosomes is high, and there is no aggregation or size change. Figure 4 Shows the results of exosome TEM detection. The left side is the overall view, showing that multiple exosomes are distributed in the background in an aggregated or dispersed form. The right side is a partial enlarged view of the left side, showing that exosomes present typical double-membrane vesicles with normal membrane structure and morphology. Figure 5The expression results of the surface specific markers TSG101 and Calnexin of exosomes were detected by Western blot. Lane Hum Exos represents exosomes derived from human umbilical cord mesenchymal stem cells, and lane 293T represents control 293T cells. The results showed that in the WB of the obtained exosomes, TSG101 was positively expressed and Calnexin was not expressed, indicating the characteristics of exosomes.

[0082] 10 mL of stem cell culture medium was used for exosome extraction with the Total Exosome Isolation Reagents kit (Catalog #4478359) from Thermo Fisher Scientific. After centrifuging and precipitating the exosomes according to the instructions, they were resuspended with 200 μL of 1×PBS, and finally 1×10 9 particles / mL could be obtained. Under the method of the present invention, 2.4×10 9 particles / mL of exosomes could be obtained, which greatly increased the extraction concentration of exosomes compared to this kit, and the exosomes derived from HUMSCs extracted had good biological activity.

[0083] 6. Preservation of exosomes derived from human umbilical cord mesenchymal stem cells

[0084] As a traditional exosome suspension, PBS often leads to the instability of exosomes in practical applications due to the lack of certain components that protect the structure and function of exosomes. This instability not only causes exosome degradation but may also result in changes in their morphology or loss of function, seriously affecting the effectiveness of clinical applications. Therefore, it is necessary to develop a more stable and effective exosome suspension.

[0085] In this example, three key components, human serum albumin (HSA), trehalose, and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), were innovatively introduced into the PBS solution to construct a PBS-HAT buffer for exosome preservation. That is, the PBS-HAT buffer is a PBS buffer containing 0.2 (wt)% HSA, 25 mM trehalose, and 25 mM HEPES. This suspension shows significant advantages in the extraction and preservation of exosomes, and can greatly improve the activity and integrity of exosomes during storage and application.

[0086] Resuspend the exosomes purified in the step of "4. Purification of Exosomes in Human Umbilical Cord Mesenchymal Stem Cell Culture Medium" in PBS-HAT buffer, and take an equal amount of exosomes and resuspend them in PBS buffer as a control group. After resuspending the exosome precipitates obtained from the same volume of HUMSCs supernatant medium with two different buffers, the measured results of the exosome concentration are shown in Figure 6 . Among them, the abscissa represents different buffers, and the ordinate represents the exosome concentration. Select the diameter range of 30-150 nm as the range of the obtained exosomes, calculate the amount of exosomes within this range, and take the exosome content resuspended in PBS as 1. The results show that a higher concentration of exosomes can be obtained by resuspending with PBS-HAT buffer.

[0087] Furthermore, resuspend the exosomes resuspended in PBS-HAT buffer and PBS buffer at normal oxygen (21% (v / v) O 2 ) or hypoxia (1% (v / v) O 2 ), and store them at 37 °C for 7 days. After the storage ends, take samples and measure the particle size range distribution of the exosomes by NTA, and calculate the exosome concentration. Figure 7 Shows the detection results of the exosome concentration after storing the exosomes resuspended in PBS buffer (left) and PBS-HAT buffer (right) for 7 days under normal oxygen and hypoxia conditions. Among them, the abscissa represents the storage conditions, and the ordinate represents the exosome concentration. The results show that when the exosomes are resuspended in PBS and stored at normal oxygen and 37 °C for 7 days, the exosome concentration drops to 90%, and under hypoxia conditions, the concentration drops to 79%; while for the exosomes resuspended in the PBS-HAT buffer of the present invention, after 7 days of normal oxygen, the concentration drops to 97%, and under hypoxia conditions, the concentration drops to 93%. It shows that regardless of whether it is under normal oxygen or hypoxia conditions, the exosomes stored using the PBS-HAT buffer of the present invention have a higher concentration and better storage effect. Therefore, the PBS-HAT buffer can effectively increase the yield of exosomes and the stability of long-term storage, and significantly extend the storage time of exosomes under normal oxygen and hypoxia conditions.

