Low-serum culture medium for maintaining cell proliferation and improving transfection efficiency and culture method

Abstract

The invention belongs to the technical field of cell culture, and relates to a low-serum culture medium for maintaining cell proliferation and improving transfection efficiency and a culture method. It is found for the first time that culture conditions are accurately regulated and controlled by providing a stable micromolecule component combination, and the method can be used for proliferation culture of various human and animal cells; the addition of serum in the original proliferation culture medium is reduced, the proliferation effect similar to that of the original culture medium is achieved, and the subsequent differentiation of cells is not influenced; experiments prove that the culture medium and the culture method have the advantages that the addition amount of serum in the culture process of porcine skeletal muscle satellite cells, mouse myoblasts, porcine kidney fibroblasts and mouse skeletal muscle cells is reduced, the biosafety risk in the experiment is reduced, the repeatability and reliability of the experiment are improved, the cost is saved, and the culture medium and the culture method are suitable for large-scale popularization and application. And a solid foundation is provided for subsequent exploration of a normal and efficient cell culture technology under a serum-free condition.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biology, in particular to a low serum culture medium and culture method for maintaining cell proliferation and improving transfection efficiency. BACKGROUND

[0002] As a key component of in vitro cell culture system, cell culture medium constitutes the key material basis for maintaining the life activities of cells. Its core function is to provide necessary nutrients (such as sugars, amino acids, vitamins) to cells, maintain osmotic pressure and acid-base balance, and regulate cell proliferation, differentiation and functional expression through growth factors (such as EGF). However, although fetal bovine serum (FBS) has multiple functions as a key additive of culture medium, the use of serum also introduces several challenges due to the particularity of the production process. Among them, fetal bovine serum is a commonly used supplement in cell culture medium. The composition of different batches of serum is not completely the same, and there is even significant variability (Gstraunthaler et al. 2013). Limited by the differences in the feeding, health process and blood collection process of the serum source animals, the vitamins, growth factors and nutrients in fetal bovine serum vary (Chelladurai et al. 2021), which makes the quality of serum in different batches uneven (Lee et al. 2022, van der Valk 2022), resulting in significant differences in cell culture effect, affecting cell proliferation and growth rate, differentiation, and may also affect the repeatability and reliability of experimental results, prompting the search for some alternatives that can reduce serum usage.

[0003] The emergence of serum-free culture systems aims to overcome the shortcomings and challenges of traditional serum culture methods, precisely regulate culture conditions by removing serum components, provide stable ingredient combinations, reduce biological safety risks in experiments, and improve the repeatability and reliability of experiments (Defendi et al. 2023). Although serum-free culture has many advantages, completely replacing serum is still a challenge in some cases. Different cell types have different nutritional and growth factor requirements, and it is necessary to continuously explore and optimize the ingredients and ratios of serum-free culture medium to ensure its applicability and effectiveness. Currently, there are few serum-free culture media that have been proven to support the sustained expansion of porcine skeletal muscle satellite cells in multiple passages (Stout et al. 2022), therefore, it is very important to optimize the process of low serum culture cells, which can ensure that the most effective culture formula is found to meet the needs of different cell types, and ultimately achieve more efficient and safer serum-free cell culture technology.

[0004] The scheme provides a low serum proliferation culture cell culture scheme, monitors the proliferation of cells in the low serum culture process, provides a new scheme for low serum culture, and has a positive significance for promoting the application of serum-free culture in cell culture. SUMMARY

[0005] In order to solve the problems in the prior art, the first object of the present application is to provide a low serum culture medium for maintaining cell proliferation and improving transfection efficiency, which can maintain the proliferation ability of cells and maintain the differentiation potential, and fuse to form muscle tubes.

[0006] In order to achieve the above technical purpose, the technical scheme is as follows:

[0007] A low serum culture medium for maintaining cell proliferation and improving transfection efficiency, comprising a basic culture medium and the following components: 10ng / mL-240ng / mL IGF-1, 1ng / mL-50ng / mL IL-6, 1ng / mL-50ng / mL TGF-β, 50μM-800μM VC, 1ng / mL-50ng / mL bFGF, 1ng / mL-50ng / mL G-CSF, 1ng / mL-50ng / mL GM-CSF, 2% penicillin-streptomycin;

[0008] The basic culture medium is DMEM culture medium containing 2%-10% fetal bovine serum or RPMI1640 culture medium containing 1% non-essential amino acid NEAA, 1% glutamine and 2%-20% fetal bovine serum.

