A culture medium for forest musk gland cells, its preparation method and application

Abstract

This application relates to the technical field of cell culture, specifically disclosing a culture medium for forest musk gland cells, its preparation method, and its application. The culture medium disclosed in this application uses DMEM / F12 as the basal medium and contains the following components as functional additives at various concentrations: penicillin 80-120 U / ml, streptomycin 0.08-0.12 mg / ml, FBS 8-12 v / v%, non-essential amino acids 0.7-1.3 v / v%, growth factors 20-40 ng / ml, and vitamins 0.05-0.2 μg / ml. This application also discloses the preparation method of the above-mentioned culture medium and its application in the culture of forest musk gland cells. When forest musk gland cells are cultured using the culture medium disclosed in this application, the cell proliferation rate is faster, the cell activity is stronger, and the cell adhesion rate is higher.

Description

Technical Field

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

[0002] The forest musk deer is a precious animal, and its fragrance is widely used in the chemical and pharmaceutical fields, including perfumes and medicines. However, due to the scarcity of forest musk deer and the extremely low yield of musk per individual, the supply of musk resources falls short of demand.

[0003] Cell culture technology refers to a method of enabling cells to survive, grow, reproduce, and maintain their main structures and functions in vitro, simulating the in vivo environment (sterility, suitable temperature, pH, and specific nutritional conditions, etc.). Cell culture technology refers to the growth of cells under in vitro conditions; during the culture process, the cultured product consists of single cells or cell populations. Cells live in an artificial environment during culture, and due to changes in the environment, cell migration, or other factors, the culture time may be prolonged, and passaging may lead to cell homogenization. Furthermore, cell culture itself is a form of cell cloning. Cell culture technology is an important and commonly used technique in cell biology research. Through cell culture, large numbers of cells can be obtained, and molecular mechanisms such as cellular signal transduction and cellular synthesis and metabolism can be studied.

[0004] However, due to the rarity and difficulty in collecting musk gland cells, and the high requirements for cell culture media, research on musk deer using cell culture techniques is relatively limited. Furthermore, currently used cell culture techniques are not suitable for the long-term growth and reproduction of musk gland cells, severely hindering research on musk deer. Summary of the Invention

[0005] In order to solve the technical problems existing in the prior art, this application provides a musk gland cell culture medium and its preparation method and application.

[0006] This application provides a culture medium using DMEM / F12 as the basal medium;

[0007] The culture medium contains the following components as functional additives at the following concentrations: penicillin 80-120 U / ml, streptomycin 0.08-0.12 mg / ml, FBS 8-12 v / v%, non-essential amino acids 0.7-1.3 v / v%, growth factors 20-40 ng / ml, and vitamins 0.05-0.2 μg / ml; the growth factors are selected from one or more of EGF, FGF, and IL-1.

[0008] This application utilizes the aforementioned concentrations of components as functional additives. The non-essential amino acids used are Gibco products, which are commonly used non-essential amino acids that can effectively improve the cell culture medium ratio and reduce the side effects of non-essential amino acids produced by the cells themselves during cell culture. Growth factors can improve survival rate, promote cell growth and division, and also promote cell proliferation. Vitamins can maintain normal cell growth and development and promote cell proliferation. This application obtains a culture medium for culturing forest musk gland cells by adjusting the concentrations of each component, which can promote cell growth and cell adhesion.

[0009] Preferably, the growth factors are EGF and IL-1 in a weight ratio of (7-11):(1-3).

[0010] EGF, also known as human oligopeptide-1, can promote cell proliferation and differentiation and increase cell renewal rate; FGF is an important morphogenetic and differentiation inducing factor, which can promote angiogenesis, promote wound healing and tissue repair, participate in nerve regeneration, and exert its effects in vitro at low concentrations; using the above-mentioned ratio of growth factors can effectively promote the division and renewal of mesenchymal stem cells and improve the speed and quality of culture.

[0011] In one specific implementation, the weight ratio of FGF to IL-1 can be 7:1, 7:2, 7:3, 9:1, 9:2, 9:3, 11:1, 11:2, or 11:3.

