CN111454892A - Tooth mesenchymal stem cell culture medium and activity verification method in dental pulp stem cells - Google Patents
Tooth mesenchymal stem cell culture medium and activity verification method in dental pulp stem cells Download PDFInfo
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Abstract
The invention discloses a culture medium of mesenchymal stem cells, which takes a-MEM as a basal medium and is added with a growth factor and small molecule inhibitor combination comprising epidermal growth factor, platelet-derived growth factor, vitamin C, TGF- β receptor inhibitor L Y3200882, gamma-secrase inhibitor L Y411575 and DNA methylase inhibitor 5-Aza-dC, wherein the content of the epidermal growth factor is 10-200 mu g/L, the content of the platelet-derived growth factor is 100 mu g/L, the content of vitamin C is 10-100 mg/L, the content of TGF- β receptor inhibitor L Y3200882 is 100 nM, the content of the gamma-secrase inhibitor L Y411575 is 50-200nM, the content of the DNA methylase inhibitor 5-Aza-dC is 1-20 nM., the invention also discloses a culture medium of the mesenchymal stem cells, which has high activity in the dental pulp stem cell culture medium, a method for conveniently verifying the quality of synthetic protein, and a method for quickly verifying the quality of the synthetic protein without the use of the abnormal stem cells.
Description
Technical Field
The invention relates to the technical field of cell engineering, in particular to a tooth mesenchymal stem cell culture medium and an activity verification method in dental pulp stem cells.
Background
The mesenchymal stem cells have multidirectional differentiation potential, can repair damaged or lost tissues, and have great clinical application potential. A variety of odontogenic mesenchymal stem cells have been found in teeth. The odontogenic stem cells currently include the following 5 types: dental pulp stem cells, periodontal ligament stem cells, dental capsule stem cells, dental papilla stem cells, deciduous tooth stem cells. Wherein the dental pulp stem cell has a plurality of potentials of osteogenic/odontoblastic differentiation, adipogenic differentiation, chondrogenic differentiation, myogenic differentiation and the like in vitro; and can form dentin-like cells and a dental pulp dentin-like complex structure in vivo. The dental pulp is an important structure for maintaining the activity and the perception capability of teeth, and the dental pulp is usually damaged or even necrosed to different degrees due to trauma, infection and the like clinically. However, as the most effective endodontic treatment means in clinical practice, root canal treatment is to replace diseased pulp with artificial material to relieve pain, which eventually results in loss of tooth activity and tooth loss. Stem cell-based tissue engineering techniques have brought new avenues for dental pulp regeneration. The dental pulp stem cells are good seed cells for dental pulp regeneration, and the transplanted dental pulp stem cells can form new dental pulp tissues with bioactivity in vivo to realize the physiological regeneration of dental pulp. However, the primary cultured dental pulp stem cells are mostly from wisdom teeth or young permanent teeth which need to be extracted due to orthodontics, and although the sources of the dental mesenchymal stem cells are wide, the number of primary stem cells obtained by a single individual is limited, and the requirement of clinical cell therapy on the number of cells is difficult to meet. Therefore, the in vitro large-scale long-term amplification culture is the premise of clinical application of the dental mesenchymal stem cells. However, the long-term culture of the stem cells under the conventional conditions can cause the defects of cell proliferation capacity reduction, cell aging, stem cell characteristic reduction, cell differentiation capacity reduction and the like, so that the development of a medium suitable for the long-term culture of the mesenchymal stem cells is a key for the clinical application of the stem cells, and the maintenance of the cell proliferation, the stem cell characteristic and the differentiation capacity of the stem cells in the in vitro long-term culture process is ensured.
At present, a culture system commonly used for in vitro cell amplification is a basal medium added with Fetal Bovine Serum (FBS) or human serum. However, there are many problems in clinical application of cells cultured in serum-containing medium, such as: the components are undefined, pathogenic microorganisms are polluted, immune reaction caused by xenogenic or xenogenic antigens, the quality is unstable, the difference between batches is large, the existence of endotoxin in cells and the like seriously influence the growth of the cells and the effectiveness and the safety of subsequent cell transplantation.
Therefore, the development of a culture medium which is well-defined, does not contain serum or animal-derived components, and can maintain the proliferation, stem cell characteristics and stem cell differentiation capacity of stem cells in a long-term culture process is a prerequisite and key point of the current clinical application of stem cells.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides a dental mesenchymal stem cell culture medium and an activity verification method thereof in dental pulp stem cells, which have the advantages that the dental pulp stem cells are expanded for a long time outside by adding a complete culture medium formed by combining artificially synthesized cell factors and small molecular compounds into a basic culture medium, the good biological activity of the stem cells in vivo and in vitro is maintained, and the regeneration of dental pulp tissues can be promoted.
