A method for preparing dental pulp stem cells

Abstract

The present invention discloses a method for preparing dental pulp stem cells, comprising the following steps: transporting the dental pulp by taking a deciduous tooth and placing it in a sampling bottle, placing the sampling bottle and medical ice pack back into a transport box for transport back to the laboratory; isolating the dental pulp stem cells by clamping the tooth crown with tweezers and then using a syringe to draw physiological saline and blow it into the dental pulp cavity from the root section to loosen the pulp; extracting the dental pulp using a pulp extraction needle; amplifying and culturing the dental pulp stem cells by taking complete culture medium, resuspending the dental pulp pellet, and transferring it to a 12-well plate; fixing the dental pulp with one pulp extraction needle and using the barb on another pulp extraction needle to peel the dental pulp into small fragments, releasing the cells therein, and then culturing them. This preparation method uses a pulp extraction needle to peel the agglomerated dental pulp, releasing the stem cells therein, avoiding damage to the dental pulp stem cells caused by the use of enzymes such as collagenase, eliminating damage to the dental pulp stem cells during the digestion process, and achieving a 100% success rate in dental pulp stem cell extraction. The method is simple and convenient to use.

Description

Technical Field

[0001] The present invention relates to the field of cell life science technology, and in particular to a method for preparing dental pulp stem cells. Background Art

[0002] Dental pulp stem cells (DPSCs) are a type of mesenchymal stem cell found in the dental pulp tissue. They are easily accessible and can be obtained from replacement deciduous teeth without compromising the function or health of the donor site. They have a strong proliferation capacity in vitro and possess many of the properties of stem cells. Under various induction conditions, they can differentiate into osteoblasts, adipocytes, neurons, endothelial cells, and other cell types. Studies comparing DPSCs with bone marrow mesenchymal stem cells have found that while they share similar immunophenotypes, DPSCs have higher colony formation and proliferation rates and exhibit a strong ability to form calcified tissue. In particular, DPSCs exhibit low expression of MHC class II molecules, resulting in low immunogenicity and immune tolerance, allowing them to evade immune surveillance and serve as seed cells for autologous or allogeneic tissue engineering. Therefore, DPSCs hold great promise as seed cells for tissue regeneration and cell therapy, particularly in autologous or allogeneic dental pulp / dentin regeneration, periodontal disease, bone tissue engineering, and nerve damage.

[0003] Although the huge potential of dental pulp stem cells in the field of regenerative medicine has attracted more and more attention, problems have also continued to emerge in its preclinical research and application: low success rate, large temperature fluctuations during transportation after the teeth are removed from the body, lack of nutritional conditions in the transportation fluid, oral microbial infection, etc., resulting in a large number of dental pulp stem cells dying during the transportation process. In addition, the content of dental pulp stem cells themselves is small, and the existing separation systems are mostly based on enzymatic digestion of dental pulp, which causes greater damage to cells. In the later culture process, aging, apoptosis or differentiation often occur, resulting in a low success rate of dental pulp stem cell culture. So far, most separation and amplification methods are not completely satisfactory. Summary of the Invention

[0004] In response to the above technical problems in the related art, the present invention proposes a method for preparing dental pulp stem cells, which can overcome the above shortcomings of the prior art.

[0005] To achieve the above technical objectives, the technical solution of the present invention is implemented as follows:

[0006] A method for preparing dental pulp stem cells, comprising the following steps:

[0007] (1) Transportation of dental pulp: The dental pulp stem cell transport box is handed over to the donor. After receiving the transport box, the donor puts the sampling bottle in the transport box into the refrigerator and the medical ice pack into the freezer. After the donor's deciduous tooth falls off, the deciduous tooth is taken and placed in the sampling bottle. The sampling bottle and medical ice pack are placed back into the transport box and transported back to the laboratory within 24 hours.

