CN105462912B - Suitable for diploid cell culture without albumen serum free medium and application - Google Patents
Suitable for diploid cell culture without albumen serum free medium and application Download PDFInfo
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- CN105462912B CN105462912B CN201610040838.2A CN201610040838A CN105462912B CN 105462912 B CN105462912 B CN 105462912B CN 201610040838 A CN201610040838 A CN 201610040838A CN 105462912 B CN105462912 B CN 105462912B
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Abstract
The present invention provides a kind of suitable for diploid cell culture without albumen serum free medium and preparation method thereof, which includes: calcium chloride, magnesium sulfate, potassium chloride, sodium dihydrogen phosphate, L R-gene, L-Asn, L-Cys-Cys dihydrochloride, L histidine monohydrochloride, L-Leu, L-Ile etc..Culture medium provided by the invention can not only meet diploid cell growth or the needs of the cell maintenance stage, and avoid with the contact of exogenous virus that may be present in albumen, serum, to reduce security risk；, can be with commercially available standard product since component is simple, clear, difference is small between product batches, while also simplifying the purification step of downstream product；Scientific basis is provided for large-scale standardized production.In addition, the present invention provides no albumen serum free mediums to apply in diploid cell growth phase or in the cell maintenance stage.
Description
Technical field
The invention belongs to cell engineering fields, are related to a kind of free serum culture of the no albumen without hydrolysate definite ingredients
Base, in particular to a kind of serum free medium suitable for diploid cell culture without albumen without hydrolysate definite ingredients and its
Preparation method and application.
Background technique
Cytotrophy is supplied when cell culture medium is cultured cell in vitro and promotes the basic substance of cell Proliferation, and thin
The living environment of intracellular growth and breeding is the basis of Cell culture invitro, is widely used in field of biological product, stem-cell therapy
Equal fields.Serum free medium of the no albumen without hydrolysate definite ingredients is one of cell culture medium, and ingredient is completely bright
Really, the substance unknown without ingredients such as albumen, hydrolysates.
It is prepared in vaccination process with diploid cell, there are two the stages need to use cell culture medium, be that cell is raw respectively
Long stage (cell expands the stage) and cell maintenance stage (virus amplification stage).
According to the prior art, during producing vaccine with diploid cell, cell culture expands the stage, mostly uses
The conventional medium of 5%-10% serum-concentration such as MEM, DMEM, or contain insulin, transferrins, albumin, hydrolysate
The serum free medium of the equal indefinite substance of ingredients, such culture medium cost is high, downstream product purifying complex, and it may
The exogenous virus of carrying can bring serious security risk, and without albumen, the serum free medium of no hydrolysate can be solved effectively
These problems.Although application No. is the patent application documents of CN200710129800.3 to disclose a kind of mad dog epidemic disease of diploid cell
The cell culture stage of the preparation method of seedling and purified rabies vaccine, the vaccine preparation method uses serum free medium culture,
Serum free medium used in this method has albumen addition.Application No. is 201310523326.8 patent application document public affairs
A kind of method for preparing cendevax with serum free medium is opened, the cell culture stage of this method uses serum-free
Culture medium, the free serum culture based component is indefinite, and contains albumen.
In the virus amplification stage, domestic production at present mostly uses the routine containing 1%-2% serum or human serum albumin
Culture medium.Although application No. is the patent application documents of CN200710129799.4 to disclose a kind of diploid somatic cell encephalitis B
The preparation method of vaccine, the cell culture in production preparation process expand the stage, have used serum free medium, but this method
Culture medium used contains albumen addition.Application No. is the patent application documents of CN200710129800.3 to disclose two times of one kind
The preparation method of body cell rabies vaccine and purified rabies vaccine, the serum free medium that the cell maintenance stage of this method uses
Equally there is albumen addition.It is disclosed application No. is the patent application document of CN201310523326.8 and a kind of uses free serum culture
The cell maintenance stage of the method that base prepares cendevax, this method uses serum free medium, the free serum culture
Based component is indefinite, and contains albumen.
