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

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...

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 ​​mucosal injury), a large number of stem cells are required. Therefore, the existing medium cannot provide a sufficient amount under the condition of a limited number of passages (i.e. expansion). stem cells

4) Regardless of whether it is a commercial culture medium or a culture system developed by an individual laboratory, the proliferation of nasal epithelial stem cells is relatively slow, and it will be a challenge to obtain a sufficient number of epithelial stem cells with stem cell characteristics in a short period of time (such as within 4 weeks). problem

[0008] To sum up, the existing nasal epithelial stem cell culture systems and methods are still unable to maintain the function and characteristics of stem cells for a long time, that is, in terms of proliferation and differentiation, it is difficult to meet the needs of clinical stem cell transplantation, which is also a common clinical application of adult stem cells. technical bottleneck

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