Mesenchymal stem cell 3D culture method based on synergistic effect of low-melting-point agarose and application

Abstract

The invention belongs to the technical field of cell culture, and particularly relates to a mesenchymal stem cell 3D culture method based on the synergistic effect of low-melting-point agarose and application, the culture method comprises the following steps: preparing a low-melting-point agarose microporous coating on the surface of a culture plate, the pore diameter is 10-50 microns, and the thickness of the coating is 10-50 microns; the method comprises the following steps: inoculating mesenchymal stem cells into a coated culture plate, adding a culture medium containing agarose, and culturing, so that the mesenchymal stem cells aggregate to form 3D mesenchymal stem cell spheres, the culture time is 24 hours, the culture temperature is 37 DEG C, and the carbon dioxide concentration is 5%. By means of the synergistic effect of the low-melting-point agarose coating and low-concentration agarose in the culture medium, efficient balling within 24 hours is achieved, the cell balls are uniform in diameter, compared with the prior art, the problems that balling is slow, the cell balls are large in diameter difference, the survival rate is low, and the survival time is short are solved, and the culture quality and the treatment effect of the mesenchymal stem cells are improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of cell culture, and particularly relates to a 3D culture method and application of mesenchymal stem cells based on the synergistic effect of low-melting-point agarose. Background Art

[0002] At the intersection of biomedicine and tissue engineering, mesenchymal stem cells (MSCs) have become a focus of regenerative medicine research due to their multidirectional differentiation potential, immunomodulatory ability, and paracrine function. They have shown great potential in the treatment of various diseases, especially osteoarthritis (OA). 3D culture technology simulates the three-dimensional microenvironment of cell growth in the body. Compared with traditional 2D culture, it can better maintain the biological characteristics of MSCs, promote cell-cell interactions, and enhance cell functional expression, providing a new direction for improving the therapeutic effects of MSCs. The development of this technology has promoted the transformation of stem cell therapy from basic research to clinical application.

[0003] However, both the MSCs culture technology and its practical application in the treatment of osteoarthritis are currently facing many challenges. In terms of culture technology, under the traditional 2D culture method, MSCs lack a three-dimensional growth environment, which leads to a serious lack of exosome secretion. 6 The amount of secretion per cell is less than 100 μg; the cell migration ability is weak, with a Transwell migration rate of less than 20%; and the survival time after transplantation is extremely short, generally less than 2 weeks. These defects make it difficult for cells to effectively home to the site of injury in the body, greatly reducing the paracrine effect and greatly limiting the therapeutic effect.

[0004] Existing 3D culture technologies also have obvious drawbacks. The hanging drop method is complicated to operate, and the spheroidization speed is slow, requiring 48 to 72 hours. In addition, the size of the cell spheres formed varies greatly, with diameters varying by more than 50%, making it difficult to meet the needs of large-scale production. Hydrogel scaffold technologies, such as sodium alginate and Matrigel, have the risk of residual materials triggering immune responses, and the degradation rate of the scaffold is not synchronized with cell metabolism, resulting in reduced cell activity and a survival rate often below 70%. Other 3D culture methods, such as microcarrier culture, are not only costly and complex, but also unable to effectively regulate the functions of cell spheres. For example, key functions such as exosome secretion are difficult to effectively improve.

[0005] In the field of osteoarthritis treatment, due to the low cell survival rate and weak paracrine effect in existing MSCs therapies, it is difficult to achieve the synergistic effects of cartilage repair and inflammation suppression at the same time, resulting in unstable clinical efficacy and an effectiveness rate that only hovers between 40% and 50%.

[0006] Based on the above problems, the present application proposes a 3D culture method and application of mesenchymal stem cells based on the synergistic effect of low-melting-point agarose to improve the above problems. Summary of the Invention

[0007] The present invention aims to provide a 3D culture method for mesenchymal stem cells based on the synergistic effect of low-melting-point agarose. Through the synergistic effect of the low-melting-point agarose coating and the low-concentration agarose in the culture medium, efficient spheroidization and uniform cell spheroid diameter are achieved within 24 hours. Compared with the existing technology, this method solves the problems of slow spheroidization, large differences in cell spheroid diameter, low survival rate, and short survival time, thereby improving the culture quality and therapeutic effect of mesenchymal stem cells.

