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Culture method of deciduous tooth pulp stem cells

A dental pulp stem cell and culture method technology, applied in the direction of non-embryonic pluripotent stem cells, animal cells, vertebrate cells, etc., can solve the problems of excessive tissue digestion, poor stability between batches, and unfavorable cells

Inactive Publication Date: 2015-11-25
GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the enzymatic hydrolysis time of this method is too long, the tissue is over-digested, and the number of dental pulp stem cells is small.
Moreover, this method adopts DMEM culture containing fetal bovine serum, which is polluting and has poor stability between batches, which is not conducive to the use of cells in animal models

Method used

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  • Culture method of deciduous tooth pulp stem cells
  • Culture method of deciduous tooth pulp stem cells
  • Culture method of deciduous tooth pulp stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Embodiment 1, the cultivation of human deciduous tooth pulp stem cells

[0038] (1) With the consent of the family members, clinically collected deciduous incisors extracted from children due to detention, and no dental pulp disease and necrosis were required. Immediately after pulling out, put it in a PBS centrifuge tube containing double antibody when it is cold at 4°C, and take it out for use within 24 hours.

[0039] (2) Take out the tooth, rinse it three times repeatedly with PBS solution containing double antibody, wrap the tooth in sterile gauze and crack the tooth with forceps to expose the pulp tissue; use sterile forceps to grasp the pulp tissue, and remove the root tip 1mm of pulp tissue. According to the size of the pulp tissue, cut the pulp tissue into 1mm with ophthalmic curved scissors 3 , placed in a 50mL centrifuge tube.

[0040] (3) Add 10 times the volume of 5g / LI type collagenase, mix well after sealing, transfer to a constant temperature air shak...

Embodiment 2

[0046] Embodiment 2, cell viability detection

[0047] The cells cultivated by the method disclosed in the application number 201210514497.X were used as a comparative example (control group), and the cells were separated according to the method in Example 1 (sample group), and the separated cells were collected and cell viability was calculated in an automatic cell counter. result like figure 1 .

[0048] Depend on figure 1 The results show that the vitality of the dental pulp stem cells obtained by the culture method of the deciduous tooth pulp stem cells of the present invention can reach more than 90%.

Embodiment 3

[0049] Embodiment 3, cell morphology observation

[0050] With the application number being the cell that the disclosed method of 201210514497.X is cultivated as a comparative example (control group), observe the cell morphology of the cell that the method (sample group) of embodiment 1 is cultivated for 7 days, the results are shown in figure 2 .

[0051] The results show that the cells cultured by the method for culturing deciduous dental pulp stem cells of the present invention can reach more than 80% cell confluence after 7 days of primary culture, while the cell confluence in the comparative example is about 50%, which is obviously lower than that of the present invention. training method.

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Abstract

The invention relates to the field of stem cells and discloses a culture method of deciduous tooth pulp stem cells. The culture method includes: taking pulp tissue, cutting the pulp tissue into pieces, and then adding I-type collagenase; digesting for 10-20min under conditions of 37 DEG C and 200rpm; stopping digestion of a serum-free culture medium; blowing and beating discrete cell mass to obtain single discrete cells; adding cell suspension to re-suspend the cells, and adjusting cell density; culturing the cells in a carbon dioxide incubator at temperature of 37 DEG C and with humidity of 95%; when the cells grow to be fused by 80-90%, using digestive liquid of trypsase to digest the cells before passage culture, wherein the cell suspension is composed of the serum-free culture medium, epidermal growth factor and basic fibroblast growth factor. The cells cultured by the method are good in shape, have tendency of fusiform cluster growing and are high in activity and quick in proliferation, good stem cell characteristics of the deciduous tooth pulp stem cells can be maintained, and the culture method is suitable for large-scale culture of the deciduous tooth pulp stem cells.

Description

technical field [0001] The invention relates to the field of stem cells, in particular to a method for culturing dental pulp stem cells, in particular to a method for cultivating dental pulp stem cells in deciduous teeth. Background technique [0002] In 2003, Miura et al. found a kind of pluripotent stem cells in the dental pulp tissue of exfoliated deciduous teeth. Compared with bone marrow mesenchymal stem cells and permanent tooth pulp stem cells, it has a higher proliferation rate, cell population doubling ability, and clone formation ability. and highly differentiated ability, they were named deciduous dental pulp stem cells (stemcellsfromdeciduousteeth, SHED). Human deciduous tooth pulp stem cells are a kind of mesenchymal stem cells (mesenchymal stem cells, MSCs), mesenchymal stem cells with self-replication ability and multi-lineage differentiation potential, can be induced to differentiate into dentin, osteoblasts, chondrocytes, adipogenic cells Cells, nerve cells...

Claims

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Application Information

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IPC IPC(8): C12N5/074C12N5/0775
Inventor 葛啸虎陈海佳王一飞冯德龙马岩岩
Owner GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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