[0009] Preferably, the culture medium comprises RPMI1640 culture medium containing 1% non-essential amino acid NEAA, 1% glutamine and 5% fetal bovine serum and the following components: 40ng / mL IGF-1, 10ng / mL IL-6, 5ng / mL TGF-β, 100μM VC, 10ng / mL bFGF, 10ng / mL G-CSF, 10ng / mL GM-CSF.

[0010] Preferably, the culture medium comprises RPMI1640 culture medium containing 1% non-essential amino acid NEAA, 1% glutamine and 5% fetal bovine serum and the following components: 40ng / mL IGF-1, 10ng / mL IL-6, 5ng / mL TGF-β, 10ng / mL bFGF, 10ng / mL G-CSF.

[0011] Preferably, the culture medium comprises DMEM culture medium containing 5% fetal bovine serum and the following components: 40ng / mL IGF-1, 10ng / mL IL-6, 5ng / mL TGF-β, 10ng / mL bFGF, 10ng / mL G-CSF.

[0012] The most suitable combination of the components of the low serum culture medium provided by the present application is the combination of IGF-1, IL-6, TGF-β, bFGF and G-CSF, which is named as proliferation synergistic factor combination (PSFC).

[0013] The second object of the present application is to provide a low serum culture method for maintaining cell proliferation and improving transfection efficiency.

[0014] In order to achieve the above technical objects, the technical solutions are as follows:

[0015] 1) Take the longest muscle and leg muscle tissue to separate the porcine skeletal muscle satellite cells PSCs; culture in RPMI1640 medium containing 20% fetal bovine serum for 2-4 days; when the PSCs cell growth density is 80%-90%, trypsin digestion is used for transfer inoculation to cell culture dish for standby;

[0016] 2) The medium is used for cell proliferation culture after transfection, and the transfection efficiency is verified.

[0017] The above method can be used for culturing human and animal cells, including porcine skeletal muscle satellite cells, mouse myoblast cells C2C12, porcine kidney fibroblasts PKFs, mouse skeletal muscle satellite cells SSCs and 293T cells.

[0018] The present application has the following beneficial effects:

[0019] The low serum culture medium provided by the present application for maintaining cell proliferation and improving transfection efficiency reduces the addition of serum in the existing common proliferation culture medium by establishing a low serum proliferation culture system for proliferating cultured cells, has similar proliferation effect as the existing common culture medium, and does not affect the subsequent differentiation of cells, helps to accurately regulate the culture conditions by reducing the serum components, provides stable component combination, reduces the biological safety risk in experiments, and improves the repeatability and reliability of experiments. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 is the Pax7 immunofluorescence staining diagram of porcine skeletal muscle satellite cells (PSCs) proliferated and cultured in Example 1, and the collection time is 1 day after the porcine skeletal muscle satellite cells are proliferated and cultured; + The immunofluorescence staining diagram of the cells is collected 1 day after the porcine skeletal muscle satellite cells are proliferated and cultured;

[0021] Figure 2 From left to right, the EdU staining diagram of the cells, the relative percentage column comparison diagram of EdU positive cells after proliferating and culturing in the proliferation culture medium containing 10 ng / mL-240 ng / mL IGF-1 in Example 2, and the collection time is 1 day after the porcine skeletal muscle satellite cells are proliferated and cultured; + The immunofluorescence staining diagram of the cells is collected 1 day after the porcine skeletal muscle satellite cells are proliferated and cultured;

[0022] Figure 3 From left to right are EdU staining images of porcine skeletal muscle satellite cells proliferated in the proliferation medium containing 1 ng / mL ~ 50 ng / mL IL-6 in Example 2 + Staining images of cells, bar comparison chart of relative percentage of EdU positive cells, the collection time is 1 day after porcine skeletal muscle satellite cells proliferated in the proliferation medium

