Isolation and processing device of high-activity dental pulp stem cells and exosome extraction and culture method
By using mechanized separation and processing devices and specific enzymatic hydrolysis methods, combined with multi-stage centrifugation and ultracentrifugation technology, the problems of damage and impurities during artificial acquisition of dental pulp tissue have been solved, achieving efficient stem cell separation and exosome extraction, and improving the activity of dental pulp stem cells and the quality of exosomes.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- GUANGDONG FOUNTAIN VALLEY BIOTECHNOLOGY CO LTD
- Filing Date
- 2026-02-28
- Publication Date
- 2026-06-05
AI Technical Summary
In existing technologies, artificially obtaining dental pulp tissue can easily cause mechanical damage and leave behind impurities, affecting stem cell activity and the quality of exosome preparation.
A mechanized, highly active dental pulp stem cell separation and processing device was used, combined with a four-enzyme synergistic digestion solution and a purification process involving multi-stage centrifugation, tangential flow filtration, and ultracentrifugation. Dental pulp tissue was precisely obtained through a clamping arm, a top-flush assembly, and a spray assembly, and resveratrol and tumor necrosis factor-α were added to the amplification culture medium for stimulation.
It avoids tissue damage and impurity residue caused by manual operation, enhances stem cell activity and proliferation capacity, and improves exosome yield, purity and biological activity.
Smart Images
Figure CN122146462A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biomedical technology, specifically to a device for separating and processing highly active dental pulp stem cells and a method for extracting and culturing exosomes. Background Technology
[0002] In the biomedical field, dental pulp stem cells possess multi-lineage differentiation potential, and their secreted exosomes show promising applications in tissue regeneration and inflammation regulation. High-quality initial dental pulp tissue is a crucial foundation for subsequent stem cell isolation, expansion, and exosome extraction and culture, directly affecting key indicators such as stem cell activity, exosome yield, and biological activity, and is of great significance for the translational application of related technologies.
[0003] Currently, the acquisition of dental pulp tissue is mostly done manually. Operators use conventional tools such as dental hammers and tweezers to break up the tooth structure and then manually peel off the pulp tissue. The precision of this method depends heavily on the operator's experience, often causing mechanical damage to the pulp tissue. Furthermore, it is difficult to completely remove tooth debris and other impurities, which may adversely affect the subsequent stem cell culture and exosome preparation. Therefore, there is an urgent need for a precise and gentle method to obtain dental pulp tissue to improve this situation. Summary of the Invention
[0004] To address the shortcomings of existing technologies, this invention provides a device for separating and processing highly active dental pulp stem cells and a method for extracting and culturing exosomes, which solves the problem that artificially obtaining dental pulp tissue can easily cause mechanical damage and impurity residues, thereby affecting the subsequent stem cell activity and the quality of exosome preparation.
[0005] In a first aspect, the present invention provides the following technical solution: a device for separating and processing highly active dental pulp stem cells, comprising a storage box, a spray assembly disposed on the upper surface of the storage box, a U-shaped frame fixedly connected to the upper surface of the storage box, a first hydraulic cylinder fixedly connected to the inner top wall of the U-shaped frame, a first connecting block fixedly connected to the output end of the first hydraulic cylinder, a second hydraulic cylinder fixedly connected to the lower surface of the first connecting block, a connecting cylinder fixedly connected to the outer wall of the second hydraulic cylinder, a sliding block fixedly connected to the bottom end of the connecting cylinder, a second connecting block fixedly connected to the output end of the second hydraulic cylinder, a punch assembly disposed on the lower surface of the second connecting block, a clamping arm rotatably connected to the outer wall of the second connecting block, a connecting rod rotatably connected to the outer wall of the clamping arm, a clamping head disposed on the outer wall of the sliding block, a transmission assembly disposed inside the connecting cylinder, and a vision module disposed on the outer wall of the transmission assembly.
[0006] By adopting the above technical solution, this application uses a mechanized, highly active dental pulp stem cell separation and processing device to obtain dental pulp tissue. Primary stem cells are efficiently separated using a four-enzyme synergistic digestion solution. Resveratrol is added to the expansion culture medium and tumor necrosis factor-α is introduced for stimulation. Combined with a purification process of multi-stage centrifugation, tangential flow filtration, and ultracentrifugation, this approach avoids tissue damage and impurity residues caused by manual operation, while also improving the activity and proliferation capacity of stem cells. At the same time, it achieves synergistic optimization of exosome yield, purity, and biological activity.
[0007] Preferably, the punching assembly includes a third hydraulic cylinder, the outer wall of which is fixedly connected to the lower surface of the second connecting block, and a miniature ejector pin is fixedly provided at the output end of the third hydraulic cylinder.
[0008] Preferably, the transmission assembly includes a first rack, the outer wall of which is slidably connected to the inside of the connecting cylinder, the bottom end of which is fixedly connected to the upper surface of the second connecting block, and the tooth ends of the first rack are meshed with a gear, the outer wall of which is rotatably connected to the inside of the second hydraulic cylinder.
[0009] Preferably, the gear teeth are meshed with a second rack, the outer wall of the second rack is slidably connected to the inside of the second hydraulic cylinder, the outer wall of the second rack is fixedly connected to the outer wall of the vision module, and the outer wall of the vision module is slidably connected to the inside of the second hydraulic cylinder.
[0010] Preferably, the spray assembly includes a fixing frame, the outer wall of which is fixedly connected to the outer wall of the storage box, and an annular frame is fixedly connected to the outer wall of the fixing frame. The outer wall of the annular frame is provided with an aperture, a nozzle, and an identification module.
[0011] Secondly, the present invention provides the following technical solution: a method for extracting and culturing exosomes of highly active dental pulp stem cells, comprising the following steps: S1. Pulp tissue acquisition: Start the separation and processing device, drive the clamping arm to clamp the tooth through the second hydraulic cylinder, and use the top punch component to crack the tooth. Then, with the assistance of the spray component, use the negative pressure suction tube to obtain the pulp tissue. S2. Stem cell isolation: The dental pulp tissue obtained in S1 is minced and enzymatically hydrolyzed using a digestive solution containing type I collagenase, type III collagenase, trypsin and hyaluronidase. The enzymatic hydrolysis is carried out under shaking and constant temperature conditions. After enzymatic hydrolysis, the solution is filtered through a cell sieve and the obtained cell suspension is washed to obtain a primary dental pulp stem cell suspension. S3. Stem cell expansion and culture medium collection: Primary dental pulp stem cells obtained in S2 were seeded in a complete culture medium supplemented with fetal bovine serum, fibroblast growth factor and resveratrol for culture and expansion; during the culture process, tumor necrosis factor-α was added to the culture medium at a predetermined time for stimulation, and the culture supernatant was collected when changing the medium and stored at low temperature. S4. Pretreatment of culture supernatant: The culture supernatant collected in S3 is centrifuged in multiple stages to remove impurities of different sizes and obtain a clear liquid; then, polyethylene glycol is added to the clear liquid for precipitation treatment, the precipitate is collected by centrifugation again and resuspended in buffer solution. S5. Exosome enrichment and purification: The resuspension obtained in S4 is concentrated by tangential flow filtration, followed by ultracentrifugation. The supernatant is discarded to obtain the exosome precipitate. The exosome precipitate is resuspended in buffer solution to obtain the exosome preparation. S6. Validation of exosome preparations: The exosome preparations obtained in S5 were co-cultured with endothelial cells, and their biological activity was evaluated through cell function experiments.
