A culture medium for promoting the formation of three-dimensional aggregates of canine mesenchymal stem cells and a three-dimensional dynamic expansion method

Abstract

The present invention belongs to the technical field of three-dimensional culture of stem cells, and specifically relates to a culture medium and a three-dimensional dynamic expansion method for promoting the formation of three-dimensional aggregates of canine mesenchymal stem cells, wherein the culture medium comprises a 2D culture medium, polyvinyl alcohol and a ROCK inhibitor Y27632, wherein the mass concentration of the polyvinyl alcohol is 0.01%-0.05%, and the concentration of Y27632 is 10 μM. No scaffold material is required, and the present invention uses PVA and ROCK inhibitor Y27632, and by means of AggreWell plate centrifugation, the effect of MSC single cells forming 3D aggregates or 3D spheres is improved, and dynamic system expansion is completed; it has been verified that the cell characteristics and functions after MSC 3D aggregate culture are better than those of MSC cultured in 2D planes, and animal experiments show that 3D MSC cells have the ability to promote the healing of diabetic refractory wounds.

Description

Technical Field

[0001] The invention relates to a culture medium for promoting the formation of three-dimensional aggregates of canine mesenchymal stem cells independent of scaffold materials and a three-dimensional dynamic amplification method, belonging to the technical field of three-dimensional culture of stem cells. Background Art

[0002] Mesenchymal stem cells (MSC) play multiple roles in cell and tissue repair therapy, and are considered to be important clinical cell therapy drugs due to their good accessibility and versatility. However, the expansion of conventional mesenchymal stem cells in a two-dimensional planar system is usually accompanied by problems such as decreased function and easy cell aging. Studies have reported that the therapeutic effect of mesenchymal cells can be optimized when the in vitro expansion environment is closer to physiological conditions (such as hypoxia, three-dimensional environment and dynamic culture). When cultured as three-dimensional cell aggregates (3D aggregates), MSCs show enhanced angiogenesis, anti-inflammatory and immunomodulatory effects, improved stemness and survival rate after transplantation, and culturing under dynamic conditions can increase their viability, proliferation and paracrine effects.

[0003] How to efficiently form mesenchymal stem cell aggregates is an important issue in the current 3D dynamic culture of MSC. The hanging drop method is laborious and time-consuming to spheroidize multiple mesenchymal stem cells, and the efficiency of single cell aggregation is not good; the low adsorption U-shaped hole centrifugation method also faces the problem of low aggregation efficiency. Nowadays, scaffold materials such as 3D microcarriers are generally used for large-scale preparation of MSC. MSC is attached to the carrier material to achieve dynamic system MSC expansion, and certain results have been achieved. However, in order to avoid adverse reactions caused by microcarriers entering the human body, high-purity MSC products without microcarriers need to be used in clinical applications. If microcarriers are not used, only relying on cell autonomous aggregation, when the aggregates are relatively loose, it is difficult to carry out subsequent dynamic suspension culture expansion, especially the stirring dynamic culture in the rotary flask. The loose cell aggregates cannot withstand shear force and cannot effectively maintain the 3D cell cluster morphology. Therefore, it is a technical problem that technicians in this field urgently need to solve to develop scaffold materials that do not rely on microcarriers, improve cell aggregation ability, efficiently form relatively dense 3D aggregates, and be compatible with orbital shakers or rotary flasks. Dynamic suspension culture system 3D expansion is compatible with orbital shakers or rotary flasks. Summary of the invention

[0004] The present invention aims to solve the problem that canine mesenchymal stem cells cannot effectively form 3D aggregates only by autonomous aggregation, are difficult to culture, and need to add scaffold materials such as microcarriers, and provide a culture medium and a three-dimensional dynamic amplification method for promoting the formation of three-dimensional aggregates of canine mesenchymal stem cells.

[0005] The technical solution of the present invention to solve the above technical problems is as follows:

[0006] One of the purposes of the present invention is to provide a culture medium for promoting the formation of three-dimensional aggregates of canine mesenchymal stem cells, comprising a 2D culture medium, polyvinyl alcohol and a ROCK inhibitor Y27632, wherein the mass concentration of the polyvinyl alcohol is 0.01%-0.05%, and the concentration of Y27632 is 10 μM.

[0007] Furthermore, the molecular weight of the polyvinyl alcohol is 30,000-70,000.

[0008] Furthermore, the composition of the 2D culture medium is basal culture medium + 10% FBS + 5 ng / mL bFGF.

[0009] Furthermore, the basal culture medium is any one of low-glucose DMEM culture medium, DMEM high-glucose culture medium, Alpha-MEM culture medium, F12 culture medium, DMEM / F12 culture medium, RPMI1640 culture medium or IMDM culture medium.

[0010] The beneficial effects of the culture medium for promoting the formation of three-dimensional aggregates of canine mesenchymal stem cells of the present invention are:

[0011] The core additives used in the culture medium of the present invention in the formation stage of 3D aggregates of mesenchymal stem cells are polyvinyl alcohol (PVA) and ROCK inhibitor Y27632. The main function of adding Y27632 is to inhibit single cell apoptosis. Adding PVA simulates extracellular matrix components. PVA material has the characteristic of cell adhesion. Adding PVA of appropriate concentration to the cell suspension can promote the connection between multiple cells and further aggregate the cells. The mass concentration of polyvinyl alcohol used in the present invention is 0.01%-0.05%. If the PVA concentration is too high, the viscosity and osmotic pressure of the culture medium will increase, which is not conducive to the survival of cell clusters. If the PVA concentration is too low, the cell aggregation effect will be poor. Within the mass concentration range of polyvinyl alcohol specified in the present invention, it can promote the efficient sphering of canine mesenchymal stem cells without damaging cell activity.

[0012] The second object of the present invention is to provide a method for three-dimensional dynamic expansion of canine mesenchymal stem cells, which uses the culture medium that promotes the formation of three-dimensional aggregates of canine mesenchymal stem cells as described above for expansion, thereby achieving efficient preparation of 3D cell aggregates of canine mesenchymal stem cells.

[0013] Furthermore, the formation of three-dimensional aggregates of canine mesenchymal stem cells was completed in the AggreWell plate.

[0014] After MSC single cells aggregate into spheres, 3D MSC cell clusters will continue to expand in a dynamic suspension culture system (such as an orbital shaker low attachment plate or a rotary shake flask reactor).

