Cell culture medium additive as well as preparation method and application thereof

Abstract

The invention discloses a cell culture medium additive as well as a preparation method and application thereof. The cell culture medium additive comprises the following components: beta-sitosterol, sericin, hydrolyzed corn starch, vitamins and a liquorice extract. Cortical neurons cultured by the cell culture medium additive provided by the invention are high in survival rate and good in growth state, and the purity reaches 90% or above; all the components in the cell culture medium additive provided by the invention have a synergistic effect, so that the cell culture medium additive plays a role in promoting the survival, growth and development of neurons and can prevent the neurons from being damaged to die; meanwhile, the antibacterial effect can be achieved without adding antibiotics, and adverse effects of the antibiotics on growth and differentiation of nerve cells are avoided; when the cell culture fluid provided by the invention is used for culturing cells, the experimental operation can be simplified, the cost can be reduced, and the stability and repeatability of experimental results can be improved.

Description

Technical Field

[0001] The invention belongs to the technical field of cell culture, and in particular relates to a cell culture medium additive and a preparation method and application thereof. Background Art

[0002] Primary neurons are the basis of neuroscience research, and their in vitro culture is a common experimental method in the field of neurobiology. At present, the commonly used neuron culture medium is Neurobasal medium, which contains B-27 cell culture additives, L-glutamic acid and penicillin-streptomycin mixture. This culture medium formula has been widely used in the field of neuroscience research and provides a stable basis for the in vitro culture of neurons.

[0003] Although the current neuronal culture medium formula is quite mature, there are still some limitations. For example, the growth environment of neurons in vivo is far more complex than that in vitro, and simple nutrients may not be able to fully simulate this complex environment, resulting in low neuronal survival rate, slow growth and unsatisfactory differentiation effect; and L-glutamate is usually added to the culture medium in the form of a solution, but the stability of glutamine solution is poor and needs to be prepared and added immediately, which increases the complexity and cost of the operation; due to the singleness of nutrients and the instability of the solution, the existing culture medium is unstable in maintaining neuronal activity, promoting neuronal growth and differentiation, affecting the reliability and repeatability of the experimental results; and, the current culture technology often adds antibiotics to the culture system to avoid pathogen contamination, but the use of antibiotics may increase the chance of invisible contamination of mycoplasma and viruses, or have a negative impression on the growth and differentiation of nerve cells. Therefore, it is necessary to further optimize and improve the existing culture medium formula. Summary of the invention

[0004] The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. To this end, the present invention proposes a cell culture medium additive that can promote the survival, growth and development of neurons and achieve good antibacterial and antimicrobial effects without adding antibiotics.

[0005] The invention also provides a cell culture solution.

[0006] The invention also provides a method for preparing the cell culture fluid.

[0007] The present invention also provides an application.

[0008] The invention also provides a neuron in vitro culture method.

[0009] According to a first aspect of the present invention, a cell culture medium additive is provided. The cell culture medium additive comprises the following components: β-sitosterol, sericin, hydrolyzed corn starch, vitamins and licorice extract.

[0010] In some embodiments of the present invention, the cell culture medium additive includes the following components at final concentrations: 0.2-5 mg / mL β-sitosterol, 0.2-8 μg / mL sericin, 0.02-1 μg / mL hydrolyzed corn starch, 0.5-10 μg / mL vitamins and 2-10 μg / mL licorice extract.

[0011] In some preferred embodiments of the present invention, the cell culture medium additive comprises the following components at final concentrations: 0.5-3 mg / mL β-sitosterol, 0.5-6 μg / mL sericin, 0.05-0.6 μg / mL hydrolyzed corn starch, 1-8 μg / mL vitamins and 3-8 μg / mL licorice extract.

[0012] In some embodiments of the present invention, the molecular weight of the sericin is 20-500 kDa.

[0013] In some preferred embodiments of the present invention, the molecular weight of the sericin is 24-400 kDa.

[0014] In some embodiments of the present invention, the hydrolyzed corn starch includes a low-molecular sugar mixture obtained by enzymatic hydrolysis of starch.

[0015] In some embodiments of the invention, the vitamins include vitamin B.

[0016] In some preferred embodiments of the present invention, the vitamin B includes riboflavin.

[0017] According to a second aspect of the present invention, a cell culture fluid is provided, wherein the cell culture fluid comprises the cell culture medium additive and a basal culture medium as described in the first aspect of the present invention.