[0012] In some specific implementations, the weight ratio of FGF to IL-1 can also be (7-9):1, (7-9):2, (7-9):3, (9-11):1, (9-11):2, (9-11):3, 11:(1-2), 11:(2-3), 11:(1-3).

[0013] Experimental analysis shows that the use of FGF and IL-1 in the above weight ratio as growth factors in the culture medium, and the application of this culture medium to culture forest musk gland cells, can further improve the culture effect of forest musk gland cells.

[0014] Preferably, the vitamin is selected from one or more of vitamin C, vitamin B12, and vitamin H.

[0015] Preferably, the culture medium further includes 0.001-0.04 μg / ml of trace elements.

[0016] Preferably, the trace element is selected from one or more of ferric ammonium citrate, sodium selenate, and sodium selenite.

[0017] Furthermore, the trace elements include ferric ammonium citrate and sodium selenate in a weight ratio of (2-6):(0.02-0.08).

[0018] In one specific implementation, the weight ratio of ferric ammonium citrate to sodium selenate can be 2:0.02, 2:0.05, 2:0.08, 4:0.02, 4:0.05, 4:0.08, 6:0.02, 6:0.05, or 6:0.08.

[0019] In some specific implementations, the weight ratio of ferric ammonium citrate and sodium selenate can also be (2-4):0.02, (2-4):0.05, (2-4):0.08, (4-6):0.02, (4-6):0.05, (4-6):0.08, 6:(0.02-0.05), 6:(0.05-0.08), 6:(0.02-0.08).

[0020] Experimental analysis shows that the use of ferric ammonium citrate and sodium selenate in the above-mentioned weight ratio as trace elements in the culture medium, and the application of this culture medium to culture forest musk gland cells, can further improve the culture effect of forest musk gland cells.

[0021] Secondly, this application also provides a method for preparing the above-mentioned musk gland cell culture medium, which specifically includes the following steps:

[0022] Weigh the corresponding amounts of each raw material and dissolve them completely in 950 mL of DMEM / F12 basal culture medium. Adjust the pH to 6.6-7.2, add sterile deionized water to a final volume of 1000 mL, and then filter through a 0.22 μm pore size filter membrane to obtain the culture medium. Store at 4°C for later use.

[0023] Thirdly, this application also provides the application of the above-mentioned forest musk gland cell culture medium in culturing forest musk gland cells.

[0024] Fourthly, this application provides a method for culturing forest musk gland cells, using the aforementioned culture medium, specifically including the following steps: adding (0.5-5)×10 to the culture medium... 3 Forest musk gland cells were inoculated at a density of cells / mL and then cultured at 37°C and 5% CO2 under saturated humidity.

[0025] In summary, the technical solution of this application has the following effects:

[0026] This application uses DMEM / F12 as the basal culture medium and penicillin, streptomycin, FBS, non-essential amino acids, growth factors, and vitamins as functional additives to prepare a culture medium for culturing forest musk gland cells. This medium can promote cell growth and cell adhesion, thereby obtaining forest musk gland cells with high cell density, high cell activity, and high adhesion rate.

[0027] This application further improves the culture effect of forest musk gland cells by screening the types of growth factors and vitamins and adding appropriate types and amounts of trace elements to the culture medium. Attached Figure Description

[0028] Figure 1 This is a microscopic observation (10x) of the forest musk gland cells at 0 h after inoculation in Example 7.

[0029] Figure 2 Microscopic observation (10x) of forest musk gland cells in Comparative Example 6 at 0 h after inoculation.

[0030] Figure 3 Microscopic observation of forest musk gland cells in Comparative Example 7 at 0 h after inoculation (10x).

[0031] Figure 4 This is a microscopic observation (10x) of the forest musk gland cells cultured for 4 days after inoculation in Example 7.

[0032] Figure 5 Microscopic observation (10x) of forest musk gland cells cultured for 4 days after inoculation in Comparative Example 6.

[0033] Figure 6 Microscopic observation (10x) of forest musk gland cells cultured for 4 days after inoculation in Comparative Example 7. Detailed Implementation

[0034] The present application will be further described in detail below with reference to embodiments, comparative examples and performance test results. These embodiments should not be construed as being included within the scope of protection claimed in this application.

