Culture method and application of mesenchymal stem cells

Abstract

The invention discloses a culture method and application of mesenchymal stem cells. The culture method comprises the following steps: dissolving swertiamarin in a DMEM (Dulbecco Modified Eagle Medium) basal culture medium to obtain the DMEM basal culture medium containing the swertiamarin; mixing the obtained DMEM basal culture medium containing swertiamarin with the P4-generation mesenchymal stem cells, performing culture, then removing supernate, and collecting the mesenchymal stem cells; the mesenchymal stem cells cultured by the method can improve the angiogenesis capability and the inflammation inhibition capability, and when the mesenchymal stem cells cultured by the method are applied to thin endometrium treatment, the regeneration of the endometrium can be promoted, and the inflammation can be inhibited at the same time.

Description

Technical Field

[0001] The present invention relates to a culture method for treating thin endometrial cells, and more specifically, to a culture method for mesenchymal stem cells and 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 sudden drop in the number of cells and glands in the endometrial epithelial and interstitial tissues, 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. Subsequently, 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 blood flow in the uterine artery, ultimately forming refractory thin endometrium.

[0003] At present, effective treatment of thin endometrium is still a major problem, and the effect of traditional treatment is limited. Mesenchymal stem cells (MSCs) have shown a bright prospect in the treatment of thin endometrium. Animal experiments and clinical trials have confirmed that mesenchymal stem cells can increase the thickness of the endometrium, revealing their great potential in the treatment of thin endometrium, and have achieved some relatively positive results. MSCs are widely available and can be isolated from a variety of tissues such as umbilical cord, placenta or fat. MSCs can inhibit the inflammatory process, reduce tissue damage and disease symptoms by releasing a variety of anti-inflammatory factors and cytokines, which makes these MSCs have potential application prospects in the treatment of inflammatory diseases; at the same time, MSCs have the ability to promote new blood vessel formation, improve blood circulation and promote tissue repair, which makes these MSCs have application prospects in the treatment of thin endometrial lesions. In clinical disease treatment, MSCs can inhibit inflammatory response and promote angiogenesis to a certain extent through paracrine and other means, thereby playing a therapeutic role in thin uterus.

[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 in vitro 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] At present, the efficiency of promoting endometrial regeneration by MSCs cultured in conventional ways is still low. If the culture process of MSCs can be improved, thereby enhancing the ability of MSCs to suppress inflammation and promote angiogenesis at the same time, the ability of MSCs to treat thin uterus may be further improved. There is no report on the method of simultaneously enhancing the ability of mesenchymal stem cells to suppress inflammation and promote angiogenesis in the prior art. Summary of the invention

[0006] The present invention overcomes the shortcomings of the prior art and provides a method for culturing mesenchymal stem cells and an implementation method of their application, in the hope of solving the problem that the mesenchymal stem cells cultured in the prior art 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 solution:

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

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

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

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

[0012] Swertimarin is one of the main components of Swertia L. plants in the Gentianaceae family. It is mainly derived from the dried whole grass of S.mileensis TNHo et WLshi and Swertia davidi Franch. S.mileensis is also known as hepatitis grass, small green fish gall, and seven jaundice medicine. Swertia davidi Franch is also known as fish gall grass, green fish gall grass, four yellow grass, and water coptis. Swertimarin is a cyclopentadiene ether terpenoid compound extracted from Swertia plants.

[0013] The active components of Swertia japonica are used in the treatment of some inflammatory diseases. For example, Chinese invention patent document CN117064933A discloses the use of active components of Swertia japonica in western Sichuan in the preparation of drugs for treating primary biliary cholangitis. It uses Swertia japonica extract to directly contact bile duct endothelial cells to treat cholangitis, but it has no ability to promote angiogenesis and cannot be directly used for the treatment of thin endometrium. The present invention uses Swertia marin to treat thin endometrial lesions, and promotes MSCs to have better inflammation inhibition ability and angiogenesis ability through the effect of Swertia marin on MSCs.

