MRC-5 serum-free medium and preparation method thereof

Abstract

The invention belongs to the field of cell culture, and relates to an MRC-5 cell serum-free medium with definite components and application thereof. Nutrient substances of the serum-free culture medium comprise amino acids, vitamins, inorganic salts, trace elements, proteins, other substances and supplements. According to the MRC-5 cell serum-free culture medium, the concentration of the amino acid, the inorganic salt, the vitamin and the serum substitute is adjusted, and meanwhile, the Chinese yam glycopeptide is added, so that adherent growth of MRC-5 cells can be effectively promoted, and the technical problem that a new variety of MRC-5 cell serum-free culture medium is lacked in the prior art is solved; the requirements of nutrient substances, carbon sources and genetic material nutrient substances required by MRC-5 cell in-vitro culture can be met.

Description

Technical Field

[0001] The invention belongs to the field of cell culture and relates to a serum-free culture medium for MRC-5 cells with defined components and application thereof. Background Art

[0002] Human diploid cells have a normal karyotype, are non-oncogenic, and are consistent with the human genome. These cells contain no exogenous animal components or neurotoxins and are therefore widely used in vaccine production. Vaccines produced using these cells require fewer injections and have relatively mild adverse reactions, making them safe and effective. As a key cell matrix for vaccine production, human diploid cells have garnered widespread attention. The main human diploid cell lines currently used in vaccine production include WI-38 (human embryonic lung cells), MRC-5 (human embryonic lung fibroblasts), 2BS (human embryonic lung cells), and KMB-17 (human embryonic lung diploid cells).

[0003] MRC-5 cells, derived from human fetal lung tissue, are a diploid human cell line with numerous advantages, including excellent proliferation capacity, robust proliferation stability, and genetic characteristics closer to those of normal human cells. They have been widely used in a variety of fields, including vaccine production, cell biology research, genetics research, and drug development. Research continues to advance both domestically and internationally, with significant results.

[0004] However, all culture media for culturing human diploid cells require the addition of bovine serum. As one of the most important components in cell culture media, bovine serum contains a variety of peptides, proteins, fats, carbohydrates, growth factors, hormones, and inorganic salts, providing essential nutrients for cell growth. However, bovine serum has a complex composition, contains pathogenic factors, and is susceptible to contamination. Exogenous factors contained in the serum can cause adverse immune responses, and differences between batches of bovine serum can affect quality control in vaccine production. Furthermore, bovine serum is expensive and requires refrigerated storage. A 10% serum concentration in growth media places high demands on vaccine manufacturers for refrigerated storage. Therefore, research and development of serum-free culture media for human diploid cells is crucial for vaccine production.

[0005] Serum-free culture medium is a synthetic culture medium developed on the basis of complete culture medium. It can maintain the growth and reproduction of cells in vitro for a long time without adding serum. It can meet the needs of cell growth while avoiding the adverse effects of animal serum. At present, in the process of producing vaccines using human diploid cells at home and abroad, conventional culture medium containing low concentration of serum is mainly used. Some serum-free culture medium production technologies have been developed at home and abroad (such as: Sigma-Aldrich, EX-CELL ®EBx™ Serum-Free Medium, Catalog No. 14531c; Gibco, Diploid Production Serum-Free Medium, Catalog No. A3969002; Gibco, Gibco™ VP-SFM, Catalog No. 11681020). Although breakthroughs have been made in using serum-free media to culture diploid cells for vaccine production, the production technology remains immature. Low-concentration serum, approximately 2%, is often added to the culture medium during use, preventing true serum-free culture and posing a safety risk to the vaccine. Some serum-free media may not provide certain nutrients, vitamins, or other molecules found in serum that support cell growth, potentially leading to poor cell function. Furthermore, these serum-free media are often expensive, potentially impacting research budgets. Summary of the Invention

[0006] In view of the current lack of new serum-free MRC-5 cell culture media, the present invention proposes a new serum-free MRC-5 cell culture media. The key technology of the present invention is to develop a human diploid serum-free culture media with appropriate nutritional components and scientifically effective concentration ratios, truly realizing human diploid cell culture under serum-free conditions, and solving the technical problem of the lack of new serum-free MRC-5 cell culture media in the existing technology.

