CN111909898A - Separation and amplification method of human dental pulp stem cells and application thereof - Google Patents
Separation and amplification method of human dental pulp stem cells and application thereof Download PDFInfo
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Abstract
The invention relates to the field of medical biology, and provides a method for separating and amplifying human dental pulp stem cells and application thereof, wherein the method for separating and amplifying the human dental pulp stem cells comprises the following steps: s1, taking human tooth marrow tissue, shearing, adding collagenase I, hyaluronidase and dispase II for digestion to obtain digested tissue and cell suspension; s2, filtering the digested tissue and cell suspension by using a filter screen, centrifuging the filtrate, and taking cell sediment to obtain human dental pulp stem cells; s3, inoculating the human dental pulp stem cells into a culture dish, coating human fibronectin in advance on the culture dish, adding a culture solution for culturing, and carrying out subculture when the confluence rate reaches 70-80%. The method of the invention has short cell adherence time, and the cell number reaches at least 4.5 multiplied by 10 after the 10 th day of culture5The purity of the cells is high, the positive expression rate of the surface markers CD73, CD90 and CD105 of the dental pulp stem cells is more than 99%, and the negative index expression rate is lower than 1.2%.
Description
Technical Field
The invention relates to the field of medical biology, in particular to a method for separating and amplifying human dental pulp stem cells and application thereof.
Background
Stem cells are a type of pluripotent cells with self-replicating ability, which can differentiate into various functional cells under certain conditions, and have the potential function of regenerating various tissues and organs and intact individuals. The sources of stem cells are many, including bone marrow, umbilical cord, cord blood, teeth, fat, skin, and hair follicles.
Gronthos et al, 2000 discovered Cells with an immunophenotype and mineralized nodule forming ability very similar to bone marrow mesenchymal Stem Cells, with fusiform cell morphology that can self-renew and differentiate in multiple directions, and these fibroblasts isolated from Pulp tissue were called Dental Pulp Stem Cells (DPSCs). The dental pulp stem cells have good osteogenic differentiation capacity, and compared with bone marrow mesenchymal stem cells, the sources of the dental pulp stem cells are rich, the natural falling teeth of children of 6-12 years old and the wisdom teeth of adults all contain rich dental pulp stem cells, and the materials are safe and convenient to obtain, so that the dental pulp stem cells become one of the hot spot cells in the field of bone regeneration medicine at present. At present, the related research of the clinical test of the dental pulp stem cells mainly focuses on the field of tooth, periodontal tissue and maxillofacial bone regeneration, and with the continuous maturation of the research of the dental pulp stem cells and the rapid development of stem cell regeneration medicine, the dental pulp stem cells also obtain good results in the treatment of nerve regeneration, diabetes and autoimmune diseases.
At present, methods such as an enzyme-linked digestion method, a tissue block culture method, a tissue block enzyme digestion method and the like are commonly used in a preparation method of dental pulp stem cells, but the prepared dental pulp stem cells are low in purity and small in quantity.
Chinese patent document CN201911173028.4 discloses a culture medium for inducing differentiation from dental pulp stem cells to osteoblasts, a preparation method and an application thereof, wherein the culture medium comprises 4-5.5 g/L BSA, 1.5mg/L reduced glutathione, 0.08-0.12 mmol/L beta-mercaptoethanol, 0.08-1.5% (V/V) SITE, 0.8-1.2M/L beta-glycerophosphoric acid, 15-25 mu M/L L-ascorbic acid and 0.5-1.2 mM/L dexamethasone, and the culture medium can optimize the method for inducing differentiation from dental pulp stem cells to osteoblasts, has short induction time and low cost, but the purity of the separated dental pulp stem cells is low. Further, chinese patent document CN201710596515.6 discloses a human dental pulp stem cell culture medium and a method for preparing human dental pulp stem cells, wherein the culture medium is based on an α -MEM culture medium, and comprises 5 to 10% by volume of human AB serum, 100 μ M ascorbic acid, 2mm l-glutamine, 100U/ml penicillin sodium and 100mg/ml streptomycin, and the culture medium contains penicillin sodium and streptomycin, and can reduce contamination by infectious microbes to some extent and improve the yield of dental pulp stem cells, but the prepared dental pulp stem cells have poor differentiation ability.
