Kit and culture medium for osteogenic induced differentiation and preparation method of culture medium

Abstract

The invention provides an osteogenic induced differentiation kit, a culture medium and a preparation method of the culture medium, and particularly relates to the technical field of stem cells. The kit comprises a DMEM (dulbecco's modified eagle medium) basal culture medium, fetal calf serum, dexamethasone, vitamin C, beta-glycerol phosphate, mesenchymal stem cell osteogenesis induced supernatant, alizarin red S staining solution and acetic acid. According to the kit provided by the invention, the problems of slow calcium nodule formation, small calcium nodule quantity and small calcium nodule in the osteogenic induced differentiation of the umbilical cord mesenchymal stem cells in the prior art are solved, so that the calcium nodule formation of the umbilical cord mesenchymal stem cells is effectively promoted, meanwhile, the calcium nodule quantity can be increased, and the osteogenic induced differentiation period is shortened.

Description

Technical Field

[0001] The present invention relates to the technical field of stem cells, and in particular to a kit and culture medium for osteogenic differentiation induction, and a method for preparing the culture medium. Background Art

[0002] From the 19th century to the present, drug development has evolved through the stages of small-molecule chemical drugs, large-molecule biological drugs, and biopharmaceuticals. Biopharmaceuticals are drugs extracted from organisms or synthesized through bioengineering methods. They are fundamentally different from traditional chemical drugs, which can be fully synthesized artificially. Traditional chemical drugs are generally small molecules, while biopharmaceuticals are generally complex mixtures or biological macromolecules. They have good efficacy and safety and can be divided into several major categories, including antibody drugs, cytokines, blood products, gene and cell therapy drugs, enzymes, and hormones. Cell therapy drugs have been one of the most popular areas of drug development globally in recent years. Cell therapy drugs can be divided into stem cell therapy and immune cell therapy based on cell type. Stem cells are cells with multidirectional differentiation potential and self-renewal ability. They are the most primitive cells at the apex of cell lineage origin and can differentiate into cells of specific tissue types in the body. The main types of stem cells used in clinical treatment include bone marrow stem cells, hematopoietic stem cells, neural stem cells, skin stem cells, pancreatic stem cells, and adipose tissue stem cells. Stem cell therapy utilizes the differentiation and repair mechanisms of human stem cells to transplant healthy stem cells into the body to repair diseased cells or rebuild functioning cells and tissues. Mesenchymal stem cells (MSCs) are stem cells with multidirectional differentiation potential. According to the International Society for Cellular Therapy's definition of the minimum standard for MSCs, MSCs should have the following characteristics: ① When cultured under standard conditions, they must have adhesion properties to plastic substrates; ② Flow cytometry detection of the positive expression rate of CD105, CD73, and CD90 surface markers in MSCs should be ≥95%, and the positive expression rate of CD45, CD34, CD14 or CD11b, CD79a or CD19, and HLA-DR should be ≤2%; ③ After induction in vitro using standard methods, MSCs can be induced to differentiate into osteoblasts, adipocytes, chondrocytes, etc. The three-way induced differentiation of mesenchymal stem cells is one of the minimum standards for identifying mesenchymal stem cells. However, in osteogenic induced differentiation, umbilical cord mesenchymal stem cells have a longer induced differentiation cycle than adipose mesenchymal stem cells and bone marrow mesenchymal stem cells, with slow formation of calcium nodules, extremely few calcium nodules and extremely small calcium nodules.

[0003] In response to the above problems, Zuk et al. proposed a classic osteogenic differentiation medium in 2001, which contains DMEM basal medium, 10% fetal bovine serum, dexamethasone, vitamin C, and β-glycerophosphate. This osteogenic differentiation medium formed more and larger calcium nodules during the osteogenic differentiation of adipose-derived mesenchymal stem cells, but formed fewer and smaller calcium nodules during the osteogenic differentiation of umbilical cord-derived mesenchymal stem cells. Even when the induction time was extended to 2-3 times that of the osteogenic differentiation of adipose-derived mesenchymal stem cells, the effect was still unsatisfactory.

[0004] In summary, how to solve the problem of fewer and smaller calcium nodules formed in the osteogenic differentiation of umbilical cord mesenchymal stem cells in the existing technology is an urgent problem to be solved in this field. Summary of the Invention

[0005] The main purpose of the present invention is to provide a kit and culture medium for osteogenic differentiation and a method for preparing the culture medium, so as to solve the problems of slow calcium nodule formation, small number of calcium nodules and small calcium nodules in the osteogenic differentiation of umbilical cord mesenchymal stem cells in the prior art, thereby effectively promoting the formation of calcium nodules of umbilical cord mesenchymal stem cells, increasing the number of calcium nodules and shortening the osteogenic differentiation cycle.

[0006] To achieve the above objectives, the present invention provides a kit and culture medium for osteogenic differentiation and a method for preparing the culture medium, thereby effectively promoting the formation of calcium nodules of umbilical cord mesenchymal stem cells, while increasing the number of calcium nodules and shortening the osteogenic differentiation cycle.

[0007] In a first aspect, the present invention provides a kit for osteogenic differentiation of umbilical cord mesenchymal stem cells, the kit comprising: DMEM basal culture medium, fetal bovine serum, dexamethasone, vitamin C, β-glycerophosphate, supernatant after osteogenic induction of mesenchymal stem cells, Alizarin Red S staining solution, and acetic acid.

[0008] In a second aspect, the present invention provides an umbilical cord mesenchymal stem cell osteogenic induction differentiation medium for use in the kit described in the first aspect, wherein the culture medium includes Dulbecco's Modified Eagle Medium (DMEM) basal culture medium, and further includes the following components and their concentrations or proportions: 5-15% by volume of fetal bovine serum, 0.01-10 μM of dexamethasone, 10-500 μg / ml of vitamin C, 1-100 mM of β-glycerophosphate, and 10-60% by volume of the supernatant after mesenchymal stem cell osteogenic induction.

