Method for enhancing immunomodulatory capacity of dental pulp mesenchymal stem cells

Abstract

Provided are a method for enhancing the immunomodulatory capacity of dental pulp mesenchymal stem cells, belonging to the field of stem cell biology and regenerative medicine. The method comprises the steps of tooth sample pretreatment, dental pulp mesenchymal stem cell primary culture and subculture, etc. In the cell culture process, a mode of a combination of adding interleukin factors and cell hypoxia culture is used, such that the anti-inflammatory capacity of the dental pulp mesenchymal stem cells can be continuously and stably improved, and cell proliferation and immunogenicity are not affected, and thus the safety of subsequent clinical application is improved.

Description

A method for enhancing the immunoregulatory ability of dental pulp mesenchymal stem cells Technical Field

[0001] The present invention relates to a method for enhancing the immunoregulatory ability of dental pulp mesenchymal stem cells, and belongs to the fields of stem cell biology and regenerative medicine. Background Art

[0002] At present, the methods used in the industry to enhance the anti-inflammatory ability of DPSC in vitro mainly include adding pro-inflammatory cytokines to the culture medium for co-culture (such as IFN-γ, TNF-α, IL-1, IL-6, etc.) and gene editing to overexpress anti-inflammatory related factors, etc., to enhance the secretion of anti-inflammatory factors to improve the anti-inflammatory function. However, there are the following disadvantages that are difficult to overcome: (1) High cost: The production cost of cytokines is high, and the price per milligram is generally more than 1,000 yuan, which directly affects the cost of cell preparation. (2) Unstable treatment effect: Cytokines have biological activity, harsh storage conditions, short shelf life, poor stability, and are easy to cause batch differences in DPSC products when used alone, and the treatment effect is poorly sustained. (3) Improved immunogenicity: Cytokine treatment time is too long, which will increase the immunogenicity of DPSC, thereby increasing the risk of DPSC transplantation process. In addition, it will affect the expression of key cell surface markers. (4) Impact on proliferation: Cytokine stimulation will affect the proliferation ability of DPSC to a certain extent and promote cell aging. (5) Safety: The gene editing process is complex, introduces a lot of exogenous substances, is difficult to control, and the safety of clinical application is questionable. Technical issues

[0003] The purpose of the present invention is to provide a method for enhancing the immunoregulatory ability of dental pulp mesenchymal stem cells by combining the addition of interleukin factors and cell hypoxia culture to continuously and stably improve the anti-inflammatory ability of dental pulp mesenchymal stem cells without affecting cell proliferation, immunogenicity, and the safety of subsequent clinical applications. Technical Solutions

[0004] In order to achieve the above object, the present invention provides the following technical solutions:

[0005] A method for enhancing the immunoregulatory ability of dental pulp mesenchymal stem cells, comprising the following steps:

[0006] (1) Pretreatment: remove the extracted teeth, clean and disinfect them;

[0007] (2) Primary culture of dental pulp mesenchymal stem cells: Extract the dental pulp from the teeth treated in step (1) and cut into pieces of 0.1-1.0 mm 3The tissue blocks are then digested and centrifuged, the supernatant is removed, and the cell pellet is left. The cells are seeded into a culture container and cultured with an appropriate amount of culture medium. After the cells crawl out, they are digested into single cells using a trypsin substitute to obtain P0 cells.

[0008] (3) Subculture of dental pulp mesenchymal stem cells: P0 cells were inoculated into a culture vessel, and lipopolysaccharide and interleukin-6 were added to the culture medium for 3-5 consecutive subcultures. Before each subculture, trypsin substitutes were used to digest them into single cells. The inoculation density for each subsequent subculture was 5000-8000 / cm 2 The cells were harvested when they reached 80%-90% confluence.

[0009] Furthermore, in step (1), the teeth are cleaned and disinfected using a physiological saline buffer solution containing penicillin-streptomycin.

[0010] Furthermore, in step (2), the digestion is performed using 0.3-0.5% collagenase for 50-70 min, the centrifugation speed is 800-1000 g, and the time is 5-8 min.

[0011] Preferably, the density of the P0 generation cells in step (3) is 4500-6500 cells / cm 2 .

[0012] . Further, the concentration of the lipopolysaccharide additive in step (3) is 1.0~2.0ng / ml.

