3D hypoxia cultured stem cell-derived exosome as well as preparation method and application thereof

Abstract

The invention discloses a 3D hypoxia cultured stem cell-derived exosome as well as a preparation method and application thereof, belongs to the technical field of derivative products of cells, and solves the problem that in the prior art, the functional difference between an in-vitro cultured stem cell-derived exosome and an in-vivo naturally generated exosome is large. Compared with the traditional 2D normal oxygen culture stem cell source exosome, the 3D low oxygen culture stem cell source exosome provided by the invention has the advantages that the particle concentration and the protein concentration are obviously improved, the abundance of miRNA (micro Ribonucleic Acid) related to heart protection contained in the 3D low oxygen culture stem cell source exosome is higher, and the 3D low oxygen culture stem cell source exosome has a stronger promotion effect on the proliferation of human umbilical vein endothelial cells; and the compound has a more obvious promotion effect on tubulation and migration of human umbilical vein endothelial cells.

Description

Technical Field

[0001] This invention relates to the field of cell-derived product technology, and more specifically to a stem cell-derived exosome cultured in 3D hypoxia, its preparation method, and its application. Background Technology

[0002] Currently, traditional exosome preparation techniques primarily employ 2D normoxic conditions. Under these conditions, stem cells grow in flat culture dishes or flasks, with the oxygen concentration maintained at atmospheric levels (approximately 19.5%-23.5% O2). Specifically, existing techniques mainly involve culturing stem cells under 2D normoxic conditions and obtaining exosomes from their culture supernatant using conventional ultracentrifugation methods.

[0003] However, 2D normoxic culture conditions cannot effectively simulate the 3D microenvironment and hypoxic conditions in vivo, affecting the functionality and bioactivity of exosomes. The in vivo environment is a complex three-dimensional structure containing various cell types, extracellular matrix, and different oxygen concentration gradients. 2D normoxic culture cannot reproduce this complex environment, resulting in significant functional differences between the prepared exosomes and naturally occurring exosomes in vivo, thus limiting their potential applications in tissue repair and regenerative medicine. Summary of the Invention

[0004] This invention provides a stem cell-derived exosome cultured in 3D hypoxia, its preparation method, and its application, in order to solve the problem of significant functional differences between exosomes cultured in vitro and naturally occurring exosomes in vivo in the prior art.

[0005] In a first aspect, the present invention provides a method for preparing stem cell-derived exosomes cultured under 3D hypoxia, comprising the following steps:

[0006] Stem cells were seeded into a 3D hypoxic culture system for expansion culture and intermittent heat shock culture, and the culture supernatant was collected; wherein the seeding density was 4000-8000 cells / cm³. 2 In the 3D hypoxia culture system, the oxygen partial pressure is 3%-8%, the pH value is 6.8-7.6, the culture medium is a serum-free stem cell culture medium containing L-ascorbic acid, and the microcarrier is a spherical microcarrier coated with poly-L-lysine.

[0007] The exosomes were obtained from the culture supernatant by tangential flow ultrafiltration.

[0008] As described above, the preparation method of stem cell-derived exosomes involves obtaining the poly-L-lysine-coated spherical microcarriers by means of the following process: placing spherical silica microcarriers in a poly-L-lysine solution and immersing them at 36-38°C for 2-4 hours to obtain poly-L-lysine-coated spherical microcarriers.

[0009] In the method for preparing stem cell-derived exosomes as described above, the filling density of the microcarrier is 0.4-0.6 kg / L.

[0010] The method for preparing stem cell-derived exosomes as described above, wherein the amplification culture temperature is 36℃-38℃ and the time is 6-9 days;

[0011] And / or, the procedure for the intermittent heat shock culture is as follows: 42℃ for 0.5h, 37℃ for 1.5h as one heat shock cycle, and a total of 12 heat shock cycles are performed.

[0012] In the method for preparing stem cell-derived exosomes as described above, the concentration of L-ascorbic acid in the serum-free culture medium containing L-ascorbic acid is 40-60 μmol / L.

[0013] The method for preparing stem cell-derived exosomes as described above, wherein the stem cells are stem cells that have been passaged in vitro for 2 to 4 generations;

[0014] The stem cells are umbilical cord mesenchymal stem cells, bone marrow mesenchymal stem cells, adipose mesenchymal stem cells, or dental pulp mesenchymal stem cells.

[0015] The method for preparing stem cell-derived exosomes as described above, wherein the tangential flow ultrafiltration method includes the following steps:

[0016] The culture supernatant was clarified and filtered sequentially through 0.45 μm and 0.22 μm filters to obtain the filtrate;

[0017] The filtrate was concentrated by tangential flow ultrafiltration through a 100-500kD hollow fiber membrane column at a concentration ratio of 1:20, and then replaced with physiological saline to obtain an exosome suspension.