[0007] The first aspect of the present invention discloses a MRC-5 serum-free culture medium with defined ingredients; the MRC-5 serum-free culture medium with defined ingredients comprises amino acids, inorganic salts, vitamins, trace elements, proteins, other substances and supplements. Amino acids include glycine, L-alanine, L-cysteine ​​dihydrochloride, glutamine, L-histidine hydrochloride, L-arginine hydrochloride, L-asparagine, L-aspartic acid, L-isoleucine, L-leucine, L-lysine hydrochloride, L-serine, L-threonine, L-tryptophan, L-methionine, L-phenylalanine, L-proline, L-tyrosine and L-valine; proteins include bovine serum albumin, human transferrin and recombinant full-chain insulin; inorganic salts include chlorine, Calcium sulfate, ferric nitrate, magnesium sulfate, potassium chloride, sodium bicarbonate, sodium chloride and sodium dihydrogen phosphate; vitamins include vitamin C, choline chloride, D-calcium pantothenate, folic acid, niacinamide, pyridoxine hydrochloride, riboflavin, ammonium sulfate hydrochloride and inositol; trace elements include ammonium metavanadate, anhydrous copper sulfate, manganese chloride and sodium selenite; other substances include: D-glucose, ethanolamine, phenol red, reduced glutathione and sodium pyruvate; supplements include yam glycopeptide, linoleic acid, lipid mixture and insulin-like growth factor. Furthermore, the concentrations of the components in the serum-free medium are as follows: glycine concentration is 1-5 mg / L, L-alanine concentration is 6-12 mg / L, L-arginine hydrochloride concentration is 60-90 mg / L, L-asparagine concentration is 9-17 mg / L, L-aspartic acid concentration is 9-17 mg / L, L-cysteine ​​dihydrochloride concentration is 56-70 mg / L, glutamine concentration is 13-15 mg / L, L-histidine hydrochloride concentration is 38-46 mg / L, L-isoleucine concentration is 100-120 mg / L, L-leucine concentration is 100-120 mg / L, L-lysine hydrochloride concentration is 130-160 mg / L, L-methionine concentration is 25-35 mg / L, L-phenylalanine concentration is 60-70 mg / L, and L-proline concentration is 10-12 mg / L, L-serine concentration is 47-60 mg / L, L-threonine concentration is 85-100 mg / L, L-tryptophan concentration is 15-18 mg / L, L-tyrosine concentration is 90-110 mg / L, L-valine concentration is 80-100 mg / L. Calcium chloride concentration is 180-220 mg / L, ferric nitrate concentration is 0.05-0.25 mg / L, magnesium sulfate concentration is 90-100 mg / L, potassium chloride concentration is 300-400 mg / L, sodium bicarbonate concentration is 3500-4000 mg / L, sodium chloride concentration is 6000-7000 mg / L, and sodium dihydrogen phosphate concentration is 100-150 mg / L.The concentration of vitamin C is 2-6 mg / L, the concentration of choline chloride is 2-6 mg / L, the concentration of D-pantothenate calcium is 2-6 mg / L, the concentration of folic acid is 2-6 mg / L, the concentration of nicotinamide is 2-6 mg / L, the concentration of pyridoxine hydrochloride is 2-6 mg / L, the concentration of riboflavin is 2-6 mg / L, the concentration of ammonium sulfate hydrochloride is 0.2-0.6 mg / L, and the concentration of inositol is 2-6 mg / L. The concentration of ammonium metavanadate is 0.1-0.3 μg / L, the concentration of anhydrous copper sulfate is 1-2 μg / L, the concentration of manganese chloride is 0.02-0.06 μg / L, the concentration of sodium selenite is 4-10 μg / L, the concentration of bovine serum albumin is 200-600 mg / L, the concentration of human transferrin is 5-10 mg / L, the concentration of recombinant full-chain insulin is 5-10 mg / L, the concentration of D-glucose is 4000-6000 mg / L, the concentration of ethanolamine is 1-3 mg / L, the concentration of reduced glutathione is 1-3 mg / L, the concentration of sodium pyruvate is 100-150 mg / L, the concentration of yam glycopeptide is 1-5 mg / L, the concentration of linoleic acid is 0.2-2 ng / mL, the concentration of lipid mixture is 0.5-5 ng / mL, and the concentration of insulin-like growth factor is 10-100 ng / mL.

