MRC-5 serum-free culture medium and preparation method thereofTechnical Field
The invention belongs to the field of cell culture, and relates to a serum-free MRC-5 cell culture medium with definite components and application thereof.
Background
Human diploid cells are cells with normal karyotypes, with non-oncogenic properties, consistent with the human genome. These cells do not contain any exogenous animal components or neurotoxins and are therefore widely used in vaccine production. The vaccine produced by using the cells requires fewer injections and has relatively mild adverse effects, thus being safe and effective. Human diploid cells are of great interest as an important cell matrix for the preparation of vaccines. The human diploid cell lines currently used for vaccine preparation are mainly 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).
MRC-5 cells are derived from human fetal lung tissue, are a human diploid cell line, and have various advantages, such as good proliferation capacity, strong proliferation stability, genetic characteristics closer to those of normal human cells, and the like. The method is widely applied to the fields of vaccine production, cytobiology research, genetics research, drug development and the like, and research at home and abroad is continuously advanced, so that the achievement is remarkable.
However, the medium for culturing human diploid cells requires the addition of bovine serum. Bovine serum, which is one of the most important components in cell culture fluid, contains various polypeptides, proteins, fats, carbohydrates, growth factors, hormones, inorganic salts, etc., and can provide necessary nutrients for cell growth. However, bovine serum is complex in components, pathogenic factors exist and are easy to pollute, exogenous factors contained in serum can cause adverse immune reaction of organisms, quality control of vaccine production can be influenced by difference of bovine serum in different batches, in addition, bovine serum is expensive and needs to be frozen for storage, and 10% serum concentration in a growth medium has high freezing storage requirement on vaccine production enterprises. It follows that the study and development of human diploid cell serum-free medium is of great importance for vaccine production.
The serum-free culture medium is a synthetic culture medium which is developed on the basis of a complete culture medium and can maintain the growth and propagation of cells in vitro for a long time without adding serum, and can not only meet the requirement of cell growth, but also avoid the adverse effect of animal serum. In the current production of vaccines with human diploid cells, conventional media containing low concentrations of serum are mainly used at home and abroad. Several techniques for producing serum-Free Medium have been developed at home and abroad (e.g., sigma-Aldrich, EX-CELL® EBxTM Serum-Free Medium, cat. No. 14531c;Gibco,Diploid Production Serum-Free Medium, cat. No. A3969002; gibco, gibcoTM VP-SFM, cat. No. 11681020). Although the technology for culturing diploid cells to produce vaccines by serum-free culture medium is broken through at present, the preparation technology is still immature, low-concentration serum with concentration of about 2% is often added in the use process of the culture medium, so that real serum-free culture cannot be realized, hidden danger is left for the safety of the vaccine, some serum-free culture medium can not provide certain nutritional ingredients, vitamins or other molecules for supporting cell growth existing in the serum, cell function can be poor, and moreover, the serum-free culture medium is usually expensive and can influence the research budget.
Disclosure of Invention
In view of the lack of a novel serum-free MRC-5 cell culture medium, the invention provides a novel serum-free MRC-5 cell culture medium, and the key technology of the invention is to develop a human diploid serum-free culture medium with proper nutrient components and scientific and effective concentration ratio, so as to truly realize the culture of human diploid cells under the serum-free condition and solve the technical problem of the lack of the novel serum-free MRC-5 cell culture medium in the prior art.
