A stem cell preparation and preparation method thereof

Abstract

The present invention relates to the field of biomedicine technology, and in particular to a stem cell preparation and a preparation method thereof. The preparation method of the stem cell preparation provided by the present invention comprises adding ingredients such as nomilin to a first culture medium and adding ingredients such as koumine to a second culture medium. The present invention proves that the preparation method provided by the present invention does not affect the proliferation activity of stem cells by setting up a stem cell proliferation test. The present invention also provides a stem cell preparation, and by setting up a human skin fibroblast proliferation ability test, a cytokine content test, and a wound healing test, it is comprehensively and multi-facetedly proved that the stem cell preparation can enhance the proliferation ability of human skin fibroblasts and promote wound healing, which is of great significance to the field of biomedicine technology.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a stem cell preparation and a preparation method thereof. Background Art

[0002] Stem cells are the source of all specialized cells that make up human organs and tissues. They are present in all multicellular tissues and can differentiate into specialized cells under specific circumstances. Compared to mature cells, stem cells possess the ability to replicate, renew, and divide, and are therefore known as "universal cells." Based on their differentiation potential, stem cells are categorized as totipotent, unipotent, and pluripotent.

[0003] Adipose-derived mesenchymal stem cells (AMSCs) are multipotent stem cells derived from the mesoderm and can be induced to differentiate into adipocytes, neurons, and other cells under specific conditions. Adipose-derived mesenchymal stem cells are typically obtained from adipose tissue and are widely used in clinical trials due to their rich content, ease of collection, more stable cell characteristics compared to other stem cell cultures, strong differentiation ability, and low immunogenicity.

[0004] With the deepening of stem cell research, stem cell preparations have attracted considerable attention in the biomedical field. Stem cell preparations are drugs or therapeutic products with specific functions and applications, produced by extracting, culturing, and processing stem cells. Numerous clinical trial results have demonstrated that stem cell preparations have the potential to promote tissue repair and regeneration, regulate inflammatory responses, and thus are used to treat a variety of diseases. However, in practical applications, existing stem cell preparations still suffer from technical issues such as poor efficacy. In light of this, the present invention proposes a method for producing stem cell preparations. Summary of the Invention

[0005] In order to overcome the deficiencies of the prior art, one of the objectives of the present invention is to provide a method for preparing a stem cell preparation, which does not affect the proliferation activity of stem cells.

[0006] The following technical solution is adopted to achieve one of the purposes of the present invention:

[0007] A method for preparing a stem cell preparation comprises the following steps:

[0008] (1) Adipose tissue is enzymatically digested and centrifuged to obtain a cell pellet, and the cell pellet is resuspended in a first culture medium for primary culture; the first culture medium comprises a basal culture medium and the following components added to the basal culture medium: 5%-10% FBS and 25-50 ng / mL of nomilin;

[0009] (2) culturing the adipose-derived mesenchymal stem cells from step (1) until the confluence reaches 80%-85% and then subculturing;

[0010] (3) The P3-P5 adipose-derived mesenchymal stem cells obtained in step (2) are inoculated into a second culture medium and cultured for 24-48 hours. The cell supernatant is collected and dialyzed by ultrafiltration to obtain a concentrate; the second culture medium comprises a basal culture medium and the following components added to the basal culture medium: 5%-10% FBS and 40-60 ng / mL of koumine.

[0011] Preferably, the method further comprises adding a freeze-drying protectant to the concentrated solution in step (3) and freeze-drying.

[0012] Preferably, the freeze-drying protective agent is: adding the following ingredients to every 100 mL of the concentrate: 5g konjac sugar, 0.01g sodium benzoate, 5g carboxymethyl cellulose, 10g hydroxyethyl starch, and 1g glutathione.

[0013] Preferably, the freeze drying is specifically: cooling to -55°C at a rate of 8°C / min, freezing for 2 hours, and placing in a vacuum freeze dryer at a pressure of 30 Pa and a temperature of -30°C for freeze drying for 30 hours.

[0014] Preferably, the basal culture medium in steps (1) and (3) is DMEM / F12 culture medium.

[0015] Preferably, the density of the cell pellet resuspended in the first culture medium in step (1) is 1.5×10 5 -6.5×10 5 pieces / mL.