[0008] The second aspect of the present invention discloses a method for preparing a MRC-5 serum-free culture medium with a defined composition, the preparation method comprising: configuring the amino acids, inorganic salts, vitamins, trace elements, proteins, other substances and supplements described in the first aspect into a high-concentration mother liquor, and completely dissolving and mixing; diluting each nutrient to a specified concentration with ultrapure water, and completely dissolving and mixing; adjusting the osmotic pressure to 290 mOSM / kg to 340 mOSM / kg with sodium chloride; adjusting the pH to 7.0-7.1 with concentrated hydrochloric acid and sodium hydroxide; and filtering and sterilizing with a 0.22 μm microporous filter membrane to obtain the MRC-5 serum-free culture medium.

[0009] The third aspect of the present invention discloses the use of the above-mentioned serum-free medium in culturing cells, which comprises adding cells to the serum-free medium and culturing the cells in vitro at 37°C and 5% CO2; The cells include MRC-5 cells.

[0010] The fourth aspect of the present invention discloses a method for preparing the above-mentioned yam glycopeptide, which comprises: weighing a certain amount of fresh yam, peeling it, adding water at a material-liquid ratio of 1:10, beating and mixing, adjusting the pH to 8.6, adding 1.5% alkaline protease by weight of the fresh yam, and reacting on a shaker at 49°C for 8 hours to obtain an enzymatic hydrolyzate; after inactivating the enzyme in a 60°C water bath, vacuum filtration is performed to remove the precipitate to obtain a crude yam glycopeptide extract; filtering with a 0.45 μm microporous filter membrane to obtain a glycopeptide extract, and using HPLC to detect the peptide distribution of the yam peptide. The peptide size of the yam peptide in the glycopeptide extract is mainly distributed in the range of 180 to 1000 Da. A TSK gel chromatography column is selected, the detection wavelength is 220 nm, the mobile phase is acetonitrile: water: trifluoroacetic acid = 45:55:0.1, the flow rate is 0.5 mL / min, and the column temperature is 30°C. The present invention selects yam glycopeptides with a peptide size of 180 to 1000 Da.

[0011] Compared with the prior art, the present invention has the following advantages: The serum-free culture medium of MRC-5 cells provided by the present invention contains L-glutamine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-threonine, L-tryptophan and L-valine, which are essential amino acids for protein synthesis. Glutamine plays an important role in cell metabolism. The nitrogen it contains is the basic source of purines and pyrimidines in the synthesis of nucleic acids, and is also an important component for the synthesis of phosphoglycerol. Other amino acid components are non-essential amino acids. When the cell density is low, these amino acids have little effect on the growth of the cells. However, when high-density cell culture is carried out in a bioreactor, the speed at which the cells synthesize these amino acids by themselves often cannot meet their growth requirements. Therefore, providing non-essential amino acids can ensure normal cell proliferation.

[0012] The serum-free culture medium for MRC-5 cells provided by the present invention includes inorganic salts, which play a crucial role in regulating metabolic pathways, enzyme, and signaling molecule activities. Calcium chloride regulates cell membrane permeability, facilitating the excretion of metabolites. Ferric nitrate maintains cell growth. Magnesium sulfate promotes nucleic acid synthesis. Potassium chloride and sodium chloride are essential for maintaining cellular material transport and energy production. Sodium bicarbonate, dipotassium hydrogen phosphate, and potassium dihydrogen phosphate maintain normal cellular osmotic pressure.

[0013] The serum-free culture medium for MRC-5 cells provided by the present invention contains folic acid, nicotinamide, pyridoxal, pantothenate, riboflavin and thiamine, all of which are components of coenzymes; choline and inositol can serve as substrates for lipid synthesis; and the high reducing ability of vitamins can also protect cells from damage by oxidative free radicals.

[0014] The serum-free culture medium for MRC-5 cells provided by the present invention contains yam glycopeptide, which is rich in antioxidant components and can help neutralize free radicals in cells, reduce oxidative stress, and thus protect cells from damage; it can also promote the proliferation of fibroblasts; stimulate anabolism, and improve cell nutrient absorption, thereby promoting cell growth and repair.

[0015] The serum-free culture medium for MRC-5 cells provided by the present invention comprises amino acid nutrients, inorganic salt nutrients and serum substitutes, and the absorbance at 450 nm (OD 450 ) as the response value, the Plackett-Burman experimental design was used to screen out some amino acid nutrients, inorganic salt nutrients and serum replacements that affect cell growth viability. After screening out the important factors, a steepest climbing experiment was performed to gradually change the concentration of the key factors and observe the effect on cell growth viability, so as to determine the optimal range of each factor. After determining the concentration range, the central combination experiment was used to explore the interaction and quadratic effects of each important factor, maximize the positive impact on cell growth, and thus establish a regression model to predict the effect of different factor combinations on cell growth viability. Finally, the optimal conditions were verified, the cell growth experiment was repeated according to the optimal conditions, and compared with the control group to verify whether the quality of the culture medium has been improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the existing methods and experiments, the following is a brief introduction to the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0017] Figure 1 This is a schematic diagram of the growth and proliferation of MRC-5 cells after five consecutive passages in a foreign serum-free culture medium product.