[0018] In some embodiments of the present invention, the basal culture medium comprises at least one of Neurobasal medium and Dulbecco's Modified Eagle (DMEM) medium.

[0019] In some preferred embodiments of the present invention, the volume ratio of the Neurobasal medium to Dulbecco's modified Eagle medium is (1-1.5):1.

[0020] In some more preferred embodiments of the present invention, the volume ratio of the Neurobasal medium to Dulbecco's modified Eagle medium is (1-1.2):1.

[0021] According to a third aspect of the present invention, a method for preparing a cell culture fluid as described in the second aspect of the present invention is provided, wherein the method comprises the following steps:

[0022] The β-sitosterol, sericin, hydrolyzed corn starch, riboflavin and licorice extract are added into the basic culture medium and mixed evenly.

[0023] According to a fourth aspect of the present invention, a use of the cell culture medium additive as described in the first aspect of the present invention or the cell culture fluid as described in the second aspect of the present invention in cell culture is proposed.

[0024] In some embodiments of the invention, the cells include neuronal cells and glial cells.

[0025] According to a fourth aspect of the present invention, a method for culturing neurons in vitro is provided, wherein the method for culturing neurons in vitro adopts the cell culture medium as described in the second aspect of the present invention for culturing.

[0026] The present invention has at least the following beneficial effects:

[0027] The cortical neurons cultured with the cell culture solution provided by the present invention have a high survival rate, a good growth state, and a purity of more than 90%; the components in the cell culture solution provided by the present invention have a synergistic effect, which promotes the survival, growth and development of neurons and can prevent neurons from being damaged and dying; at the same time, the antibacterial effect can be achieved without adding antibiotics, thereby avoiding the adverse effects of antibiotics on the growth and differentiation of nerve cells; using the cell culture solution provided by the present invention to culture cells can simplify experimental operations, reduce costs, and improve the stability and repeatability of experimental results. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0029] Figure 1 This is a diagram showing the immunofluorescence staining results of primary cortical neurons in the test example of the present invention, wherein the magnification is 10 times. DETAILED DESCRIPTION

[0030] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0031] Materials and Definitions

[0032] In the following examples and comparative examples, the β-sitosterol used was purchased from Shanghai Myrel Biochemical Technology Co., Ltd. with the trade name of β-sitosterol; the sericin used was purchased from Aladdin with the product number S488097; the hydrolyzed corn starch used was purchased from ZIBON Biomedicine Co., Ltd. with the model number BINMOIS MG 80; the riboflavin used was purchased from Nanjing Dosfu Biotechnology Co., Ltd.; and the licorice extract used was purchased from Beijing Kezhan Biology with the model number 62017.

[0033] Example 1

[0034] The present embodiment provides a cell culture medium additive, and a cell culture fluid is prepared based on the cell culture medium additive, the cell culture fluid comprising a basal culture medium and the following components at final concentrations: 0.5 mg / mL β-sitosterol, 2 μg / mL sericin, 0.05 μg / mL hydrolyzed corn starch, 5 μg / mL riboflavin and 8 μg / mL licorice extract, the basal culture medium being a mixture of DMEM culture medium and Neurobasal culture medium in a volume ratio of 1:1.2.

[0035] Example 2

[0036] The present embodiment provides a cell culture medium additive, and a cell culture fluid is prepared based on the cell culture medium additive, the cell culture fluid comprising a basal culture medium and the following components at final concentrations: 2 mg / mL β-sitosterol, 1 μg / mL sericin, 0.6 μg / mL hydrolyzed corn starch, 3 μg / mL riboflavin and 3 μg / mL licorice extract, the basal culture medium being a mixture of DMEM culture medium and Neurobasal culture medium in a volume ratio of 1:1.

[0037] Example 3

[0038] The present embodiment provides a cell culture medium additive, and a cell culture fluid is prepared based on the cell culture medium additive, the cell culture fluid comprising a basal culture medium and the following components at final concentrations: 3 mg / mL β-sitosterol, 0.5 μg / mL sericin, 0.3 μg / mL hydrolyzed corn starch, 4 μg / mL riboflavin and 3.5 μg / mL licorice extract, the basal culture medium being a mixture of DMEM culture medium and Neurobasal culture medium in a volume ratio of 0.9:1.

