Culture medium containing egg yolk extract as well as preparation method and application of culture medium

Abstract

The invention discloses a culture medium containing a yolk extract as well as a preparation method and application of the culture medium, and belongs to the technical field of cell biology. The core of the culture medium is that a basic culture medium is combined with specifically prepared yolk extract and yolk small molecule peptide. Wherein the yolk small molecule peptide is prepared by the following steps: homogenizing yolk, performing alkaline protease enzymolysis, performing enzyme deactivation, centrifuging, performing 5kDa ultrafiltration on supernate, collecting filtrate, performing 500Da nanofiltration for further treatment, and drying; the yolk extract is prepared by centrifugally filtering and sterilizing membrane-removed yolk. The culture medium is used for polyethyleneimine transfection, the cytotoxicity can be remarkably reduced through the synergistic effect of the yolk extract and the specific yolk small molecule peptide, and the cell survival rate and the transfection efficiency after transfection are improved. Therefore, the invention provides a safe, efficient and serum-free culture medium formula.

Description

A culture medium containing egg yolk extract, its preparation method and application Technical Field

[0001] This invention belongs to the field of cell biology technology, and particularly relates to a culture medium containing egg yolk extract, its preparation method, and its application. Background Technology

[0002] With the rapid development of biotechnology, especially gene therapy, protein expression, and cell engineering, gene transfection technology has become a core method for efficiently introducing exogenous nucleic acids (such as DNA and RNA) into target cells, playing a crucial role in basic research, drug development, and clinical treatment. Currently, gene transfection methods are mainly divided into two categories: viral vector methods and non-viral vector methods.

[0003] While viral vector methods offer high transfection efficiency, they also present safety risks such as strong immunogenicity, insertional mutation risk, complex preparation, and potential pathogenicity, limiting their widespread clinical application. In contrast, non-viral vector methods offer advantages such as high safety, low immunogenicity, and ease of large-scale preparation. Among these, cationic polymer polyethyleneimine (PEI), rich in amino groups, has become one of the most commonly used non-viral transfection reagents, capable of forming stable complexes with nucleic acids and promoting endocytosis and nucleic acid release. However, PEI exhibits significant cytotoxicity in practical applications, leading to a substantial decrease in cell viability after transfection. Therefore, developing serum-free or natural alternative culture media has become an industry trend. Egg yolk extract, a natural substance rich in phospholipids, proteins, peptides, and growth factors, has been explored in some serum-free media, but current research mainly focuses on its role in promoting cell proliferation and adhesion; there are no reports of its use in reducing PEI transfection toxicity. Summary of the Invention

[0004] The purpose of this invention is to provide a culture medium containing egg yolk extract and a method for preparing the same.

[0005] To achieve the above objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides a culture medium containing egg yolk extract, wherein the culture medium is composed of a basal culture medium, egg yolk extract and egg yolk small molecule peptides; the concentration of the egg yolk small molecule peptides is 200 μg / mL-1000 μg / mL; and the content of the egg yolk extract is 10-15% of the total volume of the culture medium.

[0006] Preferably, the preparation method of the egg yolk small molecule peptide is as follows: (1) After separating the egg yolk, place it in a container, add deionized water at a ratio of egg yolk mass: deionized water mass = 1:10, and stir to form a uniform egg yolk suspension; (2) Adjust the egg yolk suspension to a suitable temperature and pH for alkaline protease, add alkaline protease at 0.5% of the egg yolk raw material mass, and enzymatically hydrolyze at a constant temperature to obtain egg yolk hydrolysate; (3) Heat the egg yolk hydrolysate to inactivate the enzyme, cool it naturally to room temperature, and collect the supernatant by centrifugation; (4) Filter using a 5kDa ultrafiltration membrane and collect filtrate a; (5) Filter using a 500Da nanofiltration membrane and collect filtrate b; (6) Freeze-dry filtrate b to obtain egg yolk small molecule peptide.

