CN112522192B - Separation culture method of dental pulp mesenchymal stem cells - Google Patents
Separation culture method of dental pulp mesenchymal stem cells Download PDFInfo
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- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
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- C12N5/0664—Dental pulp stem cells, Dental follicle stem cells
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Abstract
The invention provides a method for separating and culturing dental pulp mesenchymal stem cells, which comprises the following steps: step S1, taking out and clamping a tooth sample, then taking out dental pulp tissues in the tooth sample, chopping, and immersing the chopped dental pulp tissues in a culture medium for later use; s2, placing the sterilized glass beads into a container, and soaking the glass beads in a complete culture medium for later use; s3, embedding the dental pulp tissue reserved in the S1 in the glass bead reserved in the S2, and culturing for 1-3 hours to obtain a glass bead-dental pulp tissue complex; and S4, subculturing the glass bead-dental pulp tissue complex prepared in the step S3, and collecting by using trypsin to obtain the target dental pulp mesenchymal stem cells. The invention provides a separation culture method of dental pulp mesenchymal stem cells, and aims to solve the technical problem that the scale production of the dental pulp mesenchymal stem cells cannot be carried out in the related technology.
Description
Technical Field
The invention relates to the technical field of cell culture, in particular to a separation culture method of dental pulp mesenchymal stem cells.
Background
Mesenchymal Stem Cells (MSCs) are adult stem cells with strong self-renewal capacity and multipotentiality. The source of the mesenchymal stem cells is wide, and the mesenchymal stem cells can be extracted from tissues such as dental pulp, bone marrow, umbilical cord blood, umbilical cord, fat, hair follicle and the like; under certain induction conditions, it can differentiate into cell tissues such as osteoblasts, chondroblasts, adipocytes and nerve cells. The advantages of mesenchymal stem cells are numerous: it can support hematopoiesis, promote the implantation of hematopoietic stem cells, regulate immunity, promote tissue repair by paracrine, and has simple and convenient separation and culture operation, etc.; and has higher safety performance compared with the original embryonic stem cells. Based on the advantages, the mesenchymal stem cells are widely applied to the aspects of graft-versus-host disease, rheumatoid arthritis, combined hematopoietic stem cell transplantation, hepatic fibrosis, diabetic foot and the like. Based on the advantages, the autologous mesenchymal stem cells become safe and reliable seed cells in tissue engineering and organ engineering.
Dental Pulp Stem Cells (DPSCs) are multifunctional adult Stem Cells, and are mesenchymal Stem Cells isolated from Dental Pulp tissue. At present, dental pulp stem cells have been demonstrated to have a multipotent differentiation ability, and to be able to differentiate into tissue cells such as bone, fat and vascular endothelium. Research shows that the dental pulp stem cells can be used for tissue repair and regeneration of dentin, dental pulp, tooth bodies and dental crowns, and can also be used for damage repair of skull, jaw bone and facial bone. In addition, the dental pulp stem cells also contribute greatly to the treatment of diseases such as cerebrovascular accident injury, spinal cord injury, parkinson's disease, myocardial infarction, diabetes, immunodeficiency and the like. The dental pulp stem cells have strong proliferation capacity, high histocompatibility and convenient storage, and bring great convenience to modern medicine.
In the related art, dental pulp mesenchymal stem cells are usually isolated and cultured by a method of digesting dental pulp tissue with an enzyme (hereinafter referred to as enzyme-coupled digestion method). For example, the patent with the application number of 201510209690.6 discloses a method for culturing deciduous tooth pulp stem cells, which comprises the steps of taking tooth pulp tissues, shearing the pulp tissues, adding I-type collagenase, digesting the pulp tissues at 37 ℃ and 200rpm for 10 to 20 min; the patent with the application number of 201610476143.9 discloses that the dental pulp is extracted from deciduous teeth and added with the digestive juice of the dental pulp for digestion for 1 to 3 hours; obtaining a single cell suspension; every 1mL of the dental pulp digestive juice contains 1-3 mg of collagenase, 2-4 mg of DISPASEIII enzyme and the balance of PBS buffer solution ". However, the presence of enzymes can damage cells, and the obtained primary cells are few in number and low in survival rate; in addition, the enzyme is high in price, is not beneficial to separation, culture and storage of dental pulp mesenchymal stem cells, and is not beneficial to large-scale production of the dental pulp mesenchymal stem cells.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a separation culture method of dental pulp mesenchymal stem cells, which aims to solve the technical problem that the scale production of the dental pulp mesenchymal stem cells cannot be carried out in the related technology.
