Large-scale production method of 3D-MSCs (three-dimensional mesenchymal stem cells) spheres, 3D-MSCs cell spheres and application of 3D-MSCs cell spheres

Abstract

The invention provides a 3D-MSCs sphere large-scale production method, a 3D-MSCs cell sphere and application of the 3D-MSCs cell sphere, and belongs to the technical field of biological medicine. Based on a microgravity suspension culture system, the influence of gravity factors on cells is eliminated by controlling the density of the cells and the rotating speed of a microgravity suspension culture bioreactor, so that the cells are suspended in a 3D microenvironment, collision and contact of the cells in space are increased through dynamic culture, cell aggregation is realized by utilizing the stem cell characteristics of MSCs, and the MSCs are cultured in the 3D microenvironment. Connection between cells is formed, and 3D-MSCs cell spheres can be produced in the same environment. Compared with a conventional inverted balling culture mode, the method disclosed by the invention is simple, the yield of the 3D-MSCs cells is high, the obtained 3D-MSCs cells are uniform in quality, and large-scale production of the 3D-MSCs cells can be realized.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a large-scale production method of 3D-MSCs spheres, 3D-MSCs cell spheres and applications thereof. Background Art

[0002] Mesenchymal stem cells (MSCs) are a type of multipotent stem cell belonging to the mesoderm, primarily found in connective tissue and organ mesenchyme. They have low immunogenicity and can contribute to immune reconstitution through cell-cell interactions and cytokine production. They can suppress immune responses in inflammatory environments, exerting anti-inflammatory effects. They also possess tissue repair functions, and are widely studied and applied in tissue injury and repair. MSCs also offer a potential treatment option for unresolved issues in the treatment of diabetic foot ulcers. However, the effectiveness and safety of MSC transplantation therapy pose potential challenges and risks to their clinical application.

[0003] Studies have shown that 3D culture can not only accurately simulate and reproduce the in vivo MSC growth structure, but also make the culture microenvironment closer to the in vivo level, significantly improve the paracrine capacity of MSCs, reduce the proliferation capacity of MSCs, and enhance the therapeutic potential and stability of the cells. Current studies have shown that 3D-MSC spheroids have the characteristics of promoting angiogenesis, anti-tumor formation, enhancing the paracrine secretion of pro-inflammatory and anti-inflammatory factors, improving cell survival, increasing differentiation potential and delaying replicative senescence. Compared with 2D culture, 3D culture has been shown to benefit MSCs in several aspects, including reducing cell size and reducing the occurrence of vascular occlusion; increasing self-renewal and multipotent differentiation, improving engraftment and homing ability; and producing paracrine factors, ultimately enhancing tissue repair in diseases such as myocardial infarction, stroke, angiogenesis and wound healing.

[0004] Impaired inflammatory responses and angiogenesis within the diabetic foot (DFU) microenvironment are the primary causes of poor wound healing. Therefore, modulating inflammatory responses and improving angiogenesis are promising therapeutic approaches for DFU treatment. However, the pathogenesis of DFU is complex, and conventional techniques and medications are unable to fundamentally address the problem. However, the effectiveness of 3D-MSC spheroids in DFU treatment has not been clearly documented, and the spheroids required for disease treatment are difficult to culture using conventional methods.

[0005] At present, 3D-MSCs cell spheres are mainly cultured by inverting the spheres. The cell suspension is dropped on the culture dish cover, and the surface tension and gravity are used to make the cells gather into spheres at the bottom of the droplet. However, due to the influence of gravity, this method cannot produce cell spheres of uniform quality, and the cells living on a small amount of culture medium will have the problem of insufficient nutrients. In addition, the inverted sphere method uses a pipette to drip according to the required number of cell spheres, which has the disadvantages of being time-consuming, labor-intensive, and difficult to achieve large-scale production, limiting its clinical application.