[0018] Figure 2 This is a schematic diagram of the growth and proliferation of MRC-5 cells after five consecutive passages in a domestic serum-free culture medium product.

[0019] Figure 3 This is a schematic diagram of the growth and proliferation of MRC-5 cells after five consecutive passages in the serum-free medium provided in Example 1.

[0020] Figure 4 This is a schematic diagram of the growth and proliferation of MRC-5 cells after five consecutive passages in the serum-free medium provided in Example 2.

[0021] Figure 5 This is a schematic diagram of the growth and proliferation of MRC-5 cells after five consecutive passages in the serum-free medium provided in Example 3.

[0022] Figure 6 This is a schematic diagram of the growth and proliferation of MRC-5 cells after five consecutive passages in the serum-free medium provided in Example 4.

[0023] Figure 7 These are the results of cell viability assay of MRC-5 cells in different serum-free culture media. DETAILED DESCRIPTION

[0024] The technical solutions of the present invention are described in detail below with reference to the accompanying drawings and examples, but the present invention is not limited to the scope of the examples. Experimental methods and techniques in the following examples where specific conditions are not specified are generally performed in accordance with conventional conditions in the art or the conditions recommended by the manufacturer.

[0025] Example 1 According to the first serum-free medium formula for MRC-5 cells shown in Table 1, the nutrient stock solutions were diluted to the specified concentrations with ultrapure water and completely dissolved and mixed. The osmotic pressure was adjusted to 310 mOSM / kg with sodium chloride. The pH was adjusted to 7.0 with concentrated hydrochloric acid and sodium hydroxide. The medium was sterilized by filtration using a 0.22 μm microporous filter to obtain MRC-5 serum-free medium.

[0026] Table 1: Formula of serum-free culture medium for the first MRC-5 cell line nutrients concentration nutrients concentration nutrients concentration Glycine 36 mg / L L-Tyrosine 104 mg / L Sodium dihydrogen phosphate 120 mg / L L-Alanine 9 mg / L L-Valine 94 mg / L Bovine serum albumin 100 mg / L L-Arginine hydrochloride 80 mg / L Vitamin C 2.5 mg / L Human transferrin 10 mg / L L-Asparagine 13 mg / L Choline chloride 3 mg / L Recombinant full-chain insulin 5 mg / L L-Aspartic Acid 13 mg / L D-Calcium Pantothenate 3 mg / L Ammonium metavanadate 0.2 μg / L L-Cysteine ​​dihydrochloride 60 mg / L folic acid 3 mg / L Anhydrous copper sulfate 2 μg / L glutamine 15 mg / L Niacinamide 3 mg / L Manganese chloride 0.04 μg / L L-Histidine Hydrochloride 40 mg / L Pyridoxine hydrochloride 3 mg / L Sodium selenite 4 μg / L L-Isoleucine 105 mg / L Riboflavin 0.3 mg / L D-glucose 4450 mg / L L-Leucine 105 mg / L Ammonium sulfate hydrochloride 3 mg / L Ethanolamine 1 mg / L L-Lysine Hydrochloride 140 mg / L Inositol 6 mg / L Reduced glutathione 1 mg / L L-Methionine 30 mg / L calcium chloride 150 mg / L Phenol red 15 mg / L L-Phenylalanine 60 mg / L Ferric nitrate 0.1 mg / L Sodium pyruvate 100 mg / L L-Proline 10 mg / L magnesium sulfate 95 mg / L Linoleic acid 2 ng / mL L-Serine 55 mg / L potassium chloride 380 mg / L lipid mixture 2 ng / mL L-Threonine 95 mg / L Sodium bicarbonate 3700 mg / L Insulin-like growth factor 60 ng / mL L-Tryptophan 14 mg / L Sodium chloride 6500 mg / L Yam glycopeptide 0 mg / L Example 2 Example 2 of the present invention provides a method for preparing a serum-free culture medium for MRC-5 cells, the preparation method comprising: According to the first serum-free medium formula for MRC-5 cells shown in Table 2, the nutrient stock solutions were diluted with ultrapure water to the specified concentrations and completely dissolved and mixed. The osmotic pressure was adjusted to 310 mOSM / kg with sodium chloride. The pH was adjusted to 7.0 with concentrated hydrochloric acid and sodium hydroxide. The medium was sterilized by filtration using a 0.22 μm microporous filter to obtain MRC-5 serum-free medium.