[0008] The technical solutions adopted by the present invention are as follows: A 3D culture method for mesenchymal stem cells based on the synergistic effect of low-melting-point agarose comprises the following steps: St1: preparing a low-melting-point agarose coating, and preparing a low-melting-point agarose microporous coating on the surface of the culture plate, wherein the low-melting-point agarose microporous coating has a microporous structure, a pore diameter of 10 to 50 μm, and a coating thickness of 10 to 50 μm; St2: Inoculation of MSCs. Mesenchymal stem cells are seeded into coated culture plates and cultured in a medium containing 0.01% to 0.05% agarose. Mesenchymal stem cells spontaneously aggregate to form 3D mesenchymal stem cell spheres. The culture time is 24 hours at a temperature of 37°C in a 5% CO2 atmosphere, i.e., the concentration of carbon dioxide (CO2) in the environment is 5%. Wherein, the melting point of the low melting point agarose is ≤65°C.

[0009] In a preferred embodiment, in St1, preparing a low melting point agarose microporous coating on the surface of the culture plate comprises the following steps: Step 11: Add the dissolved RPMI1640 basal medium to the low melting point agarose solution and mix well to obtain a low melting point agarose mixture; St12: Sterilize the low-melting-point agarose mixture by high-pressure steam sterilization equipment at 120°C for 20 minutes. St13: Spread the sterilized low-melting-point agarose mixture evenly on the surface of the culture plate and cool and solidify at 4-25°C to obtain a low-melting-point agarose microporous coating. Wherein, the concentration of low melting point agarose in the low melting point agarose solution is 1% to 2%.

[0010] In a preferred embodiment, in St2, the agarose concentration in the culture medium is 0.01%.

[0011] In a preferred embodiment, in St2, the spheroidization rate of 3D mesenchymal stem cell spheres is ≥90%, and the coefficient of variation (CV) of diameter distribution is <15%.

[0012] A 3D mesenchymal stem cell sphere is prepared by any of the above-mentioned mesenchymal stem cell 3D culture methods based on the synergistic effect of low-melting-point agarose, wherein the diameter of the 3D mesenchymal stem cell sphere is 50 to 200 μm.

[0013] In a preferred embodiment, the survival time of the 3D mesenchymal stem cell spheres after transplantation is ≥ 4 weeks, and the exosome secretion amount is ≥ 450 μg / 10 6 cells, the Transwell migration rate was ≥60%, and the ratio of the anti-apoptotic gene Bcl-2 / Bax increased by more than 3 times compared with that of 2D cultured cells.

[0014] In a preferred embodiment, the 3D mesenchymal stem cell spheres form a hypoxic core microenvironment by activating the HIF-1α signaling pathway, promoting the enrichment of cartilage repair factors such as TGF-β and miRNA-140 in exosomes. The content of TGF-β in the exosomes is ≥800 pg / mL, and the relative expression of miRNA-140 is ≥3.0 (with 2D culture as the control).

[0015] In a preferred embodiment, the 3D mesenchymal stem cell spheres promote chondrocyte proliferation through the exosome-mediated PI3K / Akt signaling pathway, with the proliferation rate increased by more than 40% compared with the control group, and reduce the levels of inflammatory factors such as TNF-α and IL-6 in the joint cavity by ≥60%.

[0016] In a preferred embodiment, the 3D mesenchymal stem cell spheres described in the present application are used in the preparation of a drug for treating osteoarthritis.

[0017] In a preferred embodiment, the osteoarthritis treatment drug is administered by intra-articular injection or intravenous injection, and the cell spheres are administered at a concentration of 1*10 6 ~5*10 6 cells / mL, and the single dose is 1*10 6 ~1*10 7 cells / mouse (based on mouse model).