[0015] Furthermore, the three-dimensional dynamic expansion method of canine mesenchymal stem cells specifically comprises the following steps:

[0016] (1) Preparation of culture medium to promote the formation of three-dimensional aggregates of canine mesenchymal stem cells and pretreatment of AggreWell plates:

[0017] Preparation of a culture medium for promoting the formation of three-dimensional aggregates of canine mesenchymal stem cells: adding polyvinyl alcohol and ROCK inhibitor Y27632 to a 2D culture medium at the same time, wherein the composition of the 2D culture medium is a basal culture medium + 10% FBS + 5 ng / mL bFGF, and the basal culture medium is any one of a low-glucose DMEM culture medium, a DMEM high-glucose culture medium, an Alpha-MEM culture medium, an F12 culture medium, a DMEM / F12 culture medium, an RPMI1640 culture medium or an IMDM culture medium;

[0018] AggreWell plate pretreatment: add anti-adsorption rinse solution into AggreWell and centrifuge to remove bubbles in the microwells;

[0019] (2) Single-cell digestion: Canine mesenchymal stem cells with a confluence of 85% were taken for passage, washed once with DMEM / F12 medium, digested with Accutase for 4-5 minutes, and terminated with 2D medium;

[0020] (3) After the cells in step (2) are centrifuged, the cells are resuspended in the culture medium for promoting the formation of three-dimensional aggregates of canine mesenchymal stem cells prepared in step (1), and the cells are counted;

[0021] (4) Add the cells to the AggreWell plate, pipette each well to mix, and centrifuge immediately;

[0022] (5) After centrifugation in step (4), place the cells in a cell culture incubator at 37°C and 5% CO2. After 40-50 hours, observe the cell sphering.

[0023] (6) After the 3D cell aggregates are formed, transfer the cell clusters to the well plate of the orbital shaker at a speed of 90-110 rpm and culture conditions: 37°C, 5% CO2;

[0024] (7) After culturing for 4-7 days, observe the changes in the size of the cell clusters. Change the MSC cell medium every two days. When the cell size reaches 300 μm, use Accutase to digest the cell clusters into single cells. Repeat the above steps (3)-(6). After inoculation into a new AggreWell plate to form a sphere, expand the culture in an orbital shaker system plate. If the cell amount is sufficient, transfer the 3D cell clusters to a rotary shake flask for culture.

[0025] (8) Rotary flask suspension culture: After the 3D cell aggregates in the AggreWell form spheres, transfer the cell clusters to a 500 mL rotary flask at a speed of 65-75 rpm. Culture conditions: 37°C, 5% CO2. Subculture every four days. Use Accutase to digest the cell clusters into single cells and perform suspension culture in the same manner as above.

[0026] Furthermore, in step (2), 2D culture is performed before single-cell digestion of canine mesenchymal stem cells.

[0027] Furthermore, the 2D culture in step (2) specifically includes:

[0028] ①2D culture medium preparation: basal culture medium + 10% FBS + 5 ng / mL bFGF; the basal culture medium is low-glucose DMEM medium;

[0029] ② Cell thawing: Take out the frozen canine mesenchymal stem cells and thaw them in a 37±2℃ water bath;

[0030] ③Cell centrifugation: Pipette the 2D culture medium from step ① above into a centrifuge tube, add the thawed cell suspension, centrifuge, discard the supernatant, and retain the cell pellet;

[0031] ④ Resuspend and count: Add the 2D culture medium in step ① above to the centrifuge tube to resuspend the cell pellet; use a pipette to pipette the cell suspension to mix it thoroughly, and perform cell counting and viability detection;

[0032] ⑤Cell inoculation: Inoculate the cell suspension into a T75 culture flask, add the 2D culture medium from step ① above to a total volume of 15 mL, and place the culture flask in a CO2 incubator;

[0033] ⑥ Replace the medium for 2D cell culture: Replace the medium of the cells daily by adding 15 mL of fresh 2D culture medium from step ① above until the cells proliferate to 85% confluence.

[0034] Furthermore, in step (4), the AggreWell plate has 24 wells, and 1.5 x 10 6 A single cell.

[0035] The beneficial effects of the three-dimensional dynamic expansion method of canine mesenchymal stem cells of the present invention are: using polyvinyl alcohol (PVA) and ROCK inhibitor Y27632 (Y), with the help of AggreWell plate centrifugation, the effect of MSC single cells forming 3D aggregates or 3D spheres is improved, and dynamic system expansion is successfully completed; for the first time, it is verified that the cell characteristics and functions after MSC 3D aggregate culture are superior to 2D MSC, and animal experiments have verified the ability of 3D MSC cells to promote the healing of refractory diabetic wounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 The effects of Y27632 alone and Y27632+PVA on the formation of MSC cell aggregates;

[0037] Figure 2 The changes of MSC 3D aggregates on different days;

[0038] Figure 3 To investigate the effect of adding PVA alone on the formation of MSC 3D aggregates;

[0039] Figure 4 The morphology of the MSC aggregates formed under different PVA, Y concentrations and cell inoculation amounts after being transferred to the orbital shaker dynamic culture system;

[0040] Figure 5 Results of flow cytometric detection of MSC markers for 3D cell aggregates;

[0041] Figure 6 The results of the three-lineage differentiation experiment and differentiation ability test for 3D cell aggregates;

[0042] Figure 7 The experimental results of 3D suspension culture of MSCs inhibiting lymphocyte proliferation;

[0043] Figure 8 The experimental results of 3D suspension culture of MSC on the proliferation of epidermal cells and fibroblasts are shown;

[0044] Fig. 9 The process of establishing a difficult-to-heal wound model for diabetic mice and the wound healing status;

[0045] Fig.10 The results show the effect of 3D MSC on the wound healing rate of diabetic foot ulcer model;

[0046] Fig.11 The results show the effect of 3D MSC on the wound healing rate of animals at D0 / D7 after administration;

[0047] Fig.12 The results show the effect of adding Y27632 and PVA to different basal culture media on the formation of MSC aggregates. DETAILED DESCRIPTION

[0048] The principles and features of the present invention are described below in conjunction with the accompanying drawings. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0049] 1. 2D planar system culture of canine mesenchymal stem cells:

[0050] ① Preparation of MSC 2D culture medium: basal culture medium + 10% FBS + 5 ng / mL bFGF. The culture medium used is marked as "culture medium 1";