[0027] Table 2: Second serum-free culture medium formula for MRC-5 cells nutrients concentration nutrients concentration nutrients concentration Glycine 36 mg / L L-Tyrosine 104 mg / L Sodium dihydrogen phosphate 120 mg / L L-Alanine 9 mg / L L-Valine 94 mg / L Bovine serum albumin 100 mg / L L-Arginine hydrochloride 80 mg / L Vitamin C 2.5 mg / L Human transferrin 10 mg / L L-Asparagine 13 mg / L Choline chloride 3 mg / L Recombinant full-chain insulin 5 mg / L L-Aspartic Acid 13 mg / L D-Calcium Pantothenate 3 mg / L Ammonium metavanadate 0.2 μg / L L-Cysteine ​​dihydrochloride 60 mg / L folic acid 3 mg / L Anhydrous copper sulfate 2 μg / L glutamine 15 mg / L Niacinamide 3 mg / L Manganese chloride 0.04 μg / L L-Histidine Hydrochloride 40 mg / L Pyridoxine hydrochloride 3 mg / L Sodium selenite 4 μg / L L-Isoleucine 105 mg / L Riboflavin 0.3 mg / L D-glucose 4450 mg / L L-Leucine 105 mg / L Ammonium sulfate hydrochloride 3 mg / L Ethanolamine 1 mg / L L-Lysine Hydrochloride 140 mg / L Inositol 6 mg / L Reduced glutathione 1 mg / L L-Methionine 30 mg / L calcium chloride 150 mg / L Phenol red 15 mg / L L-Phenylalanine 60 mg / L Ferric nitrate 0.1 mg / L Sodium pyruvate 100 mg / L L-Proline 10 mg / L magnesium sulfate 95 mg / L Linoleic acid 2 ng / mL L-Serine 55 mg / L potassium chloride 380 mg / L lipid mixture 2 ng / mL L-Threonine 95 mg / L Sodium bicarbonate 3700 mg / L Insulin-like growth factor 60 ng / mL L-Tryptophan 14 mg / L Sodium chloride 6500 mg / L Yam glycopeptide 1 mg / L Example 3 Example 3 of the present invention provides a method for preparing a serum-free culture medium for MRC-5 cells, the preparation method comprising: According to the first serum-free medium formula for MRC-5 cells shown in Table 3, the nutrient stock solutions were diluted with ultrapure water to the specified concentrations and completely dissolved and mixed. The osmotic pressure was adjusted to 310 mOSM / kg with sodium chloride. The pH was adjusted to 7.0 with concentrated hydrochloric acid and sodium hydroxide. The medium was sterilized by filtration using a 0.22 μm microporous filter to obtain MRC-5 serum-free medium.