[0023] Figure 4 From left to right are EdU staining images of porcine skeletal muscle satellite cells proliferated in the proliferation medium containing 1 ng / mL ~ 50 ng / mL TGF-β in Example 2 + Staining images of cells, bar comparison chart of relative percentage of EdU positive cells, the collection time is 1 day after porcine skeletal muscle satellite cells proliferated in the proliferation medium

[0024] Figure 5 From left to right are EdU staining images of porcine skeletal muscle satellite cells proliferated in the proliferation medium containing 50 μM ~ 800 μM VC in Example 2 + Staining images of cells, bar comparison chart of relative percentage of EdU positive cells, the collection time is 1 day after porcine skeletal muscle satellite cells proliferated in the proliferation medium

[0025] Figure 6 From left to right are EdU staining images of porcine skeletal muscle satellite cells proliferated in the proliferation medium containing 1 ng / mL ~ 50 ng / mL bFGF in Example 2 + Staining images of cells, bar comparison chart of relative percentage of EdU positive cells, the collection time is 1 day after porcine skeletal muscle satellite cells proliferated in the proliferation medium

[0026] Figure 7 From left to right are EdU staining images of porcine skeletal muscle satellite cells proliferated in the proliferation medium containing 1 ng / mL ~ 50 ng / mL G-CSF in Example 2 + Staining images of cells, bar comparison chart of relative percentage of EdU positive cells, the collection time is 1 day after porcine skeletal muscle satellite cells proliferated in the proliferation medium

[0027] Figure 8 From left to right are EdU staining images of porcine skeletal muscle satellite cells proliferated in the proliferation medium containing 1 ng / mL ~ 50 ng / mL GM-CSF in Example 2 + Staining images of cells, bar comparison chart of relative percentage of EdU positive cells, the collection time is 1 day after porcine skeletal muscle satellite cells proliferated in the proliferation medium

[0028] Figure 9 From left to right are EdU staining images of porcine skeletal muscle satellite cells proliferated in the proliferation medium containing different material combinations in the first screening in Example 3 + Staining images of cells, bar comparison chart of relative percentage of EdU positive cells, the collection time is 1 day after porcine skeletal muscle satellite cells proliferated in the proliferation medium

[0029] Figure 10 From left to right are EdU of seven substances in Example 3 after VC removal, the second screening of the proliferation medium of different substance combinations after proliferation culture + Cell staining chart, relative percentage column chart of EdU positive cells, collection time is 1 day after proliferation culture of porcine skeletal muscle satellite cells;

[0030] Figure 11 From left to right are EdU of the obtained optimal substance combination PSFC (IGF-1, IL-6, TGF-β, bFGF, G-CSF) in Example 4 after setting different serum concentration gradients of the proliferation medium for proliferation culture + Cell staining chart, relative percentage column chart of EdU positive cells, collection time is 1 day after proliferation culture of porcine skeletal muscle satellite cells;

[0031] Figure 12 Cell counting column chart after setting different serum concentration gradients of the proliferation medium of the obtained optimal combination PSFC (IGF-1, IL-6, TGF-β, bFGF, G-CSF) in Example 4 for proliferation culture, collection time is the second generation, the fourth generation, the sixth generation of porcine skeletal muscle satellite cells after proliferation culture;

[0032] Figure 13 Relative mRNA expression column chart after 5% FBS proliferation culture of the obtained optimal combination PSFC (IGF-1, IL-6, TGF-β, bFGF, G-CSF) in Example 4, collection time is 2 days after proliferation culture of porcine skeletal muscle satellite cells;

[0033] Figure 14 From left to right are Western blot result chart and relative protein level column chart after 5% FBS proliferation culture of the obtained optimal combination PSFC (IGF-1, IL-6, TGF-β, bFGF, G-CSF) in Example 4, collection time is 2 days after proliferation culture of porcine skeletal muscle satellite cells;

[0034] Figure 15 From left to right are EdU of C2C12 cells after proliferation culture by the obtained low serum medium in Example 4 + Cell staining chart, relative percentage column chart of EdU positive cells, collection time is 1 day after proliferation culture of C2C12 cells;

[0035] Figure 16 From left to right are EdU of PKFs cells after proliferation culture by the obtained low serum medium in Example 4 +Cell staining images, relative percentage of EdU positive cells bar graph, collection time is 1 day after proliferation culture of PKFs cells;