The invention provides a method for separating and culturing dental pulp mesenchymal stem cells, which comprises the following steps:
step S1, taking out and clamping a tooth sample, then taking out dental pulp tissues in the tooth sample, chopping the dental pulp tissues, and immersing the chopped dental pulp tissues in a culture medium for later use;
s2, placing the sterilized glass beads into a container, and soaking the glass beads in a complete culture medium for later use;
s3, shallow burying the dental pulp tissue prepared in the step S1 in the glass microspheres prepared in the step S2, and culturing for 1-3 hours to obtain a glass microsphere-dental pulp tissue complex;
and S4, subculturing the glass bead-dental pulp tissue complex prepared in the step S3, and collecting by using trypsin to obtain the target dental pulp mesenchymal stem cells.
Optionally, in step S1, the medium is DMEM/F12 medium; and/or the presence of a gas in the atmosphere,
in step S2, the complete medium is DMEM/F12 medium with fetal bovine serum at a volume concentration of 5-15%.
Optionally, in step S2, the particle size of the glass beads is 70 to 90um, and the pH of the glass beads is 7.8 to 8.0; and/or the presence of a gas in the gas,
the glass beads are hollow glass beads.
Alternatively, in step S3, the specific step of obtaining the glass bead-dental pulp tissue composite by culturing for 1 to 3 hours includes:
culturing for 1-3 hours in an environment with the ambient temperature of 37 ℃, the ambient concentration of 5 percent of carbon dioxide and the saturated humidity of more than or equal to 95 percent until 60-75 percent of the glass microspheres are adhered to the surface of the dental pulp tissue to obtain the glass microsphere-dental pulp tissue complex.
Optionally, in step S4, the specific steps of subculturing the glass bead-dental pulp tissue complex prepared in step S3 and collecting the subcultured glass bead-dental pulp tissue complex with trypsin to obtain the target dental pulp mesenchymal stem cells include:
step S41, transferring the glass bead-dental pulp tissue complex prepared in the step S3 to another complete culture medium, culturing the container in an environment with an ambient temperature of 37 ℃, a carbon dioxide ambient concentration of 5% and a saturation humidity of greater than or equal to 95% for 7-10 days, replacing the completely new complete culture medium every 4 days, and collecting the mixture by trypsin after the cell mass at the bottom of the container is fused to 65-75%, so as to obtain the first-generation dental pulp mesenchymal stem cells;
step S42, repeating the culture operation in the step S41 until the cell mass of the first-generation dental pulp mesenchymal stem cells at the bottom of the container is fused to be more than or equal to 95%, and collecting the first-generation dental pulp mesenchymal stem cells by using trypsin to obtain second-generation dental pulp mesenchymal stem cells;
and S43, repeating the culture operation in the step S41 until the cell mass of the second-generation dental pulp mesenchymal stem cells at the bottom of the container is fused to be greater than or equal to 95%, and collecting the second-generation dental pulp mesenchymal stem cells by using trypsin to obtain the target dental pulp mesenchymal stem cells.
Alternatively, in step S41, after the cell mass is grown around the glass bead-dental pulp tissue complex to cover 65 to 75% of the area, the container is gently shaken to change the position of the glass bead-dental pulp tissue complex and to sink the cell mass grown around the glass bead-dental pulp tissue complex into the bottom of the container; then, shaking the container every 4 days until the cell mass at the bottom of the container is fused to 65-75%; and/or the presence of a gas in the atmosphere,
in steps S42 and S43, the concentration of seeded cells was 0.5 x 10 4 ～1.5*10 4 Per cm 3 。
Optionally, the mass concentration of the trypsin is 0.20-0.3 g/cm 3 。
Optionally, before step S1, the method further includes:
and S0, pretreating the tooth sample, placing the treated tooth sample in a protective solution, and preserving at 4-8 ℃ for later use.