[0012] Preferably, in step S2, the mass percentage concentrations of each enzyme component in the digestive solution are as follows: type I collagenase 0.2-0.4%, type III collagenase 0.05-0.15%, trypsin 0.05-0.15%, and hyaluronidase 0.01-0.05%; the mass ratio of type I collagenase, type III collagenase, trypsin, and hyaluronidase is (4-8):(1-3):(1-3):1; the enzymatic hydrolysis conditions are: temperature 36-38℃, oscillation speed 180-220rpm, and duration 10-20min; the pore size of the cell sieve is 70-80μm.
[0013] Preferably, in step S3, the complete culture medium is based on DMEM / F12, wherein the volume concentration of fetal bovine serum is 8-12%, the concentration of fibroblast growth factor is 8-12 ng / mL, and the concentration of resveratrol is 1-5 μM; the resveratrol is added by dimethyl sulfoxide as a solubilizer, and the final volume concentration of dimethyl sulfoxide in the culture medium is not higher than 0.1%; the predetermined time for adding tumor necrosis factor-α for stimulation is 12-24 hours before each passage, and the added concentration of tumor necrosis factor-α is 5-10 ng / mL.
[0014] Preferably, in step S4, the multi-stage centrifugation includes: first, centrifuging at 300-500×g for 8-12 min at 4°C and collecting the supernatant; then, centrifuging at 2000-3000×g for 15-25 min at 4°C and collecting the supernatant again; subsequently, centrifuging at 10000-12000×g for 30-40 min at 4°C to obtain the clarified liquid; the polyethylene glycol is polyethylene glycol 6000, the final concentration of which is added is 5-10%, and after standing at 4°C, the second centrifugation is performed.
[0015] Preferably, in step S5, the tangential flow filtration uses a filter membrane with a molecular weight cutoff of 3-5 kDa and a concentration factor of 50-100 times; the ultracentrifugation conditions are 4℃, 100,000-120,000×g, and centrifugation for 60-80 min; the buffer solution is PBS; in step S6, the endothelial cells are human umbilical vein endothelial cells; and the cell function experiment is a cell scratch assay or a tubule formation assay.
[0016] Working principle: After the device is started, the first hydraulic cylinder drives the clamping assembly to move down to the appropriate height, and the operator places the tooth sample to be processed between the clamping heads. The storage box is in standby mode, and its smooth, non-stick inner wall is ready to receive the buffer solution, tooth debris, and pulp tissue sprayed later, while also providing support for the subsequent collection and unified treatment of waste liquid.
[0017] The second hydraulic cylinder is activated, causing the second connecting block to move downwards. Through the transmission action of the connecting rod, the sliding block is pulled to slide within the connecting cylinder, thereby driving the clamping arm to retract inwards. The replaceable clamping head at the end of the clamping arm adaptively conforms to the tooth surface according to the size and shape of the tooth sample, achieving uniform force clamping and preventing the tooth sample from breaking due to excessive clamping. Simultaneously with the clamping action, the second connecting block drives the first rack to slide within the connecting cylinder. Through the meshing transmission between the gear and the second rack, the vision module extends synchronously from inside the second hydraulic cylinder, providing real-time monitoring support for subsequent operations.
[0018] The vision module captures tooth position information via a high-definition macro camera and transmits it to the control system. The operator adjusts the output stroke of the third hydraulic cylinder based on feedback parameters. This third hydraulic cylinder drives a diamond-coated miniature ejector pin downwards, precisely impacting the natural gaps in the tooth structure to achieve uniform cracking while preventing excessive penetration that could damage the pulp chamber. Throughout the impaction process, the vision module monitors the ejector pin position and the tooth cracking status in real time, ensuring precise and controllable operation.
[0019] After the tooth cracks, the first hydraulic cylinder drives the clamping assembly to move downwards, allowing the cracked tooth to extend into the spray area above the storage box. The nozzles on the ring frame spray PBS buffer containing 2% bispecific antibodies in a ring array, thoroughly rinsing away tooth debris and surface impurities. The aperture automatically adjusts its brightness according to the ambient light, providing a stable light source for image acquisition. The recognition module uses an infrared scanning sensor to locate the specific position of the dental pulp tissue and simultaneously detects the cleanliness of the tooth surface. Once the cleanliness meets the standard, the next step of the operation is performed.
[0020] Based on the coordinates of the dental pulp tissue provided by the identification module, the operator manipulates the negative pressure suction tube to precisely insert into the cracks in the tooth, and completely aspirates the dental pulp tissue under the set negative pressure value. The spray component continuously sprays buffer solution to reduce the probability of dental pulp tissue adhering to the tooth wall and improve the extraction rate. The aspirated dental pulp tissue is retained in the storage box, and the waste liquid is collected and processed through the storage box, completing the entire non-destructive dental pulp tissue acquisition process.
[0021] This invention provides an apparatus for isolating and processing highly active dental pulp stem cells and a method for extracting and culturing exosomes. It has the following beneficial effects: 1. This application uses mechanized pulp tissue acquisition. The second hydraulic cylinder drives the clamping arm to firmly clamp the tooth, the top punch component precisely splits the tooth, the spray component washes away impurities, and the negative pressure suction tube completely aspirates the tissue, avoiding tissue damage and impurity residue caused by manual operation, thus achieving the effect of improving the quality of the initial pulp tissue.
[0022] 2. In this application, a four-enzyme synergistic digestion solution containing type I collagenase, type III collagenase, trypsin and hyaluronidase is preferred. Each enzyme is combined in a specific ratio to target different components of the extracellular matrix of dental pulp tissue, achieving mild and efficient enzymatic hydrolysis, and thus improving the viability and harvesting efficiency of primary dental pulp stem cells.
[0023] 3. This application achieves the effect of enhancing stem cell proliferation capacity and exosome secretion by adding resveratrol and tumor necrosis factor-α to the stem cell expansion culture medium. Resveratrol maintains the stemness of dental pulp stem cells, and tumor necrosis factor-α specifically stimulates exosome secretion.