[0008] (2) Isolation of dental pulp stem cells: Take the deciduous tooth out of the sampling bottle, clean the deciduous tooth with saline, clamp the crown of the tooth with tweezers, and then use a syringe to draw saline from the root section to blow the pulp cavity inward to rinse the loose pulp; use a pulp extraction needle to insert it into the pulp cavity from the root section, slowly twist it, gently pull it outward, remove the pulp, and place it in a centrifuge tube. Use a syringe to draw saline again and rinse the pulp cavity once. Collect the saline used to rinse the pulp cavity twice and add it to the centrifuge tube where the pulp is placed. Centrifuge to remove the saline and collect the pulp;

[0009] (3) For the expansion and culture of dental pulp stem cells, complete culture medium was taken, the pulp pellet was resuspended, and the pellet was transferred to a 12-well plate. The pulp was fixed with a pulp extraction needle, and the barb on another pulp extraction needle was used to peel the pulp to form small fragments, releasing the cells therein, and then cultured at 37°C, 5% CO2, and saturated humidity.

[0010] Furthermore, a transport fluid is placed in the sampling bottle in step (1), and the transport fluid formula is as follows: 0.1 ml of calcium glucose for injection, 5 ml of human serum albumin for injection, 44.9 ml of dextran 40 glucose injection for injection, 50 ml of adenosine triphosphate for injection, 20,000 units of sodium penicillin, and 20,000 units of streptomycin sulfate per 100 ml.

[0011] Furthermore, in step (3), the pulp is stripped to 1-2 mm using the barb on the pulp extraction needle. 3 Small fragments.

[0012] Furthermore, in step (3), after culturing for 2 days, the medium is replaced by half with fresh medium, and the induction culture is continued, with the medium replaced by half every 2 days.

[0013] Furthermore, in step (3), the first passage is performed when the cell confluence reaches 80%.

[0014] The beneficial effects of the present invention are as follows: calcium ions are used to promote blood coagulation in the dental pulp during transportation, forming a fibrin net to trap the dental pulp cells and retain them in the teeth, thereby maintaining the original dental pulp microenvironment; glucose and adenosine triphosphate are used to provide nutrition for the dental pulp, effectively reducing cell damage and apoptosis of dental pulp stem cells during transportation, and ensuring the survival rate of cells in the dental pulp; pulp extraction is used to peel off the coagulated dental pulp, releasing the stem cells therein, avoiding the damage to dental pulp stem cells caused by the use of collagenase and the like, and eliminating the damage to dental pulp stem cells caused by the digestion process, so that the success rate of dental pulp stem cell extraction reaches 100%, and the application is simple and convenient. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0016] Figure 1 This is a photo of primary cells crawling out of the dental pulp;

[0017] Figure 2 This is a photo of 90% cell fusion;

[0018] Figure 3 Flow cytometry was used to detect cell phenotype. DETAILED DESCRIPTION

[0019] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention are within the scope of protection of the present invention.

[0020] Example 1: Transportation of dental pulp

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

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

[0023] 3) After the donor's deciduous teeth fall off on their own, take the deciduous teeth and place them in a sampling bottle;

[0024] 4) Place the sampling bottle and medical ice pack back into the small sealed transport box and transport it back to the laboratory within 24 hours;

[0025] Example 2: Isolation of dental pulp stem cells

[0026] 1) Use medical tweezers to remove the tooth from the sampling bottle and place it in a small medical stainless steel dish. Use a 20ml syringe to draw 100ml of normal saline solution to rinse the tooth twice, and then pour out the cleaning solution.

[0027] 3) Grasp the crown of the tooth with tweezers, use a 5mL syringe to draw 5mL of normal saline solution and blow it into the pulp cavity from the root section (if the section is small, you can cut it with scissors) to flush out the loose pulp.

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

[0029] 5) Use a 5mL syringe to draw up 5mL of normal saline solution and rinse the pulp cavity once.

[0030] 6) Collect 5 mL of normal saline twice and add it to the centrifuge tube containing the dental pulp;

[0031] 7) Centrifuge at 1300 rpm for 10 min, remove the saline solution, and collect the dental pulp.

[0032] Example 3: Proliferation and culture of dental pulp stem cells

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

[0034] 2) One extraction needle fixes the pulp, and the barb on the other extraction needle is used to slowly peel off the pulp, leaving a 1-2mm pulp. 3 Small fragments, separation is completed, and cultured at 37℃, 5% CO2, saturated humidity; the primary cells crawl out of the dental pulp as shown in the photo. Figure 1 As shown;

[0035] 3) Two days after inoculation, replace half of the medium with fresh medium and continue induction culture, replacing half of the medium every two days;

[0036] 4) On the 4th to 6th day after isolation, a large number of dental pulp stem cells can be seen growing monoclonally. When the cell confluence reaches about 80%, the first passage can be carried out. When the cell confluence reaches 90%, the following picture is shown: Figure 2 As shown;

[0037] 5) Gently blow to suspend the cells for passage, count the cells, and adjust the cell concentration to 7000-10000 / cm2 Passage into new wells of a 12-well plate;

[0038] 6) When the cell confluence reaches about 80% again, perform the second passage;

[0039] 7) Gently blow to suspend the cells for passage, count the cells, and adjust the cell concentration to 3000-5000 / cm 2 Transfer the cells into new T175 culture flasks.