In a first aspect, the invention discloses a defined MRC-5 serum-free medium, which comprises amino acids, inorganic salts, vitamins, trace elements, proteins, other substances and supplements. Amino acids including 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 including bovine serum albumin, human transferrin and recombinant whole chain insulin, inorganic salts including calcium chloride, ferric nitrate, magnesium sulfate, potassium chloride, sodium bicarbonate, sodium chloride and sodium dihydrogen phosphate, vitamins including vitamin C, Choline chloride, D-calcium pantothenate, folic acid, nicotinamide, pyridoxine hydrochloride, riboflavin, ammonium sulfate hydrochloride and inositol, microelements comprise ammonium metavanadate, anhydrous copper sulfate, manganese chloride and sodium selenite, other substances comprise D-glucose, ethanolamine, phenol red, reduced glutathione and sodium pyruvate, and supplements comprise yam glycopeptide, linoleic acid, lipid mixture and insulin-like growth factor. Further, the concentration of each component in the serum-free medium is 1-5 mg/L, 6-12mg/L of L-alanine, 60-90 mg/L of L-arginine hydrochloride, 9-17mg/L of L-asparagine, 9-17mg/L of L-aspartic acid, 56-70mg/L of L-cysteine dihydrochloride, 13-15 mg/L of glutamine, 38-46 mg/L of L-histidine hydrochloride, 100-120 mg/L of L-isoleucine, 100-120 mg/L of L-leucine, 130-160 mg/L of L-lysine hydrochloride, 25-35 mg/L of L-methionine, 60-70 mg/L of L-proline, 10-12/L of L-proline, 47-60 mg/L of L-threonine, 85-100/L-mg/L of tryptophan, 15-mg/L of tyrosine, and 110-80/L of tyrosine. 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 300-400mg/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. Vitamin C2-6 mg/L, choline chloride 2-6mg/L, D-calcium pantothenate 2-6mg/L, folic acid 2-6mg/L, nicotinamide 2-6mg/L, pyridoxine hydrochloride 2-6mg/L, riboflavin 2-6mg/L, ammonium sulfate hydrochloride 0.2-0.6 mg/L, inositol 2-6 mg/L. The concentration of ammonium metavanadate is 0.1-0.3 mug/L, the concentration of anhydrous copper sulfate is 1-2 mug/L, the concentration of manganese chloride is 0.02-0.06 mug/L, the concentration of sodium selenite is 4-10 mug/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/L, and the concentration of insulin-like growth factor is 10-100ng/mL.
The invention discloses a preparation method of a defined MRC-5 serum-free culture medium, which comprises the steps of preparing high-concentration mother liquor from amino acid, inorganic salt, vitamin, trace elements, protein, other substances and supplements in the first aspect, completely dissolving and uniformly mixing, diluting each nutrient substance to a specified concentration by ultrapure water, completely dissolving and uniformly mixing, regulating osmotic pressure to 290 mOSM/kg-340 mOSM/kg by sodium chloride, regulating pH to 7.0-7.1 by concentrated hydrochloric acid and sodium hydroxide, and filtering and sterilizing by using a 0.22 mu m microporous filter membrane to obtain the MRC-5 serum-free culture medium.
The third aspect of the invention discloses the use of the serum-free medium in culturing cells, wherein the use comprises the steps of adding cells into the serum-free medium, and performing in-vitro adherence culture of the cells under the conditions of 37 ℃ and 5% CO2;
the cells include MRC-5 cells.
The fourth aspect of the invention discloses a preparation method of yam glycopeptide, which comprises the steps of weighing a certain amount of fresh yam, peeling, adding water according to a feed liquid ratio of 1:10, pulping and uniformly mixing, adjusting pH value to 8.6, adding alkaline protease accounting for 1.5% of the weight of the fresh yam, performing shaking table reaction at 49 ℃ to obtain enzymolysis liquid, performing enzyme deactivation in a 60 ℃ water bath, removing sediment by vacuum filtration to obtain yam glycopeptide crude extract, filtering by using a microporous filter membrane with the size of 0.45 mu m to obtain glycopeptide refined extract, detecting peptide fragment distribution of yam peptide by HPLC, wherein the peptide fragment size of yam peptide in the glycopeptide refined extract is mainly distributed in 180-1000 Da, selecting TSK gel chromatographic column with the detection wavelength of 220 nm, and the mobile phase acetonitrile water of trifluoroacetic acid of 45:55:0.1 with the flow rate of 0.5mL/min and the column temperature of 30 ℃, wherein the peptide fragment size of yam is 180-1000 Da.
