CN110628697A - Serum-free culture medium for VERO serum-free cell culture and corresponding virus production - Google Patents

Serum-free culture medium for VERO serum-free cell culture and corresponding virus production Download PDF

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CN110628697A
CN110628697A CN201910900379.4A CN201910900379A CN110628697A CN 110628697 A CN110628697 A CN 110628697A CN 201910900379 A CN201910900379 A CN 201910900379A CN 110628697 A CN110628697 A CN 110628697A
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serum
sodium
vero
free
cell culture
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齐智
孟丹丹
房圆瑗
刘海英
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Shandong Oracle Biotechnology Co Ltd
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Shandong Oracle Biotechnology Co Ltd
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Abstract

The invention discloses a serum-free culture medium for VERO serum-free cell culture and corresponding virus production, which comprises the following components: amino acids, vitamins, inorganic salts, auxiliary components and proteins, wherein the amino acids comprise glycine, alanine, arginine hydrochloride, cystine dihydrochloride, glutamic acid, glutamine, histidine monohydrochloride, isoleucine, leucine, lysine hydrochloride, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine disodium salt dihydrate, valine, hydroxyproline, ornithine and taurine; the protein comprises human transferrin and recombinant insulin; when the culture medium prepared by the invention is used for VERO cell culture, the use of animal serum is not relied on, and animal source protein is not introduced, so that the production cost is greatly reduced, the stable product quality is fully ensured, and the long-time culture and passage can be realized without increasing the apoptosis ratio.

Description

Serum-free culture medium for VERO serum-free cell culture and corresponding virus production
Technical Field
The invention relates to the field of culture media, in particular to a serum-free culture medium for VERO serum-free cell culture and corresponding virus production.
Background
The VERO cell serum-free culture production vaccine has become the current mainstream trend, accords with the adherence characteristic of the VERO cell and improves the cell density, and the design and optimization of the serum-free chemically defined culture medium are the key of the VERO cell serum-free culture technology.
But most of the current commercial serum-free culture media have low universality, are not widely applied, have no universality and have high manufacturing cost, and are not beneficial to commercial development and application; meanwhile, VERO cells grow in a small quantity in a serum-free culture medium, the later-stage continuity is poor, and the cells are easy to die during continuous passage.
Disclosure of Invention
The object of the present invention is to provide a serum-free medium for VERO serum-free cell culture and production of the corresponding virus, which solves the problems set forth in the background art mentioned above.
In order to achieve the purpose, the invention provides the following technical scheme:
a serum-free medium for VERO serum-free cell culture and production of the corresponding virus, comprising the following components: amino acid, vitamin, inorganic salt, auxiliary component and protein, wherein the amino acid comprises glycine 15-20mg/L, alanine 2-5mg/L, arginine hydrochloride 150-300mg/L, asparagine monohydrate 20-60mg/L, aspartic acid 5-10mg/L, cysteine monohydrate 15-20mg/L, cystine dihydrochloride 25-75mg/L, glutamic acid 5-10mg/L, glutamine 100-200mg/L, histidine monohydrate 20-40mg/L, isoleucine 25-75mg/L, leucine 25-75mg/L, lysine hydrochloride 60-95mg/L, methionine 50-75mg/L, phenylalanine 20-40mg/L, arginine hydrochloride, methionine, 15-20mg/L of proline, 180mg/L of serine, 40-60mg/L of threonine, 5-10mg/L of tryptophan, 200mg/L of tyrosine disodium salt dihydrate, 25-60mg/L of valine, 10-15mg/L of hydroxyproline, 40-60mg/L of ornithine and 50-75mg/L of taurine; the protein comprises 10-15mg/L of human transferrin, 5-10mg/L of recombinant insulin, 4000mg/L, KERRY of 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) 2000-500 mg/L of ultrafiltration phytone peptone (key 4601N)100-500mg/L and 500mg/L of Angel yeast powder (Angel ultra).