[0036] Figure 17 From left to right are EdU staining images of SSCs cells after proliferation culture with obtained low serum medium in Example 4, MYHC staining images after changing differentiation medium, collection time is 2 days after differentiation culture of SSCs cells. + Cell staining images, relative percentage of EdU positive cells bar graph, collection time is 1 day after proliferation culture of SSCs cells;

[0037] Figure 18 From left to right are MYHC staining images of PSCs cells after proliferation culture with obtained low serum medium in Example 4, MYHC staining images after changing differentiation medium, collection time is 2 days after differentiation culture of PSCs cells. + Cell immunofluorescence staining images, percentage of differentiation index bar graph, collection time is 2 days after differentiation culture of PSCs cells.

[0038] Figure 19 From left to right are MYHC staining images of SSCs cells after proliferation culture with obtained low serum medium in Example 4, MYHC staining images after changing differentiation medium, collection time is 2 days after differentiation culture of SSCs cells. + Cell immunofluorescence staining images, percentage of differentiation index bar graph, collection time is 2 days after differentiation culture of SSCs cells.

[0039] Figure 20 From left to right are MYHC staining images of C2C12 cells after proliferation culture with obtained low serum medium in Example 4, MYHC staining images after changing differentiation medium, collection time is 5 days after differentiation culture of C2C12 cells. + Cell immunofluorescence staining images, percentage of differentiation index bar graph, collection time is 5 days after differentiation culture of C2C12 cells.

[0040] Figure 21 Relative mRNA expression level bar graph of C2C12 cells after proliferation culture with obtained low serum medium in Example 4, after changing differentiation medium, collection time is 5 days after differentiation culture of C2C12 cells.

[0041] Figure 22 From left to right are Western blot results and relative protein level bar graph of C2C12 cells after proliferation culture with obtained low serum medium in Example 4, after changing differentiation medium, collection time is 5 days after differentiation culture of C2C12 cells.

[0042] Figure 23 From left to right are fluorescence microscope images and flow cytometry histogram of PSCs cells transfected with plasmid after 2 days of proliferation culture with obtained low serum medium in Example 5, collection time is 1 day after cell transfection.

[0043] Figure 24From left to right are fluorescence microscope images and flow cytometry histograms of transfected plasmid after proliferative culture of C2C12 cells for 2 days using the obtained low serum medium in Example 5, and the acquisition time is 1 day after cell transfection.

[0044] Figure 25 From left to right are fluorescence microscope images and flow cytometry histograms of transfected plasmid after proliferative culture of PKFs cells for 2 days using the obtained low serum medium in Example 5, and the acquisition time is 1 day after cell transfection.

[0045] Figure 26 From left to right are fluorescence microscope images and flow cytometry histograms of transfected plasmid after proliferative culture of 293T cells for 2 days using the obtained low serum medium in Example 5, and the acquisition time is 1 day after cell transfection.

[0046] Figure 27 From left to right are columnar comparison diagrams of fluorescence positive cells after transfection of different plasmids after proliferative culture of PSCs, C2C12, PKFs, 293T cells for 2 days using the obtained low serum medium in Example 5, and the acquisition time is 1 day after cell transfection. DETAILED DESCRIPTION

[0047] The technical solutions of the present application are further illustrated below in combination with examples.

[0048] Trypsin-EDTA (0.25%) (Gibco, #25200114)

[0049] Collagenase type I (Gibco, #17018029)

[0050] RPMI 1640 medium (Gibco, #6124030)

[0051] Fetal bovine serum (FBS, Gibco, #A5670701)

[0052] PBS (Gibco, #10010023)

[0053] Penicillin-streptomycin (Gibco, #15070063)

[0054] Glutamax (Gibco, #35050061)

[0055] NEAA (Gibco, #11140050)

[0056] IGF-1 (Aladdin, #P05019)

[0057] bFGF (Aladdin, #P09038)

[0058] IL-6 (GenScript, #Z03034-10)

[0059] TGF-β (Aladdin, #P01137)

[0060] Ascorbic acid (Aladdin, #A103539)

[0061] G-CSF (GenScript, #Z02974-10)