Compared with the prior art, the invention has the following advantages:
The serum-free culture medium of MRC-5 cells provided by the invention is added with 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 synthesizing proteins, glutamine plays an important role in cell metabolism, and nitrogen contained in the glutamine is a basic source of purine and pyrimidine in synthetic nucleic acid and is an important component for synthesizing phosphatidylglycerol. Other amino acid components are optional amino acids, and in the case of low cell density, these amino acids have little effect on the growth of cells. However, when high density cell cultures are performed in bioreactors, the rate at which the cells synthesize these amino acids themselves often fails to meet their growth requirements, and thus, the provision of nonessential amino acids ensures normal proliferation of the cells.
The addition of inorganic salts into the serum-free medium of MRC-5 cells provided by the invention can play a very critical role in regulating the activities of metabolic pathways, enzymes and signal molecules. Calcium chloride can regulate cell membrane permeability, is favorable for metabolite discharge, ferric nitrate can maintain cell growth state, magnesium sulfate promotes synthesis of nucleic acid, potassium chloride and sodium chloride are substances essential for maintaining cell mass transportation and productivity, and sodium bicarbonate, dipotassium hydrogen phosphate and potassium dihydrogen phosphate can maintain normal osmotic pressure of cells.
In the serum-free culture medium of MRC-5 cells, folic acid, nicotinamide, pyridoxal, pantothenate, riboflavin and thiamine are all components of coenzyme, choline and inositol can be used as matrixes for lipid synthesis, and the high reducing power of vitamins can also protect cells from being damaged by oxidative free radicals.
The serum-free culture medium of MRC-5 cells provided by the invention is added with yam glycopeptide which is rich in antioxidant components, so that free radicals in the cells can be neutralized, the oxidation pressure can be reduced, the cells can be protected from damage, the proliferation of fibroblasts can be promoted, anabolism can be stimulated, and the nutrient absorption of the cells can be improved, thereby promoting the growth and repair of the cells.
The serum-free culture medium for MRC-5 cells comprises amino acid nutrient substances, inorganic salt nutrient substances and serum substitutes according to the formula proportion, wherein absorbance (OD450) at 450 nm measured after CCK-8 is added is taken as a response value, and the Plackett-Burman experimental design is used for respectively screening out the amino acid nutrient substances, the inorganic salt nutrient substances and the serum substitutes which affect the growth activity of the cells. After the important factors are screened, the steepest climbing experiment is carried out, and the influence on the growth activity of the cells is observed by gradually changing the concentration of the key factors, so that the optimal range of each factor is determined. After the concentration range is determined, the interaction and secondary effect of all important factors are explored by utilizing a central combination experiment, the positive influence of the probe on the cell growth is maximized, and a regression model is built to predict the influence of different factor combinations on the cell growth activity. And finally, verifying the optimal condition, repeating the cell growth experiment according to the optimal condition, and comparing with a control group to verify whether the quality of the culture medium is improved.
Drawings
For a clearer description of embodiments of the invention or of the prior art methods and technical solutions in experiments, the accompanying drawings, which are used in the embodiments, will be briefly described below, it being understood that the following drawings only illustrate certain embodiments of the invention and should not be considered as limiting the scope, and that other related drawings can be obtained from these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic diagram showing the growth and proliferation of MRC-5 cells after 5 passages of serial passage culture in foreign serum-free medium products.
FIG. 2 is a schematic representation of growth and proliferation after 5 passages of serial subculture in MRC-5 cells in a domestic serum-free medium product.
FIG. 3 is a schematic representation of the growth and proliferation of MRC-5 cells after 5 passages of serial subculture in serum-free medium provided in example 1.
FIG. 4 is a schematic representation of the growth and proliferation of MRC-5 cells after 5 passages of serial subculture in serum-free medium as provided in example 2.
FIG. 5 is a schematic representation of the growth and proliferation of MRC-5 cells after 5 passages of serial subculture in serum-free medium provided in example 3.