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

[0015] Optionally, the culture conditions of 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 amount of P4 mesenchymal stem cells in step (2) is 7-8 million cells / T175 culture bottle, and the DMEM basal medium containing swertiamarin in the 175 culture bottle is at least 20 ml.

[0016] Preferably, the P4 generation mesenchymal stem cells 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. This culturing allows the P4 generation mesenchymal stem cells to grow fully.

[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 / 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 can fully provide the nutrients needed for the cells to grow from 1.5 million cells / T175 culture flask to 7-8 million cells / T175 culture flask. After the cells grow to 7-8 million cells / T175 culture flask, swertiamarin is used to influence the mesenchymal stem cells to secrete factors, and then the mesenchymal stem cells affected by swertiamarin are collected and tested.

[0022] Optionally, in step 2.1, the complete medium is at least 20 mL, and the complete medium is placed in a T175 culture flask. After the cells are cultured with the complete medium and the confluence reaches 70%-80%, the complete medium is removed, and the cells are continued to be cultured with a DMEM basal medium containing swertiamarin in an equal volume to the complete medium, that is, if the complete medium is 20 mL, the DMEM basal medium containing swertiamarin is also 20 mL. The amount of the adjusted medium has a certain effect on the final number of cells grown 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% CO2, and 80%-95% humidity. Preferably, the culture conditions are 37° C., 5% CO2, 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 in 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 are still left in the culture flask. The cells are washed with physiological saline to 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 culture medium to terminate digestion, wherein the complete culture medium is DMEM basal culture medium containing 10% volume fraction of fetal bovine serum;

[0027] 3.3. Absorb the liquid at the bottom of the culture flask and blow on the side of the cells adhering to the wall 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 bottle after the culture in step (2) is performed in a biological safety cabinet in step 3.1. In this step, the supernatant is first poured out so that the cells remain in the culture bottle, which is convenient for subsequent washing and dispersion of the cells.

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

[0032] In step 3.3, the liquid at the bottom of the culture bottle can be sucked with a pipette; blowing the side of the culture bottle where the cells adhere to the wall means sucking the liquid with a pipette and blowing gently, and the number of blowing can be 5-10 times; blowing gently can disperse and suspend the cells without destroying the cells. Blowing 5-10 times can usually make the cells fully dispersed.

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

[0034] The present invention obtains a mesenchymal stem cell through the above-mentioned culture method, and uses the mesenchymal stem cell 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 the 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 factors that inhibit inflammation (IL-10, IDO1, iNOS) increase. At the same time, the culture supernatant of the mesenchymal stem cells to which scutellaria baicalensis is added is co-cultured with umbilical vein epithelial cells (HuVECs). It is found that the mesenchymal stem cells to which scutellaria baicalensis is added can promote the increase in the number of vascular cavities formed by umbilical vein epithelial cells (HUVECs), and can also promote the inflammatory inhibition 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 concentration of pro-angiogenic factors (VEGF, bFGF, HGF) expressed outside the cells of group A and group B;

[0038] Figure 3 The q-PCR technique was used to detect the relative expression of angiogenic factors (VEGF, bFGF, HGF) mRNA in cells of group A and group B;

[0039] Figure 4The q-PCR technique was used to detect the relative mRNA expression of inflammatory inhibitors (IL-10, IDO1, and iNOS) in cells of group A and group B;

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

[0041] Figure 6 The in vitro inflammatory cell model experiment was used to detect the contents 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 solution 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 used to limit the present invention.

[0043] Example 1

[0044] Prepare cells: add 20 mL of complete medium into a T175 culture flask, add P4 mesenchymal stem cells (1.5 million / T175 culture flask), and then place the T175 culture flask in a cell culture incubator for culture. The complete medium includes DMEM basal medium and 10% volume fraction of fetal bovine serum. The culture conditions of the cell culture incubator are 37° C., 5% CO2, and humidity of about 90%. During the culture process, observe with an inverted fluorescence microscope. When the confluence of the cells reaches 75%, stop the culture, wash the cells twice with physiological saline, and set aside.