[0016] Preferably, the seeding density of adipose-derived mesenchymal stem cells in the second culture medium in step (3) is 2×10 4 -5×10 4 pieces / mL.

[0017] Preferably, the ultrafiltration dialysis in step (3) is specifically: ultrafiltration dialysis is performed using an ultrafiltration membrane with a molecular weight of 40KD to obtain a dialysate, and then the dialysate is passed through an ultrafiltration membrane with a molecular weight of 100D to obtain a concentrated solution.

[0018] Preferably, the culturing in steps (1), (2) and (3) is carried out at 37°C and 5% CO2.

[0019] A second object of the present invention is to provide a stem cell preparation that can enhance the proliferation capacity of human fibroblasts and promote wound healing.

[0020] The second object of the present invention is achieved by adopting the following technical solution:

[0021] A stem cell preparation prepared by the above preparation method

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] (1) The preparation method of the stem cell preparation provided by the present invention comprises adding ingredients such as nomilin to a first culture medium and adding ingredients such as koumine to a second culture medium;

[0024] (2) The present invention demonstrates that the preparation method provided by the present invention does not affect the proliferation activity of stem cells by setting up a stem cell proliferation test;

[0025] (3) The present invention also provides a stem cell preparation, and by setting up a human skin fibroblast proliferation ability test, a cytokine content test, and a wound healing test, it is comprehensively and multi-facetedly demonstrated that the stem cell preparation can enhance the proliferation ability of human skin fibroblasts and promote wound healing, which is of great significance to the field of biomedicine technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Graph showing the effects of the preparation methods of Examples 2-4 and Comparative Examples 1-5 on the proliferation activity of adipose-derived mesenchymal stem cells;

[0027] Figure 2 This figure shows the effects of the stem cell preparations prepared in Example 2 and Comparative Examples 1-5 on the proliferation capacity of skin fibroblasts;

[0028] Figure 3 Graph showing the fibroblast factor content in the stem cell preparations prepared in Example 1 and Comparative Examples 1-5. DETAILED DESCRIPTION

[0029] The present invention will be further described below in conjunction with the accompanying drawings and specific implementation methods. It should be noted that, under the premise of no conflict, the various embodiments or technical features described below can be arbitrarily combined to form new embodiments.

[0030] Example 1

[0031] This embodiment provides a stem cell preparation and a preparation method thereof, the method is as follows:

[0032] (1) Adipose tissue was washed with D-Hanks solution, centrifuged, and then 1% type II collagenase (3 times the volume) was added for digestion for 25 minutes. The cell pellet was then diluted with D-Hanks solution (1.5 times the volume) and centrifuged to obtain a cell pellet. The cell pellet was resuspended in the first culture medium (DMEM / F12 culture medium supplemented with 10% FBS and 36 ng / mL of normiline). The cell pellet was resuspended in the first culture medium at a density of 4 × 10 5 / mL, and placed in an incubator at 37°C and 5% CO2 saturated humidity for primary culture;

[0033] (2) culturing the adipose-derived mesenchymal stem cells obtained in step (1) in an incubator at 37° C. and 5% CO 2 saturated humidity until the confluence reaches 80%, and then subculturing the cells at a ratio of 1:2 to obtain P3 adipose-derived mesenchymal stem cells;

[0034] (3) The P3 adipose-derived mesenchymal stem cells obtained in step (2) were inoculated into the second culture medium (DMEM / F12 culture medium supplemented with 10% FBS and 50 ng / mL of koumine). The inoculation density of the P3 adipose-derived mesenchymal stem cells in the second culture medium was 3×10 4 Cells were cultured at 37°C in an incubator with 5% CO2 saturated humidity for 36 h. The cell supernatant was collected and the supernatant was dialyzed through an ultrafiltration membrane with a molecular weight of 40 kD to obtain a dialysate. The dialysate was then passed through an ultrafiltration membrane with a molecular weight of 100 D to obtain a concentrate. A lyoprotectant was added to the concentrate (the following ingredients were added to every 100 mL of concentrate: 5 g konjac sugar, 0.01 g sodium benzoate, 5 g carboxymethyl cellulose, 10 g hydroxyethyl starch, and 1 g glutathione). The concentrate was cooled to -55°C at a rate of 8°C / min, frozen for 2 h, and lyophilized in a vacuum freeze dryer at a pressure of 30 Pa and a temperature of -30°C for 30 h to obtain a stem cell preparation.