[0006] Therefore, it is crucial to achieve large-scale production of 3D-MSCs spheroids and improve their therapeutic effect on diabetic foot. Summary of the Invention

[0007] The purpose of the present invention is to provide a large-scale production method of 3D-MSCs spheroids, 3D-MSCs cell spheroids and their applications. 3D-MSCs cell spheroids are produced on a large scale through a microgravity suspension culture system. The method is simple, the yield of 3D-MSCs cell spheroids is large, and the quality is uniform. The obtained 3D-MSCs cell spheroids have high stability and safety for in vivo transplantation and good therapeutic effect.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions: The present invention provides a method for large-scale production of 3D-MSCs spheroids, comprising the following steps: (1) Digest MSCs and adjust the density to obtain a cell suspension; (2) The cell suspension is inoculated into a microgravity suspension culture bioreactor for culture.

[0009] Preferably, in step (1), the density of the cell suspension is 0.4-0.8×10 6 / mL.

[0010] Preferably, the density of the cell suspension is 0.6×10 6 / mL.

[0011] Preferably, in step (2), the rotation speed of the microgravity suspension culture bioreactor is 10-20 rpm, and the culture time is 24-72 hours.

[0012] Preferably, the rotation speed of the microgravity suspension culture bioreactor is 15 rpm, and the culture time is 48 hours.

[0013] The present invention also provides a 3D-MSCs cell sphere prepared based on the above-mentioned 3D-MSCs sphere large-scale production method.

[0014] The present invention also provides a use of the above-mentioned 3D-MSCs cell spheres in preparing a drug for treating diabetic foot.

[0015] The present invention also provides an application of the above-mentioned 3D-MSCs sphere large-scale production method in the preparation of a drug for treating diabetic foot.

[0016] The beneficial effects of the present invention compared with the prior art are: (1) The present invention is based on a microgravity suspension culture system. By controlling the cell density and the rotation speed of the microgravity suspension culture bioreactor, the influence of gravity on the cells is eliminated, causing them to suspend in a 3D microenvironment. Dynamic culture increases the collision and contact of cells in space, utilizes the stem cell properties of MSCs to achieve cell aggregation, and forms intercellular connections, thereby producing 3D-MSCs cell spheres in the same environment. Compared with conventional inverted sphere culture methods, the method of the present invention is simple, has a high yield of 3D-MSCs cell spheres, and the obtained 3D-MSCs cell spheres are of uniform quality, which can achieve large-scale production of 3D-MSCs cell spheres.

[0017] (2) The 3D-MSCs spheroids prepared by the present invention meet the identification standards of MSCs, and compared with conventional MSCs cells, the proliferation ability and particle size of the 3D-MSCs spheroids of the present invention are significantly reduced, thereby improving the stability and safety of in vivo transplantation. The exosome paracrine capacity of the 3D-MSCs spheroids is significantly improved, thereby enhancing their therapeutic potential in diseases. In addition, the 3D-MSCs spheroids can improve the proliferation ability, survival ability, and migration ability of HUVEC under high glucose conditions, and promote angiogenesis, thereby promoting the healing of diabetic foot wounds. It is a new product for the treatment of diabetic foot wounds, which has achieved significant therapeutic effects and provides a new idea for the treatment of diabetic foot wounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0019] Figure 1 Graph showing the characterization results of the large-scale production of 3D-MSCs spheroids (3D-MSCs (scaled)), 2D-MSCs, and 3D-MSCs (inverted into spheres) in Experimental Example 1 of the present invention; Figure 2 This is a graph showing the survival of 3D-MSCs (scaled), 2D-MSCs, and 3D-MSCs (inverted into spheres) in Experimental Example 1 of the present invention; Figure 3 This is a graph showing the results of flow-through detection of MSCs markers in 3D-MSCs (scaled) and 2D-MSCs in Experimental Example 2 of the present invention; Figure 4 This is a graph showing the changes in particle size and proliferation capacity of 3D-MSCs (scaled) and 2D-MSCs in Experimental Example 3 of the present invention; Figure 5 This is a graph showing the changes in the exosome secretion capacity of 3D-MSCs (scaled) and 2D-MSCs in Experimental Example 4 of the present invention; Figure 6 This is a graph showing the effects of 3D-MSCs (scaled) and 2D-MSCs on the survival, proliferation, migration, and tube formation of HUVECs damaged by high glucose in Experimental Example 5 of the present invention; Figure 7 This is a graph showing the results of cell retention in the wounds of DFU mice treated with 3D-MSCs (scaled) and 2D-MSCs transplantation in Experimental Example 6 of the present invention; Figure 8 This is a diagram showing the wound healing of DFU mice treated with 3D-MSCs (scaled) and 2D-MSCs transplantation in Experimental Example 6 of the present invention; Figure 9 This is the result of histopathological staining of the wound surface of DFU mice treated with 3D-MSCs (scaled) and 2D-MSCs transplantation in Experimental Example 6 of the present invention; Figure 10 This is the result of CD31 immunofluorescence staining of the wounds of DFU mice treated with 3D-MSCs (scaled) and 2D-MSCs transplantation in Experimental Example 6 of the present invention; Figure 11 This is the result of Krt14 immunofluorescence staining of 3D-MSCs (scaled) and 2D-MSCs transplantation to treat DFU mouse wounds in Experimental Example 6 of the present invention. DETAILED DESCRIPTION