[0035] Preparation Example

[0036] Preparation Examples 1-12

[0037] Preparation Examples 1-12 each provide a culture medium.

[0038] The differences between the above preparation examples lie in the composition and concentration of growth factors in the culture medium. See Table 1 for details.

[0039] The method for preparing the culture medium includes the following steps:

[0040] Based on a 1L culture medium, weigh each raw material according to the final concentrations of penicillin 100U / ml, streptomycin 0.1mg / ml, FBS 10v / v%, non-essential amino acids (Gibco) 1v / v%, and vitamin H 0.1μg / ml. Then, according to Table 1, weigh the corresponding weights of growth factors, fully dissolve them in 950mL of basal culture medium DMEM / F12, adjust the pH to 6.6-7.2, add sterile deionized water to a final volume of 1000mL, and then filter through a 0.22μm pore size filter membrane to obtain the culture medium, which should be stored at 4℃ for later use.

[0041] Table 1. Types and amounts of growth factors added in Preparation Examples 1-12

[0042]

[0043] Preparation Examples 13-14

[0044] Preparation Examples 13-14 each provide a culture medium.

[0045] The difference between the above preparation examples and Preparation Example 8 lies in the types of vitamins in the culture medium, specifically:

[0046] The vitamin used in the culture medium prepared in Example 13 was vitamin C;

[0047] The vitamin used in the culture medium prepared in Example 14 was vitamin B12.

[0048] Preparation Examples 15-29

[0049] Preparation Examples 15-29 each provide a culture medium.

[0050] The difference between the above preparation examples and preparation example 8 is that the culture medium also includes trace elements, and the composition and concentration of the trace elements are shown in Table 2.

[0051] Table 2 shows the composition and concentration of trace elements in preparation examples 8 and 15-29.

[0052]

[0053]

[0054] Preparation Examples 30-33

[0055] Preparation Examples 30-34 each provide a culture medium.

[0056] The specific differences between the above preparation examples and preparation example 8 are as follows:

[0057] The concentration of growth factor in the culture medium of Preparation Example 30 was 10 ng / ml;

[0058] The concentration of growth factor in the culture medium of Preparation Example 31 was 55 ng / ml;

[0059] The growth factor in the culture medium of Preparation Example 32 was PDGF.

[0060] The culture medium in Preparation Example 33 does not contain vitamins.

[0061] The culture medium in Preparation Example 34 does not contain non-essential amino acids.

[0062] Preparation Example 35

[0063] Preparation Example 35 provides a culture medium.

[0064] The preparation method of the culture medium in this example includes the following steps:

[0065] Based on a 1L culture medium, weigh out each ingredient according to the final concentrations of 1% penicillin-streptomycin, 20v / v% FBS, 10ng / mL EGF, 2mM glutamine, and 0.1mM Mβ-mercaptoethanol. Dissolve each ingredient thoroughly in 950mL of Ham's F12 basal medium and adjust the pH to 6.6-7.2. Add sterile deionized water to a final volume of 1000mL, then filter through a 0.22μm pore size filter membrane to obtain the culture medium. Store at 4℃ for later use.

[0066] Preparation Example 36

[0067] Preparation Example 36 provides a culture medium.

[0068] The preparation method of the culture medium in this example includes the following steps:

[0069] Based on a 1L culture medium, weigh out the following raw materials according to the final concentrations: bovine insulin 0.5 mg / mL, casein 0.5 mg / mL, transferrin 1.5 μg / mL, bovine insulin 15 μg / mL, linoleic acid 5 μg / mL, testosterone 400 nmol / L, vitamin A 0.15 mmol / L, L-ascorbic acid 0.05 mol / L, polylysine 0.26 mmol / L, putrescine 1 nmol / L, and 25 μmol / L 2-mercaptoethanol.

