Donkey mammary epithelial cell in-vitro culture method based on specific enzyme liquid and culture medium
In vitro culture of donkey breast epithelial cells through specific enzyme fluid and culture medium solves the problem of lack of efficient and reliable cell isolation and culture methods in the prior art, and achieves efficient cell isolation and stable cell growth, providing a powerful tool for the study of physiological functions and disease mechanisms of donkey breasts.
Patent Information
- Application Number
- CN202411481711.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-23
- Publication Date
- 2025-03-04
- Estimated Expiration
- Not applicable · inactive patent
AI Technical Summary
The lack of efficient and reliable in vitro isolation and culture methods for donkey breast epithelial cells in the prior art cannot meet the needs of in-depth research.
The in vitro culture of donkey breast epithelial cells was carried out using specific enzyme solution and culture medium, including cleaning and disinfection treatment, enzyme solution digestion, careful design of cell culture medium and regular liquid change operations.
It improves cell separation efficiency and culture stability, provides a suitable growth environment, promotes cell growth and proliferation, fills the gaps in the existing technology, and provides new ideas and methods for research in related fields.
Smart Images
Figure CN119552801A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of cell separation and culture, in particular to an in vitro culture method of donkey mammary epithelial cells based on a specific enzyme solution and a culture medium. Background Art
[0002] With the continuous development of biotechnology, the research on various animal cells has become increasingly in-depth. As an important livestock, the study of donkey mammary epithelial cells is of great significance for understanding the physiological function of donkey mammary glands, the pathogenesis of mammary diseases, and the development of new dairy products. However, the in vitro isolation and culture of donkey mammary epithelial cells is still imperfect, and there is a lack of efficient and reliable technical means. Traditional methods have shortcomings in cell separation efficiency and optimization of culture conditions, and cannot meet the needs of in-depth research. Summary of the invention
[0003] 1. Technical issues to be resolved
[0004] In view of the deficiencies of the prior art, the present invention provides an in vitro culture method of donkey mammary epithelial cells based on a specific enzyme solution and a culture medium.
[0005] (II) Technical solution
[0006] The in vitro culture method of donkey mammary epithelial cells based on specific enzyme solution and culture medium comprises the following steps:
[0007] S1. Mammary gland tissue of healthy donkeys was collected and cleaned and disinfected. During the cleaning process, sterile saline was used for preliminary washing to remove surface impurities, and then a specific cleaning solution was used for secondary washing. The cleaning solution contained antibacterial ingredients and cell protectants, which could effectively kill possible bacteria and fungi while reducing damage to cells. The disinfection treatment used a composite solution containing antibiotics for immersion disinfection to ensure the sterility of the tissue.
[0008] S2. Digesting the breast tissue with a specific enzyme solution to obtain a single cell suspension, wherein the enzyme solution is a mixed solution of collagenase, trypsin and hyaluronidase, and the ratio of the three enzymes is determined through a large number of experiments, which can efficiently digest the breast tissue in the shortest time while minimizing damage to the cells;
[0009] S3, inoculating the single cell suspension into a culture container, adding a specific cell culture medium for culture, the cell culture medium contains growth factors and nutrients in a specific ratio, the growth factors include epidermal growth factor and insulin-like growth factor, and the nutrients are carefully screened and optimized to provide a suitable growth environment for the cells;
[0010] S4. Regularly observe the cell growth status and perform medium change operations. During the medium change process, use special medium change devices and operation methods to avoid mechanical damage to the cells and ensure that the new culture medium can evenly cover the cells.
[0011] Preferably, the antibacterial components in the cleaning solution include but are not limited to penicillin, streptomycin and gentamicin, and the cell protectant is glutathione or vitamin C.
[0012] Preferably, the ratio of collagenase, trypsin and hyaluronidase in the enzyme solution is 1:0.4-0.6:0.8-1.2.