[0035] Example 2

[0036] This embodiment provides a stem cell preparation and a preparation method thereof, the method is as follows:

[0037] (1) Adipose tissue was washed with D-Hanks solution, centrifuged, and then 1% type II collagenase (3 times the volume) was added for digestion for 25 minutes. The cell pellet was then diluted with D-Hanks solution (1.5 times the volume) and centrifuged to obtain a cell pellet. The cell pellet was resuspended in the first culture medium (DMEM / F12 culture medium supplemented with 10% FBS and 36 ng / mL of normiline). The cell pellet was resuspended in the first culture medium at a density of 4 × 10 5 / mL, and placed in an incubator at 37°C and 5% CO2 saturated humidity for primary culture;

[0038] (2) culturing the adipose-derived mesenchymal stem cells obtained in step (1) in an incubator at 37° C. and 5% CO 2 saturated humidity until the confluence reaches 80%, and then subculturing the cells at a ratio of 1:2 to obtain P3 adipose-derived mesenchymal stem cells;

[0039] (3) The P3 adipose-derived mesenchymal stem cells obtained in step (2) were inoculated into the second culture medium (DMEM / F12 culture medium supplemented with 10% FBS and 50 ng / mL of koumine). The inoculation density of the P3 adipose-derived mesenchymal stem cells in the second culture medium was 3×10 4The cells were cultured at 37°C in an incubator with 5% CO2 saturated humidity for 36 h, and the cell supernatant was collected. The supernatant was subjected to ultrafiltration and dialyzed through an ultrafiltration membrane with a molecular weight of 40 KD to obtain a dialysate. The dialysate was then passed through an ultrafiltration membrane with a molecular weight of 100 D to obtain a concentrated solution, which is the stem cell preparation.

[0040] Example 3

[0041] This embodiment provides a stem cell preparation and a preparation method thereof, the method is as follows:

[0042] (1) Adipose tissue was washed with D-Hanks solution, centrifuged, and then 1% type II collagenase (3 times the volume) was added for digestion for 25 minutes. The cell pellet was then diluted with D-Hanks solution (1.5 times the volume) and centrifuged to obtain a cell pellet. The cell pellet was resuspended in the first culture medium (DMEM / F12 culture medium supplemented with 5% FBS and 25 ng / mL of normiline). The cell pellet was resuspended in the first culture medium at a density of 1.5×10 5 / mL, and placed in an incubator at 37°C and 5% CO2 saturated humidity for primary culture;

[0043] (2) culturing the adipose-derived mesenchymal stem cells obtained in step (1) in an incubator at 37° C. and 5% CO 2 saturated humidity until the confluence reaches 85%, and then subculturing the cells at a ratio of 1:2 to obtain P4 adipose-derived mesenchymal stem cells;

[0044] (3) The P4 adipose-derived mesenchymal stem cells obtained in step (2) were inoculated into the second culture medium (DMEM / F12 culture medium supplemented with 5% FBS and 40 ng / mL of koumine). The inoculation density of the P4 adipose-derived mesenchymal stem cells in the second culture medium was 2×10 4 The cells were cultured at 37°C in an incubator with 5% CO2 saturated humidity for 24 h, and the cell supernatant was collected. The supernatant was subjected to ultrafiltration and dialyzed through an ultrafiltration membrane with a molecular weight of 40 KD to obtain a dialysate. The dialysate was then passed through an ultrafiltration membrane with a molecular weight of 100 D to obtain a concentrated solution, which is the stem cell preparation.

[0045] Example 4

[0046] This embodiment provides a stem cell preparation and a preparation method thereof, the method is as follows:

[0047] (1) Adipose tissue was washed with D-Hanks solution, centrifuged, and then 1% type II collagenase (3 times the volume) was added for digestion for 25 minutes. The cell pellet was then diluted with D-Hanks solution (1.5 times the volume) and centrifuged to obtain a cell pellet. The cell pellet was resuspended in the first culture medium (DMEM / F12 culture medium supplemented with 8% FBS and 50 ng / mL of normiline). The cell pellet was resuspended in the first culture medium at a density of 6.5×10 5 / mL, and placed in an incubator at 37°C and 5% CO2 saturated humidity for primary culture;