[0009] Optionally, the umbilical cord mesenchymal stem cell osteogenic induction differentiation medium includes DMEM basal medium and the following components and their concentrations or proportions: 6-12% by volume of fetal bovine serum, 0.05-5 μM of dexamethasone, 20-300 μg / ml of vitamin C, 2-50 mM of β-glycerophosphate, and 15-50% by volume of the supernatant after mesenchymal stem cell osteogenic induction.

[0010] Optionally, the umbilical cord mesenchymal stem cell osteogenic induction differentiation medium includes DMEM basal medium and the following components and their concentrations or proportions: 8-10% by volume of fetal bovine serum, 0.1-2 μM dexamethasone, 50-80 μg / ml of vitamin C, 5-15 mM of β-glycerophosphate, and 20-30% by volume of the supernatant after mesenchymal stem cell osteogenic induction.

[0011] Optionally, the umbilical cord mesenchymal stem cell osteogenic induction differentiation medium includes: mesenchymal stem cells, the mesenchymal stem cells are placed in the umbilical cord mesenchymal stem cell osteogenic induction differentiation medium, and the umbilical cord mesenchymal stem cell osteogenic induction differentiation medium is used to culture the mesenchymal stem cells, wherein the mesenchymal stem cells include at least one of the following: umbilical cord mesenchymal stem cells, adipose mesenchymal stem cells, bone marrow mesenchymal stem cells, amniotic mesenchymal stem cells, and chorionic mesenchymal stem cells.

[0012] In a third aspect, the present invention provides a method for preparing an umbilical cord mesenchymal stem cell osteogenic induction differentiation medium, comprising: adding the fetal bovine serum, the dexamethasone, the vitamin C, the β-glycerophosphate, and the supernatant after mesenchymal stem cell osteogenic induction to the DMEM basal medium in sequence according to the concentration or proportion described in the second aspect, mixing them evenly and then using the culture medium.

[0013] In a fourth aspect, the present invention provides an induction method for osteogenic differentiation of umbilical cord mesenchymal stem cells using the osteogenic differentiation medium of the second aspect, comprising the following steps:

[0014] a. Inoculation of umbilical cord mesenchymal stem cells;

[0015] b. After the cells grow to a preset degree of confluence, discard the original culture medium and replace it with the umbilical cord mesenchymal stem cell osteogenic differentiation medium;

[0016] c. Replace the umbilical cord mesenchymal stem cell osteogenic differentiation medium with fresh one every 3-4 days;

[0017] d. End differentiation induction when brown granular aggregates of a preset area are visible in the cells under a microscope;

[0018] e. Alizarin Red S was used to stain the calcium nodules after differentiation, and the differentiated osteoblasts were observed based on the dark red calcium nodules.

[0019] Optionally, the induction time required to complete the steps in the induction method of the umbilical cord mesenchymal stem cell osteogenic differentiation medium is: starting from step b in the step to completing the differentiation induction in step d in the step, a total of 14-21 days.

[0020] In a fifth aspect, the present invention provides a use of the umbilical cord mesenchymal stem cell osteogenic differentiation medium as described in the second aspect in the osteogenic differentiation of umbilical cord mesenchymal stem cells.

[0021] In a sixth aspect, the present invention provides a method for inducing osteogenic differentiation of umbilical cord mesenchymal stem cells, wherein the method utilizes the kit for inducing osteogenic differentiation of umbilical cord mesenchymal stem cells described in the first aspect to obtain bone cells.

[0022] The present application provides a kit and culture medium for osteogenic differentiation, as well as a method for preparing the culture medium. The kit comprises: DMEM basal medium, fetal bovine serum, dexamethasone, vitamin C, β-glycerophosphate, supernatant after osteogenic induction of mesenchymal stem cells, Alizarin Red S staining solution, and acetic acid. The culture medium comprises DMEM basal medium and also comprises the following components and their concentrations or ratios: 5-15% by volume of fetal bovine serum, 0.01-10 μM dexamethasone, 10-500 μg / ml vitamin C, 1-100 mM β-glycerophosphate, and 10-60% by volume of supernatant after osteogenic induction of mesenchymal stem cells. The umbilical cord mesenchymal stem cell osteogenic differentiation culture medium provided by the present invention is prepared by sequentially adding the fetal bovine serum, dexamethasone, vitamin C, β-glycerophosphate, and supernatant after osteogenic induction of mesenchymal stem cells to DMEM basal medium at the concentrations or ratios described, and mixing them evenly to obtain the culture medium. The invention solves the problems of slow formation of calcium nodules, small number of calcium nodules and small size of calcium nodules in the osteogenic differentiation of umbilical cord mesenchymal stem cells in the prior art, thereby effectively promoting the formation of calcium nodules of umbilical cord mesenchymal stem cells, increasing the number of calcium nodules and shortening the osteogenic differentiation cycle. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The accompanying drawings, which constitute part of this application, are intended to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are intended to explain the present invention and do not constitute an undue limitation of the present invention. In the accompanying drawings:

[0024] Figure 1 This is a cell image of the uninduced group under a 10× objective microscope after Alizarin Red S staining provided in this application;

[0025] Figure 2 This is a cell image of the uninduced group under a 10× objective microscope after Alizarin Red S staining provided in this application;

[0026] Figure 3 Cells under a 10× objective microscope after Alizarin Red S staining of the induced group provided in this application Figure 1 ;

[0027] Figure 4 The cells under a 20× objective microscope after Alizarin Red S staining of the induced group provided in this application Figure 1 ;

[0028] Figure 5 Comparison of OD540nm values ​​between the induced group and the uninduced group after calcium nodule elution provided in this application Figure 1 ;