In summary, the existing technology for separating dental pulp stem cells from dental tissues still has the problems of insufficient cell quantity, insufficient cell purity and insufficient differentiation capability. Therefore, the invention provides a technical method for separating and amplifying dental pulp stem cells, and the prepared dental pulp stem cells have the advantages of large quantity, high purity and strong differentiation capability, and can fully meet the requirements of clinical application.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a method for separating and amplifying human dental pulp stem cells, which has high efficiency and a large number of separated dental pulp stem cells, wherein the improved culture solution can effectively promote the growth of the human dental pulp stem cells, keeps higher activity and has stronger differentiation capability.
In order to achieve the purpose, the invention provides the following technical scheme:
a method for separating and amplifying human dental pulp stem cells comprises the following steps:
s1, taking human tooth marrow tissue, shearing, adding collagenase I, hyaluronidase and dispase II for digestion, and obtaining digested tissue and cell suspension;
s2, after digestion is finished, filtering digested tissues and cell suspension by using a filter screen with the diameter of 60-80 mu m, and centrifuging filtrate to obtain cell sediment to obtain human dental pulp stem cells;
s3, inoculating the human dental pulp stem cells obtained in the step S2 into a culture dish, wherein the culture dish is coated with 1-5 mu g/cm2Adding human fibronectin into culture dishAnd (3) culturing the dental pulp stem cells, and digesting by adopting trypsin and ethylenediamine tetraacetic acid when the confluence rate of the human dental pulp stem cells reaches 70-80%, and carrying out subculture.
Preferably, the mass volume concentration of the collagenase I is 2-5 mg/ml, the mass volume concentration of the hyaluronidase is 0.1-0.3%, and the mass volume concentration of the dispase II is 2-6 mg/ml.
More preferably, the mass ratio of the collagenase I to the hyaluronidase to the dispase II is (2-5): (0.1-0.3): (2-6).
Preferably, the culture solution comprises an alpha-MEM culture medium containing 5-10% of human AB serum, 80-120 mu M L-ascorbic acid phosphate, 5-10 nM dexamethasone, 0.5-2 mu M berberine, 10-30 ng/ml tumor suppressor M, 1-2 mu M esculin, 8-15 mM beta-sodium glycerophosphate, 1-5 mM L-glutamine and a final concentration of 0.5-1.5% streptomycin double antibody.
More preferably, the culture medium comprises an alpha-MEM medium containing 5-10% human AB serum, 100. mu. M L-ascorbic acid phosphate ester, 10nM dexamethasone, 1. mu.M berberine, 20ng/ml oncostatin M, 1.6. mu.M esculin, 10mM beta-sodium glycerophosphate, 2mM L-glutamine and 1% final concentration of streptomycin diabody.
Preferably, the culture solution comprises an alpha-MEM culture medium containing 5-10% of human AB serum, 80 mu M L-ascorbic acid phosphate, 10nM dexamethasone, 2 mu M berberine, 10ng/ml of antitumor agent M, 1 mu M esculin, 15mM beta-sodium glycerophosphate, 2mM L-glutamine and 1% final concentration of streptomycin diabody.
Preferably, the diameter of the screen is 70 μm.
Preferably, in the step S3, the human dental pulp stem cells are 5 × 103cells/cm2Inoculated in a petri dish.
Preferably, in the step S3, the mass volume concentration of the trypsin is 0.1-0.25%, and the mass volume concentration of the ethylenediaminetetraacetic acid is 1-2 mM.
The invention also provides an application of the human dental pulp stem cells in human tissue and organ repair materials.
In the technical scheme of the invention, the collagenase I, the hyaluronidase and the dispase II are jointly used for digestion, so that the fibers and the matrixes in human dental pulp tissues can be effectively decomposed, the number of the human dental pulp stem cells obtained by separation is large, the efficiency is high, and the separated human dental pulp stem cells are inoculated into a culture dish coated with human fibronectin in advance, so that the growth and the proliferation of the human dental pulp stem cells can be effectively promoted. And a filter screen with the diameter of 60-80 mu m is used for filtering, so that the dental pulp stem cells can be obtained to the maximum extent without damaging the dental pulp stem cells.