[0070] Dissolve each of the above raw materials thoroughly in 950 mL of DMEM / F12 basal culture medium, adjust the pH to 6.6-7.2, add sterile deionized water to bring the volume to 1000 mL, and then filter through a filter membrane with a pore size of 0.22 μm to obtain the culture medium, which should be stored at 4℃ for later use.

[0071] Example

[0072] Examples 1-29

[0073] Examples 1-29 provide a method for culturing forest musk gland cells.

[0074] The difference between the above embodiments is that the culture medium used in the culture of forest musk gland cells is different. The culture medium used in Examples 1-29 is derived from Preparation Examples 1-29, respectively.

[0075] The above embodiments of the method for culturing forest musk gland cells specifically include the following steps:

[0076] Cells were isolated from the musk gland tissue of the forest musk deer: After anesthetizing the forest musk deer with teletamin hydrochloride and zolazepam hydrochloride, the deer was laid flat, and the skin above the musk gland was cut open with ophthalmic scissors to obtain the gland tissue. The obtained tissue sample was immediately transferred to physiological saline containing 100 U / ml penicillin and 100 μg / ml streptomycin.

[0077] Digestion: Use sterile ophthalmic scissors to cut the sample tissue into pieces of about 1 mm, and add 0.25 mg / mL trypsin containing EDTA for about 3-10 minutes to digest.

[0078] Cell preparation: Digestion was terminated using phosphate-buffered saline (PBS) containing 100 IU / ml penicillin-streptomycin. The cells were pipetted into centrifuge tubes and centrifuged at 1500 rpm for 5 min. The supernatant was discarded, and the obtained cell suspension was added to complete culture medium and centrifuged again. This process was repeated once to collect the dispersed cells.

[0079] Cell culture: Dilute the prepared single-cell solution to 3×10⁻⁶. 4 Cells / mL were then seeded into 25T cell culture flasks and cultured in an incubator at 37°C and 5% CO2 saturated humidity. After the cells adhered, the medium was replaced with a new complete medium. Cells began to migrate out after about 5 days. The medium was then replaced every 48 hours thereafter.

[0080] Comparative Example

[0081] Comparative Examples 1-7

[0082] Comparative Examples 1-7 each provide a method for culturing forest musk gland cells.

[0083] The difference between the above comparative examples lies in the culture medium used in the musk gland cell culture method. The cultures used in comparative examples 1-7 were derived from preparation examples 30-36, respectively.

[0084] Performance testing

[0085] Using the detection methods provided in Examples 1-29 and Comparative Examples 1-7 as the detection objects, the cell density, cell activity, and adhesion rate of forest musk gland cells were determined during the culture process.

[0086] Detection methods: On days 1, 2, 4 and 8 after culturing forest musk gland cells, one sample of cells was taken daily and digested with 0.25% trypsin containing EDTA. The supernatant was collected, and the cells were counted using a hemocytometer to determine the cell density. The cell viability was determined using trypan blue staining. The cell adhesion rate was detected by visual observation under a microscope.

[0087] Test results:

[0088] like Figure 1-5 Microscopic images of forest musk gland cells, in which, Figure 1 This is a microscopic observation (10x) of the musk gland cells in Example 7 at 0 h after inoculation. Figure 2 This is a microscopic observation (10x) of the forest musk gland cells in Comparative Example 6 at 0 h after inoculation. Figure 3 This is a microscopic observation (10x) of the forest musk gland cells in Comparative Example 7 at 0 h after inoculation. Figure 4 This is a microscopic observation (10x) of the forest musk gland cells cultured for 4 days after inoculation in Example 7. Figure 5 This is a microscopic observation (10x) of the forest musk gland cells in Comparative Example 6 after 4 days of inoculation and culture. Figure 6 Microscopic observation of forest musk gland cells in Comparative Example 7 after 4 days of inoculation and culture (10x);

[0089] The cell density, cell activity, and adhesion rate of the forest musk gland cells in Examples 1-29 and Comparative Examples 1-7 are shown in Table 3.