In order to achieve the purpose, the invention adopts the technical scheme that the culture medium of the dental mesenchymal stem cells is formed by adding a composition of growth factors including epidermal growth factor, platelet-derived growth factor, vitamin C, TGF- β receptor inhibitor L Y3200882, gamma-secretase inhibitor L Y411575 and DNA methylase inhibitor 5-Aza-dC and a small molecule inhibitor into a basic culture medium of a-MEM, wherein the composition comprises 10-200 mu g/L of the epidermal growth factor, 100-500 mu g/L of the platelet-derived growth factor, 10-100 mg/L of the vitamin C, 100 nM of the TGF- β receptor inhibitor L Y3200882, 50-200nM of the gamma-secretase inhibitor L Y411575 and 1-20nM of the DNA methylase inhibitor 5-Aza-1 dC-20 nM.
A method for verifying the cell activity of the dental mesenchymal stem cell culture medium in dental pulp stem cells comprises the following steps:
(1) the pretreatment of the cells is carried out,
separating and culturing primary dental pulp stem cells, and dividing the primary dental pulp stem cells into a primary cell Control Group (Con), a conventional culture medium long-term culture Group (L ong-term culture with continuous culture medium, CM) and a tooth mesenchymal stem cell culture medium long-term culture Group (L ong-term culture with SFM), wherein the primary dental pulp stem cells are used as the primary dental pulp stem cells, and the long-term culture Group is formed by passage of the dental pulp stem cells in the conventional culture medium and the tooth mesenchymal stem cell culture medium to the 9 th generation;
(2) the evaluation of the biological properties of cells in vitro,
2.1 evaluation of morphology and surface markers of cells;
2.2 evaluation of cell proliferation and senescence;
2.3 evaluation of the characteristics of stem cells;
2.4 evaluation of the migration ability of cells;
2.5 evaluation of odontoblastic differentiation ability of dental pulp stem cells;
(3) evaluation of the ability to regenerate dental pulp in an animal,
injecting the dental pulp stem cells obtained in the step 2.1 after compounding collagen into a prepared dentin matrix casing, implanting the dental pulp stem cells into a nude mouse subcutaneous tissue, sampling 2 months later, fixing 4% paraformaldehyde, decalcifying with 10% EDTA, preparing a conventional tissue slice, performing HE and MASSON staining, and analyzing the dental pulp regeneration condition after transplanting each group of cells.
Preferably, in the step 2.1, the morphology of the cells and the morphology of the cells of each group are observed through a cell optical microscope in the evaluation of the surface markers, and the cells of the primary cell control group and the tooth mesenchymal stem cell culture medium long-term culture group are smaller and are in a long spindle shape or a spindle shape compared with the cells of the conventional culture medium long-term culture group; compared with the other two groups of cells, the cells of the long-term culture group of the conventional culture medium are larger and have flat shapes; flow cytometry was used to detect expression of cell surface markers with no difference between groups.
Preferably, the step 2.2 evaluates the proliferation capacity and cell senescence of the cells by CCK-8 experimental cell senescence β -galactosidase (SA- β -Gal) staining, wherein the cell proliferation capacity of the dental mesenchymal stem cell culture medium long-term culture group is not different from that of the primary cell control group but higher than that of the conventional culture medium long-term culture group, the cell senescence of the dental mesenchymal stem cell culture medium long-term culture group is not different from that of the primary cell control group, and the cell senescence of the conventional culture medium long-term culture group is higher than that of the dental mesenchymal stem cell culture medium long-term culture group and the primary cell control group.
The results show that the dental pulp stem cells cultured by the culture medium for a long time can effectively maintain the cell proliferation capacity and inhibit the occurrence of cell aging.
Preferably, the step 2.3 is to evaluate the characteristics of the stem cells through a cloning experiment and detection of expression of the sternness gene of the cells, wherein, the cell clone forming capability and the expression level of the sternness gene SOX2 of the dental mesenchymal stem cell culture medium long-term culture group are higher than those of the conventional culture medium long-term culture group.
The culture medium of the invention can effectively maintain the dryness of the dental pulp stem cells after long-term culture.