[0051] In the 2D culture medium used in this embodiment, the basic culture medium is a low-glucose DMEM culture medium, the full name of the DMEM culture medium is Dulbecco's Modified Eagle's Medium, and the culture medium used in this embodiment is Thermo Fisher, catalog number 11885084; FBS is fetal bovine serum; bFGF is basic fibroblast growth factor;

[0052] ② Cell thawing: Take out the frozen canine mesenchymal stem cells from the liquid nitrogen tank, quickly transfer the cryopreserved tubes to a preheated water bath at 37±2℃, shake the frozen cells continuously to thaw them quickly, and transfer the cell cryopreserved tubes to a biosafety cabinet;

[0053] In the biosafety cabinet, transfer 9.0 mL of culture medium to a 15 mL centrifuge tube, take out the thawed cell suspension with a pipette and add it to the centrifuge tube, cover the centrifuge tube, and move out of the biosafety cabinet for subsequent centrifugation operations;

[0054] ③Cell centrifugation: fully mix the cell suspension in the centrifuge tube, tighten the centrifuge tube cap, transfer the centrifuge tube to the centrifuge, centrifuge at 300g for 5 minutes, discard the supernatant, and retain the cell pellet;

[0055] ④ Resuspend and count: Add 1 mL of culture medium 1 to each centrifuge tube to resuspend the cell pellet; use a 10 mL pipette to gently pipette the cell suspension to mix it thoroughly, and then perform cell counting and viability testing;

[0056] ⑤Cell seeding: 2.0x10 6 cells / cm2, inoculate the cell suspension into a T75 culture flask, add medium I to a total volume of 15 mL, and place the culture flask in a CO2 incubator at 37.0°C and a CO2 concentration of 5.0%;

[0057] ⑥2D cell culture medium replacement: Cell medium is replaced daily with 15 mL of fresh culture medium. When the cells proliferate to about 85% confluence, they are digested. Depending on the cell condition, you can choose to continue 2D system subculture (see step ⑦), or prepare 3D cell aggregates for subsequent 3D cell culture (see part 2);

[0058] In step ⑥, after cell recovery, the cells are subcultured for 3 generations in a 2D system to restore the cell state and expand the cell number. If the cell state is not good, the 3D spheroidization efficiency will decrease. After the P3 generation, 3D spheroidization and dynamic suspension culture are performed to continue the expansion of canine mesenchymal stem cells.

[0059] ⑦2D system passaging operation: cells were washed once with DMEM / F12 medium (Gibco, catalog number: 21331-020), and digested with Accutase cell digestion solution (STEMCELL, catalog number: 07922) at 37°C for 4-5 minutes, and terminated by adding medium 1. After centrifugation at 200g for 4 minutes, the cells were resuspended with medium 1, the cells were counted, and the cells were passaged at a ratio of 1:4. After cell inoculation, the culture flask was placed in a CO2 incubator at a temperature of 37.0°C and a CO2 concentration of 5.0%.

[0060] 2. Preparation of 3D cell aggregates using AggreWell plates:

[0061] (1) Preparation of 3D dynamic expansion medium: Medium 1 (low-glucose DMEM + 10% FBS + 5 ng / mL bFGF) + PVA (0.05% concentration, sigma, catalog number P8136-250G) + Y27632 (10 μM), freshly prepared;

[0062] AggreWell plate pretreatment: 500 μL / well Anti-Adherence Rinsing Solution (StemCell) was added to the AggreWell plate and centrifuged at 1300 g for 5 min to remove bubbles in the microwells.

[0063] (2) Single-cell digestion of MSC cells: When the degree of confluence of canine mesenchymal stem cells reaches about 85%, they are passaged; washed once with DMEM / F12 medium, and digested with Accutase cell digestion solution at 37°C for 4-5 minutes, and then terminated by adding medium 1;

[0064] (3) After centrifugation at 200 g for 4 min, the cells were resuspended in the culture medium formed in step (1) and the cells were counted;

[0065] (4) Add 1.5x10 6 Single cells: Mix each well by pipetting with a Pasteur pipette, 3 mL / well for 24-well plates, and immediately centrifuge at 100 g for 3 min.

[0066] (5) After centrifugation, place the cells in a cell culture incubator at 37°C and 5% CO2. Observe the cell sphering after 48 hours.

[0067] (6) After the 3D cell aggregates were sphericalized, the cell aggregates from four wells were transferred to a 6-well plate in an orbital shaker at 100 rpm, with a culture volume of 5.5 mL and culture conditions at 37°C and 5% CO2.

[0068] (7) After culturing for 4-7 days, observe the changes in the size of the cell clusters. Change the MSC cell medium every two days and replace with fresh 2D culture medium. If the cell size is close to 300 μm, use Accutase to digest the cell clusters into single cells. Repeat steps (3)-(6) in part 2 above. After the cells form spheres in the AggreWell plate, transfer them to new wells for expanded culture. If the cell quantity is sufficient, transfer the 3D cell clusters to a rotary shake flask for culture. The cell clusters can be transferred to a 500 mL rotary shake flask with 200-300 mL of culture medium in the shake flask.

[0069] In the above step (7), during the culture process of the cell clusters, the size of the clusters will gradually increase due to cell proliferation. When the cell clusters are cultured for 4-7 days, the size is too large, which will affect the nutrient supply of the cells in the center of the clusters. Therefore, the cell clusters are digested into single cells and re-clustered. After forming small clusters, the clusters continue to proliferate and the size gradually increases.

[0070] (8) Rotary flask suspension culture: After the 3D cell aggregates in the AggreWell form spheres, transfer sufficient cell clusters to a 500 mL rotary flask at 65-75 rpm. Culture conditions: 37°C, 5% CO2. Subculture every four days. Use Accutase to digest the cell clusters into single cells. Perform suspension culture in the same manner as above. Generally, the cell clusters are expanded for 8-10 generations in vitro.