[0020] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0021] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any intermediate value within a stated value or stated range and any other stated value or intermediate value within the stated range is also encompassed by the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0022] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0023] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0024] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0025] Example 1

[0026] In Example 1 of the present invention, 3D-MSCs spheroids were cultured using a microgravity suspension culture bioreactor, and the specific steps were as follows: Well-grown MSCs were digested with 0.25% trypsin (containing EDTA) at 37°C for about 1 minute until the cells began to fall off the bottom of the dish when the dish was gently tapped. The digestion was terminated with twice the volume of DMEM complete medium. The cell suspension was collected and centrifuged at 1000 rpm for 3 minutes, after which the supernatant was discarded. The cells were resuspended in complete medium and the cell density was adjusted to 0.6 × 10 by counting. 6 / mL to obtain a cell suspension.

[0027] The cell suspension was inoculated into a microgravity suspension culture bioreactor, cultured at 15 rpm in a 37°C, 5% CO2 incubator. After 48 hours of culture, well-defined, compact 3D-MSC spheroids (3D-MSCs (Scaled-Up)) were obtained.

[0028] In the microgravity suspension culture system, 50 mL of culture volume can produce 5,000 to 10,000 cell spheroids with a diameter of 150-300 μm, which can realize the large-scale production of 3D-MSCs cell spheroids.

[0029] Comparative Example 1 In Comparative Example 1 of the present invention, a conventional inverted spheroid culture method was used to obtain 3D-MSCs spheroids (inverted spheroids), and the specific steps were as follows: Well-grown MSCs were harvested by trypsinization and resuspended in complete DMEM. After counting, the cell density was adjusted to 5,000 cells / 30 μL / drop. The cell suspension was then placed on the lid of a culture dish. PBS buffer was added to the dish to provide humidity. The lid of the dish, filled with the cell suspension droplet, was then carefully flipped over and placed on the dish. The dish was then incubated in a 37°C, 5% CO2 incubator. After 48 to 72 hours, the cells aggregated at the lowest point of the droplet, forming 3D-MSC spheroids (inverted spheres).

[0030] In conventional inverted spheroid culture, a maximum of 100 cell spheroids can be obtained by inverted culture in a 10 cm culture dish. In contrast, in a microgravity suspension culture system, 50 mL of culture volume can produce 5,000 to 10,000 cell spheroids with a diameter of 150 to 300 μm. To produce cell spheroids of this magnitude using a traditional inverted spheroid culture system, at least 5,000 to 10,000 droplets and 50 to 100 10 cm culture dishes are required for culture.