[0062] GM-CSF (GenScript, #Z05361-100)

[0063] 4% paraformaldehyde solution (Servicebio, #G1101)

[0064] EdU Cell Proliferation Detection Kit (Beyotime, #C0075S)

[0065] Myosin heavy chain primary antibody (MyHC, Santa Cruz, #sc-376157)

[0066] Myogenin primary antibody (MyoG, Santa Cruz, #sc-52903)

[0067] Cy3 labeled goat anti-mouse IgG (Servicebio, #GB21401)

[0068] Example 1: Acquisition of PSCs seed cells

[0069] After blood sampling of 1-3 day old piglets, the piglets were washed and disinfected, and the longissimus dorsi muscle and leg muscle tissue were taken to isolate PSCs cells. The muscle tissue was cut into a paste with surgical scissors, and PBS solution was added to prepare a suspension. Centrifugation was performed at 2500 rpm for 5 min, and the supernatant was discarded. This step was repeated until the supernatant was clear. A 2 mg / mL collagenase type I solution was prepared and mixed with the muscle precipitate, and incubated at 37°C on a shaking table for about 2 hours. After 2 hours of digestion, DMEM medium containing 10% FBS was added to terminate digestion, and 100 μm, 70 μm and 40 μm cell filters were used in sequence for filtration. After differential centrifugation for 2 hours, PSCs cells were obtained. The PSCs cells were cultured in new RPMI1640 medium containing 1% non-essential amino acids (NEAA), 1% glutamine, 2% penicillin-streptomycin and 20% fetal bovine serum (FBS) for 2-4 days. When the PSCs cells grew to 80%-90% of the density, they were trypsinized and transferred to a 10 CM cell culture dish. Figure 1 is a Pax7 immunofluorescence staining diagram of PSCs cells isolated in Example 1.

[0070] Example 2: Study of optimal concentration of additives

[0071] In order to explore the optimal culture concentration of each additive for the culture of porcine skeletal muscle satellite cells. The research design includes setting different concentration gradients of each substance. First, RPMI1640 medium containing 1% non-essential amino acids (NEAA), 1% glutamine, 2% penicillin-streptomycin is used as the basic component (the basic component for subsequent culture of PSCs and SSCs cells is the same, and the experimental group and the control group are optimized on the basis of the basic component), then after adding 20% fetal bovine serum (FBS), different concentrations of each substance are added to prepare proliferation medium containing 10 ng / mL-240 ng / mL IGF-1, proliferation medium containing 1 ng / mL-50 ng / mL IL-6, proliferation medium containing 1 ng / mL-50 ng / mL TGF-β, proliferation medium containing 50 μM-800 μM VC, proliferation medium containing 1 ng / mL-50 ng / mL bFGF, proliferation medium containing 1 ng / mL-50 ng / mL G-CSF, and proliferation medium containing 1 ng / mL-50 ng / mL GM-CSF. Each medium is used as a test group, and the control group is RPMI1640 medium containing 1% non-essential amino acids (NEAA), 1% glutamine, 2% penicillin-streptomycin, and 20% fetal bovine serum (FBS) for the culture of porcine skeletal muscle satellite cells. EdU cell proliferation staining is used to determine the optimal culture concentration of each substance for the culture of porcine skeletal muscle satellite cells.

[0072] Figure 2 is the EdU cell proliferation staining and quantification chart of porcine skeletal muscle satellite cells cultured with 10 ng / mL-240 ng / mL IGF-1 proliferation medium in Example 2. Figure 3 is the EdU cell proliferation staining and quantification chart of porcine skeletal muscle satellite cells cultured with 1 ng / mL-50 ng / mL IL-6 proliferation medium in Example 2. Figure 4 is the EdU cell proliferation staining and quantification chart of porcine skeletal muscle satellite cells cultured with 1 ng / mL-50 ng / mL TGF-β proliferation medium in Example 2. Figure 5 is the EdU cell proliferation staining and quantification chart of porcine skeletal muscle satellite cells cultured with 50 μM-800 μM VC proliferation medium in Example 2. Figure 6 is the EdU cell proliferation staining and quantification chart of porcine skeletal muscle satellite cells cultured with 1 ng / mL-50 ng / mL bFGF proliferation medium in Example 2. Figure 7 is the EdU cell proliferation staining and quantification chart of porcine skeletal muscle satellite cells cultured with 1 ng / mL-50 ng / mL G-CSF proliferation medium in Example 2.Figure 8 EdU cell proliferation staining and quantification of porcine skeletal muscle satellite cells cultured in the proliferation medium with 1 ng / mL ~ 50 ng / mL GM-CSF in Example 2.