[0013] Furthermore, the added concentration of interleukin factor in step (3) is 2-6 ng / ml.

[0014] Preferably, the added concentration of interleukin factor in step (3) is 4 ng / ml.

[0015] Furthermore, the seeding density of the cells in step (3) is 5000-8000 cells / cm 2 .

[0016] Furthermore, the cells in step (3) are cultured under hypoxic conditions.

[0017] Furthermore, the hypoxic culture condition is culture in a three-gas incubator with 5% O2, 5% CO2 and 37°C. Beneficial effects

[0018] The beneficial effects of the present invention are:

[0019] (1) The method of enhancing the immunoregulatory capacity of dental pulp mesenchymal stem cells described in the present invention more stably enhances the anti-inflammatory immunoregulatory capacity of DPSCs, and has stronger anti-inflammatory function than the addition of interleukin alone, better sustainability, and more stable batch-to-batch processing. (2) While enhancing their anti-inflammatory capacity and immune activity, it does not affect the changes in DPSC surface markers, thereby ensuring cell quality and reducing risks during application. (3) The use of a low-oxygen culture environment during MSC cell culture can better exert the immunoregulatory effect of MSCs. Best Mode for Carrying Out the Invention

[0020] Example 1

[0021] A method for enhancing the immunoregulatory capacity of dental pulp mesenchymal stem cells, comprising:

[0022] (1) Primary cell culture: Take the extracted deciduous teeth, disinfect them, take the dental pulp, rinse them 2-3 times with physiological saline buffer containing penicillin-streptomycin, add 8 ml of 2 mg / ml type I collagenase, 3 mg / ml protease and complete human mesenchymal culture medium, and digest them in a 37°C, 5% CO2 incubator for 1 hour to form a dental pulp suspension; after digestion, collect the dental pulp suspension in a 15 mL centrifuge tube, place it in a centrifuge at 800 g / min at room temperature for 5 minutes, and remove the supernatant. Resuspend the cell pellet with 10 mL of complete human mesenchymal culture medium, gently blow to make a single cell suspension, and transfer to a 75 cm 2 The cells were placed in a culture flask at 5% O2, 5% CO2, and 37°C in a three-gas incubator. After 72 hours of culture, the cells were observed to crawl out under an inverted microscope every day. During this period, half of the medium was changed every three days. After 7-13 days, the cells were fused to 70-80%. Trypsin substitute was used to digest them into single cells for passage, which was recorded as P1.

[0023] (2) Additives: Cells were centrifuged at 6000 / cm 2 Inoculate the cells into a culture flask, add IL-6 at a concentration of 2-4 ng / ml to the culture medium and mix thoroughly. Then, culture the cells in a three-gas incubator with hypoxic culture conditions of 5% O2, 5% CO2, and 37°C.

[0024] (3) Cell passaging: After 3-5 days, observe the morphology and growth of cells under an inverted microscope. It is found that the cells proliferate rapidly in the form of colonies, growing in a spindle-shaped or polygonal shape attached to the wall. Cells are passaged when the degree of confluence reaches 80-90%.

[0025] (4) Cell harvesting: When the cells reach the P5 generation and the cell confluence reaches the required level, the cells are harvested. This is the dental pulp mesenchymal stem cells used in the experiment. Modes for Carrying Out the Invention

[0026] Comparative Example 1

[0027] On the basis of the above Example 1, neither lipopolysaccharide nor IL-6 was added, and the cells were cultured for the same number of days.

[0028] Comparative Example 2

[0029] Based on the above Example 1, only lipopolysaccharide was added and cultured for the same number of days.

[0030] Comparative Example 3

[0031] Based on the above Example 1, only IL-6 was added and cultured for the same number of days.

[0032] Result analysis:

[0033] (1) An in vitro experimental model was established by co-culturing peripheral blood mononuclear cells (PBMC) with dental pulp mesenchymal stem cells (DPSC) from different groups in Example 1 and Comparative Examples 1-3, and detecting the secretion of inflammatory factor TNF-α in the supernatant.

[0034] The results showed that the combined treatment group (implemented in step 1) significantly suppressed the secretion of the inflammatory factor TNF-α compared with the other groups. This suggests that the method of the present invention can more consistently and stably enhance the anti-inflammatory immunomodulatory capacity of MSCs.