Optionally, in step S0, the preprocessing specifically includes:
taking deciduous teeth or adult wisdom teeth as a tooth sample, firstly cleaning the tooth sample by using an isotonic solution, soaking the tooth sample in a 75% alcohol solution for 30-120 seconds for disinfection, and then cleaning the disinfected tooth sample by using the isotonic solution again to obtain the pretreated tooth sample.
Optionally, the protective solution comprises streptomycin with the mass concentration of 100-500 mg/L and a DMEM/F12 culture medium containing streptomycin with the mass concentration of 60-300 mg/L; and/or the presence of a gas in the gas,
the isotonic solution is one of normal saline, D-Hank' S solution or PBS solution.
Compared with the prior art, the invention has the following beneficial effects:
in the technology of the invention, the glass beads are adhered to the surface of the dental pulp tissue to form a glass bead-dental pulp tissue complex, and then the glass bead-dental pulp tissue complex is subjected to cell culture, so that the separation success rate of cultured cells is effectively improved, and the number of cell cultures is increased; in addition, under the method provided by the invention, the separation success rate of the dental pulp mesenchymal stem cells can reach more than 95%. In the separation culture method provided by the invention, the utilization of enzymes such as collagenase, dispase and the like is not involved, so that the damage to cells is effectively avoided, and the quality of the cells subjected to separation culture is improved. The whole operation is simple and efficient, and the large-scale production of separation, culture and storage of dental pulp mesenchymal stem cells is realized.
Drawings
FIG. 1 is an electron microscope image of a glass bead-dental pulp tissue complex in accordance with an embodiment of the present invention;
FIG. 2 is an electron microscope image of a large number of mesenchymal stem cells growing around the glass bead-dental pulp tissue complex according to an embodiment of the present invention;
fig. 3 is an electron micrograph of the first generation of dental pulp mesenchymal stem cells growing to 95% confluency in an embodiment of the present invention;
fig. 4 and 5 are graphs showing the flow detection result of the dental pulp mesenchymal stem cells in the embodiment of the present invention;
fig. 6 is a graph of growth of dental pulp mesenchymal stem cells in an embodiment of the present invention.
Detailed Description
In order to make the objects, technical solutions and beneficial effects of the present invention more clearly apparent, the technical solutions of the present invention are further described below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
The invention provides a method for separating and culturing dental pulp mesenchymal stem cells, which comprises the following steps:
s0, taking deciduous teeth or adult wisdom teeth as a tooth sample, firstly cleaning the tooth sample by using an isotonic solution, soaking the tooth sample in a 75% alcohol solution for 30-120 seconds for disinfection, then cleaning the disinfected tooth sample again by using the isotonic solution to obtain a pretreated tooth sample, placing the treated tooth sample in a protective solution, and preserving at 4-8 ℃ for later use;
the protective solution comprises streptomycin with the mass concentration of 100-500 mg/L and a DMEM/F12 culture medium containing streptomycin with the mass concentration of 60-300 mg/L, and the isotonic solution is normal saline;
step S1, taking out and clamping a tooth sample, then taking out dental pulp tissues in the tooth sample, chopping, and immersing the chopped dental pulp tissues in a DMEM/F12 culture medium for later use;
s2, placing the sterilized glass beads into a container, and soaking the glass beads in a complete culture medium for later use;
wherein the complete culture medium is a DMEM/F12 culture medium with the volume concentration of fetal calf serum of 5-15%; the particle size of the glass beads is 70-90 um, and the pH value of the glass beads is 7.8-8.0; the glass beads are hollow glass beads, and the explanation is that the glass beads are hollow regular spheres, have high sphericity and strong hydrophilicity, are made of ultra-light inorganic materials, and have low cost and stable chemical properties; the water content of the glass beads is less than or equal to 0.3%, and the density of the glass beads is greater than or equal to 0.25g/cm 3 ；
