A culture medium for forest musk gland cells and a method for culturing forest musk gland cells

Abstract

The application relates to the technical field of cell culture, and particularly discloses a culture medium for musk deer musk gland cells and a culture method for musk deer musk gland cells. The culture medium for the musk deer musk gland cells comprises a DMEM / F12 culture medium and the following components: 80-120 U / mL penicillin, 0.08-0.12 mg / mL streptomycin, 8-12% fetal bovine serum, 40-60 ng / mL epidermal growth factor, 80-120 ng / mL fibroblast growth factor, 0.06-0.15 ug / mL hydrocortisone, 0.4-0.6 uM / mL TGF-beta inhibitor, 1.6-2.4 mM / mL glutamine and 3.5-6 mM / mL acetyl-L-cysteine. The application further provides a culture method for the musk deer musk gland cells, which comprises the following steps: digestion, cell preparation, primary culture and subculture. The culture medium for the musk deer musk gland cells is used to culture the musk deer musk gland cells, the subculture effect of the musk deer musk gland cells is good, the proliferation capacity of the musk deer musk gland cells is strong, the morphological change of the musk deer musk gland cells is small, and the musk deer musk gland cells can be well kept dry during the subculture.

Description

Technical Field

[0001] This application relates to the field of cell culture technology, specifically to a culture medium for forest musk gland cells and a method for culturing forest musk gland cells. Background Technology

[0002] The forest musk deer is a small mammal belonging to the Musk Deer family, native to Asia. It typically inhabits forests or mountainous areas, feeding on grasses, leaves, fruits, and insects. Research has found that the musk gland epithelial cells in male forest musk deer can secrete musk, a substance known for its properties of refreshing the mind, promoting blood circulation, reducing swelling, and relieving pain. Therefore, it is often used in the production of perfumes and medicines. However, the yield of natural musk is extremely low; each male forest musk deer produces only about 15 grams per year, making it exceptionally precious.

[0003] To meet market demand for musk, researchers are actively seeking methods to obtain musk through in vitro cell culture. However, when isolating and culturing forest musk gland epithelial cells using culture media prepared with existing technologies, problems arise such as poor cell growth, undirected cell differentiation during subculturing, and inability to maintain stemness effectively. These issues severely hinder research on the biomolecules of forest musk gland cells and the application of musk. Summary of the Invention

[0004] To overcome the problems of poor growth, easy cell differentiation, and inability to maintain stemness in the culture of forest musk gland epithelial cells, this application provides a culture medium and a method for culturing forest musk gland cells.

[0005] In a first aspect, this application provides a culture medium for forest musk gland cells, employing the following technical solution:

[0006] A culture medium for forest musk gland cells, comprising DMEM / F12 medium and further comprising the following components in concentrations: 80-120 U / mL penicillin, 0.08-0.12 mg / mL streptomycin, 8-12% fetal bovine serum, 40-60 ng / mL epidermal growth factor, 80-120 ng / mL fibroblast growth factor, 0.06-0.15 μg / mL hydrocortisone, 0.4-0.6 μM / mL TGF-β inhibitor, 1.6-2.4 mM / mL glutamine, and 3.5-6 mM / mL acetyl-L-cysteine.

[0007] This application provides a culture medium for forest musk gland cells. This medium is based on DMEM / F12 medium, but with the addition of epidermal growth factor, fibroblast growth factor, hydrocortisone, and other components, resulting in a suitable medium for culturing forest musk gland cells. When forest musk gland cells are passaged using this medium, the cells exhibit good passage efficiency, strong proliferation capacity, minimal morphological changes, and excellent retention of stemness.

[0008] In this application, fibroblast growth factor (GF) is a growth factor that regulates cell growth, proliferation, and differentiation, and can participate in maintaining cell stemness by regulating cell growth through multiple signaling pathways. Epidermal growth factor (EGF) can promote the growth, proliferation, and directional differentiation of musk gland cells. Hydrocortisone is a glucocorticoid extracted from the adrenal cortex. Hydrocortisone can promote lipid synthesis in musk cells and maintain the tightness of cell connections. Experimental studies in this application have found that both EGF and fibroblast growth factor have a certain promoting effect on the growth and directional differentiation of musk gland cells, while hydrocortisone has a smaller promoting effect on the proliferation of musk gland cells and a certain inhibitory effect on their differentiation. However, experimental studies in this application have found that when hydrocortisone is used in combination with EGF and fibroblast growth factor, the growth and proliferation of musk gland cells are more significant, while cell differentiation is less during passage culture, indicating a synergistic effect between hydrocortisone and EGF / fibroblast growth factor. Therefore, the culture medium prepared by this application using epidermal growth factor, fibroblast growth factor and hydrocortisone can maintain the good growth status and stemness of forest musk gland epithelial cells during culture, reduce cell differentiation of forest musk gland cells during passage, and ultimately promote the growth and proliferation of forest musk gland cells.