The inventors of the present invention found that the number of dental pulp stem cells prepared was small and the number of human dental pulp stem cells cultured for 10 days was only 2.5X 10 in the conventional culture system (alpha-MEM medium containing 10% fetal bovine serum) at the early stage of the experiment5The activity of the cells is low, the differentiation function is not strong, and the cells are very easy to be polluted by mixed bacteria in the preparation process; therefore, the inventor creates a culture solution with a new formula through a large number of experimental operations, wherein the culture solution comprises an alpha-MEM culture medium containing 5-10% of human AB serum, 80-120 mu M L-ascorbic acid phosphate, 5-10 nM dexamethasone, 0.5-2 mu M berberine, 10-30 ng/ml of tumor suppressor M, 8-15 mM beta-sodium glycerophosphate, 1-5 mM L-glutamine and a streptomycin double antibody with the final concentration of 0.5-1.5%, wherein the human AB serum can be added to avoid the heterologous pollution of animal serum and the like, so that the immunological rejection reaction of a human body is reduced; l-ascorbic acid phosphate is involved in the vital activities of human dental pulp stem cells such as protein metabolism, fat metabolism, carbohydrate metabolism and the like; dexamethasone and beta-sodium glycerophosphate can promote the differentiation capacity of the human dental pulp stem cells; glutamine is used as an organic nitrogen source, can promote the growth of human dental pulp stem cells and maintain the life activity of the cells; the pollution of mixed bacteria in the preparation process of the dental pulp stem cells can be reduced by adding the streptomycin double antibody into the culture solution, but the effect of inhibiting the mixed bacteria is poor, so that the inventor introduces berberine which has the effects of inhibiting and killing various gram-positive and gram-negative bacteria, fungi, moulds, viruses, protozoa and nematodes, has high safety and high stability, and introduces the tumor suppressor M which can promote the differentiation capability of the human dental pulp stem cells. The culture solution formula can improve the purity of human dental pulp stem cells, and dental pulp stem cells are detected by cell purityThe positive expression rate of stem cell surface markers CD73, CD90 and CD105 is more than 99%, and the negative index expression rate is lower than 1.2%. On the basis, the invention unexpectedly adds the esculin, and the esculin and the human fibronectin which is added in the culture dish in advance act together, thereby shortening the cell adherence time and effectively promoting the increase of the number of the human dental pulp stem cells.
The culture system can effectively promote the growth of human dental pulp stem cells, obtain more cell number, maintain higher activity, have high cell purity and short cell adherence time, and show stronger differentiation potential.
Compared with the prior art, the invention has the following beneficial effects:
1. the method for separating and amplifying the human dental pulp stem cells obtains more cells, keeps higher activity, has short cell adherence time, and ensures that the cell number of the human dental pulp stem cells reaches at least 4.5 multiplied by 10 at the 10 th day of culture5The number of the dental pulp stem cells is far higher than that of dental pulp stem cells obtained by a conventional culture system;
2. according to the method for separating and amplifying the human dental pulp stem cells, the separation process is not easily polluted by mixed bacteria, the differentiation capacity is strong, the purity of the cultured human dental pulp stem cells is high, the positive expression rate of surface markers CD73, CD90 and CD105 of the dental pulp stem cells is more than 99%, and the negative index expression rate is lower than 1.2%;
3. the human dental pulp stem cell provided by the invention is applied to the repair of human tissues and organs, has a wide application range, and fully meets the requirements of clinical application.
Drawings
FIG. 1 is a schematic view of flow cytometry detection of human dental pulp stem cells prepared in example 5 of the present invention.
Detailed Description
The present invention will be described in further detail with reference to the accompanying drawings and examples.
In the following examples, the components used are all common commercial products, all of which are commercially available.
Berberine was purchased from Sigma; oncostatin M was purchased from PeproTech.
Sanchinoside is purchased from Nanjing Benyuan Biotechnology Ltd under CAS number 88105-29-7.
