Stem cell preparation and preparation method thereof

Abstract

The invention relates to the technical field of biological medicine, in particular to a stem cell preparation and a preparation method thereof. The preparation method of the stem cell preparation provided by the invention comprises the following steps: adding nomilin and other components into the first culture medium, and adding koumine and other components into the second culture medium. A stem cell proliferation test proves that the preparation method provided by the invention does not influence the proliferation activity of the stem cells. The invention also provides a stem cell preparation, and through a human skin fibroblast proliferation ability test, a cytokine content test and a wound healing test, it is proved comprehensively and from multiple angles that the stem cell preparation can improve the proliferation ability of human skin fibroblasts, promote wound healing and promote wound healing. The method is of great significance to the technical field of biological medicine.

Description

Technical Field

[0001] The present invention relates to the field of biomedical 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 exist in all multicellular tissues and can differentiate into specific cells under specific circumstances. Compared with mature cells, stem cells have the ability to self-replicate, renew, and divide, and are called "universal cells." Based on their differentiation potential, stem cells are divided into: totipotent stem cells, unipotent stem cells, and pluripotent stem cells.

[0003] Adipose-derived mesenchymal stem cells are a type of multipotent stem cell that originates from the mesoderm and can be induced to differentiate into adipocytes, neurons, etc. under specific conditions. Adipose-derived mesenchymal stem cells can usually be obtained from adipose tissue and are widely used in clinical trials because of their rich content, easy access, more stable cell properties than other stem cell cultures, strong differentiation ability, and low immunogenicity.

[0004] With the deepening of stem cell research, stem cell preparations have attracted much attention in the biomedical field. Stem cell preparations are drugs or therapeutic products with specific functions and application values ​​obtained by extracting, culturing and processing stem cells. A large number of clinical experimental results show that stem cell preparations have the characteristics of promoting tissue repair and regeneration, regulating inflammatory responses, and are thus used to treat a variety of diseases. However, in actual applications, stem cell preparations obtained by existing technologies still have technical problems such as poor application effects. In view of this, the present invention proposes a method for 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 preparation method will not affect the proliferation activity of stem cells.

[0006] The following technical solution is used 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) The 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 includes a basal culture medium and the following components added to the basal culture medium: 5%-10% FBS, 25-50 ng / mL of nomilin;

[0009] (2) culturing the adipose-derived mesenchymal stem cells in 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 a concentrated solution is obtained 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.

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

[0012] Preferably, the lyophilization protective agent is: adding the following ingredients to every 100 mL of the concentrated solution: 5 g konjac, 0.01 g sodium benzoate, 5 g carboxymethyl cellulose, 10 g hydroxyethyl starch, and 1 g 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% CO 2 .

[0019] The second purpose of the present invention is to provide a stem cell preparation, which can enhance the proliferation ability 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 provides a stem cell proliferation test to prove that the preparation method provided by the present invention does not affect the proliferation activity of stem cells;

[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 proved from multiple angles 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 biomedical technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The effect of the preparation methods of Examples 2-4 and Comparative Examples 1-5 on the proliferation activity of adipose-derived mesenchymal stem cells is shown in FIG.

[0027] Figure 2 This is a graph showing 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 The figure shows the content of fibroblast factors in the stem cell preparations prepared in Example 1 and Comparative Examples 1-5. DETAILED DESCRIPTION

[0029] The present invention is 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 a new embodiment.

[0030] Example 1

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

[0032] (1) The adipose tissue was washed with D-Hanks solution, centrifuged, and then 1% type II collagenase (3 volumes) was added for digestion for 25 min. The cell pellet was then diluted with D-Hanks solution (1.5 volumes) 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 density of the cell pellet resuspended in the first culture medium was 4×10 5 / mL, placed in an incubator at 37°C and 5% CO2 saturated humidity for primary culture;

[0033] (2) culturing the adipose-derived mesenchymal stem cells of 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 subcultured P3 adipose-derived mesenchymal stem cells;