[0009] Preferably, the culture medium comprises DMEM / F12 medium, and further comprises the following components in the following amounts: 80-120 U / mL penicillin, 0.08-0.12 mg / mL streptomycin, 8-12% fetal bovine serum, 40-60 ng / mL epidermal growth factor, 90-110 ng / mL fibroblast growth factor, 0.08-0.12 μg / mL hydrocortisone, 0.4-0.6 μM / mL TGF-β inhibitor, 1.6-2.4 mM / mL glutamine, and 3.5-6 mM / mL acetyl-L-cysteine.

[0010] In some specific implementations, the content of the fibroblast growth factor is 80 ng / mL, 90 ng / mL, 100 ng / mL, 110 ng / mL, or 120 ng / mL.

[0011] In some specific embodiments, the hydrocortisone content is 0.06 μg / mL, 0.08 μg / mL, 0.1 μg / mL, 0.12 μg / mL, or 0.15 μg / mL.

[0012] Preferably, the culture medium comprises DMEM / F12 medium, and further comprises the following components in the following amounts: 100 U / mL penicillin, 0.1 mg / mL streptomycin, 10% fetal bovine serum, 50 ng / mL epidermal growth factor, 100 ng / mL fibroblast growth factor, 0.1 μg / mL hydrocortisone, 0.5 μM / mL TGF-β inhibitor, 2 mM / mL glutamine, and 5 mM / mL acetyl-L-cysteine.

[0013] In this application, the fibroblast growth factor is selected from FGF2, FGF8, FGF17 and FGF18.

[0014] Preferably, the fibroblast growth factor is a mixture of FGF2 and FGF8; the weight ratio of FGF2 to FGF8 is 1:(0.3-0.7).

[0015] In this application, FGF2 can maintain the stemness of neural stem cells, while FGF8, FGF17, and FGF18 can all promote cell growth, proliferation, and passage. Through experimental research, this application found that using a mixture of FGF2 and FGF8 at a weight ratio of 1:(0.3-0.7) as fibroblast growth factor resulted in a culture medium more suitable for the passage of *Muscaria lucida* gland cells, thus enhancing their proliferative capacity.

[0016] Secondly, the application of the culture medium for forest musk gland cells provided in this application in the culture of forest musk gland cells.

[0017] Thirdly, this application provides a method for culturing forest musk gland cells, employing the following technical solution:

[0018] A method for culturing forest musk gland cells includes the following steps: digestion, cell preparation, primary culture, and subculture; wherein the primary culture and subculture use a culture medium for forest musk gland cells.

[0019] Preferably, the digestion step is as follows: the sac gland tissue sample is cut into fragments and then digested with type I collagenase at 37°C for 4-5 hours.

[0020] In this application, collagenase is a protease capable of degrading natural collagen and reticular fibers, and also effectively hydrolyzing other proteins in the extracellular matrix of connective tissue and epithelial tissue. Through experimental research, this application has found that using type I collagenase to digest forest musk gland tissue can break down the collagen in the forest musk gland tissue into smaller molecules. These smaller molecules can be fully absorbed and utilized by forest musk gland cells, thereby promoting the growth and metabolism of forest musk gland cells.

[0021] Preferably, the cell preparation step is as follows: DMEM / F12 medium is added to the material obtained in the digestion step to terminate the digestion; the DMEM / F12 medium contains 10% fetal bovine serum, 2mM glutamine and 100IU / mL penicillin-streptomycin.

[0022] In summary, this application has the following beneficial effects:

[0023] 1. The present application provides a culture medium for forest musk gland cells, which contains epidermal growth factor, fibroblast growth factor, hydrocortisone and other components. When forest musk gland cells are cultured using this culture medium, the cells have good passage effect, strong proliferation ability, small morphological changes, and can maintain good cell stemness.

[0024] 2. This application further controls the content of fibroblast growth factor in the culture medium to be between 90-110 ng / mL and the content of hydrocortisone to be between 0.06-0.12 μg / mL. After culturing forest musk gland cells for 4 days, the cell adhesion rate is above 85% and the cell number is 2.5 × 10⁻⁶. 6 More than one hole per hole.