[0018] The technical effects achieved by the present invention are: The present invention achieves efficient spheroidization within 24 hours and uniform cell spheroid diameter through the synergistic effect of a low-melting-point agarose coating and a low-concentration agarose in the culture medium. Compared with the existing technology, it solves the problems of slow spheroidization, large differences in cell spheroid diameter, low survival rate, and short survival time. At the same time, it also improves the secretion amount and migration rate of exosomes and their survival time in the body. When used in the treatment of osteoarthritis, it can effectively promote cartilage repair and inhibit inflammation, comprehensively solving the shortcomings of the existing technology and greatly improving the culture quality and treatment effect of mesenchymal stem cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a schematic diagram of cells under a microscope of the 3D mesenchymal stem cell sphere in Test Example 1 of the present invention; Figure 2 Schematic diagram of bisection of pathological tissue in Test Example 3 of the present invention; Figure 3 This is an electron micrograph of the therapeutic effect of the pathological tissue in Test Example 3 of the present invention; Figure 4 Schematic diagram of the treatment effects of three pathological tissues in the test example of the present invention. DETAILED DESCRIPTION

[0020] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific embodiments of the present invention are described in detail below with reference to the accompanying drawings.

[0021] In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention may also be implemented in other ways different from those described herein. Those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0022] Secondly, the term "one embodiment" or "embodiment" herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in a preferred embodiment" appearing in various places throughout this specification does not necessarily refer to the same embodiment, nor does it constitute a separate or selective embodiment that is mutually exclusive of other embodiments.

[0023] Furthermore, the present invention is described in detail with reference to schematic diagrams. For ease of illustration, when describing the embodiments of the present invention, cross-sectional views illustrating device structures may be partially enlarged and not to scale. Furthermore, the schematic diagrams are merely illustrative and should not limit the scope of protection of the present invention. Furthermore, in actual production, the three-dimensional dimensions of length, width, and depth should be included.

[0024] Preparation Example The third generation human umbilical cord MSCs were digested with trypsin and then 1*10 6cells / mL, and the cell suspension was inoculated on the coated culture dish (5×10 5 cells / dish), and cultured in a 37°C, 5% CO2 incubator. After 24 hours, the cell sphere formation was observed. The medium was changed every 2 days, and the culture period was 7 days to obtain mesenchymal stem cells.

[0025] Example A 1% low-melting-point agarose solution was melted in an 80°C water bath. The dissolved RPMI1640 basal medium was added to the 1% low-melting-point agarose solution and mixed thoroughly to obtain a low-melting-point agarose mixture. The low-melting-point agarose mixture was sterilized by high-pressure steam sterilization at 120°C for 20 min. 10 ml of the sterilized low-melting-point agarose mixture was evenly spread on the surface of a 15-cm culture plate and cooled and solidified at 4°C to obtain a low-melting-point agarose microporous coating with a microporous structure (pore size of 10-50 μm and coating thickness of approximately 20 μm). Mesenchymal stem cells were seeded on the coated culture plate, and medium containing 0.01% agarose was added. The cells were cultured in an incubator at 37°C with a carbon dioxide concentration of 5% for 24 h. The mesenchymal stem cells spontaneously aggregated to form 3D mesenchymal stem cell spheres.

[0026] Among them, the low melting point agarose is preferably Sigma-A9414 low melting point agarose (melting point ≤ 65 ° C, gel strength ≥ 200g / cm 2 ).

[0027] It's important to note that during culture, the low-melting-point agarose coating provides mechanical support and chemical signals for cells. Its microporous structure, similar to collagen in tissue, mimics the in vivo physical environment, facilitates cell attachment, and enhances exosome secretion and inflammatory cytokine regulation. Furthermore, agarose mimics naturally occurring polysaccharides, modulating inflammatory responses and cell secretion. Furthermore, low-melting-point agarose may activate the YAP / TAZ pathway through physical or chemical signals, promoting cell growth and survival, further modulating inflammation and supporting cell growth. In the treatment of osteoarthritis, the hypoxic core formed within 3D-cultured cell spheroids mimics the in vivo stem cell niche, activating the HIF-1α signaling pathway and enhancing cell resistance to apoptosis. Exosomes secreted by spheroids are rich in cartilage repair factors such as TGF-β and miRNA-140. These factors promote chondrocyte proliferation through the PI3K / Akt pathway while reducing the levels of inflammatory factors in the joint cavity, achieving dual cartilage repair and anti-inflammatory benefits, effectively treating osteoarthritis.

[0028] Test Case It should be noted that in the following tests, 3D mesenchymal stem cell spheres prepared according to the operating steps of the examples in this application without special grouping instructions were recorded as the experimental group, and mesenchymal stem cell spheres prepared according to other existing technologies were recorded as the control group.