[0045] 20 mL of DMEM basal medium containing swertiamarin was prepared, wherein the concentration of swertiamarin in the medium was 50 ng / ml, and the preparation method was as follows: weighing swertiamarin and dissolving it in DMEM basal medium;

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

[0047] Collect cell supernatant: Take out the T175 culture flask in the cell culture incubator, and pour the supernatant in the T175 culture flask directly into a centrifuge tube in the biosafety cabinet. The resulting supernatant is named supernatant B for subsequent testing.

[0048] Collecting mesenchymal stem cells: After the 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 culture medium + 10% fetal bovine serum) is added to terminate the digestion. The liquid at the bottom of the T175 culture flask is sucked with a pipette, and the cell-adherent growth side of the culture flask is gently blown 5 times to suspend the cells, and then all the liquid is sucked out, and the resulting liquid is centrifuged at 500 g centrifugal force and 20° C. After removing the supernatant, the remaining suspension is cells, and 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 substantially 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 technology to detect the concentration of inflammatory inhibitory factors (IL-10, IDO1, iNOS) expressed outside the cells. 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 in supernatant A, indicating that the inflammation-inhibiting ability of the mesenchymal stem cells cultured in Example 1 using the 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 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 intracellular 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 using q-PCR technology. The detection results are as follows: Figure 3 As shown, the relative expression of inflammatory inhibitors (IL-10, IDO1, iNOS) in the cells was 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 of the mesenchymal stem cell suspension B obtained in Example 1 are significantly greater than the relative expression levels of inflammatory inhibitory factors (IL-10, IDO1, iNOS) in cells of the mesenchymal stem cell suspension A 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] The in vitro angiogenesis experiment was used to analyze the effect of the mesenchymal stem cells cultured in Example 1 and Comparative Example 1 on the vascular cavity formation of HuVECs cells, and the steps were as follows:

[0062] 1. Establish three in vitro angiogenesis models of groups A, B, and C. Evenly spread Matrigel (50 μL / well) on the bottom of a 96-well cell culture plate precooled at -20°C, and incubate the cell culture plate at 37°C, 5% CO2, and 90% humidity for 1 h to solidify the Matrigel;

[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, 50 μL of basal culture medium was added to Group A as a blank control group, 50 μL of the mesenchymal stem cell suspension obtained in Comparative Example 1 was added to Group B, and 50 μL of the mesenchymal stem cell suspension obtained in Example 1 was added to Group C.

[0064] 3. The above three groups of cells 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 the endometrial inflammation model cells stimulated by lipopolysaccharide (LPS) was verified as follows:

[0068] 1. Select four groups of 6-well culture plates, named blank control group A, normal control group B, mesenchymal stem cell group C, and swertiamarin group D, and add 2 mL of basal medium containing 10% bovine serum to the culture wells of each group of culture plates, then inoculate the endometrial epithelial cell suspension (inoculation amount 400,000 / 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] The 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 given 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, and 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 are 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 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 into a T175 culture flask, add P4 mesenchymal stem cells (1.5 million / T175 culture flask), and then place the T175 culture flask in a cell culture incubator for culture. The complete medium includes DMEM basal medium and 10% volume fraction of fetal bovine serum. The culture conditions of the cell culture incubator are 37° C., 5% CO2, and humidity of about 85%. During the culture process, observe with an inverted fluorescence microscope. When the confluence of the cells reaches 75%, stop the culture, wash the cells twice with physiological saline, and set aside.

[0078] 20 mL of DMEM basal medium containing swertiamarin was prepared, wherein the concentration of swertiamarin in the medium was 55 ng / ml, and the preparation method was as follows: weighing swertiamarin and dissolving it in DMEM basal medium;

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

[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 culture medium + 10% fetal bovine serum) is added to terminate the digestion, and the liquid at the bottom of the T175 culture flask is sucked out with a pipette, and the cell-adherent growth side of the culture flask is gently blown 5 times to suspend the cells, and then all the liquid is sucked out, and the resulting liquid is centrifuged at 500g centrifugal force and 20°C temperature conditions. After removing the supernatant, the remaining suspension is cells, and the remaining suspension is gently flicked to obtain a mesenchymal stem cell suspension.