[0025] 3. This application further selects FGF2 and FGF8 in a weight ratio of 1:(0.3-1) as fibroblast growth factors, and the resulting culture medium has a more significant promoting effect on the growth and proliferation of forest musk gland cells; after culturing forest musk gland cells for 4 days, the cell adhesion rate is over 90%, and the cell number is 3.4×10⁻⁶. 6 One or more holes. Attached Figure Description

[0026] Figure 1 This is a schematic diagram of the forest musk gland cells after 4 days of growth under a 10x microscope in Example 3.

[0027] Figure 2 This is a schematic diagram of the forest musk gland cells in Comparative Example 1 after 4 days of growth under a ×10 (magnification) microscope.

[0028] Figure 3 This is a schematic diagram of the forest musk gland cells in Comparative Example 2 after 4 days of growth under a 10x microscope.

[0029] Figure 4 This is a schematic diagram of the forest musk gland cells in Comparative Example 3 after 4 days of growth under a 10x microscope.

[0030] Figure 5 This is a schematic diagram of the forest musk gland cells in Comparative Example 4 after 4 days of growth under a 10x microscope.

[0031] Figure 6 This is a schematic diagram of forest musk gland cells in Comparative Example 9 after 4 days of growth under a 10x microscope.

[0032] Figure 7 This is a schematic diagram of forest musk gland cells in Comparative Example 10 after 4 days of growth under a 10x microscope.

[0033] Figure 8 This is a schematic diagram of the forest musk gland cells in Comparative Example 11 after 4 days of growth under a 10x microscope.

[0034] Figure 9 This is a schematic diagram of the forest musk gland cells after Oil Red O staining under a 10x microscope in Example 3.

[0035] Figure 10 This is a schematic diagram of the forest musk gland cells in Comparative Example 1 after Oil Red O staining under a 10x microscope.

[0036] Figure 11 This is a schematic diagram of the forest musk gland cells in Comparative Example 2 after Oil Red O staining under a 10x microscope.

[0037] Figure 12 This is a schematic diagram of the forest musk gland cells in Comparative Example 3 after Oil Red O staining under a 10x microscope.

[0038] Figure 13 This is a schematic diagram of the forest musk gland cells in Comparative Example 4 after Oil Red O staining under a 10x microscope.

[0039] Figure 14 This is a schematic diagram of the forest musk gland cells in Comparative Example 9 after Oil Red O staining under a 10x microscope.

[0040] Figure 15 This is a schematic diagram of the forest musk gland cells in Comparative Example 10 after Oil Red O staining under a 10x microscope.

[0041] Figure 16 This is a schematic diagram of the forest musk gland cells in Comparative Example 11 after Oil Red O staining under a 10x microscope. Detailed Implementation

[0042] This application provides a method for culturing forest musk gland cells, including the following steps:

[0043] (1) Sample collection: After anesthetizing the musk deer with tledamine hydrochloride and zolpidem hydrochloride, the musk deer was made to lie flat; then the skin above the musk gland was cut open with ophthalmic scissors to obtain gland tissue; a small piece of musk gland tissue was cut off and immediately transferred to phosphate-buffered saline (PBS) containing 100 U / mL penicillin and 100 μg / mL streptomycin to wash it and remove the outer membrane and connective tissue.

[0044] (2) Digestion: Cut the sac gland tissue sample into fragments about 1 mm long and wide using ophthalmic scissors, and then digest it with type I collagenase at 37°C for 4-5 hours.

[0045] (3) Cell preparation: Digestion was terminated using DMEM / F12 medium. The digestion solution was filtered and centrifuged at 600 r / min for 3 min. The supernatant was discarded, and the cells were resuspended in complete medium. The cells were washed three times and the dispersed cells were collected. The DMEM / F12 medium contained 10% fetal bovine serum, 2 mM glutamine, and 100 IU / mL penicillin-streptomycin.

[0046] (4) Primary culture: Adjust the cell density to 1×10⁶ 4 ~5×10 4 Cells were cultured at 100 cells / mL and then seeded into 25mL cell culture flasks. The flasks were incubated at 37°C and 5% CO2 with saturated humidity. Once the cells adhered, the culture medium was replaced with fresh complete medium. The medium was then replaced every 24 hours.

[0047] (5) Subculture: When the cell growth density reaches about 70%, use complete culture medium (culture medium for forest musk gland cells) for subculture, and change the culture medium every 48 hours.

