Culturing method of periodontal ligament stem cell

A technology of periodontal ligament stem cells and culture methods, which is applied in the field of periodontal ligament stem cell culture, can solve the problems of low cell number and differences in biological properties, and achieve the effect of promoting growth and proliferation and good stem cell characteristics

Active Publication Date: 2016-01-20
GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Most of the existing periodontal ligament stem cells are cultured by monolayer cell culture, the number of cells obtained is low, and the biological properties are different from those in vivo.

Method used

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  • Culturing method of periodontal ligament stem cell
  • Culturing method of periodontal ligament stem cell
  • Culturing method of periodontal ligament stem cell

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Embodiment 1, culture method of periodontal ligament stem cell

[0052] A culture method of periodontal ligament stem cells, comprising the following steps:

[0053] S1. Isolation and purification of periodontal ligament stem cells

[0054] Collect the wisdom teeth that have fallen out and been extracted from people under 30 years old. After cleaning the dirt on the tooth surface with PBS, scrape 1 / 3 of the periodontal ligament tissue in the root with a sterile blade, cut it into pieces and place it in a 50mL centrifuge tube, add 10mL of 2g / Type LI collagenase solution, mix well, transfer to a constant temperature air shaker, digest at 37°C, 200rpm for about 45min, add an equal volume of complete medium to stop digestion, centrifuge at 1000rpm for 5min, discard the supernatant, and repeatedly blow and beat the discrete cells with 40mL PBS Agglomerates were filtered through a 70 μm cell mesh, the filtrate was centrifuged at 1000 rpm for 5 min, and the sediment was resu...

Embodiment 2

[0060] Embodiment 2, culture method of periodontal ligament stem cell

[0061] A culture method of periodontal ligament stem cells, substantially the same as in Example 1, comprising the following steps:

[0062] S1. Isolation and purification of periodontal ligament stem cells

[0063] The isolated periodontal ligament stem cells were divided into 1×10 4 / well was inoculated into a six-well plate, and 2 mL of serum-free medium for two-dimensional culture was added to each well. 2 , in a carbon dioxide incubator with a humidity of 95%. The medium was changed every 3 days, and the formation of colonies could be observed under a microscope around 7-14 days. When the cells grew to 80%-90% confluence, the cells were digested with 0.25m / v% trypsin solution and subcultured. Human periodontal ligament stem cells were cloned and cultured by the first-generation cells according to the limited dilution method. When the clones grew to about 30-60% of the bottom of the well, the cells...

Embodiment 3

[0068] Embodiment 3, culture method of periodontal ligament stem cell

[0069] A culture method of periodontal ligament stem cells, substantially the same as in Example 1, comprising the following steps:

[0070] S1. Isolation and purification of periodontal ligament stem cells

[0071] The isolated periodontal ligament stem cells were divided into 1×10 4 / well was inoculated into a six-well plate, and 2 mL of serum-free medium for two-dimensional culture was added to each well. 2 , Humidity 95% in a carbon dioxide incubator. The medium was changed every 3 days, and the formation of colonies could be observed under a microscope around 7-14 days. When the cells grew to 80%-90% confluence, the cells were digested with 0.25m / v% trypsin solution and subcultured. Human periodontal ligament stem cells were cloned and cultured by the first-generation cells according to the limited dilution method. When the clones grew to about 30-60% of the bottom of the well, the cells were dige...

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Abstract

The invention discloses a culturing method of a periodontal ligament stem cell. The culturing method comprises the following steps of inoculating the periodontal ligament stem cell onto a hydrogel, and carrying out three-dimensional culturing by using a serum-free medium containing BMP-2. The periodontal ligament stem cell used for three-dimensional culturing and inoculating is cultured by using a serum-free medium containing bFGF when being two-dimensional cultured. Compared with the prior art, according to the culturing method of the periodontal ligament stem cell, a cell and hydrogel culturing system can better maintain the proliferation and cell activity of the periodontal ligament stem cell; the used three-dimensional culturing system is simple and easy to operate, and meanwhile, the favorable stem cell characteristics can be maintained.

Description

technical field [0001] The invention relates to the field of cell culture, in particular to a culture method of periodontal ligament stem cells. Background technique [0002] Periodontal ligament stem cells (PDLSCs) are a kind of fibroblast-like stem cells with self-renewal, strong proliferation ability and multilineage differentiation ability existing in tooth periodontal tissue. Periodontal ligament stem cells have the potential of multidirectional differentiation. They can differentiate into adipocytes, osteoblast-like cells, cementoblast-like cells and neural precursor cells in vitro, and can differentiate into cementoblast-like and periodontal cells in vivo. membranous tissue. Therefore, periodontal ligament stem cells are the seed cells for periodontal tissue regeneration, and the periodontal ligament stem cells cultured in vitro are reinfused into the human body to treat periodontal disease and other diseases. The current culture of periodontal ligament stem cells m...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/074
Inventor 葛啸虎陈海佳王一飞冯德龙张维敏
Owner GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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