[0048] (2) culturing the adipose-derived mesenchymal stem cells obtained in step (1) in an incubator at 37° C. and 5% CO 2 saturated humidity until the confluence reaches 80%, and then subculturing the cells at a ratio of 1:2 to obtain P5 adipose-derived mesenchymal stem cells;

[0049] (3) The P5 adipose-derived mesenchymal stem cells obtained in step (2) were inoculated into the second culture medium (DMEM / F12 culture medium supplemented with 8% FBS and 60 ng / mL of koumine). The inoculation density of the P5 adipose-derived mesenchymal stem cells in the second culture medium was 5×10 4 The cells were cultured at 37°C in an incubator with 5% CO2 saturated humidity for 48 h, and the cell supernatant was collected. The supernatant was subjected to ultrafiltration and dialyzed through an ultrafiltration membrane with a molecular weight of 40 KD to obtain a dialysate. The dialysate was then passed through an ultrafiltration membrane with a molecular weight of 100 D to obtain a concentrate, which was the stem cell preparation.

[0050] Comparative Example 1

[0051] This comparative example provides a stem cell preparation and a preparation method thereof, which differs from Example 2 in that no nomilin is added to the first culture medium, and the rest is the same as Example 2.

[0052] Comparative Example 2

[0053] This comparative example provides a stem cell preparation and a preparation method thereof, which differs from Example 2 in that the amount of nomilin in the first culture medium is adjusted to 70 ng / mL, and the rest is the same as Example 2.

[0054] Comparative Example 3

[0055] This comparative example provides a stem cell preparation and a preparation method thereof, which differs from Example 2 in that no koumine is added to the second culture medium, and the rest is the same as Example 2.

[0056] Comparative Example 4

[0057] This comparative example provides a stem cell preparation and a preparation method thereof, which differs from Example 2 in that the amount of koumine in the second culture medium is adjusted to 85 ng / mL, and the rest is the same as Example 2.

[0058] Comparative Example 5

[0059] This comparative example provides a stem cell preparation and a preparation method thereof, which differs from Example 2 in that: no nomilin is added to the first culture medium, and no koumine is added to the second culture medium. The rest is the same as Example 2.

[0060] Test Example 1

[0061] This test example explores the effects of the preparation methods of Examples 2-4 and Comparative Examples 1-5 on the proliferation activity of adipose-derived mesenchymal stem cells:

[0062] The cell pellet collected from step (3) of Examples 2-4 and Comparative Examples 1-5 was resuspended in complete culture medium and then inoculated into 96-well plates (DMEM / F12 culture medium containing 10% FBS) at a density of 1×10 4 After culturing for 48 h in an incubator at 37°C and 5% CO2 saturated humidity, the OD value at 450 nm was detected using the CCK-8 method. The OD value represents the proliferation activity of mesenchymal stem cells. Figure 1 shown.

[0063] Figure 1 The figure shows the effect of the preparation methods of Examples 2-4 and Comparative Examples 1-5 on the proliferation activity of mesenchymal stem cells. Figure 1 It can be seen that there is no significant difference in the OD values of mesenchymal stem cells obtained using the preparation methods of Examples 2-4 and Comparative Examples 1-5, that is, there is no significant difference in the proliferation activity of mesenchymal stem cells in Examples 2-4 and Comparative Examples 1-5. This shows that the preparation method of the present invention does not affect the proliferation activity of mesenchymal stem cells.

[0064] Test Example 2

[0065] This test example explores the effects of the stem cell preparations prepared in Example 2 and Comparative Examples 1-5 on the proliferation capacity of human skin fibroblasts:

[0066] (1) Preparation of culture medium

[0067] Preparation of a culture medium containing the stem cell preparation: Add the stem cell preparations of Example 2 and Comparative Examples 1-5 to DMEM / F12 medium at a concentration of 50 μg / mL, and add 10% by volume of FBS to prepare a culture medium containing the stem cell preparations of Example 2 and Comparative Examples 1-5;

[0068] Prepare the culture medium for control group 1: add 10% FBS by volume to DMEM / F12 culture medium to prepare the culture medium;

[0069] Prepare the culture medium for control group 2: add 10% FBS and 36 ng / mL nomilin to DMEM / F12 culture medium to prepare the culture medium;

[0070] Prepare the culture medium for control group 3: add 10% FBS and 50 ng / mL koumine to DMEM / F12 culture medium to prepare the culture medium;