Example 1
A method for separating and amplifying human dental pulp stem cells comprises the following steps:
s1, collecting third molar of an adult 18-25 years old, wherein tooth tissue is required to be complete, free of caries, free of dental pulp disease, periodontal disease and the like, after the tooth is taken down, cleaning and disinfecting the surface of the tooth tissue by using PBS (phosphate buffered saline) buffer solution, then placing the tooth into a collecting tube containing DMEM (DMEM) culture medium with 1% of final concentration of the dual-antibody against streptomycin, sending the tooth into a laboratory by using a constant-temperature storage box at 2-8 ℃, placing 2 10cm sterile culture dishes in a biological safety cabinet, and adding a proper amount of PBS buffer solution with 1% of final concentration of the dual-antibody against streptomycin. Taking out the teeth in the collecting tube with sterile forceps, cleaning and sterilizing in the above culture dish for 2-3min to remove periodontal soft tissue and blood stain, and sterilizing the sterilized teeth in a second culture dish. Placing the cleaned tooth on sterile gauze, wrapping the tooth with the gauze, clamping enamel and dentin of the tooth with a bench vice, opening the sterile gauze, and finding out dental pulp tissue from the dental pulp cavity with forceps;
s2, placing human tooth marrow tissue into an EP tube containing an alpha-MEM culture medium, and shearing the human tooth marrow tissue to be about 0.5mm by using a sterile ophthalmic scissors3Centrifuging the tissue blocks with the size for 5min at the centrifugal rotating speed of 800rpm/min to remove the alpha-MEM culture medium, adding collagenase I with the mass volume concentration of 2mg/ml, hyaluronidase with the mass volume concentration of 0.1% and dispase II with the mass volume concentration of 2mg/ml for digestion for 1h to obtain digested tissue and cell suspension;
s3, after digestion is finished, filtering the digestive juice by using a filter screen with the diameter of 60 mu m, and centrifuging the filtrate to obtain cell sediment to obtain the human dental pulp stem cells;
s4, the human tooth marrow stem cells obtained in the step S3 are arranged according to the proportion of 5 multiplied by 103cells/cm2Inoculating into a culture dish, wherein the culture dish is coated with 1 mu g/cm in advance2Washing human fibronectin with PBS 2 times before using a culture dish, adding human dental pulp stem cell culture solution into the culture dish to culture human dental pulp stem cells, changing the solution every 3 days,when the confluence rate of human dental pulp stem cells reaches 70%, the cells are digested by trypsin with the mass volume concentration of 0.1% and ethylenediamine tetraacetic acid with the mass volume concentration of 1mM, and subculture is carried out.
In this example, the mass ratio of collagenase I, hyaluronidase, and dispase II was 2:0.1:2, and the total volume of collagenase I, hyaluronidase, and dispase II was 5 times the total volume of the tissue mass.
In this example, the culture medium included alpha-MEM medium containing 5% human AB serum, 80. mu. M L-ascorbic acid phosphate, 5nM dexamethasone, 0.5. mu.M berberine, 10ng/ml oncostatin M, 1. mu.M esculin, 8mM sodium beta-glycerophosphate, 1mM L-glutamine, and a final concentration of 0.5% streptomycin diabody.
Example 2
A method for separating and amplifying human dental pulp stem cells comprises the following steps:
s1, the method is the same as the embodiment 1;
s2, placing human tooth marrow tissue into an EP tube containing an alpha-MEM culture medium, and shearing the human tooth marrow tissue to about 1.0mm by using a sterile ophthalmic scissors3Centrifuging the tissue blocks with the size for 5min at the centrifugal rotating speed of 1000rpm/min to remove the alpha-MEM culture medium, adding collagenase I with the mass volume concentration of 3mg/ml, hyaluronidase with the mass volume concentration of 0.15% and dispase II with the mass volume concentration of 4mg/ml for digestion for 1h to obtain digested tissue and cell suspension;
s3, after digestion is finished, filtering the digestive juice by using a filter screen with the diameter of 70 microns, and centrifuging the filtrate to obtain cell sediment to obtain human dental pulp stem cells;
s4, the human tooth marrow stem cells obtained in the step S3 are arranged according to the proportion of 5 multiplied by 103cells/cm2Inoculating into a culture dish, wherein the culture dish is pre-coated with 2 mu g/cm2Washing human fibronectin with PBS 2 times before using a culture dish, adding human dental pulp stem cell culture solution into the culture dish to culture human dental pulp stem cells, changing the culture solution every 3 days, digesting with trypsin with the mass volume concentration of 0.125% and ethylenediamine tetraacetic acid with the mass volume concentration of 1.25mM when the confluence rate of the human dental pulp stem cells reaches 80%, and carrying out subculture.