[0018] The exosome suspension was sterilized by passing it through a 0.22 μm filter to obtain the exosomes.

[0019] Secondly, the present invention provides a stem cell-derived exosome cultured in 3D hypoxia, which is prepared by the aforementioned preparation method.

[0020] Thirdly, the present invention provides the application of exosomes prepared by the preparation method described above or stem cell-derived exosomes cultured in 3D hypoxia in the preparation of reagents that promote endothelial cell tube formation and migration.

[0021] As described above, the exosomes meet the following conditions: (1) the particle size distribution of the exosomes is 30-150 nm; (2) the protein concentration of the exosomes is greater than 2 μg / μL; (3) the particle concentration of the exosomes is greater than 1.0 × 10⁻⁶. 11 Particles / mL.

[0022] As described above, the exosomes contain miRNAs associated with cardiac repair; the miRNAs are at least one of miR-16, miR-34a, miR-138, and miR-1271.

[0023] As described above, the tissue repair is achieved by resisting hypoxia damage and / or regulating cell proliferation and / or promoting angiogenesis.

[0024] Compared with the prior art, the present invention has the following advantages:

[0025] Compared with stem cell-derived exosomes cultured in conventional 2D normoxic culture, stem cell-derived exosomes cultured on a large scale in 3D hypoxia show little difference in morphology, particle size, and particle size distribution, but their particle concentration and protein concentration are significantly higher (the particle concentration of 3D-Exos is 1.8 times that of 2D-Exos). RNA sequencing results show that the abundance of miRNAs closely related to the cardiac repair process in 3D-Exos is significantly higher than that in 2D-Exos, such as miR-16, miR-34a, miR-138, and miR-1271. The target genes of these upregulated miRNAs are mainly enriched in a series of signaling pathways that regulate cell proliferation, promote angiogenesis, resist hypoxia damage, and activate other pathways, such as PI3K / AKT, MAPK, mTOR, and TGF-β. Furthermore, compared with stem cell-derived exosomes cultured in conventional 2D normoxic culture, stem cell-derived exosomes cultured on a large scale in 3D hypoxia have a stronger promoting effect on the proliferation of human umbilical vein endothelial cells and a more significant promoting effect on the tube formation and migration of human umbilical vein endothelial cells.

[0026] While existing methods for culturing stem cell-derived exosomes under 2D normoxic conditions are relatively mature and simple, they are difficult to scale up for production, resulting in low batch yields, high costs, and significant differences from the in vivo environment in terms of cell growth environment, nutrient supply, and oxygen concentration. This invention utilizes microcarriers and microchannels to construct a 3D culture environment, simulating the hypoxic environment in vivo by adjusting conditions such as oxygen concentration in a bioreactor. This promotes stem cell growth and differentiation closer to their natural state, thereby improving the quality and function of stem cell exosomes. The key difference between this patent and existing technologies lies in the use of a 3D hypoxic culture method and the implementation of large-scale production via a bioreactor. This improvement significantly increases the exosome content (particle concentration) and protein concentration per unit volume, while optimizing the miRNA composition within the exosomes, particularly increasing the abundance of miRNAs closely related to the heart repair process. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0028] Figure 1 TEM images of 2D-Exos and 3D-Exos provided in embodiments of the present invention, wherein Figure 1 In the image, A is the TEM image of 2D-Exos. Figure 1 B in the image is a TEM image of 3D-Exos.

[0029] Figure 2 The image shows the particle size distribution detection results of 2D-Exos and 3D-Exos provided in the embodiments of the present invention. Figure 2 In the figure, A represents the particle size distribution detection result of 2D-Exos. Figure 2 In the figure, B represents the particle size distribution detection result of 3D-Exos.

[0030] Figure 3 The protein content and particle concentration detection results of 2D-Exos and 3D-Exos provided in the embodiments of the present invention are as follows: Figure 3 In the figure, A represents the protein content detection results of 2D-Exos and 3D-Exos. Figure 3 B in the figure represents the particle concentration detection results for 2D-Exos and 3D-Exos.

[0031] Figure 4 Volcano plot of differential metabolites in miRNA analysis of 2D-Exos and 3D-Exos provided for embodiments of the present invention.

[0032] Figure 5 Metabolic biomarker heatmap clustering analysis of miRNAs contained in 2D-Exos and 3D-Exos provided in the embodiments of the present invention.