As can be seen from the above analysis, there are problems that at least two parties face in current production technology, be on the one hand culture
It needs to add serum in base；It on the other hand is to increase the risk of culture containing indefinite components such as albumen in culture medium, it is also unfavorable
In product purification.
Summary of the invention
The purpose of the present invention is to provide a kind of suitable for diploid cell culture without albumen serum free medium, has
Without albumen, the advantage clear without hydrolysate, serum-free, component, the cell amplification or virus of diploid cell culture can be supported
Amplification.
Another object of the present invention is that providing above-mentioned training without albumen serum-free suitable for diploid cell culture
Support the preparation method of base.
A further object of the present invention is to provide above-mentioned serum free medium of the no albumen without hydrolysate definite ingredients, specifically
Cell growth phase in diploid cell culture or the application in the cell maintenance stage.
In order to achieve the above object, the present invention takes following technical scheme to realize.
The present invention provides a kind of suitable for diploid cell culture without albumen serum free medium, by component in table 1
It is prepared:
Table 1: suitable for diploid cell culture without albumen serum free medium component
Invention further provides the above-mentioned preparations without albumen serum free medium suitable for diploid cell culture
1-10mg Tween 80 is dissolved in 50ml water for injection by method, and 0.0005-0.15mg linoleic acid, 0.0005- is then added
0.85mg linolenic acid, 0.0001-0.03mg arachidonic acid, 0.2-2mg vitamin E, 0.2-0.8mg myristic acid, 0.18-
0.5mg stearic acid sufficiently to its dissolution with water for injection fluid infusion to 500ml, adds 4.5-10g sodium chloride, 1-20g grape
Sugar, 0-6g fructose, stir to clarify, with water for injection fluid infusion to 900ml, are eventually adding component in table 2:
Table 2: Tween 80, linoleic acid, linolenic acid, flower are removed without albumen serum free medium suitable for diploid cell culture
Component other than raw tetraenoic acid, vitamin E, myristic acid, stearic acid, sodium chloride, glucose, fructose
After addition, continue stirring to being completely dissolved, then use water for injection fluid infusion to 1000ml, 0.22 μm of filter filters
Degerming, 4 DEG C are kept in dark place.
By the metabolic analysis to diploid cell, L-Asn, L-Ile, L-Leu, L-Val etc. are determined
Several amino acid are to rapidly deplete type amino acid, increase the dosage of these amino acid, grow to cell and maintain to have very well
Effect.
Vitamin is as ingredient indispensable in serum free medium, generally as coenzyme in cellular process
Or prothetic group participates in the metabolic regulation of cell, the constituent such as biotin as some specific hydroxylases, participate in glycometabolism and
The synthesis of fatty acid；Folic acid is used for cell tetrahydrobiopterin synthesis folic acid, and tetrahydrofolic acid is important one carbon unit, in nucleic acid and protein
Synthesis in play an important role；D calcium pantothenate is transformed into acyl carrier protein and coenzyme in the cell, participates in carbohydrate, lipid, albumen
Catalysis reaction in matter metabolism.By having been determined using plackett-burman design method and center combination design method etc.
The vitamin proportion for growing and maintaining suitable for diploid cell.
Addition cell versus glucose is not easy the fructose utilized, can reduce the generation of lactic acid in metabolic process, to stablize
PH value；Linoleic acid, linolenic acid, arachidonic acid are fatty acid necessary to mammalian cell；The glucose and paddy ammonia of optimization
Amide proportion, can effectively control the production quantity of lactic acid and ammonia in cellular process.
Citric acid, ferrous sulfate and ferric nitrate, which are applied in combination, in diploid cell incubation can be very good substitution iron
Albumen, effect is better than exclusive use ironic citrate or ferric nitrate；Zinc sulfate can substitute the effect of insulin, promote glycogen
With the synthesis of fatty acid；Sodium carboxymethylcellulose can substitute albumin as microelement, the carrier of essential fatty acid；β-mercapto
Base ethyl alcohol then can direct stimulating cellular growth, have the partial action of serum.