[0007] Preferably, the preparation method of the egg yolk extract is as follows: 1) Take fresh egg yolks, remove the outer yolk membrane, add 1-2 times the weight of the egg yolks in sterile water, and stir evenly; 2) Use a homogenizer to homogenize at 10,000-12,000 rpm for 2-3 minutes until the mixture forms a uniform emulsion, and obtain a homogenate; 3) Freeze the homogenate at -20℃ for more than 12 hours, and then thaw at 4℃, repeating twice; 4) Place it in a centrifuge and centrifuge at 8000-10000 rpm for 15-20 minutes, and collect the supernatant; 5) Pre-filter the supernatant through a 0.45μm filter membrane, and then filter it through a 0.22μm filter membrane for sterilization to obtain the egg yolk extract.

[0008] Preferably, the culture medium is used to reduce the decrease in cell viability caused by polyethyleneimine transfection and / or to improve the efficiency of polyethyleneimine transfection.

[0009] Preferably, the basal culture medium consists of MEM medium, L-glutamine, HEPES and N-acetylcysteine.

[0010] Preferably, in the basal culture medium, the concentration of L-glutamine is 2 mM, the concentration of HEPES is 10 mM, and the concentration of N-acetylcysteine ​​is 0.5 mM.

[0011] Secondly, the present invention provides the use of a composition in preparing a culture medium that reduces cytotoxicity during polyethyleneimine transfection and improves cell survival and / or transfection efficiency after transfection, the composition comprising egg yolk small molecule peptides and egg yolk extract; the egg yolk small molecule peptides are prepared by the preparation method of claim 2; the egg yolk extract is prepared by the preparation method of claim 3.

[0012] Preferably, the concentration of the egg yolk small molecule peptide in the culture medium is 200 μg / mL.

[0013] Preferably, the content of the egg yolk extract in the culture medium is 10%.

[0014] Thirdly, the present invention provides a method for preparing a culture medium containing egg yolk extract, wherein each 100 ml of the method comprises the following steps: (a) in a biosafety cabinet, take 90 mL of MEM into a sterile bottle, slowly add 20 mg of egg yolk small molecule peptide, and stir until completely dissolved; (b) add auxiliary components: L-glutamine to a final concentration of 2 mM, HEPES to a final concentration of 10 mM, and N-acetylcysteine ​​to a final concentration of 0.5 mM, and filter and sterilize; (c) add 10 mL of egg yolk extract, gently mix, and obtain the culture medium.

[0015] The beneficial effects of this invention are as follows: This invention provides a culture medium containing egg yolk extract, which consists of a basal medium, a specifically prepared egg yolk extract, and egg yolk small molecule peptides, and exhibits significant advantages during polyethyleneimine (PEI) transfection. On the one hand, it significantly reduces the cytotoxicity of PEI transfection and greatly improves cell survival rate, significantly outperforming the control group which was supplemented with egg yolk extract, egg yolk small molecule peptides, or fetal bovine serum alone. This indicates that the specific combination of egg yolk extract and egg yolk small molecule peptides has a significant synergistic protective effect.

[0016] On the other hand, the culture medium of the present invention enhances cell survival while increasing PEI transfection efficiency to 89.50%, overcoming the drawback of traditional culture media (such as those containing FBS) that may reduce transfection efficiency. In summary, the present invention provides a highly efficient and low-toxicity transfection culture medium that can significantly and synergistically improve cell survival and transfection efficiency in PEI-mediated gene transfection. Attached Figure Description

[0017] Figure 1 shows the cell survival rate of HEK293 cells transfected with PEI under different culture conditions; Figure 2 shows the transfection efficiency of HEK293 cells transfected with PEI under different culture conditions. Detailed Implementation

[0018] Example 1: A culture medium a containing egg yolk extract. The composition of culture medium a is as follows: 90 mL MEM basal medium (Gibco), 10 mL egg yolk extract, 200 μg / mL egg yolk small molecule peptide a, 2 mM glutamine, 10 mM HEPES, 0.5 mM N-acetylcysteine.