[0033] Figure 6 Scatter plot of metabolic pathway enrichment for miRNA analysis of 2D-Exos and 3D-Exos provided in embodiments of the present invention.

[0034] Figure 7 The comparison results of the effects of different concentrations of 2D-Exos and 3D-Exos on HUVEC proliferation provided in the embodiments of the present invention.

[0035] Figure 8This is a comparison of the effects of 2D-Exos and 3D-Exos on the in vitro tubular formation of HUVECs provided in this embodiment of the invention. Control refers to the group without exosomes, 2D-Exos refers to the group with 2D-Exos, and 3D-Exos refers to the group with 3D-Exos. Figure 8 In the image, A represents the representative images of tubular formation in each group of HUVECs. Figure 8 B in the figure is a quantitative analysis chart of the length (cumulative tube length) of each component tube.

[0036] Figure 9 This is a comparison of the effects of 2D-Exos and 3D-Exos on the wound healing ability of HUVEC scratch simulation provided in this embodiment of the invention. Control refers to the group without exosomes, 2D-Exos refers to the group with 2D-Exos, and 3D-Exos refers to the group with 3D-Exos. Figure 9 In the image, A represents a representative image of wound healing in each group of HUVECs. Figure 9 B in the figure represents the quantitative analysis of cell migration rate in each group. Detailed Implementation

[0037] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0038] In the following description, the term "and / or" is used to describe the relationship between related objects, indicating that there can be three relationships. For example, A and / or B can mean: A exists alone, B exists alone, and A and B exist simultaneously. A and B can be singular or plural.

[0039] In the following description, the terms “including,” “containing,” “having,” and “containing” are open-ended terms, meaning that they include but are not limited to.

[0040] Those skilled in the art should understand that, in the following description of the embodiments of this application, the sequence of numbers does not imply the order of execution. Some or all steps may be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0041] Those skilled in the art will understand that the numerical ranges in the embodiments of this application should be understood to specifically disclose each intermediate value between the upper and lower limits of the range. Each smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this application. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0042] Unless otherwise stated, the technical / scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. While this application describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this application. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0043] Current technologies primarily employ culturing stem cells under 2D normoxic conditions and obtaining exosomes from the culture supernatant using traditional ultracentrifugation. However, 2D normoxic culture conditions cannot effectively simulate the 3D microenvironment and hypoxic conditions in vivo, affecting the functionality and bioactivity of stem cell exosomes. The in vivo environment is a complex three-dimensional structure containing multiple cell types, extracellular matrix, and varying oxygen concentration gradients. 2D normoxic culture cannot reproduce this complex environment, resulting in significant functional differences between the prepared exosomes and naturally occurring exosomes in vivo, thus limiting their potential applications in tissue repair and regenerative medicine.

[0044] Based on this, in a first aspect, the present invention provides a method for preparing stem cell-derived exosomes cultured under 3D hypoxia, comprising the following steps:

[0045] Stem cells were seeded into a 3D hypoxic culture system for expansion culture and intermittent heat shock culture, and the culture supernatant was collected; wherein the seeding density was 4000-8000 cells / cm³. 2 In the 3D hypoxia culture system, the oxygen partial pressure is 3%-8%, the pH value is 6.8-7.6, the culture medium is a serum-free stem cell culture medium containing L-ascorbic acid, and the microcarrier is a spherical microcarrier coated with poly-L-lysine.

[0046] The exosomes were obtained from the culture supernatant by tangential flow ultrafiltration.

[0047] In this invention, both the amplification culture and the intermittent heat shock culture processes are carried out in a bioreactor. In some embodiments, the bioreactor used is a stainless steel bioreactor BRSTMCELL-2.6L.

[0048] It is understandable that in the 3D hypoxic culture system, the oxygen partial pressure is achieved by introducing high-purity nitrogen or high-purity oxygen into the bioreactor; and the pH is achieved by introducing carbon dioxide or sodium bicarbonate into the bioreactor.

[0049] The inventors' research shows that the method provided by this invention can produce exosomes with higher particle and protein concentrations (i.e., stem cell-derived exosomes cultured in 3D hypoxia). Furthermore, the abundance of miRNAs closely related to the heart repair process in these exosomes is significantly increased, and they have a stronger promoting effect on the proliferation of human umbilical vein endothelial cells (HUVECs), as well as a more significant promoting effect on the tube formation and migration of human umbilical vein endothelial cells.

[0050] In some embodiments, the poly-L-lysine-coated spherical microcarriers are obtained by a preparation method comprising the following steps: placing spherical silica microcarriers in a poly-L-lysine solution and immersing them at 36-38°C for 2-4 hours to obtain poly-L-lysine-coated spherical microcarriers.

