Method for differentiating sustentacular cell into inner ear hair cell and application

A technology of inner ear hair cells and supporting cells, applied in new medical fields, can solve problems such as inability to fundamentally treat deafness, and achieve the effect of high differentiation efficiency

Inactive Publication Date: 2017-11-24
SHANDONG XINRUI BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, sensorineural deafness is mainly treated clinically by using hearing aids, cochlear implants or drugs, but these methods cannot fundamentally treat deafness

Method used

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  • Method for differentiating sustentacular cell into inner ear hair cell and application
  • Method for differentiating sustentacular cell into inner ear hair cell and application
  • Method for differentiating sustentacular cell into inner ear hair cell and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment example 1

[0028] Example 1: Culture of Sertoli cells in the mouse inner ear

[0029] Cochlear epithelial cells of mice (purchased from Guangzhou University of Traditional Chinese Medicine, female, belonging to SPF (III) grade animals) were isolated, cultured for 10 days, and the medium was changed every 3 days to obtain single cells. Cells were transferred to DMEM+10 containing: 50ng / mL Epidermal Growth Factor (EGF), 50ng / mL Fibroblast Factor (bFGF), 50ng / mL Insulin Factor-1 (IGF-1) i.e. EFI mix %FBS (purchased from Gbico) medium; 50ng / mL epidermal growth factor (EGF), 50ng / mL fibroblast factor (bFGF), 50ng / mL insulin factor-1 (IGF-1), 3μM glycogen kinase Inhibitor-3 (CHIR99021 or C), 1 mM valproic acid (VPA or V), 100 μg / mL 2-phospho-L-ascorbic acid (pVc or P), 2 μM ALK5 inhibitor (616452 or 6) i.e. EFICUP6 mixed factor combination DMEM+10% FBS medium, at 37°C, 5% CO 2 In the incubator, culture for 10 days, change the medium every day, and observe the number of Lgr5+ cells. Sertoli ...

Embodiment example 2

[0030] Example 2: Effects of different factor combinations on differentiation into Lgr5+ cells

[0031] Cochlear epithelial cells of mice (purchased from Guangzhou University of Traditional Chinese Medicine, female, belonging to SPF (III) grade animals) were isolated, cultured for 10 days, and the medium was changed every 3 days to obtain single cells. Inoculate to contain:

[0032] DMEM+10% FBS medium of EFICVP6 mixed factor combination;

[0033] The DMEM+10%FBS medium of the FICVP6 (-EFI) mixed factor combination without adding EGF;

[0034] EICVP6 (-bFGF) mixed factor combination DMEM+10% FBS medium without adding bFGF;

[0035] EFCVP6 (-IGF-1) mixed factor combination DMEM+10% FBS medium without adding IGF-1;

[0036] EFIVP6 (-CHIR) mixed factor combination DMEM+10% FBS medium without adding CHIR99021;

[0037] The DMEM+10%FBS medium of the EFICP6 (-VPA) mixed factor combination without adding VPA;

[0038] The DMEM+10% FBS medium of the EFICV6 (-bFGF) mixed factor comb...

Embodiment example 3

[0040] Example 3: Differentiation of Lgr5+ cells into inner ear hair cells

[0041] The best EFICVP6 medium obtained in Example 2 was selected to amplify Sertoli cells, and cultured for 10 days to obtain Lgr5+ cells. Then add the cells to the induction medium, the induction medium is: DMEM+10% FBS medium; add 5 μM LY411575 (LY) factor DMEM+10% FBS medium; add CHIR99021 (C) factor DMEM+10% FBS culture base; add LY411575 and CHIR99021 (LYC) factor DMEM+10% FBS medium, induce differentiation and culture for 10 days, count the number of hair cells. according to Figure 4 , it was concluded that the number of hair cells induced by DMEM+10%FBS medium added with LYC factor was better than that of medium without factor added and only added with LY or C. Therefore, DMEM+10% FBS medium supplemented with LYC factor was selected as the optimal induction differentiation medium.

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Abstract

The invention discloses a method for differentiating a sustentacular cell into an inner ear hair cell. The method is characterized by comprising the steps of cultivating a cochlear epithelial cell into a single cell, adding the single cell to an expansion culture medium containing an epidermal cell growth factor, a fiber forming mother cell factor, an insulin factor-1, a glycogen kinase inhibitor-3, valproic acid, 2-phosphoric acid-L-ascorbic acid and an ALK5 inhibitor, expanding the sustentacular cell to obtain an Lgr5+ cell, adding an induction culture medium containing an LY411575 factor and the glycogen kinase inhibitor-3, performing induced differentiation cultivation for 10 days to obtain the inner ear hair cell, and detecting a gene level and a protein level of the inner ear hair cell subjected to induced differentiation to confirm the inner ear hair cell. According to the method, division and multiplication of the sustentacular cell are promoted by the culture medium containing the factors, and then the sustentacular cell is induced to be differentiated into the inner ear hair cell by the induction culture medium, so that the high-purity and high-ratio inner ear hair cell can be obtained.

Description

technical field [0001] The invention relates to the field of new medical technology, more specifically, it relates to a method and application for supporting cells to differentiate into inner ear hair cells. Background technique [0002] Deafness is one of the common chronic diseases that affect the quality of human life. There are about 360 million people with deafness in the world. Among them, sensorineural deafness is very common in clinical practice, and the loss of hair cells caused by noise exposure, ototoxic drugs, bacterial or viral infection, and aging is the main cause of sensorineural deafness. Because the number of cochlear hair cells is limited, about 15,000, and there is no spontaneous regeneration ability after injury, it often causes permanent hearing loss, which is a difficult problem in current clinical treatment. At present, sensorineural deafness is mainly treated clinically by using hearing aids, cochlear implants or drugs, but these methods cannot fund...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071
CPCC12N5/0627C12N2500/30C12N2500/38C12N2501/11C12N2501/115C12N2501/33C12N2501/727
Inventor 刘明录金海锋强邦明万磊韩庆梅冯建海张传鹏马洪华
Owner SHANDONG XINRUI BIOTECH CO LTD
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