[0024] 4. This application removes impurities of different sizes from the culture supernatant through multi-stage centrifugation pretreatment, concentrates it by tangential flow filtration, and then purifies it by ultracentrifugation. Impurities are separated step by step while preserving the structural integrity of exosomes, thus achieving the effect of improving the purity and particle size uniformity of exosome preparations. Attached Figure Description
[0025] Figure 1 This is a perspective view of the present invention; Figure 2 This is a schematic diagram of the second hydraulic cylinder of the present invention; Figure 3 This is a schematic diagram of the connecting cylinder of the present invention; Figure 4 This is a schematic diagram of the first connecting block of the present invention; Figure 5 This is a schematic diagram of the gear of the present invention; Figure 6 This is a schematic diagram of the fixing frame of the present invention; Figure 7 This is a schematic diagram of the nozzle of the present invention; Figure 8 This is a flowchart of a method for extracting and culturing exosomes from highly active dental pulp stem cells according to the present invention.
[0026] The components are as follows: 1. Storage box; 2. U-shaped frame; 3. First hydraulic cylinder; 4. First connecting block; 5. Second hydraulic cylinder; 6. Second connecting block; 7. Clamping arm; 8. Connecting rod; 9. Sliding block; 10. Clamping head; 11. Connecting cylinder; 12. Third hydraulic cylinder; 13. Miniature ejector pin; 14. First rack; 15. Gear; 16. Second rack; 17. Vision module; 18. Fixing frame; 19. Ring frame; 20. Aperture; 21. Nozzle; 22. Recognition module. Detailed Implementation
[0027] The technical solution of the present invention will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0028] Example 1: Please refer to the appendix Figure 1 -Appendix Figure 7 This invention provides a device for separating and processing highly active dental pulp stem cells, including a storage box 1. A spray assembly is provided on the upper surface of the storage box 1. A U-shaped frame 2 is fixedly connected to the upper surface of the storage box 1. A first hydraulic cylinder 3 is fixedly connected to the inner top wall of the U-shaped frame 2. A first connecting block 4 is fixedly connected to the output end of the first hydraulic cylinder 3. A second hydraulic cylinder 5 is fixedly connected to the lower surface of the first connecting block 4. A connecting cylinder 11 is fixedly connected to the outer wall of the second hydraulic cylinder 5. A sliding block 9 is fixedly connected to the bottom end of the connecting cylinder 11. A second connecting block 6 is fixedly connected to the output end of the second hydraulic cylinder 5. A punch assembly is provided on the lower surface of the second connecting block 6. A clamping arm 7 is rotatably connected to the outer wall of the second connecting block 6. A connecting rod 8 is rotatably connected to the outer wall of the clamping arm 7. The outer wall of the connecting rod 8 is rotatably connected to the outer wall of the sliding block 9. A clamping head 10 is provided on the outer wall of the sliding block 9. A transmission assembly is provided inside the connecting cylinder 11. A vision module 17 is provided on the outer wall of the transmission assembly.
[0029] Specifically, the storage box 1 serves to store the buffer solution sprayed out and the pulp flushed out. The inner wall of the storage box 1 is made of a smooth, non-stick material to prevent pulp tissue from adhering to the box wall and causing loss, and it also collects waste liquid for subsequent unified treatment. The storage box 1 also supports and fixes the U-shaped frame 2, whose stable structure provides a stable mounting base for all components above. The first hydraulic cylinder 3 can move the entire clamping assembly up and down, flexibly adjusting the relative position of the tooth sample, the spraying assembly, and the negative pressure suction tube to adapt to the height requirements of different operating stages. The second hydraulic cylinder 5 supports and fixes the connecting cylinder 11. The second hydraulic cylinder 5, through the second connecting block 6, can pull the clamping arm 7 to rotate. The connecting cylinder 11 supports and fixes the sliding block 9, which provides a rotation base point for the connecting rod 8 and also supports and limits the connecting rod 8. When the clamping arm 7 is pulled, it drives the connecting rod 8 to rotate around the sliding block 9. The clamping arm 7 clamps and fixes the tooth through the sliding block 9. The force is evenly distributed during the clamping process, which can prevent the tooth sample from breaking due to excessive clamping. When the tooth is clamped and fixed, the second connecting block 6 extends the vision module 17 through the transmission component. The replaceable clamping head 10 can adapt to tooth samples of different sizes and shapes, such as deciduous teeth and wisdom teeth, which greatly improves the versatility and applicability of the device. After the tooth is clamped and fixed stably, the tooth can be cracked by the punch component and then inserted into the storage box 1. The spray component can thoroughly wash away tooth debris and impurities, and at the same time spray PBS buffer containing 2% double antibiotics to achieve secondary disinfection and sterilization of the tooth surface, reducing the probability of subsequent pulp tissue contamination. Then, a negative pressure suction tube is precisely inserted into the fractured tooth crevices to efficiently and completely extract the dental pulp tissue, avoiding tissue tearing caused by manual operation and preserving the integrity of the dental pulp tissue to the greatest extent, laying the foundation for the subsequent separation of highly active stem cells.
[0030] Please see the appendix Figure 2 -Appendix Figure 4 The top punch assembly includes a third hydraulic cylinder 12, the outer wall of which is fixedly connected to the lower surface of the second connecting block 6, and a miniature ejector pin 13 is fixedly provided at the output end of the third hydraulic cylinder 12.
[0031] Specifically, the second connecting block 6 supports and fixes the third hydraulic cylinder 12, which in turn drives the miniature ejector pin 13 to move, thereby fracturing the fixed tooth. The miniature ejector pin 13 is made of high-strength diamond, with high hardness and a sharp tip. Under the precise drive of the third hydraulic cylinder 12, it can quickly and accurately impact the natural gaps in the tooth structure, achieving uniform fracturing and avoiding damage to the pulp tissue caused by violent breakage, thus preserving the initial viability of the pulp tissue. Simultaneously, the output stroke of the third hydraulic cylinder 12 can be precisely adjusted, allowing for adjustment of the impact depth according to the thickness of different tooth samples, further improving the safety and stability of the fracturing operation and preventing the ejector pin from excessively penetrating and damaging the internal structure of the pulp cavity.
[0032] Please see the appendix Figure 2 -Appendix Figure 5 The transmission assembly includes a first rack 14, the outer wall of which is slidably connected to the inside of the connecting cylinder 11. The bottom end of the first rack 14 is fixedly connected to the upper surface of the second connecting block 6. The tooth ends of the first rack 14 are meshed with a gear 15. The outer wall of the gear 15 is rotatably connected to the inside of the second hydraulic cylinder 5. The tooth ends of the gear 15 are meshed with a second rack 16. The outer wall of the second rack 16 is slidably connected to the inside of the second hydraulic cylinder 5. The outer wall of the second rack 16 is fixedly connected to the outer wall of the vision module 17. The outer wall of the vision module 17 is slidably connected to the inside of the second hydraulic cylinder 5.