[0048] In the above culture method, the complete culture medium is the culture medium for forest musk gland cells. This culture medium is based on DMEM / F12 medium and contains 80-120 U / mL penicillin, 0.08-0.12 mg / mL streptomycin, 8-12% fetal bovine serum, 40-60 ng / mL epidermal growth factor, 80-120 ng / mL fibroblast growth factor, 0.06-0.15 μg / mL hydrocortisone, 0.4-0.6 μM / mL TGF-β inhibitor, 1.6-2.4 mM / mL glutamine, and 3.5-6 mM / mL acetyl-L-cysteine.

[0049] In this application, DMEM / F12 culture medium was purchased from Beijing Solarbio Science & Technology Co., Ltd.; the CAS number of epidermal growth factor was 62229-50-9, purchased from Qiyuan (Guangdong) Pharmaceutical & Chemical Co., Ltd.; fibroblast growth factors included FGF2, FGF8, FGF17, and FGF18, all of which were purchased from Shanghai Qiyuan Biotechnology Co., Ltd., with FGF2 catalog number 4038-10 and FGF17 catalog number 7150-10. The product code for FGF18 is 4082-1000; the product code for FGF8 is YKW-11597, purchased from Shanghai Youkewei Biotechnology Co., Ltd.; fetal bovine serum, TGF-β inhibitor, and hydrocortisone were all purchased from MCE, with the product code for fetal bovine serum being HY-T1000, the product code for TGF-β inhibitor (A83-01) being HY-10432, and the product code for hydrocortisone being HY-B0983; the remaining raw materials, reagents, solvents, etc. of this application can all be obtained commercially.

[0050] The present application will be further described in detail below with reference to embodiments, performance testing tests and accompanying drawings.

[0051] Examples 1-5

[0052] Examples 1-5 provide a method for culturing forest musk gland cells, the difference being that the amount of fibroblast growth factor used in the culture medium for forest musk gland cells is as shown in Table 1.

[0053] The above-mentioned method for culturing forest musk gland cells includes the following steps:

[0054] (1) Sample collection: After anesthetizing the musk deer with tledamine hydrochloride and zolpidem hydrochloride, the musk deer was made to lie flat; then the skin above the musk gland was cut open with ophthalmic scissors to obtain gland tissue; a small piece of musk gland tissue was cut off and immediately transferred to phosphate-buffered saline (PBS) containing 100 U / mL penicillin and 100 μg / mL streptomycin to wash it and remove the outer membrane and connective tissue.

[0055] (2) Digestion: The sac gland tissue sample was cut into fragments about 1 mm long and wide using ophthalmic scissors, and then digested with type I collagenase at 37°C for 5 hours.

[0056] (3) Cell preparation: Digestion was terminated using DMEM / F12 medium. The digestion solution was filtered and centrifuged at 600 r / min for 3 min. The supernatant was discarded, and the cells were resuspended in complete medium. The cells were washed three times and the dispersed cells were collected. The DMEM / F12 medium contained 10% fetal bovine serum, 2 mM glutamine, and 100 IU / mL penicillin-streptomycin.

[0057] (4) Primary culture: Adjust the cell density to 1×10⁶4 ~5×10 4 Cells / mL were seeded into 25mL cell culture flasks and cultured at 37℃ with 5% CO2 and saturated humidity. After the cells adhered, the culture medium (the medium for forest musk gland cells) was replaced, and then the culture medium was replaced every 24 hours.

[0058] (5) Subculture: When the cell growth density reaches about 70%, use complete culture medium (culture medium for forest musk gland cells) for subculture, and change the culture medium every 48 hours.

[0059] Table 1. Components and contents of the culture medium for forest musk gland cells in Examples 1-5

[0060]

[0061]

[0062] Examples 6-9

[0063] Examples 6-9 were carried out according to the method of Example 3, except that the amount of hydrocortisone used in the culture medium for the forest musk gland cells was as shown in Table 2.

[0064] Table 2 shows the hydrocortisone content in the culture medium of Examples 3 and 6-9.

[0065] Example Hydrocortisone (μg / mL) 3 0.1 6 0.06 7 0.08 8 0.12 9 0.15

[0066] Examples 10-17

[0067] Examples 10-17 were carried out according to the method of Example 3, except that the fibroblast growth factor components in the culture medium for forest musk gland cells were as shown in Table 3.

[0068] Components of fibroblast growth factor in the culture medium of Examples 3 and 10-17

[0069]

[0070] Comparative Examples 1-4

[0071] Comparative Examples 1-4 were performed according to the method of Example 3, except that the content of each component in the culture medium for the forest musk gland cells was as shown in Table 4.