S3, shallow burying the dental pulp tissue reserved in the step S1 in the glass beads reserved in the step S2, and culturing for 1-3 hours in an environment with the ambient temperature of 37 ℃, the ambient concentration of 5% of carbon dioxide and the saturated humidity of more than or equal to 95% until 60-75% of the glass beads are adhered to the surface of the dental pulp tissue to obtain a glass bead-dental pulp tissue complex;
step S41, transferring the glass bead-dental pulp tissue complex prepared in the step S3 to another complete culture medium, culturing the container in an environment with an ambient temperature of 37 ℃, a carbon dioxide ambient concentration of 5% and a saturation humidity of greater than or equal to 95% for 7-10 days, replacing the completely new complete culture medium every 4 days, and collecting the mixture by trypsin after the cell mass at the bottom of the container is fused to 65-75%, so as to obtain the first-generation dental pulp mesenchymal stem cells;
after cell clusters grow out around the glass bead-dental pulp tissue complex, the container is slightly shaken to change the position of the glass bead-dental pulp tissue complex; then, shaking the container every 4 days until the cell mass at the bottom of the container is fused to 65-75%;
step S42, repeating the culture operation in the step S41 until the cell mass of the first-generation dental pulp mesenchymal stem cells at the bottom of the container is fused to be more than or equal to 95%, and collecting the first-generation dental pulp mesenchymal stem cells by using trypsin to obtain second-generation dental pulp mesenchymal stem cells;
and S43, repeating the culture operation in the step S41 until the cell mass of the second-generation dental pulp mesenchymal stem cells at the bottom of the container is fused to be greater than or equal to 95%, and collecting the second-generation dental pulp mesenchymal stem cells by using trypsin to obtain the target dental pulp mesenchymal stem cells. Wherein the concentration of the seeded cells is 0.5 x 10 in steps S42 and S43 4 ～1.5*10 4 Per cm 3 The mass concentration of the trypsin is 0.2-0.3 g/cm 3 . For example, but not limited to, the trypsin is present at a mass concentration of 0.25g/cm 3 。
Due to the characteristics of smooth surface and strong hydrophilicity, the glass microballoon has the capability of generating tight adhesion with tissue blocks and cells. Based on the above, in the technical scheme provided by the invention, a large amount of glass beads are directly adhered to the surface of the dental pulp tissue to prepare and form the glass bead-dental pulp tissue complex, so that the surface contact area of the dental pulp tissue and the glass beads can be increased to the maximum extent, mesenchymal stem cells in the dental pulp tissue can be adhered to the surface of the glass beads in a large amount to grow and proliferate, and finally migrate to the bottom of the container under the action of gravity. In addition, the contact position between the dental pulp tissue and the glass beads is changed by assisting the slight shaking operation of the container, thereby increasing the contact position between the dental pulp mesenchymal stem cells and the glassThe contact area of the micro-beads improves the number of cell cultures. In addition, under the method provided by the invention, the separation success rate of the dental pulp mesenchymal stem cells can reach more than 95 percent, and the number of the cultured cells can reach 4 to 10 when the dental pulp mesenchymal stem cells are used as the target dental pulp mesenchymal stem cells 7 Per cm 3 。
Meanwhile, in the separation culture method provided by the invention, the utilization of enzymes such as collagenase and dispase is not involved, the damage to cells is effectively avoided, and the quality of the cells subjected to separation culture is improved.
Moreover, the whole operation is simple and efficient, so that the large-scale production of separation, culture and storage of dental pulp mesenchymal stem cells is realized.
In order to further explain the culture effect of the method for isolated culture of dental pulp mesenchymal stem cells according to the present invention, the following examples are selected for further explanation. It should be understood that the following examples are only for illustrating the effect of isolated culture of dental pulp mesenchymal stem cells in the present invention, and do not limit the method of isolated culture of dental pulp mesenchymal stem cells in the present invention.
It should be noted that the experimental methods used in the following examples are all conventional methods unless otherwise specified. Materials, reagents, equipment and the like used in the following examples are commercially available unless otherwise specified.
