A method for culturing mesenchymal stem cells and its application

Abstract

The present invention discloses a culture method and application of mesenchymal stem cells. The method comprises the following steps: dissolving swetiamarin in a DMEM basal culture medium to obtain a DMEM basal culture medium containing swetiamarin; mixing the obtained DMEM basal culture medium containing swetiamarin with P4 generation mesenchymal stem cells, culturing the mixture, removing the supernatant, and collecting the mesenchymal stem cells. The mesenchymal stem cells cultured by the present invention can enhance angiogenesis ability and inflammation inhibition ability, and applying the cultured mesenchymal stem cells by the present invention to the treatment of thin endometrium can promote endometrial regeneration while inhibiting inflammation.

Description

Technical Field

[0001] The present invention relates to a culture method for treating thin endometrial cells, and more particularly to a culture method for mesenchymal stem cells and an application thereof. Background Art

[0002] The female uterus is prone to thinning of the endometrium due to endocrine disorders, infections, intrauterine operations and other reasons. In addition, factors such as uterine inflammation can easily cause pathological phenomena in the uterus. The pathology of thin endometrium (thickness ≤ 7mm) refers to severe inflammatory reactions or damage to the basal layer of the uterus, which can cause a sharp drop in the number of cells and glands in the endometrial epithelium and interstitial tissue, and lose the physiological secretory function of the endometrium. Inflammatory factors invade the endometrium and engulf the epithelial cells on its surface, resulting in interstitial exposure, reduced interstitial cell elasticity, increased fiber activity, and the normal boundary between the functional layer and the basal layer of the endometrium becomes blurred and difficult to identify, or even disappears completely. Then there is a lack of epithelial cell regeneration, the epithelial layer does not respond to hormonal stimulation, inflammatory factors promote adhesion of fibrous tissue, damaged and deficient blood vessels in the interstitium, and high resistance to uterine artery blood flow, ultimately forming refractory thin endometrium.

[0003] Currently, effective treatment for thin endometrium remains a major challenge, with traditional treatments having limited efficacy. Mesenchymal stem cells (MSCs) have shown promising promise in the treatment of thin endometrium. Both animal studies and clinical trials have demonstrated their ability to increase endometrial thickness, revealing their significant potential for treating thin endometrium and yielding some promising results. MSCs are widely available and can be isolated from various tissues, including the umbilical cord, placenta, and adipose tissue. By releasing a variety of anti-inflammatory factors and cytokines, MSCs can inhibit inflammation, alleviate tissue damage, and alleviate disease symptoms, making them promising candidates for the treatment of inflammatory diseases. MSCs also have the ability to promote angiogenesis, improving blood circulation and promoting tissue repair, making them promising candidates for the treatment of thin endometrial lesions. In clinical treatment, MSCs can inhibit inflammation and promote angiogenesis through paracrine and other mechanisms, thereby potentially treating thin endometrium.

[0004] Chinese invention patent document CN115364119A discloses the use of adipose-derived mesenchymal stem cells in the preparation of a drug for treating thin endometrium. The drug extracts adipose-derived mesenchymal stem cells from ex vivo adipose tissue and treats thin endometrium in patients by intrauterine administration or intraperitoneal injection. This treatment method can achieve a certain effect on the regeneration of the endometrium, but it cannot solve the root cause of thin uterus, namely uterine inflammation.

[0005] Currently, conventionally cultured MSCs are still inefficient in promoting endometrial regeneration. Improving the MSC culture process, thereby simultaneously enhancing their ability to suppress inflammation and promote angiogenesis, could further enhance the ability of MSCs to treat thin uteri. However, methods for simultaneously enhancing the anti-inflammatory and angiogenic abilities of mesenchymal stem cells have not been reported. Summary of the Invention

[0006] The present invention overcomes the shortcomings of the existing technology and provides a method for culturing mesenchymal stem cells and an implementation method for their application, in the hope of solving the problem that the mesenchymal stem cells cultured in the existing technology are difficult to simultaneously have good inflammation inhibition ability and angiogenesis promotion ability, thereby resulting in insufficient ability to treat thin uterus.