In this example, the mass ratio of collagenase I, hyaluronidase, and dispase II was 3:0.15:4, and the total volume of collagenase I, hyaluronidase, and dispase II was 6 times the total volume of the tissue mass.
In this example, the culture medium included α -MEM medium containing 7% human AB serum, 80 μ M L-ascorbyl phosphate, 10nM dexamethasone, 2 μ M berberine, 10ng/ml oncostatin M, 1 μ M esculin, 15mM β -sodium glycerophosphate, 2mM L-glutamine, and 1.0% final concentration of streptomycin diabody.
Example 3
A method for separating and amplifying human dental pulp stem cells comprises the following steps:
s1, the method is the same as the embodiment 1;
s2, placing human tooth marrow tissue into an EP tube containing an alpha-MEM culture medium, and shearing the human tooth marrow tissue to about 1.0mm by using a sterile ophthalmic scissors3Centrifuging the tissue blocks with the size for 10min at the centrifugal rotating speed of 1200rpm/min to remove the alpha-MEM culture medium, adding collagenase I with the mass volume concentration of 4mg/ml, hyaluronidase with the mass volume concentration of 0.2% and dispase II with the mass volume concentration of 5mg/ml for digestion for 1h to obtain digested tissue and cell suspension;
s3, after digestion is finished, filtering the digestive juice by using a filter screen with the diameter of 80 microns, and centrifuging the filtrate to obtain cell sediment to obtain human dental pulp stem cells;
s4, the human tooth marrow stem cells obtained in the step S3 are arranged according to the proportion of 5 multiplied by 103cells/cm2Inoculating into a culture dish, wherein the culture dish is pre-coated with 4 mu g/cm2Washing human fibronectin with PBS 3 times before using a culture dish, adding human dental pulp stem cell culture solution into the culture dish to culture human dental pulp stem cells, changing the culture solution every 3 days, digesting with trypsin with the mass volume concentration of 0.15% and ethylenediamine tetraacetic acid with the mass volume concentration of 1.5mM when the confluence rate of the human dental pulp stem cells reaches 80%, and carrying out subculture.
In this example, the mass ratio of collagenase I, hyaluronidase, and dispase II was 4:0.2:5, and the total volume of collagenase I, hyaluronidase, and dispase II was 6 times the total volume of the tissue mass.
In this example, the culture medium included alpha-MEM medium containing 9% human AB serum, 110. mu. M L-ascorbic acid phosphate, 8nM dexamethasone, 1.5. mu.M berberine, 25ng/ml oncostatin M, 1.8. mu.M esculin, 13mM beta-sodium glycerophosphate, 4mM L-glutamine and 1.5% final concentration of the streptomycin diabody.
Example 4
A method for separating and amplifying human dental pulp stem cells comprises the following steps:
s1, the method is the same as the embodiment 1;
s2, placing human tooth marrow tissue into an EP tube containing an alpha-MEM culture medium, and shearing the human tooth marrow tissue to about 1.0mm by using a sterile ophthalmic scissors3Centrifuging the tissue blocks with the size for 10min at the centrifugal rotating speed of 1200rpm/min to remove the alpha-MEM culture medium, adding collagenase I with the mass volume concentration of 5mg/ml, hyaluronidase with the mass volume concentration of 0.3% and dispase II with the mass volume concentration of 6mg/ml for digestion for 1h, and obtaining digested tissue and cell suspension;
s3, after digestion is finished, filtering the digestive juice by using a filter screen with the diameter of 70 microns, and centrifuging the filtrate to obtain cell sediment to obtain human dental pulp stem cells;
s4, the human tooth marrow stem cells obtained in the step S3 are arranged according to the proportion of 5 multiplied by 103cells/cm2Inoculating into a culture dish, and coating the culture dish with 5 μ g/cm2Washing human fibronectin with PBS 2 times before using a culture dish, adding human dental pulp stem cell culture solution into the culture dish to culture human dental pulp stem cells, changing the culture solution every 3 days, digesting with trypsin with the mass volume concentration of 0.2% and ethylenediamine tetraacetic acid with the mass volume concentration of 2mM when the confluence rate of the human dental pulp stem cells reaches 80%, and subculturing.