[0082] Example 3

[0083] Prepare cells: add 20 mL of complete medium into a T175 culture flask, add P4 mesenchymal stem cells (1.5 million / T175 culture flask), and then place the T175 culture flask in a cell culture incubator for culture. The complete medium includes DMEM basal medium and 10% volume fraction of fetal bovine serum. The culture conditions of the cell culture incubator are 37° C., 5% CO2, and humidity of about 80%. During the culture process, observe with an inverted fluorescence microscope. When the confluence of the cells reaches 75%, stop the culture, wash the cells twice with physiological saline, and set aside.

[0084] 20 mL of DMEM basal medium containing swertiamarin was prepared, wherein the concentration of swertiamarin in the medium was 60 ng / ml, and the preparation method was as follows: weighing swertiamarin and dissolving it in DMEM basal medium;

[0085] Culture: Pour 20 ml of DMEM basal medium containing swertiamarin into a T175 culture flask containing the spare cells, and continue to culture 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 culture medium + 10% fetal bovine serum) is added to terminate the digestion, and the liquid at the bottom of the T175 culture flask is sucked out with a pipette, and the cell-adherent growth side of the culture flask is gently blown 5 times to suspend the cells, and then all the liquid is sucked out, and the resulting liquid is centrifuged at 500g centrifugal force and 20°C temperature conditions. After removing the supernatant, the remaining suspension is cells, and the remaining suspension is gently flicked to obtain a mesenchymal stem cell suspension.

[0088] Although the present invention is described herein with reference to the illustrative embodiments of the present invention, it should be understood that those skilled in the art can design many other modifications and implementations, which will fall within the scope and spirit of the principles disclosed in the present application. More specifically, within the scope disclosed in the present application, multiple variations and improvements can be made to the components and / or layout of the subject combination layout. In addition to the variations and improvements made to the components and / or layout, other uses will also 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 medium to obtain a DMEM basal medium containing swertiamarin; (2) culturing the P4 mesenchymal stem cells using the DMEM basal medium containing swertiamarin; (3) Collect the cultured mesenchymal stem cells.

2. The method for culturing mesenchymal stem cells according to claim 1, characterized in that: In the DMEM basal culture medium containing swertiamarin in step (1), the content of swertiamarin is 50-60 ng / ml.

3. The method for culturing mesenchymal stem cells according to claim 2, characterized in that: In the DMEM basal culture medium containing swertiamarin, the content of swertiamarin is 50 ng / ml.

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

5. The method for culturing mesenchymal stem cells according to claim 4, characterized in that: The complete culture medium in step 2.1 is at least 20 mL; the DMEM basal culture medium containing swertiamarin in step (2) is equal in volume to the complete culture medium.

6. The method for culturing mesenchymal stem cells according to claim 5, characterized in that: The culture conditions in step (2) and step 2.3 are both 37° C., 5% CO 2 , and 80%-95% humidity; the culture time in step (2) is at least 24 hours.

7. The method for culturing mesenchymal stem cells according to claim 6, characterized in that: 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.

8. The method for culturing mesenchymal stem cells according to claim 7, characterized in that: 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), 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 digestion; 3.

3. Absorb the liquid at the bottom of the T175 culture flask and blow on the side of the T175 culture flask where the cells adhere to the wall 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.

9. The method for culturing mesenchymal stem cells according to claim 8, characterized in that: In step 3.2, the cells in the T175 culture flask are digested, the amount of trypsin digestion solution used is 2.5-3.0 ml, the incubation condition is 37° C. for 2 minutes, and the amount of complete culture medium used is 5-6.0 mL; in step 3.4, centrifugation is performed at a centrifugal force of 450-550 g.

10. Use of mesenchymal stem cells obtained by the culture method according to any one of claims 1 to 9 in a preparation for treating thin endometrium.

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