[0071] See also Figure 1 The effects of adding Y27632 alone and adding Y27632 and PVA at the same time on cell sphering were tested (the amount of cells was 3000 / microwell). The specific test process is as follows:

[0072] (1) 2D culture of mesenchymal stem cells: The process is the same as the 2D planar culture of canine mesenchymal stem cells in 1 above;

[0073] (2) Test the effects of PVA and Y27632 on the spheroidization of canine mesenchymal stem cells:

[0074] ① Prepare different culture media, each of which is freshly prepared:

[0075] Conventional culture medium: low-glucose DMEM +10% FBS+5 ng / mL bFGF

[0076] Add Y27632 culture medium: low-glucose DMEM + 10% FBS + 5 ng / mL bFGF + Y27632 (10 μM);

[0077] Y27632 and PVA culture medium: low-glucose DMEM + 10% FBS + 5 ng / mL bFGF + PVA (0.05%) + Y27632 (10 μM);

[0078] AggreWell plate pretreatment: 500 μL / well anti-adsorption wash solution (StemCell) was added to the AggreWell plate and centrifuged at 1300 g for 5 min to remove bubbles in the microwells;

[0079] ② Single-cell digestion of MSC cells: When the degree of fusion of canine mesenchymal stem cells reaches about 85%, they are passaged; washed once with DMEM / F12 medium, digested with Accutase at 37°C for 4-5 minutes, and terminated by adding medium (low-glucose DMEM +10% FBS +5 ng / mL bFGF);

[0080] ③ After centrifugation at 200g for 4 min, remove the supernatant, resuspend the cells in the culture medium prepared in step ①, and count the cells;

[0081] ④Add 1.5x10 6 Single cells: Mix each well by pipetting with a Pasteur pipette, 3 mL / well for 24-well plates, and immediately centrifuge at 100 g for 3 min.

[0082] ⑤ After centrifugation, place the cells in a cell culture incubator at 37°C and 5% CO2. After 48 hours, observe the cell sphering under different culture medium conditions.

[0083] Results Figure 1 The results showed that when PVA+Y was used, the cell sphere formation efficiency was the best, while when neither Y nor PVA was added or only Y was added, no obvious sphere formation was observed.

[0084] See also Figure 2 , the aggregate change process of different days in the PVA+Y system was investigated. The specific experimental process is as follows:

[0085] (1) 2D culture of mesenchymal stem cells: The process is the same as the 2D planar culture of canine mesenchymal stem cells in 1 above;

[0086] (2) Study on the optimal ball formation time in AggreWell holes:

[0087] ①Configure a culture medium that promotes 3D spheroidization:

[0088] Spheroidization medium A (PVA 0.01%): low-glucose DMEM + 10% FBS + 5 ng / mL bFGF + PVA (0.01% concentration) + Y27632 (10 μM);

[0089] Spheroidization medium B (PVA0.05%): low-glucose DMEM + 10% FBS + 5 ng / mL bFGF + PVA (0.05% concentration) + Y27632 (10 μM);

[0090] AggreWell plate pretreatment: 500 μL / well anti-adsorption wash solution (StemCell) was added to the AggreWell plate and centrifuged at 1300 g for 5 min to remove bubbles in the microwells;

[0091] ② Single-cell digestion of MSC cells: When the degree of fusion of canine mesenchymal stem cells reaches about 85%, they are passaged; washed once with DMEM / F12 medium, digested with Accutase at 37°C for 4-5 minutes, and terminated by adding medium (low-glucose DMEM +10% FBS +5 ng / mL bFGF);

[0092] ③ After centrifugation at 200g for 4 min, remove the supernatant, resuspend the cells in the culture medium prepared in step ①, and count the cells;

[0093] ④Add 1.5x10 6 Single cells: Mix each well by pipetting with a Pasteur pipette, 3 mL / well for 24-well plates, and immediately centrifuge at 100 g for 3 min.

[0094] ⑤After centrifugation, take a photo to form a D0 photo and place it in a cell culture incubator. The culture conditions are: 37℃, 5% CO2; take photos at D1 (24 hours), D2 (48 hours) and D3 (72 hours) to record the cell sphering situation at each time point.

[0095] Results Figure 2 The results showed that when Y27632+PVA culture medium was used, the cell sphere formation efficiency was good, and 3D cell spheres were formed after 24 hours and maintained until 72 hours. The sphere formation efficiency was also good when PVA was added at concentrations of 0.01% and 0.05%.

[0096] See also Figure 3 , investigate the effect of adding PVA alone on the sphering effect of mesenchymal stem cells in the AggreWell system. The experimental process is as follows:

[0097] ① Prepare the culture medium that promotes 3D sphering: domestic PVA is Aladdin P434363, sigma PVA is sigma, P8136;

[0098] Spheroidization medium C (domestic PVA 0.05%): low-glucose DMEM + 10% FBS + 5 ng / mL bFGF + PVA (0.05% concentration);

[0099] Spheroidization medium D (domestic PVA 0.01%): low-glucose DMEM + 10% FBS + 5 ng / mL bFGF + PVA (0.01% concentration);

[0100] Spheroidization medium E (sigma PVA0.05%): low-glucose DMEM + 10% FBS + 5 ng / mL bFGF + PVA (0.05% concentration);

[0101] Spheroid medium F (sigma PVA0.01%): low-glucose DMEM + 10% FBS + 5 ng / mL bFGF + PVA (0.01% concentration);

[0102] AggreWell plate pretreatment: 500 μL / well anti-adsorption wash solution (StemCell) was added to the AggreWell plate and centrifuged at 1300 g for 5 min to remove bubbles in the microwells;

[0103] ② Single-cell digestion of MSC cells: When the degree of fusion of canine mesenchymal stem cells reaches about 85%, they are passaged; washed once with DMEM / F12 medium, digested with Accutase at 37°C for 4-5 minutes, and terminated by adding medium (low-glucose DMEM +10% FBS +5 ng / mL bFGF);

[0104] ③ After centrifugation at 200g for 4 min, remove the supernatant, resuspend the cells in the culture medium prepared in step ①, and count the cells;

[0105] ④Add 0.9x10 6 , 1.2x10 6 and 1.4x10 6 Single cells: Mix each well by pipetting with a Pasteur pipette, 3 mL / well for 24-well plates, and immediately centrifuge at 100 g for 3 min.

[0106] ⑤After centrifugation, place the cells in a cell culture incubator at 37°C, 5% CO2; take photos on D1 (24 hours) to record the cell sphering.

[0107] Results Figure 3 ,The results showed that when PVA was added alone, the cells failed to form spheres, and different cell seeding densities, different brands of PVA, and different concentrations of PVA were all unsuccessful.