[0039] Example 4

[0040] The present embodiment provides a cell culture medium additive, and a cell culture fluid is prepared based on the cell culture medium additive, the cell culture fluid comprising a basal culture medium and the following components at final concentrations: 1.5 mg / mL β-sitosterol, 1 μg / mL sericin, 0.2 μg / mL hydrolyzed corn starch, 8 μg / mL riboflavin and 5 μg / mL licorice extract, and the basal culture medium is DMEM culture medium.

[0041] Example 5

[0042] The present embodiment provides a cell culture medium additive, and a cell culture fluid is prepared based on the cell culture medium additive, the cell culture fluid comprising a basal culture medium and the following components at final concentrations: 0.8 mg / mL β-sitosterol, 6 μg / mL sericin, 0.3 μg / mL hydrolyzed corn starch, 1 μg / mL riboflavin and 6 μg / mL licorice extract, and the basal culture medium is Neurobasal culture medium.

[0043] Comparative Example 1

[0044] This comparative example provides a cell culture medium additive, and a cell culture fluid is prepared based on the cell culture medium additive. The cell culture fluid is different from Example 3 only in that β-sitosterol is omitted, and other components are the same.

[0045] Comparative Example 2

[0046] This comparative example provides a cell culture medium additive, and a cell culture fluid is prepared based on the cell culture medium additive. The cell culture fluid is different from Example 3 only in that sericin is omitted, and other components are the same.

[0047] Comparative Example 3

[0048] This comparative example provides a cell culture medium additive, and a cell culture fluid is prepared based on the cell culture medium additive. The cell culture fluid is different from Example 3 only in that the hydrolyzed corn starch is omitted, and other components are the same.

[0049] Comparative Example 4

[0050] This comparative example provides a cell culture medium additive, and a cell culture fluid is prepared based on the cell culture medium additive. The cell culture fluid is different from Example 3 only in that riboflavin is omitted, and other components are the same.

[0051] Comparative Example 5

[0052] This comparative example provides a cell culture medium additive, and a cell culture fluid is prepared based on the cell culture medium additive. The cell culture fluid is different from Example 3 only in that the licorice extract is omitted, and other components are the same.

[0053] Comparative Example 6

[0054] This comparative example provides a cell culture medium additive, and a cell culture fluid is prepared based on the cell culture medium additive. The cell culture fluid is different from Example 3 only in that β-sitosterol and licorice extract are omitted, and other components are the same.

[0055] Comparative Example 7

[0056] This comparative example provides a cell culture medium additive, and a cell culture fluid is prepared based on the cell culture medium additive. The cell culture fluid is different from Example 3 only in that silk fibroin, riboflavin and hydrolyzed corn starch are omitted, and other components are the same.

[0057] Comparative Example 8

[0058] This comparative example provides a cell culture solution, which uses Neurobasal culture medium as a basic culture medium and is added with the following components by volume fraction: 2% B-27 cell culture additive, 1% L-glutamic acid and 1% penicillin-streptomycin.

[0059] Comparative Example 9

[0060] This comparative example provides a cell culture solution, which is a mixture of DMEM culture medium and Neurobasal culture medium in a volume ratio of 0.9:1.

[0061] Test example

[0062] This test example tests multiple properties of the cell culture fluids provided in Examples 1 to 5 and Comparative Examples 1 to 9, including the effect on the in vitro survival rate of neural cells, the effect on the purity of neurons, the stability of the culture medium, and the antibacterial properties of the culture medium; the formulas of the cell culture fluids provided in Examples 1 to 5 and Comparative Examples 1 to 9 are shown in Table 1; the test methods and results of the above tests are as follows:

[0063] Table 1 Cell culture fluid formula of Examples 1 to 5 and Comparative Examples 1 to 9

[0064]

[0065] 1. Test the effect of cell culture medium on the survival rate of neural cells in vitro:

[0066] 1) Extraction of primary neural cells:

[0067] C57BL / 6 female and male mice were selected for mating. After the pups were born, the brain tissues of C57BL / 6J pups within 48 hours of birth were isolated. The bilateral hippocampus and cortex were isolated under a microscope. The digested and purified cell suspension (1×105 The cells were inoculated into pre-coated culture flasks or well plates, shaken evenly, and cultured in a 37°C, 5% CO2 constant temperature incubator.