[0072] Table 4. Components and contents of the culture medium for forest musk gland cells in Example 3 and Comparative Examples 1-4

[0073]

[0074]

[0075] Comparative Examples 5-8

[0076] Comparative Examples 5-8 were performed according to the method of Example 3, except that the amount of hydrocortisone used in the culture medium for the forest musk gland cells was as shown in Table 5.

[0077] Table 5. Hydrocortisone content in the culture media of Examples 3 and Comparative Examples 5-8

[0078] / Hydrocortisone (μg / mL) Example 3 0.1 Comparative Example 5 0.01 Comparative Example 6 0.03 Comparative Example 7 0.20 Comparative Example 8 0.25

[0079] Comparative Example 9

[0080] Comparative Example 9 was carried out according to the method of Example 3, except that the composition and content of the culture medium for the forest musk gland cells were different.

[0081] The culture medium for the forest musk gland cells in Comparative Example 9 was DEME / F12 basal medium containing 10% fetal bovine serum, 100 U / mL penicillin, 0.1 mg / mL streptomycin, 50 ng / mL epidermal growth factor, 10 mM / mL insulin, 2 mM / mL GlutaMAX dipeptide, 5 μg / mL transferrin, 5 μg / mL T3 triiodothyronine, and 400 ng / mL hydrcortisone.

[0082] Comparative Example 10

[0083] Comparative Example 10 was carried out according to the method of Example 3, except that the composition and content of the culture medium for the forest musk gland cells were different.

[0084] The culture medium for the forest musk gland cells in Comparative Example 10 was Ham's F12 basal medium containing 20% ​​fetal bovine serum, 1% penicillin-streptomycin, 10 ng / mL epidermal growth factor, 2 mM glutamine, and 0.1 mM β-mercaptoethanol.

[0085] Comparative Example 11

[0086] Comparative Example 11 was carried out according to the method of Example 3, except that the composition and content of the culture medium for the forest musk gland cells were different.

[0087] The culture medium for the forest musk gland cells in Comparative Example 11 was DMEM-based medium containing 10% fetal bovine serum, 1% penicillin-streptomycin, and 10 ng / mL EPC factor.

[0088] Cell stemness test

[0089] (I) Morphological identification

[0090] Forest musk gland cells were cultured using the methods of Examples 1-17, Comparative Examples 1-4, and Comparative Examples 9-11. After 4 days of subculturing, the morphology and structure of the cells were observed under an optical microscope.

[0091] Observations revealed that during the growth of the forest musk gland cells in Examples 1-17, they appeared as spindle-shaped or irregular triangles on the surface of the support, with an oval nucleus in the center of the cell and protrusions of varying lengths extending outward from the cytoplasm. After passage, the cells were arranged in a homogeneous, whorled pattern.

[0092] A schematic diagram of the forest musk gland cells after 4 days of growth under a 10× microscope in Example 3 is shown below. Figure 1 As shown.

[0093] A schematic diagram of forest musk gland cells from Comparative Example 1 after 4 days of growth under a 10× microscope is shown below. Figure 2 As shown.

[0094] A schematic diagram of forest musk gland cells from Comparative Example 2 after 4 days of growth under a 10× microscope is shown below. Figure 3 As shown.

[0095] A schematic diagram of forest musk gland cells from Comparative Example 3 after 4 days of growth under a 10× microscope is shown below. Figure 4 As shown.

[0096] A schematic diagram of forest musk gland cells from Comparative Example 4 after 4 days of growth under a 10× microscope is shown below. Figure 5 As shown.

[0097] A schematic diagram of forest musk gland cells from Comparative Example 9 after 4 days of growth under a 10× microscope is shown below. Figure 6 As shown.

[0098] A schematic diagram of forest musk gland cells (Comparative Example 10) after 4 days of growth under a 10× microscope is shown below. Figure 7 As shown.

[0099] A schematic diagram of forest musk gland cells from Comparative Example 11 after 4 days of growth under a 10× microscope is shown below. Figure 8 As shown.

[0100] (II) Oil Red O Staining Method

[0101] Forest musk gland cells were cultured using the methods described in Examples 1-17, Comparative Examples 1-4, and Comparative Examples 9-11. After 4 days of subculturing, the adipogenic differentiation potential of each forest musk gland cell was identified using an Oil Red O staining kit (cell-specific, purchased from Beijing Solarbio Science & Technology Co., Ltd.).