[0019] The preparation method of egg yolk small molecule peptide a is as follows: (1) Take fresh eggs (Hy-Line brown eggs, laying date less than 3 days), wipe the shell with disinfectant alcohol, separate the egg yolk with egg yolk and egg white separator and weigh it; (2) Place the separated egg yolk in a clean beaker, slowly add deionized water according to the mass ratio of egg yolk mass: deionized water = 1:10, stir thoroughly to form a uniform egg yolk suspension; (3) Heat the egg yolk suspension to 55℃, adjust the pH to 8.5, add alkaline protease according to 0.5% of the egg yolk raw material mass, and enzymatically hydrolyze for 4 hours at constant temperature to obtain egg yolk hydrolysate; (4) After the enzymatic hydrolysis is completed, heat the egg yolk hydrolysate to 95℃ and keep it for 15 minutes to completely inactivate the protease; (5) After naturally cooling to room temperature, obtain the hydrolysate, transfer the cooled hydrolysate to a centrifuge, centrifuge at 4000 rpm for 15 minutes, and collect the supernatant; (6) Add the supernatant to the ultrafiltration system, use 5kDa (7) Filter the solution using a Merck Millipore ultrafiltration membrane (25°C, 0.25 MPa) and collect filtrate a; (8) Add the filtrate to a nanofiltration system and filter it using a 500 Da nanofiltration membrane (Synder Filtration) (25°C, 1.0 MPa) and collect filtrate b; (9) Freeze-dry the collected filtrate b to obtain egg yolk small molecule peptide a.

[0020] The preparation method of egg yolk extract is as follows: 1) Take fresh egg yolks, remove the outer yolk membrane, add sterile water at twice the weight of the egg yolks, and stir evenly; 2) Homogenize at 10,000 rpm for 3 minutes using a homogenizer until the mixture forms a uniform emulsion; 3) Freeze the homogenate at -20℃ for more than 12 hours, and then thaw at 4℃, repeating twice; 4) Place in a centrifuge and centrifuge at 8000 rpm for 20 minutes, collecting the supernatant; 5) Pre-filter the supernatant through a 0.45μm filter membrane, and then filter through a 0.22μm filter membrane for sterilization to obtain egg yolk extract.

[0021] The preparation method of culture medium a is as follows: (1) In a biosafety cabinet, take 90 mL of MEM into a sterile bottle, slowly add 20 mg of egg yolk small molecule peptide a, and stir until completely dissolved; (2) Add auxiliary components: L-glutamine to a final concentration of 2 mM, HEPES to a final concentration of 10 mM, N-acetylcysteine ​​to a final concentration of 0.5 mM, filter and sterilize; (3) Add 10 mL of egg yolk extract, mix gently to obtain culture medium a.

[0022] Example 2 A culture medium b containing egg yolk extract. The composition of culture medium b is as follows: 90 mL MEM basal medium, 10 mL egg yolk extract, 200 μg / mL egg yolk small molecule peptide b, 2 mM L-glutamine, 10 mM HEPES, 0.5 mM N-acetylcysteine.

[0023] The preparation method of egg yolk small molecule peptide B is as follows: (1) Take fresh eggs (Hy-Line Brown eggs, laying date less than 3 days), wipe the shell with disinfectant alcohol, separate the egg yolk with egg yolk and egg white separator and weigh it; (2) Place the separated egg yolk in a clean beaker, slowly add deionized water according to the mass ratio of egg yolk mass: deionized water = 1:10, stir thoroughly to form a uniform egg yolk suspension; (3) Heat the egg yolk suspension to 55℃, adjust the pH to 8.0, add alkaline protease according to 0.5% of the egg yolk raw material mass, and enzymatically hydrolyze for 4 hours at constant temperature to obtain egg yolk hydrolysate; (4) After the enzymatic hydrolysis is completed, heat the egg yolk hydrolysate to 95℃ and keep it for 15 minutes to completely inactivate the protease; (5) After naturally cooling to room temperature, obtain the hydrolysate, transfer the cooled hydrolysate to a centrifuge, centrifuge at 4000 rpm for 15 minutes, and collect the supernatant; (6) Add the supernatant to the ultrafiltration system, use 3kDa (7) The filtrate was filtered using an ultrafiltration membrane (Merck Millipore) at 25°C and 0.25 MPa, and the filtrate was collected. The filtrate was then added to a nanofiltration system and filtered using a nanofiltration membrane (Synder Filtration) at 1000 Da (25°C and 1.0 MPa), and the concentrate was collected. The collected concentrate was then freeze-dried to obtain egg yolk small molecule peptides.

[0024] The preparation method of culture medium b is the same as in Example 1.

[0025] Example 3 A culture medium c containing egg yolk extract The composition of culture medium c is as follows: 90 mL MEM basal medium, 10 mL egg yolk extract, 200 μg / mL egg yolk small molecule peptide c, 2 mM L-glutamine, 10 mM HEPES, 0.5 mM N-acetylcysteine.