[0029] Figure 6 Cells under a 10× objective microscope after Alizarin Red S staining of the induced group provided in this application Figure 2 ;

[0030] Figure 7 The cells under a 20× objective microscope after Alizarin Red S staining of the induced group provided in this application Figure 2 ;

[0031] Figure 8 Comparison of OD540nm values ​​between the induced group and the uninduced group after calcium nodule elution provided in this application Figure 2 ;

[0032] Figure 9 Cells under a 10× objective microscope after Alizarin Red S staining of the induced group provided in this application Figure 3 ;

[0033] Figure 10 The cells under a 20× objective microscope after Alizarin Red S staining of the induced group provided in this application Figure 3 ;

[0034] Figure 11 Comparison of OD540nm values ​​between the induced group and the uninduced group after calcium nodule elution provided in this application Figure 3 .

[0035] The above drawings illustrate specific embodiments of the present application, which will be described in more detail below. These drawings and the textual description are not intended to limit the scope of the present application in any way, but rather to illustrate the concepts of the present application to those skilled in the art by reference to specific embodiments. DETAILED DESCRIPTION

[0036] To make the objectives, technical solutions, and advantages of this application more clear, the technical solutions in this application will be clearly and completely described below in conjunction with the accompanying drawings. Obviously, the embodiments described are only part of the embodiments of this application, not all of them. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of this application.

[0037] In the present invention, words such as "exemplary" or "for example" are used to indicate examples, illustrations, or descriptions. Any embodiment or design described in this application as "exemplary" or "for example" should not be construed as being preferred or advantageous over other embodiments or designs. Rather, the use of words such as "exemplary" or "for example" is intended to present the relevant concepts in a concrete manner.

[0038] Mesenchymal stem cells (MSCs) are stem cells with multidirectional differentiation potential. According to the International Society for Cellular Therapy's definition of the minimum standard for MSCs, MSCs should have the following characteristics: ① When cultured under standard conditions, they must have adhesion properties to plastic substrates; ② Flow cytometry detection of the positive expression rate of CD105, CD73, and CD90 surface markers in MSCs should be ≥95%, and the positive expression rate of CD45, CD34, CD14 or CD11b, CD79a or CD19, and HLA-DR should be ≤2%; ③ After induction in vitro using standard methods, MSCs can be induced to differentiate into osteoblasts, adipocytes, chondrocytes, etc. The three-way induced differentiation of mesenchymal stem cells is one of the minimum standards for identifying mesenchymal stem cells. However, in osteogenic induced differentiation, umbilical cord mesenchymal stem cells have a longer induced differentiation cycle than adipose mesenchymal stem cells and bone marrow mesenchymal stem cells, with slow formation of calcium nodules, extremely few calcium nodules and extremely small calcium nodules.

[0039] In response to the above problems, Zuk et al. proposed a classic osteogenic differentiation medium in 2001, which contains DMEM basal medium, 10% fetal bovine serum, dexamethasone, vitamin C, and β-glycerophosphate. This osteogenic differentiation medium formed more and larger calcium nodules during the osteogenic differentiation of adipose-derived mesenchymal stem cells, but formed fewer and smaller calcium nodules during the osteogenic differentiation of umbilical cord-derived mesenchymal stem cells. Even when the induction time was extended to 2-3 times that of the osteogenic differentiation of adipose-derived mesenchymal stem cells, the effect was still unsatisfactory.

[0040] In summary, how to solve the problem of fewer and smaller calcium nodules formed in the osteogenic differentiation of umbilical cord mesenchymal stem cells in the existing technology is an urgent problem to be solved in this field.

[0041] In response to the above problems, the present application provides a kit and culture medium for osteogenic differentiation and a method for preparing the culture medium, wherein the culture medium includes Dulbecco's Modified Eagle Medium (DMEM) basal culture medium, and also includes the following components and their concentrations or proportions: 5-15% by volume of fetal bovine serum, 0.01-10 μM dexamethasone, 10-500 μg / ml of vitamin C, 1-100 mM of β-glycerophosphate, and 10-60% by volume of the supernatant after osteogenic induction of mesenchymal stem cells. The umbilical cord mesenchymal stem cell osteogenic differentiation culture medium and kit provided by the present invention solve the problems of slow formation of calcium nodules, small number of calcium nodules, and small size of calcium nodules in the osteogenic differentiation of umbilical cord mesenchymal stem cells in the prior art, thereby effectively promoting the formation of calcium nodules of umbilical cord mesenchymal stem cells, while being able to increase the number of calcium nodules and shortening the osteogenic differentiation cycle.

[0042] The following specific embodiments describe in detail the technical solution of the present application and how the technical solution of the present application solves the above-mentioned technical problems. The following specific embodiments can be combined with each other, and the same or similar concepts or processes may not be repeated in some embodiments. The embodiments of the present application will be described below in conjunction with the accompanying drawings.

[0043] Example 1: Kit 1

[0044] This embodiment provides a kit for inducing osteogenic differentiation of umbilical cord mesenchymal stem cells, comprising: DMEM basal medium, fetal bovine serum, dexamethasone, vitamin C, β-glycerophosphate, supernatant from mesenchymal stem cell osteogenic induction, Alizarin Red S staining solution, and acetic acid. Preferably, the fetal bovine serum comprises 10% by volume and the acetic acid comprises 1% by volume.

[0045] Optionally, this embodiment provides an umbilical cord mesenchymal stem cell osteogenic induction differentiation medium, and the induction differentiation medium provided in this embodiment is used in the induction differentiation kit provided in this embodiment, wherein the induction differentiation medium includes DMEM basal medium and the following components and their concentrations or proportions: fetal bovine serum 5-15% by volume, dexamethasone 0.01-10 μM, vitamin C 10-500 μg / ml, β-glycerophosphate 1-100 mM, and supernatant after mesenchymal stem cell osteogenic induction 10-60% by volume.