[0102] After being stained with Oil Red O, the forest musk gland cells cultured by the methods provided in Examples 1-17 were observed under a microscope and found to contain red oil, indicating that the above cell culture method can effectively maintain the stemness of forest musk gland cells.

[0103] Example 3 shows a schematic diagram of the forest musk gland cells cultured using the method described in Example 3, after being stained with Oil Red O, under a microscope. Figure 9 As shown. By Figure 9 It can be seen that the cell density and adhesion rate in Example 3 are high, and a large amount of lipid substances are clearly found.

[0104] A schematic diagram of the forest musk gland cells cultured using the method provided in Comparative Example 1, after being stained with Oil Red O, is shown under a microscope. Figure 10 As shown. By Figure 10 It can be seen that the cell density, cell activity and adhesion rate of Comparative Example 1 were all low, and only a small amount of lipid substances were found after staining.

[0105] A schematic diagram of the forest musk gland cells cultured using the method provided in Comparative Example 2, after being stained with Oil Red O, is shown under a microscope. Figure 11 As shown. By Figure 11 It can be seen that the cells in Comparative Example 2 had lower density and adhesion rate, and staining revealed only a small amount of lipid substances.

[0106] A schematic diagram of the forest musk gland cells cultured using the method provided in Comparative Example 3, after being stained with Oil Red O, is shown under a microscope. Figure 12 As shown. By Figure 12 It can be seen that the cells in Comparative Example 3 have higher density, activity, and adhesion rate, and contain more lipid substances after staining.

[0107] A schematic diagram of the forest musk gland cells cultured using the method provided in Comparative Example 4, after being stained with Oil Red O, is shown under a microscope. Figure 13 As shown. By Figure 13 It can be seen that the cells in Comparative Example 4 grew very slowly, had weak cell activity, and were difficult to stain.

[0108] A schematic diagram of the forest musk gland cells cultured using the method provided in Comparative Example 9, stained with Oil Red O, under a microscope is shown below. Figure 14 As shown. By Figure 14 As can be seen, under a microscope, Comparative Example 9 showed lower cell density and adhesion rate, indicating a relatively poor culture effect. Staining revealed only a small amount of lipid substances.

[0109] A schematic diagram of the forest musk gland cells cultured using the method provided in Comparative Example 10, stained with Oil Red O, under a microscope is shown below. Figure 15 As shown. By Figure 15 It can be seen that the cell density of Comparative Example 10 is low and the staining effect is poor.

[0110] A schematic diagram of the forest musk gland cells cultured using the method provided in Comparative Example 11, stained with Oil Red O, under a microscope is shown below. Figure 16 As shown. By Figure 16 It can be seen that the cells in Comparative Example 11 have low cell density and poor staining effect.

[0111] Performance testing

[0112] The culture methods for forest musk gland cells provided in Examples 1-17 and Comparative Examples 1-11 were tested, and the results are shown in Table 6. The specific testing process is as follows:

[0113] (1) Adhesion rate: Forest musk gland cells were cultured according to the methods of Examples 1-17 and Comparative Examples 1-11. On the 4th day of culture, the growth status of the cells was observed under a microscope to estimate the cell adhesion rate.

[0114] (2) Cell growth and counting: Cells were seeded in 6-well plates (1×10⁶ cells / wells). 4 Cells were seeded in 6-well plates (1×10⁶ cells / well) and cultured for 24 hours according to the methods of Examples 1-17 and Comparative Examples 1-11, respectively. Cell counts were then performed under a microscope using a hemocytometer to assess cell proliferation. (Known information: the hemocytometer squares are 1mm × 1mm, the culture dish size is 35mm, and the culture medium volume is 2ml.) (3) Cell growth rate: Cells were seeded in 6-well plates (1×10⁶ cells / well). 4 Cells were cultured in wells (cells / well) for 24 hours according to the methods of Examples 1-17 and Comparative Examples 1-11, respectively. The OD values ​​before and after 24 hours of cell culture were measured using an OD value analyzer, and the cell growth rate was calculated according to the following cell growth rate formula. Note: The initial OD value before cell culture was used as the OD value of the control group, and the OD value after 24 hours of cell culture was used as the OD value of the experimental group.