[0029] Test Example 1: 3D ball formation rate and uniformity test 3D mesenchymal stem cell spheres were prepared according to the steps in Example 1 and the hanging drop method. The number of cell spheres with diameters of 50 to 200 μm was counted under a microscope at 24 and 48 hours, and the sphere formation rate was calculated. The diameters of 100 cell spheres were then measured, and the number of cell spheres with diameters <50 μm and >μm was counted. The coefficient of variation (CV) was calculated, and the test results were recorded. The test results are shown in Table 1.

[0030] The calculation formula for spheroidization rate is: spheroidization rate = number of cell spheres with target diameter / total number of cell spheres * 100%; The coefficient of variation was calculated as follows: coefficient of variation = number of spheres with non-target diameters / 100*100%.

[0031] Furthermore, the method of preparing 3D mesenchymal stem cell spheres by the hanging drop method is an existing mature technology. The specific operation steps are referred to the existing technology and are not further described here.

[0032] Table 1: Experimental group control group 24h ball formation rate 92% 60% 48h ball formation rate 95% 75% Cell spheroid diameter (μm) 50～200 50～300 Coefficient of variation 12% 45% Please refer to Table 1 and Figure 1 ,in, Figure 1 A and B are enlarged schematic diagrams of 3D mesenchymal stem cell spheres in the experimental group, and C and D are enlarged schematic diagrams of 3D mesenchymal stem cell spheres in the control group. Figure 1 It can be seen that the preparation method in this example achieves efficient spheroidization within 24 hours, and the cell spheroids are uniform in size. Compared with the preparation method using the hanging drop method, the spheroidization efficiency is greatly improved, and the uniformity of the 3D mesenchymal stem cell spheroids is ensured.

[0033] Test Example 2: Exosome Secretion Detection The third generation human umbilical cord mesenchymal stem cells (MSCs) were cultured using the culture method described in the example (coated culture plate with 0.01% agarose medium) and the traditional 2D culture method (ordinary culture plate with conventional medium). The cell seeding density was 5×10 5Cells were plated in 6-well plates and cultured in a 37°C, 5% CO2 incubator for 7 days. The supernatant was collected and exosomes were extracted by ultracentrifugation (4°C, 300×g for 10 minutes to remove cell debris, 10,000×g for 30 minutes to remove apoptotic bodies, and 100,000×g for 70 minutes to enrich exosomes. The cells were resuspended in PBS and ultracentrifuged twice repeatedly). The total protein concentration of the exosomes was determined using a BCA protein quantification kit. The expression of exosome surface markers CD63 and CD81 was detected using an ELISA kit, and the content of the cartilage repair factor TGF-β in the exosomes was quantitatively detected. The relative expression of miRNA-140 in exosomes was detected by qRT-PCR. Three replicates were set for each sample, and the experiment was repeated three times. The test data were recorded. The test data are shown in Table 2.

[0034] Table 2: Experimental group control group <![CDATA[Exosome production (μg / 10 6 cells)]]> 580±25 160±18 CD63 positive rate (%) 98.5±0.8 72.3±3.5 CD81 positive rate (%) 97.3±1.2 68.9±2.8 TGF-β content (pg / mL) 880±40 220±30 Relative expression of miRNA-140 3.5±0.2 1.0±0.1 As can be seen from Table 2, compared with the traditional 2D culture method, the culture method in this example increased the exosome yield by 3.6 times, significantly increased the positive rate of exosome surface markers, increased the content of the cartilage repair factor TGF-β by 4 times, and increased the expression of miRNA-140 by 3.5 times. The culture method in this example significantly enhanced the exosome secretion capacity and functional factor enrichment effect of MSCs through synergistic effects, providing a highly active source of exosomes for the treatment of osteoarthritis.