[0033] Specifically, the connecting cylinder 11 supports and limits the first rack 14, gear 15, and second rack 16. The closed structure of the connecting cylinder 11 prevents external impurities from entering the transmission components, ensuring smooth and stable transmission. When the miniature ejector pin 13 is moved downward by the second connecting block 6, the second connecting block 6 pulls the first rack 14 to slide within the connecting cylinder 11. The first rack 14 drives the gear 15 to rotate, which in turn drives the second rack 16 to slide within the connecting cylinder 11. The second rack 16 then extends the vision module 17 synchronously. The second connecting block 6 supports and fixes the first rack 14, and the second rack 16 supports and fixes the vision module 17. The vision module 17 is equipped with a high-definition macro camera and image recognition algorithm, which can capture the tooth clamping status and ejector pin position in real time and transmit the image synchronously to the control system. This allows operators to monitor the process in real time, adjust parameters promptly, and avoid problems such as unstable clamping or ejector pin position deviation, ensuring that every step of the operation is precise and controllable.
[0034] Please see the appendix Figure 6 and attached Figure 7 The spray assembly includes a fixed frame 18, the outer wall of which is fixedly connected to the outer wall of the storage box 1. An annular frame 19 is fixedly connected to the outer wall of the fixed frame 18. An aperture 20, a nozzle 21, and an identification module 22 are provided on the outer wall of the annular frame 19.
[0035] Specifically, the storage box 1 supports and fixes the mounting bracket 18, which in turn supports and fixes the ring bracket 19. The ring bracket 19 then supports and fixes the aperture 20, nozzle 21, and recognition module 22. The aperture 20 automatically adjusts its brightness according to the ambient light intensity, providing sufficient and soft light for the recognition module 22 and vision module 17, avoiding strong light reflection that could affect image acquisition accuracy and ensuring the accuracy of subsequent recognition and observation. The nozzles 21 are arranged in a ring array inside the ring bracket 19, allowing for all-around, no-dead-angle spraying of buffer solution onto the tooth surface. This not only washes away debris and impurities generated after tooth breakage but also moistens the inside of the tooth before pulp tissue extraction, reducing the probability of pulp tissue adhering to the tooth wall and improving the pulp tissue extraction rate. The identification module 22 is equipped with an infrared scanning sensor, which can quickly locate the specific location of the pulp tissue inside the fractured tooth, providing coordinate guidance for the precise suction of the negative pressure suction tube, further improving the extraction efficiency and integrity of the pulp tissue. At the same time, the identification module 22 can also detect the cleanliness of the tooth surface, ensuring that the spray disinfection step meets the standards before proceeding with subsequent operations.
[0036] Please see the appendix Figure 8 Example 2: This example provides a method for extracting and culturing exosomes of highly active dental pulp stem cells, used in the isolation and processing apparatus for highly active dental pulp stem cells according to any one of claims 1-5, comprising the following steps: S1. Pulp tissue acquisition: Start the separation and processing device, drive the clamping arm 7 to clamp the tooth through the second hydraulic cylinder 5, and use the top punch assembly to crack the tooth. Then, with the assistance of the spray assembly, use the negative pressure suction tube to obtain the pulp tissue.
[0037] The negative pressure suction tube uses a negative pressure of 0.05 MPa for aspiration, which can quickly and completely aspirate dental pulp tissue, avoiding tissue residue in the gaps of the tooth. The spray assembly continuously sprays PBS buffer containing 2% bispecific antibodies to rinse the inside of the cracked tooth, remove tooth debris and surface impurities, and reduce the probability of contamination in subsequent cell culture. The micro-pin 13 of the punching assembly controls the punching depth to 1 / 2 of the tooth thickness to prevent excessive punching from damaging the integrity of the dental pulp tissue.
[0038] S2. Stem cell isolation: The dental pulp tissue obtained in S1 is minced and enzymatically hydrolyzed using a digestive solution containing type I collagenase, type III collagenase, trypsin and hyaluronidase. The enzymatic hydrolysis is carried out under shaking and constant temperature conditions. After enzymatic hydrolysis, the solution is filtered through a cell sieve and the obtained cell suspension is washed to obtain a primary dental pulp stem cell suspension.
[0039] The mass percentage concentrations of each enzyme component in the digestion solution were as follows: type I collagenase 0.3%, type III collagenase 0.1%, trypsin 0.1%, and hyaluronidase 0.03%; the mass ratio of type I collagenase, type III collagenase, trypsin, and hyaluronidase was 6:2:2:1; the enzymatic hydrolysis conditions were: temperature 37℃, shaking speed 200 rpm, duration 15 min; the pore size of the cell sieve was 75 μm; the enzymatic hydrolysis process was carried out in a constant temperature shaking incubator with vortex shaking to ensure sufficient contact between the digestion solution and the dental pulp tissue block, thereby improving the efficiency of enzymatic hydrolysis; the cell suspension was washed twice with PBS buffer, and after each wash, it was centrifuged at 265×g for 4 min, the supernatant was discarded, and the cell pellet was retained to reduce the impact of enzyme residue on cell viability.
[0040] S3. Stem cell expansion and culture medium collection: Primary dental pulp stem cells obtained in S2 were seeded in a complete culture medium supplemented with fetal bovine serum, fibroblast growth factor and resveratrol for culture and expansion. During the culture process, tumor necrosis factor-α was added to the culture medium at a predetermined time for stimulation, and the culture supernatant was collected when changing the medium and stored at low temperature.
[0041] The complete culture medium was based on DMEM / F12, containing 10% fetal bovine serum, 10 ng / mL fibroblast growth factor, and 3 μM resveratrol. Resveratrol was added using dimethyl sulfoxide (DMSO) as a solubilizer, with a final DMSO concentration of 0.05% in the medium. Tumor necrosis factor-α (TNF-α) stimulation was initiated 18 hours before each passage, at a concentration of 7.5 ng / mL. Primary dental pulp stem cells were seeded at a density of 1 × 10⁻⁶ cells / mL. 4 The cells were cultured in a constant temperature incubator at 37°C, 5% CO2, and 95% relative humidity. Half of the medium was changed every 3 days. The culture supernatant collected during the medium change was immediately placed in an ultra-low temperature freezer at -80°C to prevent exosome degradation. The cells were passaged when the confluence reached 80%.
[0042] S4. Pretreatment of culture supernatant: The culture supernatant collected in S3 is centrifuged in multiple stages to remove impurities of different sizes and obtain a clear liquid. Then, polyethylene glycol is added to the clear liquid for precipitation treatment, and the precipitate is collected by centrifugation again and resuspended in buffer solution.
[0043] The multi-stage centrifugation process included: first, centrifuging at 400×g for 10 min at 4℃ and collecting the supernatant; then, centrifuging at 2500×g for 20 min at 4℃ and collecting the supernatant again; subsequently, centrifuging at 11000×g for 35 min at 4℃ to obtain a clear solution; the polyethylene glycol used was polyethylene glycol 6000, with a final concentration of 7.5%, and after standing at 4℃ for 12 h, it was centrifuged again; the second centrifugation conditions were 4℃, 3000×g for 20 min; the buffer was PBS buffer with pH 7.2-7.4, and the precipitate was gently pipetted during resuspension to avoid exosome rupture.
[0044] S5. Exosome enrichment and purification: The resuspension obtained in S4 is concentrated by tangential flow filtration, followed by ultracentrifugation. The supernatant is discarded to obtain the exosome precipitate. The exosome precipitate is resuspended in buffer solution to obtain the exosome preparation.