Example 1 isolation and culture of mesenchymal Stem cells of deciduous tooth pulp
1. And (3) experimental operation:
s0, taking deciduous teeth as a deciduous tooth sample, firstly cleaning the deciduous tooth sample by using normal saline, placing the deciduous tooth sample in 75% alcohol solution for soaking for 60 seconds for disinfection, then cleaning the disinfected deciduous tooth sample by using normal saline again to obtain a preprocessed deciduous tooth sample, placing the preprocessed deciduous tooth sample in a protective solution, and preserving at 4-8 ℃ for later use; wherein the protective solution comprises streptomycin with the mass concentration of 100-500 mg/L and a DMEM/F12 culture medium containing the streptomycin with the mass concentration of 60-300 mg/L;
s1, taking out a deciduous tooth sample and carrying out fracture, then taking out dental pulp tissues in the deciduous tooth sample, spreading out the dental pulp tissues with the length of about 6-8 mm, cutting the dental pulp tissues into 5-10 parts by using a scalpel, wherein the length of each part is about 1mm, and then immersing the dental pulp tissues in a DMEM/F12 culture medium for later use;
s2, taking 20mL of sterilized glass beads in a culture dish, and soaking the glass beads in a complete culture medium with the volume twice that of the glass beads for later use; wherein the complete culture medium is a DMEM/F12 culture medium with volume concentration of fetal bovine serum of 5-15%, and it is understood that the complete culture medium is a liquid culture medium; the particle size of the glass beads is 70-90 um, and the pH value of the glass beads is 7.8-8.0; the glass beads are hollow glass beads, the water content of the glass beads is less than or equal to 0.3%, and the density of the glass beads is greater than or equal to 0.25g/cm 3 ；
S3, shallow burying the dental pulp tissue reserved in the step S1 in the glass beads reserved in the step S2, and culturing for 1-3 hours in an environment with an ambient temperature of 37 ℃, a carbon dioxide ambient concentration of 5% and a saturation humidity of 95% until 60% of the glass beads are adhered to the surface of the dental pulp tissue to obtain a glass bead-dental pulp tissue complex; as shown in fig. 1, fig. 1 is an electron microscope image of the glass bead-dental pulp tissue complex;
step S41, transferring the glass bead-dental pulp tissue complex prepared in the step S3 to another complete culture medium, culturing the container in an environment with an ambient temperature of 37 ℃, an ambient concentration of 5% of carbon dioxide and a saturation humidity of 95% for 7-10 days, and then replacing the brand-new complete culture medium every 4 days; after the cell clusters growing out around the glass bead-dental pulp tissue complex cover 70% (as shown in fig. 2, fig. 2 is an electron microscope image of a large amount of dental pulp mesenchymal stem cells growing out around the glass bead-dental pulp tissue complex), the container is slightly shaken to change the position of the glass bead-dental pulp tissue complex and enable the cell clusters growing out around the glass bead-dental pulp tissue complex to sink to the bottom of the container; then, shaking the container once every 4 days until the cell mass at the bottom of the container reaches 70% fusion, and collecting with trypsin to obtain the first-generation dental pulp mesenchymal stem cells;
step S42, repeating the culturing operation in step S41 to the first generation of dental pulp mesenchymeCollecting the stem cells at the bottom of the container with trypsin after the cell mass reaches 95% fusion (as shown in fig. 3, fig. 3 is an electron microscope image of the first-generation dental pulp mesenchymal stem cells after growing to 95% fusion state), so as to obtain the second-generation dental pulp mesenchymal stem cells; wherein the concentration of the inoculated cells is 0.5 x 10 4 ～1.5*10 4 Per cm 3 The mass concentration of the trypsin is 0.25g/cm 3 ；
Step S43, repeating the culture operation in the step S41 until the cell mass of the second-generation dental pulp mesenchymal stem cells at the bottom of the container is fused to 95%, and collecting the second-generation dental pulp mesenchymal stem cells by using trypsin to obtain the target dental pulp mesenchymal stem cells; wherein the concentration of the inoculated cells is 0.5 x 10 4 ～1.5*10 4 Per cm 3 The mass concentration of the trypsin is 0.25g/cm 3 。
2. And (4) analyzing results: the number of dental pulp mesenchymal stem cells cultured by the culture method reaches 4 multiplied by 10 7 The separation success rate of the dental pulp mesenchymal stem cells can reach more than 95 percent. Thus, the method for culturing and separating dental pulp mesenchymal stem cells provided by the invention is beneficial to industrial production of dental pulp mesenchymal stem cells.