[0108] See also Figure 4 The 3D suspension culture of mesenchymal stem cells after sphering under different sphering conditions was investigated. The specific test process is as follows:

[0109] ① Prepare a culture medium that promotes 3D spheroidization: sigma PVA is sigma, P8136;

[0110] Spheroid formation medium G (sigma PVA 0.05%): low-glucose DMEM + 10% FBS + 5 ng / mL bFGF + PVA (0.05% concentration);

[0111] Spheroidization medium H (sigma PVA 0.01%): low-glucose DMEM + 10% FBS + 5 ng / mL bFGF + PVA (0.01% concentration);

[0112] AggreWell plate pretreatment: 500 μL / well anti-adsorption wash solution (StemCell) was added to the AggreWell plate and centrifuged at 1300 g for 5 min to remove bubbles in the microwells;

[0113] ② Single-cell digestion of MSC cells: When the degree of fusion of canine mesenchymal stem cells reaches about 85%, they are passaged; washed once with DMEM / F12 medium, digested with Accutase at 37°C for 4-5 minutes, and terminated by adding medium (low-glucose DMEM +10% FBS +5 ng / mL bFGF);

[0114] ③ After centrifugation at 200g for 4 min, remove the supernatant, resuspend the cells in the culture medium prepared in step ①, and count the cells;

[0115] ④Add 1.2x10 6 or 1.5x10 6 Single cells: Mix each well by pipetting with a Pasteur pipette, 3 mL / well for 24-well plates, and immediately centrifuge at 100 g for 3 min.

[0116] ⑤ After centrifugation, place in a cell culture incubator at 37°C, 5% CO2; after the cells are pelleted, transfer to a 6-well plate orbital shaker on D4: transfer 4 wells of cell pellets to a 6-well plate in an orbital shaker at 100 rpm, culture volume 5.5 mL, culture conditions: 37°C, 5% CO2;

[0117] ⑥ Culture in an orbital shaker for 2 days and take photos to observe the size of cell clusters.

[0118] Results Figure 4 The results showed that after cells with different inoculation densities formed spheres in a suitable culture system, they could maintain 3D dynamic culture and 3D cell cluster morphology in an orbital shaker system.

[0119] See also Figure 1-4In this example, it was found that the use of PVA alone without the addition of Y27632 could not support the formation of canine mesenchymal stem cells into spheres. Figure 3 When only PVA was added, the effect of canine mesenchymal stem cells in the single-cell state to form 3D cell clusters was extremely poor. Different brands of PVA and different concentrations of PVA conditions failed to successfully form spheres; see Figure 1 When Y27632 is added alone, the particle size of the 3D sphere is too small and the effect is not ideal; see Figure 2 When PVA and Y27632 are added at the same time, canine mesenchymal stem cells have the best effect in forming 3D cell clusters. Mesenchymal stem cells are mesenchymal cells derived from the mesoderm. During 2D culture, there is no obvious connection between cells. Through the above verification, it can be seen that MSCs need to be added with PVA and Y27632 at the same time and centrifuged with the help of AggreWell plate to effectively form spheres.

[0120] 3. After multiple generations of 3D expansion, MSC indicators and in vitro functional tests were performed:

[0121] The flow detection process is as follows:

[0122] (1) Collect P3 MSC and P10 MSC single cells. Each antibody detection group for CD34, CD29, CD90, and CD44 requires 1x10 cells per group. 6 indivual;

[0123] (2) Collect cells by centrifugation at 500g for 5 min and discard the supernatant; resuspend in 400 μL PBS and centrifuge at 500g for 5 min. Repeat twice and discard the supernatant.

[0124] (3) The resuspended cells were divided into two portions, each with 200 μL cell suspension. One portion was for the isotype group: according to the instructions for use of each antibody, the corresponding volume of isotype was added and resuspended; the other portion was for the experimental group: the corresponding volume of anti-CD90 / CD44 / CD29 / CD34 antibodies were added and incubated at 4°C in the dark for 30 min; after incubation, at 4°C, 500g, 5min, centrifuged to collect the cells and discard the supernatant; 500 μL PBS was added to resuspend, at 4°C, 500g, 5min, centrifuged to collect the cells and discard the supernatant, repeated 3 times, and 200 μL PBS was added to resuspend for flow cytometry detection of the expression of each marker. Results are shown in Figure 5 shown.

[0125] Figure 5 The results showed that under 3D subculture conditions, P10 cells still maintained good expression of MSC marker proteins (positive markers CD29 / CD44 / CD90 were all greater than 90%, and negative marker CD34 was less than 2%).

[0126] The sources of the antibodies and related reagents used are shown in Table 1 below.

[0127] Table 1 Sources of reagents

[0128]

[0129] 4. 3D dynamic culture of MSCs for osteogenic, adipogenic and chondrogenic differentiation detection:

[0130] (1) Osteogenic induction (using Cyagen, CUXMD-90021, OriCell adipose-derived mesenchymal stem cell osteogenic differentiation kit):

[0131] ① Add 1 mL of 0.1% gelatin to the six-well plate and shake it well to evenly cover the bottom of each well;

[0132] ② Place the six-well plate covered with 0.1% gelatin in a clean bench or CO2 incubator for at least 30 minutes;

[0133] ③After 30 minutes, remove the gelatin and use it to inoculate cells, or wait for the six-well plate to dry before inoculation;

[0134] ④ The adipose-derived mesenchymal stem cells to be induced were divided into 2x10 4 cells / cm 2 The cells were seeded at a density of 100 μg / mL in a six-well plate, and 2 mL of normal complete medium (low-glucose DMEM + 10% FBS + 5 ng / mL bFGF) was added to each well.

[0135] ⑤ The cells were cultured in a CO2 incubator at 37°C, 5% CO2, and saturated humidity;

[0136] ⑥ When the cell confluence reaches 70%, aspirate the complete medium in the well and add 2mL of Osteogenic Differentiation Medium of Richel Adipose-derived Mesenchymal Stem Cells to the six-well plate;

[0137] ⑦ Replace with fresh OriCell adipose-derived mesenchymal stem cell osteogenic differentiation medium every 3 days;

[0138] ⑧After 2-4 weeks of induction, the cells were stained with Alizarin Red depending on their morphological changes and growth.