[0031] Comparative Example 2 MSCs in good growth condition were digested with 0.25% trypsin (containing EDTA) at 37°C for 1 minute until the cells fell off the bottom of the dish. The digestion was then terminated with two volumes of DMEM complete medium. The cell suspension was collected and centrifuged at 1000 rpm for 3 minutes, the supernatant was discarded, and the cells were resuspended in complete medium. The cell density was adjusted to 0.6×10 by counting. 6 The cell suspension was then inoculated into magnetic stirring suspension culture flasks, cultured at 15 rpm in a 37°C, 5% CO2 incubator, and observed for different cell densities and spheroid formation after 24 and 48 hours. It was found that MSCs cultured in magnetic stirring flasks had difficulty aggregating to form 3D-MSC spheroids.

[0032] Test Example 1 In Experimental Example 1 of the present invention, the 3D-MSCs spheres prepared in Example 1 and Comparative Example 1 were characterized. The specific steps are as follows: The appearance and survival of 3D-MSCs spheres prepared in Example 1 and Comparative Example 1 were characterized by comparing with the traditional 2D monolayer cultured MSCs single cells (2D-MSCs). Figure 1 、 2 shown.

[0033] Figure 1 It was shown that the microgravity suspension culture system can generate a large number of 3D-MSCs (scaled) spheroids. Figure 2 Calcein / PI staining results showed that MSCs in three culture systems could survive normally.

[0034] Test Example 2 Experimental Example 2 of the present invention is to investigate whether the 3D-MSCs cell spheroids produced by the large-scale spheroid culture system of Example 1 meet the identification criteria of MSCs. Marker identification is performed on the MSCs surface. The specific steps are as follows: The MSCs single cells of the traditional 2D monolayer culture were collected; the cell spheres of Example 1 were taken out after being cultured in the microgravity bioreactor for 48 hours, and the cell spheres were digested into single cells using trypsin, and then 10 5 / 100μL was dispensed into flow cytometry tubes and flow cytometry staining was performed to analyze the expression of MSCs negative complexes and positive markers CD73, CD90, and CD105. The results were as follows Figure 3 shown.

[0035] Figure 3 The results showed that the 3D-MSCs spheroids produced by the large-scale spheroid culture system (3D-MSCs (scaled)) were consistent with the traditional 2D cultured MSCs, did not express the negative markers for MSCs identification, and highly expressed the MSCs positive markers CD73, CD90, and CD105, indicating that the 3D-MSCs spheroids produced in Example 1 of the present invention met the identification standards for MSCs application.

[0036] Test Example 3 Experimental Example 3 of the present invention detected the changes in the size and proliferation capacity of 3D-MSCs spheroids produced by the large-scale spheroid culture system of Example 1. The specific steps are as follows: The safety risks of MSCs in preclinical research and clinical application mainly come from problems such as cell blockage leading to embolism after transplantation and uncertain proliferation in the body. Therefore, it is necessary to further test the proliferation ability and cell particle size of the 3D-MSCs cell spheroids produced by the present invention.

[0037] We collected single cells from traditional 2D monolayer culture of MSCs and single cells from 3D-MSCs spheroids produced by large-scale spheroid culture system, and further measured their cell size. The results are as follows: Figure 4 shown.

[0038] Figure 4The results showed that the average particle size of traditional 2D cultured MSCs was 14μm, while the average particle size of MSCs derived from 3D-MSCs spheroids produced by the large-scale spheroid culture system was significantly reduced to 9.8μm. Flow cytometry was further used to detect the proportion of cells in the G2 / M phase. The proportion of cells in 2D cultured MSCs (2D-MSCs) in G2 / M was 18.8%, while the proportion of cells in 3D-MSCs spheroids (3D-MSCs) in G2 / M was 9.22%, indicating a significant reduction in the proliferation capacity of MSCs spheroids, thereby improving their safety in vivo transplantation. Figure 4 ).

[0039] Test Example 4 The therapeutic effect of MSCs mainly comes from its paracrine capacity, and the main active ingredient is the exosomes secreted by them. Experimental Example 4 of the present invention tested the ability of 3D-MSCs spheroids to secrete exosomes produced by the large-scale spheroid culture system of Example 1. The specific steps are as follows: MSCs were cultured using traditional 2D monolayer and scaled sphere culture systems, with a total cell inoculation volume of 1×10 7 After culturing in normal DMEM complete medium for 48 hours, the culture medium was replaced with exosome-free complete medium for 48 hours, and then the cell culture supernatant was collected. Exosomes were then collected by density gradient centrifugation. Half of the collected exosomes were extracted with protein lysis buffer and the protein content was determined, and the other half was resuspended in PBS and traced with nanoparticles to detect the number and diameter of exosomes. The results are as follows Figure 5 shown.