[0051] The present invention does not impose a particular limitation on the specific diameter of the above-mentioned spherical silica microcarriers, which can be selected according to actual needs. In some embodiments, the diameter of the spherical silica microcarriers can be 1-1.5 mm.

[0052] The use of poly-L-lysine-coated spherical microcarriers was to promote the adherent growth of stem cells.

[0053] In some embodiments, the filling density of the microcarrier is 0.4-0.6 kg / L, preferably 0.5 kg / L.

[0054] When the microcarrier packing density is within the above range, the optimal balance between cell yield and cell viability can be achieved. If the microcarrier packing density is too high (>0.6 kg / L), cell yield increases, but cell viability decreases; if the microcarrier packing density is too low (<0.4 kg / L), cell viability remains unchanged, but cell yield decreases.

[0055] For example, the filling density of the microcarrier can be any one of 0.4 g / L, 0.5 g / L, 0.6 kg / L, or any combination of both.

[0056] In some embodiments, the amplification culture is carried out at a temperature of 36°C-38°C for 6-9 days.

[0057] In some embodiments, the intermittent heat shock culture procedure is as follows: 42°C for 0.5 h, 37°C for 1.5 h constitutes one heat shock cycle, and a total of 12 heat shock cycles are performed.

[0058] In this invention, the role of intermittent heat shock culture is to promote the secretion of exosomes by stem cells, increase exosome production, and enhance exosome function (such as a stronger effect on promoting the proliferation of human umbilical vein endothelial cells, and a stronger effect on promoting the tube formation and migration of human umbilical vein endothelial cells).

[0059] In some embodiments, the concentration of L-ascorbic acid in the serum-free culture medium containing L-ascorbic acid is 40-60 μmol / L, preferably 50 μmol / L.

[0060] In this invention, adding L-ascorbic acid to serum-free culture medium and controlling its concentration at 40-60 μmol / L can promote stem cell secretion of exosomes and enhance exosome function (such as a stronger effect on promoting the proliferation of human umbilical vein endothelial cells and a stronger effect on promoting the tube formation and migration of human umbilical vein endothelial cells).

[0061] In some embodiments, the stem cells are stem cells that have been passaged in vitro for 2 to 4 generations; the stem cells are umbilical cord mesenchymal stem cells, bone marrow mesenchymal stem cells, adipose mesenchymal stem cells or dental pulp mesenchymal stem cells, preferably umbilical cord mesenchymal stem cells.

[0062] In some embodiments, the tangential flow ultrafiltration method includes the following steps:

[0063] The culture supernatant was clarified and filtered sequentially through 0.45 μm and 0.22 μm filters to obtain the filtrate;

[0064] The filtrate was concentrated by tangential flow ultrafiltration through a 100-500kD hollow fiber membrane column at a concentration ratio of 1:20, and then replaced with physiological saline to obtain an exosome suspension.

[0065] The exosome suspension was sterilized by passing it through a 0.22 μm filter to obtain the exosomes.

[0066] Tangential flow ultrafiltration can efficiently concentrate and purify exosomes in culture supernatant under mild conditions, while maintaining their structure and biological activity. The stem cell-derived exosomes obtained by this method have high purity and stable recovery rate, enabling standardized and large-scale production.

[0067] Secondly, the present invention provides a stem cell-derived exosome cultured in 3D hypoxia, which is prepared by the aforementioned preparation method.

[0068] Thirdly, the present invention provides the application of exosomes prepared by the preparation method described above or stem cell-derived exosomes cultured in 3D hypoxia in the preparation of reagents that promote endothelial cell tube formation and migration.

[0069] In some embodiments, the exosomes satisfy the following conditions: (1) the particle size distribution of the exosomes is 30-150 nm; (2) the protein concentration of the exosomes is greater than 2 μg / μL; (3) the particle concentration of the exosomes is greater than 1.0 × 10⁻⁶. 11 Particles / mL.

[0070] In some embodiments, the endothelial cells are human umbilical vein endothelial cells.

[0071] In some embodiments, the exosomes contain miRNAs associated with cardiac repair; the miRNAs are at least one of miR-16, miR-34a, miR-138, and miR-1271.

[0072] In some embodiments, the tissue repair is achieved by resisting hypoxia damage and / or regulating cell proliferation and / or promoting angiogenesis.