[0026] The preparation method of egg yolk small molecule peptide C is as follows: (1) Take fresh eggs (Hy-Line brown eggs, laying date less than 3 days), wipe the shell with disinfectant alcohol, separate the egg yolk with egg yolk and egg white separator and weigh it; (2) Place the separated egg yolk in a clean beaker, slowly add deionized water according to the mass ratio of egg yolk mass: deionized water = 1:10, stir thoroughly to form a uniform egg yolk suspension; (3) Heat the egg yolk suspension to 55℃, adjust the pH to 8.0, add alkaline protease according to 0.5% of the egg yolk raw material mass, and enzymatically hydrolyze for 4 hours at constant temperature to obtain egg yolk hydrolysate; (4) After the enzymatic hydrolysis is completed, heat the egg yolk hydrolysate to 95℃ and keep it for 15 minutes to completely inactivate the protease; (5) After naturally cooling to room temperature, obtain the hydrolysate, transfer the cooled hydrolysate to a centrifuge, centrifuge at 4000 rpm for 15 minutes, and collect the supernatant; (6) Add the supernatant to the ultrafiltration system, use 1kDa The solution was filtered through an ultrafiltration membrane (Merck Millipore) at 25°C and 0.25 MPa to collect the concentrate; (7) the collected concentrate was freeze-dried to obtain egg yolk small molecule peptide c.

[0027] The preparation method of culture medium c is the same as in Example 1.

[0028] Comparative Example 1: Basal Control Culture Medium. The composition of the basal control culture medium is as follows: 90 mL MEM basal culture medium, 10 mL fetal bovine serum, 2 mM L-glutamine, 10 mM HEPES, and 0.5 mM N-acetylcysteine.

[0029] The preparation method of the basic control culture medium is the same as in Example 1.

[0030] Comparative Example 2: Control medium a. The composition of control medium a is as follows: 90 mL MEM basal medium, 10 mL egg yolk extract, 2 mM L-glutamine, 10 mM HEPES, and 0.5 mM N-acetylcysteine.

[0031] The preparation method of control culture medium a is the same as in Example 1.

[0032] Comparative Example 3: Control medium b. The composition of control medium b is as follows: 100 mL MEM basal medium, 200 μg / mL egg yolk small molecule peptide a, 2 mM L-glutamine, 10 mM HEPES, 0.5 mM N-acetylcysteine.

[0033] The preparation method of control culture medium b is the same as in Example 1.

[0034] Comparative Example 4: The composition of control medium c is as follows: 90 mL MEM basal medium, 200 μg / mL egg yolk small molecule peptide a, 10 mL FBS, 2 mM L-glutamine, 10 mM HEPES, and 0.5 mM N-acetylcysteine.

[0035] Example 4 Experimental materials: Cell line: HEK293 cells (ATCC CRL-1573), cultured in DMEM containing 10% FBS.

[0036] Transfection reagent: PEI (Sigma-Aldrich, 408719).

[0037] Plasmid: pEGFP-N1 (4.7 kb, Clontech), concentration 1 mg / mL.

[0038] Experimental methods: (1) HEK293 cells were inoculated at 1×10 4 Cells / well were seeded at a density of 6 replicates per well in 96-well plates and cultured in DMEM containing 10% FBS for 24 hours until confluence reached 70-80%. The old culture medium was discarded and the cells were washed once with PBS.

[0039] (2) Prepare a mixture containing 20 μg pEGFP-N1 plasmid and 100 μg in a stepwise manner. 5000 μl of serum-free MEM was vortexed and incubated at room temperature for 20 minutes to form a PEI / DNA complex; (3) The culture medium was discarded and the PEI / DNA complex was added to a 96-well plate, 50 μl per well. The culture medium was supplemented according to the following groups: Experimental treatment group 1: 50 μL of culture medium a was added; Experimental treatment group 2: 50 μL of culture medium b was added; Experimental treatment group 3: 50 μL of culture medium c was added; Control treatment group 1: 50 μL of serum-free MEM culture medium was added; Control treatment group 2: 50 μL of control culture medium a was added; Control treatment group 3: 50 μL of basic control culture medium was added; Control treatment group 4: 50 μL of control culture medium b was added; Control treatment group 5: 50 μL of control culture medium c was added; At the same time, a non-transfected control group was set up, which only added MEM culture medium and did not add PEI / DNA; 4 hours after transfection, the culture medium was discarded, washed once with PBS, and the corresponding culture medium was added again and cultured for another 20 hours.

