Nose epithelial stem cell culture method and nose epithelial stem cell proliferation medium

A stem cell culture and stem cell proliferation technology, applied in the field of nasal epithelial stem cell culture method and nasal epithelial stem cell proliferation medium, can solve the problems that cells are difficult to differentiate into ciliated or goblet epithelial cells, the differentiation ability is reduced, and the differentiation ability is difficult to guarantee. Achieve the effect of maintaining proliferation ability, maintaining differentiation ability, and breaking through key technical bottlenecks

Active Publication Date: 2018-11-06
THE FIRST AFFILIATED HOSPITAL OF SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The existing culture systems and methods have the following limitations: 1) The commercial medium can only maintain the proliferation and culture of undifferentiated nasal epithelial stem cells for 2-3 generations, and generally after the 2nd generation, the cells are difficult to differentiate into ciliated or goblet epithelial cells
2) Although the culture system developed by the applicant in the early stage can maintain the proliferation and culture of 3-5 generations of undifferentiated epithelial stem cells, it is difficult to guarantee the differentiation ability of the cells after more than 3 generations of proliferation
Although certain stem cell proliferation characteristics can be maintained, the reduction in differentiation ability makes it difficult to maintain the functional characteristics (mucociliary clearance function) of epithelial stem cells
3) In the expected clinical application, in some cases (such as a large area of...

Method used

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  • Nose epithelial stem cell culture method and nose epithelial stem cell proliferation medium
  • Nose epithelial stem cell culture method and nose epithelial stem cell proliferation medium
  • Nose epithelial stem cell culture method and nose epithelial stem cell proliferation medium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0093] Nasal epithelial stem cells were derived from sinusitis tissue samples from a patient diagnosed with chronic sinusitis.

[0094] 1) Treat trophoblast cells

[0095] ①The trophoblast cells were 3T3 cells, and the culture conditions were DMEM medium, 10% fetal bovine serum and 1x penicillin-streptomycin complex solution.

[0096] ②The 3T3 cells were grown to 100% density and treated with mitomycin to inhibit the growth of 3T3 cells. The concentration of mitomycin was 0.5-20ug / mL.

[0097] ③The trophoblast cells treated with mitomycin were digested according to a certain density (0.1-1x10 4 cells / cm 2 ) were pre-plated in cell culture dishes.

[0098] ④The 3T3 cells treated with mitomycin can adhere to the wall smoothly within 12 hours to 24 hours.

[0099] 2) Proliferation and cultivation of nasal epithelial stem cells (respectively used as the medium of the present invention and the BEGM medium)

[0100] ① After the trophoblast cells adhere to the wall, the epitheli...

Embodiment 2

[0125] Nasal epithelial stem cells were derived from nasal polyp tissue samples, and the patient was diagnosed with nasal polyps.

[0126] 1) Treat trophoblast cells

[0127] ①The trophoblast cells were 3T3 cells, and the culture conditions were DMEM medium, 10% fetal bovine serum and 1x penicillin-streptomycin complex solution.

[0128] ②The 3T3 cells were grown to 100% density and treated with mitomycin to inhibit the growth of 3T3 cells. The concentration of mitomycin was 0.5-20ug / mL.

[0129] ③The trophoblast cells treated with mitomycin were digested according to a certain density (0.1-1x10 4 cells / cm 2 ) were pre-plated in cell culture dishes.

[0130] ④The 3T3 cells treated with mitomycin can adhere to the wall smoothly within 12 hours to 24 hours.

[0131] 2) Proliferation and cultivation of nasal epithelial stem cells (respectively used as the medium of the present invention and the BEGM medium)

[0132] ① After the trophoblast cells adhere to the wall, the epith...

Embodiment 3

[0156] Nasal epithelial stem cells were derived from inferior turbinate tissue samples, and the patient was diagnosed with inferior turbinate hypertrophy.

[0157] 1) Treat trophoblast cells

[0158] ①The trophoblast cells were 3T3 cells, and the culture conditions were DMEM medium, 10% fetal bovine serum and 1x penicillin-streptomycin complex solution.

[0159] ②The 3T3 cells were grown to 100% density and treated with mitomycin to inhibit the growth of 3T3 cells. The concentration of mitomycin was 0.5-20ug / mL.

[0160] ③The trophoblast cells treated with mitomycin were digested according to a certain density (0.1-1x10 4 cells / cm 2 ) were pre-plated in cell culture dishes.

[0161] ④The 3T3 cells treated with mitomycin can adhere to the wall smoothly within 12 hours to 24 hours.

[0162] 2) Proliferation and cultivation of nasal epithelial stem cells (respectively used as the medium of the present invention and the BEGM medium)

[0163] ① After the trophoblast cells adhe...

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Abstract

The invention discloses a nose epithelial stem cell culture method which comprises the following steps: 1) laying nourishing layer cells into a culture dish; 2) separating primary cells of nose epithelial stem cells from nasal mucosa; 3) laying the primary cells of the nose epithelial stem cells on the nourishing layer cells, replacing a medium by a nose epithelial stem cell proliferation medium,and removing off residual tissue cells; 4) when the nose epithelial stem cells are proliferated to a density of 80%, carrying out trypsinization, and carrying out continuous passage amplification; 5)transferring the nose epithelial stem cells into a gas-liquid phase culture system and a PneumaCult-ALI differentiation medium, and carrying out differentiation culture. The invention further discloses a nose epithelial stem cell proliferation medium which comprises the following components: DMEM/F12, fetal calf serum, penicillin, streptomycin, insulin, an epidermal growth factor, hydrocortisone,3,3',5-triiodo-L-sodium adenoglycosine and an ROCK inhibitor. By adopting the method, a key application base is provided for transformation application of clinical transplanting of the nose epithelialstem cells.

Description

technical field [0001] The invention belongs to the technical field of adult stem cells, and in particular relates to a nasal epithelial stem cell culture method and a nasal epithelial stem cell proliferation medium. Background technique [0002] Adult stem cells refer to undifferentiated cells that exist in tissues. These cells can self-renew, and under certain conditions, differentiate according to a certain program to form new functional cells, so that the tissue or organ maintains a dynamic balance of growth and decline. . Embryonic stem cells are totipotent, can be established and passed on, can be immortalized and have strong proliferation ability, but their disadvantages are obvious, such as immune rejection, non-localized differentiation, and the possibility of causing cancer. In contrast, adult stem cells are derived from the individual, have no histocompatibility problems, avoid transplant rejection, and have tissue-specific proliferation and directional different...

Claims

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Application Information

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IPC IPC(8): C12N5/071
CPCC12N5/0625C12N2501/11C12N2501/33C12N2501/30
Inventor 李春炜洪玥何根谢曦
Owner THE FIRST AFFILIATED HOSPITAL OF SUN YAT SEN UNIV
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