Above-mentioned serum free medium of the no albumen without hydrolysate definite ingredients, the cell growth phase for diploid cell
With the cell maintenance stage.After culture medium provided by the invention, in diploid cell culture and maintenance stage, blood is not used
Cleer and peaceful albumin can be used for people with the production of the vaccines such as mad dog, varicella, polio.
It is provided by the invention suitable for diploid cell culture without albumen serum free medium and preparation method thereof, have
At least one of below the utility model has the advantages that
(1) serum free medium without albumen without hydrolysate definite ingredients prepared, can not only meet diploid cell
Culture or the demand maintained, and due to without adding serum, albumen and hydrolysate etc., thus avoid in albumen, serum
The contact of exogenous virus that may be present, to reduce security risk；
(2), can be with commercially available standard product since component is simple, clear, difference is small between product batches,
The purification step of downstream product is also simplified simultaneously；Scientific basis is provided for large-scale standardized production；
(3) since without containing indefinite additives of ingredients such as albumen, component is clear, the separation of vaccine later period is significantly reduced
Difficulty and cost are purified, allergic reaction risk is reduced；
(4) available to have serum with conventional with serum-free provided by the invention, protein-free medium culture diploid cell
The comparable cell density of culture medium culture is being applied to epidemic cerebrospinal meningitis B virus, rabies viruses, poliovirus preparation
When, the virus titer of acquisition and the conventional virus titer for having blood serum medium culture to obtain are without significant difference.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described, it should be apparent that, be described below in attached drawing be only this
Some embodiments of invention for those of ordinary skills without creative efforts, can be with
Illustrated embodiment obtains other embodiments and its attached drawing according to these attached drawings.
Figure 1A is that the MEM culture MRC-5 cell that culture medium and serum-concentration provided in this embodiment are 10% is respectively adopted
Cell growth curve figure；
Figure 1B is the cell state figure using culture medium culture MRC-5 cell 72h provided in this embodiment；
Fig. 1 C is the cell state figure for using serum-concentration to cultivate MRC-5 cell 72h for 10% MEM；
Fig. 2A is that the MEM culture 2BS cell that culture medium and serum-concentration provided in this embodiment are 10% is respectively adopted
Cell growth curve figure；
Fig. 2 B is the cell state figure using culture medium culture 2BS cell 72h provided in this embodiment；
Fig. 2 C is the cell state figure for using serum-concentration to cultivate 2BS cell 72h for 10% MEM.
Specific embodiment
Clear, complete description is carried out below with reference to technical solution of the attached drawing to various embodiments of the present invention, it is clear that is retouched
The embodiment stated is only a part of the embodiments of the present invention, instead of all the embodiments.Based on the embodiments of the present invention,
Those of ordinary skill in the art's obtained all other embodiment without making creative work belongs to this
Invent protected range.
Embodiment 1
Manufactured in the present embodiment is the culture medium suitable for MRC-5 cell culture, dosage of each component be referred in table 3 to
Data out, the specific steps are as follows:
Tween 80 is added in the reaction vessel that capacity is 1000ml, adds 50ml water for injection and is dissolved, then
Linoleic acid, linolenic acid, arachidonic acid, vitamin E, myristic acid, stearic acid are sequentially added, sufficiently to its dissolution, uses injection
Water fluid infusion sequentially adds sodium chloride, glucose, fructose, stirs to clarify to 500ml, with water for injection fluid infusion to 900ml,
Finally sequentially add calcium chloride, magnesium sulfate, potassium chloride, disodium hydrogen phosphate, potassium dihydrogen phosphate, L R-gene, L-Asp,
L-Asn, L-Cys-Cys dihydrochloride, L histidine monohydrochloride, L-Leu, L-Ile, glycine, L lysine hydrochloride, L
Methionine, L-Phe, L-Pro, L-Ser, L-Thr, L-Trp, L-Tyr disodium salt, L-Val, L bird
Propylhomoserin hydrochloride, L citrulling, L-Glu, oxaloacetic acid, biotin, choline chloride, vitamin C, folic acid, inositol, niacinamide,
P-aminobenzoic acid, D calcium pantothenate, pyridoxal hydrochloride, puridoxine hydrochloride, riboflavin and vitamin B12, sodium carboxymethylcellulose, phenol
Red, ethanol amine, L glutamine, sodium bicarbonate, zinc sulfate, copper sulphate, ferric nitrate, ammonium metavanadate, potassium iodide, stannic chloride, sulfuric acid
Nickel, sodium metasilicate, lipoic acid, vitamin E, sodium selenite, cholesterol, progesterone, thymidine, myristic acid, stearic acid, L-cysteine,
Magnesium chloride, sodium butyrate, hypoxanthine, adenine, ferrous sulfate, putrescine, taurine, citric acid, beta -mercaptoethanol, fill in rice
Pine, bismuth ammonium citrate, HEPES, caffeic acid, the third dipeptides of paddy, cobalt nitrate, Boratex, ammonium molybdate；After addition, continue to stir
To being completely dissolved, then with water for injection fluid infusion to 1000ml, 0.22 μm of filter filtration sterilization, 4 DEG C are kept in dark place.