Example 2 detection of dental pulp mesenchymal stem cells
1. And (3) experimental operation:
CD34, CD45, HLA-DR, CD73, CD105, CD90 of the dental pulp mesenchymal stem cells of interest were fluorescently labeled, and then the fluorescently labeled dental pulp mesenchymal stem cells of interest were detected by flow cytometry, and the result data shown in fig. 4 and 5 were obtained.
2. And (4) analyzing results: as can be seen from fig. 4, CD34, CD45, and HLA-DR of the dental pulp mesenchymal stem cells were negative, and the positive expression rate was less than 2%. As can be seen from fig. 5, all of CD73, CD105, and CD90 of the dental pulp mesenchymal stem cells were positive, and the positive expression rate was higher than 95%. It can be known that dental pulp mesenchymal stem cells cultured by the method for isolated culture of dental pulp mesenchymal stem cells of the present invention meet the international standards for mesenchymal stem cells; it has a guiding function in the substantial meaning for the industrialized production of the dental pulp mesenchymal stem cells.
Example 3 growth experiment of dental pulp mesenchymal stem cells
1. And (3) experimental operation:
an appropriate amount of the objective dental pulp mesenchymal stem cells were taken to perform a cell growth experiment to determine the growth curve of the objective dental pulp mesenchymal stem cells, and a growth curve graph as shown in fig. 6 was obtained.
2. And (4) analyzing results: as can be seen from FIG. 6, the number of cells rapidly increased with the increase of the culture time, and the number of cells reached up to 4X 10 7 It can be seen that the dental pulp mesenchymal stem cells have strong proliferation ability and strong cell viability.
In conclusion, the dental pulp stem cells are cultured in a mode of forming the glass bead-dental pulp tissue complex, so that the cell separation rate is improved, and the successful probability of cell separation can reach more than 95%; the number of separated cells is increased to 4 x 10 7 A plurality of; in addition, the obtained target dental pulp mesenchymal stem cells have strong cell activity and proliferation capacity and meet the international standards of mesenchymal stem cells.
Finally, the above embodiments are only for illustrating the technical solutions of the present invention and not for limiting, although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all of them should be covered in the claims of the present invention.
Claims (8)
1. A method for isolated culture of dental pulp mesenchymal stem cells, the method comprising:
step S1, taking out and clamping a tooth sample, then taking out dental pulp tissues in the tooth sample, chopping, and immersing the chopped dental pulp tissues in a culture medium for later use;
s2, placing the sterilized glass beads into a container, and soaking the glass beads in a complete culture medium for later use;
s3, embedding the dental pulp tissue reserved in the S1 in the glass bead reserved in the S2, and culturing for 1-3 hours to obtain a glass bead-dental pulp tissue complex;
s4, subculturing the glass bead-dental pulp tissue complex prepared in the step S3, and collecting by using trypsin to obtain the target dental pulp mesenchymal stem cells;
in step S3, the specific step of obtaining the glass bead-dental pulp tissue complex by culturing for 1 to 3 hours includes:
culturing for 1-3 hours in an environment with the ambient temperature of 37 ℃, the carbon dioxide ambient concentration of 5% and the saturation humidity of more than or equal to 95% until 60% -75% of the glass microspheres are adhered to the surface of the dental pulp tissue to obtain a glass microsphere-dental pulp tissue complex;
the specific steps of subculturing the glass bead-dental pulp tissue complex prepared in the step S3 and collecting the subcultured glass bead-dental pulp tissue complex by using trypsin to obtain the target dental pulp mesenchymal stem cells comprise:
step S41, transferring the glass bead-dental pulp tissue complex prepared in the step S3 to another complete culture medium, culturing the container in an environment with an ambient temperature of 37 ℃, a carbon dioxide ambient concentration of 5% and a saturation humidity of greater than or equal to 95% for 7-10 days, replacing the completely new complete culture medium every 4 days, and collecting the mixture by trypsin after the cell mass at the bottom of the container is fused to 65-75%, so as to obtain the first-generation dental pulp mesenchymal stem cells;
step S42, repeating the culture operation in the step S41 until the cell mass of the first-generation dental pulp mesenchymal stem cells at the bottom of the container is fused to be more than or equal to 95%, and collecting the first-generation dental pulp mesenchymal stem cells by using trypsin to obtain second-generation dental pulp mesenchymal stem cells;
and S43, repeating the culture operation in the step S41 until the cell mass of the second-generation dental pulp mesenchymal stem cells at the bottom of the container is fused to be greater than or equal to 95%, and collecting the second-generation dental pulp mesenchymal stem cells by using trypsin to obtain the target dental pulp mesenchymal stem cells.