[0013] Preferably, the growth factors in the cell culture medium are epidermal growth factor and insulin-like growth factor in a mass ratio of 1:1, and the nutrients are amino acids, inorganic salts, and vitamins. The amino acids are complex amino acids composed of twenty kinds of amino acids required by the human body, and the inorganic salts are sodium chloride, potassium dihydrogen phosphate, ferrous sulfate, magnesium sulfate, and calcium sulfate in a mass ratio of 1:0.1-0.3:0.1-0.3:0.1-0.3:0.1-0.3, and the vitamin is vitamin C; the mass ratio of the epidermal growth factor, amino acids, inorganic salts, and vitamins is 1:2-4:0.6-0.8:0.6-0.8.
[0014] Preferably, the liquid replacement device is a pipette with a fine filter, and the operation method includes slowly aspirating the old culture medium and adding the new culture medium at a uniform speed.
[0015] Preferably, the cultured cells are identified as donkey mammary epithelial cells using a specific identification method, and the identification method includes morphological observation, immunocytochemical staining and RT-PCR detection; morphological observation observes the shape, size and arrangement of the cells under a microscope; immunocytochemical staining uses specific antibodies to stain the cells and detects the expression of epithelial cell markers; RT-PCR detection determines the type of cells by detecting the expression of epithelial cell-specific genes.
[0016] (III) Beneficial technical effects
[0017] 1. The use of optimized cleaning solution and disinfection method improves the sterility of the tissue, reduces damage to cells, and provides a good foundation for subsequent cell culture.
[0018] 2. The innovative enzyme solution formula can efficiently digest breast tissue, obtain more single-cell suspension, and improve cell separation efficiency.
[0019] 3. The carefully designed cell culture medium components and proportions provide cells with a suitable growth environment, promoting cell growth and proliferation.
[0020] 4. The special fluid replacement device and operation method avoid mechanical damage to cells and ensure the stability of cell culture.
[0021] 5. This method provides a powerful tool for studying the physiological function and disease mechanism of donkey mammary gland, which will help promote the development of donkey industry and dairy products.
[0022] 6. It fills the gap in the current in vitro isolation and culture methods of donkey mammary epithelial cells, and provides new ideas and methods for research in related fields. BRIEF DESCRIPTION OF THE DRAWINGS
[0023] Figure 1 This is a diagram of donkey mammary epithelial cells.
[0024] Figure 2 This is an agarose gel electrophoresis image of donkey mammary epithelial cells.
[0025] Figure 3 These are the immunofluorescence staining identification pictures of donkey mammary epithelial cells; A is the immunofluorescence staining identification picture of keratin 14 intracellular expression, B is the immunofluorescence staining identification picture of cell nucleus DAPI, and C is the merged picture. DETAILED DESCRIPTION
[0026] Example 1
[0027] S1. Collect mammary tissue from healthy donkeys, clean and disinfect it. During the cleaning process, use sterile saline for preliminary rinsing to remove surface impurities, and then use a specific cleaning solution for secondary rinsing. The cleaning solution contains antibacterial ingredients such as penicillin, streptomycin and gentamicin and a cell protector, glutathione, which can effectively kill possible bacteria and fungi while reducing damage to cells. Disinfection is carried out by immersion disinfection using a composite solution containing antibiotics to ensure the sterility of the tissue.
[0028] S2. Use a specific enzyme solution to digest the breast tissue to obtain a single cell suspension. The enzyme solution is a mixed solution of collagenase, trypsin and hyaluronidase in a ratio of 1:0.4:1.2, which can efficiently digest the breast tissue in the shortest time while minimizing damage to the cells.
[0029] S3. Inoculate the single cell suspension into a culture container and add a specific cell culture medium for culture. The cell culture medium contains growth factors and nutrients in specific proportions. The growth factors include epidermal growth factor and insulin-like growth factor. The nutrients are carefully screened and optimized to provide a suitable growth environment for the cells.
[0030] The growth factors in the cell culture medium are epidermal growth factor and insulin-like growth factor in a mass ratio of 1:1, and the nutrients are amino acids, inorganic salts, and vitamins. The amino acids are complex amino acids composed of twenty kinds of amino acids required by the human body, the inorganic salts are sodium chloride, potassium dihydrogen phosphate, ferrous sulfate, magnesium sulfate, and calcium sulfate in a mass ratio of 1:0.1:0.3:0.1:0.2, and the vitamin is vitamin C; the mass ratio of the growth factors, amino acids, inorganic salts, and vitamins is 1:3:0.8:0.6.