As a further scheme of the invention: the vitamins comprise 40-60mg/L ascorbic acid, 1-5mg/L sodium ascorbyl phosphonate, 0.1-0.5mg/L biotin, 5-10mg/L, D-calcium pantothenate, 1-5mg/L folic acid, 1-5mg/L nicotinic acid, 1-5mg/L pyridoxal hydrochloride, 0.1-0.5mg/L, B12 riboflavin, 1-5mg/L, i-inositol, 10-15mg/L vitamin K and 30.05-0.2mg/L vitamin K.
As a still further scheme of the invention: the inorganic salts comprise 150-200mg/L of anhydrous calcium chloride, 50-100mg/L of anhydrous magnesium sulfate, 200-400mg/L of potassium chloride, 1000-3000mg/L of sodium bicarbonate, 5000-6000mg/L of sodium chloride, 130-150mg/L of monobasic sodium phosphate, 50-80mg/L of anhydrous disodium hydrogen phosphate, 0.001-0.003mg/L of blue vitriol, 0.5-1mg/L of ferrous sulfate heptahydrate, 50-60mg/L of anhydrous magnesium chloride and 0.1-0.6mg/L of zinc sulfate heptahydrate.
As a still further scheme of the invention: the auxiliary components comprise 3000mg/L of D-glucose 1000-1, 5-10mg/L of phenol red, 0.005-0.01mg/L of putrescine dihydrochloride, 90-120mg/L of sodium pyruvate, 1-5mg/L of reduced glutathione, 0.0001-0.0005mg/L of ammonium metavanadate, 0.00001-0.00005mg/L of manganese dichloride, 0.005-0.01mg/L of sodium selenite, 0.01-0.5mg/L of adenine, 0.01-0.5mg/L of sodium lactate, 1-10mg/L of serotonin, 200mg/L of fructose 100-1, 0.005-0.01mg/L of oligopeptide-1 (BGF), 0.005-0.01mg/L of human oligopeptide-1 (EGF), 0.005-0.01mg/L of platelet derived growth factor-BB (PDGF-BB), 15-20mg/L of urea and thymidine
0.1-0.5mg/L。
As a still further scheme of the invention: comprises the following components: 18.75mg/L glycine, 4.45mg/L alanine, 247.5mg/L arginine hydrochloride, 57.5mg/L asparagine monohydrate, 6.65mg/L aspartic acid, 17.56mg/L cysteine hydrochloride monohydrate, 46.29mg/L cystine dihydrochloride, 7.35mg/L glutamic acid, 190mg/L glutamine, 31.48mg/L histidine hydrochloride monohydrate, 54.47mg/L isoleucine, 59.05mg/L leucine, 91.25mg/L lysine hydrochloride, 67.24mg/L methionine, 35.48mg/L phenylalanine, 17.25mg/L proline, 176.25mg/L serine, 53.45mg/L threonine, 9.02mg/L tryptophan, 275.79mg/L tyrosine disodium salt dihydrate, 52.85mg/L valine, 13.1mg/L hydroxyproline, 50.58mg/L ornithine, Taurine 62.5mg/L, ascorbic acid 50mg/L, sodium ascorbyl phosphonate 2.5mg/L, biotin 0.5mg/L, choline chloride 8.98mg/L, D-calcium pantothenate 6.24mg/L, folic acid 2.65mg/L, nicotinic acid 2.02mg/L, pyridoxal hydrochloride 2.013mg/L, riboflavin 0.219mg/L, B12 vitamin 2.17mg/L, i-inositol 12.6mg/L, vitamin K30.1mg/L, anhydrous calcium chloride 170mg/L, anhydrous magnesium sulfate 97.67mg/L, potassium chloride 311.8mg/L, sodium bicarbonate 1200mg/L, sodium chloride 5800mg/L, monosodium phosphate monohydrate 140mg/L, disodium hydrogen phosphate 71.02mg/L, cupric sulfate pentahydrate 0.0023mg/L, ferrous sulfate heptahydrate 0.917mg/L, anhydrous magnesium chloride 53.64mg/L, 0.532mg/L, D mg of zinc sulfate heptahydrate-1000 mg/L of glucose, 8.1mg/L of phenol red, 0.0081mg/L of putrescine dihydrochloride, 110mg/L of sodium pyruvate, 4mg/L of reduced glutathione, 0.0003mg/L of ammonium metavanadate, 0.00005mg/L of manganese dichloride, 0.01mg/L of sodium selenite, 0.365mg/L of adenine, 0.2mg/L of sodium lactate, 5.5mg/L of serotonin, 196.87mg/L of fructose, 0.01mg/L of oligopeptide-1 (BGF), 0.01mg/L of human oligopeptide-1 (EGF), 0.01mg/L of platelet-derived growth factor-BB (PDGF-BB), and 17mg/L of urea
0.365mg/L of thymidine, 15mg/L of human transferrin, 10mg/L of recombinant insulin, 3574.5mg/L, kerry4601N 300mg/L of 4-hydroxyethyl piperazine ethanesulfonic acid (HEPES) and 150mg/L of Angel ultra.