[0028] Table 3: Formula of serum-free culture medium for the third type of MRC-5 cells nutrients concentration nutrients concentration nutrients concentration Glycine 34 mg / L L-Tyrosine 104 mg / L Sodium dihydrogen phosphate 110 mg / L L-Alanine 9 mg / L L-Valine 94 mg / L Bovine serum albumin 100 mg / L L-Arginine hydrochloride 75 mg / L Vitamin C 2.5 mg / L Human transferrin 10 mg / L L-Asparagine 13 mg / L Choline chloride 3 mg / L Recombinant full-chain insulin 5 mg / L L-Aspartic Acid 13 mg / L D-Calcium Pantothenate 3 mg / L Ammonium metavanadate 0.2 μg / L L-Cysteine ​​dihydrochloride 60 mg / L folic acid 3 mg / L Anhydrous copper sulfate 2 μg / L glutamine 15 mg / L Niacinamide 3 mg / L Manganese chloride 0.04 μg / L L-Histidine Hydrochloride 50 mg / L Pyridoxine hydrochloride 3 mg / L Sodium selenite 4 μg / L L-Isoleucine 105 mg / L Riboflavin 0.3 mg / L D-glucose 4450 mg / L L-Leucine 105 mg / L Ammonium sulfate hydrochloride 3 mg / L Ethanolamine 1 mg / L L-Lysine Hydrochloride 140 mg / L Inositol 6 mg / L Reduced glutathione 1 mg / L L-Methionine 30 mg / L calcium chloride 140 mg / L Phenol red 15 mg / L L-Phenylalanine 60 mg / L Ferric nitrate 0.1 mg / L Sodium pyruvate 100 mg / L L-Proline 10 mg / L magnesium sulfate 95 mg / L Linoleic acid 2 ng / mL L-Serine 65 mg / L potassium chloride 350 mg / L lipid mixture 3 ng / mL L-Threonine 95 mg / L Sodium bicarbonate 3700 mg / L Insulin-like growth factor 70 ng / mL L-Tryptophan 12 mg / L Sodium chloride 6550 mg / L Yam glycopeptide 1 mg / L Example 4 According to the first serum-free medium formula for MRC-5 cells shown in Table 4, the nutrient stock solutions were diluted with ultrapure water to the specified concentrations and completely dissolved and mixed. The osmotic pressure was adjusted to 310 mOSM / kg with sodium chloride. The pH was adjusted to 7.0 with concentrated hydrochloric acid and sodium hydroxide. The medium was sterilized by filtration using a 0.22 μm microporous filter to obtain MRC-5 serum-free medium.

[0029] Table 4: Formula of serum-free culture medium for the fourth type of MRC-5 cells nutrients concentration nutrients concentration nutrients concentration Glycine 36 mg / L L-Tyrosine 104 mg / L Sodium dihydrogen phosphate 120 mg / L L-Alanine 9 mg / L L-Valine 94 mg / L Bovine serum albumin 100 mg / L L-Arginine hydrochloride 80 mg / L Vitamin C 2.5 mg / L Human transferrin 10 mg / L L-Asparagine 13 mg / L Choline chloride 3 mg / L Recombinant full-chain insulin 5 mg / L L-Aspartic Acid 13 mg / L D-Calcium Pantothenate 3 mg / L Ammonium metavanadate 0.2 μg / L L-Cysteine ​​dihydrochloride 60 mg / L folic acid 3 mg / L Anhydrous copper sulfate 2 μg / L glutamine 15 mg / L Niacinamide 3 mg / L Manganese chloride 0.04 μg / L L-Histidine Hydrochloride 40 mg / L Pyridoxine hydrochloride 3 mg / L Sodium selenite 4 μg / L L-Isoleucine 105 mg / L Riboflavin 0.3 mg / L D-glucose 4450 mg / L L-Leucine 105 mg / L Ammonium sulfate hydrochloride 3 mg / L Ethanolamine 1 mg / L L-Lysine Hydrochloride 140 mg / L Inositol 6 mg / L Reduced glutathione 1 mg / L L-Methionine 30 mg / L calcium chloride 150 mg / L Phenol red 15 mg / L L-Phenylalanine 60 mg / L Ferric nitrate 0.1 mg / L Sodium pyruvate 100 mg / L L-Proline 10 mg / L magnesium sulfate 95 mg / L Linoleic acid 2 ng / mL L-Serine 55 mg / L potassium chloride 380 mg / L lipid mixture 2 ng / mL L-Threonine 95 mg / L Sodium bicarbonate 3700 mg / L Insulin-like growth factor 60 ng / mL L-Tryptophan 14 mg / L Sodium chloride 6500 mg / L Yam glycopeptide 2 mg / L Experimental methods MRC-5 cells were cultured at a rate of 1×10 5 cell / cm 2 The cells were seeded at a density of 100 μg / cm2 in a 6 cm dish, and the culture media of Example 1, Example 2, Example 3, Example 4, domestic commercial serum-free culture medium, and foreign commercial serum-free culture medium were added for continuous subculture. Cell morphology was observed every day starting from the second day. After five consecutive subcultures, cell images were collected at the same growth time.

[0030] To verify the effect of the culture medium on cell growth stability, MRC-5 cells were subcultured. Based on a large amount of experimental data and general experience in the field of cell culture, the effect of the culture medium on cell growth stability will gradually become apparent as the number of subcultures increases during the subculture process. When the cells are subcultured to the fifth generation, the cells have adapted to the culture environment and the potential effect of the culture medium on cell growth has accumulated to a level that can be effectively observed. Therefore, after the MRC-5 cells are subcultured to the fifth generation, their growth status is observed using conventional cell observation methods such as microscopy to evaluate the stability of the culture medium on cell growth.