[0068] 2) After culturing for 12 hours, the cell culture medium provided in Examples 1 to 5 and Comparative Examples 1 to 9 was used to replace the entire medium, and then half of the medium was replaced with the cell culture medium provided in Examples 1 to 5 and Comparative Examples 1 to 9 every 2 to 3 days; the cell survival rate of the primary neural cells after culturing for 4 days was counted, and the results are shown in Table 2.

[0069] Table 2 Survival rate of primary neural cells

[0070] Group Survival rate / % Example 1 94.27 Example 2 95.34 Example 3 97.49 Example 4 96.57 Example 5 96.56 Comparative Example 1 72.15 Comparative Example 2 85.34 Comparative Example 3 80.42 Comparative Example 4 83.81 Comparative Example 5 82.62 Comparative Example 6 55.37 Comparative Example 7 60.49 Comparative Example 8 89.16 Comparative Example 9 40.27

[0071] From Table 2, we can see that:

[0072] 1) Compared with Comparative Example 8, the cell culture fluids provided in Examples 1 to 5 of the present invention can make the cell viability of neuronal cells in vitro culture reach more than 95%, while that of Comparative Example 8 is about 90%, which shows that the culture medium of the present invention has a better ability to maintain cell activity than the currently conventional neuronal culture medium;

[0073] 2) Compared with Comparative Examples 1 to 5, the cell culture solutions provided in Examples 1 to 5 of the present invention can improve the cell survival rate of neuronal cells cultured in vitro, while Comparative Examples 1 to 5 lack a key component respectively, which indicates that the ratio of the cell culture solution provided by the present invention is the best combination obtained after optimization, and the lack of any one of the components will affect cell growth and activity;

[0074] 3) According to the results of Comparative Example 6, Comparative Example 7 and Example 3, based on Jin Zhengjun's formula: The synergistic effect between the components was evaluated; A+B Refers to the survival rate of Example 3, E A Refers to the survival rate of comparative example 6, E B Referring to the survival rate of comparative example 7, it was calculated that q>1.15, indicating that the five components in the cell culture medium have a synergistic effect and none of the five components can be missing.

[0075] 2. Immunofluorescence identification and purity identification of characteristic proteins of neuronal cells:

[0076] In this test example, the cell culture medium provided in Example 3 was used to culture primary cortical neurons, and the characteristic protein MAP-2 in the cultured primary cortical neurons was immunofluorescently stained to further identify the purity of the neurons. The specific steps are as follows:

[0077] 1) Cell fixation: After discarding the culture medium, add PBS to wash, then add paraformaldehyde to fix for 10 minutes at room temperature, and then wash with PBS;

[0078] 2) Cell membrane disruption: Add PBS containing 0.3% triton-100 to the 6-well plate for 10 minutes, discard the membrane disruption solution, add PBS and wash on a shaker;

[0079] 3) Blocking: Add 5% BSA and block at room temperature for one hour;

[0080] 4) Primary antibody incubation: After taking out the 6-well plate slide from the well plate, place it on a glass slide, draw a circle with an immunohistochemistry pen, add the corresponding antibody on the cell slide and place it in a 4C refrigerator overnight;

[0081] 5) Secondary antibody incubation: discard the primary antibody, wash with PBS, add the secondary antibody, and incubate at room temperature in the dark for 1 hour;

[0082] 6) Staining cell nuclei: discard the secondary antibody, wash with PBS, add DAPI and incubate for 5 minutes in the dark, and wash with PBS;

[0083] 7) Sealing: After sealing, take photos and observe. The results are as follows Figure 1 As shown;

[0084] Purity (%) = (number of target molecule positively stained cells / total number of cells) × 100. The calculated results are shown in Table 3.

[0085] Table 3 Neuronal purity results

[0086]

[0087]

[0088] Depend on Figure 1 As shown in Table 3, after culturing primary cortical neurons with the cell culture medium provided in Examples 1 to 5 of the present invention, immunofluorescence staining of neuronal cell slides cultured for 5 days revealed that the cortical neurons had high purity, few foreign cells, a clear and clean background, and few tissue fragments, with a purity as high as 95%; MAP-2 was labeled as a neuron-specific antigen, labeled as green fluorescence, and DAPI labeled the cell nucleus.