[0031] S4. Regularly observe the cell growth status and perform fluid change operations. During the fluid change process, use a special fluid change device, a pipette with a fine filter, and an operating method that includes slowly aspirating the old culture medium and adding the new culture medium at a uniform speed to avoid mechanical damage to the cells and ensure that the new culture medium can evenly cover the cells.
[0032] Comparative Example 1
[0033] S1. Mammary gland tissue of healthy donkeys was collected, and the tissue was cleaned and disinfected. During the cleaning process, sterile saline was used to rinse the tissue first to remove surface impurities.
[0034] S2. Use a specific enzyme solution to digest the breast tissue to obtain a single cell suspension. The enzyme solution is a mixed solution of collagenase, trypsin and hyaluronidase in a ratio of 1:0.4:1.2, which can efficiently digest the breast tissue in the shortest time while minimizing damage to the cells.
[0035] S3. Inoculate the single cell suspension into a culture container and add a specific cell culture medium for culture. The cell culture medium contains growth factors and nutrients in specific proportions. The growth factors include epidermal growth factor and insulin-like growth factor. The nutrients are carefully screened and optimized to provide a suitable growth environment for the cells.
[0036] The growth factors in the cell culture medium are epidermal growth factor and insulin-like growth factor in a mass ratio of 1:1, and the nutrients are amino acids, inorganic salts, and vitamins. The amino acids are complex amino acids composed of twenty kinds of amino acids required by the human body, the inorganic salts are sodium chloride, potassium dihydrogen phosphate, ferrous sulfate, magnesium sulfate, and calcium sulfate in a mass ratio of 1:0.1:0.3:0.1:0.2, and the vitamin is vitamin C; the mass ratio of the growth factors, amino acids, inorganic salts, and vitamins is 1:3:0.8:0.6.
[0037] S4. Regularly observe the cell growth status and perform fluid change operations. During the fluid change process, use a special fluid change device, a pipette with a fine filter, and an operating method that includes slowly aspirating the old culture medium and adding the new culture medium at a uniform speed to avoid mechanical damage to the cells and ensure that the new culture medium can evenly cover the cells.
[0038] Comparative Example 2
[0039] S1. Collect mammary tissue from healthy donkeys, clean and disinfect it. During the cleaning process, use sterile saline for preliminary rinsing to remove surface impurities, and then use a specific cleaning solution for secondary rinsing. The cleaning solution contains antibacterial ingredients such as penicillin, streptomycin and gentamicin and a cell protector, glutathione, which can effectively kill possible bacteria and fungi while reducing damage to cells. Disinfection is carried out by immersion disinfection using a composite solution containing antibiotics to ensure the sterility of the tissue.
[0040] S2. Use a specific enzyme solution to digest the breast tissue to obtain a single cell suspension. The enzyme solution is a mixed solution of collagenase, trypsin and hyaluronidase in a ratio of 1:0.4:1.2, which can efficiently digest the breast tissue in the shortest time while minimizing damage to the cells.
[0041] S3. Inoculate the single cell suspension into a culture container and add beef extract peptone agar medium for culture.
[0042] S4. Regularly observe the cell growth status and perform fluid change operations. During the fluid change process, use a special fluid change device, a pipette with a fine filter, and an operating method that includes slowly aspirating the old culture medium and adding the new culture medium at a uniform speed to avoid mechanical damage to the cells and ensure that the new culture medium can evenly cover the cells.
[0043] Cell viability detection: The cell viability detection kit BB-4122 was used for detection.
[0044] Usage: Take 100μL of reagent B and add 900μL of deionized water to dilute and mix to obtain a cell suspension. Collect the cell sample, wash the cells twice with PBS, resuspend the cells with the cell suspension, take 180μL of the resuspended cells, add 20μL of staining solution A, mix gently, and stain for three minutes. Take a small amount of stained cells and count them with a cell counting plate.