[0115] Cell growth rate = (OD value of experimental group - OD value of control group) / OD value of control group

[0116] Table 6. Results of cell adhesion rate and cell proliferation in Examples 1-17 and Comparative Examples 1-11

[0117]

[0118]

[0119] Observations on the growth of musk deer gland cells obtained in Examples 1-17 of this application revealed that these cells exhibited rapid growth, strong cell activity, high adhesion rate, and good passage efficiency. After 4 days of detailed cell culture using the methods described in Examples 1-17 of this application, the adhesion rate of the musk deer gland cells was ≥70%; after 24 hours of cell culture, the cell number increased from 1×10⁶ cells / year. 4The number of holes / well increased to (1.5-4.8)×10 6 Cells per well, with a cell growth rate of 70-86%.

[0120] In Comparative Example 1, observation of cell growth revealed that the forest musk gland cells exhibited slow growth, weak activity, and low adhesion rate. Cell count and proliferation rate measurements showed that, using the culture method in Comparative Example 1, after 4 days of culture, the forest musk gland cells achieved an adhesion rate of only 41%, and after 24 hours of culture, the cell count was only 5.0 × 10⁻⁶. 5 Cells per well, cell growth rate only 38%.

[0121] In Comparative Example 2, observation of cell growth revealed that the forest musk gland cells exhibited slow growth, weak activity, and low adhesion rate. Cell count and proliferation rate measurements showed that, using the culture method in Comparative Example 2, after 4 days of culture, the forest musk gland cells achieved an adhesion rate of only 44%, and after 24 hours of culture, the cell count was only 6.1 × 10⁻⁶. 5 Cells per well, cell growth rate 39%.

[0122] In Comparative Example 3, observation of cell growth revealed that the forest musk gland cells exhibited rapid growth, high activity, and a high adhesion rate. Cell count and proliferation rate measurements showed that, using the culture method in Comparative Example 3, after 4 days of culture, the forest musk gland cells achieved a 49% adhesion rate, and after 24 hours of culture, the cell count was only 7.5 × 10⁻⁶. 5 Cells per well, cell growth rate of 40%.

[0123] In Comparative Example 4, observation of cell growth revealed that the growth rate of *Musk deer* gland cells was extremely slow. Cell count and proliferation rate measurements showed that, using the culture method in Comparative Example 4, after 4 days of cell culture, the adhesion rate of *Musk deer* gland cells was only 13%, and after 24 hours of culture, the cell count was only 7 × 10⁻⁶. 4 Cells per well, cell growth rate 11%.

[0124] In Comparative Example 9, observation of cell growth revealed that the forest musk gland cells exhibited slow growth, weak activity, and low adhesion rate. Cell count and proliferation rate measurements showed that, using the culture method of Comparative Example 9, after 4 days of culture, the forest musk gland cells achieved a 45% adhesion rate, and after 24 hours of culture, the cell count was only 5.2 × 10⁻⁶. 5 Cells per well, cell growth rate 38%.

[0125] In Comparative Example 10, observation of cell growth revealed that the forest musk gland cells exhibited slow growth, weak activity, and low adhesion rate. Cell count and proliferation rate measurements showed that, using the culture method in Comparative Example 10, after 4 days of culture, the forest musk gland cells achieved a adhesion rate of only 38%, and after 24 hours of culture, the cell count was only 1.8 × 10⁻⁶. 5 Cells per well, cell growth rate of 25%.

[0126] In Comparative Example 11, observation of cell growth revealed that the forest musk gland cells exhibited slow growth, weak activity, and low adhesion rate. Cell count and proliferation rate measurements showed that, using the culture method of Comparative Example 10, after 4 days of culture, the forest musk gland cells achieved a adhesion rate of only 37%, and after 24 hours of culture, the cell count was only 1.4 × 10⁻⁶. 5 Cells per well, cell growth rate 23%.

[0127] Based on the cell growth count and cell proliferation rate detection results of Examples 1-5, it was found that with the increase of fibroblast growth factor addition, the proliferation of forest musk gland cells showed a trend of first accelerating and then slowing down. Further comparison revealed that Examples 2-4, which further controlled the fibroblast growth factor content between 90-110 ng / mL, achieved a cell adhesion rate of over 85%, and after 24 hours of culture, the cell number was 2.5 × 10⁻⁶. 6 The number of cells per well is above 90-110 ng / mL. Therefore, this indicates that by controlling the fibroblast growth factor content within the range of 90-110 ng / mL, the culture medium prepared has a more significant effect on promoting the growth of forest musk gland cells and maintaining cell stemness.