[0034] (3) The P3 adipose-derived mesenchymal stem cells obtained in step (2) were inoculated into a 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 / mL, cultured in an incubator at 37°C and 5% CO2 saturated humidity for 36 hours, the cell supernatant was collected, the supernatant was ultrafiltered and dialyzed through an ultrafiltration membrane with a molecular weight of 40KD to obtain a dialysate, the dialysate was then passed through an ultrafiltration membrane with a molecular weight of 100D to obtain a concentrate, a lyophilization protectant was added to the concentrate (the following ingredients were added to every 100mL of the concentrate: 5g konjac sugar, 0.01g sodium benzoate, 5g carboxymethyl cellulose, 10g hydroxyethyl starch, and 1g glutathione), the temperature was lowered to -55°C at a rate of 8°C / min, frozen for 2 hours, and freeze-dried in a vacuum freeze dryer at a pressure of 30Pa and a temperature of -30°C for 30 hours 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) The adipose tissue was washed with D-Hanks solution, centrifuged, and then 1% type II collagenase (3 volumes) was added for digestion for 25 min. The cell pellet was then diluted with D-Hanks solution (1.5 volumes) 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 density of the cell pellet resuspended in the first culture medium was 4×10 5 / mL, placed in an incubator at 37°C and 5% CO2 saturated humidity for primary culture;

[0038] (2) culturing the adipose-derived mesenchymal stem cells of 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 subcultured P3 adipose-derived mesenchymal stem cells;

[0039] (3) The P3 adipose-derived mesenchymal stem cells obtained in step (2) were inoculated into a 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 / mL, cultured in an incubator at 37°C and 5% CO2 saturated humidity for 36 hours, the cell supernatant was collected, the supernatant was ultrafiltered and dialyzed through an ultrafiltration membrane with a molecular weight of 40KD to obtain a dialysate, and the dialysate was then passed through an ultrafiltration membrane with a molecular weight of 100D 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) The adipose tissue was washed with D-Hanks solution, centrifuged, and then 1% type II collagenase (3 volumes) was added for digestion for 25 min. The cell pellet was then diluted with D-Hanks solution (1.5 volumes) 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 density of the cell pellet resuspended in the first culture medium was 1.5×10 5 / mL, placed in an incubator at 37°C and 5% CO2 saturated humidity for primary culture;

[0043] (2) culturing the adipose-derived mesenchymal stem cells of 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 subcultured P4 adipose-derived mesenchymal stem cells;

[0044] (3) The P4 adipose-derived mesenchymal stem cells obtained in step (2) were inoculated into a 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 / mL, cultured in an incubator at 37°C and 5% CO2 saturated humidity for 24 hours, the cell supernatant was collected, the supernatant was ultrafiltered and dialyzed through an ultrafiltration membrane with a molecular weight of 40KD to obtain a dialysate, and the dialysate was then passed through an ultrafiltration membrane with a molecular weight of 100D 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) The adipose tissue was washed with D-Hanks solution, centrifuged, and then 1% type II collagenase (3 volumes) was added for digestion for 25 min. The cell pellet was then diluted with D-Hanks solution (1.5 volumes) 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 density of the cell pellet resuspended in the first culture medium was 6.5×10 5 / mL, placed in an incubator at 37°C and 5% CO2 saturated humidity for primary culture;

[0048] (2) culturing the adipose-derived mesenchymal stem cells of 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 subcultured P5 adipose-derived mesenchymal stem cells;

[0049] (3) The P5 adipose-derived mesenchymal stem cells obtained in step (2) were inoculated into a 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 / mL, cultured in an incubator at 37°C and 5% CO2 saturated humidity for 48 hours, the cell supernatant was collected, the supernatant was ultrafiltered and dialyzed through an ultrafiltration membrane with a molecular weight of 40KD to obtain a dialysate, and the dialysate was then passed through an ultrafiltration membrane with a molecular weight of 100D to obtain a concentrated solution, that is, the stem cell preparation.

[0050] Comparative Example 1

[0051] This comparative example provides a stem cell preparation and a preparation method thereof, which is different from Example 2 in that: no nomiline 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 is different 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 is different 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 is different 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 is different from Example 2 in that: no nomiline is added to the first culture medium, and no koumine is added to the second culture medium, and 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 the supernatant in step (3) of Examples 2-4 and Comparative Examples 1-5 was resuspended in complete culture medium and then inoculated in a 96-well plate (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 by CCK-8 method. The OD value represents the proliferation activity of mesenchymal stem cells. Figure 1 shown.