2. The isolated culture method of dental pulp mesenchymal stem cells according to claim 1, wherein, in step S1, the medium is DMEM/F12 medium; and/or the presence of a gas in the gas,
in step S2, the complete medium is DMEM/F12 medium with fetal bovine serum at a volume concentration of 5-15%.
3. The isolated culture method of dental pulp mesenchymal stem cells according to claim 1, wherein in step S2, the glass beads have a particle size of 70 to 90um and a pH of 7.8 to 8.0; and/or the presence of a gas in the gas,
the glass beads are hollow glass beads.
4. The method for isolated culture of dental pulp mesenchymal stem cells according to claim 1, wherein in step S41, after the cell mass growing around the glass bead-dental pulp tissue complex covers 65 to 75% of the area, the container is gently shaken to change the position of the glass bead-dental pulp tissue complex and to allow the growing cell mass around the glass bead-dental pulp tissue complex to sink to the bottom of the container; then, shaking the container every 4 days until the cell mass at the bottom of the container is fused to 65-75%; and/or the presence of a gas in the gas,
in steps S42 and S43, the concentration of seeded cells was 0.5 x 10 4 ～1.5*10 4 Per cm 3 。
5. The method for isolated culture of dental pulp mesenchymal stem cells according to claim 4, wherein the trypsin is present at a mass concentration of 0.20 to 0.3g/cm 3 。
6. The isolated culture method of dental pulp mesenchymal stem cells according to any one of claims 1 to 5, further comprising, before step S1:
and S0, pretreating the tooth sample, placing the treated tooth sample in a protective solution, and preserving at 4-8 ℃ for later use.
7. The isolated culture method of dental pulp mesenchymal stem cells according to claim 6, wherein in the step S0, the specific step of the pretreatment comprises:
taking deciduous teeth or adult wisdom teeth as a tooth sample, firstly cleaning the tooth sample by using an isotonic solution, soaking the tooth sample in a 75% alcohol solution for 30-120 seconds for disinfection, and then cleaning the disinfected tooth sample by using the isotonic solution again to obtain the pretreated tooth sample.
8. The isolated culture method of dental pulp mesenchymal stem cells according to claim 7, wherein the protective solution comprises streptomycin at a mass concentration of 100 to 500mg/L and DMEM/F12 medium containing streptomycin at a mass concentration of 60 to 300 mg/L; and/or the presence of a gas in the atmosphere,
the isotonic solution is one of normal saline, D-Hank' S solution or PBS solution.
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| CN103849595A (en) * | 2012-12-05 | 2014-06-11 | 上海坤爱生物科技有限公司 | Large scale production technology for dental pulp stem cell |
| WO2018143378A1 (en) * | 2017-02-03 | 2018-08-09 | 国立大学法人九州大学 | Method for preparing dental pulp stem cells from cells derived from dental pulp tissue |
| CN109251889A (en) * | 2018-09-18 | 2019-01-22 | 中晶生物技术股份有限公司 | A kind of transplanting preparation system of dental pulp mescenchymal stem cell microballoon |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103849595A (en) * | 2012-12-05 | 2014-06-11 | 上海坤爱生物科技有限公司 | Large scale production technology for dental pulp stem cell |
| WO2018143378A1 (en) * | 2017-02-03 | 2018-08-09 | 国立大学法人九州大学 | Method for preparing dental pulp stem cells from cells derived from dental pulp tissue |
| CN109251889A (en) * | 2018-09-18 | 2019-01-22 | 中晶生物技术股份有限公司 | A kind of transplanting preparation system of dental pulp mescenchymal stem cell microballoon |
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| 人体牙髓干细胞制备、保存及分析鉴定的研究进展;滕肇慧等;《国际老年医学杂志》;20200320(第02期);67-70 * |
| 改良组织块酶消化法原代培养人牙髓干细胞的研究;刘影等;《口腔疾病防治》;20180320(第03期);36-40 * |
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