As a still further scheme of the invention: the human transferrin is saturated iron human transferrin.
As a still further scheme of the invention: the recombinant insulin is a full-chain recombinant insulin.
Compared with the prior art, the invention has the beneficial effects that:
when the culture medium prepared by the invention is used for VERO cell culture, the use of animal serum is not relied on, and animal source protein is not introduced, so that the production cost is greatly reduced, the stable product quality is fully ensured, and the long-time culture and passage can be realized without increasing the apoptosis ratio.
Drawings
FIG. 1 shows the growth of VERO cells in SFM serum-free medium in a medium assay.
FIG. 2 shows VERO SFM serum-free medium passage data in medium experiments.
FIG. 3 is the VERO SFM serum-free medium proliferation fold in the medium experiment.
Detailed Description
The technical solution of the present invention will be described in further detail with reference to specific embodiments.
Example 1
A serum-free medium for VERO serum-free cell culture and production of the corresponding virus, comprising the following components: 20mg/L glycine, 5mg/L alanine, 300mg/L arginine hydrochloride, 60mg/L asparagine monohydrate, 10mg/L aspartic acid, 20mg/L cysteine hydrochloride monohydrate, 75mg/L cystine dihydrochloride, 10mg/L glutamic acid, 200mg/L glutamine, 40mg/L histidine hydrochloride monohydrate, 75mg/L isoleucine, 75mg/L leucine, 95mg/L lysine hydrochloride, 75mg/L methionine, 40mg/L phenylalanine, 20mg/L proline, 180mg/L serine, 60mg/L threonine, 10mg/L tryptophan, 300mg/L tyrosine disodium salt dihydrate, 60mg/L valine, 15mg/L hydroxyproline, 60mg/L ornithine, 75mg/L taurine, 15mg/L human transferrin, 10mg/L recombinant insulin, 4000 mg/L4-hydroxyethyl piperazine ethanesulfonic acid (HEPES)4000mg/L, KERRY ultrafiltration plant peptone (kerry4601N)500mg/L Angel yeast powder (Angel ultra), 60mg/L ascorbic acid, 5mg/L sodium ascorbyl phosphonate, 0.5mg/L biotin, 10mg/L, D-calcium chloride 10mg/L folic acid 5mg/L, 5mg/L nicotinic acid, 5mg/L pyridoxal hydrochloride, 0.5mg/L, B12 vitamin 5mg/L, i-inositol 15mg/L riboflavin, K30.2mg/L vitamin, 200mg/L anhydrous calcium chloride, 100mg/L anhydrous magnesium sulfate, 400mg/L potassium chloride, 5mg/L anhydrous calcium chloride, 3000mg/L sodium bicarbonate, 6000mg/L sodium chloride, 150mg/L sodium dihydrogen phosphate monohydrate, 80mg/L disodium hydrogen phosphate anhydrous, 0.003mg/L copper sulfate pentahydrate, 1mg/L ferrous sulfate heptahydrate, 60mg/L magnesium chloride anhydrous, 0.6mg/L, D-glucose heptahydrate, 3000mg/L phenol red, 0.01mg/L putrescine dihydrochloride, 120mg/L sodium pyruvate, 5mg/L reduced glutathione, 0.0005mg/L ammonium metavanadate, 0.00005mg/L manganese dichloride, 0.01mg/L sodium selenite, 0.5mg/L adenine, 0.5mg/L sodium lactate, 10mg/L serotonin, 200mg/L fructose, 0.01mg/L oligopeptide-1 (BGF), 0.01mg/L human oligopeptide-1 (EGF), 0.01mg/L, Platelet derived growth factor-BB (PDGF-BB)0.01mg/L, urea 20mg/L, thymidine 0.5 mg/L.