[0045] The tangential flow filtration used a filter membrane with a molecular weight cutoff of 4 kDa and a concentration factor of 75 times. The ultracentrifugation conditions were 4℃, 110,000×g, and 70 min. The buffer solution was PBS buffer with a pH of 7.2-7.4. The flow rate was controlled at 10 mL / min during the tangential flow filtration process to avoid damage to the exosome structure due to excessive flow rate. An ultracentrifuge was used for ultracentrifugation, and the centrifuge tubes were pre-sterilized by autoclaving to ensure the sterility of the exosome preparation.
[0046] S6. Validation of exosome preparations: The exosome preparations obtained in S5 were co-cultured with endothelial cells, and their biological activity was evaluated through cell function experiments.
[0047] The endothelial cells were human umbilical vein endothelial cells; the cell function experiment was a cell scratch assay; the cell scratch assay procedure was as follows: human umbilical vein endothelial cells were seeded in 6-well plates, and when the cell confluence reached 90%, the cells were scratched with a 200 μL sterile pipette tip. After washing twice with PBS to remove detached cells, serum-free culture medium containing exosome preparations was added.
[0048] Please see the appendix Figure 8 Example 3: This example provides a method for extracting and culturing exosomes of highly active dental pulp stem cells, used in the isolation and processing apparatus for highly active dental pulp stem cells according to any one of claims 1-5, comprising the following steps: S1. Pulp tissue acquisition: Start the separation and processing device, drive the clamping arm 7 to clamp the tooth through the second hydraulic cylinder 5, and use the top punch assembly to crack the tooth. Then, with the assistance of the spray assembly, use the negative pressure suction tube to obtain the pulp tissue.
[0049] The negative pressure suction tube uses a negative pressure of 0.04 MPa to gently aspirate dental pulp tissue, avoiding tissue rupture caused by excessive negative pressure. The spray assembly continuously sprays PBS buffer containing 2% bispecific antibodies to rinse the inside of the cracked tooth, removing tooth debris and surface impurities, reducing the probability of contamination in subsequent cell culture. The micro-pin 13 of the punching assembly controls the punching depth to 1 / 3 of the tooth thickness to prevent excessive punching from damaging the integrity of the dental pulp tissue.
[0050] S2. Stem cell isolation: The dental pulp tissue obtained in S1 is minced and enzymatically hydrolyzed using a digestive solution containing type I collagenase, type III collagenase, trypsin and hyaluronidase. The enzymatic hydrolysis is carried out under shaking and constant temperature conditions. After enzymatic hydrolysis, the solution is filtered through a cell sieve and the obtained cell suspension is washed to obtain a primary dental pulp stem cell suspension.
[0051] The mass percentage concentrations of each enzyme component in the digestion solution were as follows: type I collagenase 0.2%, type III collagenase 0.05%, trypsin 0.05%, and hyaluronidase 0.01%; the mass ratio of type I collagenase, type III collagenase, trypsin, and hyaluronidase was 4:1:1:1; the enzymatic hydrolysis conditions were: temperature 36℃, shaking speed 180 rpm, duration 10 min; the pore size of the cell sieve was 70 μm; the enzymatic hydrolysis process was carried out in a constant temperature shaking incubator, and the shaking mode was vortex shaking, which could ensure sufficient contact between the digestion solution and the dental pulp tissue block and improve the enzymatic hydrolysis efficiency; the cell suspension was washed twice with PBS buffer, and after each washing, it was centrifuged at 265×g for 4 min, the supernatant was discarded and the cell pellet was retained to reduce the impact of enzyme residue on cell viability.
[0052] S3. Stem cell expansion and culture medium collection: Primary dental pulp stem cells obtained in S2 were seeded in a complete culture medium supplemented with fetal bovine serum, fibroblast growth factor and resveratrol for culture and expansion. During the culture process, tumor necrosis factor-α was added to the culture medium at a predetermined time for stimulation, and the culture supernatant was collected when changing the medium and stored at low temperature.
[0053] The complete culture medium was based on DMEM / F12, containing 8% fetal bovine serum, 8 ng / mL fibroblast growth factor, and 1 μM resveratrol. Resveratrol was added using dimethyl sulfoxide (DMSO) as a solubilizer, with a final DMSO concentration of 0.02%. Tumor necrosis factor-α (TNF-α) stimulation was initiated 12 hours before each passage, at a concentration of 5 ng / mL. Primary dental pulp stem cells were seeded at a density of 1 × 10⁻⁶ cells / mL. 4The cells were cultured in a constant temperature incubator at 37°C, 5% CO2, and 95% relative humidity. Half of the medium was changed every 3 days. The culture supernatant collected during the medium change was immediately placed in an ultra-low temperature freezer at -80°C to prevent exosome degradation. The cells were passaged when the confluence reached 75%.
[0054] S4. Pretreatment of culture supernatant: The culture supernatant collected in S3 is centrifuged in multiple stages to remove impurities of different sizes and obtain a clear liquid. Then, polyethylene glycol is added to the clear liquid for precipitation treatment, and the precipitate is collected by centrifugation again and resuspended in buffer solution.
[0055] The multi-stage centrifugation process included: first, centrifuging at 300×g for 8 min at 4℃ and collecting the supernatant; then, centrifuging at 2000×g for 15 min at 4℃ and collecting the supernatant again; subsequently, centrifuging at 10000×g for 30 min at 4℃ to obtain a clear solution; the polyethylene glycol used was polyethylene glycol 6000, with a final concentration of 5%, and after standing at 4℃ for 8 h, it was centrifuged again; the second centrifugation conditions were 4℃, 3000×g for 20 min; the buffer was PBS buffer with pH 7.2-7.4, and the precipitate was gently pipetted during resuspension to avoid exosome rupture.
[0056] S5. Exosome enrichment and purification: The resuspension obtained in S4 is concentrated by tangential flow filtration, followed by ultracentrifugation. The supernatant is discarded to obtain the exosome precipitate. The exosome precipitate is resuspended in buffer solution to obtain the exosome preparation.
[0057] The tangential flow filtration used a filter membrane with a molecular weight cutoff of 3 kDa and a concentration factor of 50 times. The ultracentrifugation conditions were 4℃, 100,000×g, and 60 min. The buffer solution was PBS buffer with a pH of 7.2-7.4. The flow rate was controlled at 8 mL / min during the tangential flow filtration process to avoid damage to the exosome structure due to excessive flow rate. An ultracentrifuge was used for ultracentrifugation, and the centrifuge tubes were pre-sterilized by autoclaving to ensure the sterility of the exosome preparation.
[0058] S6. Validation of exosome preparations: The exosome preparations obtained in S5 were co-cultured with endothelial cells, and their biological activity was evaluated through cell function experiments.