[0073] This invention provides a characterization experiment of stem cell exosomes. It shows that the 3D hypoxic cultured stem cell-derived exosomes provided by this invention have little difference in shape and particle size from traditional 2D normoxic cultured stem cell-derived exosomes, but their particle concentration and protein concentration are significantly higher (the particle concentration of 3D-Exos is 1.8 times that of 2D-Exos). Furthermore, RNA sequencing results show that the abundance of miRNAs closely related to the cardiac repair process in 3D-Exos is significantly higher than that in 2D-Exos, such as miR-16, miR-34a, miR-138, and miR-1271. The target genes of these upregulated miRNAs are mainly enriched in a series of signal regulation mechanisms such as PI3K / AKT, MAPK, mTOR, and TGF-β, which regulate cell proliferation, promote angiogenesis, resist hypoxia injury, and activate other signals.

[0074] This invention provides an experimental analysis of the mechanism of action of exosomes. It shows that, compared with stem cell-derived exosomes cultured in conventional 2D normoxic culture, stem cell-derived exosomes cultured on a large scale in 3D hypoxia have a stronger promoting effect on the proliferation of human umbilical vein endothelial cells and a more significant promoting effect on the tube formation and migration of human umbilical vein endothelial cells.

[0075] The technical solution of the present invention will be further described below with reference to specific embodiments.

[0076] In the following examples, the bioreactor used is a stainless steel bioreactor BRSTMCELL-2.6L, sourced from BiRui biotech; L-ascorbic acid (HY-B0166, medchemexpress); serum-free mesenchymal stem cell culture medium (SC2013-G, TBD); spherical silica microcarriers (1.1 mm in diameter, 5001-89-S1, Tiantai Precision); and poly-L-lysine solution (0.1 mg / mL, 60717ES50, Yisheng Biotechnology).

[0077] Example 1

[0078] This embodiment provides an experiment for the preparation of exosomes.

[0079] 1. Preparation of P2-P4 generation umbilical cord mesenchymal stem cells: The two ends of the human umbilical cord tissue were tied off and immersed in a bottle containing physiological saline. The tube was then transported to the laboratory. The bottle was opened in a clean bench, and the preservation solution was collected (if cells were contaminated, this liquid was used for testing to locate the source of contamination). After discarding the excess preservation solution, 20 mL of 75% alcohol was added to the bottle (enough to submerge the umbilical cord). The bottle was tightened, and the tube was shaken 7 times from side to side before soaking for 2 minutes. The alcohol was discarded, and the tube was washed twice with 20 mL of physiological saline to remove any alcohol residue. The umbilical cord was removed with sterile forceps and placed in a sterile 100 mm culture dish. 10 mL of physiological saline was added. Using sterile scissors, divide the umbilical cord into 3-4 cm segments. Grasp the center of the cord with forceps and push it towards both ends to wash away blood. Locate the umbilical vein, bluntly peel the cord apart from the vein, and remove the vein to obtain a smooth tissue block. Separate the umbilical cord mesenchyme (gelatinous), and promptly place the separated mesenchyme into physiological saline. Remove the umbilical arteries (2). Place the tissue block into a 50 mL centrifuge tube and cut it into 1-3 mm pieces using long scissors. 3 Small segments. Serum-free culture medium was dispensed into T75 cell culture flasks at a rate of 10 mL / flask. The shredded tissue pieces were then dispensed into these flasks; each umbilical cord could be divided into 10 flasks. After evenly distributing the tissue pieces, the flasks were placed in an incubator at 37°C, 5% CO2, and saturated humidity for 5 days, followed by medium replacement. The tissue pieces were observed under a microscope. The culture medium was changed every 5 days. After 9 days, cells began to emerge from around the tissue pieces. Once 80% of the cells had merged, the cells were collected and passaged. 2D culture was performed to passages 2-4, yielding passage 2 (P2) to passage 4 (P4) umbilical cord mesenchymal stem cells.

[0080] 2. Preparation of exosome A: P3 generation umbilical cord mesenchymal stem cells were seeded in a 3D hypoxic culture system for expansion culture and intermittent heat shock culture, and the culture supernatant was collected; wherein, the seeding density was 5000 cells / cm³. 2In the 3D hypoxic culture system, the oxygen partial pressure was 5%, the pH was 7.2, and the culture medium used was serum-free mesenchymal stem cell culture medium containing 50 μmol / L L-ascorbic acid. The microcarriers used were spherical microcarriers modified with poly-L-lysine, and the packing density of the microcarriers was 0.5 kg / L. The amplification culture temperature was 37℃, and the time was 7 days. The intermittent heat shock culture program was as follows: 0.5 h of culture at 42℃ and 1.5 h of culture at 37℃ constituted one heat shock cycle, for a total of 12 heat shock cycles. Both the amplification culture and the intermittent heat shock culture were carried out in a BRSTMCELL-2.6L stainless steel bioreactor. The serum-free medium for mesenchymal stem cells containing L-ascorbic acid was prepared by the following steps: 500 mL of serum-free medium for mesenchymal stem cells was taken, and L-ascorbic acid was added to a final concentration of 50 μmol / L; the poly-L-lysine-coated spherical microcarriers were prepared by the following steps: 1.1 mm diameter spherical silica microcarriers were placed in a poly-L-lysine solution and soaked at 37 °C for 2 h. The coating solution was then discarded to obtain poly-L-lysine-coated spherical microcarriers.