Wherein the human transferrin is saturated iron human transferrin; the recombinant insulin is a full-chain recombinant insulin.
Example 2
A serum-free medium for VERO serum-free cell culture and production of the corresponding virus, comprising the following components: 15mg/L glycine, 2mg/L alanine, 150mg/L arginine hydrochloride, 20mg/L asparagine monohydrate, 5mg/L aspartic acid, 15mg/L cysteine hydrochloride monohydrate, 25mg/L cystine dihydrochloride, 5mg/L glutamic acid, 100mg/L glutamine, 20mg/L histidine hydrochloride monohydrate, 25mg/L isoleucine, 25mg/L leucine, 60mg/L lysine hydrochloride, 50mg/L methionine, 20mg/L phenylalanine, 15mg/L proline, 150mg/L serine, 40mg/L threonine, 5mg/L tryptophan, 200mg/L tyrosine disodium salt dihydrate, 25mg/L valine, 10mg/L hydroxyproline, 40mg/L ornithine, Taurine 50mg/L, human transferrin 10mg/L, recombinant insulin 5mg/L, 4-hydroxyethyl piperazine ethanesulfonic acid (HEPES)2000mg/L, KERRY ultrafiltration plant peptone (kerry4601N)100mg/L, Angel yeast powder (Angel ultra)100mg/L, ascorbic acid 40mg/L, sodium ascorbyl phosphonate 1mg/L, biotin 0.1mg/L, choline chloride 5mg/L, D-calcium pantothenate 5mg/L, folic acid 1mg/L, nicotinic acid 1mg/L, pyridoxal hydrochloride 1mg/L, riboflavin 0.1mg/L, B12 vitamin 1mg/L, i-inositol 10mg/L, vitamin K30.05mg/L, anhydrous calcium chloride 150mg/L, anhydrous magnesium sulfate 50mg/L, potassium chloride 200mg/L, potassium chloride, 1000mg/L sodium bicarbonate, 5000mg/L sodium chloride, 130mg/L sodium dihydrogen phosphate monohydrate, 50mg/L disodium hydrogen phosphate anhydrous, 0.001mg/L copper sulfate pentahydrate, 0.5mg/L ferrous sulfate heptahydrate, 50mg/L magnesium chloride anhydrous, 0.1mg/L, D-glucose heptahydrate, 1000mg/L phenol red, 0.005mg/L putrescine dihydrochloride, 90mg/L sodium pyruvate, 1mg/L reduced glutathione, 0.0001mg/L ammonium metavanadate, 0.00001mg/L manganese dichloride, 0.005mg/L sodium selenite, 0.01mg/L adenine, 0.01mg/L sodium lactate, 1mg/L serotonin, 100mg/L fructose, 0.005mg/L oligopeptide-1 (BGF), 0.005mg/L human oligopeptide-1 (EGF), 0.005mg/L EGF, Platelet derived growth factor-BB (PDGF-BB)0.005mg/L, urea 15mg/L, thymidine 0.1 mg/L.
Wherein the human transferrin is saturated iron human transferrin; the recombinant insulin is a full-chain recombinant insulin.