[0046] Optionally, the umbilical cord mesenchymal stem cell osteogenic differentiation medium comprises: mesenchymal stem cells, wherein the mesenchymal stem cells are placed in the umbilical cord mesenchymal stem cell osteogenic differentiation medium, and the umbilical cord mesenchymal stem cell osteogenic differentiation medium is used to culture the mesenchymal stem cells, wherein the mesenchymal stem cells include at least one of the following: umbilical cord mesenchymal stem cells, adipose-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, amniotic membrane-derived mesenchymal stem cells, and chorionic villus-derived mesenchymal stem cells. Umbilical cord mesenchymal stem cells are preferred.

[0047] The preparation method of the umbilical cord mesenchymal stem cell osteogenic induction differentiation medium described in this embodiment is as follows: to the DMEM basal medium, 5-15% by volume of the fetal bovine serum, 0.01-10 μM of dexamethasone, 10-500 μg / ml of vitamin C, 1-100 mM of β-glycerophosphate are added in sequence according to the concentrations or proportions described above, and 10-60% by volume of the supernatant after mesenchymal stem cell osteogenic induction is added. The mixture is mixed evenly and the medium is ready for use.

[0048] The induction method of the umbilical cord mesenchymal stem cell osteogenic differentiation medium described in this embodiment comprises the following steps:

[0049] a. Inoculation of umbilical cord mesenchymal stem cells;

[0050] b. After the cells grow to a preset degree of confluence, discard the original culture medium and replace it with the umbilical cord mesenchymal stem cell osteogenic differentiation medium;

[0051] c. Replace the umbilical cord mesenchymal stem cell osteogenic differentiation medium with fresh one every 3-4 days;

[0052] d. When brown granular aggregates of a predetermined area (60%-95% of the visible intracellular area) are visible under a microscope, differentiation induction is terminated.

[0053] e. Alizarin Red S was used to stain the calcium nodules after differentiation, and the differentiated osteoblasts were observed based on the dark red calcium nodules.

[0054] Specifically, the method includes the following steps:

[0055] 1. Prepare the umbilical cord mesenchymal stem cell osteogenic induction differentiation medium described in this embodiment, and add 5% fetal bovine serum, 6μM dexamethasone, 400μg / ml vitamin C, 80mM β-glycerophosphate and 50% volume percentage of the supernatant after mesenchymal stem cell osteogenic induction to the DMEM basal medium. Among them, the full name of DMEM basal medium is Dulbecco's Modified Eagle Medium basal medium, which is a culture medium widely used in cell culture; the supernatant after mesenchymal stem cell (MSC) osteogenic induction refers to when mesenchymal stem cells are induced to differentiate into osteoblasts in vitro, they will secrete a variety of growth factors, cytokines and other bioactive substances into the culture medium, and these substances exist in a dissolved state in the culture medium to form the so-called "supernatant". The supernatant after mesenchymal stem cell osteogenic induction can be obtained by the following steps:

[0056] Step 1: Cell seeding and culture: MSCs in the logarithmic growth phase are seeded into coated culture vessels at a specific cell density (e.g., 2×104 cells / cm2). Culture at 37°C, 5% CO2 until confluence is achieved (e.g., 60-70% or higher).

[0057] Step 2: Osteogenic differentiation: Discard the original culture medium and add osteogenic differentiation medium. Replace the osteogenic differentiation medium every 2-3 days for approximately 14-21 days or longer to induce MSC differentiation into osteoblasts. The osteogenic differentiation medium can be the umbilical cord mesenchymal stem cell osteogenic differentiation medium described in this example, or other conventional osteogenic differentiation medium, such as DMEM basal medium.

[0058] Step 3: Supernatant Collection: During osteogenic differentiation, the supernatant in the culture medium will continuously accumulate various bioactive substances secreted by MSCs. When it is time to collect the supernatant, discard the cells and attached materials in the culture vessel and retain only the supernatant.

[0059] 2. Digestion and collection of human UC-MSCs and counting at 5000-8000 cells / cm 2The cells were inoculated at a density of 100 μg / well in a 6-well cell culture plate with 2 ml / well of human UC-MSCs complete medium, and cultured in a 37°C, 5% CO2 incubator. Human UC-MSCs complete medium refers to human umbilical cord mesenchymal stem cells (UC-MSCs) complete medium, which is a medium specially designed for culturing human umbilical cord mesenchymal stem cells. It contains various nutrients, growth factors and other necessary components required for cell growth. Human UC-MSCs complete medium is usually composed of basal culture medium, serum, growth factors, antibiotics and other additives. The specific ingredients may vary depending on the formula of different brands or laboratories, but generally include the following key elements:

[0060] Basal culture medium: such as DMEM (Dulbecco's Modified Eagle's Medium), α-MEM (AlphaMinimum Essential Medium), etc., provides basic nutrients required for cell growth.

[0061] Serum: Fetal bovine serum (FBS) is commonly used, which is rich in growth factors, hormones and other biologically active substances that are essential for cell growth and differentiation.

[0062] Growth factors: such as bone morphogenetic proteins (BMPs) and transforming growth factor-β (TGF-β), these growth factors play an important role in inducing cell differentiation and promoting cell proliferation.

[0063] Antibiotics: such as penicillin and streptomycin, used to prevent bacterial contamination in culture media.

[0064] Other additives: such as glutamine, non-essential amino acids, vitamins, etc. These additives help maintain healthy cell growth.

[0065] 3. When the cell fusion reaches 70-90%, discard the culture supernatant and add 2 ml of the prepared umbilical cord mesenchymal stem cell osteogenic differentiation medium described in this example to each well. Add 2 ml of the control group cell culture medium to the wells and culture in a 37°C, 5% CO2 incubator.