[0007] In order to solve the above technical problems, one embodiment of the present invention adopts the following technical solutions:

[0008] A method for culturing mesenchymal stem cells comprises the following steps:

[0009] (1) dissolving swertiamarin in DMEM basal culture medium to obtain a DMEM basal culture medium containing swertiamarin;

[0010] (2) culturing the P4 mesenchymal stem cells with the DMEM basal medium containing swertiamarin;

[0011] (3) Collect the cultured mesenchymal stem cells.

[0012] Swertimarin is a major component of Swertia L. plants in the Gentianaceae family. It is primarily derived from the dried whole herb of Swertia mileensis (TNHo et WLshi) and Swertia davidi Franch (Swertia davidi Franch). Swertimarin is also known as hepatitis grass, small herring gall, and seven-jaundice medicine. Swertia davidi Franch is also known as fish gall grass, herring gall grass, four-yellow grass, and water coptis root. Swertimarin is an iridoid terpenoid compound extracted from Swertia plants.

[0013] Active components of Swertia japonica are used in the treatment of some inflammatory diseases. For example, Chinese invention patent CN117064933A discloses the use of active components of Swertia japonica from western Sichuan in the preparation of a drug for treating primary biliary cholangitis. This drug uses a Swertia japonica extract in direct contact with bile duct endothelial cells to treat cholangitis. However, Swertia japonica extract lacks the ability to promote angiogenesis and cannot be directly applied to the treatment of thin endometrium. The present invention uses Swertiamarin to treat thin endometrial lesions. Through its effect on MSCs, Swertiamarin promotes MSCs to have both better inflammation suppression and angiogenesis.

[0014] Optionally, the content of scutamarin in the DMEM basal medium containing scutamarin in step (1) is 50-60 ng / ml. Preferably, the content of scutamarin is 50 ng / ml. The culture can be carried out in a T175 culture flask, or other culture flasks can be used instead.

[0015] Optionally, the culture conditions in step (2) are 37° C., 5% CO2, and 80%-95% humidity; preferably, the culture conditions are 37° C., 5% CO2, and 90% humidity. After the culture is completed, the number of P4 mesenchymal stem cells in step (2) is 7-8 million per T175 culture flask, and the DMEM basal medium containing swertiamarin in the 175 culture flask is at least 20 ml.

[0016] Preferably, the P4 generation mesenchymal stem cells in step (2) are cultured in a complete medium, wherein the complete medium is obtained by adding 10% by volume of fetal bovine serum to DMEM basal medium. This culture allows the P4 generation mesenchymal stem cells to fully grow.

[0017] The steps of culturing the P4 mesenchymal stem cells used in step (2) with complete medium are as follows:

[0018] 2.1. Prepare complete culture medium;

[0019] 2.2. Add P4 mesenchymal stem cells at a rate of 1.5 million cells / T175 culture flask;

[0020] 2.3. Cultivate until the cell confluence reaches 70%-80% and then stop culturing.

[0021] The complete culture medium contains fetal bovine serum, which provides the nutrients necessary for cell growth from 1.5 million cells per T175 flask to 7-8 million cells per T175 flask. After the cells reach 7-8 million cells per T175 flask, scutellaria serrata is used to stimulate the secretion of factors by the mesenchymal stem cells, which are then collected and tested.

[0022] Optionally, in step 2.1, the volume of complete medium is at least 20 mL, and the complete medium is placed in a T175 culture flask. After the cells reach 70%-80% confluence in the complete medium, the complete medium is removed and the cells are cultured again with an equal volume of DMEM basal medium containing scutellarin. That is, if the volume of complete medium is 20 mL, the volume of DMEM basal medium containing scutellarin should also be 20 mL. Adjusting the amount of medium used will have a certain effect on the final cell number after growth in the T175 culture flask.

[0023] Preferably, the P4 mesenchymal stem cells in step 2.3 are cultured in a cell culture incubator at 37° C., 5% CO 2 , and 80%-95% humidity. Preferably, the culture conditions are 37° C., 5% CO 2 , and 90% humidity.

[0024] The method of collecting mesenchymal stem cells in step (3) comprises the following steps:

[0025] 3.1. Pour out the supernatant from the culture flask after the culture in step (2) is completed, and wash the cells in the culture flask once with physiological saline. In this step, the supernatant is poured out, and the cells remain in the culture flask. Use physiological saline to wash the cells and remove the residual culture medium.