Example 3
A serum-free medium for VERO serum-free cell culture and production of the corresponding virus, comprising the following components: 18.75mg/L glycine, 4.45mg/L alanine, 247.5mg/L arginine hydrochloride, 57.5mg/L asparagine monohydrate, 6.65mg/L aspartic acid, 17.56mg/L cysteine hydrochloride monohydrate, 46.29mg/L cystine dihydrochloride, 7.35mg/L glutamic acid, 190mg/L glutamine, 31.48mg/L histidine hydrochloride monohydrate, 54.47mg/L isoleucine, 59.05mg/L leucine, 91.25mg/L lysine hydrochloride, 67.24mg/L methionine, 35.48mg/L phenylalanine, 17.25mg/L proline, 176.25mg/L serine, 53.45mg/L threonine, 9.02mg/L tryptophan, 275.79mg/L tyrosine disodium salt dihydrate, 52.85mg/L valine, 13.1mg/L hydroxyproline, 50.58mg/L ornithine, Taurine 62.5mg/L, ascorbic acid 50mg/L, sodium ascorbyl phosphonate 2.5mg/L, biotin 0.5mg/L, choline chloride 8.98mg/L, D-calcium pantothenate 6.24mg/L, folic acid 2.65mg/L, nicotinic acid 2.02mg/L, pyridoxal hydrochloride 2.013mg/L, riboflavin 0.219mg/L, B12 vitamin 2.17mg/L, i-inositol 12.6mg/L, vitamin K30.1mg/L, anhydrous calcium chloride 170mg/L, anhydrous magnesium sulfate 97.67mg/L, potassium chloride 311.8mg/L, sodium bicarbonate 1200mg/L, sodium chloride 5800mg/L, monosodium phosphate monohydrate 140mg/L, disodium hydrogen phosphate 71.02mg/L, cupric sulfate pentahydrate 0.0023mg/L, ferrous sulfate heptahydrate 0.917mg/L, anhydrous magnesium chloride 53.64mg/L, 0.532mg/L, D mg of zinc sulfate heptahydrate-1000 mg/L of glucose, 8.1mg/L of phenol red, 0.0081mg/L of putrescine dihydrochloride, 110mg/L of sodium pyruvate, 4mg/L of reduced glutathione, 0.0003mg/L of ammonium metavanadate, 0.00005mg/L of manganese dichloride, 0.01mg/L of sodium selenite, 0.365mg/L of adenine, 0.2mg/L of sodium lactate, 5.5mg/L of serotonin, 196.87mg/L of fructose, 0.01mg/L of oligopeptide-1 (BGF), 0.01mg/L of human oligopeptide-1 (EGF), 0.01mg/L of platelet-derived growth factor-BB (PDGF-BB), 17mg/L of urea, 0.365mg/L of thymidine, 15mg/L of human transferrin, 10mg/L of recombinant insulin, 3574.5mg/L of 4-hydroxyethyl piperazine ethanesulfonic acid (ES), key 4601N 300mg/L, Angel ultra150 mg/L.
Wherein the human transferrin is saturated iron human transferrin; the recombinant insulin is a full-chain recombinant insulin.
Example 4
A serum-free medium for VERO serum-free cell culture and production of the corresponding virus, comprising the following components: 15mg/L glycine, 2mg/L alanine, 150mg/L arginine hydrochloride, 20mg/L asparagine monohydrate, 5mg/L aspartic acid, 15mg/L cysteine hydrochloride monohydrate, 25mg/L cystine dihydrochloride, 5mg/L glutamic acid, 100mg/L glutamine, 20mg/L histidine hydrochloride monohydrate, 25mg/L isoleucine, 25mg/L leucine, 60mg/L lysine hydrochloride, 50mg/L methionine, 20mg/L phenylalanine, 15mg/L proline, 150mg/L serine, 40mg/L threonine, 5mg/L tryptophan, 200mg/L tyrosine disodium salt dihydrate, 25mg/L valine, 10mg/L hydroxyproline, 40mg/L ornithine, 50mg/L taurine, 50mg/L ascorbic acid, 2.5mg/L sodium ascorbyl phosphonate, 0.5mg/L biotin, 8.98mg/L, D mg/L choline chloride, 6.24mg/L calcium pantothenate, 2.65mg/L folic acid, 2.02mg/L nicotinic acid, 2.013mg/L pyridoxal hydrochloride, 0.219mg/L, B12 riboflavin, 2.17mg/L, i-inositol, 12.6mg/L vitamin K30.1mg/L anhydrous calcium chloride, 170mg/L anhydrous