[0139] (2) Adipogenic differentiation (using Cyagen, CUXMD-90031, OriCell adipose-derived mesenchymal stem cell adipogenic differentiation kit):

[0140] ① Add 1 mL of 0.1% gelatin to the six-well plate and shake it well to evenly cover the bottom of each well;

[0141] ② Place the six-well plate covered with 0.1% gelatin in a clean bench or CO2 incubator for at least 30 minutes;

[0142] ③After 30 minutes, remove the gelatin and use it to inoculate cells, or wait for the six-well plate to dry before inoculation;

[0143] ④ The adipose-derived mesenchymal stem cells to be induced were divided into 2x10 4 cells / cm 2 The cells were seeded at a density of 100 μg / mL in a six-well plate, and 2 mL of normal complete medium (low-glucose DMEM + 10% FBS + 5 ng / mL bFGF) was added to each well.

[0144] ⑤ The cells were cultured in a CO2 incubator at 37°C, 5% CO2, and saturated humidity;

[0145] ⑥ When the cell confluence reaches 100%, remove the complete medium in the well and add 2 mL of medium A of the adipogenic differentiation kit to the six-well plate;

[0146] ⑦ After 3 days of induction, remove the A solution in the six-well plate and add 2 mL of medium B of the Oricell adipose-derived mesenchymal stem cell adipogenic differentiation kit;

[0147] ⑧After 1 day, remove solution B and replace it with solution A for induction;

[0148] ⑨ Solution A and solution B are used alternately, and the cell status needs to be observed every day. If cell shrinkage or death occurs during the induction process of solution A, it needs to be replaced with solution B in time until the cell status recovers;

[0149] ⑩ Repeat the induction and maintenance process until obvious lipid droplets appear and then staining can be performed.

[0150] (3) Chondrogenic induction (Cyagen, CUXMD-90041, OriCell Adipose-derived Mesenchymal Stem Cell Chondrogenic Differentiation Kit):

[0151] ① 3×10 5 -4×10 5 Transfer the cells to be induced into a 15 mL centrifuge tube and centrifuge at 250 g for 4 min at 20°C.

[0152] ② Aspirate the supernatant, add 0.5 mL of the premixed solution of the chondrogenic differentiation kit, resuspend the precipitate obtained by centrifugation in the previous step, and centrifuge at 20°C, 150g for 5 minutes;

[0153] ③Repeat step ② and wash the cells again;

[0154] ④ Addition of Supplement II (hereinafter referred to as Supplement II) of the OriCell Adipose-derived Mesenchymal Stem Cell Chondrogenic Differentiation Kit. According to the ratio (10 μL of Supplement II is added to 1 mL of Chondrogenic Differentiation Premix), pipette the required dose of Supplement II for the experiment, add it to the corresponding volume of premix to prepare a complete chondrogenic differentiation medium, and mix it by gently pipetting.

[0155] ⑤ Resuspend the cell pellet obtained in step ③ with 0.5 mL of complete chondrogenic differentiation medium and centrifuge at 20°C, 150 g for 5 min;

[0156] ⑥ Loosen the cap of the centrifuge tube to facilitate gas exchange, and place it upright in a CO2 incubator at 37°C, 5% CO2, and saturated humidity;

[0157] ⑦ When the cells begin to aggregate (usually after 24 or 48 hours), gently tap the bottom of the centrifuge tube to detach the chondrocytes from the bottom of the tube and suspend them in the liquid;

[0158] ⑧ Starting from the inoculation, replace the cells with fresh chondrogenic differentiation complete medium every 2-3 days, about 0.5 mL per tube;

[0159] ⑨ Continue induction until a chondrocyte ball with a diameter of 1.5-2 mm is formed in the tube, and then prepare for sectioning and staining.

[0160] Results Figure 6 As shown, Figure 6 It shows that mesenchymal stem cells after 3D culture can effectively complete adipogenic, osteogenic and chondrogenic differentiation, and the three differentiation abilities remain unchanged.

[0161] 5. Detection of the ability of 3D cultured MSCs to inhibit lymphocyte proliferation (using Invitrogen, CellTraceCFSE Cell Proliferation Kit, Catalog No. C34554):

[0162] The steps are as follows:

[0163] (1) Prepare CellTrace™ stock solution according to the instructions: add 18 μl DMSO (Component B) (concentration 5 mM) to one portion of CellTrace™ reagent (Component A), mix well, and dilute the CellTrace™ stock solution with preheated PBS to a final concentration of 5 μM (PBS dye solution);

[0164] (2) Prepare PBS suspension of PBMC cells, centrifuge, and discard the supernatant; resuspend the cells in PBS dye solution at a concentration of 1*10 6 / mL;

[0165] (3) Incubate at room temperature in the dark for 20 min;

[0166] (4) Add 5 times the volume of dye to RPMI 1640 complete medium (containing at least 1% FBS) (GIBCO, Catalog No. 11875093) and incubate for 5 min to remove residual dye in the solution;

[0167] (5) Centrifuge at 300 g for 8 min, discard the supernatant, and resuspend in RPMI 1640 medium + 10% FBS (FBS inactivated at 56°C for 30 min); incubate for at least 10 min to allow the dye to be hydrolyzed in the cells;

[0168] (6) As shown in Table 2 below, add PBMC, standard control cells or cells to be tested to a 12-well plate, with 3 parallel wells for each group. The culture medium is RPMI 1640 + 10% FBS + 300 IU / mL IL2; add CD3 / CD28 antibody magnetic beads to activate PBMC; PBMC: MSC = 5:1 ratio, co-culture the two for 7 days and then perform flow cytometry. D3 half-change the medium, gently aspirate 1 mL of supernatant, add 1 mL of fresh RPMI 1640 + 10% FBS + 300 IU / mL IL2 medium, and gently blow away the aggregated cell clusters in the PBMC single culture well, then change the medium every other day, and do not continue to add magnetic beads when changing the medium;

[0169] Table 2 Grouping of each group and the amount of each reagent and cell used in the lymphocyte proliferation inhibition test

[0170]

[0171] (7) One day after staining, the initial CFSE fluorescence intensity (maximum, control group B) was evaluated, and the fluorescence intensity of the unstained group was detected to exclude background interference (control group A);

[0172] (8) Result calculation

[0173] Under the premise of the test being effective, if the percentage of proliferating lymphocytes in the test cell group is less than that in the control group C, it is judged that the test cells have an inhibitory effect on the proliferation of lymphocytes, and the inhibition rate of 2D cultured MSC and 3D cultured MSC on lymphocyte proliferation is calculated using the following formula:

[0174] Lymphocyte proliferation inhibition rate = (CX) / C×100%, where X is the percentage of proliferating lymphocytes in the MSC cell group, and C is the percentage of proliferating lymphocytes in the PBMC magnetic bead cell group;

[0175] See also Figure 7 The results showed that all groups had the effect of inhibiting PBMC proliferation. Among them, after calculation, the inhibition rate of 3D MSC was 60.81%, and the inhibition rate of 2D MSC was 52.64%.

