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Method for culturing and amplifying odontogenic epithelial cells

An epithelial cell and odontogenic technology, applied in the field of cell culture, can solve the problems of difficulty in culturing and expanding epithelial cells, incomplete understanding of the molecular mechanism of tooth development, etc., and achieve the effect of easy mastery, high standardization, and wide application prospects.

Active Publication Date: 2011-04-06
成都世联康健生物科技有限公司
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  • Summary
  • Abstract
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AI Technical Summary

Problems solved by technology

However, the study of tooth development and regeneration only starts from the study of mesenchymal cells, and cannot fully understand the molecular mechanism of tooth development. Therefore, the study of odontogenic epithelial cells has to first solve the problem of difficult cultivation and expansion of epithelial cells

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 Culture of epithelial stem cells from rat lower incisor cervical ring:

[0044] (1) According to the law of incisor development in rats, SD rats were selected from 1 day to 10 days after birth, and the experimental mice were drowned in alcohol, and their heads were cut from the back edge of the ears, and the bilateral mandibular ramus was used as the Free the mandible with a sharp scalpel and micro forceps, remove the tongue and surrounding soft tissues; fix in 4% paraformaldehyde at 4°C for 7 days, decalcify with 10% g / mL EDTA for 1 month; rinse with running water overnight , gradient ethanol dehydration, xylene transparent, paraffin embedding, continuous 5μm sections, HE staining, observation and photography under a microscope; according to the most enriched time of incisor cervical ring cells, it is concluded that 4-5 days after birth is the rat cervical ring epithelium Optimal time point for cell sampling.

[0045] (2) Take 10 SD rats in the most enriche...

Embodiment 2

[0048] Embodiment 2, Minipig first molar HERS cell culture:

[0049] (1) According to the rules of molar development of minipigs, the first molars of minipigs were selected on the 100th, 110th, 120th, and 130th day of embryos and 1st, 10th, 20th, and 30th day after birth; fixed in 4% paraformaldehyde at 4°C for 14 1 day, 10% g / mL EDTA decalcification for 1 month; washed overnight with running water, dehydrated with graded ethanol, cleared with xylene, embedded in paraffin, sliced ​​at 5 μm in length, stained with HE, observed and photographed under a microscope; according to the HERS of the first molar just formed Period is the best time point for HERS collection, so it is concluded that 1-2 days after birth is the best time point for collection of minipig molar HERS cells.

[0050] (2) Take 10 first molars of minipigs in the HERS formation period (1-2 days after birth); rinse with PBS, and use a sharp surgical knife in a petri dish containing 10% fetal bovine serum DMEM cul...

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Abstract

The invention discloses a method for culturing and amplifying odontogenic epithelial cells, and belongs to the technical field of cell culture. The method for culturing and amplifying the odontogenic epithelial cells of the invention comprises the steps of obtaining and culturing primary epithelial cells and amplifying the epithelial cells, wherein the primary culture comprises the steps of digesting odontogenic chyle-shaped tissues by using mixed enzyme digestive juice consisting of dispersing enzyme, I-type collagenase and DNA enzyme, collecting cells and culturing; and the mixed enzyme comprises the dispersing enzyme, the I-type collagenase and the DNA enzyme; and sub-culturing comprises the steps of digesting the cells by combining a cell scraper scrapping method and a 0.025 to 0.25 percent trypsin digestive juice, suspending the cells again with a culture medium and culturing. By the method, the cells are sub-cultured for 2 to 3 times to form a great number of purified odontogenic epithelial cells which are identified to be epithelial cells by an epithelial cell expression marker CK14. The method solves the problem of difficulty in culturing the odontogenic epithelial cells, and a great number of stable and purified odontogenic epithelial cells are obtained and the source of the epithelial cells is provided for the teeth development and regeneration research.

Description

technical field [0001] The invention relates to a method for culturing and expanding epithelial cells, in particular to a method for culturing and expanding dental epithelial cells, and belongs to the technical field of cell culture. Background technique [0002] Tooth loss is a common and frequently-occurring disease in humans, which has a significant impact on patients' chewing, speech, aesthetics and psychology. According to WHO statistics, dental disease is one of the three non-communicable diseases with the highest incidence rate in humans, and cases of tooth loss caused by various dental diseases are very common. Although the existing denture restoration methods are mature, they are difficult to compare with natural teeth due to their lack of biological activity, and cannot realize the vision of humans having a "third set of teeth". Therefore, tooth development regenerative medicine has become the most important scientific problem in stomatology. [0003] The develop...

Claims

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Application Information

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IPC IPC(8): C12N5/071
Inventor 郭维华田卫东孔子任郭永文葛雅能
Owner 成都世联康健生物科技有限公司
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