[0071] (2) Cultivation of human skin fibroblasts

[0072] Human skin fibroblasts were seeded in 96-well plates at a cell seeding density of 2×10 3 100 μL of DMEM / F12 medium (containing 10% FBS) was added to each well, and the cells were cultured in an incubator at 37°C and 5% CO2 saturated humidity for 24 h. The medium was removed, and the culture medium containing the stem cell preparations of Example 2 and Comparative Examples 1 to 5, the culture medium of Control Group 1, the culture medium of Control Group 2, and the culture medium of Control Group 3 were added, respectively, and the cells were cultured in an incubator at 37°C and 5% CO2 saturated humidity for 2 days.

[0073] (3) Testing samples

[0074] 10 μL CCK-8 reagent was added to each well and incubated at 37°C in the dark for 1 hour. The OD value at 450 nm was measured. The OD value represents the degree of effect of the stem cell preparation on the proliferation of skin fibroblasts. The results are as follows: Figure 2 shown.

[0075] Figure 2 The figure shows the effect of the stem cell preparations prepared in Example 2 and Comparative Examples 1-5 on the proliferation ability of human skin fibroblasts. Figure 2 As can be seen, the OD value of Example 2 was significantly higher than that of Control 1. Compared with Example 2, the proliferation capacity of human skin fibroblasts cultured with the stem cell preparations of Comparative Examples 1-5 was reduced. These results demonstrate that the stem cell preparations prepared by the present invention can effectively enhance the proliferation capacity of skin fibroblasts.

[0076] There was no significant difference in the proliferation capacity of human skin fibroblasts between control groups 2 and 3 compared to control group 1. Compared to control group 1, the culture medium of control group 2 also contained nomilin, and the culture medium of control group 3 also contained koumine. This suggests that nomilin or koumine do not affect the proliferation capacity of human skin fibroblasts.

[0077] Test Example 3

[0078] This test example uses enzyme-linked immunosorbent assay to explore the content of fibroblast growth factor (FGF) in the stem cell preparations prepared in Example 1 and Comparative Examples 1-5:

[0079] A lyoprotectant was added to the stem cell preparations prepared in Comparative Examples 1-5 (5 g konjac, 0.01 g sodium benzoate, 5 g carboxymethyl cellulose, 10 g hydroxyethyl starch, and 1 g glutathione per 100 mL of concentrate). The mixture was cooled to -55°C at a rate of 8°C / min, frozen for 2 h, and freeze-dried in a vacuum freeze dryer at a pressure of 30 Pa and a temperature of -30°C for 30 h to obtain the freeze-dried stem cell preparations of Comparative Examples 1-5.

[0080] The stem cell preparations of Example 1 and the freeze-dried comparative examples 1-5 were mixed with a solvent (5 g VC, 6 g hyaluronic acid dissolved in 100 mL purified water) at a ratio of 1:3. The fibroblast growth factor (FGF) content was detected using an ELISA kit. The results were as follows: Figure 3 shown.

[0081] Figure 3 The figure shows the content of fibroblast factors in the stem cell preparations prepared in Example 1 and Comparative Examples 1-5. Figure 3 It can be seen that the content of fibroblast factor in the stem cell preparation prepared in Example 1 is higher than that in the stem cell preparations prepared in Comparative Examples 1-5.

[0082] Analysis shows that in Comparative Example 1, no nomilin was added to the first culture medium, in Comparative Example 2, the amount of nomilin was increased in the first culture medium, in Comparative Example 3, no koumine was added to the second culture medium, in Comparative Example 4, the amount of koumine was increased in the second culture medium, and in Comparative Example 5, no nomilin was added to the first culture medium and no koumine was added to the second culture medium. It can be seen that adjusting the amount of nomilin or koumine significantly affects the content of fibroblast factors in the prepared stem cell preparation.

[0083] Test Example 4

[0084] This test example explores the effects of the stem cell preparations prepared in Example 1 and Comparative Examples 1-5 on wound healing. The preparation process of the freeze-dried stem cell preparations in Comparative Examples 1-5 is the same as that in Test Example 3:

[0085] (1) Healthy Japanese white rabbits (18 rabbits, 3 rabbits per group) were selected and injected with sodium pentobarbital solution (3%) into the ear vein. After complete anesthesia, the subsequent experiments were performed.

