Separation and amplification method and application of human dental pulp stem cells

A technology of dental pulp stem cells and dental pulp, which is applied in the field of separation and expansion of human dental pulp stem cells, can solve the problems of short induction time, small quantity, and low cost, and achieve the promotion of growth and proliferation, short adherence time, and differentiation powerful effect

Active Publication Date: 2020-11-10
SHENZHEN WINGOR BIO TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the commonly used methods for the preparation of dental pulp stem cells include enzymatic combined digestion, tissue block culture, and tissue block enzyme digestion methods, but the purity of the prepared dental pulp stem cells is not high and the number is small
[0005] Chinese patent document CN201911173028.4 discloses a medium for inducing differentiation of dental pulp stem cells into osteoblasts and its preparation method and application. The medium includes 4-5.5g/L BSA, 1.5mg/L reduced glutathione , 0.08~0.12mmol/L β-mercaptoethanol, 0.08%~1.5% (V/V) SITE, 0.8~1.2M/L β-glycerophosphate, 15~25μM/L L-ascorbic acid, 0.5~1.2mM/L dextrose Measone, this medium can optimize the method of inducing differentiation of dental pulp stem cells into osteoblasts, the induction time is short and the cost is low, but the purity of the is...

Method used

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  • Separation and amplification method and application of human dental pulp stem cells
  • Separation and amplification method and application of human dental pulp stem cells
  • Separation and amplification method and application of human dental pulp stem cells

Examples

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Effect test

Embodiment 1

[0035]A method for separating and expanding human dental pulp stem cells, comprising the following steps:

[0036] S1. The third molars of adults aged 18-25 are collected, and the tooth tissue is required to be complete, free of caries, pulp disease and periodontal disease, etc. After the tooth is removed, the surface of the tooth tissue is cleaned and disinfected with PBS buffer, and then Put the teeth in a collection tube containing DMEM medium with a final concentration of 1% penicillin-streptomycin, send it to the laboratory with a constant temperature preservation box at 2-8°C, and place two 10cm sterile tubes in a biological safety cabinet. Add an appropriate amount of PBS buffer containing a final concentration of 1% penicillin-streptomycin double antibody to the petri dish. Use sterile tweezers to take out the teeth in the collection tube, put them into the above petri dish for cleaning and disinfection for 2-3 minutes, fully remove the periodontal soft tissue and bloo...

Embodiment 2

[0043] A method for separating and expanding human dental pulp stem cells, comprising the following steps:

[0044] S1. is the same as embodiment 1;

[0045] S2. Take the human dental pulp tissue and put it in the EP tube containing α-MEM medium, and cut the human dental pulp tissue into about 1.0mm with sterile ophthalmic scissors 3 Tissue pieces of different sizes, at a centrifugation speed of 1000rpm / min, centrifuge for 5min to remove the α-MEM medium, add collagenase I with a mass volume concentration of 3mg / ml, hyaluronidase with a mass volume concentration of 0.15%, and mass volume Dispase II with a concentration of 4 mg / ml was digested for 1 hour to obtain digested tissue and cell suspension;

[0046] S3. After the digestion, filter the digestion liquid with a filter with a diameter of 70 μm, centrifuge the filtrate to obtain cell pellets, and obtain human dental pulp stem cells;

[0047] S4. The human dental pulp stem cells obtained in step S3 were divided into 5×10 ...

Embodiment 3

[0051] A method for separating and expanding human dental pulp stem cells, comprising the following steps:

[0052] S1. is the same as embodiment 1;

[0053] S2. Take the human dental pulp tissue and put it in the EP tube containing α-MEM medium, and cut the human dental pulp tissue into about 1.0mm with sterile ophthalmic scissors 3 The size of the tissue pieces, at a centrifugation speed of 1200rpm / min, centrifuge for 10min to remove the α-MEM medium, add collagenase I with a mass volume concentration of 4mg / ml, hyaluronidase with a mass volume concentration of 0.2%, and mass volume Digest with dispase II at a concentration of 5 mg / ml for 1 hour to obtain digested tissue and cell suspension;

[0054] S3. After the digestion, filter the digestion liquid with a filter with a diameter of 80 μm, centrifuge the filtrate to obtain cell pellets, and obtain human dental pulp stem cells;

[0055] S4. The human dental pulp stem cells obtained in step S3 were divided into 5×10 3 cel...

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Abstract

The invention relates to the field of medical biology, and provides a separation and amplification method and application of human dental pulp stem cells. The separation and amplification method comprises the following steps: S1, taking human dental pulp tissue, cutting the human dental pulp tissue into pieces, and adding collagenase I, hyaluronidase and dispase II for digestion to obtain digestedtissue and cell suspension; S2, filtering the digested tissue and cell suspension by using a filter screen, centrifuging the filtrate, and taking cell precipitate to obtain human dental pulp stem cells; and S3, inoculating the human dental pulp stem cells into a culture dish pre-coated with human fibronectin, adding a culture solution for culture, and performing subculture when convergence rate reaches 70-80%. The method has a short time for cell adherence, number of cells cultured to the 10th day of at least 4.5x105, high cell purity, positive expression rate of dental pulp stem cell surfacemarkers CD73, CD90 and CD105 above 99%, and negative index expression rate lower than 1.2%.

Description

technical field [0001] The invention relates to the field of medicine and biology, in particular to a method for separating and expanding human dental pulp stem cells and its application. Background technique [0002] Stem cells are a kind of pluripotent cells with self-replication ability. Under certain conditions, they can differentiate into various functional cells, and have the potential function of regenerating various tissues, organs and complete individuals. Stem cells come from many sources, including bone marrow, umbilical cord, cord blood, teeth, fat, skin, and hair follicles. [0003] In 2000, Gronthos et al. discovered a cell with a very similar immune phenotype and the ability to form mineralized nodules with bone marrow mesenchymal stem cells. The cell shape was spindle-shaped, capable of self-renewal and multi-directional differentiation. Fibroblasts isolated from dental pulp are called dental pulp stem cells (Dental Pulp Stem Cells, DPSCs). Dental pulp stem...

Claims

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Application Information

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IPC IPC(8): C12N5/0775A61L27/38
CPCA61L27/3834A61L27/3895C12N5/0664C12N2500/32C12N2500/35C12N2500/38C12N2501/237C12N2501/39C12N2501/999C12N2509/00C12N2533/52
Inventor 姜舒张芸纪惜銮谢亮
Owner SHENZHEN WINGOR BIO TECH
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