[0026] 3.2. Add trypsin digestion solution to the culture flask, gently shake the culture flask for incubation, and add complete medium to terminate the digestion. The complete medium is DMEM basal medium containing 10% volume fraction of fetal bovine serum;

[0027] 3.3. Aspirate the liquid at the bottom of the culture flask and blow on the adherent side of the culture flask to suspend the cells;

[0028] 3.4. Aspirate all the cell suspension, centrifuge at 18-22℃, pour out the supernatant, and gently flick to obtain the cell suspension.

[0029] The culture bottle can be a T175 culture bottle.

[0030] Optionally, the operation of pouring out the supernatant in the culture flask after the culture in step (2) is performed in a biological safety cabinet in step 3.1. In this step, the supernatant is poured out first, so that the cells remain in the culture flask, which is convenient for subsequent washing and dispersion of the cells.

[0031] In step 3.2, for digestion of cells in a T175 culture flask, use 2.5-3.0 ml of trypsin digestion solution. The incubation conditions are 37°C for 2 minutes. The amount of complete medium used is twice the volume of trypsin, that is, 5-6.0 ml.

[0032] In step 3.3, you can use a pipette to aspirate the liquid from the bottom of the culture flask. To pipette the adherent side of the flask, pipette the liquid and gently pipette 5-10 times. Gently pipette to disperse and suspend the cells without damaging them. 5-10 pipettings usually allow the cells to fully disperse.

[0033] In step 3.4, the centrifugation is performed at a centrifugal force of 450-550 g, preferably at a centrifugal force of 500 g, and the centrifugation temperature is 20°C.

[0034] The present invention obtains mesenchymal stem cells through the above-mentioned culture method, and uses the mesenchymal stem cells in a preparation for treating thin endometrium.

[0035] Compared with the prior art, the present invention has at least the following beneficial effects: the present invention adds scutellaria baicalensis to the culture medium and then cultures mesenchymal stem cells. After testing, it is found that the concentrations of angiogenesis-related factors (bFGF, VEGF, HGF) of the mesenchymal stem cells increase, and the concentrations of inflammation-inhibiting factors (IL-10, IDO1, iNOS) increase. At the same time, the culture supernatant of the mesenchymal stem cells with scutellaria baicalensis is co-cultured with umbilical vein epithelial cells (HuVECs). It is found that the mesenchymal stem cells with the addition of scutellaria baicalensis can promote the increase in the number of vascular cavities formed by umbilical vein epithelial cells (HUVECs), and can also promote the inflammatory inhibitory ability of the mesenchymal stem cells in the endometrial cell inflammation model caused by lipopolysaccharide (LPS) (reducing the concentrations of TNF-α, IL-6, and IL-1β secreted by the inflammatory endometrial model cells). Therefore, this method can obtain umbilical cord mesenchymal stem cells that have both the ability to promote angiogenesis and the ability to inhibit inflammation. The use of the mesenchymal stem cells cultured in the present invention in the treatment of thin endometrium can promote endometrial regeneration while inhibiting inflammation. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 The ELISA technique was used to detect the concentrations of inflammatory inhibitory factors (IL-10, IDO1, and iNOS) expressed outside the cells of group A and group B;

[0037] Figure 2 The ELISA technique was used to detect the concentrations of pro-angiogenic factors (VEGF, bFGF, HGF) expressed outside the cells of group A and group B;

[0038] Figure 3 The relative expression of angiogenic factors (VEGF, bFGF, HGF) mRNA in cells of group A and group B was detected by q-PCR technology;

[0039] Figure 4The relative mRNA expression of inflammatory inhibitors (IL-10, IDO1, and iNOS) in cells of group A and group B was detected by q-PCR technology;

[0040] Figure 5 is the number of vascular lumens formed in each group detected by in vitro angiogenesis assay;

[0041] Figure 6 An in vitro inflammatory cell model experiment was used to detect the levels of inflammatory factors IL-6, IL-1β and TNF-α in the cell culture supernatant of each group. DETAILED DESCRIPTION

[0042] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0043] Example 1

[0044] Prepare cells: Add 20 mL of complete medium to a T175 culture flask, add P4 mesenchymal stem cells (1.5 million cells / T175 culture flask), and then culture the T175 culture flask in a cell culture incubator. The complete medium consists of DMEM basal medium and 10% fetal bovine serum by volume. The incubator is maintained at 37°C, 5% CO2, and approximately 90% humidity. Observe the culture under an inverted fluorescence microscope during the culture process. Stop the culture when the cell confluence reaches 75%. Wash the cells twice with saline and set aside.