magnesium sulfate, 97.67mg/L anhydrous magnesium sulfate, 311.8mg/L potassium chloride, 1200mg/L sodium bicarbonate, 5800mg/L sodium chloride, 140mg/L sodium dihydrogen phosphate monohydrate, 71.02mg/L anhydrous disodium hydrogen phosphate, 0.0023mg/L cupric sulfate pentahydrate, 0.917mg/L ferrous sulfate heptahydrate, 53.64mg/L anhydrous magnesium chloride, 0.532mg/L, D mg of zinc sulfate heptahydrate-1000 mg/L of glucose, 8.1mg/L of phenol red, 0.0081mg/L of putrescine dihydrochloride, 110mg/L of sodium pyruvate, 4mg/L of reduced glutathione, 0.0003mg/L of ammonium metavanadate, 0.00005mg/L of manganese dichloride, 0.01mg/L of sodium selenite, 0.365mg/L of adenine, 0.2mg/L of sodium lactate, 5.5mg/L of serotonin, 196.87mg/L of fructose, 0.01mg/L of oligopeptide-1 (BGF), 0.01mg/L of human oligopeptide-1 (EGF), 0.01mg/L of platelet-derived growth factor-BB (PDGF-BB), 17mg/L of urea, 0.365mg/L of thymidine, 15mg/L of human transferrin, 10mg/L of recombinant insulin, 3574.5mg/L of 4-hydroxyethyl piperazine ethanesulfonic acid (ES), key 4601N 300mg/L, Angel ultra150 mg/L.
Wherein the human transferrin is saturated iron human transferrin; the recombinant insulin is a full-chain recombinant insulin.
Example 5
A serum-free medium for VERO serum-free cell culture and production of the corresponding virus, comprising the following components: 18.75mg/L glycine, 4.45mg/L alanine, 247.5mg/L arginine hydrochloride, 57.5mg/L asparagine monohydrate, 6.65mg/L aspartic acid, 17.56mg/L cysteine hydrochloride monohydrate, 46.29mg/L cystine dihydrochloride, 7.35mg/L glutamic acid, 190mg/L glutamine, 31.48mg/L histidine hydrochloride monohydrate, 54.47mg/L isoleucine, 59.05mg/L leucine, 91.25mg/L lysine hydrochloride, 67.24mg/L methionine, 35.48mg/L phenylalanine, 17.25mg/L proline, 176.25mg/L serine, 53.45mg/L threonine, 9.02mg/L tryptophan, 275.79mg/L tyrosine disodium salt dihydrate, 52.85mg/L valine, 13.1mg/L hydroxyproline, 50.58mg/L ornithine, Taurine 62.5mg/L, ascorbic acid 50mg/L, sodium ascorbyl phosphonate 2.5mg/L, biotin 0.5mg/L, choline chloride 8.98mg/L, D-calcium pantothenate 6.24mg/L, folic acid 2.65mg/L, nicotinic acid 2.02mg/L, pyridoxal hydrochloride 2.013mg/L, riboflavin 0.219mg/L, B12 vitamin 2.17mg/L, i-inositol 12.6mg/L, vitamin K30.1mg/L, anhydrous calcium chloride 200mg/L, anhydrous magnesium sulfate 100mg/L, potassium chloride 400mg/L, sodium bicarbonate 3000mg/L, sodium chloride 6000mg/L, sodium dihydrogen phosphate monohydrate 150mg/L, anhydrous disodium hydrogen phosphate 80mg/L, cupric sulfate pentahydrate 0.003mg/L, ferrous sulfate heptahydrate 1mg/L, anhydrous magnesium chloride 60mg/L, Zinc sulfate heptahydrate 0.6mg/L, D-glucose 3000mg/L, phenol red 10mg/L, putrescine dihydrochloride 0.01mg/L, sodium pyruvate 120mg/L, reduced glutathione 5mg/L, ammonium metavanadate 0.0005mg/L, manganese dichloride 0.00005mg/L, sodium selenite 0.01mg/L, adenine 0.5mg/L, sodium lactate 0.5mg/L, serotonin 10mg/L, fructose 200mg/L, oligopeptide-1 (BGF)0.01mg/L, human oligopeptide-1 (EGF)0.01mg/L, platelet derived growth factor-BB (PDGF-BB)0.01mg/L, urea 20mg/L, thymidine 0.5mg/L, human transferrin 10mg/L, recombinant insulin 5mg/L, 4-hydroxyethyl piperazine ethanesulfonic acid (HEPES)2000mg/L, KERRY ultrafiltration phytone (KERRY4601N)100mg/L, Angel yeast powder (Angel ultra)100 mg/L.