Table 3: the serum free medium component in embodiment 1 and embodiment 2 without albumen without hydrolysate definite ingredients
In order to investigate performance of the culture medium provided in this embodiment in cell growth phase, by training provided in this embodiment
It supports the MEM that base and serum-concentration are 10% and conventionally cultivates MRC-5 cell respectively.The specific implementation that the present embodiment is taken
Mode are as follows: take cell generation in the MRC-5 cell in 10-40 generation, with 0.05% trypsase and 0.02%EDTA (ethylenediamine tetrem
Acid) cell monolayer digest by solution from culture bottle, respectively with prepared the present embodiment culture medium and containing 10% serum
Cell is resuspended in the MEM of concentration, and with 0.6*106Cells/ml inoculation, adds a certain amount of corresponding culture medium and is placed in 37 DEG C of trainings
It supports, is counted per digestion for 24 hours.
Serum is contained to culture medium provided in this embodiment and tradition below with reference to cell growth curve figure and cell state figure
The MRC-5 cell of culture medium culture is compared.Figure 1A, which gives, is respectively adopted culture medium and MEM culture provided in this embodiment
The cell growth curve figure of MRC-5 cell, it can be seen from the figure that when using culture medium provided by the embodiment, growth rate
Faster, maximum cell density can be reached earlier.Figure 1B is to use culture medium culture MRC-5 cell 72h provided in this embodiment
Cell state figure, Fig. 1 C be use concentration for 10% MEM cultivate MRC-5 cell 72h cell state figure.From Figure 1B and figure
The comparison of 1C is seen, conventionally cultivates MRC-5 cell, growth conditions and use with culture medium provided in this embodiment
MEM culture medium is suitable.
In order to investigate culture medium provided by the embodiment in the performance of cell maintenance stage, culture provided in this embodiment is utilized
Then base culture MRC-5 cell is inoculated with varicella virus (Oka plants of VZV and rabies viruses PM strain) amplification cultivation afterwards, harvest respectively
Virus liquid finds that the virus titer obtained and virus harvest amount are suitable with conventional medium used in the prior art, virus
Titre is shown in Table 4.
Table 4: the culture medium culture MRC-5 cell and conventional medium culture MRC-5 cell that embodiment provides prepare varicella
The virus titer of virus and rabies viruses
| Culture medium | Varicella (Oka plants of VZV) | Rabies viruses (PM rabies virus strain) |
| The present embodiment culture medium | 7.3 | 8.4 |
| MEM (10% serum-concentration) | 7.25 | 8.5 |
Embodiment 2
Manufactured in the present embodiment is the culture medium suitable for 2BS cell culture, and dosage of each component is referred to provide in table 3
Data, specific steps are in the same manner as in Example 1.