[0045] 20 mL of DMEM basal medium containing scutamarin was prepared, wherein the concentration of scutamarin in the medium was 50 ng / mL. The preparation method was as follows: scutamarin was weighed and dissolved in DMEM basal medium;

[0046] Culture: Pour 20 ml of DMEM basal medium containing swedemarin into a T175 culture flask containing the spare cells, and continue culturing for 24 hours in a cell culture incubator at 37° C., 5% CO 2 , and 90% humidity.

[0047] Collect cell supernatant: Remove the T175 culture flask from the cell culture incubator and pour the supernatant directly into a centrifuge tube in a biosafety cabinet. Name the resulting supernatant Supernatant B for subsequent testing.

[0048] Collecting mesenchymal stem cells: After supernatant B is collected, the remaining mesenchymal stem cells in the T175 culture flask are washed once with physiological saline, and then 2.5 mL of trypsin digestion solution is added to the culture flask, and the culture flask is gently shaken and incubated at 37°C for 2 minutes. Finally, 5 mL of complete culture medium (DMEM basal medium + 10% fetal bovine serum) is added to terminate digestion. The liquid at the bottom of the T175 culture flask is aspirated with a pipette, and the cell-adherent growth side of the culture flask is gently blown 5 times to suspend the cells. Then all the liquid is aspirated, and the resulting liquid is centrifuged at 500g centrifugal force and 20°C temperature. After removing the supernatant, the remaining suspension is cells. The remaining suspension is gently flicked to obtain a mesenchymal stem cell suspension. The mesenchymal stem cell suspension obtained in this example is named mesenchymal stem cell suspension B.

[0049] Comparative Example 1

[0050] Comparative Example 1 is basically the same as Example 1, except that: the DMEM basal medium used to replace the complete medium does not contain swertiamarin; the collected supernatant is named supernatant A; and the collected mesenchymal stem cell suspension is named mesenchymal stem cell suspension A.

[0051] 1. ELISA technology to detect extracellular expression

[0052] The supernatant B obtained in Example 1 and the supernatant A obtained in Comparative Example 1 were tested by ELISA to detect the concentration of extracellular inflammatory inhibitory factors (IL-10, IDO1, iNOS). The test results are as follows: Figure 1 As shown, the concentration of angiogenic factors (bFGF, VEGF, HGF) expressed outside the cells was detected at the same time. The test results are as follows Figure 2 shown.

[0053] from Figure 1 It can be seen that the concentration of inflammation-inhibiting related factors (IL-10, IDO1, iNOS) contained in supernatant B is significantly higher than that contained in supernatant A, indicating that the inflammation-inhibiting ability of the mesenchymal stem cells cultured in Example 1 using a culture medium containing swertiamarin is stronger than that of the mesenchymal stem cells cultured in Comparative Example 1.

[0054] from Figure 2 It can be seen that the concentration of angiogenesis-related factors (bFGF, VEGF, HGF) contained in supernatant B is significantly higher than that contained in supernatant A, indicating that the mesenchymal stem cells cultured in the culture medium containing swertiamarin in Example 1 have a stronger ability to promote angiogenesis than the mesenchymal stem cells cultured in Comparative Example 1, and can more effectively promote tissue growth and wound healing.

[0055] 2. q-PCR technology to detect intracellular expression

[0056] The relative expression levels of angiogenic factors (bFGF, VEGF, HGF) in the mesenchymal stem cell suspension B obtained in Example 1 and the mesenchymal stem cell suspension A obtained in Comparative Example 1 were detected by q-PCR. Figure 3 As shown, the relative expression levels of inflammatory inhibitors (IL-10, IDO1, iNOS) in the cells were detected. The test results are shown in Figure 4 shown.