Preferably, in the step 2.4, the evaluation of the cell migration ability of the three groups is evaluated through a cell scratch test and a Transwell test, wherein the migration ability of the dental pulp stem cell group of the primary cell control group and the migration ability of the dental pulp stem cell of the dental mesenchymal stem cell culture medium long-term culture group are higher than the migration ability of the dental pulp stem cell group of the conventional culture medium long-term culture group.
The culture medium of the invention is proved to be capable of effectively maintaining the cell migration capability when used for culturing dental pulp stem cells for a long time.
Preferably, the step 2.5 is performed by quantitatively analyzing a L P activity and mineralized nodule formation ability of three groups of stem cells, wherein a L P activity and mineralized nodule formation ability of the dental pulp stem cell group of the primary cell control group and a L P activity and mineralized nodule formation ability of the dental pulp stem cell group of the dental mesenchymal stem cell culture medium long-term culture group are higher than a L P activity and mineralized nodule formation ability of the dental pulp stem cell group of the conventional culture medium long-term culture group.
The culture medium of the invention can effectively maintain the tooth differentiation capability of the dental pulp stem cells for long-term culture.
Preferably, in step 3, the ability of the animal to form dental pulp tissue in the animal is evaluated by transplanting three groups of dental pulp stem cells in the animal, wherein the dental pulp stem cell group of the primary cell control group and the dental pulp stem cell group of the dental mesenchymal stem cell culture medium long-term culture group can form new dental pulp dentin tissue, while the cells of the conventional medium long-term culture group are not formed by dental pulp dentin tissue.
The culture medium of the invention can effectively maintain the regeneration capability of cell tissues when used for culturing dental pulp stem cells for a long time.
The invention has the beneficial effects that:
the invention applies a novel tooth mesenchymal stem cell culture medium combined by cell factors and small molecular compounds with definite components, has no foreign proteins or serum-free components, expands tooth mesenchymal stem cells in vitro (taking dental pulp stem cells as an example), not only avoids the defects of undefined components, large batch difference, potential safety risk, ethical problems and the like caused by serum in the traditional culture medium, but also has the characteristics of cell proliferation, stem cell characteristics, tooth differentiation capacity and the like similar to primary cells of the stem cells obtained after the long-term culture of the tooth mesenchymal stem cells, and is a culture medium with good precursor external expansion in clinical application of the stem cells.
The cell factor and the small molecular compound used in the invention have wide sources, high safety, clear biological function, stable quality, no problems of immunological rejection and the like caused by pathogens and foreign proteins, and are convenient for popularization and large-scale use.
The invention is beneficial to the cell amplification before clinic or the experiment with high requirement on the quality stability of the experimental cells.
Drawings
Fig. 1A is a schematic diagram of cell morphology of a method for verifying cell activity of a dental mesenchymal stem cell culture medium in dental pulp stem cells according to the present invention;
fig. 1B is a schematic diagram of cell surface marker detection in the verification method of cell activity of dental mesenchymal stem cell culture medium in dental pulp stem cells according to the present invention;
fig. 2A is a schematic diagram illustrating the evaluation of cell proliferation ability of the method for verifying the cell activity of the dental mesenchymal stem cell culture medium in dental pulp stem cells according to the present invention;
fig. 2B is a schematic diagram of cell aging of the verification method of cell activity of the dental mesenchymal stem cell culture medium in dental pulp stem cells according to the present invention;
fig. 2C is a schematic diagram of a cell aging quantitative analysis of the method for verifying the cell activity of the dental mesenchymal stem cell culture medium in the dental pulp stem cells according to the present invention;
fig. 3A is a schematic diagram illustrating the cell cloning capacity of the method for verifying the cell activity of the dental mesenchymal stem cell culture medium in dental pulp stem cells according to the present invention;
fig. 3B is a stem cell characteristic diagram of quantitative PCR of stem cell marker proteins OCT4 and SOX2 according to the method for verifying cell activity of dental mesenchymal stem cell culture medium in dental pulp stem cells;
fig. 3C is a schematic diagram of immunofluorescent-stained stem cell characteristics of a method for verifying cell activity of a dental mesenchymal stem cell culture medium in dental pulp stem cells according to the present invention;
fig. 4A is a graph illustrating the influence of the mesenchymal stem cell culture medium on the dryness of the dental pulp stem cells evaluated by a cell scratch test in the method for verifying the cell activity of the mesenchymal stem cell culture medium in the dental pulp stem cells;
fig. 4B is a graph illustrating the influence of the dryness of the dental pulp stem cell evaluated by the dental mesenchymal stem cell culture medium through a Transwell experiment in the method for verifying the cell activity of the dental mesenchymal stem cell culture medium in the dental pulp stem cell according to the present invention;
fig. 5A is a schematic diagram of activity of cells in dental mesenchymal stem cell culture medium a L P in a method for verifying cell activity of dental mesenchymal stem cell culture medium in dental pulp stem cells according to the present invention;
fig. 5B is a schematic diagram illustrating the alizarin red staining mineralization nodule forming ability of cells in the dental mesenchymal stem cell culture medium in the method for verifying the cell activity of the dental mesenchymal stem cell culture medium in dental pulp stem cells according to the present invention;
FIG. 6A is a schematic diagram of HE of stem cell tissue section after cells are cultured in a conventional culture medium in the method for verifying the cell activity of the dental mesenchymal stem cell culture medium in dental pulp stem cells;
fig. 6B is a schematic diagram of a tooth mesenchymal stem cell culture medium after MASSON staining in a method for verifying cell activity in a dental pulp stem cell according to the present invention;
Detailed Description
Embodiments of the present invention will be described in detail below with reference to the accompanying drawings.