[0063] Figure 1 The figure is a graph showing 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 value of the mesenchymal stem cells obtained by the preparation methods of Examples 2-4 and Comparative Examples 1-5, that is, there is no significant difference in the proliferation activity of the mesenchymal stem cells of 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 the 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 ability of human skin fibroblasts:

[0066] (1) Preparation of culture medium

[0067] Prepare a culture medium containing stem cell preparations: add the stem cell preparations of Example 2 and Comparative Examples 1-5 to DMEM / F12 culture medium, respectively, the content of the stem cell preparations in the culture medium is 50 μg / mL, add FBS with a volume fraction of 10%, and 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 into 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 in 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 into 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 / well, add 100 μL DMEM / F12 medium (containing 10% FBS) to each well, and culture in an incubator at 37°C and 5% CO2 saturated humidity for 24 hours, remove the medium, and add the medium containing the stem cell preparations of Example 2 and Comparative Examples 1 to 5, the medium of Control Group 1, the medium of Control Group 2, and the medium of Control Group 3 respectively, and culture in an incubator at 37°C and 5% CO2 saturated humidity for 2 days;

[0073] (3) Test samples

[0074] 10 μL CCK-8 reagent was added to each well, incubated at 37°C in the dark for 1 h, and the OD value at 450 nm was detected. The OD value represents the 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 It can be seen that the OD value of Example 2 is significantly higher than that of Control Group 1; and the proliferation ability of human skin fibroblasts cultured with the stem cell preparations of Comparative Examples 1-5 is reduced compared with Example 2. The above results show that the stem cell preparations prepared by the present invention can effectively enhance the proliferation ability of skin fibroblasts.

[0076] There was no significant difference in the proliferation ability of human skin fibroblasts between control group 2 and control group 3 and control group 1. Compared with control group 1, the culture medium of control group 2 also added nomilin, and the culture medium of control group 3 also added koumine. This shows that nomilin or koumine does not affect the proliferation ability 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 lyophilization protective agent was added to the stem cell preparation prepared in Comparative Example 1-5 (the following ingredients were added to every 100 mL of the concentrate: 5 g konjac, 0.01 g sodium benzoate, 5 g carboxymethyl cellulose, 10 g hydroxyethyl starch, and 1 g glutathione), the temperature was cooled to -55 ° C at a rate of 8 ° C / min, frozen for 2 h, and placed in a vacuum freeze dryer at a pressure of 30 Pa and a temperature of -30 ° C for lyophilization for 30 h to obtain the lyophilized stem cell preparation of Comparative Example 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, and the content of fibroblast factor (FGF) was detected using an ELISA kit. The results are as follows: Figure 3 shown.

[0081] Figure 3 The figure is a graph showing 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] It is known from the analysis that in the first culture medium of comparative example 1, no nomilin was added, in the first culture medium of comparative example 2, the amount of nomilin was increased, in the second culture medium of comparative example 3, no koumine was added, in the second culture medium of comparative example 4, the amount of koumine was increased, and in the first culture medium of comparative example 5, no nomilin was added and koumine was not added to the second culture medium. It can be seen that adjusting the amount of nomilin or koumine will significantly affect the content of fibroblast factor 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, wherein 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 in total, 3 in each group) were selected and injected with sodium pentobarbital solution (concentration 3%) into the ear vein. Subsequent experiments were performed after complete anesthesia.

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

[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 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 can be seen from Table 1, when the stem cell preparation prepared in Example 1 is used for wound healing in rabbits, the average number of days for wound healing in rabbits is significantly less than that in Comparative Examples 1-5, which indicates that the growth factor activity in the stem cell preparation in Example 1 is higher than that in Comparative Examples 1-5. Adjusting the dosage of nomilin or koumine in Comparative Examples 1-5 will result in the growth factor activity of the prepared stem cell preparation being lower than that in Example 1, which in turn results in a longer average time for wound healing.

[0093] The above-mentioned 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 substitutions made by technicians in this field on the basis of the present invention shall 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) The 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 includes a basal culture medium and the following components added to the basal culture medium: 5%-10% FBS, 25-50 ng / mL of nomilin; (2) culturing the adipose-derived mesenchymal stem cells in step (1) until the confluence reaches 80%-85% and then subculturing; (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 a concentrated solution is obtained 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.

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

3. The method for preparing a stem cell preparation according to claim 2, characterized in that: The freeze-drying protective agent is: the following ingredients are added to every 100 mL of the concentrated solution: 5 g konjac, 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, characterized in that: 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, characterized in that: 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, characterized in that: 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, characterized in that: 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, characterized in that: The ultrafiltration dialysis in step (3) is specifically as follows: 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.

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

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

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