[0066] 4. The induction group was fed with fresh osteogenic differentiation medium described in this example every 3-4 days, and the control group was fed with an equal volume of control medium and cultured in a 37°C, 5% CO2 incubator.

[0067] 5. After 2-3 weeks of induction, a large area of ​​brown particles (60%-95% of the visible intracellular area) can be seen under the microscope, and the induction culture is terminated.

[0068] 6. After the induction culture is completed, the culture supernatant is discarded, the cells are washed twice with PBS, and the cells are fixed with 1 ml / well of 4% neutral paraformaldehyde solution at room temperature for 30 minutes (performed in a fume hood).

[0069] 7. Discard the fixative solution, wash twice with PBS, add 1 ml of Alizarin Red S staining solution to each well, and stain for 10 minutes at room temperature.

[0070] 8. Discard the Alizarin Red S staining solution and wash three times with PBS to thoroughly remove excess staining solution.

[0071] 9. Add 1 ml of PBS to each well, observe under a microscope and take pictures. Figure 1 、 Figure 2 、 Figure 3 、 Figure 4 As shown in the figure, the uninduced group is used to indicate the control group in this embodiment, and the dark red calcium nodules colored in the figure are used to indicate the induced differentiation effect. The more and larger the dark red calcium nodules are, the more calcium nodules there are and the larger the calcium nodules are.

[0072] 10. Semi-quantitative detection of osteogenic differentiation: Add 0.6 ml of cetylpyridinium chloride to each well, elute on a shaker at room temperature for 30 minutes, and take 0.2 ml of the eluate from each well for detection at 540 nm using a microplate reader. Figure 5 As shown in the figure, the blue control is used to indicate the OD of the uninduced group 540nm The red SG medium indicates the OD of the induced group. 540nm The specific values ​​are shown in Table 1, where the OD540nm value is used to indicate the content or concentration of calcium nodules. The larger the OD540nm value, the higher the content or concentration of calcium nodules.

[0073] Table 1: OD after calcium nodule elution in this example 540nm value

[0074]

[0075] Optionally, the induction time required to complete the steps required by the above induction method is: starting from step b in the step to the end of inducing differentiation in step d in the step, a total of 14-21 days.

[0076] Optionally, the umbilical cord mesenchymal stem cell osteogenic differentiation medium described in this embodiment can be used in the osteogenic differentiation of umbilical cord mesenchymal stem cells.

[0077] Optionally, this embodiment provides a kit for osteogenic differentiation of umbilical cord mesenchymal stem cells, the kit comprising an umbilical cord mesenchymal stem cell osteogenic differentiation medium, and the medium is the umbilical cord mesenchymal stem cell osteogenic differentiation medium described in this embodiment.

[0078] Optionally, this embodiment provides a method for inducing osteogenic differentiation of umbilical cord mesenchymal stem cells, and bone cells are obtained using the kit for inducing osteogenic differentiation of umbilical cord mesenchymal stem cells provided in this embodiment.

[0079] Example 2: Kit 2

[0080] This embodiment provides a kit for inducing osteogenic differentiation of umbilical cord mesenchymal stem cells, comprising: DMEM basal culture medium, fetal bovine serum, dexamethasone, vitamin C, β-glycerophosphate, supernatant after osteogenic induction of mesenchymal stem cells, Alizarin Red S staining solution, and acetic acid.

[0081] Optionally, this embodiment provides an umbilical cord mesenchymal stem cell osteogenic induction differentiation medium, and the induction differentiation medium provided in this embodiment is used in the induction differentiation kit provided in this embodiment, wherein the induction differentiation medium includes DMEM basal medium and the following components and their concentrations or proportions: fetal bovine serum 6-12% by volume, dexamethasone 0.05-5 μM, vitamin C 20-300 μg / ml, β-glycerophosphate 2-50 mM, and supernatant after mesenchymal stem cell osteogenic induction 15-50% by volume.

[0082] Optionally, the umbilical cord mesenchymal stem cell osteogenic differentiation medium comprises: mesenchymal stem cells, wherein the mesenchymal stem cells are placed in the umbilical cord mesenchymal stem cell osteogenic differentiation medium, and the umbilical cord mesenchymal stem cell osteogenic differentiation medium is used to culture the mesenchymal stem cells, wherein the mesenchymal stem cells include at least one of the following: umbilical cord mesenchymal stem cells, adipose-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, amniotic membrane-derived mesenchymal stem cells, and chorionic villus-derived mesenchymal stem cells. Umbilical cord mesenchymal stem cells are preferred.

[0083] The preparation method of the umbilical cord mesenchymal stem cell osteogenic induction differentiation medium described in this embodiment is as follows: to the DMEM basal medium, 6-12% by volume of the fetal bovine serum, 0.05-5 μM of dexamethasone, 20-300 μg / ml of vitamin C, and 2-50 mM of β-glycerophosphate are added in sequence according to the concentrations or proportions described above, and 15-50% by volume of the supernatant after mesenchymal stem cell osteogenic induction is added. The mixture is mixed evenly and the medium is ready for use.

[0084] The induction method of the umbilical cord mesenchymal stem cell osteogenic differentiation medium described in this embodiment comprises the following steps:

[0085] a. Inoculation of umbilical cord mesenchymal stem cells;

[0086] b. After the cells grow to a preset degree of confluence, discard the original culture medium and replace it with the umbilical cord mesenchymal stem cell osteogenic differentiation medium;

[0087] c. Replace the umbilical cord mesenchymal stem cell osteogenic differentiation medium with fresh one every 3-4 days;

[0088] d. When brown granular aggregates of a predetermined area (60%-95% of the visible intracellular area) are visible under a microscope, differentiation induction is terminated.