Wherein the human transferrin is saturated iron human transferrin; the recombinant insulin is a full-chain recombinant insulin.
Experiment in culture Medium
Experimental cell lines VERO serum-free cells
Culture bottle T25 bottle
Density of inoculation 1.0 × 105 cells/bottle
Culture conditions Humidified culture at 37 ℃ with 5% CO2
Days of growth 3 days
When the culture medium prepared by the invention is used for VERO cell culture, the use of animal serum is not relied on, and animal source protein is not introduced, so that the production cost is greatly reduced, the stable product quality is fully ensured, and the long-time culture and passage can be realized without increasing the apoptosis ratio.
While the preferred embodiments of the present invention have been described in detail, the present invention is not limited to the above embodiments, and various changes can be made without departing from the spirit of the present invention within the knowledge of those skilled in the art.

Claims (7)

1. A serum-free medium for VERO serum-free cell culture and production of the corresponding virus, comprising the following components: amino acid, vitamin, inorganic salt, auxiliary component and protein, wherein the amino acid comprises glycine 15-20mg/L, alanine 2-5mg/L, arginine hydrochloride 150-300mg/L, asparagine monohydrate 20-60mg/L, aspartic acid 5-10mg/L, cysteine monohydrate 15-20mg/L, cystine dihydrochloride 25-75mg/L, glutamic acid 5-10mg/L, glutamine 100-200mg/L, histidine monohydrate 20-40mg/L, isoleucine 25-75mg/L, leucine 25-75mg/L, lysine hydrochloride 60-95mg/L, methionine 50-75mg/L, phenylalanine 20-40mg/L, arginine hydrochloride, methionine, 15-20mg/L of proline, 180mg/L of serine, 40-60mg/L of threonine, 5-10mg/L of tryptophan, 200mg/L of tyrosine disodium salt dihydrate, 25-60mg/L of valine, 10-15mg/L of hydroxyproline, 40-60mg/L of ornithine and 50-75mg/L of taurine; the protein comprises 10-15mg/L of human transferrin, 5-10mg/L of recombinant insulin, 2000-4000mg/L, KERRY of 4-hydroxyethyl piperazine ethanesulfonic acid, 100-500mg/L of ultrafiltration plant peptone and 500mg/L of Angel yeast powder.
2. The serum-free medium for VERO serum-free cell culture and production of a corresponding virus according to claim 1, wherein the vitamins comprise ascorbic acid 40-60mg/L, sodium ascorbyl phosphonate 1-5mg/L, biotin 0.1-0.5mg/L, choline chloride 5-10mg/L, D-calcium pantothenate 5-10mg/L, folic acid 1-5mg/L, nicotinic acid 1-5mg/L, pyridoxal hydrochloride 1-5mg/L, riboflavin 0.1-0.5mg/L, B12, vitamin 1-5mg/L, i-inositol 10-15mg/L, vitamin K30.05-0.2 mg/L.
3. The serum-free medium for VERO serum-free cell culture and production of a corresponding virus of claim 1, wherein the inorganic salts comprise anhydrous calcium chloride 150-200mg/L, anhydrous magnesium sulfate 50-100mg/L, potassium chloride 200-400mg/L, sodium bicarbonate 1000-3000mg/L, sodium chloride 5000-6000mg/L, monobasic sodium phosphate 130-150mg/L, anhydrous dibasic sodium phosphate 50-80mg/L, anhydrous copper sulfate 0.001-0.003mg/L, ferrous sulfate heptahydrate 0.5-1mg/L, anhydrous magnesium chloride 50-60mg/L, and zinc sulfate heptahydrate 0.1-0.6 mg/L.