[0040] pass Figure 5 BCA protein quantification and nanoparticle tracing technology found that under the same cell number, the ability of 3D-MSCs spheroids in the large-scale spheroid culture system to secrete exosomes was significantly higher than that of traditional 2D monolayer cultured MSCs ( Figure 5 ).

[0041] Test Example 5 Experimental Example 5 of the present invention examined the effect of 3D-MSCs spheroids produced by the large-scale spheroid culture system prepared in Example 1 on the function of HUVECs damaged by high glucose. The specific steps were as follows: HUVECs were expanded and cultured in vitro and pretreated with 30 mM glucose for 72 hours to simulate the high-glucose environment of diabetes. Equal numbers of 2D and 3D-MSC spheroids (3D-MSCs) from Example 1 were co-cultured in transwell chambers to investigate their effects on the functions of HUVECs damaged by high glucose, including proliferation, survival, migration, and tube formation. The specific procedures were as follows: HUVEC proliferation capacity: HUVEC cells that were in good growth condition and pretreated with high glucose for 72 hours were collected by trypsin digestion and resuspended in complete medium (30 mM glucose) at a concentration of 5 × 10 4 cells / well were seeded in a 12-well plate. After 48 hours, when the cell confluence reached about 70%, a transwell chamber was added and 5×10 4 A number of 2D-MSCs and 3D-MSCs spheres (3D-MSCs) were co-cultured, EdU-labeled cells were added 48 hours later, EdU staining was performed 12 hours later, and EdU-positive cells were further observed by confocal microscopy to evaluate the proliferation ability of HUVEC.

[0042] HUVEC survival: HUVEC cells that were in good growth condition and pretreated with high glucose for 72 hours were collected by trypsin digestion and resuspended in complete medium (30 mM glucose) at a concentration of 5 × 10 4 cells / well were seeded in a 12-well plate. After 48 hours, when the cell confluence reached about 70%, a transwell chamber was added and 5×10 4 A number of 2D-MSCs and 3D-MSCs spheres were co-cultured, and Calcein / PI staining was performed 48 hours later. Calcein-positive and PI-positive cells were further observed under a confocal microscope to evaluate the survival of HUVEC.

[0043] HUVEC migration ability: HUVEC cells that were in good growth condition and pretreated with high glucose for 72 hours were collected by trypsin digestion and resuspended in complete medium (30 mM glucose) at 6×10 4 cells / well were seeded in a 12-well plate. After 48 hours, when the cell confluence reached about 90%, a 200 μL pipette tip was used to scratch the well chamber. Then, a transwell chamber was added, and 6×10 4 The researchers co-cultured a large number of 2D-MSCs and 3D-MSC spheroids, and after 24 hours, the width of the scratches was observed under a microscope after crystal violet staining to assess HUVEC migration. The results showed that 3D-MSC spheroids significantly promoted the migration of HUVEC under high glucose conditions.

[0044] HUVEC tube-forming ability: HUVEC cells with good growth status were collected by trypsin digestion and resuspended in complete medium (30 mM glucose) at a density of 6 × 10 4 cells / well were seeded in a 24-well plate coated with Matrigel, and then transwell chambers were added, placing 6×10 4A number of 2D-MSCs and 3D-MSCs spheres were co-cultured, and the network formation was observed under a microscope after 24 hours to evaluate the HUVEC tube-forming ability. Figure 6 shown.

[0045] Figure 6 It was shown that 3D-MSCs spheres significantly promoted the proliferation, survival, migration and angiogenesis ability of HUVECs under high glucose conditions.