In this example, the mass ratio of collagenase I, hyaluronidase, and dispase II was 5:0.3:6, and the total volume of collagenase I, hyaluronidase, and dispase II was 7 times the total volume of the tissue mass.
In this example, the culture medium included alpha-MEM medium containing 10% human AB serum, 120. mu. M L-ascorbyl phosphate, 10nM dexamethasone, 2. mu.M berberine, 30ng/ml oncostatin M, 2. mu.M esculin, 15mM sodium beta-glycerophosphate, 5mM L-glutamine, and 1.5% final concentration of streptomycin diabody.
Example 5
A method for separating and amplifying human dental pulp stem cells comprises the following steps:
s1, the method is the same as the embodiment 1;
s2, placing human tooth marrow tissue into an EP tube containing an alpha-MEM culture medium, and shearing the human tooth marrow tissue to about 1.0mm by using a sterile ophthalmic scissors3Centrifuging the tissue blocks with the size at the centrifugation speed of 1000rpm/min for 10min to remove the alpha-MEM culture medium, adding collagenase I with the mass volume concentration of 3mg/ml, hyaluronidase with the mass volume concentration of 0.25% and dispase II with the mass volume concentration of 4mg/ml, and digesting for 1h to obtain digested tissue and cell suspension;
s3, after digestion is finished, filtering the digestive juice by using a filter screen with the diameter of 70 microns, and centrifuging the filtrate to obtain cell sediment to obtain human dental pulp stem cells;
s4, the human tooth marrow stem cells obtained in the step S3 are arranged according to the proportion of 5 multiplied by 103cells/cm2Inoculating into a culture dish, and coating the culture dish with 5 μ g/cm2Washing human fibronectin with PBS 2 times before using a culture dish, adding human dental pulp stem cell culture solution into the culture dish to culture human dental pulp stem cells, changing the culture solution every 3 days, digesting with trypsin with the mass volume concentration of 0.25% and ethylenediamine tetraacetic acid with the mass volume concentration of 1mM when the confluence rate of the human dental pulp stem cells reaches 80%, and subculturing.
In this example, the mass ratio of collagenase I, hyaluronidase, and dispase II was 3:0.25:4, and the total volume of collagenase I, hyaluronidase, and dispase II was 6 times the total volume of the tissue mass.
In this example, the culture medium included alpha-MEM medium containing 10% human AB serum, 100. mu. M L-ascorbic acid phosphate, 10nM dexamethasone, 1. mu.M berberine, 20ng/ml oncostatin M, 1.6. mu.M esculin, 10mM sodium beta-glycerophosphate, 2mM L-glutamine and 1.0% final concentration of the streptomycin diabody.
In the technical scheme of the invention, the human dental pulp stem cells prepared in the embodiments 1-5 can be applied to human tissue and organ repair materials, and have strong differentiation potential.
Comparative example 1
Compared to example 5, the comparative example differs only in that, without hyaluronidase, collagenase I and dispase II are present in a mass ratio of 4: 5.
Comparative example 2
The comparative example differs from example 5 only in that it does not contain esculin and human fibronectin, and the remaining raw materials and parameters are the same as example 5.
Comparative example 3
The comparative example differs from example 5 only in that no esculin is present and the remaining raw materials and parameters are the same as in example 5.
Comparative example 4
The comparative example differs from example 5 only in that no human fibronectin is present and the remaining raw materials and parameters are the same as in example 5.
Comparative example 5
Compared with example 5, the comparative example only differs in that berberine is not contained and the rest of the raw materials and parameters are the same as example 5.
Comparative example 6
The comparative example differs from example 5 only in that no oncostatin M was present and the remaining raw materials and parameters were the same as in example 5.
Test example I detection of adherence time of human dental pulp Stem cells
The preparation systems of the groups 1 to 5 of examples and the groups 1 to 4 of comparative examples were set, freshly prepared human dental pulp stem cells were inoculated in a 24-well plate, 3 multiple wells per group, placed in a cell culture chamber, and the cell adherence time was observed, as shown in table 1.