4. The serum-free medium for VERO serum-free cell culture and production of a corresponding virus according to claim 1, wherein the auxiliary components comprise 3000mg/L of D-glucose 1000-, Platelet derived growth factor-bb 0.005-0.01mg/L, urea 15-20mg/L, and thymidine 0.1-0.5 mg/L.
5. Serum-free medium for VERO serum-free cell culture and corresponding virus production according to claim 1, characterized in that it comprises the following components: 18.75mg/L glycine, 4.45mg/L alanine, 247.5mg/L arginine hydrochloride, 57.5mg/L asparagine monohydrate, 6.65mg/L aspartic acid, 17.56mg/L cysteine hydrochloride monohydrate, 46.29mg/L cystine dihydrochloride, 7.35mg/L glutamic acid, 190mg/L glutamine, 31.48mg/L histidine hydrochloride monohydrate, 54.47mg/L isoleucine, 59.05mg/L leucine, 91.25mg/L lysine hydrochloride, 67.24mg/L methionine, 35.48mg/L phenylalanine, 17.25mg/L proline, 176.25mg/L serine, 53.45mg/L threonine, 9.02mg/L tryptophan, 275.79mg/L tyrosine disodium salt dihydrate, 52.85mg/L valine, 13.1mg/L hydroxyproline, 13.1mg/L tyrosine, 275.79mg/L tyrosine, and the like, 50.58mg/L ornithine, 62.5mg/L taurine, 50mg/L ascorbic acid, 2.5mg/L sodium ascorbyl phosphonate, 0.5mg/L biotin, 8.98mg/L, D-calcium chloride 6.24mg/L choline chloride, 2.65mg/L folic acid, 2.02mg/L nicotinic acid, 2.013mg/L pyridoxal hydrochloride, 0.219mg/L, B12 riboflavin, 2.17mg/L, i-inositol, 12.6mg/L vitamin K30.1mg/L, 170mg/L anhydrous calcium chloride, 97.67mg/L anhydrous magnesium sulfate, 311.8mg/L potassium chloride, 1200mg/L sodium bicarbonate, 5800mg/L sodium chloride, 140mg/L sodium dihydrogen phosphate monohydrate, 71.02mg/L anhydrous sodium hydrogen phosphate, 0.0023mg/L copper sulfate pentahydrate, 0.917mg/L ferrous sulfate heptahydrate, 0.917mg/L ferrous sulfate, 53.64mg/L of anhydrous magnesium chloride, 0.532mg/L, D mg/L of zinc sulfate heptahydrate, 8.1mg/L of phenol red, 0.0081mg/L of putrescine dihydrochloride, 110mg/L of sodium pyruvate, 4mg/L of reduced glutathione, 0.0003mg/L of ammonium metavanadate, 0.00005mg/L of manganese dichloride, 0.01mg/L of sodium selenite, 0.365mg/L of adenine, 0.2mg/L of sodium lactate, 5.5mg/L of serotonin, 196.87mg/L of fructose, 10.01 mg/L of oligopeptide, 10.01 mg/L of human oligopeptide, 0.01mg/L of platelet-derived growth factor-bb, 17mg/L of urea, 0.365mg/L of thymidine, 15mg/L of human transferrin, 10mg/L of recombinant insulin, 3574.5mg/L of 4-hydroxyethyl piperazine ethanesulfonic acid, KERRY ultrafiltration plant peptone 300mg/L, Angel yeast powder 150 mg/L.
6. The serum-free medium for VERO serum-free cell culture and production of the corresponding virus according to claim 1, wherein the human transferrin is saturated iron human transferrin.
7. Serum-free medium for VERO serum-free cell culture and corresponding virus production according to claim 1, wherein the recombinant insulin is a whole-chain recombinant insulin.
CN201910900379.4A 2019-09-23 2019-09-23 Serum-free culture medium for VERO serum-free cell culture and corresponding virus production Pending CN110628697A (en)

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Application publication date: 20191231