[0176] 6. Detection of the ability to promote proliferation of epidermal cells and fibroblasts:

[0177] (1) Immortalized human epidermal cells (HaCaT cells) and human lung fibroblasts (HFL1 cells) were cultured at 1×10 4 The cells / well were seeded in a 12-well plate containing regular culture medium (10% FBS DMEM-high glucose medium) and cultured for 24 h to allow the cells to adhere.

[0178] (2) After cells adhered overnight, AO / PI was used to measure the cell number to obtain the baseline value (0) day.

[0179] (3) Collect the supernatant culture medium of 2D cultured MSC P5 and the supernatant culture medium of 3D dynamic suspension cultured MSC P5. Filter the cell supernatant with a 0.25 μm filter before use.

[0180] (4) After obtaining the values ​​on day 0, randomly select wells and treat them with 2D MSC conditioned medium (2D MSC supernatant: MSC fresh medium at a ratio of 1:1), 3D MSC conditioned medium (3D MSC supernatant: MSC fresh medium at a ratio of 1:1), or completely fresh medium. The group treated with fresh medium is the control group (Control group). The fresh medium is low-glucose DMEM + 10% FBS + 5 ng / mL bFGF.

[0181] (5) After 3 days of treatment, the cell number was measured again (Day 3).

[0182] Test results see Figure 8 As shown, compared with the control group treated with fresh culture medium, the conditioned medium prepared from 2D MSC and 3D MSC culture supernatants can effectively promote the proliferation of HFL1 and HaCaT cells, and the supernatant prepared from 3D suspension cultured MSCs is more suitable for epidermal cell proliferation.

[0183] 7. Detection of the ability of canine 3D MSC to promote the healing of difficult-to-heal wounds:

[0184] (1) 3D passaging operation: dynamic suspension culture of cell aggregates:

[0185] ① Small-scale system orbital shaker dynamic suspension culture: After the 3D cell aggregates are spherical, transfer the cell clusters from 4 wells to a 6-well plate in an orbital shaker. The orbital shaker speed is 100 rpm, the culture volume is 5.5 mL, and the culture conditions are: 37°C, 5% CO2;

[0186] ② After culturing for 4-7 days, observe the changes in the size of the cell clusters and change the MSC cell medium every two days. When the cell size is close to 300 μm, use Accutase to digest the cell clusters into single cells. The same operation as the "3D cell aggregate formation process" is performed. After the cells are spheroidized in AggreWell, they are transferred to shake flasks for expanded culture.

[0187] ③ When the cell quantity is sufficient, use Accutase to digest the cell clusters into single cells for subsequent animal experiments.

[0188] (2) Optimization process of diabetic mouse wound model:

[0189] In order to screen suitable STZ (streptozotocin) diabetic wound model mice, skin wound models were performed on 9-week-old and 16-week-old diabetic mice, and the wound recovery speed was tested compared with the skin model of non-STZ diabetic mice (Ctrl). The specific operations are as follows:

[0190] ① A single intraperitoneal injection of 55 mg / kg of STZ (streptozotocin) solution was given; the subjects were fasted for 1 hour after administration, and 5% glucose water and feed were given; blood sugar and body weight changes were monitored after administration; a circular wound (d=8 mm) was artificially made on the back;

[0191] ② Take photos of the wounds and measure the wound area on days 0, 4, 8 / 9, 13 / 14, and 18 after modeling until the wounds are completely healed.

[0192] The skin healing status of mice in each group was as follows Fig. 9 , showing that the 16-week-old mouse Model group was more suitable for establishing a refractory diabetic mouse wound model than the 9-week-old Model group.

[0193] (3) Construction and operation of diabetic mouse wound model:

[0194] 16-week-old mice were intraperitoneally injected with a single dose of 55 mg / kg STZ solution. After administration, they were fasted for 1 hour and given 5% glucose water and feed. After administration, blood sugar and body weight changes were monitored. A circular wound (d=8 mm) was artificially made on the back.

[0195] (4) Drug administration in mouse wounds:

[0196] On D0 day after modeling, the MSC administration group was treated with 3D MSC cell clusters at a rate of 4x10 6The cells were administered subcutaneously at multiple points near the wound of the cell cluster, and no other administration was performed in the untreated group. Wounds were photographed and wound areas were measured on days 0, 2, 4, 7, 9, 11, 14, 16, and 18 after modeling until the wound was completely healed;

[0197] Results Fig.10 and Fig.11 . Fig.10 The figure shows the wound healing curves of different treatment groups. After administration of 3D mesenchymal stem cells, the wound healing speed of mice can be effectively accelerated. Fig.11 Typical wound photos of mice in the 3D mesenchymal stem cell administration group and the control non-treatment group on different days showed that the wounds in the 3D MSC administration group were significantly smaller than those in the non-treatment group.

[0198] 8. Test the effects of spheroidization medium containing PVA and Y in other different basal medium configurations on MSC single cell aggregation:

[0199] ① Prepare a culture medium that promotes 3D spheroidization: Sigma PVA is Sigma, P8136;

[0200] Spheroidization medium I (sigma PVA 0.05%): Alpha-MEM + 10% FBS + 5 ng / mL bFGF + PVA (0.05% concentration) + Y27632 (10 μM);

[0201] Spheroidization medium J (sigma PVA 0.05%): DMEM-high glucose + 10% FBS + 5 ng / mL bFGF + PVA (0.05% concentration) + Y27632 (10 μM);

[0202] Spheroidization medium K (sigma PVA 0.05%): F12 + 10% FBS + 5 ng / mL bFGF + PVA (0.05% concentration) + Y27632 (10 μM);

[0203] Spheroidization medium L (sigma PVA 0.05%): DMEM / F12+10% FBS+5 ng / mL bFGF+PVA (0.05% concentration)+Y27632 (10 μM);

[0204] Spheroidization medium M (sigma PVA 0.05%): RPMI1640 + 10% FBS + 5 ng / mL bFGF + PVA (0.05% concentration) + Y27632 (10 μM);