TABLE 1
| Group of | Time of cell adherence |
| Example 1 | 52h |
| Example 2 | 51h |
| Example 3 | 49h |
| Example 4 | 48h |
| Example 5 | 48h |
| Comparative example 1 | 48h |
| Comparative example 2 | 60h |
| Comparative example 3 | 50h |
| Comparative example 4 | 58h |
| Control group for conventional culture | 60h |
As can be seen from the data in Table 1, compared with the conventional culture control group, the preparation system of the invention using the groups of examples 1-5 can significantly shorten the time for the human dental pulp stem cells to adhere to the wall, and the time for the cells to adhere to the wall can be shortened to 48-52 h.
Compared with example 5, comparative example 1 does not contain hyaluronidase, the mass ratio of collagenase I and dispase II is changed, and different enzymes have no significant influence on shortening of cell adherence time; the comparison 2 lacks esculin and human fibronectin, the cell attachment time of the human dental pulp stem cells is long, the comparison 3 or the comparison 4 lacks esculin and human fibronectin, the cell attachment time of the esculin and the human fibronectin is longer, but the cell attachment time of the esculin and the human fibronectin is shorter than that of the comparison 2, so that the combination of esculin and human fibronectin added in advance in a culture dish can shorten the cell attachment time, and effectively promote the increase of the number of the human dental pulp stem cells.
Test example two, detection of cell proliferation of human dental pulp stem cells
The results of counting human dental pulp stem cells on day 10 in examples 1 to 5 and comparative examples 1 to 4 in a conventional culture control group (. alpha. -MEM + 10% fetal bovine serum) are shown in Table 2.
TABLE 2
According to Table 2, the number of human dental pulp stem cells on the 10 th day of culture in examples 1 to 5 was at least 4.50X 105The stem cell number of human tooth marrow can reach 4.86X 105The growth and proliferation of human dental pulp stem cells are fast, and example 5 is the best example.
The comparative example 1 does not contain hyaluronidase, the mass ratio of the collagenase I to the dispase II is changed, the growth of the human dental pulp stem cells is influenced, the number of the human dental pulp stem cells is less than that of the examples 1-5, the collagenase I, the hyaluronidase and the dispase II are jointly used for digestion, the fibers and the matrix in the human dental pulp tissue can be effectively decomposed, the number of the human dental pulp stem cells obtained through separation is large, and the efficiency is high.
The number of human dental pulp stem cells was lower on day 10 of culture in comparative examples 2 to 4, and was not higher than that of human dental pulp stem cells in examples 1 to 5, but was still higher than that of human dental pulp stem cells cultured in the conventional culture control group on day 10.
Test example III detection of purity of human dental pulp Stem cells
Human dental pulp stem cells cultured up to the 3 rd generation in examples 1 to 5 and comparative examples 5 to 6 were subjected to cell purity measurement, and surface markers CD73, CD90, and CD105 positive and negative indicators (CD14, CD19, CD34, CD45, and HLA-DR) of the dental pulp stem cells were measured and measured, and the results are shown in table 3.
TABLE 3
In the embodiments 1-5 of the invention, the human dental pulp stem cells cultured to the 3 rd generation are subjected to cell purity detection, the purity of the human dental pulp stem cells is high, the positive expression rate of the dental pulp stem cell surface markers CD73, CD90 and CD105 is more than 99%, and the negative index expression rate is less than 1.2%, wherein FIG. 1 is a schematic diagram of flow cytometry detection in the embodiment 5, and the embodiment 5 is a best embodiment.
The comparative example 5 lacks berberine or the comparative example 6 lacks tumor suppressor M, the purity of the human dental pulp stem cells is lower than that of the human dental pulp stem cells cultured to the 3 rd generation in examples 1-5, which indicates that the purity of the human dental pulp stem cells can be obviously improved when the berberine and the tumor suppressor M are compounded, the expression rate of CD73 is as high as 100%, and the negative index is lower than 0.55%.