Example 1:
a dental mesenchymal stem cell culture medium is prepared by taking a-MEM as a basic culture medium and adding a composition of growth factors including epidermal growth factor, platelet-derived growth factor, vitamin C, TGF- β receptor inhibitor L Y3200882, gamma-secretase inhibitor L Y411575 and DNA methylase inhibitor 5-Aza-dC and a small molecule inhibitor to form the dental mesenchymal stem cell culture medium, wherein the composition comprises the following components in an amount of 10-200 mu g/L of the epidermal growth factor, 500 mu g/L of the platelet-derived growth factor, 10-100 mg/L of vitamin C, 500nM of 100 nM of TGF- β receptor inhibitor L Y3200882, 50 nM-200 nM of the gamma-secretase inhibitor L Y411575 and 1-20nM of the DNA methylase inhibitor 5-Aza-dC.
Example 2:
a method for verifying cell activity of a dental mesenchymal stem cell culture medium in dental pulp stem cells as described in example 1, comprising the following steps:
(1) pretreatment of cells
Separating and culturing primary dental pulp stem cells, and dividing the primary dental pulp stem cells into a primary cell Control Group (Con), a conventional culture medium long-term culture Group (L ong-term culture with continuous culture medium, CM) and a tooth mesenchymal stem cell culture medium long-term culture Group (L ong-term culture with SFM), wherein the primary dental pulp stem cells are used as the primary dental pulp stem cells, and the long-term culture Group is formed by passage of the dental pulp stem cells in the conventional culture medium and the tooth mesenchymal stem cell culture medium to the 9 th generation;
(2) evaluation of biological Properties in vitro of cells
2.1 evaluation of morphology and surface markers of cells;
the Primary cell Control Group (Control Group of Primary mesenchymal stem cells, Con), the conventional medium long-term culture Group (L ong-term culture with continuous culture medium, CM) and the tooth mesenchymal stem cell culture medium long-term culture Group (L ong-term culture with SFM) were observed under an inverted microscope, and the cells of the Primary cell Control Group are typical tooth mesenchymal stem cells, clear in cell outline, long spindle type or spindle type, the cell morphology of the conventional medium long-term culture Group is obviously changed, the cells are large and flat, the outline is unclear, the tooth mesenchymal stem cell culture medium long-term culture Group maintains better tooth pulp mesenchymal stem cell morphology, is spindle type (figure 1A), the cell surface markers are detected by flow cytometry, and the results show that the Primary cell Control Group (Control Group of Primary mesenchymal stem cells, Congest stem cell culture medium cells, CD44, CD medium cell culture medium surface markers (CD) and the blood vessel surface markers (CD 355631, CD 27, CD 21, 365631, CD 21, 3631, 27, 3655) and CD 21, 365631, 3631, CD3, CD 21, CD3, CD mark and CD mark expression of three groups of blood stem cell culture medium, CD mark of a blood stem cell culture medium, CD mark and CD mark of a blood stem cell culture medium
2.2 evaluation of cell proliferation and senescence;
CCK-8 experiments show that the primary dental pulp stem cells have good cell proliferation capacity, the cell proliferation capacity of the primary dental pulp stem cells after long-term culture in a conventional culture medium is remarkably reduced, and the proliferation capacity of the dental pulp stem cells cultured by using the dental mesenchymal stem cell culture medium is remarkably higher than that of cells in the conventional culture medium and is close to that of the primary dental pulp stem cells (figure 2A); cell senescence staining showed that dental pulp stem cells cultured for a long period using the medium of the present invention senesced significantly less than cells of the conventional medium group, close to primary cells (fig. 2B, C). The culture medium can maintain the proliferation capacity of the dental pulp stem cells and delay cell aging in the long-term culture process.