FIG. 6 is a schematic representation of the growth and proliferation of MRC-5 cells after 5 passages of serial subculture in serum-free medium provided in example 4.
FIG. 7 shows the results of cell viability assays of MRC-5 cells in different serum-free media.
Detailed Description
The technical solution of the present invention will be described in detail below with reference to the drawings and examples, but the present invention is not limited to the scope of the examples. The experimental methods and techniques, for which specific conditions are not noted in the following examples, are generally performed according to conventional conditions in the art or according to conditions suggested by manufacturers.
Example 1
According to the first formula of MRC-5 cell serum-free medium shown in Table 1, each nutrient mother liquor was diluted with ultrapure water to a specified concentration, completely dissolved and mixed uniformly, 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, and the culture was sterilized by filtration using a 0.22 μm microporous filter membrane to obtain MRC-5 serum-free medium.
TABLE 1 serum free Medium formulation of first MRC-5 cells
| Nutrient substances | Concentration of | Nutrient substances | Concentration of | Nutrient substances | Concentration of |
| Glycine (Gly) | 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-pantothenic acid calcium salt | 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 | Nicotinamide | 3 mg/L | Manganese chloride | 0.04 μg/L |
| L-histidine hydrochloride | 40 mg/L | Pyridoxine hydrochloride | 3 mg/L | Sodium selenite (Se-Na) | 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 (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 | Pyruvic acid sodium salt | 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 | Rhizoma Dioscoreae glycopeptide | 0 mg/L |
Example 2
The embodiment 2 of the invention provides a preparation method of a serum-free culture medium of MRC-5 cells, which comprises the following steps:
according to the first formula of MRC-5 cell serum-free medium shown in Table 2, each nutrient mother liquor was diluted with ultrapure water to a specified concentration, completely dissolved and mixed, osmotic pressure was adjusted to 310 mOSM/kg with sodium chloride, pH was adjusted to 7.0 with concentrated hydrochloric acid and sodium hydroxide, and filtered and sterilized with a 0.22 μm microporous filter membrane to obtain MRC-5 serum-free medium.
TABLE 2 serum free Medium formulation of the second MRC-5 cells
| Nutrient substances | Concentration of | Nutrient substances | Concentration of | Nutrient substances | Concentration of |
| Glycine (Gly) | 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-pantothenic acid calcium salt | 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 | Nicotinamide | 3 mg/L | Manganese chloride | 0.04 μg/L |
| L-histidine hydrochloride | 40 mg/L | Pyridoxine hydrochloride | 3 mg/L | Sodium selenite (Se-Na) | 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 (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 | Pyruvic acid sodium salt | 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 | Rhizoma Dioscoreae glycopeptide | 1 mg/L |
Example 3
The embodiment 3 of the invention provides a preparation method of a serum-free culture medium of MRC-5 cells, which comprises the following steps:
According to the first formula of MRC-5 cell serum-free medium shown in Table 3, each nutrient mother liquor was diluted with ultrapure water to a specified concentration, completely dissolved and mixed, osmotic pressure was adjusted to 310 mOSM/kg with sodium chloride, pH was adjusted to 7.0 with concentrated hydrochloric acid and sodium hydroxide, and filtered and sterilized with a 0.22 μm microporous filter membrane to obtain MRC-5 serum-free medium.
TABLE 3 serum free Medium formulation of third MRC-5 cells
| Nutrient substances | Concentration of | Nutrient substances | Concentration of | Nutrient substances | Concentration of |
| Glycine (Gly) | 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-pantothenic acid calcium salt | 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 | Nicotinamide | 3 mg/L | Manganese chloride | 0.04 μg/L |
| L-histidine hydrochloride | 50 mg/L | Pyridoxine hydrochloride | 3 mg/L | Sodium selenite (Se-Na) | 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 (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 | Pyruvic acid sodium salt | 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 | Rhizoma Dioscoreae glycopeptide | 1 mg/L |
Example 4
According to the first MRC-5 cell serum-free medium formulation shown in Table 4, each nutrient stock solution was diluted with ultrapure water to a specified concentration, completely dissolved and mixed, osmotic pressure was adjusted to 310 mOSM/kg with sodium chloride, pH was adjusted to 7.0 with concentrated hydrochloric acid and sodium hydroxide, and filtered and sterilized with a 0.22 μm microporous filter membrane to obtain MRC-5 serum-free medium.