[0045] Cell viability = (total number of cells - number of blue cells) / total number of cells × 100%.
[0046] Table 1
[0047] Group Cell survival rate (%) Example 1 96.5 Comparative Example 1 83.0 Comparative Example 2 85.2
[0048] As can be seen from Table 1, the survival rate of donkey mammary epithelial cells cultured by the process of Example 1 is the highest.
[0049] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.
Claims
1. A method for culturing donkey mammary epithelial cells in vitro based on a specific enzyme solution and a culture medium, characterized in that: The steps include: S1. Mammary gland tissue of healthy donkeys was collected and cleaned and disinfected. During the cleaning process, sterile saline was used for preliminary washing to remove surface impurities, and then a specific cleaning solution was used for secondary washing. The cleaning solution contained antibacterial ingredients and cell protectants, which could effectively kill possible bacteria and fungi while reducing damage to cells. The disinfection treatment used a composite solution containing antibiotics for immersion disinfection to ensure the sterility of the tissue. S2. Digesting the breast tissue with a specific enzyme solution to obtain a single cell suspension, wherein the enzyme solution is a mixed solution of collagenase, trypsin and hyaluronidase, and the ratio of the three enzymes is determined through a large number of experiments, which can efficiently digest the breast tissue in the shortest time while minimizing damage to the cells; S3, inoculating the single cell suspension into a culture container, adding a specific cell culture medium for culture, the cell culture medium contains growth factors and nutrients in a specific ratio, the growth factors include epidermal growth factor and insulin-like growth factor, and the nutrients are carefully screened and optimized to provide a suitable growth environment for the cells; S4. Regularly observe the cell growth status and perform medium change operations. During the medium change process, use special medium change devices and operation methods to avoid mechanical damage to the cells and ensure that the new culture medium can evenly cover the cells.
2. The method for in vitro culture of donkey mammary epithelial cells based on a specific enzyme solution and a culture medium according to claim 1, characterized in that: The antibacterial components in the cleaning solution include penicillin, streptomycin and gentamicin, and the cell protectant is glutathione or vitamin C.
3. The in vitro culture method of donkey mammary epithelial cells based on specific enzyme solution and culture medium according to claim 1, characterized in that: The ratio of collagenase, trypsin and hyaluronidase in the enzyme solution is 1:0.4-0.6:0.8-1.
2.
4. The in vitro culture method of donkey mammary epithelial cells based on specific enzyme solution and culture medium according to claim 1, characterized in that: The growth factors in the cell culture medium are epidermal growth factor and insulin-like growth factor in a mass ratio of 1:1, and the nutrients are amino acids, inorganic salts, and vitamins. The amino acids are complex amino acids composed of twenty kinds of amino acids required by the human body, the inorganic salts are sodium chloride, potassium dihydrogen phosphate, ferrous sulfate, magnesium sulfate, and calcium sulfate in a mass ratio of 1:0.1-0.3:0.1-0.3:0.1-0.3:0.1-0.3, and the vitamin is vitamin C; the mass ratio of the epidermal growth factor, amino acids, inorganic salts, and vitamins is 1:2-4:0.6-0.8:0.6-0.
8.
5. The in vitro culture method of donkey mammary epithelial cells based on specific enzyme solution and culture medium according to claim 1, characterized in that: The liquid replacement device is a pipette with a fine filter, and the operation method includes slowly aspirating the old culture medium and adding the new culture medium at a uniform speed.
6. A donkey mammary epithelial cell cultured by the in vitro culture method of donkey mammary epithelial cells based on a specific enzyme solution and a culture medium as claimed in claim 1, characterized in that: The cultured cells were identified as donkey mammary epithelial cells using specific identification methods, including morphological observation, immunocytochemical staining and RT-PCR detection. Morphological observation involves observing the shape, size and arrangement of the cells under a microscope. Immunocytochemical staining uses specific antibodies to stain the cells and detect the expression of epithelial cell markers. RT-PCR detection determines the cell type by detecting the expression of epithelial cell-specific genes.