[0057] from Figure 3 It can be seen that the relative expression levels of angiogenic factors (bFGF, VEGF, HGF) in cells B of the mesenchymal stem cell suspension obtained in Example 1 are significantly greater than the relative expression levels of angiogenic factors (bFGF, VEGF, HGF) in cells A of the mesenchymal stem cell suspension obtained in Comparative Example 1.

[0058] from Figure 4 It can be seen that the relative expression levels of inflammatory inhibitory factors (IL-10, IDO1, iNOS) in cells B of the mesenchymal stem cell suspension obtained in Example 1 are significantly greater than the relative expression levels of inflammatory inhibitory factors (IL-10, IDO1, iNOS) in cells A of the mesenchymal stem cell suspension obtained in Comparative Example 1.

[0059] Figures 1 to 4 It can be seen that, whether extracellularly or intracellularly, the relative expression levels of angiogenic factors (bFGF, VEGF, HGF) and the relative expression levels of inflammatory inhibitors (IL-10, IDO1, iNOS) in Example 1 are higher than those in Comparative Example 1, indicating that culturing mesenchymal stem cells with a culture medium containing swertiamarin promotes the expression of inflammatory inhibitors and angiogenic factors by mesenchymal stem cells.

[0060] 3. In vitro angiogenesis assay

[0061] An in vitro angiogenesis experiment was performed to analyze the effect of the mesenchymal stem cells cultured in Example 1 and Comparative Example 1 on the vascular lumen formation of HuVECs cells. The steps were as follows:

[0062] 1. Establish three in vitro angiogenesis models (A, B, and C). Matrigel (50 μL / well) was evenly spread on the bottom of a 96-well cell culture plate pre-cooled at -20°C. The cell culture plate was incubated at 37°C, 5% CO2, and 90% humidity for 1 h to allow the Matrigel to solidify.

[0063] 2. Inoculate HuVECs single cell suspension in the culture wells of the three groups of cell culture plates A, B, and C. Inoculate 3×10 HuVECs single cell suspension in each well.5 In addition, Group A was added with 50 μL of basal culture medium as a blank control group, Group B was added with 50 μL of the mesenchymal stem cell suspension obtained in Comparative Example 1, and Group C was added with 50 μL of the mesenchymal stem cell suspension obtained in Example 1.

[0064] 3. The cells in groups A, B, and C were incubated together at 37°C, 5% CO2, and 90% humidity for 6 hours, and then photographed using a fluorescence microscope to observe and count the number of vascular cavities formed.

[0065] from Figure 5 It can be seen that the number of blood vessel cavities formed in group B is significantly greater than that in group A, indicating that the mesenchymal stem cells cultured in comparative example 1 can promote angiogenesis, and the number of blood vessel cavities formed in group C is significantly greater than that in group B, indicating that the mesenchymal stem cell suspension prepared in Example 1 can further promote the angiogenesis-promoting ability of mesenchymal stem cells for HuVECs cells.

[0066] 4. Verification of anti-inflammatory effects

[0067] The anti-inflammatory effect of lipopolysaccharide (LPS)-stimulated endometrial inflammation model cells was verified as follows:

[0068] 1. Select four groups of 6-well culture plates and designate them as blank control group A, normal control group B, mesenchymal stem cell group C, and swertiamarin group D. First, add 2 mL of basal medium containing 10% bovine serum to the culture wells of each culture plate, then inoculate the endometrial epithelial cell suspension (inoculation size 400,000 cells / well) and culture overnight at 37°C, 5% CO2, and 90% humidity.

[0069] 2. The blank control group A culture plate was given 100 μL of basal culture medium and 100 μL of normal saline;

[0070] Normal control group B was given 100 μL of basal medium and 100 μL of 25 μg / mL LPS;

[0071] Mesenchymal stem cell group C was given 100 μL of 25 μg / mL LPS and 100 μL of supernatant A obtained in comparative example 1;

[0072] The swertiamarin group D was administered with 100 μL of 25 μg / mL LPS and 100 μL of the supernatant B containing swertiamarin obtained in Example 1;

[0073] 3. The above four groups of cells were cultured at 37°C, 5% CO2, and 90% humidity for 24 hours. Then the ELISA method was used to determine the content of IL-6, IL-1β, and TNF-α in the culture supernatant of each group of cells. The test results were as follows: Figure 6 .