[0046] Test Example 6 The present invention tested the effect of 3D-MSCs spheroids produced by the large-scale spheroid culture system of Example 1 on the wound healing ability of diabetic mice. The specific steps are as follows: Construction of diabetic mouse model: 8-week-old C57BL / J male mice were intraperitoneally injected with 50 mg / kg streptozotocin for 5 consecutive days. One week later, the fasting blood glucose was greater than 16.7 mM, which was identified as a diabetic model. After 8 weeks of stable high glucose, the diabetic trauma model can be constructed.

[0047] GFP-labeled cell construction: To investigate the retention of MSC 3D spheroids at the wound site, MSCs were infected with lentivirus and labeled with GFP. The specific procedure was as follows: Well-grown MSCs were transfected with GFP-labeled lentivirus at an MOI of 30, with a confluency of approximately 50%. 2 μg / mL polybrene was added as a transfection aid. After 24 hours, the culture medium was replaced with complete culture medium and cultured for 48 hours. Fluorescence microscopy revealed that the percentage of GFP-positive cells approached 100%. The MSCs-GFP cells were then expanded and cultured for subsequent in vivo imaging to observe the retention of 3D MSC spheroids at the wound site of diabetic mice.

[0048] Construction of diabetic trauma mouse model and MSCs transplantation therapy: After 8 weeks of high glucose stabilization, the back hair was shaved after anesthesia. Then, two full dermal wounds were constructed on the back using an 8 mm skin biopsy device. Then, 1×10 6 The amount of cells was transplanted in situ at the wound site with or without GFP-labeled 2D and 3D-MSCs spheres for treatment. In vivo imaging was performed to analyze the cell retention at the wound site in mice transplanted with GFP-labeled cells. Wound healing was recorded by photographing on days 1, 3, 5, 7, 10, and 12. At the end of treatment, on day 12, the mice were euthanized and skin tissue from the wound was collected for pathological staining and immunofluorescence staining to evaluate the healing status. The results are shown in the figure below. Figures 7 to 11 shown.

[0049] Figure 7The results showed that the ability of 3D-MSCs spheres (3D-MSCs) to stay in wounds was significantly higher than that of 2D-MSCs by detecting GFP fluorescence intensity through in vivo imaging; by recording the wound healing status, it was found that the ability of 3D-MSCs spheres to promote diabetic wound healing was significantly higher than that of 2D-MSCs ( Figure 8 ); HE and Masson staining of skin tissue revealed that 3D-MSCs spheres promoted diabetic wound tissue regeneration and collagen production, thereby promoting wound healing ( Figure 9 ); Through skin tissue immunofluorescence staining, it was found that 3D-MSCs spheres can significantly improve the angiogenesis ability of diabetic mouse wounds ( Figure 10 ) and re-epithelialization capacity ( Figure 11 ), thereby accelerating wound healing.

[0050] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for large-scale production of 3D-MSCs spheroids, characterized in that: The steps include: (1) Digest MSCs and adjust the density to obtain a cell suspension; (2) The cell suspension is inoculated into a microgravity suspension culture bioreactor for culture.

2. The method for large-scale production of 3D-MSCs spheroids according to claim 1, characterized in that: In step (1), the density of the cell suspension is 0.4~0.8×10 6 / mL.

3. The method for large-scale production of 3D-MSCs spheroids according to claim 2, characterized in that: The density of the cell suspension was 0.6×10 6 / mL.

4. The method for large-scale production of 3D-MSCs spheroids according to claim 1, characterized in that: In step (2), the rotation speed of the microgravity suspension culture bioreactor is 10-20 rpm, and the culture time is 24-72 hours.

5. The method for large-scale production of 3D-MSCs spheroids according to claim 4, characterized in that: The rotation speed of the microgravity suspension culture bioreactor is 15 rpm, and the culture time is 48 hours.

6. A 3D-MSCs spheroid prepared based on the large-scale production method of 3D-MSCs spheroids according to any one of claims 1 to 5.

7. Use of the 3D-MSCs spheroids according to claim 6 in preparing a drug for treating diabetic foot.

8. Use of the large-scale production method of 3D-MSCs spheroids according to any one of claims 1 to 5 in the preparation of drugs for treating diabetic foot.

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