[0081] The culture supernatant was sequentially filtered through 0.45 μm and 0.22 μm filters to remove cell debris and large impurity particles, yielding a filtrate. The filtrate was then concentrated by tangential flow ultrafiltration through a 300 kD hollow fiber membrane column at a concentration ratio of 1:20, followed by replacement with physiological saline to obtain an exosome suspension. The exosome suspension was then sterilized by filtration through a 0.22 μm filter to obtain exosome A.

[0082] 3. Preparation of exosome B: P2 generation umbilical cord mesenchymal stem cells were seeded in a 3D hypoxic culture system for expansion culture and intermittent heat shock culture, and the culture supernatant was collected; wherein, the seeding density was 5000 cells / cm³. 2In the 3D hypoxic culture system, the oxygen partial pressure was 3%, the pH was 7.2, and the culture medium was serum-free mesenchymal stem cell culture medium containing 50 μmol / L L-ascorbic acid. The microcarriers used were spherical microcarriers modified with poly-L-lysine, with a packing density of 0.5 kg / L. The amplification culture was conducted at 37°C for 7 days. The intermittent heat shock culture program consisted of 0.5 h at 42°C followed by 1.5 h at 37°C as one heat shock cycle, for a total of 12 heat shock cycles. Both the amplification culture and the intermittent heat shock culture were carried out in a BRSTMCELL-2.6L stainless steel bioreactor. The serum-free medium for mesenchymal stem cells containing L-ascorbic acid was prepared by the following steps: 500 mL of serum-free medium for mesenchymal stem cells was taken, and L-ascorbic acid was added to a final concentration of 50 μmol / L; the poly-L-lysine-coated spherical microcarriers were prepared by the following steps: 1.1 mm diameter spherical silica microcarriers were placed in a poly-L-lysine solution and soaked at 37 °C for 2 h. The coating solution was then discarded to obtain poly-L-lysine-coated spherical microcarriers.

[0083] The culture supernatant was sequentially filtered through 0.45 μm and 0.22 μm filters to remove cell debris and large impurity particles, yielding a filtrate. The filtrate was then concentrated by tangential flow ultrafiltration through a 300 kD hollow fiber membrane column at a concentration ratio of 1:20, followed by replacement with physiological saline to obtain an exosome suspension. The exosome suspension was then sterilized by filtration through a 0.22 μm filter to obtain exosome B.

[0084] 4. Preparation of exosomes C: P4 generation umbilical cord mesenchymal stem cells were seeded in a 3D hypoxic culture system for expansion culture and intermittent heat shock culture, and the culture supernatant was collected; wherein, the seeding density was 5000 cells / cm³. 2In the 3D hypoxic culture system, the oxygen partial pressure was 5%, the pH was 7.2, and the culture medium used was serum-free mesenchymal stem cell culture medium containing 50 μmol / L L-ascorbic acid. The microcarriers used were spherical microcarriers modified with poly-L-lysine, and the packing density of the microcarriers was 0.5 kg / L. The amplification culture temperature was 37℃, and the time was 7 days. The intermittent heat shock culture program was as follows: 0.5 h of culture at 42℃ and 1.5 h of culture at 37℃ constituted one heat shock cycle, for a total of 12 heat shock cycles. Both the amplification culture and the intermittent heat shock culture were carried out in a BRSTMCELL-2.6L stainless steel bioreactor. The serum-free medium for mesenchymal stem cells containing L-ascorbic acid was prepared by the following steps: 500 mL of serum-free medium for mesenchymal stem cells was taken, and L-ascorbic acid was added to a final concentration of 50 μmol / L; the poly-L-lysine-coated spherical microcarriers were prepared by the following steps: 1.1 mm diameter spherical silica microcarriers were placed in a poly-L-lysine solution and soaked at 37 °C for 2 h. The coating solution was then discarded to obtain poly-L-lysine-coated spherical microcarriers.