[0073] Example 3: Study on optimal combination of additives

[0074] In order to explore the optimal combination of additives for culturing porcine skeletal muscle satellite cells, the optimal concentrations of each substance obtained in Example 2 for culturing porcine skeletal muscle satellite cells were selected, i.e., the additive concentrations were 40 ng / mL IGF-1, 10 ng / mL IL-6, 5 ng / mL TGF-β, 100 μM VC, 10 ng / mL bFGF, 10 ng / mL G-CSF, and 10 ng / mL GM-CSF, and a combination screening was performed. In each round of screening, a control group without adding any substance combination was set, and an experimental group was set by adding all substances or sequentially reducing one substance. The serum concentration of the control group and the experimental group was 20%. Figure 9 EdU cell proliferation staining and quantification of porcine skeletal muscle satellite cells cultured in the proliferation medium in the first screening. According to the experimental results of EdU, the cell proliferation after removing VC was higher than that of the combination of all substances, indicating that VC had no significant promoting effect on cell growth in this round of combination, so VC was removed from the combination of substances through the first screening. The second screening was similar to the first screening. Figure 10 EdU cell proliferation staining and quantification of porcine skeletal muscle satellite cells cultured in the proliferation medium in the second screening. The results showed that the cell proliferation of the group without GM-CSF was greater than that of the control group, indicating that this substance had no promoting effect on cell growth in the combination and could be screened out. After two rounds of screening, a relatively optimal substance combination of 40 ng / mL IGF-1 + 10 ng / mL IL-6 + 5 ng / mL TGF-β + 10 ng / mL bFGF + 10 ng / mL G-CSF was obtained.

[0075] Example 4: Exploration of low serum culture concentration

[0076] In order to explore the low serum culture effect of the optimal combination of additives for culturing porcine skeletal muscle satellite cells, the best combination for culturing porcine skeletal muscle satellite cells obtained in Example 3 was selected. The control group was 20% serum without adding any of the above-mentioned substance combinations, and the experimental group was set by setting the serum concentration as a single variable under the condition of adding the same substance combination (40 ng / mL IGF-1 + 10 ng / mL IL-6 + 5 ng / mL TGF-β + 10 ng / mL bFGF + 10 ng / mL G-CSF). The serum concentration gradient was 20%, 15%, 10%, 5%, and 2%. Figure 11is a low serum culture EdU cell proliferation staining graph and a quantitative graph. The results show that after adding 20% serum, 15% serum, 10% serum, and 5% serum to the composition for culture, the proliferation of porcine skeletal muscle satellite cells can be promoted. In order to further evaluate long-term proliferation, continuous passage culture was carried out, serum concentration gradients of 20%, 15%, 10%, 5%, and 2% were set, the composition was added respectively, and cell counting experiments were carried out, Figure 12 is a cell counting result quantification graph. The results show that compared with the control group, after adding 5% serum to the composition for culturing cells, the number of cells can be promoted or maintained.

[0077] In order to verify the effect of adding 5% serum to the composition on the proliferation of porcine skeletal muscle satellite cells, RT-qPCR experiments and Western blot experiments were carried out, Figure 13 is an RT-qPCR experiment result graph. The results show that after culturing porcine skeletal muscle satellite cells with 5% serum and the composition, there is no significant difference in the expression of genes related to proliferation compared with the control group. Figure 14 is a Western blot experiment result and quantification graph. The results show that after culturing porcine skeletal muscle satellite cells with 5% serum and the composition, there is no significant difference in the expression of Ki67 protein compared with the control group.