[0035] Test case 3: Verification of in vivo survival time and therapeutic effect Sixty healthy female ICR mice weighing 20-25 g were randomly divided into three groups: A (3D modeling treatment group), B (2D modeling treatment group), and C (normal modeling group), with 20 mice in each group. 30 mg of type II collagenase at a concentration of 0.4 mg / mL was injected into the right hind knee joint of the mice to establish an osteoarthritis model. In group A, the 3D mesenchymal stem cell spheres of the present invention labeled with PKH26 red fluorescent dye were injected into the joint cavity at a cell concentration of 1*10 6 cells / mouse; Group B was injected with traditional 2D cultured MSCs labeled with the same fluorescent dye, with a cell concentration of 1*10 6Cells / mouse; Group C was injected with an equal volume of 0.9% saline as a control group. Mice were imaged using an imaging system on days 7, 14, 28, and 56 after injection to observe the distribution and survival of fluorescently labeled cells. Changes in cell fluorescence intensity over time were recorded to determine cell survival. On day 56 after surgery, all mice were assessed using the WOMAC score, which assesses pain (e.g., degree of lameness when walking, response to pain stimuli), stiffness (limited joint movement, stiffness after waking up or resting), and joint function (ability to perform daily activities such as climbing a cage, standing, and walking). Following scoring, knee joint tissue was harvested and cut into 5 μm-thick sections. The sections were stained with hematoxylin and eosin (HE), and the morphology and structure of the sections were observed under a microscope to assess the extent of cartilage repair and calculate the cartilage repair rate. The test data are shown in Table 3.

[0036] The calculation method of cartilage repair rate is: cartilage repair rate = cartilage repair area / total cartilage damage area × 100%.

[0037] Table 2: Group A Group B Group C Survival time in the body >4 weeks: The fluorescence signal is obvious at 4 weeks and weak at 8 weeks <2 weeks, weak fluorescence signal after 2 weeks - WOMAC score 85±5 50±8 30±7 Cartilage repair rate 75%±5%, cartilage tissue defects are significantly repaired, extracellular matrix increases, and chondrocyte morphology tends to be normal 30%±6%, cartilage has been repaired to some extent, but there are still many defective areas 10%±3%, minimal cartilage repair and large defect area pass Figure 2 It can be seen that 3D cell therapy can effectively reduce the degree of cartilage damage in the knee joint of mice and inhibit the formation of osteophytes. Compared with 2D cell therapy, the effect is better. The 3D culture environment can better simulate the cell growth microenvironment in the body, maintain cell activity and function, promote cartilage repair, and reduce osteophyte formation. Figure 4 It can be seen that 3D cell therapy can effectively repair cartilage tissue defects, while 2D cell therapy still has more defect areas. Figures 2 to 4 It can be seen that the survival time of the 3D mesenchymal stem cell spheres prepared in the examples in mice is significantly longer than that of traditional 2D cultured MSCs, and they can exert their effects more sustainably. In terms of therapeutic effect, the improvement rate of the WOMAC score reaches 80% to 85%, and the cartilage repair effect is significantly better than that of traditional methods. In summary, the 3D mesenchymal stem cell spheres prepared in the examples have good application prospects in the treatment of osteoarthritis, and can effectively repair damaged cartilage tissue, improve joint function, and reduce osteoarthritis symptoms.

[0038] Test Example 4: Cell Migration Ability and Signaling Pathway Analysis Mesenchymal stem cells in the logarithmic growth phase were used to prepare 3D mesenchymal stem cell spheres according to the steps in Example 1 and the traditional 2D method. In the Transwell experiment, 1*10 5Cells cultured in different ways were cultured, and culture medium containing 10% FBS was added to the lower chamber as a chemotactic factor. The Transwell chamber was placed in an incubator at 37°C and a carbon dioxide concentration of 5% and incubated for 24 hours. After the incubation, the chamber was removed, the culture medium in the upper chamber was discarded, and the cells were gently rinsed 3 times with PBS to remove the non-migrated cells. The cells on the bottom surface of the upper chamber were carefully wiped off with a cotton swab; the chamber was immersed in 4% paraformaldehyde for 20 minutes, rinsed 3 times with PBS, and then stained with 0.1% crystal violet for 15 minutes. After the staining, it was slowly rinsed again with PBS 3 times until the rinsing fluid became colorless. Randomly select under a microscope Five fields of view were taken, and the number of cells that migrated to the bottom of the lower chamber was counted to calculate the number of migrating cells. At the same time, the two groups of cells were collected, and total RNA was extracted using the Trizol method. The RNA was reverse transcribed into cDNA using a reverse transcription kit. Then, qPCR technology was used to detect the mRNA expression levels of migration-related genes MMP2 and CXCR4 using GAPDH as an internal reference gene. In addition, Western blot was used to detect the expression of YAP / TAZ pathway-related proteins (such as YAP, p-YAP, etc.) using β-actin as an internal reference. The pathway activation status was analyzed, and the test results were recorded. The results are shown in Table 4.