[0031] After the MRC-5 cells were passaged to the fifth generation, 1×10 5 cell / cm 2The cells were inoculated into a 96-well plate using Example 1, Example 2, Example 3, Example 4, a domestic commercial serum-free culture medium, and a foreign commercial serum-free culture medium, respectively. After the cells grew to 70%-90%, CCK-8 reagent was added to detect the absorbance at 450 nm as a blank. After 2 h, the absorbance value was detected again, and the difference between the two was taken as the cell viability.

[0032] Experimental results The results are as follows Figures 1-6 As shown, normal cell growth was observed in the culture media of Examples 1, 2, 3, and 4. Under a microscope, the cells exhibited elongated, fibrous growth, reaching or even exceeding the levels of serum-free culture media both domestically and internationally. Cell viability assay results were consistent with those of cell growth. Compared to Example 1, Example 2 added yam glycopeptide at a concentration of 1 mg / L, while maintaining the same concentrations of other nutrients. Comparison of the cell culture conditions and cell viability of Example 2 and Example 1 under the same experimental incubation time revealed that the cell density and cell viability in the cell culture system corresponding to Example 2 were significantly higher than those in Example 1. This demonstrates that, under the conditions established in this experiment, the addition of yam glycopeptide has a positive effect on promoting cell proliferation and increasing cell density. In Examples 3 and 4, the concentration of yam glycopeptide was adjusted, and the concentrations of the corresponding amino acid nutrients were adjusted to maintain the balance between the system's osmotic pressure and nutrients. The experimental results showed that the cells in Examples 3 and 4 also exhibited better growth (the same applies here) and viability compared to those in Example 1, demonstrating that yam peptide can be a nutrient-enhancing component in the culture medium.

[0033] The above embodiments are preferred implementations of the present invention, but the implementations of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and also fall within the scope of protection of the present invention and are included in the scope of protection of the present invention.

Claims

1. A serum-free medium with defined composition, characterized in that The serum-free medium with defined composition includes amino acids, proteins, inorganic salts, vitamins, trace elements, other substances and supplements; Amino acids include glycine, L-alanine, L-cysteine ​​dihydrochloride, glutamine, L-histidine hydrochloride, L-arginine hydrochloride, L-asparagine, L-aspartic acid, L-isoleucine, L-leucine, L-lysine hydrochloride, L-serine, L-threonine, L-tryptophan, L-methionine, L-phenylalanine, L-proline, L-tyrosine, and L-valine; Proteins included bovine serum albumin, human transferrin, and recombinant full-chain insulin; Inorganic salts include calcium chloride, ferric nitrate, magnesium sulfate, potassium chloride, sodium bicarbonate, sodium chloride, and sodium dihydrogen phosphate; Vitamins include vitamin C, choline chloride, D-calcium pantothenate, folic acid, niacinamide, pyridoxine hydrochloride, riboflavin, ammonium sulfate hydrochloride, and inositol; Trace elements include ammonium metavanadate, anhydrous copper sulfate, manganese chloride, and sodium selenite; Other substances include: D-glucose, ethanolamine, phenol red, reduced glutathione, and sodium pyruvate; The supplement includes yam glycopeptides, linoleic acid, a lipid blend, and insulin-like growth factor.

2. The serum-free culture medium according to claim 1, wherein The concentration of each amino acid component is: The concentration of glycine is 1-5 mg / L, the concentration of L-alanine is 6-12 mg / L, the concentration of L-arginine hydrochloride is 60-90 mg / L, the concentration of L-asparagine is 12-14 mg / L, the concentration of L-aspartic acid is 12-14 mg / L, the concentration of L-cysteine ​​dihydrochloride is 56-70 mg / L, the concentration of glutamine is 13-15 mg / L, the concentration of L-histidine hydrochloride is 38-46 mg / L, the concentration of L-isoleucine is 94-116 mg / L, the concentration of L-leucine is 94-116 mg / L, the concentration of L-lysine hydrochloride is 130-160 mg / L, the concentration of L-methionine is 25-35 mg / L, the concentration of L-phenylalanine is 60-70 mg / L, the concentration of L-proline is 10-12 mg / L, and the concentration of L-serine is 47-60 mg / L, L-threonine concentration is 85-100 mg / L, L-tryptophan concentration is 15-18 mg / L, L-tyrosine concentration is 90-110 mg / L, and L-valine concentration is 80-100 mg / L.