[0059] The endothelial cells were human umbilical vein endothelial cells; the cell function experiment was a tubule formation experiment; the tubule formation experiment procedure was as follows: Matrigel was spread in a 24-well plate and incubated at 37°C for 30 min to solidify it, then human umbilical vein endothelial cells were inoculated and serum-free culture medium containing exosome preparation was added.
[0060] Please see the appendix Figure 8Example 4: This example provides a method for extracting and culturing exosomes of highly active dental pulp stem cells, used in the isolation and processing apparatus for highly active dental pulp stem cells according to any one of claims 1-5, comprising the following steps: S1. Pulp tissue acquisition: Start the separation and processing device, drive the clamping arm 7 to clamp the tooth through the second hydraulic cylinder 5, and use the top punch assembly to crack the tooth. Then, with the assistance of the spray assembly, use the negative pressure suction tube to obtain the pulp tissue.
[0061] The negative pressure suction tube uses a negative pressure of 0.06 MPa for aspiration, which can quickly aspirate large volumes of dental pulp tissue and improve tissue acquisition efficiency; the spray assembly continuously sprays PBS buffer containing 2% bispecific antibodies to rinse the inside of the cracked tooth, remove tooth debris and surface impurities, and reduce the probability of contamination in subsequent cell culture; the micro-pin 13 of the punching assembly controls the punching depth to 2 / 3 of the tooth thickness to prevent excessive punching from damaging the integrity of the dental pulp tissue.
[0062] S2. Stem cell isolation: The dental pulp tissue obtained in S1 is minced and enzymatically hydrolyzed using a digestive solution containing type I collagenase, type III collagenase, trypsin and hyaluronidase. The enzymatic hydrolysis is carried out under shaking and constant temperature conditions. After enzymatic hydrolysis, the solution is filtered through a cell sieve and the obtained cell suspension is washed to obtain a primary dental pulp stem cell suspension.
[0063] The mass percentage concentrations of each enzyme component in the digestion solution were as follows: type I collagenase 0.4%, type III collagenase 0.15%, trypsin 0.15%, and hyaluronidase 0.05%; the mass ratio of type I collagenase, type III collagenase, trypsin, and hyaluronidase was 8:3:3:1; the enzymatic hydrolysis conditions were: temperature 38℃, shaking speed 220 rpm, duration 20 min; the pore size of the cell sieve was 80 μm; the enzymatic hydrolysis process was carried out in a constant temperature shaking incubator, and the shaking mode was vortex shaking, which could ensure sufficient contact between the digestion solution and the dental pulp tissue block and improve the enzymatic hydrolysis efficiency; the cell suspension was washed twice with PBS buffer, and after each washing, it was centrifuged at 265×g for 4 min, the supernatant was discarded and the cell pellet was retained to reduce the impact of enzyme residue on cell viability.
[0064] S3. Stem cell expansion and culture medium collection: Primary dental pulp stem cells obtained in S2 were seeded in a complete culture medium supplemented with fetal bovine serum, fibroblast growth factor and resveratrol for culture and expansion. During the culture process, tumor necrosis factor-α was added to the culture medium at a predetermined time for stimulation, and the culture supernatant was collected when changing the medium and stored at low temperature.
[0065] The complete culture medium was based on DMEM / F12, containing 12% fetal bovine serum, 12 ng / mL fibroblast growth factor, and 5 μM resveratrol. Resveratrol was added using dimethyl sulfoxide (DMSO) as a solubilizer, with a final DMSO concentration of 0.08% in the medium. Tumor necrosis factor-α (TNF-α) stimulation was initiated 24 hours before each passage at a concentration of 10 ng / mL. Primary dental pulp stem cells were seeded at a density of 1 × 10⁻⁶ cells / mL. 4 The cells were cultured in a constant temperature incubator at 37°C, 5% CO2, and 95% relative humidity. Half of the medium was changed every 3 days. The culture supernatant collected during the medium change was immediately placed in an ultra-low temperature freezer at -80°C to prevent exosome degradation. The cells were passaged when the confluence reached 85%.
[0066] S4. Pretreatment of culture supernatant: The culture supernatant collected in S3 is centrifuged in multiple stages to remove impurities of different sizes and obtain a clear liquid. Then, polyethylene glycol is added to the clear liquid for precipitation treatment, and the precipitate is collected by centrifugation again and resuspended in buffer solution.
[0067] The multi-stage centrifugation process included: first, centrifuging at 500×g for 12 min at 4℃ and collecting the supernatant; then, centrifuging at 3000×g for 25 min at 4℃ and collecting the supernatant again; subsequently, centrifuging at 12000×g for 40 min at 4℃ to obtain a clear solution; the polyethylene glycol used was polyethylene glycol 6000, with a final concentration of 10%, and after standing at 4℃ for 16 h, it was centrifuged again; the second centrifugation conditions were 4℃, 3000×g for 20 min; the buffer was PBS buffer with pH 7.2-7.4, and the precipitate was gently agitated during resuspension to avoid exosome rupture.
[0068] S5. Exosome enrichment and purification: The resuspension obtained in S4 is concentrated by tangential flow filtration, followed by ultracentrifugation. The supernatant is discarded to obtain the exosome precipitate. The exosome precipitate is resuspended in buffer solution to obtain the exosome preparation.
[0069] The tangential flow filtration used a filter membrane with a molecular weight cutoff of 5 kDa and a concentration factor of 100 times. The ultracentrifugation conditions were 4℃, 120,000×g, and centrifugation for 80 min. The buffer solution was PBS buffer with a pH of 7.2-7.4. The flow rate was controlled at 12 mL / min during the tangential flow filtration process to avoid damage to the exosome structure due to excessive flow rate. An ultracentrifuge was used for ultracentrifugation, and the centrifuge tubes were pre-sterilized by autoclaving to ensure the sterility of the exosome preparation.
[0070] S6. Validation of exosome preparations: The exosome preparations obtained in S5 were co-cultured with endothelial cells, and their biological activity was evaluated through cell function experiments.
[0071] The endothelial cells were human umbilical vein endothelial cells; the cell function experiment was a cell scratch assay; the cell scratch assay procedure was as follows: human umbilical vein endothelial cells were seeded in 6-well plates, and when the cell confluence reached 90%, the cells were scratched with a 200 μL sterile pipette tip. After washing twice with PBS to remove detached cells, serum-free culture medium containing exosome preparations was added.
[0072] Comparative Example 1: The only difference between this comparative example and Example 2 is that the dental pulp tissue was not obtained using the separation and processing device for the highly active dental pulp stem cells described above. Instead, it was obtained manually, specifically by manually breaking the teeth with a dental hammer and manually peeling off the dental pulp tissue. During the process, the buffer solution of the spray component was not used for rinsing, and the dental pulp tissue was directly picked up with tweezers.
[0073] Comparative Example 2: The only difference between this comparative example and Example 2 is that the digestion solution used for stem cell separation is a traditional dual-enzyme system that does not contain type III collagenase and hyaluronidase. Specifically, the digestion solution contains only 0.3% type I collagenase and 0.1% trypsin, without type III collagenase and hyaluronidase components. The temperature, shaking speed, and duration of the enzymatic hydrolysis are the same as in Example 2.