[0040] (4) 24 hours after transfection, add 20 μL of MTT solution to each well and incubate at 37°C for 4 hours; (5) carefully aspirate the supernatant, add 150 μL of DMSO to each well to dissolve the crystals, shake on a shaker for 10 minutes, and measure the absorbance at 570 nm wavelength using an enzyme-linked immunosorbent assay reader.

[0041] Table 1. Cell viability of PEI-transfected HEK293 cells under different culture conditions

[0042] As shown in Table 1, the survival rate of the serum-free control was 75.58%. The results of control groups 2, 3 and 4 show that egg yolk extract, FBS and egg yolk small molecule peptide a can all reduce PEI-induced cytotoxicity to a certain extent, but the effect of adding them alone is relatively limited.

[0043] The cell survival rate of control group 5 increased by 18.62%, providing better protection, but it still exhibited high cytotoxicity.

[0044] In experimental treatment groups 1, 2, and 3, the cell survival rate of experimental treatment group 1 was significantly improved, while the improvement in cell survival rate of experimental treatment groups 2 and 3 was significantly reduced. This indicates that among the three egg yolk small molecule peptides, only egg yolk small molecule peptide a has a significant synergistic effect when used in combination with egg yolk extract, while egg yolk small molecule peptides b and c do not have this effect. Simultaneously, the cell survival rate of experimental treatment group 1 was also significantly higher than that of control treatment group 5, indicating that egg yolk small molecule peptide a needs to be present simultaneously with the egg yolk extract of this invention to exert a significant protective effect.

[0045] Experimental materials for Example 5: Same as those for Example 4.

[0046] Experimental methods (1) HEK293 cells were inoculated at 1×10 5 Seed cells per well into 12-well plates, add 1 mL of DMEM containing 10% FBS to each well and incubate for 24 hours until confluence reaches 70-80%. Discard the old culture medium and wash once with PBS.

[0047] (2) Prepare 10 ml of serum-free MEM containing 40 μg pEGFP-N1 plasmid and 200 μg PEI in a stepwise manner, vortex mix, and incubate at room temperature for 20 minutes to form PEI / DNA complex; (3) Discard the culture medium, add 200 μl of PEI / DNA complex to each well of a 12-well plate, and supplement the culture medium according to the following groups: Experimental treatment group 1: supplement 200 μL of culture medium a; Experimental treatment group 2: supplement 200 μL of culture medium b; Experimental treatment group 3: supplement 200 μL of culture medium c; Control treatment group 1: supplement 200 μL of serum-free MEM culture medium; Control treatment group 2: supplement 200 μL of control culture medium a; Control treatment group 3: supplement 200 μL of basal control culture medium; Control treatment group 4: supplement 200 μL of control culture medium b; Control treatment group 5: supplement 200 μL of control culture medium c; At the same time, set up a non-transfected control group, which only adds MEM culture medium and does not add PEI / DNA; Each group is set up with 3 replicates, and the experiment is repeated 3 times. Four hours after the first transfection, the culture medium was discarded, the cells were washed once with PBS, and 1 mL of the corresponding culture medium was added again. The cells were then cultured for another 44 hours.

[0048] (4) Remove the culture medium, wash the cells once with pre-cooled PBS, add 100 μL of EDTA-free trypsin to each well, and incubate at 37°C for 2 minutes until the cells detach.

[0049] (5) Add 500 μL of PBS containing 1% BSA to each well to stop digestion and transfer the cell suspension to a round-bottom tube.

[0050] (6) Centrifuge the cell tubes at 300g for 5 minutes, discard the supernatant, and resuspend the cells in 500μL of flow cytometry buffer; (7) Use a flow cytometer, set the 488nm laser and FITC / GFP channel, and calculate the transfection positive cell rate.

[0051] Table 2. Transfection efficiency of HEK293 cells with PEI under different culture conditions.