[0205] Sphere formation medium N (sigma PVA 0.05%): IMDM + 10% FBS + 5 ng / mL bFGF + PVA (0.05% concentration) + Y27632 (10 μM);

[0206] AggreWell plate pretreatment: 500 μL / well anti-adsorption wash solution (StemCell) was added to the AggreWell plate and centrifuged at 1300 g for 5 min to remove bubbles in the microwells;

[0207] ② Single-cell digestion of MSC cells: When the degree of fusion of canine mesenchymal stem cells reaches about 85%, they are passaged; washed once with DMEM / F12 medium, digested with Accutase at 37°C for 4-5 minutes, and terminated by adding medium (low-glucose DMEM +10% FBS +5 ng / mL bFGF);

[0208] ③ After centrifugation at 200g for 4 min, remove the supernatant, resuspend the cells in the culture medium prepared in step ①, and count the cells;

[0209] ④Add 1.5x10 6 Single cells: Mix each well by pipetting with a Pasteur pipette, 3 mL / well for 24-well plates, and immediately centrifuge at 100 g for 3 min.

[0210] ⑤ After centrifugation, place the cells in a cell culture incubator at 37°C, 5% CO2; take photos on D2 after the cells have formed clusters.

[0211] Each group is named according to the different basal culture media in the spheroid formation culture medium. The effect of MSC single cell aggregate formation under different basal culture media conditions can be found in Fig.12 The results showed that under the conditions of adding PVA (0.05% concentration) and Y27632 (10 μM), MSC single cells successfully formed 3D cell clusters in multiple basic culture media.

[0212] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A three-dimensional dynamic expansion method of canine mesenchymal stem cells, characterized in that: The canine mesenchymal stem cells were amplified in an AggreWell plate using a culture medium that promotes the formation of three-dimensional aggregates of canine mesenchymal stem cells. The culture medium that promotes the formation of three-dimensional aggregates of canine mesenchymal stem cells consists of a 2D culture medium, polyvinyl alcohol, and a ROCK inhibitor, Y27632, wherein the mass concentration of polyvinyl alcohol is 0.01%-0.05%, and the concentration of Y27632 is 10 μM. The molecular weight of the polyvinyl alcohol is 30,000-70,000. The composition of the 2D culture medium is basal medium + 10% FBS + 5 ng / mL bFGF, The basic culture medium is any one of low-glucose DMEM culture medium, DMEM high-glucose culture medium, Alpha-MEM culture medium, F12 culture medium, DMEM / F12 culture medium, RPMI1640 culture medium or IMDM culture medium.

2. The three-dimensional dynamic expansion method of canine mesenchymal stem cells according to claim 1, characterized in that: The specific steps include: (1) Preparation of culture medium to promote the formation of three-dimensional aggregates of canine mesenchymal stem cells and pretreatment of AggreWell plates: Preparation of a culture medium for promoting the formation of three-dimensional aggregates of canine mesenchymal stem cells: polyvinyl alcohol and ROCK inhibitor Y27632 are added to the 2D culture medium for preparation; AggreWell plate pretreatment: add anti-adsorption rinse solution into the AggreWell plate and centrifuge to remove bubbles in the microwells; (2) Single-cell digestion: Take canine mesenchymal stem cells with a confluence of 85% for subculture, wash once with DMEM / F12 medium, add Accutase cell digestion solution to digest for 4 to 5 minutes, and terminate with 2D medium; (3) Centrifuging the cells in step (2), then resuspending the cells in the medium for promoting the formation of three-dimensional aggregates of canine mesenchymal stem cells prepared in step (1), and counting the cells; (4) Add the resuspended cells from step (3) to the AggreWell plate, pipette each well to mix, and centrifuge immediately; (5) After centrifugation in step (4), place the cells in a cell culture incubator at 37°C and 5% CO2. After 40-50 hours, observe the cell sphering. (6) After the 3D cell aggregates are spherical, transfer the cell clusters to the well plate of an orbital shaker at a speed of 90-110 rpm and culture conditions: 37°C, 5% CO2; (7) After culturing for 4-7 days, observe the changes in the size of the cell clusters. Change the MSC cell medium every two days. When the cell size reaches 300 μm, use Accutase to digest the cell clusters into single cells. Repeat the above steps (3)-(6). After inoculation into a new AggreWell plate to form a sphere, expand the culture in the orbital shaker system plate, and transfer the 3D cell clusters to a rotary shake flask for culture. (8) Rotary flask suspension culture: After the 3D cell aggregates in the AggreWell plate form spheres, transfer the cell clusters to a rotary flask at a rotation speed of 65-75 rpm. Culture conditions: 37°C, 5% CO2. Subculture every four days. Use Accutase to digest the cell clusters into single cells and perform suspension culture in the same manner as above.

3. The three-dimensional dynamic expansion method of canine mesenchymal stem cells according to claim 2, characterized in that: In step (2), 2D culture is performed before single-cell digestion of canine mesenchymal stem cells.

4. The three-dimensional dynamic expansion method of canine mesenchymal stem cells according to claim 3, characterized in that: In step (2), 2D culture specifically includes: ①2D culture medium preparation: basal culture medium + 10% FBS + 5 ng / mL bFGF, wherein the basal culture medium is low-glucose DMEM culture medium; ② Cell thawing: Take out the frozen canine mesenchymal stem cells and thaw them in a 37±2℃ water bath; ③Cell centrifugation: Pipette the 2D culture medium from step ① above into a centrifuge tube, add the thawed cell suspension, centrifuge, discard the supernatant, and retain the cell pellet; ④ Resuspend and count: Add the 2D culture medium in step ① above to the centrifuge tube to resuspend the cell pellet; use a pipette to pipette the cell suspension to mix it thoroughly, and perform cell counting and viability detection; ⑤Cell inoculation: Inoculate the cell suspension into a T75 culture flask, add the 2D culture medium from step ① above to a total volume of 15 mL, and place the culture flask in a CO2 incubator; ⑥ Replace the medium for 2D cell culture: Replace the medium of the cells daily by adding 15 mL of fresh 2D culture medium from step ① above until the cells proliferate to 85% confluence.

5. The three-dimensional dynamic expansion method of canine mesenchymal stem cells according to claim 2, characterized in that: In step (4), the AggreWell plate has 24 wells, and 1.5x10 6 A single cell.

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