Test example four measurement of osteogenic differentiation Capacity of human dental pulp Stem cells
The dental pulp stem cells cultured in examples 1 to 5 and comparative example 6 to the 3 rd generation were collected, and the alkaline phosphatase activity was measured by performing the procedure according to the instructions of the alkaline phosphatase measurement kit and measuring the OD value of the sample at a wavelength of 405nm using a microplate reader. The alkaline phosphatase activity of the human dental pulp stem cells cultured in the embodiments 1-5 can reach 2mmol/L, while the alkaline phosphatase activity of the human dental pulp stem cells cultured in the comparative example 6 can not reach 2mmol/L but only reach 1.47mmol/L, and the osteogenic differentiation capability is poor.
The embodiments of the present invention are preferred embodiments of the present invention, and the scope of the present invention is not limited by these embodiments, so: all equivalent changes made according to the structure, shape and principle of the invention are covered by the protection scope of the invention.
Claims (10)
1. A method for separating and amplifying human dental pulp stem cells is characterized by comprising the following steps:
s1, taking human tooth marrow tissue, shearing, adding collagenase I, hyaluronidase and dispase II for digestion to obtain digested tissue and cell suspension;
s2, after digestion is finished, filtering digested tissues and cell suspension by using a filter screen with the diameter of 60-80 mu m, centrifuging filtrate, and taking cell sediment to obtain human dental pulp stem cells;
s3, inoculating the human dental pulp stem cells obtained in the step S2 into a culture dish, wherein the culture dish is coated with 1-5 mu g/cm2And (3) adding a culture solution into the culture dish to culture the human dental pulp stem cells, and digesting by adopting trypsin and ethylenediamine tetraacetic acid when the confluence rate of the human dental pulp stem cells reaches 70-80%, and carrying out subculture.
2. The method for isolating and amplifying human dental pulp stem cells according to claim 1, wherein the mass volume concentration of collagenase I is 2 to 5mg/ml, the mass volume concentration of hyaluronidase is 0.1 to 0.3%, and the mass volume concentration of dispase II is 2 to 6 mg/ml.
3. The method for separating and amplifying human dental pulp stem cells according to claim 2, wherein the mass ratio of collagenase I, hyaluronidase and dispase II is (2-5): (0.1-0.3): (2-6).
4. The method for separating and amplifying human dental pulp stem cells according to claim 1, wherein the culture solution comprises an alpha-MEM culture medium containing 5-10% of human AB serum, 80-120 μ M L-ascorbic acid phosphate, 5-10 nM dexamethasone, 0.5-2 μ M berberine, 10-30 ng/ml tumor suppressor M, 1-2 μ M triceptadenin, 8-15 mM beta-sodium glycerophosphate, 1-5 mM L-glutamine and a final concentration of 0.5-1.5% streptomycin diantibody.
5. The method for separating and expanding human dental pulp stem cells according to claim 4, wherein the culture solution comprises an α -MEM medium containing 5% to 10% human AB serum, 100 μ M L-ascorbyl phosphate, 10nM dexamethasone, 1 μ M berberine, 20ng/ml oncostatin M, 1.6 μ M tricuspin, 10mM β -sodium glycerophosphate, 2mM L-glutamine and 1% final concentration of streptomycin diabody.
6. The method for separating and expanding human dental pulp stem cells according to claim 1, wherein the culture solution comprises α -MEM medium containing 5% to 10% human AB serum, 80 μ M L-ascorbyl phosphate, 10nM dexamethasone, 2 μ M berberine, 10ng/ml oncostatin M, 1 μ M tricuspid, 15mM sodium β -glycerophosphate, 2mM L-glutamine and 1% final concentration of streptomycin diabody.
7. The method for separating and expanding human dental pulp stem cells according to claim 1, wherein the diameter of the mesh is 70 μm.
8. The method for separating and expanding human dental pulp stem cells according to claim 1, wherein the human dental pulp stem cells are separated into 5 x 10 cells in the step S33cells/cm2Inoculated in a petri dish.
9. The method for isolating and amplifying human dental pulp stem cells according to claim 1, wherein the concentration of trypsin in step S3 is 0.1-0.25% by mass and the concentration of EDTA is 1-2 mM by mass.
10. An application of human tooth marrow stem cell in human tissue and organ repairing material.
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