[0035] The expression rates of CD73, CD105, and CD90, which are surface markers of mesenchymal stem cells, were detected.

[0036] According to the literature published by the International Society for Cell Therapy in 2006, the positive expression rate of CD73, CD105, and CD90 is greater than 95% as the identification and quality control standard for mesenchymal stem cells.

[0037] The results showed that the method adopted by the present invention enhanced the anti-inflammatory and immune activities without affecting the changes of key markers on the surface of MSC cells, thereby ensuring cell quality while reducing the risks of the application process.

[0038] The above examples and comparative examples demonstrate that the addition of lipopolysaccharide and IL-6 can be used in in vitro and in vivo experiments to understand molecular pathways and immune response models. This can enhance the immunomodulatory capacity of dental pulp mesenchymal stem cells without affecting normal cell expression or the safety of subsequent clinical applications. Industrial Applicability

[0039] The present invention adopts a combination of adding interleukin factors and cell hypoxia culture during the cell culture process, which can continuously and stably improve the anti-inflammatory ability of dental pulp mesenchymal stem cells without affecting cell proliferation and immunogenicity, thereby improving the safety of subsequent clinical applications. This method can be widely used in various types of cell culture processes.

Claims

1. A method for enhancing the immunomodulatory ability of dental pulp stem mesenchymal cells, characterized in that It includes the following steps: (1) Pretreatment: Take the extracted teeth, clean and disinfect them; (2)Primary culture of dental pulp mesenchymal stem cells: Extract the dental pulp from the teeth treated in step (1), cut it into tissue blocks of 0.1-1.0 mm 3 , then digest, centrifuge, remove the supernatant, leave the cell pellet, inoculate the cells in a culture container, add an appropriate amount of culture medium for culture. After the cells crawl out, use a trypsin substitute to digest them into single cells to obtain P0 generation cells; (3)Subculture of dental pulp mesenchymal stem cells: Seed the P0 cells in a culture vessel, add lipopolysaccharide and interleukin factor IL-6 to the culture medium for culturing, and passage continuously for 3-5 times. Before each passage, it is necessary to digest them into single cells using a trypsin substitute. The seeding density for each subsequent passage is 5000-8000 / cm 2 , and harvest the cells when they reach 80%-90% confluence.

2. The method for enhancing the immunomodulatory ability of dental pulp mesenchymal stem cells according to claim 1, wherein In the step (1), a physiological saline buffer containing penicillin-streptomycin is used to clean and disinfect the teeth.

3. A method for enhancing the immunomodulatory ability of dental pulp mesenchymal stem cells according to claim 1, characterized in that In the step (2), the digestion is carried out using 0.3 - 0.5% collagenase for 50 - 70 min, and the centrifugation speed is 800 - 1000 g for 5 - 8 min.

4. A method for enhancing the immunomodulatory ability of dental pulp mesenchymal stem cells according to claim 1, characterized in that The density of the P0 generation cells in step (3) is 4,500 - 6,500 cells / cm 2 .

5. A method for enhancing the immunomodulatory ability of dental pulp stem mesenchymal cells according to claim 1, characterized in that In the step (3), the concentration of the lipopolysaccharide additive is 1.0 - 2.0 ng / ml.

6. A method for enhancing the immunomodulatory ability of dental pulp mesenchymal stem cells according to claim 1, characterized in that, In the step (3), the added concentration of the interleukin factor is 2 - 6 ng / ml.

7. A method for enhancing the immunomodulatory ability of dental pulp stem mesenchymal cells according to claim 6, characterized in that, In the step (3), the added concentration of the interleukin factor is preferably 4 ng / ml.

8. A method for enhancing the immunomodulatory ability of dental pulp mesenchymal stem cells according to claim 1, characterized in that, The seeding density of cell passage in the step (3) is 5000-8000 cells / cm 2 .

9. A method for enhancing the immunomodulatory ability of dental pulp mesenchymal stem cells according to claim 1, characterized in that, The cells in the step (3) are cultured under hypoxic conditions.

10. A method for enhancing the immunomodulatory ability of dental pulp mesenchymal stem cells according to claim 9, characterized in that, The hypoxic culture conditions are culturing in a three-gas incubator with 5% O2, 5% CO2, and 37°C.