3. The serum-free culture medium according to claim 1, wherein The concentration of each component of the inorganic salt is: The concentration of calcium chloride is 180-220 mg / L, the concentration of ferric nitrate is 0.05-0.25 mg / L, the concentration of magnesium sulfate is 90-100 mg / L, the concentration of potassium chloride is 300-400 mg / L, the concentration of sodium bicarbonate is 3500-4000 mg / L, the concentration of sodium chloride is 6000-7000 mg / L, and the concentration of sodium dihydrogen phosphate is 100-150 mg / L.

4. The serum-free culture medium according to claim 1, wherein The concentration of each vitamin component is: The concentration of vitamin C is 2-6 mg / L, the concentration of choline chloride is 2-6 mg / L, the concentration of D-pantothenate calcium is 2-6 mg / L, the concentration of folic acid is 2-6 mg / L, the concentration of nicotinamide is 2-6 mg / L, the concentration of pyridoxine hydrochloride is 2-6 mg / L, the concentration of riboflavin is 2-6 mg / L, the concentration of ammonium sulfate hydrochloride is 0.2-0.6 mg / L, and the concentration of inositol is 2-6 mg / L.

5. The serum-free culture medium according to claim 1, wherein The concentration of each component of the trace elements is: The concentration of ammonium metavanadate is 0.1-0.3 μg / L, the concentration of anhydrous copper sulfate is 1-2 μg / L, the concentration of manganese chloride is 0.02-0.06 μg / L, and the concentration of sodium selenite is 4-10 μg / L.

6. The serum-free culture medium according to claim 1, wherein The concentrations of the various components of the protein are: The concentration of bovine serum albumin is 200-600 mg / L, the concentration of human transferrin is 5-10 mg / L, and the concentration of recombinant full-chain insulin is 5-10 mg / L.

7. The serum-free culture medium according to claim 1, wherein The concentration of each component of the other substances is: The concentration of D-glucose is 4000-6000 mg / L, the concentration of ethanolamine is 1-3 mg / L, the concentration of reduced glutathione is 1-3 mg / L, and the concentration of sodium pyruvate is 100-150 mg / L.

8. The serum-free culture medium according to claim 1, wherein The concentrations of the ingredients in the supplement are: The concentration of yam glycopeptide is 1-5 mg / L, the concentration of linoleic acid is 0.2-2 ng / mL, the concentration of lipid mixture is 0.5-5 ng / mL, and the concentration of insulin-like growth factor is 10-100 ng / mL.

9. The method for preparing the serum-free medium according to any one of claims 1 to 8, wherein: The specific steps are as follows: (1) Glycine, L-alanine, L-cysteine ​​dihydrochloride, glutamine, L-histidine hydrochloride, L-arginine hydrochloride, L-asparagine, L-aspartic acid, L-isoleucine, L-leucine, L-lysine hydrochloride, L-serine, L-threonine, L-tryptophan, L-methionine, L-phenylalanine, L-proline, L-tyrosine and L-valine; inorganic salts including sodium bicarbonate, sodium chloride, sodium dihydrogen phosphate, calcium chloride, ferric nitrate, magnesium sulfate, potassium chloride, vitamin C, choline chloride, D-calcium pantothenate, folic acid, nicotinamide, pyridoxine hydrochloride, riboflavin, ammonium sulfate hydrochloride, inositol, ammonium metavanadate, anhydrous copper sulfate, manganese chloride, sodium selenite, bovine serum albumin, human transferrin, recombinant full-chain insulin, D-glucose, ethanolamine, phenol red, reduced glutathione and sodium pyruvate are prepared into a high concentration stock solution and completely dissolved and mixed; (2) Dilute each nutrient with ultrapure water to the specified concentration and completely dissolve and mix; (3) Adjust the osmotic pressure to 290 mOSM / kg to 340 mOSM / kg with sodium chloride; (4) Adjust the pH to 7.0-7.1 with concentrated hydrochloric acid and sodium hydroxide; (5) Filter and sterilize using a 0.22 μm microporous filter to obtain serum-free culture medium, which was stored at 4°C.

10. Use of the serum-free medium according to any one of claims 1 to 8 in culturing cells, characterized in that: The use includes adding cells to the serum-free culture medium and performing in vitro cell adherence culture at 37° C. and 5% CO 2 ; The cells are MRC-5 cells.

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