[0039] Table 4: Experimental group control group Number of migrated cells (per field of view) 650±40 280±30 MMP2 expression (relative value) 2.8±0.4 1.0±0.1 CXCR4 expression (relative value) 2.5±0.3 1.0±0.1 YAP phosphorylation level (%) 35±3 15±3 As can be seen from Table 4, the 3D mesenchymal stem cell spheres prepared in the examples have a strong migration rate, the expression of migration-related genes is significantly upregulated, and the YAP / TAZ pathway is activated, proving that it optimizes cell function through mechanical signaling pathways.

[0040] In summary, this application achieves a 3D mesenchymal stem cell sphere formation rate of greater than 90% within 24 hours through the synergistic effect of the low-melting-point agarose microporous coating on the culture plate surface and the low-concentration agarose in the culture medium. It also has the advantages of high exosome secretion and long survival time. This culture method significantly enhances the migration ability and anti-apoptosis properties of 3D mesenchymal stem cell spheres. The exosomes secreted by them promote cartilage repair through the PI3K / Akt pathway, with a clinical efficacy of 80% to 85%, providing an innovative solution for the treatment of osteoarthritis. The foregoing is merely a preferred embodiment of the present invention. It should be noted that those skilled in the art may make various improvements and modifications without departing from the principles of the present invention, and such improvements and modifications are also within the scope of protection of the present invention. Structures, devices, and operating methods not specifically described or explained herein shall, unless otherwise specified or limited, be implemented in accordance with conventional means in the art.

Claims

1. A 3D culture method for mesenchymal stem cells based on the synergistic effect of low-melting-point agarose, characterized by: The following steps are involved: St1: preparing a low-melting-point agarose microporous coating on the surface of the culture plate, wherein the low-melting-point agarose microporous coating has a microporous structure, a pore diameter of 10 to 50 μm, and a coating thickness of 10 to 50 μm; St2: Mesenchymal stem cells were seeded into the coated culture plate and cultured in agarose-containing medium. The mesenchymal stem cells aggregated to form 3D mesenchymal stem cell spheres. The culture time was 24 hours at a temperature of 37°C and a carbon dioxide concentration of 5%. Wherein, the melting point of the low melting point agarose is ≤65°C.

2. The method for 3D culture of mesenchymal stem cells based on the synergistic effect of low-melting-point agarose according to claim 1, characterized in that: In the step 1, a low melting point agarose microporous coating is prepared on the surface of the culture plate, comprising the following steps: Step 11: Add the dissolved RPMI1640 basal medium to the low melting point agarose solution and mix well to obtain a low melting point agarose mixture; St12: Sterilize the low-melting-point agarose mixture by high-pressure steam sterilization equipment at 120°C for 20 minutes. St13: Spread the sterilized low-melting-point agarose mixture evenly on the surface of the culture plate and cool and solidify at 4-25°C to obtain a low-melting-point agarose microporous coating. Wherein, the concentration of the low melting point agarose solution is 1% to 2%.

3. The method for 3D culture of mesenchymal stem cells based on the synergistic effect of low-melting-point agarose according to claim 1, characterized in that: In St2, the agarose concentration in the culture medium is 0.01%.

4. The method for 3D culture of mesenchymal stem cells based on the synergistic effect of low-melting-point agarose according to claim 1, characterized in that: In the St2, the spheroidization rate of 3D mesenchymal stem cell spheres is ≥90%, and the coefficient of variation of diameter distribution is <15%.

5. A 3D mesenchymal stem cell sphere prepared by the mesenchymal stem cell 3D culture method based on the synergistic effect of low-melting-point agarose according to any one of claims 1 to 4, characterized in that: The diameter of the 3D mesenchymal stem cell sphere is 50 to 200 μm.

6. Use of the 3D mesenchymal stem cell sphere according to claim 5 in the preparation of a drug for treating osteoarthritis.

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