[0089] e. Alizarin Red S was used to stain the calcium nodules after differentiation, and the differentiated osteoblasts were observed based on the dark red calcium nodules.

[0090] Specifically, the method includes the following steps:

[0091] 1. Prepare umbilical cord mesenchymal stem cell osteogenic differentiation medium: Dulbecco's Modified Eagle Medium (DMEM), 6% fetal bovine serum, 4 μM dexamethasone, 200 μg / ml vitamin C, 40 mM β-glycerophosphate, and 40% by volume of the supernatant after osteogenic induction of mesenchymal stem cells.

[0092] 2. Digestion and collection of human UC-MSCs and counting at 5000-8000 cells / cm 2 The cells were seeded at a density of 100 μg / well in a 6-well cell culture plate with 2 ml of human UC-MSCs complete medium per well and cultured in a 37°C, 5% CO2 incubator.

[0093] 3. When the cell fusion reaches 70-90%, discard the culture supernatant and add 2 ml of the prepared umbilical cord mesenchymal stem cell osteogenic differentiation medium described in this example to each well. Add 2 ml of the control group cell culture medium to the wells and culture in a 37°C, 5% CO2 incubator.

[0094] 4. The induction group was fed with fresh osteogenic differentiation medium described in this example every 3-4 days, and the control group was fed with an equal volume of control medium and cultured in a 37°C, 5% CO2 incubator.

[0095] 5. After 2-3 weeks of induction, a large area of ​​brown particles (60%-95% of the visible intracellular area) can be seen under the microscope, and the induction culture is terminated.

[0096] 6. After the induction culture is completed, the culture supernatant is discarded, the cells are washed twice with PBS, and the cells are fixed with 1 ml / well of 4% neutral paraformaldehyde solution at room temperature for 30 minutes (performed in a fume hood).

[0097] 7. Discard the fixative solution, wash twice with PBS, add 1 ml of Alizarin Red S staining solution to each well, and stain for 10 minutes at room temperature.

[0098] 8. Discard the Alizarin Red S staining solution and wash three times with PBS to thoroughly remove excess staining solution.

[0099] 9. Add 1 ml PBS to each well, observe under a microscope and take pictures. Figure 6 、 Figure 7 As shown in the figure, the dark red calcium nodules are used to indicate the induced differentiation effect. The more and larger the dark red calcium nodules are, the more and larger the number of calcium nodules is, which indicates that the calcium nodules are large and large.

[0100] 10. Semi-quantitative detection of osteogenic differentiation: Add 0.6 ml of cetylpyridinium chloride to each well, elute on a shaker at room temperature for 30 minutes, and take 0.2 ml of the eluate from each well for detection at 540 nm using a microplate reader. Figure 8 As shown, the blue control in the figure indicates the OD of the uninduced group 540nm The red SG medium indicates the OD of the induced group. 540nm The specific values ​​are shown in Table 2, where the OD540nm value is used to indicate the content or concentration of calcium nodules. The larger the OD540nm value, the higher the content or concentration of calcium nodules.

[0101] Table 2: OD540nm values ​​after calcium nodules elution in this example

[0102]

[0103] Optionally, the induction time required to complete the steps required by the above induction method is: starting from step b in the step to the end of inducing differentiation in step d in the step, a total of 14-21 days.

[0104] Optionally, the umbilical cord mesenchymal stem cell osteogenic differentiation medium described in this embodiment can be used in the osteogenic differentiation of umbilical cord mesenchymal stem cells.

[0105] Optionally, this embodiment provides a kit for osteogenic differentiation of umbilical cord mesenchymal stem cells, the kit comprising an umbilical cord mesenchymal stem cell osteogenic differentiation medium, and the medium is the umbilical cord mesenchymal stem cell osteogenic differentiation medium described in this embodiment.

[0106] Optionally, this embodiment provides a method for inducing osteogenic differentiation of umbilical cord mesenchymal stem cells, and bone cells are obtained using the kit for inducing osteogenic differentiation of umbilical cord mesenchymal stem cells provided in this embodiment.

[0107] Example 3: Kit 3

[0108] This embodiment provides a kit for inducing osteogenic differentiation of umbilical cord mesenchymal stem cells, comprising: DMEM basal culture medium, fetal bovine serum, dexamethasone, vitamin C, β-glycerophosphate, supernatant after osteogenic induction of mesenchymal stem cells, Alizarin Red S staining solution, and acetic acid.

[0109] Optionally, this embodiment provides an umbilical cord mesenchymal stem cell osteogenic induction differentiation medium, and the induction differentiation medium provided in this embodiment is used in the induction differentiation kit provided in this embodiment, wherein the induction differentiation medium includes DMEM basal medium and the following components and their concentrations or proportions: fetal bovine serum 8-10% by volume, dexamethasone 0.1-2 μM, vitamin C 50-80 μg / ml, β-glycerophosphate 5-15 mM, and supernatant after mesenchymal stem cell osteogenic induction 20-30% by volume.

[0110] Optionally, the umbilical cord mesenchymal stem cell osteogenic differentiation medium comprises: mesenchymal stem cells, wherein the mesenchymal stem cells are placed in the umbilical cord mesenchymal stem cell osteogenic differentiation medium, and the umbilical cord mesenchymal stem cell osteogenic differentiation medium is used to culture the mesenchymal stem cells, wherein the mesenchymal stem cells include at least one of the following: umbilical cord mesenchymal stem cells, adipose-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, amniotic membrane-derived mesenchymal stem cells, and chorionic villus-derived mesenchymal stem cells. Umbilical cord mesenchymal stem cells are preferred.