[0074] Comparative Example 3: The only difference between this comparative example and Example 2 is that: the complete culture medium used for stem cell expansion did not contain resveratrol, and tumor necrosis factor-α was not added for stimulation during the culture process. The complete culture medium was based on DMEM / F12, with 10% fetal bovine serum and 10 ng / mL fibroblast growth factor added. The stem cell seeding density, culture environment, medium change and passage time were the same as in Example 2.
[0075] Test Item 1: Detection of dental pulp stem cell activity and proliferation capacity. The experiment was conducted according to WS / T777-2021 "General Requirements for Identification of Mesenchymal Stem Cells". Primary dental pulp stem cell suspensions obtained in step S2 of Examples 2, 3, and 4, and Comparative Examples 1, 2, and 3, as well as dental pulp stem cells passaged to the third generation in step S3, were used to detect cell viability using trypan blue staining. Specifically, 100 μL of cell suspension was thoroughly mixed with 100 μL of 0.4% trypan blue staining solution, allowed to stand for 3 minutes, and then... The number of live and dead cells was counted under a microscope to calculate cell viability. Cell proliferation capacity was detected using a CCK-8 assay kit. During the procedure, third-generation dental pulp stem cells from the above examples and comparative examples were seeded in 96-well plates at a density of 1×10³ cells / well. 100 μL of complete culture medium was added to each well, and the plates were incubated at 37°C in a 5% CO2 incubator. 10 μL of CCK-8 solution was added to each well on days 1, 3, 5, and 7, and the plates were incubated for another 2 hours. The absorbance at 450 nm was then measured using a microplate reader.
[0076] Test Item 2: Exosome yield and particle size uniformity detection. The experiment was conducted in accordance with T / CSCB0003-2021 "Technical Specification for Exosome Isolation, Purification and Identification". The exosome preparations obtained in step S5 of Examples 2, 3, and 4, as well as Comparative Examples 1, 2, and 3, were diluted with PBS buffer to appropriate concentrations and detected using nanoparticle tracking analysis. Specifically, the diluted exosome preparations were injected into the NTA detection cell, the detection temperature was set to 25℃, the capture time was 60s, and each sample was tested three times. The concentration and particle size distribution of exosomes were recorded, and the average concentration and particle size variation coefficient of exosomes in the examples and comparisons were calculated. The higher the average concentration and the smaller the particle size variation coefficient, the higher the exosome yield and the better the particle size uniformity.
[0077] Test Item 3: Exosome biological activity assay. The experiment was conducted according to T / CSTM00097-2020 "Technical Specifications for Cell Migration Experiments" and T / CSTM00098-2020 "Technical Specifications for Angiogenesis Experiments". Exosome preparations obtained in step S5 of Examples 2, 3, and 4, as well as Comparative Examples 1, 2, and 3, were co-cultured with human umbilical vein endothelial cells in the logarithmic growth phase. A cell scratch assay was performed first, with human umbilical vein endothelial cells cultured at 5 × 10⁻⁶ cells / year. 4 Cells were seeded at a density of 2 cells / well in 6-well plates. When cell confluence reached 90%, scratches were made using a 200 μL sterile pipette tip. After washing twice with PBS to remove detached cells, serum-free medium containing exosome preparations from different embodiments and control groups was added. A blank control group without exosomes was also included. The plates were incubated at 37°C in a 5% CO2 incubator. The scratched areas were photographed and recorded at 0h, 24h, and 48h to calculate the scratch healing rate. Subsequently, a tube formation experiment was performed. Matrigel was plated in 24-well plates and incubated at 37°C for 30 min to solidify. Human umbilical vein endothelial cells were seeded at a density of 2 × 10⁶ cells / well. 4 The cells were seeded onto the matrix gel at a density of cells / well, and serum-free culture medium containing estrogens from different examples and control samples was added. After culturing for 12 hours, the formation of tubules was observed by taking pictures, and the number of tubule branching points and the total length of the tubules were counted.
[0078] Table 1: Results of detection of dental pulp stem cell activity and proliferation capacity
[0079] Table 2: Results of Exosome Yield and Particle Size Uniformity Testing
[0080] Table 3: Results of Exosome Biological Activity Detection
[0081] As can be seen from Examples 2 to 4 and Comparative Example 1, and in conjunction with Tables 1-3, the method of obtaining dental pulp tissue directly affects the subsequent activity of stem cells and the related properties of exosomes. Mechanized separation and processing devices can protect the integrity of dental pulp tissue through precise clamping and thrusting actions. Combined with buffer rinsing from the spray assembly, this reduces residual tooth debris and impurities, preventing contamination during subsequent cell culture. This method provides excellent initial conditions for stem cell separation and expansion. Manual operation not only easily causes mechanical damage to dental pulp tissue but also introduces more impurities due to the lack of a cleaning step, thus affecting the activity and proliferation capacity of stem cells. It also has a chain reaction effect on the yield, particle size uniformity, and biological activity of subsequent exosomes.
[0082] As can be seen from Examples 2 to 4 and Comparative Example 2, and in conjunction with Tables 1-3, the four-enzyme synergistic digestion system is more suitable for the isolation of dental pulp stem cells compared to the traditional two-enzyme system. Different enzyme components can target different components of the extracellular matrix of dental pulp tissue, achieving efficient and gentle degradation. This improves cell isolation efficiency while reducing damage to stem cells and helps maintain their high activity. In contrast, a digestion system using only two enzymes cannot adequately degrade the complex extracellular matrix structure, which not only reduces cell acquisition efficiency but may also affect stem cell activity due to incomplete or excessive digestion, thereby impacting stem cell proliferation. Furthermore, it can adversely affect the yield, particle size uniformity, and biological activity of subsequent exosomes.
[0083] As can be seen from Examples 2 to 4 and Comparative Example 3, and in conjunction with Tables 1-3, the combined stimulation strategy of resveratrol and tumor necrosis factor-α plays a crucial role in stem cell proliferation and exosome secretion. Resveratrol helps maintain the stemness of dental pulp stem cells and reduces cell activity decline during passage, while tumor necrosis factor-α specifically stimulates stem cells to secrete more biologically active exosomes. The synergistic effect of the two can improve overall performance from two aspects: maintaining stem cell activity and stimulating exosome secretion. A culture system lacking these two substances cannot provide adequate conditions for maintaining stemness and stimulating exosome secretion, thus affecting stem cell proliferation and reducing exosome yield and biological activity, negatively impacting the overall performance of the product.
[0084] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.