[0078] In order to verify whether the system is suitable for other cells, mouse myoblast cells (C2C12), porcine kidney fibroblasts (PKFs), and mouse skeletal muscle satellite cells (SSCs) were selected for low serum proliferation culture experiments. Mouse skeletal muscle satellite cells (SSCs) were cultured. The control group was RPMI1640 medium containing 1% non-essential amino acids (NEAA), 1% glutamine, 2% penicillin-streptomycin, and 20% fetal bovine serum (FBS) without adding the composition, and the experimental group was RPMI1640 medium containing 1% non-essential amino acids (NEAA), 1% glutamine, 2% penicillin-streptomycin, and 5% FBS with the addition of the composition (40 ng / mL IGF-1+10 ng / mL IL-6+5 ng / mL TGF-β+10 ng / mL bFGF+10 ng / mL G-CSF);

[0079] C2C12 cells and PKFs cells were cultured in DMEM medium containing 2% penicillin-streptomycin and 10% fetal bovine serum (FBS) without the addition of the composition as the control group, and in DMEM medium containing 2% penicillin-streptomycin and 5% FBS with the addition of the composition (40 ng / mL IGF-1 + 10 ng / mL IL-6 + 5 ng / mL TGF-β + 10 ng / mL bFGF + 10 ng / mL G-CSF) as the experimental group, Figure 15 EdU cell proliferation staining and quantification of C2C12 cells cultured in low serum, and the results showed that 5% serum plus the composition could maintain normal cell proliferation, Figure 16 EdU cell proliferation staining and quantification of PKFs cells cultured in low serum, and the results showed that 5% serum plus the composition could maintain normal cell proliferation compared with the control group, Figure 17 EdU cell proliferation staining and quantification of SSCs cells cultured in low serum, and the results showed that 5% serum plus the composition could maintain normal cell proliferation compared with the control group.

[0080] To verify whether the differentiation ability of cells cultured in low serum proliferation medium is changed, the control group was set to 20% serum, and the experimental group was set to 5% serum plus the composition for proliferation culture of different cells, and then the differentiation medium was replaced for differentiation culture. Figure 18 PSCs cell immunofluorescence and quantification, and the results showed that there was no significant difference in myotube production between the experimental group and the control group. Figure 19 SSCs cell immunofluorescence and quantification, and the results showed that low serum proliferation culture could promote the differentiation ability of SSCs cells. Figure 20 C2C12 cell immunofluorescence and quantification, and the results showed that low serum proliferation culture could maintain normal differentiation ability of cells compared with the control group. Figure 21 RT-qPCR experimental results of C2C12 cells cultured in low serum proliferation medium and then differentiated, and the results showed that there was no significant difference in the expression of MyHC, MyOG and MyOD between the experimental group and the control group after C2C12 cells were cultured in 5% serum plus the composition. Figure 22 Western blot experimental results and quantification, and the results showed that there was no significant difference in the expression of differentiation marker proteins MyHC and MyOG between the experimental group and the control group.

[0081] Example 5 Study of transfection efficiency

[0082] To verify whether the transfection efficiency of cells is changed after low serum proliferation culture of the cells. When culturing PSCs cells, the control group is set as 20% serum RPMI1640 culture medium without adding 1% non-essential amino acid (NEAA), 1% glutamine, 2% penicillin-streptomycin, and two experimental groups are respectively 20% serum RPMI1640 culture medium added with 1% non-essential amino acid (NEAA), 1% glutamine, 2% penicillin-streptomycin of the composition substance verified in Example 3 and 5% serum RPMI1640 culture medium added with 1% non-essential amino acid (NEAA), 1% glutamine, 2% penicillin-streptomycin of the composition substance verified in Example 3. When culturing C2C12, PKFs, 293T, the control group is set as 10% serum DMEM culture medium without adding 2% penicillin-streptomycin of the composition substance, and two experimental groups are respectively 10% serum DMEM culture medium added with 2% penicillin-streptomycin of the composition substance and 5% serum DMEM culture medium added with 2% penicillin-streptomycin of the composition substance. After proliferation culture of different cells, different plasmids are transfected into the cells.