[0111] The preparation method of the umbilical cord mesenchymal stem cell osteogenic induction differentiation medium described in this embodiment is as follows: to the DMEM basal medium, 8-10% by volume of the fetal bovine serum, 0.1-2 μM of dexamethasone, 50-80 μg / ml of vitamin C, 5-15 mM of β-glycerophosphate are added in sequence according to the concentrations or proportions described above, and 20-30% by volume of the supernatant after mesenchymal stem cell osteogenic induction is added, and the mixture is mixed evenly before use.

[0112] The induction method of the umbilical cord mesenchymal stem cell osteogenic differentiation medium described in this embodiment comprises the following steps:

[0113] a. Inoculation of umbilical cord mesenchymal stem cells;

[0114] b. After the cells grow to a preset degree of confluence, discard the original culture medium and replace it with the umbilical cord mesenchymal stem cell osteogenic differentiation medium;

[0115] c. Replace the umbilical cord mesenchymal stem cell osteogenic differentiation medium with fresh one every 3-4 days;

[0116] d. When brown granular aggregates of a predetermined area (60%-95% of the visible intracellular area) are visible under a microscope, differentiation induction is terminated.

[0117] e. Alizarin Red S was used to stain the calcium nodules after differentiation, and the differentiated osteoblasts were observed based on the dark red calcium nodules.

[0118] Specifically, the method includes the following steps:

[0119] 1. Prepare umbilical cord mesenchymal stem cell osteogenic differentiation medium: Dulbecco's Modified Eagle Medium (DMEM), 10% fetal bovine serum, 0.5 μM dexamethasone, 60 μg / ml vitamin C, 5 mM β-glycerophosphate, and 30% volume percentage of the supernatant after mesenchymal stem cell osteogenic induction.

[0120] 2. Digestion and collection of human UC-MSCs and counting at 5000-8000 cells / cm 2 The cells were seeded at a density of 100 μg / well in a 6-well cell culture plate with 2 ml of human UC-MSCs complete medium per well and cultured in a 37°C, 5% CO2 incubator.

[0121] 3. When the cell fusion reaches 70-90%, discard the culture supernatant and add 2 ml of the prepared umbilical cord mesenchymal stem cell osteogenic differentiation medium described in this example to each well. Add 2 ml of the control group cell culture medium to the wells and culture in a 37°C, 5% CO2 incubator.

[0122] 4. The induction group was fed with fresh osteogenic differentiation medium described in this example every 3-4 days, and the control group was fed with an equal volume of control medium and cultured in a 37°C, 5% CO2 incubator.

[0123] 5. After 2-3 weeks of induction, a large area of ​​brown particles (60%-95% of the visible intracellular area) can be seen under the microscope, and the induction culture is terminated.

[0124] 6. After the induction culture is completed, the culture supernatant is discarded, the cells are washed twice with PBS, and the cells are fixed with 1 ml / well of 4% neutral paraformaldehyde solution at room temperature for 30 minutes (performed in a fume hood).

[0125] 7. Discard the fixative solution, wash twice with PBS, add 1 ml of Alizarin Red S staining solution to each well, and stain for 10 minutes at room temperature.

[0126] 8. Discard the Alizarin Red S staining solution and wash three times with PBS to thoroughly remove excess staining solution.

[0127] 9. Add 1 ml PBS to each well, observe under a microscope and take pictures. Figure 9 、 Figure 10As shown in the figure, the dark red calcium nodules are used to indicate the induced differentiation effect. The more and larger the dark red calcium nodules are, the more and larger the number of calcium nodules is, which indicates that the calcium nodules are large and large.

[0128] 10. Semi-quantitative detection of osteogenic differentiation: Add 0.6 ml of cetylpyridinium chloride to each well, elute on a shaker at room temperature for 30 minutes, and take 0.2 ml of the eluate from each well for detection at 540 nm using a microplate reader. Figure 11 As shown, the blue control in the figure indicates the OD of the uninduced group 540nm The red SG medium indicates the OD of the induced group. 540nm The specific values ​​are shown in Table 3, where the OD540nm value is used to indicate the content or concentration of calcium nodules. The larger the OD540nm value, the higher the content or concentration of calcium nodules.

[0129] Table 3: OD after calcium nodule elution in this example 540nm value

[0130]

[0131] Optionally, the induction time required to complete the steps required by the above induction method is: starting from step b in the step to the end of inducing differentiation in step d in the step, a total of 14-21 days.

[0132] Optionally, the umbilical cord mesenchymal stem cell osteogenic differentiation medium described in this embodiment can be used in the osteogenic differentiation of umbilical cord mesenchymal stem cells.

[0133] Optionally, this embodiment provides a kit for osteogenic differentiation of umbilical cord mesenchymal stem cells, the kit comprising an umbilical cord mesenchymal stem cell osteogenic differentiation medium, and the medium is the umbilical cord mesenchymal stem cell osteogenic differentiation medium described in this embodiment.

[0134] Optionally, this embodiment provides a method for inducing osteogenic differentiation of umbilical cord mesenchymal stem cells, and bone cells are obtained using the kit for inducing osteogenic differentiation of umbilical cord mesenchymal stem cells provided in this embodiment.

[0135] It is worth mentioning that compared with the classic osteogenic induction differentiation medium, the advantages of the present invention are: the umbilical cord mesenchymal stem cell high-efficiency osteogenic induction differentiation medium provided by the present invention is not only for the problem of human umbilical cord mesenchymal stem cell osteogenesis difficulty, but also for other species (rats, mice, rhesus monkeys, beagles, etc.) and mesenchymal stem cells from other tissue sources (fat, bone marrow, placenta, dental pulp, etc.) osteogenic induction differentiation is also applicable, and compared to the calcium nodules formed after umbilical cord induction differentiation, mesenchymal stem cells from other species and other tissue sources have better effects, i.e., more and larger calcium nodules and shorter induction differentiation test cycles. In addition, the preparation method provided by the present invention is simple and easy, and the prepared high-efficiency osteogenic induction differentiation medium can achieve stability, efficiency and repeatability of osteogenic induction differentiation.