[0040] Example 4: Cryopreservation of Dental Pulp Stem Cells

[0041] 1) Digest the cells with 0.1% (M / V) trypsin (purchased from Armesco) and collect the suspended cells;

[0042] 2) Centrifuge at 1000 rpm for 10 minutes. Discard the supernatant and resuspend the cells in MSC cryoprotectant solution to adjust the cell density to 1–5 × 10⁶ cells / mL.

[0043] 3) Aliquot the cell suspension into cryopreservation tubes and label them as needed.

[0044] 4) Place the cell cryovials directly into a -80°C freezer. After 24 hours, transfer to liquid nitrogen for storage.

[0045] 5) In the later stage, dental pulp stem cells can be revived, expanded or clinically applied as needed.

[0046] Figure 3 The cell phenotype results of dental pulp stem cells obtained by flow cytometry were as follows: CD90 result was 99.85%; CD73 result was 98.52%; CD105 result was 99.8%; CD45 result was 0.47%; and CDHLA-DR result was 0.67%.

[0047] In summary, with the help of the above-mentioned technical scheme of the present invention, calcium ions are used to promote blood coagulation in the dental pulp during transportation, forming a fibrin net to trap the dental pulp cells and keep them in the teeth, thereby maintaining the original dental pulp microenvironment; glucose and adenosine triphosphate are used to provide nutrition for the dental pulp, effectively reducing cell damage and apoptosis of dental pulp stem cells during transportation, and ensuring the survival rate of cells in the dental pulp; pulp extraction is used to peel off the coagulated dental pulp, releasing the stem cells therein, avoiding the damage to dental pulp stem cells by the use of collagenase, etc., and eliminating the damage to dental pulp stem cells by the digestion process, so that the success rate of dental pulp stem cell extraction reaches 100%, and the application is simple and convenient.

[0048] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for preparing dental pulp stem cells, characterized in that: The steps include: (1) Transportation of dental pulp: The dental pulp stem cell transport box is handed over to the donor. After receiving the transport box, the donor puts the sampling bottle in the transport box into the refrigerator and the medical ice pack into the freezer. After the donor's deciduous teeth fall off, the deciduous teeth are taken and placed in the sampling bottle. The sampling bottle and the medical ice pack are returned to the transport box and transported back to the laboratory within 24 hours. The sampling bottle contains a transport solution. The formula of the transport solution is that every 100 ml contains: 0.1 ml of calcium glucose injection, 5 ml of human serum albumin injection, 44.9 ml of dextran 40 glucose injection, 50 ml of adenosine triphosphate injection, 20,000 units of sodium penicillin, and 20,000 units of streptomycin sulfate; (2) Isolation of dental pulp stem cells: Take the deciduous tooth out of the sampling bottle, clean the deciduous tooth with saline, clamp the crown of the tooth with tweezers, and then use a syringe to draw saline from the root section to blow the pulp cavity inward to rinse the loose pulp; use a pulp extraction needle to insert it into the pulp cavity from the root section, slowly twist it, gently pull it outward, remove the pulp, and place it in a centrifuge tube. Use a syringe to draw saline again and rinse the pulp cavity once. Collect the saline used to rinse the pulp cavity twice and add it to the centrifuge tube where the pulp is placed. Centrifuge to remove the saline and collect the pulp; (3) Proliferation and culture of dental pulp stem cells: Take complete culture medium, resuspend the dental pulp pellet, and transfer it to a 12-well plate; fix the dental pulp with a pulp extraction needle and peel the dental pulp with the barb on the other pulp extraction needle to form a 1-2 mm 3 The cells were then separated into small fragments and the cells were released, and then cultured at 37°C, 5% CO2 and saturated humidity.

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