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

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...

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 isolated dental pulp stem cells is low

In addition, Chinese patent document CN201710596515.6 discloses a human dental pulp stem cell culture medium and a preparation method of human dental pulp stem cells. The culture medium is based on α-MEM medium and contains 5% to 10% by volume Human AB serum, 100μM ascorbic acid, 2mML-glutamine, 100U / ml penicillin sodium and 100mg / ml streptomycin, the medium contains penicillin sodium and streptomycin, which can reduce the contamination of miscellaneous bacteria to a certain extent, Improve the production of dental pulp stem cells, but the prepared dental pulp stem cells have poor differentiation ability

[0006] To sum up, the current technology for isolating dental pulp stem cells from dental tissue still has the problems of insufficient cell number, insufficient cell purity, and insufficient differentiation ability.

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