[0090] Table 3. Cell density, cell viability, and adhesion rate of forest musk gland cells in Examples 1-29 and Comparative Examples 1-7

[0091]

[0092]

[0093]

[0094] Based on Table 3, by comparing the cell density, cell activity, and adhesion rate of forest musk gland cells in Examples 1-29 and Comparative Examples 1-7, it can be seen that using the culture medium prepared in this application to culture forest musk gland cells can promote cell growth and cell adhesion, thereby obtaining forest musk gland cells with high cell density, high cell activity, and high adhesion rate.

[0095] Combination Figure 3 , Figure 6Compared with Table 3, Comparative Example 7 used a commonly used culture medium to culture forest musk gland cells. After 4 days of culture, the adhesion rate of the forest musk gland cells was as low as 0, resulting in cell failure and the death of most cells; after 8 days of culture, the cell density of the forest musk gland cells was as low as 0.1 × 10⁻⁶. 4 The cell count / mL was as low as 21.8%.

[0096] Combination Figure 2 , Figure 5 Compared with Table 3, the culture medium used in Comparative Example 6 for culturing forest musk gland cells showed poor cell density, cell viability, and adhesion rate.

[0097] Comparative Example 4 did not use vitamins as functional additives, and Comparative Example 5 did not use non-essential amino acids as functional additives. When the culture medium prepared in this way was used to culture forest musk gland cells, the cell culture effect was poor.

[0098] By comparing the test results of Examples 1-12 with those of Comparative Examples 1-3, it can be seen that in Comparative Examples 1-3, excessively low or excessively high concentrations of growth factors in the culture medium, or the use of other types of growth factors, will all lead to a deterioration in the culture effect of *Musk deer* gland cells. Therefore, this application selects to use growth factors at a concentration of 20-40 ng / ml in the culture medium. Furthermore, by comparing the test results of Examples 1-10, this application selects EGF and IL-1 at a weight ratio of (7-11):(1-3) as growth factors, which can further improve the culture effect of *Musk deer* gland cells.

[0099] By comparing the test results of Examples 8 and 13-14, it can be seen that, compared with the use of vitamin C and vitamin B12 to add to the culture medium, the culture effect of forest musk gland cells is improved by using vitamin H to add to the culture medium in this application.

[0100] By comparing the test results of Examples 8 and 15-29, it can be seen that compared with using copper sulfate pentahydrate or zinc sulfate heptahydrate as trace elements added to the culture medium, the culture effect of forest musk gland cells is better improved by using one or more of ferric citrate, sodium ammonium selenate, and sodium selenite as trace elements added to the culture medium in this application. Furthermore, this application selects ferric ammonium citrate and sodium selenate in a weight ratio of (2-6):(0.02-0.08) as trace elements added to the culture medium.

[0101] 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 forest musk gland cells, characterized in that, The culture medium takes DMEM / F12 as a base medium; The culture medium contains the following components as functional additives: penicillin 80-120 U / ml, streptomycin 0.08-0.12 mg / ml, FBS 8-12 v / v%, non-essential amino acids 0.7-1.3 v / v%, growth factors 20-40 ng / ml, vitamin H 0.05-0.2 μg / ml, trace elements 0.001-0.04 μg / ml. The growth factors are EGF and IL-1 with a weight ratio of (7-11):(1-3), and the trace elements are composed of ammonium ferric citrate and sodium selenate with a weight ratio of (2-6):(0.02-0.08).

2. The method for preparing the musk gland cell culture medium as described in claim 1, characterized in that, Specifically comprising the following steps: The corresponding weights of each raw material are weighed, dissolved in 950 mL of base medium DMEM / F12, adjusted to pH 6.6-7.2, added with sterile deionized water to 1000 mL, and then filtered with a filter membrane with a pore size of 0.22 μm to obtain the culture medium, which is stored at 4 ℃ for use.

3. Application of the musk deer scent gland cell culture medium of claim 1 in culturing musk deer scent gland cells.

4. A method of culturing musk deer gland cells, characterized by, The culture is carried out using the culture medium of claim 1, specifically comprising the following steps: inoculating musk deer scent gland cells in the culture medium at an inoculation density of (0.4-4) x 10 5 cells / mL, and then culturing the musk deer scent gland cells at 37°C under a saturated humidity condition with 5% CO2.

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