Claims
1. A device for separating and processing highly active dental pulp stem cells, comprising a storage box (1), characterized in that: The upper surface of the storage box (1) is provided with a spray assembly. A U-shaped frame (2) is fixedly connected to the upper surface of the storage box (1). A first hydraulic cylinder (3) is fixedly connected to the inner top wall of the U-shaped frame (2). A first connecting block (4) is fixedly connected to the output end of the first hydraulic cylinder (3). A second hydraulic cylinder (5) is fixedly connected to the lower surface of the first connecting block (4). A connecting cylinder (11) is fixedly connected to the outer wall of the second hydraulic cylinder (5). A sliding block (9) is fixedly connected to the bottom end of the connecting cylinder (11). The output end of the cylinder (5) is fixedly connected to a second connecting block (6). The lower surface of the second connecting block (6) is provided with a punching assembly. The outer wall of the second connecting block (6) is rotatably connected to a clamping arm (7). The outer wall of the clamping arm (7) is rotatably connected to a connecting rod (8). The outer wall of the connecting rod (8) is rotatably connected to the outer wall of the sliding block (9). The outer wall of the sliding block (9) is provided with a clamping head (10). The inside of the connecting cylinder (11) is provided with a transmission assembly. The outer wall of the transmission assembly is provided with a vision module (17).
2. The device for separating and processing highly active dental pulp stem cells according to claim 1, characterized in that: The top punch assembly includes a third hydraulic cylinder (12), the outer wall of which is fixedly connected to the lower surface of the second connecting block (6), and a miniature ejector pin (13) is fixedly provided at the output end of the third hydraulic cylinder (12).
3. The device for separating and processing highly active dental pulp stem cells according to claim 1, characterized in that: The transmission assembly includes a first rack (14), the outer wall of which is slidably connected to the inside of the connecting cylinder (11), the bottom end of which is fixedly connected to the upper surface of the second connecting block (6), and the tooth ends of the first rack (14) are meshed with a gear (15), the outer wall of which is rotatably connected to the inside of the second hydraulic cylinder (5).
4. The device for separating and processing highly active dental pulp stem cells according to claim 3, characterized in that: The gear (15) is meshed with a second rack (16), the outer wall of the second rack (16) is slidably connected to the inside of the second hydraulic cylinder (5), the outer wall of the second rack (16) is fixedly connected to the outer wall of the vision module (17), and the outer wall of the vision module (17) is slidably connected to the inside of the second hydraulic cylinder (5).
5. The device for separating and processing highly active dental pulp stem cells according to claim 1, characterized in that: The spray assembly includes a fixed frame (18), the outer wall of which is fixedly connected to the outer wall of the storage box (1), and an annular frame (19) is fixedly connected to the outer wall of the fixed frame (18). The outer wall of the annular frame (19) is provided with an aperture (20), a nozzle (21) and an identification module (22).
6. A method for extracting and culturing exosomes of highly active dental pulp stem cells, characterized in that, An apparatus for separating and processing highly active dental pulp stem cells according to any one of claims 1-5, comprising the following steps: S1. Obtaining dental pulp tissue: Start the separation and processing device, drive the clamping arm (7) to clamp the tooth through the second hydraulic cylinder (5), and use the top punch assembly to crack the tooth. Then, with the assistance of the spray assembly, use the negative pressure suction tube to obtain dental pulp tissue. S2. Stem cell isolation: The dental pulp tissue obtained in S1 is minced and enzymatically hydrolyzed using a digestive solution containing type I collagenase, type III collagenase, trypsin and hyaluronidase. The enzymatic hydrolysis is carried out under shaking and constant temperature conditions. After enzymatic hydrolysis, the solution is filtered through a cell sieve and the obtained cell suspension is washed to obtain a primary dental pulp stem cell suspension. S3. Stem cell expansion and culture medium collection: Primary dental pulp stem cells obtained in S2 were seeded in a complete culture medium supplemented with fetal bovine serum, fibroblast growth factor and resveratrol for culture and expansion; during the culture process, tumor necrosis factor-α was added to the culture medium at a predetermined time for stimulation, and the culture supernatant was collected when changing the medium and stored at low temperature. S4. Pretreatment of culture supernatant: The culture supernatant collected in S3 is centrifuged in multiple stages to remove impurities of different sizes and obtain a clear liquid; then, polyethylene glycol is added to the clear liquid for precipitation treatment, the precipitate is collected by centrifugation again and resuspended in buffer solution. S5. Exosome enrichment and purification: The resuspension obtained in S4 is concentrated by tangential flow filtration, followed by ultracentrifugation. The supernatant is discarded to obtain the exosome precipitate. The exosome precipitate is resuspended in buffer solution to obtain the exosome preparation. S6. Validation of exosome preparations: The exosome preparations obtained in S5 were co-cultured with endothelial cells, and their biological activity was evaluated through cell function experiments.
7. The method for extracting and culturing exosomes of highly active dental pulp stem cells according to claim 6, characterized in that: In step S2, the mass percentage concentrations of each enzyme component in the digestion solution are as follows: type I collagenase 0.2-0.4%, type III collagenase 0.05-0.15%, trypsin 0.05-0.15%, and hyaluronidase 0.01-0.05%; the mass ratio of type I collagenase, type III collagenase, trypsin, and hyaluronidase is (4-8):(1-3):(1-3):1; the enzymatic hydrolysis conditions are: temperature 36-38℃, oscillation speed 180-220rpm, and duration 10-20min; the pore size of the cell sieve is 70-80μm.
8. The method for extracting and culturing exosomes of highly active dental pulp stem cells according to claim 6, characterized in that: In step S3, the complete culture medium is based on DMEM / F12, wherein the volume concentration of fetal bovine serum is 8-12%, the concentration of fibroblast growth factor is 8-12 ng / mL, and the concentration of resveratrol is 1-5 μM; the resveratrol is added with dimethyl sulfoxide as a solubilizer, and the final volume concentration of dimethyl sulfoxide in the culture medium is not higher than 0.1%; the predetermined time for adding tumor necrosis factor-α for stimulation is 12-24 hours before each passage, and the added concentration of tumor necrosis factor-α is 5-10 ng / mL.
9. The method for extracting and culturing exosomes of highly active dental pulp stem cells according to claim 6, characterized in that: In step S4, the multi-stage centrifugation includes: first, centrifuging at 300-500×g for 8-12 min at 4℃ and collecting the supernatant; then, centrifuging at 2000-3000×g for 15-25 min at 4℃ and collecting the supernatant again; subsequently, centrifuging at 10000-12000×g for 30-40 min at 4℃ to obtain the clarified liquid; the polyethylene glycol is polyethylene glycol 6000, the final concentration of which is added is 5-10%, and after standing at 4℃, the second centrifugation is performed.
10. The method for extracting and culturing exosomes of highly active dental pulp stem cells according to claim 6, characterized in that: In step S5, the tangential flow filtration uses a filter membrane with a molecular weight cutoff of 3-5 kDa and a concentration factor of 50-100 times; the ultracentrifugation conditions are 4℃, 100,000-120,000 × g, and centrifugation for 60-80 min; the buffer solution is PBS; in step S6, the endothelial cells are human umbilical vein endothelial cells; the cell function experiment is a cell scratch assay or a tubule formation assay.