[0136] According to the above embodiments, it can be understood that the osteogenic differentiation kit and culture medium and the preparation method of the culture medium provided by the present application include Dulbecco's Modified Eagle Medium (DMEM) basal culture medium, and also include the following components and their concentrations or proportions: 5-15% volume percentage of fetal bovine serum, 0.01-10μM dexamethasone, 10-500μg / ml of vitamin C, 1-100mM of β-glycerophosphate, and 10-60% volume percentage of the supernatant after osteogenic induction of mesenchymal stem cells. The umbilical cord mesenchymal stem cell osteogenic differentiation culture medium provided by the present invention solves the problems of slow formation of calcium nodules, small number of calcium nodules and small calcium nodules in the osteogenic differentiation of umbilical cord mesenchymal stem cells in the prior art, thereby effectively promoting the formation of calcium nodules of umbilical cord mesenchymal stem cells, while being able to increase the number of calcium nodules and shortening the osteogenic differentiation cycle.

[0137] Those skilled in the art will readily appreciate other embodiments of the present application after considering the specification and practicing the invention disclosed herein. This application is intended to cover any variations, uses, or adaptations of the present application that follow the general principles of the present application and include common knowledge or customary techniques in the art not disclosed herein. The description and examples are to be considered as exemplary only, and the true scope and spirit of the present application are indicated by the following claims.

[0138] It should be understood that the present application is not limited to the exact structure described above and shown in the drawings, and that various modifications and changes may be made without departing from the scope thereof. The scope of the present application is limited only by the appended claims.

Claims

1. A kit for osteogenic differentiation of umbilical cord mesenchymal stem cells, characterized in that: The kit comprises: DMEM basal culture medium, fetal bovine serum, dexamethasone, vitamin C, beta-glycerophosphate, supernatant after osteogenic induction of mesenchymal stem cells, Alizarin Red S staining solution, and acetic acid.

2. An umbilical cord mesenchymal stem cell osteogenic differentiation medium used in the kit according to claim 1, characterized in that: The culture medium includes DMEM basal culture medium and the following components and their concentrations or proportions: 5-15% by volume of fetal bovine serum, 0.01-10 μM of dexamethasone, 10-500 μg / ml of vitamin C, 1-100 mM of beta-glycerophosphate, and 10-60% by volume of supernatant after osteogenic induction of mesenchymal stem cells.

3. The umbilical cord mesenchymal stem cell osteogenic differentiation medium according to claim 2, characterized in that The fetal bovine serum is 6-12% by volume, the dexamethasone is 0.05-5 μM, the vitamin C is 20-300 μg / ml, the beta-glycerophosphate is 2-50 mM, and the supernatant after osteogenic induction of mesenchymal stem cells is 15-50% by volume.

4. The osteogenic differentiation medium for umbilical cord mesenchymal stem cells according to claim 2, characterized in that The fetal bovine serum is 8-10% by volume, the dexamethasone is 0.1-2 μM, the vitamin C is 50-80 μg / ml, the beta-glycerol phosphate is 5-15 mM, and the supernatant after osteogenic induction of mesenchymal stem cells is 20-30% by volume.

5. The umbilical cord mesenchymal stem cell osteogenic differentiation medium according to any one of claims 2 to 4, characterized in that The umbilical cord mesenchymal stem cell osteogenic induction differentiation medium also includes: mesenchymal stem cells, the mesenchymal stem cells are placed in the umbilical cord mesenchymal stem cell osteogenic induction differentiation medium, and the umbilical cord mesenchymal stem cell osteogenic induction differentiation medium is used to culture the mesenchymal stem cells, wherein the mesenchymal stem cells include at least one of the following: umbilical cord mesenchymal stem cells, adipose mesenchymal stem cells, bone marrow mesenchymal stem cells, amniotic mesenchymal stem cells, and chorionic mesenchymal stem cells.

6. A method for preparing an umbilical cord mesenchymal stem cell osteogenic differentiation medium according to any one of claims 2 to 4, characterized in that: include: The fetal bovine serum, the dexamethasone, the vitamin C, the β-glycerophosphate, and the supernatant of the mesenchymal stem cells after osteogenic induction are added to the DMEM basal medium in sequence according to the concentrations or proportions, and the mixture is evenly mixed before use.

7. A method for inducing osteogenic differentiation of umbilical cord mesenchymal stem cells using the osteogenic differentiation medium according to any one of claims 2 to 4, characterized in that: The following steps are involved: a. Inoculation of umbilical cord mesenchymal stem cells; b. After the cells grow to a preset degree of confluence, discard the original culture medium and replace it with the umbilical cord mesenchymal stem cell osteogenic differentiation medium; c. Replace the umbilical cord mesenchymal stem cell osteogenic differentiation medium with fresh one every 3-4 days; d. End differentiation induction when brown granular aggregates of a preset area are visible in the cells under a microscope; e. Alizarin Red S was used to stain the calcium nodules after differentiation, and the differentiated osteoblasts were observed based on the dark red calcium nodules.

8. The induction method according to claim 7, characterized in that The induction time required to complete the steps is: starting from step b in the steps to the end of inducing differentiation in step d in the steps, a total of 14-21 days.

9. Use of the umbilical cord mesenchymal stem cell osteogenic differentiation medium according to any one of claims 2 to 4 in the osteogenic differentiation of umbilical cord mesenchymal stem cells.

10. A method for inducing osteogenic differentiation of umbilical cord mesenchymal stem cells, characterized by: Osteocytes are obtained using the kit for osteogenic differentiation of umbilical cord mesenchymal stem cells as claimed in claim 1.

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