Dental pulp stem cell separation and culture method

Abstract

The invention discloses a dental pulp stem cell separation and culture method. The method comprises steps as follows: separation of dental pulp tissue, enzymolysis of the dental pulp tissue with IV type collagenase, V type collagenase and pancreatin mixed enzyme with the mass volume concentration of 0.05%-0.5%, primary culture of dental pulp stem cells and adding of cell growth factors, glutamine,decavitamin, insulin, tremella polysaccharide, a codonopsis tangshen extract, a dendrobium officinale extract and other nutritional ingredients. The method has the advantages as follows: the dental pulp tissue is more thoroughly digested, and the total number of the obtained dental pulp stem cells is larger; the total number of the obtained dental pulp stem cells is increased, the cell viabilityof the dental pulp stem cells is increased, besides, the passage time of the stem cells is shortened, and the isolated culture cycle of dental pulp stem cells is greatly shortened.

Description

technical field

[0001] The invention relates to the technical field of cell culture, in particular to a method for isolating and culturing dental pulp stem cells. Background technique

[0002] In recent years, odontogenic stem cells, as seed cells for tissue engineering, have attracted widespread attention and attention. They can be used not only for the regeneration of oral tissues, but also for the regeneration of non-oral tissues such as bone and nerves. Dental stem cells not only have superior stem cell characteristics, but can also be efficiently obtained from medical waste (such as orthodontic teeth, wisdom teeth, etc.).

[0003] The study found that dental pulp-derived cells have similar functions to bone marrow mesenchymal cells, which can repair and regenerate the pulp-dentin complex in vivo. Comparing dental pulp-derived cells with bone marrow mesenchymal stem cells, it was found that the rate of cell proliferation and clonal cell formation was significantly highe...

Examples

Embodiment 1

[0028] (1) Extraction of dental pulp tissue: Healthy white rabbits were selected as experimental subjects, male and female were randomly selected, and the age of the rabbits was 6 months. A molar was extracted from a white rabbit under local anesthesia, transferred to pre-cooled high-concentration double-antibody (double-antibody: streptomycin and penicillin), soaked for 10 minutes, disinfected, rinsed with PBS solution for 3 times, and then broken. Dental crown, extract the pulp from the pulp cavity of the tooth sample, and cut the pulp tissue into 0.4×0.4mm blocks.

[0029] (2) Put 2 times the volume of mixed enzyme in the above-mentioned dental pulp tissue for enzymatic hydrolysis; the mixed enzyme is a mixed solution of type IV collagenase, type V collagenase and trypsin with a mass volume concentration of 0.05% ; Digest for 4 hours at 37°C until the pulp tissue is completely digested, then centrifuge at 200 rpm for 5 minutes to remove the supernatant, and obtain enzymatic...

Embodiment 2

[0035] The only difference between this example and Example 1 is that in step (2), 10 times the volume of mixed enzyme is added to the cleaned pulp tissue.

Embodiment 3

[0037] The only difference between this example and Example 1 is that in the step (2), 20 times the volume of the mixed enzyme is added to the cleaned pulp tissue.

technical field

[0001] The invention relates to the technical field of cell culture, in particular to a method for isolating and culturing dental pulp stem cells. Background technique

[0002] In recent years, odontogenic stem cells, as seed cells for tissue engineering, have attracted widespread attention and attention. They can be used not only for the regeneration of oral tissues, but also for the regeneration of non-oral tissues such as bone and nerves. Dental stem cells not only have superior stem cell characteristics, but can also be efficiently obtained from medical waste (such as orthodontic teeth, wisdom teeth, etc.).

[0003] The study found that dental pulp-derived cells have similar functions to bone marrow mesenchymal cells, which can repair and regenerate the pulp-dentin complex in vivo. Comparing dental pulp-derived cells with bone marrow mesenchymal stem cells, it was found that the rate of cell proliferation and clonal cell formation was significantly highe...

Examples

Embodiment 1

[0028] (1) Extraction of dental pulp tissue: Healthy white rabbits were selected as experimental subjects, male and female were randomly selected, and the age of the rabbits was 6 months. A molar was extracted from a white rabbit under local anesthesia, transferred to pre-cooled high-concentration double-antibody (double-antibody: streptomycin and penicillin), soaked for 10 minutes, disinfected, rinsed with PBS solution for 3 times, and then broken. Dental crown, extract the pulp from the pulp cavity of the tooth sample, and cut the pulp tissue into 0.4×0.4mm blocks.

[0029] (2) Put 2 times the volume of mixed enzyme in the above-mentioned dental pulp tissue for enzymatic hydrolysis; the mixed enzyme is a mixed solution of type IV collagenase, type V collagenase and trypsin with a mass volume concentration of 0.05% ; Digest for 4 hours at 37°C until the pulp tissue is completely digested, then centrifuge at 200 rpm for 5 minutes to remove the supernatant, and obtain enzymatic...

Embodiment 2

[0035] The only difference between this example and Example 1 is that in step (2), 10 times the volume of mixed enzyme is added to the cleaned pulp tissue.

Embodiment 3

[0037] The only difference between this example and Example 1 is that in the step (2), 20 times the volume of the mixed enzyme is added to the cleaned pulp tissue.

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