[0128] Based on the cell growth count and cell proliferation rate detection results of Examples 3, 6-9, and Comparative Examples 5-8, it can be seen that the cell adhesion rate obtained in Comparative Examples 5-8 is below 50%, and the cell number is 1.5 × 10⁻⁶ after 24 hours of culture. 6 The cell count was less than 10 cells / well; while the cell adhesion rate obtained in Examples 3 and 6-9 was over 80%, and the cell number was 1.5 × 10⁻⁶ after 24 hours of culture. 6 Cells per well or higher were observed, and the proliferation of forest musk gland cells initially accelerated and then slowed down with increasing hydrocortisone content. Further comparison revealed that in Examples 3 and 7-8, where the hydrocortisone content was controlled between 0.06-0.12 μg / mL, the cell adhesion rate was above 85%, and the cell number reached 2.5 × 10⁶ cells after 24 hours of culture. 6 The number of cells per well is above 0.06-0.12 μg / mL. Therefore, it is shown that by controlling the hydrocortisone content within the range of 0.06-0.12 μg / mL, the culture medium prepared has a more significant effect on promoting the growth of forest musk gland cells and maintaining cell stemness.

[0129] According to the test results of Examples 3 and 10-17, the use of FGF2 and FGF8 in a weight ratio of 1:(0.3-1) as fibroblast growth factors in the culture medium of Examples 12-15 resulted in a cell adhesion rate ≥90% and a cell number as high as (3.4-4.8)×10⁻⁶. 6 Cells / well, cell growth rate >60%. Therefore, it is evident that the use of FGF2 and FGF8 in a weight ratio of 1:(0.3-1) as fibroblast growth factors in this application results in a culture medium with better culture effect on forest musk gland cells and better cell passage effect.

[0130] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A culture medium for musk deer scent gland cells, characterized by comprising: The culture medium comprises DMEM / F12 medium, and further comprises the following components: 80-120 U / mL penicillin, 0.08-0.12 mg / mL streptomycin, 8-12% fetal bovine serum, 40-60 ng / mL epidermal growth factor, 80-120 ng / mL fibroblast growth factor, 0.06-0.15 μg / mL hydrocortisone, 0.4-0.6 uM / mL TGF-β inhibitor, 1.6-2.4 mM / mL glutamine and 3.5-6 mM / mL acetyl-L-cysteine; The fibroblast growth factor is selected from FGF2, FGF8, FGF17 and FGF18.

2. The culture medium of musk deer scent gland cells according to claim 1, characterized in that, The culture medium comprises DMEM / F12 medium, and further comprises the following components: 80-120 U / mL penicillin, 0.08-0.12 mg / mL streptomycin, 8-12% fetal bovine serum, 40-60 ng / mL epidermal growth factor, 80-120 ng / mL fibroblast growth factor, 0.06-0.15 μg / mL hydrocortisone, 0.4-0.6 uM / mL TGF-β inhibitor, 1.6-2.4 mM / mL glutamine and 3.5-6 mM / mL acetyl-L-cysteine.

3. The culture medium of musk deer scent gland cells according to claim 1, characterized in that, The culture medium comprises DMEM / F12 medium, and further comprises the following components: 80-120 U / mL penicillin, 0.08-0.12 mg / mL streptomycin, 8-12% fetal bovine serum, 40-60 ng / mL epidermal growth factor, 80-120 ng / mL fibroblast growth factor, 0.06-0.15 μg / mL hydrocortisone, 0.4-0.6 uM / mL TGF-β inhibitor, 1.6-2.4 mM / mL glutamine and 3.5-6 mM / mL acetyl-L-cysteine.

4. The culture medium of musk deer scent gland cells according to claim 1, wherein, The fibroblast growth factor is a mixture of FGF2 and FGF8; the weight ratio of FGF2 to FGF8 is 1: (0.3-0.7).

5. Use of the culture medium of the musk deer scent gland cells according to any one of claims 1-4 in the culture of musk deer scent gland cells.

6. A culture method of musk deer scent gland cells, characterized by, The method comprises the following steps: Digestion, cell preparation, primary culture and subculture; the primary culture and subculture use the culture medium of the musk deer scent gland cells according to any one of claims 1-4.

7. The culture method of the musk deer scent gland cells according to claim 6, wherein, The digestion step is to cut the sachet gland tissue sample into fragments, and then digest at 37°C for 4-5 h using collagenase type I.

8. The culture method of the musk deer scent gland cells according to claim 7, wherein, The cell preparation step is to terminate the digestion by adding DMEM / F12 medium to the material obtained in the digestion step; the DMEM / F12 medium contains 10% fetal bovine serum, 2 mM glutamine and 100 IU / mL penicillin-streptomycin.

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