2.3 evaluation of the characteristics of stem cells;
cell clone formation experiments show that the cell clone formation capability of primary dental pulp stem cells is higher than that of cells cultured in a conventional culture medium for a long time, and the clone formation capability of the dental pulp stem cells cultured by the dental mesenchymal stem cell culture medium is higher than that of the dental pulp stem cells cultured in the conventional culture medium for a long time (figure 3A). Quantitative PCR (fig. 3B) and immunofluorescence staining (fig. 3C) of stem cell marker proteins OCT4 and SOX2 showed that dental pulp stem cells cultured in dental mesenchymal stem cell medium for a long period positively expressed OCT4 and SOX2, with a higher percentage of positive cells than the conventional medium group, close to the primary dental pulp stem cell group. It was shown that SFM medium can maintain the dryness of dental pulp stem cells during long-term culture.
2.4 evaluation of the migration ability of cells;
transwell (fig. 4A) and cell scratch experiments (fig. 4B) showed that the cell migration capacity of the dental mesenchymal stem cell culture medium long-term cultured group cells was higher than that of the conventional culture medium long-term cultured group, indicating that dental pulp stem cells cultured in the dental mesenchymal stem cell culture medium long-term can maintain the migration capacity close to that of primary cells.
2.5 evaluation of tooth-Forming differentiation Capacity of dental pulp Stem cells
As shown in fig. 5, the cells in the long-term culture group of the dental mesenchymal stem cells are superior to the cells in the conventional culture medium group in both a L P activity (fig. 5A) and mineralized nodule formation ability (fig. 5B), and quantitative analysis shows that the a L P activity and mineralized nodule formation level of the cells are close to those of the primary dental pulp stem cells, which indicates that the culture medium of the present invention can maintain the odontoblastic differentiation ability of the dental pulp stem cells in the long-term culture process of the dental pulp stem cells.
(3) Evaluation of regeneration Capacity of dental pulp in animal body
Injecting the dental pulp stem cells obtained in the step 2.1 after compounding collagen into a prepared dentin matrix casing, implanting the dental pulp stem cells into a nude mouse subcutaneous tissue, sampling 2 months later, fixing 4% paraformaldehyde, decalcifying with 10% EDTA, preparing a conventional tissue section, performing HE staining and MASSON staining, and analyzing the dental pulp regeneration condition after transplanting each group of cells. The primary dental pulp stem cell group and the dental pulp stem cell group cultured by the dental mesenchymal stem cell culture medium for a long time can form a new dental pulp dentin tissue, and the conventional culture group cells are not formed by the dental pulp dentin tissue, so that the dental pulp stem cells cultured by the serum-free culture medium for a long time can effectively maintain the regeneration capability of the cell tissue.
The above-mentioned embodiments only express the specific embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention.
Claims (8)
1. A dental mesenchymal stem cell culture medium is characterized in that a-MEM is used as a basal medium, and a composition of a growth factor including an epidermal growth factor, a platelet-derived growth factor, a vitamin C, TGF- β receptor inhibitor L Y3200882, a gamma-secrase inhibitor L Y411575 and a DNA methylase inhibitor 5-Aza-dC and a small molecule inhibitor is added to form the dental mesenchymal stem cell culture medium, wherein the composition comprises the components of 10-200 mu g/L of the epidermal growth factor, 500 mu g/L of the platelet-derived growth factor, 10-100 mg/L of the vitamin C, 100 mu g/3200882 of the TGF- β receptor inhibitor L Y3200882, 50-200nM of the gamma-secrase inhibitor L Y411575 and 1-20nM of the DNA methylase inhibitor 5-Aza-dC-20.