TABLE 4 serum free Medium formulation for fourth MRC-5 cells
| Nutrient substances | Concentration of | Nutrient substances | Concentration of | Nutrient substances | Concentration of |
| Glycine (Gly) | 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-pantothenic acid calcium salt | 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 | Nicotinamide | 3 mg/L | Manganese chloride | 0.04 μg/L |
| L-histidine hydrochloride | 40 mg/L | Pyridoxine hydrochloride | 3 mg/L | Sodium selenite (Se-Na) | 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 (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 | Pyruvic acid sodium salt | 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 | Rhizoma Dioscoreae glycopeptide | 2 mg/L |
Experimental method
MRC-5 cells were inoculated into 6 cm dish at a density of 1X 105cell/cm2, and serial subcultures were performed in each of the groups of medium including example 1, example 2, example 3, example 4, domestic commercial serum-free medium, and foreign commercial serum-free medium, and cell morphology was observed every day from day 2, and after serial passage for 5 passages, cell images were collected at the same growth time.
To verify the effect of the 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, in general, in the process of passage of cells, the influence of a culture medium on the growth stability of the cells gradually appears along with the increase of passage times. When the MRC-5 cells are passaged to the 5 th generation, the cells are adapted to the culture environment, and the potential influence of the culture medium on the cell growth is accumulated to the extent that the cell growth can be effectively observed, so that after the MRC-5 cells are passaged to the 5 th generation, the growth state of the MRC-5 cells is observed by adopting conventional cell observation means such as a microscope, so that the stability of the culture medium on the cell growth is evaluated.
MRC-5 cells were passaged to 5 passages, and then inoculated into 96-well plates according to a density of 1X 105cell/cm2, using the commercial serum-free medium of example 1, example 2, example 3, example 4, domestic commercial serum-free medium, and foreign commercial serum-free medium, respectively, until the cells grew to 70% -90%, adding CCK-8 reagent to detect absorbance at 450: 450 nm as a blank, detecting absorbance values again after 2h, and taking the difference between the two as cell viability.
Experimental results
As shown in FIGS. 1-6, the normal growth of cells can be observed in the culture media of example 1, example 2, example 3 and example 4, the cells show fiber-length and fiber-forming state under a microscope, the growth state reaches or even exceeds the domestic and foreign serum-free culture medium level, the cell viability measurement result is consistent with the cell growth state result, and the yam glycopeptide with the concentration of 1 mg/L is additionally added under the premise of keeping the concentration of other nutrient substances consistent in example 2 compared with example 1. Comparison of the cell culture conditions and cell viability of example 2 with those of example 1 under the same experimental culture time conditions shows that the cell density and cell viability in the cell culture system corresponding to example 2 are significantly higher than those of example 1. From this, it was confirmed that the addition of yam glycopeptides has a positive effect on promoting cell proliferation and increasing cell density under the conditions set in the present experiment. Examples 3 and 4 adjusted the concentration of yam glycopeptides added, and also adjusted the concentration of the corresponding amino acid nutrients in order to maintain the balance of osmotic pressure and nutrients in the system, and experimental results show that examples 3 and 4 also showed better growth status (as well as here) and growth activity of the cells relative to example 1, indicating that yam peptides can become promoting components of nutrients in the culture medium.
The foregoing examples are illustrative of the present invention, but the present invention is not limited to the foregoing examples, and any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principles of the present invention should be made in equivalent manner, and are also within the scope of the present invention.