[0083] Figure 23 The fluorescence microscope images and flow cytometry histograms of plasmid transfection of PSCs cells after low serum proliferation culture are shown. The results show that the cell transfection efficiency can be improved after culturing PSCs cells in 5% serum plus the composition substance. Figure 24 The fluorescence microscope images and flow cytometry histograms of plasmid transfection of C2C12 cells after low serum proliferation culture are shown. The results show that the cell transfection efficiency can be improved after culturing C2C12 cells in 5% serum plus the composition substance. Figure 25 The fluorescence microscope images and flow cytometry histograms of plasmid transfection of PKFs cells after low serum proliferation culture are shown. The results show that the cell transfection efficiency can be improved after culturing PKFs cells in 5% serum plus the composition substance. Figure 26 The fluorescence microscope images and flow cytometry histograms of plasmid transfection of 293T cells after low serum proliferation culture are shown. The results show that the cell transfection efficiency can be improved after culturing 293T cells in 5% serum plus the composition substance. Figure 27 The fluorescence positive cell columnar comparison chart of PSCs, C2C12, PKFs, 293T cells after transfection of different plasmids is shown. The results show that the cell transfection efficiency can be improved after low serum proliferation culture compared with the control group.

[0084] The above is a further detailed description of the present application, which cannot be regarded as a limitation on the specific implementation of the present application. For ordinary skilled persons in the technical field to which the present application belongs, simple deduction or replacement without departing from the concept of the present application is within the protection scope of the present application.

Claims

1. A low serum medium for maintaining cell proliferation and improving transfection efficiency, characterized in that, The basic culture medium is DMEM medium containing 2%-10% fetal bovine serum or RPMI1640 medium containing 1% non-essential amino acid NEAA, 1% glutamine and 2%-20% fetal bovine serum. The basic culture medium is DMEM medium containing 2%-10% fetal bovine serum or RPMI1640 medium containing 1% non-essential amino acid NEAA, 1% glutamine and 2%-20% fetal bovine serum.

2. The low serum medium for maintaining cell proliferation and increasing transfection efficiency according to claim 1, wherein, The culture medium comprises RPMI1640 medium containing 1% non-essential amino acid NEAA, 1% glutamine and 5% fetal bovine serum and the following components: 40 ng / mL IGF-1, 10 ng / mL IL-6, 5 ng / mL TGF-β, 100 μM VC, 10 ng / mL bFGF, 10 ng / mL G-CSF, 10 ng / mL GM-CSF.

3. The low serum medium for maintaining cell proliferation and increasing transfection efficiency according to claim 1, wherein, The culture medium comprises RPMI1640 medium containing 1% non-essential amino acid NEAA, 1% glutamine and 5% fetal bovine serum and the following components: 40 ng / mL IGF-1, 10 ng / mL IL-6, 5 ng / mL TGF-β, 10 ng / mL bFGF, 10 ng / mL G-CSF.

4. The low serum medium for maintaining cell proliferation and increasing transfection efficiency according to claim 1, wherein, The culture medium comprises DMEM medium containing 5% fetal bovine serum and the following components: 40 ng / mL IGF-1, 10 ng / mL IL-6, 5 ng / mL TGF-β, 10 ng / mL bFGF, 10 ng / mL G-CSF.

5. A low serum culture method for maintaining cell proliferation and improving transfection efficiency, comprising the following steps: 1) Take the longissimus dorsi muscle and leg muscle tissue to isolate porcine skeletal muscle satellite cells PSCs; culture in RPMI1640 medium containing 20% fetal bovine serum for 2-4 days; when the PSCs cell growth density is 80%-90%, use trypsin digestion to transfer and inoculate in a cell culture dish for standby; 2) Use the culture medium of claim 1 or 2 or 3 for cell proliferation culture after transfection, and verify the transfection efficiency.

6. The culture method according to claim 5, wherein The culture medium of claim 4 is used in step 2).

7. Use of a medium according to any one of claims 1 to 4 or based on the culture method according to claim 5 or 6, characterized in that The application is for culturing human and animal cells, including porcine skeletal muscle satellite cells, mouse myoblast cells C2C12, porcine kidney fibroblasts PKFs, mouse skeletal muscle satellite cells SSCs, 293T cells.

8. Use according to claim 7, characterized in that, It is also used for long-term proliferation and continuous passage culture of human and animal cells. It is also used for long-term proliferation and continuous passage culture of human and animal cells.

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