[0052] As can be seen from the results in Table 2, compared with control group 1, the transfection efficiency of control group 3 with added FBS was significantly lower than that of the control group, while control group 2 with added egg yolk extract was slightly higher than that of control group 1. This indicates that the egg yolk extract used in this invention effectively improves cell survival rate without affecting transfection efficiency.

[0053] Meanwhile, compared with control group 1, the transfection effect of experimental group 1 was significantly improved, indicating that the combined use of egg yolk small molecule peptide a and egg yolk extract can not only effectively enhance the survival ability of transfected cells, but also effectively enhance the transfection efficiency.

Claims

1. A culture medium containing egg yolk extract, characterized in that, The culture medium consists of a basal culture medium, egg yolk extract, and egg yolk small molecule peptides; the concentration of the egg yolk small molecule peptides is 200 μg / mL-1000 μg / mL; and the content of the egg yolk extract is 10-15% of the total volume of the culture medium.

2. The culture medium according to claim 1, characterized in that, The preparation method of the egg yolk small molecule peptide is as follows: (1) After separating the egg yolk, place it in a container and add deionized water at a ratio of 1:10 (egg yolk mass: deionized water mass = 1:10). Stir to form a uniform egg yolk suspension; (2) Adjust the egg yolk suspension to the appropriate temperature and pH for alkaline protease, add alkaline protease at 0.5% of the egg yolk raw material mass, and enzymatically hydrolyze at a constant temperature to obtain egg yolk hydrolysate; (3) Heat the egg yolk hydrolysate to inactivate the enzyme, cool it naturally to room temperature, and collect the supernatant by centrifugation; (4) Filter using a 5kDa ultrafiltration membrane and collect filtrate a. (5) Filter using a nanofiltration membrane with a molecular weight of 500 Da and collect filtrate b; (6) Freeze-dry filtrate b to obtain egg yolk small molecule peptides.

3. The culture medium according to claim 2, characterized in that, The preparation method of the egg yolk extract is as follows: 1) Take fresh egg yolks, remove the outer yolk membrane, add 1-2 times the weight of the egg yolks in sterile water, and stir evenly; 2) Use a homogenizer to homogenize at 10,000-12,000 rpm for 2-3 minutes until the mixture forms a uniform emulsion, and obtain a homogenate; 3) Freeze the homogenate at -20℃ for more than 12 hours, and then thaw at 4℃, repeating twice; 4) Place it in a centrifuge and centrifuge at 8000-10000 rpm for 15-20 minutes, and collect the supernatant; 5) Pre-filter the supernatant through a 0.45μm filter membrane, and then filter it through a 0.22μm filter membrane for sterilization to obtain the egg yolk extract.

4. The culture medium according to claim 3, characterized in that, The culture medium is used to improve the cell viability reduction caused by polyethyleneimine transfection and / or improve the efficiency of polyethyleneimine transfection.

5. The culture medium according to claim 4, characterized in that, The basal culture medium consists of MEM medium, L-glutamine, HEPES and N-acetylcysteine.

6. The culture medium according to claim 5, characterized in that, In the basal culture medium, the concentration of L-glutamine is 2 mM, the concentration of HEPES is 10 mM, and the concentration of N-acetylcysteine ​​is 0.5 mM.

7. The use of a composition in the preparation of a culture medium that reduces cytotoxicity during polyethyleneimine transfection and improves cell viability and / or transfection efficiency after transfection, characterized in that, The composition comprises egg yolk small molecule peptides and egg yolk extract; the egg yolk small molecule peptides are prepared by the preparation method of claim 2; the egg yolk extract is prepared by the preparation method of claim 3.

8. The application according to claim 7, characterized in that, In the culture medium, the concentration of the egg yolk small molecule peptide is 200 μg / mL.

9. The application according to claim 8, characterized in that, The egg yolk extract content in the culture medium is 10%.

10. A method for preparing a culture medium containing egg yolk extract, characterized in that, The method per 100 ml comprises the following steps: (a) In a biosafety cabinet, take 90 mL of MEM into a sterile bottle, slowly add 20 mg of egg yolk small molecule peptides, and stir until completely dissolved; (b) Add auxiliary components: L-glutamine to a final concentration of 2 mM, HEPES to a final concentration of 10 mM, and N-acetylcysteine ​​to a final concentration of 0.5 mM, and filter and sterilize; (c) Add 10 mL of egg yolk extract, gently mix, and obtain the culture medium.

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