[0074] from Figure 6 It can be seen that the mesenchymal stem cells (Group D) obtained by culturing with the addition of swertiamarin can promote the inflammatory inhibitory ability of mesenchymal stem cells in the LPS-induced endometrial cell inflammation model (reducing the concentration of TNF-α, IL-6, and IL-1β secreted by the inflammatory endometrial model cells), and the effect is significantly better than that of Group C.

[0075] The above experimental results show that compared with stem cells cultured by conventional methods, the mesenchymal stem cells obtained by the method of the present invention have more obvious abilities to promote angiogenesis and inhibit inflammation. The application of mesenchymal stem cells cultured by the present invention in the treatment of thin endometrium can promote endometrial regeneration while inhibiting inflammation.

[0076] Example 2

[0077] Prepare cells: Add 20 mL of complete medium to a T175 culture flask, add P4 mesenchymal stem cells (1.5 million cells / T175 culture flask), and then culture the T175 culture flask in a cell culture incubator. The complete medium consists of DMEM basal medium and 10% fetal bovine serum by volume. The incubator is maintained at 37°C, 5% CO2, and approximately 85% humidity. Observe the cells under an inverted fluorescence microscope during the culture process. Stop the culture when the cell confluence reaches 75%. Wash the cells twice with saline and set aside.

[0078] 20 mL of DMEM basal medium containing scutamarin was prepared, wherein the concentration of scutamarin in the medium was 55 ng / mL. The preparation method was as follows: scutamarin was weighed and dissolved in DMEM basal medium;

[0079] Culture: Pour 20 ml of DMEM basal medium containing swedemarin into a T175 culture flask containing the spare cells, and continue to culture in a cell culture incubator at 37° C., 5% CO 2 , and 85% humidity for 24 hours.

[0080] Remove the cell supernatant: Take out the T175 culture flask from the cell culture incubator and pour out the supernatant in the T175 culture flask directly in the biosafety cabinet.

[0081] Collecting mesenchymal stem cells: After the supernatant is removed, the remaining mesenchymal stem cells in the T175 culture flask are washed once with physiological saline, and then 2.5 mL of trypsin digestion solution is added to the culture flask, and the culture flask is gently shaken and incubated at 37°C for 2 minutes. Finally, 5 mL of complete culture medium (DMEM basal medium + 10% fetal bovine serum) is added to terminate the digestion. Use a pipette to aspirate the liquid at the bottom of the T175 culture flask, and gently blow the cell-adherent growth side of the culture flask 5 times to suspend the cells. Then all the liquid is aspirated and the resulting liquid is centrifuged at 500g centrifugal force and 20°C temperature. After removing the supernatant, the remaining suspension is cells. Gently flick the remaining suspension to obtain a mesenchymal stem cell suspension.

[0082] Example 3

[0083] Prepare cells: Add 20 mL of complete medium to a T175 culture flask, add P4 mesenchymal stem cells (1.5 million cells / T175 culture flask), and then culture the T175 culture flask in a cell culture incubator. The complete medium consists of DMEM basal medium and 10% fetal bovine serum by volume. The incubator is maintained at 37°C, 5% CO2, and approximately 80% humidity. Observe the cells under an inverted fluorescence microscope during the culture process. Stop the culture when the cell confluence reaches 75%. Wash the cells twice with saline and set aside.

[0084] 20 mL of DMEM basal medium containing scutamarin was prepared, wherein the concentration of scutamarin in the medium was 60 ng / mL. The preparation method was as follows: scutamarin was weighed and dissolved in DMEM basal medium;

[0085] Culture: Pour 20 ml of DMEM basal medium containing swedemarin into a T175 culture flask containing the spare cells, and continue culturing for 24 hours in a cell culture incubator at 37° C., 5% CO 2 , and 80% humidity.