[0089] 3. Test the stability of cell culture medium:

[0090] The cell culture fluids provided in Example 3 and Comparative Examples 1 to 7 and 9 were subjected to an accelerated stability test and a normal temperature test, respectively. The specific test steps and results are as follows:

[0091] 1) Accelerated test:

[0092] The cell culture fluid was placed at a temperature of 40°C ± 2°C and a relative humidity of 75% ± 5% for 6 months. Samples were taken at the end of 1 month, 2 months, 3 months and 6 months during the test to detect the properties, color and odor of the cell culture fluid.

[0093] The results showed that all indicators of the cell culture medium provided in Example 3 did not change significantly; while the cell culture medium provided in Comparative Examples 1 to 7 became darker in color and had a small amount of precipitation, and Comparative Example 9 had a large amount of precipitation.

[0094] 2) Long-term test:

[0095] The above cell culture fluids were placed at a temperature of 25°C ± 2°C and a relative humidity of 60% ± 10% for 12 months. Samples were taken at the end of 0 month, 3 months, 6 months, 9 months and 12 months during the test period to detect the properties, color and odor of the cell culture fluids.

[0096] The results showed that all indicators of the cell culture solution provided in Example 3 did not change significantly; while the cell culture solutions provided in Comparative Examples 1 to 7 showed precipitation and darkening of color.

[0097] From the results of the above-mentioned accelerated tests and long-term tests, it can be seen that the cell culture fluid provided by the present invention can significantly improve the stability of the basal culture medium and avoid problems such as precipitation, stratification and discoloration of the basal culture medium. If one component, or two or three components are reduced based on the formula of Example 3, the stability of the neuronal culture medium decreases.

[0098] 4. Test the antibacterial properties of cell culture fluid:

[0099] The cell culture fluids provided in Example 3, Comparative Example 1, Comparative Example 7, and Comparative Example 9 were sealed and stored for 12 months at a temperature of 25°C ± 2°C and a relative humidity of 60% ± 10%, and then the total number of bacteria in each group of cell culture fluids was detected. The results are shown in Table 4.

[0100] Table 4 Statistics of total bacteria in neuron culture medium

[0101] Group Total colony count (cfu / mL) Example 3 20 Comparative Example 1 106 Comparative Example 7 175 Comparative Example 9 764

[0102] It can be seen from Table 4 that the cell culture fluid provided in Example 3 can effectively prevent nerve cells from being infected by bacteria, effectively inhibit the growth and reproduction of bacteria in the culture system, and ensure the safety of the use of the cell culture fluid; if one or more components are reduced on the basis of the formula of Example 3, the antibacterial ability of the cell culture fluid will be affected.

[0103] The embodiments of the present invention are described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. A cell culture medium additive, characterized in that The cell culture medium additive comprises the following components: β-sitosterol, sericin, hydrolyzed corn starch, vitamins and licorice extract.

2. The cell culture medium additive according to claim 1, characterized in that The cell culture medium additive comprises the following components at final concentrations: 0.2-5 mg / mL beta-sitosterol, 0.2-8 μg / mL sericin, 0.02-1 μg / mL hydrolyzed corn starch, 0.5-10 μg / mL vitamins and 2-10 μg / mL licorice extract.

3. The cell culture medium additive according to claim 1, characterized in that The vitamins include vitamin B.

4. The cell culture medium additive according to claim 3, characterized in that The B vitamins include riboflavin.

5. A cell culture medium, characterized in that: The cell culture solution comprises the cell culture medium additive as claimed in any one of claims 1 to 4 and a basal culture medium.

6. The cell culture solution according to claim 5, characterized in that The basic culture medium includes at least one of Neurobasal medium and Dulbecco's modified Eagle medium.

7. The cell culture solution according to claim 6, characterized in that The volume ratio of the Neurobasal culture medium to the Dulbecco's modified Eagle culture medium is (1-1.5):

1.

8. A method for preparing a cell culture fluid as claimed in any one of claims 5 to 7, characterized in that: The preparation method comprises the following steps: The β-sitosterol, sericin, hydrolyzed corn starch, riboflavin and licorice extract are added into the basic culture medium and mixed evenly.

9. Use of the cell culture medium additive according to any one of claims 1 to 4 or the cell culture solution according to any one of claims 5 to 7 in cell culture.

10. A method for culturing neurons in vitro, characterized in that: The in vitro neuron culture method uses the cell culture medium additive as described in any one of claims 1 to 4 or the cell culture fluid as described in any one of claims 5 to 7 for culture.

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