[0086] (2) Shave the abdomen of the rabbit, disinfect it, and then make a wound (1 cm × 1 cm) using a scalpel;

[0087] (3) The stem cell preparations of Example 1 and Comparative Examples 1-5 were mixed with a solvent (5 g VC, 6 g hyaluronic acid dissolved in 100 mL purified water) at a ratio of 1:3 to prepare a solution. The solution of each test group was applied to the wound and bandaged with gauze, which was changed once a day.

[0088] (4) The changes in the wounds and the healing status of each wound were observed every day, and the wound healing days of the Japanese big-eared white rabbits were counted. The results are shown in Table 1.

[0089] Table 1

[0090]

[0091]

[0092] Table 1 shows the effects of the stem cell preparations prepared in Example 1 and Comparative Examples 1-5 on wound healing. As shown in Table 1, when the stem cell preparation prepared in Example 1 was used to heal rabbit wounds, the average wound healing time was significantly shorter than that in Comparative Examples 1-5. This indicates that the growth factor activity in the stem cell preparation in Example 1 was higher than that in Comparative Examples 1-5. Adjusting the dosage of nomilin or koumine in Comparative Examples 1-5 resulted in stem cell preparations with lower growth factor activity than in Example 1, leading to longer average wound healing times.

[0093] The above embodiments are only preferred embodiments of the present invention and cannot be used to limit the scope of protection of the present invention. Any non-substantial changes and replacements made by technicians in this field on the basis of the present invention fall within the scope of protection required by the present invention.

Claims

1. A method for preparing a stem cell preparation, characterized in that: The following steps are involved: (1) Adipose tissue is enzymatically digested and centrifuged to obtain a cell pellet, and the cell pellet is resuspended in a first culture medium for primary culture; the first culture medium comprises a basal culture medium and the following components added to the basal culture medium: 5%-10% FBS and 25-50 ng / mL of nomilin; (2) culturing the adipose-derived mesenchymal stem cells from step (1) until the confluence reaches 80%-85% and then subculturing; (3) inoculating the P3-P5 adipose-derived mesenchymal stem cells obtained in step (2) into a second culture medium and culturing for 24-48 hours, collecting the cell supernatant, and obtaining a concentrate by ultrafiltration and dialysis; the second culture medium comprises a basal culture medium and the following components added to the basal culture medium: 5%-10% FBS and 40-60 ng / mL of koumine; Ultrafiltration dialysis specifically involves: using an ultrafiltration membrane with a molecular weight of 40KD to perform ultrafiltration dialysis to obtain dialysate, and then passing it through an ultrafiltration membrane with a molecular weight of 100D to obtain a concentrated solution.

2. The method for preparing a stem cell preparation according to claim 1, characterized in that: The method further comprises adding a freeze-drying protective agent to the concentrated solution of step (3) and freeze-drying.

3. The method for preparing the stem cell preparation according to claim 2, characterized in that: The freeze-drying protective agent is: adding the following ingredients to every 100 mL of the concentrated solution: 5 g konjac sugar, 0.01 g sodium benzoate, 5 g carboxymethyl cellulose, 10 g hydroxyethyl starch, and 1 g glutathione.

4. The method for preparing a stem cell preparation according to claim 2, wherein: The freeze drying specifically comprises: cooling to -55°C at a rate of 8°C / min, freezing for 2 hours, and placing in a vacuum freeze dryer at a pressure of 30 Pa and a temperature of -30°C for freeze drying for 30 hours.

5. The method for preparing a stem cell preparation according to claim 1, wherein: The basic culture medium in steps (1) and (3) is DMEM / F12 culture medium.

6. The method for preparing a stem cell preparation according to claim 1, wherein: In step (1), the cell pellet is resuspended in the first culture medium at a density of 1.5×10 5 -6.5×10 5 pieces / mL.

7. The method for preparing a stem cell preparation according to claim 1, wherein: In step (3), the seeding density of adipose-derived mesenchymal stem cells in the second culture medium is 2×10 4 -5×10 4 pieces / mL.

8. The method for preparing a stem cell preparation according to claim 1, wherein: The culturing in steps (1), (2) and (3) is carried out at 37° C. and 5% CO 2 .

9. A stem cell preparation, characterized in that The preparation method is described in any one of claims 1 to 8.

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