A kind of nasal epithelial stem cell culture method and nasal epithelial stem cell proliferation medium

Abstract

The invention discloses a method for cultivating nasal epithelial stem cells, which comprises the following steps: 1) paving trophoblast cells in a culture dish; 2) isolating primary nasal epithelial stem cells from nasal mucosa tissue; 3) separating primary nasal epithelial stem cells into The generation cells are placed on the trophoblast cells, the culture medium is replaced with nasal epithelial stem cell proliferation medium, and the rest of the tissue cells are removed; 4) when the nasal epithelial stem cells proliferate to 80% density, they are digested with trypsin and passed on to continue to expand; 5) Transfer nasal epithelial stem cells to gas-liquid phase culture system and PneumaCult-ALI differentiation medium for differentiation culture. The invention also discloses a nasal epithelial stem cell proliferation medium, comprising the following components: DMEM / F12, fetal bovine serum, penicillin, streptomycin, insulin, epidermal growth factor, hydrocortisone, 3,3',5 ‑triiodo‑L‑thyronine sodium, ROCK inhibitors. The invention provides a key implementation basis for the transformation application of nasal epithelial stem cell clinical transplantation.

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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