[0085] The culture supernatant was sequentially filtered through 0.45 μm and 0.22 μm filters to remove cell debris and large impurity particles, yielding a filtrate. The filtrate was then concentrated by tangential flow ultrafiltration through a 300 kD hollow fiber membrane column at a concentration ratio of 1:20, followed by replacement with physiological saline to obtain an exosome suspension. The exosome suspension was then sterilized by filtration through a 0.22 μm filter to obtain exosome C.

[0086] 5. Preparation of exosome D: Exosomes cultured under normoxic conditions for 2D were used as exosome D. The culture conditions were as follows: P3 generation umbilical cord mesenchymal stem cells were cultured at 5000 cells / cm³. 2 The cells were inoculated at the desired density in T175 cell culture flasks with an oxygen concentration of 20% and cultured at 37°C in a 5% CO2 incubator for 72 hours. The culture supernatant was collected, and exosomes, namely exosome D, were extracted from the culture supernatant by ultracentrifugation.

[0087] Example 2

[0088] This embodiment provides a characterization experiment for exosomes.

[0089] This embodiment characterizes exosomes cultured under 2D normoxic conditions (2D-Exos) and exosomes cultured under 3D hypoxic conditions (3D-Exos). Exosome A (3D-Exos) prepared in Example 1 is used as the representative of exosomes cultured under 3D hypoxic conditions, and exosome D (2D-Exos) prepared in Example 1 is used as the representative of exosomes cultured under 2D normoxic conditions. Morphological identification, concentration determination, particle size determination and miRNA differential analysis are performed.

[0090] 2D-Exos and 3D-Exos were observed using transmission electron microscopy (TEM). Sample preparation for TEM involved uranium acetate staining; the exosomes were fixed onto a copper grid for staining and observation. Results were obtained as follows: Figure 1 The TEM results are shown. (From...) Figure 1 It is evident that a clear double-layered membrane structure can be observed in both.

[0091] The particle size distributions of 2D-Exos and 3D-Exos were detected using the ZetaView Nanoparticle Tracking Analysis (NTA) system manufactured by Particle Metrix, as shown below. Figure 2 The results are shown. (By...) Figure 2 It can be seen that the particle size of both is between 30-150 nm, which is consistent with the size of mesenchymal stem cell-derived exosomes (MSC-exos).

[0092] The protein content of 2D-Exos and 3D-Exos was detected using the BCA method, and the results were as follows: Figure 3 The results are shown in A; the particle concentrations of 2D-Exos and 3D-Exos were detected using nanoparticle tracking analysis (NTA) technology, as shown in Figure A. Figure 3 The result shown in B is from... Figure 3 It is evident that the particle concentration and protein concentration of 3D-Exos are significantly higher than those of 2D-Exos.

[0093] Differential analysis of miRNAs contained in 2D-Exos and 3D-Exos was performed using miRNA sequencing, yielding the following results: Figure 4 , Figure 5 and Figure 6 The results are shown. (By...) Figure 4 It can be seen that, compared with 2D-Exos, 3D-Exos has 22 upregulated and 16 downregulated miRNAs; Figure 5 It can be seen that, compared with 2D-Exos, the abundance of some miRNAs closely related to the cardiac repair process, such as miR-16, miR-34a, miR-138, and miR-1271, is increased in 3D-Exos; Figure 6 It is known that the target genes of upregulated miRNAs (miR-16, miR-34a, miR-138, miR-1271, etc.) are mainly enriched in a series of signaling pathways that regulate cell proliferation, angiogenesis, anti-hypoxia injury, and inflammation, such as PI3K / AKT, MAPK, mTOR, and TGF-β.

[0094] Under 2D culture conditions, the differentiation potential and self-renewal capacity of stem cells are limited. Stem cells grow in a planar environment and cannot form the complex three-dimensional interactions with surrounding cells and matrix as they do in vivo, thus affecting their key biological characteristics. 2D culture cannot effectively simulate the 3D microenvironment and hypoxic conditions in vivo, impacting the functionality and bioactivity of exosomes. The in vivo environment is a complex three-dimensional structure containing multiple cell types, extracellular matrix, and varying oxygen concentration gradients. 2D culture cannot reproduce this complex environment, resulting in differences in morphology, function, protein and particle concentrations, and miRNA content between exosomes and naturally occurring exosomes in vivo, thus limiting their potential applications in tissue repair and regenerative medicine.

[0095] Example 3

[0096] This embodiment provides an experimental analysis of the mechanism of action of exosomes.

[0097] This embodiment analyzes the mechanism of exosomes cultured under 2D normoxic conditions (2D-Exos) and exosomes cultured under 3D hypoxic conditions (3D-Exos). The effects of exosome A (3D-Exos) prepared in Example 1 on HUVECs are analyzed.