2. A method for verifying cell activity of the dental mesenchymal stem cell culture medium in dental pulp stem cells according to claim 1, comprising the steps of:
(1) the pretreatment of the cells is carried out,
separating and culturing the primary dental pulp stem cells into a primary cell control group, a conventional culture medium long-term culture group and a tooth mesenchymal stem cell culture medium long-term culture group; wherein the primary cell control group is primary dental pulp stem cells, and the long-term culture group is that the dental pulp stem cells are passaged to the 9 th generation in a conventional culture medium and a tooth mesenchymal stem cell culture medium;
(2) the evaluation of the biological properties of cells in vitro,
2.1 evaluation of morphology and surface markers of cells;
2.2 evaluation of cell proliferation and senescence;
2.3 evaluation of the characteristics of stem cells;
2.4 evaluation of the migration ability of cells;
2.5 evaluation of odontoblastic differentiation ability of dental pulp stem cells;
(3) evaluation of the ability to regenerate dental pulp in an animal,
injecting the dental pulp stem cells obtained in the step 2.1 after compounding collagen into a prepared dentin matrix casing, implanting the dental pulp stem cells into a nude mouse subcutaneous tissue, sampling 2 months later, fixing 4% paraformaldehyde, decalcifying with 10% EDTA, preparing a conventional tissue slice, performing HE and MASSON staining, and analyzing the dental pulp regeneration condition after transplanting each group of cells.
3. The method for verifying cell activity of a dental mesenchymal stem cell culture medium in dental pulp stem cells according to claim 2, wherein in the step 2.1, the morphology of the cells and the surface marker evaluation are evaluated by a cell optical microscope, and the cells of the primary cell control group and the dental mesenchymal stem cell culture medium long-term culture group are smaller and have a long spindle shape or a spindle shape compared with the cells of the conventional culture medium long-term culture group; compared with the other two groups of cells, the cells of the long-term culture group of the conventional culture medium are larger and have flat shapes; flow cytometry was used to detect expression of cell surface markers with no difference between groups.
4. The method for verifying cell activity of the tooth mesenchymal stem cell culture medium in the tooth pulp stem cells according to claim 2, wherein in the step 2.2, the proliferation capacity and the cell senescence of the cells are evaluated by CCK-8 experimental cell senescence β -galactosidase (SA- β -Gal) staining, wherein the cell proliferation capacity of the tooth mesenchymal stem cell culture medium long-term culture group is not different from that of the primary cell control group but higher than that of the conventional culture medium long-term culture group, and the cell senescence of the tooth mesenchymal stem cell culture medium long-term culture group is not different from that of the primary cell control group, while the cell senescence of the conventional culture medium long-term culture group is higher than that of the tooth mesenchymal stem cell culture medium long-term culture group and the primary cell control group.
5. The method for verifying the cell activity of the dental mesenchymal stem cell culture medium in the dental pulp stem cells according to claim 2, wherein the step 2.3 is to evaluate the characteristics of the stem cells through a cloning experiment and the detection of the expression of the sternness gene of the cells, wherein the cell clone formation ability and the expression level of the sternness gene SOX2 of the dental mesenchymal stem cell culture medium long-term culture group are higher than those of the conventional culture medium long-term culture group.
6. The method for verifying cell activity of dental pulp stem cells maintained by a mesenchymal stem cell culture medium according to claim 2, wherein the evaluation of cell migration ability of the three groups is performed by a cell scratch test and a Transwell in step 2.4, wherein the migration ability of the dental pulp stem cell group of the primary cell control group and the migration ability of the dental pulp stem cells of the dental pulp stem cell culture medium long-term group are higher than the migration ability of the dental pulp stem cell group of the conventional medium long-term culture group.
7. The method for verifying cell activity of dental mesenchymal stem cell culture medium in dental pulp stem cells according to claim 2, wherein the step 2.5 is performed by quantitatively analyzing a L P activity and mineralized nodule formation ability of three groups of stem cells, wherein a L P activity and mineralized nodule formation ability of dental pulp stem cell group of primary cell control group and a L P activity and mineralized nodule formation ability of dental pulp stem cells of dental mesenchymal stem cell culture medium long-term culture group are higher than a L P activity and mineralized nodule formation ability of dental pulp stem cell group of conventional culture medium long-term culture group.
8. The method for verifying cell activity of mesenchymal stem cell culture medium in dental pulp stem cells according to claim 2, wherein in step 3, the ability of three groups of dental pulp stem cells to form dental pulp tissue in an animal body is evaluated by transplanting three groups of dental pulp stem cells in the animal body, wherein the dental pulp stem cell group of the primary cell control group and the dental pulp stem cell group of the long-term culture group of the dental mesenchymal stem cell culture medium can form new dental pulp dentin tissue, while the cells of the long-term culture group of the conventional culture medium are not formed by dental pulp dentin tissue.
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