Pluripotent stem cells MDPSCs for dental pulp regeneration, and isolated culture method and application thereof
A technology for pluripotent stem cells, separation and culture, applied in the field of pluripotent stem cells MDPSCs for dental pulp regeneration and their separation and culture, which can solve the differences in biomechanical environment and tissue physiological conditions, large batch differences in regeneration efficiency, and multipotency of molecularly labeled cells. The heterogeneity of sex mesenchymal cells is difficult to control and other problems
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Embodiment 1
[0047] Example 1 Isolation and culture of pluripotent stem cells in mouse dental papilla
[0048] In this example, the three-dimensional sphere-forming experiment model diagram of the separation and culture of dental papilla cells is as follows figure 1 shown. The isolation and culture of pluripotent sphere-forming stem cells in the mouse dental papilla specifically includes the following steps:
[0049] 1) Isolation and culture of primary dental papilla cells
[0050] Select 10 C57BL / 6 mouse suckling mice born 1-3 days after birth, kill them by decapitation, soak them in 75% ethanol for 5-10 minutes for disinfection, separate the upper and lower mandibles, and use microtweezers to strip the first and second molars intact under a stereomicroscope. For the tooth germ, the dental papilla tissue was taken out from under the calcified edge of the crown, rinsed with PBS containing antibiotics, and cut into chylus with ophthalmic scissors;
[0051] Digest the tissue with collagen...
Embodiment 2
[0059] Example 2 Detection of Pluripotent Stem Cells
[0060] 1. Immunofluorescence detection
[0061] The DPC spheres were inoculated in a confocal small dish, and after 30-60 min, the cell pellets settled and adhered to the wall, fixed with 4% paraformaldehyde, and immunofluorescent staining was used to identify the pluripotency of the DPC spheres.
[0062] Immunofluorescence staining steps are:
[0063] a. Cell pellets were fixed with 4% paraformaldehyde for 1 h, and washed 3 times with PBS;
[0064] b. Add 0.5% TritionX-100 to punch holes at room temperature for 30 minutes, wash with PBS 3 times;
[0065] c. Block with 5% BSA at room temperature for 30 minutes;
[0066] d. Discard the blocking solution, add the primary antibody and incubate overnight at 4°C;
[0067] e. Rewarm for 10 minutes, wash with PBS for 5 minutes / time, a total of 3 times, add the corresponding fluorescent secondary antibody, and incubate at 37°C for 1 hour;
[0068] f. Wash with PBS for 3 times...
Embodiment 3
[0106] The result is as Figure 5 As shown, after 4 weeks of transplantation under the renal capsule, only collagen fiber-like structures can be seen in monolayer cells, and the expression of DMP1 and DSPP is relatively weak, while DPC spheres can form a mineralized structure with obvious positive expression of DMP1 and DSPP, which shows that DPC spheres Shows greater osteogenic / odontogenic differentiation capacity in vivo. Example 3 DPC spheres promote functional regeneration of pulp-dentin complex in vivo
[0107] 1. Subcutaneous transplantation in nude mice, the flow chart is as follows Figure 6 As shown, the specific operation process is as follows:
[0108] a. TDM preparation: extract the central incisor from the pig mandible; carefully scrape the periodontal ligament tissue, grind away the cementum, pulp tissue and dentin layer; cut the root canal into 2-3 Small segments of millimeters; ultrasonic cleaning machine vibration cleaning root canal 3 times, each time 5 ~ ...
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