[0098] The effects of different concentrations (0, 10 μg / mL, 20 μg / mL, 40 μg / mL, 50 μg / mL) of 2D-Exos and 3D-Exos on the proliferation of HUVECs (human umbilical vein endothelial cells, model cells) were detected using the CCK-8 assay. The results are as follows: Figure 7 The results are shown. (By...) Figure 7 It was found that at low concentrations, there was no significant difference in the effects of 2D-Exos and 3D-Exos on HUVEC proliferation. At a concentration of 50 μg / mL, the relative viability of HUVECs treated with 3D-Exos was significantly enhanced compared to those treated with 2D-Exos.

[0099] The effects of 2D-Exos and 3D-Exos on the in vitro tubular formation of HUVECs were observed through angiogenesis experiments, and the results were as follows: Figure 8 The results are shown. (By...) Figure 8It can be seen that the angiogenesis-promoting effect of 3D-Exos is more significant than that of 2D-Exos.

[0100] The effects of 2D-Exos and 3D-Exos on the wound healing ability of HUVEC scratch simulation were evaluated, and the results were as follows: Figure 9 The results are shown. (By...) Figure 9 It can be seen that the HUVEC mobility after 3D-Exos treatment is significantly greater than that of the 2D-Exos treatment group.

[0101] The above results indicate that, compared with 2D-Exos, 3D-Exos has a stronger promoting effect on the proliferation of human umbilical vein endothelial cells, and a more significant promoting effect on the tube formation and migration of human umbilical vein endothelial cells.

[0102] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method of preparing 3D hypoxically cultured stem cell-derived exosomes, characterized by, comprising the following steps: The stem cells are inoculated in a 3D low-oxygen culture system for expansion culture and intermittent heat shock culture, and culture supernatant is collected; wherein, the inoculation density is 4000-8000 cells / cm 2 ; in the 3D low-oxygen culture system, the oxygen partial pressure is 3%-8%, the pH value is 6.8-7.6, the culture medium is a stem cell serum-free culture medium containing L-ascorbic acid, and the microcarrier is a spherical microcarrier modified by poly-L-lysine coating. obtaining the exosomes from the culture supernatant by a tangential flow ultrafiltration method.

2. The production method according to claim 1, characterized by, The poly-L-lysine coated modified spherical microcarriers are obtained by a preparation method comprising the following process: placing spherical silica microcarriers in a poly-L-lysine solution, soaking and coating at 36-38℃ for 2-4h to obtain poly-L-lysine coated modified spherical microcarriers.

3. The production method according to claim 1, characterized by, The packing density of the microcarriers is 0.4-0.6kg / L.

4. The method of claim 1, wherein, The temperature of the expansion culture is 36-38℃, and the time is 6-9 days. And / or, the program of the intermittent heat shock culture is: 42℃ culture for 0.5h, 37℃ culture for 1.5h as one heat shock cycle, and a total of 12 heat shock cycles.

5. The preparation method according to claim 1, characterized in that, The concentration of L-ascorbic acid in the L-ascorbic acid containing serum-free medium is 40-60μmol / L.

6. The method of claim 1, wherein, The stem cells are stem cells cultured in vitro for 2-4 generations. The stem cells are umbilical cord mesenchymal stem cells, bone marrow mesenchymal stem cells, adipose tissue mesenchymal stem cells or dental pulp mesenchymal stem cells.

7. The preparation method according to claim 1, characterized in that, The tangential flow ultrafiltration method comprises the following steps: The culture supernatant is sequentially subjected to clarification filtration through 0.45μm and 0.22μm filters to obtain a filtrate; The filtrate is subjected to tangential flow ultrafiltration concentration through a 100-500kD hollow fiber column membrane, the concentration ratio is 1:20, and physiological saline is used for displacement to obtain an exosome suspension; The exosome suspension is subjected to sterilization filtration through a 0.22μm filter to obtain the exosomes.

8. A 3D hypoxically cultured stem cell-derived exosome, characterized in that, It is obtained by the preparation method of any one of claims 1-7.

9. Use of the exosomes prepared by the preparation method of any one of claims 1-7 or the 3D hypoxic culture stem cell derived exosomes of claim 8 in the preparation of a reagent for promoting endothelial cell tube formation and migration.

10. Use according to claim 9, characterized in that, The exosomes satisfy the following conditions: (1) the particle size distribution of the exosomes is 30-150 nm; (2) the protein concentration of the exosomes is greater than 2 μg / uL; (3) the particle concentration of the exosomes is greater than 1.0 x 10 11 particles / mL.

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