[0086] Remove the cell supernatant: Take out the T175 culture flask from the cell culture incubator and pour out the supernatant in the T175 culture flask in the biosafety cabinet.

[0087] Collecting mesenchymal stem cells: After the supernatant is removed, the remaining mesenchymal stem cells in the T175 culture flask are washed once with physiological saline, and then 2.5 mL of trypsin digestion solution is added to the culture flask, and the culture flask is gently shaken and incubated at 37°C for 2 minutes. Finally, 5 mL of complete culture medium (DMEM basal medium + 10% fetal bovine serum) is added to terminate the digestion. Use a pipette to aspirate the liquid at the bottom of the T175 culture flask, and gently blow the cell-adherent growth side of the culture flask 5 times to suspend the cells. Then all the liquid is aspirated and the resulting liquid is centrifuged at 500g centrifugal force and 20°C temperature. After removing the supernatant, the remaining suspension is cells. Gently flick the remaining suspension to obtain a mesenchymal stem cell suspension.

[0088] Although the present invention has been described herein with reference to illustrative embodiments of the present invention, it will be appreciated that those skilled in the art may devise numerous other modifications and implementations that fall within the scope and spirit of the principles disclosed herein. More specifically, within the scope disclosed herein, various variations and improvements may be made to the components and / or layout of the subject combination layout. In addition to variations and improvements made to the components and / or layout, other uses will be apparent to those skilled in the art.

Claims

1. A method for culturing mesenchymal stem cells, characterized in that: The steps include: (1) dissolving swertiamarin in DMEM basal culture medium to obtain DMEM basal culture medium containing swertiamarin; (2) culturing the P4 mesenchymal stem cells using the DMEM basal medium containing swertiamarin; (3) Collecting cultured mesenchymal stem cells; In the DMEM basal medium containing scutellaria serrata in step (1), the content of scutellaria serrata is 50 ng / ml; The incubation time in step (2) is at least 24 hours.

2. The method for culturing mesenchymal stem cells according to claim 1, wherein The P4 generation mesenchymal stem cells used in step (2) are obtained by culturing with a complete medium, wherein the complete medium is obtained by adding 10% volume fraction of fetal bovine serum to DMEM basal medium; the steps of culturing the P4 generation mesenchymal stem cells used in step (2) with the complete medium include: 2.

1. Prepare complete culture medium; 2.

2. Add P4 mesenchymal stem cells; 2.

3. Cultivate until the cell confluence reaches 70%-80% and then stop culturing.

3. The method for culturing mesenchymal stem cells according to claim 2, wherein: The complete culture medium in step 2.1 is at least 20 mL; the volume of the DMEM basal culture medium containing swertiamarin in step (2) is equal to the volume of the complete culture medium.

4. The method for culturing mesenchymal stem cells according to claim 3, wherein: The culture conditions in step (2) and step 2.3 are both 37°C, 5% CO2, and 80%-95% humidity.

5. The method for culturing mesenchymal stem cells according to claim 4, wherein: The culture in step 2.3 is carried out in a T175 culture flask, and the amount of P4 mesenchymal stem cells added to the T175 culture flask is 1.5 million / T175 culture flask.

6. The method for culturing mesenchymal stem cells according to claim 5, wherein: The method of collecting mesenchymal stem cells in step (3) comprises the following steps: 3.

1. Pour out the supernatant in the T175 culture flask after the culture in step (2) is completed, and wash the cells in the T175 culture flask with physiological saline; 3.

2. Add trypsin digestion solution to the T175 culture flask, shake the T175 culture flask for incubation, and add complete culture medium to terminate the digestion; 3.

3. Aspirate the liquid at the bottom of the T175 culture flask and pipette the cells on the adherent side of the flask to suspend the cells. 3.

4. Aspirate all the cell suspension, centrifuge at 18-22℃, pour out the supernatant, and flick to obtain the cell suspension.

7. The method for culturing mesenchymal stem cells according to claim 6, wherein: In step 3.2, the cells in the T175 culture flask are digested with 2.5-3.0 ml of trypsin digestion solution, the incubation condition is 37° C. for 2 minutes, and the amount of complete culture medium is 5-6.0 mL; in step 3.4, centrifugation is performed at a centrifugal force of 450-550 g.

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