Dental pulp stem cell preparation method

A technology of dental pulp stem cells and dental pulp, applied in cell dissociation methods, biochemical equipment and methods, animal cells, etc., can solve the problems of dental pulp stem cell death, low success rate, and large cell damage, and reduce cell damage and apoptosis, simple and convenient application, and the effect of ensuring survival rate

Pending Publication Date: 2019-11-19
北京中瑞联合生物科技有限公司
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AI Technical Summary

Problems solved by technology

[0003] Although the great potential of dental pulp stem cells in the field of regenerative medicine has attracted more and more attention, problems in its preclinical research and application are also constantly emerging: the success rate is low, and after the teeth are removed from the body, the temperature fluctuates greatly due to transportation. , the lack of nutritional conditions in the transport fluid, oral microbial infection, etc., lead to the death of a large number of dental pulp stem cells during the transportation process. In addition, the content of dental pulp stem cells is small, and most of the existing separation systems are mainly based on enzymatic digestion of dental pulp, which causes great damage to the cells. , the late culture process often leads to aging, apoptosis or differentiation, resulting in a low success rate of dental pulp stem cell culture. So far, most isolation and expansion methods are not completely satisfactory.

Method used

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Examples

Experimental program
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Effect test

Embodiment 1

[0020] Embodiment 1, transportation of dental pulp

[0021] 1) Hand over the dental pulp stem cell transport box to the donor. There are sampling bottles and medical ice packs in the transport box. There is transport liquid in the sampling bottle. The formula of the transport liquid is that every 100ml contains: calcium glucose for injection 0.1ml (calcium content is 10mg / 100ml), human albumin for injection 5ml, dextran 40 glucose injection for injection 44.9ml, adenosine triphosphate for injection 50ml (adenosine triphosphate content is 10mg / 100ml), penicillin sodium 20,000 units, streptomycin sulfate 20,000 units;

[0022] 2) After the donor receives the transport box, put the sampling bottle in the refrigerator, and the medical ice pack in the refrigerator;

[0023] 3) After the deciduous teeth of the donor fall off by themselves, the deciduous teeth are collected and put into the sampling bottle;

[0024] 4) Put the sampling bottle and medical ice pack back into the smal...

Embodiment 2

[0025] Embodiment 2, the separation of dental pulp stem cells

[0026] 1) Use medical tweezers to take out the tooth from the sampling bottle, put it into a small medical stainless steel plate, use a 20ml syringe to draw 100mL normal saline to clean the teeth twice, and pour out the cleaning solution.

[0027] 3) Clamp the crown of the tooth with tweezers, use a 5mL syringe to suck 5mL of saline from the root section (if the section is small, you can cut it with scissors) and blow the pulp cavity inward to rinse and loosen the pulp.

[0028] 4) Use a pulp extraction needle to insert into the pulp cavity from the broken root, twist it slowly, pull it outward gently, take out the pulp, and place it in a 15mL centrifuge tube.

[0029] 5) Use a 5mL syringe to re-absorb 5mL of normal saline, and rinse the pulp cavity once.

[0030] 6) Collect 5mL normal saline twice and add it to the centrifuge tube where the pulp is placed;

[0031] 7) Centrifuge at 1300rpm for 10min, remove the...

Embodiment 3

[0032] Embodiment 3, expansion culture of dental pulp stem cells

[0033] 1) Take 2 mL of complete medium, resuspend the pulp pellet, and transfer it to a 12-well plate;

[0034] 2) Fix the pulp with one pulp extraction needle, and slowly peel off the pulp with the barb on the other pulp extraction needle to form a 1-2mm pulp 3 Fine fragments, end separation, 37°C, 5% CO 2 , cultured under saturated humidity conditions; the primary cells crawled out of the dental pulp figure 1 shown;

[0035] 3) After 2 days of inoculation, replace the half volume of medium with fresh medium, continue the induction culture, and change the medium once every 2 days;

[0036] 4) On the 4th to 6th day after separation, many dental pulp stem cells can be seen growing monoclonally. When the cell fusion degree reaches about 80%, the first passage can be carried out; the photo of the cell fusion 90% is as follows figure 2 shown;

[0037] 5) Gently blow and suspend the cells for passage, count th...

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Abstract

The invention discloses a dental pulp stem cell preparation method. The dental pulp stem cell preparation method includes the following steps of: transporting dental pulp, wherein a deciduous tooth isplaced in a sampling bottle, the sampling bottle and a medical ice bag are placed back into a transportation box to be transported back to a laboratory; separating dental pulp stem cells, wherein tweezers are adopted to clamp the crown of the tooth, then, a syringe is used for sucking up normal saline to blow a dental pulp cavity inwards from a fracture surface of the root of the tooth to wash and loosen the dental pulp, and using a broach for taking out the dental pulp; and proliferating and culturing the dental pulp stem cells, wherein a complete culture medium is adopted, a dental pulp precipitate is resuspended and transferred to a 12-well microplate, one broach is used for fixing the dental pulp, barbs on another broach are used for stripping the dental pulp to make the dental pulp become fine pieces, the cells in the dental pulp are released, and then culture is conducted. According to the dental pulp stem cell preparation method, the broach is adopted to strip the agglomerate dental pulp to release the stem cells in the dental pulp, damage caused by using collagenase and the like on the dental pulp stem cells is avoided, damage on the dental pulp stem cells during digestioncan be eliminated, therefore, the extraction success rate of the dental pulp stem cells reaches 100%, and application is simple and convenient.

Description

technical field [0001] The invention relates to the technical field of cell life science, in particular to a method for preparing dental pulp stem cells. Background technique [0002] Dental pulp stem cells (DPSC) are a type of mesenchymal stem cells that exist in dental pulp tissue. They are easy to obtain and can be obtained from replaced deciduous teeth without causing functional and health damage to the donor site. It has strong proliferative ability and possesses multiple properties of stem cells. Under different induction conditions, it can differentiate into osteoblasts, adipocytes, nerve cells, endothelial cells, etc. Studies have compared DPSCs with bone marrow mesenchymal stem cells and found that they have similar immunophenotypes, but dental pulp stem cells have higher clone formation and proliferation rates, and exhibit strong calcified tissue formation capabilities. In particular, DPSCs have low expression of MHC-II molecules, etc., which have low immunogenici...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775
CPCC12N5/0663C12N2509/10
Inventor 张彦茹黄宇唐雯
Owner 北京中瑞联合生物科技有限公司
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