[0088] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing stem cell-derived exosomes, characterized in that: The following steps are involved: S1. Cut the umbilical cord tissue into small pieces, add a digestion solution containing collagenase I and hyaluronidase for digestion, centrifuge the digested suspension, collect the tissue precipitate, inoculate it in α-MEM culture medium for culture, and obtain a cell culture medium containing human umbilical cord mesenchymal stem cells; S2, centrifuging the cell culture fluid of step S1, collecting the cell precipitate and resuspending it, then adding fluorescent group-coupled positive antibodies and / or negative antibodies to label the human umbilical cord mesenchymal stem cells, wherein the positive antibodies include anti-CD73 antibodies, anti-CD90 antibodies, anti-CD105 antibodies and anti-CD44 antibodies, and the negative antibodies include anti-HLA-DR antibodies, anti-CD34 antibodies and anti-CD45 antibodies, sorting out the cell population that is positive for CD73, CD90, CD105, and CD44 and negative for HLA-DR, CD34, and CD45, to obtain the human umbilical cord mesenchymal stem cells; S3, culturing the human umbilical cord mesenchymal stem cells, collecting stem cell culture fluid, and extracting exosomes in the stem cell culture fluid by differential centrifugation.

2. The method for preparing stem cell-derived exosomes according to claim 1, characterized in that: In step S1, the digestion solution includes 1 mg / mL collagenase I and 1 U / mL hyaluronidase, and digestion is performed at 37°C for 1-2 hours.

3. The method for preparing stem cell-derived exosomes according to claim 1, characterized in that: The method for labeling the human umbilical cord mesenchymal stem cells comprises: The cell culture medium in step S1 was centrifuged, the cell pellet was collected and resuspended with PBS, the cell density was adjusted to 1×106 cells / mL, and the fluorescent group-conjugated positive antibody and / or negative antibody were added. After mixing, the mixture was incubated at 4°C in the dark for 30 minutes; then, the mixture was centrifuged at 300×g for 5 minutes, the supernatant was discarded, and the mixture was resuspended with PBS.

4. The method for preparing stem cell-derived exosomes according to claim 1, characterized in that: In step S2, the positive rate of the positive antibodies is greater than 99%, and the positive rate of the negative antibodies is less than 1%.

5. The method for preparing stem cell-derived exosomes according to any one of claims 1 to 4, characterized in that: Step S2 also includes the step of inducing osteogenic and adipogenic differentiation of the human umbilical cord mesenchymal stem cells.

6. The method for preparing stem cell-derived exosomes according to claim 5, characterized in that: The osteogenic adipogenic differentiation induction comprises the following steps: Subculturing the human umbilical cord mesenchymal stem cells to 80%-90% confluence, adding osteogenic induction medium, and continuing culturing for 21 days, during which the medium was replaced every 3 days, and detecting osteogenic differentiation by Alizarin red staining; Then, the osteogenic induction medium was replaced with an adipogenic induction medium, and the culture was continued for 14 days, during which the medium was replaced every 2 days, and the formation of lipid droplets was detected by Oil Red O staining; The human umbilical cord mesenchymal stem cells having osteogenic and adipogenic differentiation capabilities are selected for subsequent culture.

7. The method for preparing stem cell-derived exosomes according to claim 1, characterized in that: In step S3, extracting exosomes from the stem cell culture medium by differential centrifugation comprises the following steps: The human umbilical cord mesenchymal stem cells were subcultured to 80%-90% confluence, and the stem cell culture medium was collected. The stem cell culture medium was centrifuged at 300×g for 10 minutes, then at 2000×g for 20 minutes, and then at 10000×g for 30 minutes. The supernatant was collected and filtered through a 0.22 μm filter membrane. The filtrate was centrifuged at 100000×g for 70 minutes, the exosome precipitate was resuspended with PBS, and then centrifuged at 100000×g for 70 minutes, the supernatant was discarded, and the exosome precipitate was collected.

8. A method for preserving stem cell-derived exosomes, characterized in that: The following steps are involved: The exosome precipitate prepared by the method for preparing stem cell-derived exosomes according to any one of claims 1 to 7 is added to a PBS buffer containing human serum albumin, trehalose and 4-hydroxyethylpiperazineethanesulfonic acid for storage.

9. The method for preserving stem cell-derived exosomes according to claim 8, characterized in that: In the PBS buffer, the mass concentration of human serum albumin is 0.2%, the molar concentration of trehalose is 25 mM, and the molar concentration of 4-hydroxyethylpiperazineethanesulfonic acid is 25 mM.

10. The method for preserving stem cell-derived exosomes according to claim 8, characterized in that: The storage conditions include normoxic and / or hypoxic conditions, and the hypoxic conditions are those in which the oxygen content is greater than or equal to 1% and less than 21%.

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