In order to investigate performance of the culture medium provided in this embodiment in cell growth phase, by training provided in this embodiment
It supports base and concentration and cultivates 2BS cell respectively for 10% MEM.The specific embodiment that the present embodiment is taken are as follows: take cell generation
In the 2BS cell in 10-40 generation, with 0.05% trypsase and 0.02%EDTA (ethylenediamine tetra-acetic acid) solution by cell monolayer from
It digesting in culture bottle, cell is resuspended in the MEM with prepared the present embodiment culture medium and containing 10% serum-concentration respectively, and
With 0.55*106Cells/ml inoculation, adds a certain amount of corresponding culture medium and is placed in 37 DEG C of cultures, counts per digestion for 24 hours.
Serum is contained to culture medium provided in this embodiment and tradition below with reference to cell growth curve figure and cell state figure
The 2BS cell of culture medium culture is compared.Fig. 2A, which gives, is respectively adopted culture medium and MEM culture provided in this embodiment
The cell growth curve figure of 2BS cell, Fig. 2 B are using the cellular of culture medium culture 2BS cell 72h provided in this embodiment
State figure, Fig. 2 C are the cell state figure for using concentration to cultivate MRC-5 cell 72h for 10% MEM.From pair of Fig. 2A, 2B and 2C
Than seeing, 2BS cell, growth conditions and use MEM culture medium are conventionally cultivated with culture medium provided in this embodiment
Quite, maximum cell density is suitable.
In order to investigate culture medium provided by the embodiment in the performance of cell maintenance stage, culture provided in this embodiment is utilized
Base culture 2BS cell, then be inoculated with poliovirus (sabine plant II types), through detection discovery acquisition virus titer and
Virus harvest amount is suitable with conventional medium used in the prior art, and virus titer is shown in Table 5.
Table 5: the culture medium culture 2BS cell and conventional medium culture 2BS cell that embodiment provides prepare gray nucleus
The virus titer of scorching virus
| Culture medium | Poliovirus (II type of sabine strain) |
| Culture medium of the present invention | 7.56 |
| MEM (10% serum-concentration) | 7.52 |
From the analysis to embodiment 1 and implementation 2 as can be seen that the training provided by the invention suitable for diploid cell culture
Feeding base can substitute completely conventional in the prior art has blood serum medium either protein-contg serum free medium；Due to it
Itself serum-free, without albumen, without hydrolysate, definite ingredients, which has unrivaled advantage, has in culture medium field
There are very big application space and development space.
Those of ordinary skill in the art will understand that the embodiments described herein, which is to help reader, understands this hair
Bright principle, it should be understood that protection scope of the present invention is not limited to such specific embodiments and embodiments.This field
Those of ordinary skill disclosed the technical disclosures can make according to the present invention and various not depart from the other each of essence of the invention
The specific variations and combinations of kind, these variations and combinations are still within the scope of the present invention.
Claims (4)
1. it is a kind of suitable for diploid cell culture without albumen serum free medium, which is characterized in that prepared by following component
It forms:
Calcium chloride 100-300mg/L
Magnesium sulfate 50-150mg/L
Potassium chloride 300-800mg/L
Disodium hydrogen phosphate 100-200mg/L
Sodium chloride 4.5-10g/L
Potassium dihydrogen phosphate 20-150mg/L
L R-gene 100-600mg/L
L-Asp 20-100mg/L
L-Asn 200-600mg/L
L-Cys-Cys dihydrochloride 100-300mg/L
L histidine monohydrochloride 10-200mg/L
L-Leu 100-200mg/L
L-Ile 20-200mg/L
Glycine 0-80mg/L
L lysine hydrochloride 60-200mg/L
L-Met 5-200mg/L
L-Phe 15-150mg/L
L-Pro 0-50mg/L
L-Ser 80-200mg/L
L-Thr 30-100mg/L
L-Trp 8-45mg/L
L-Tyr disodium salt 10-130mg/L
L-Val 100-200mg/L
L ornithine hydrochloride 5-25mg/L
L citrulling 10-50mg/L
L-Glu 0-80 mg/L
Oxaloacetic acid 10-80mg/L
Biotin 0.004-2mg/L
Choline chloride 5-80mg/L
Vitamin C 0.3-15mg/L
Folic acid 2.5-13mg/L
Inositol 2-30mg/L
Niacinamide 1-18mg/L
P-aminobenzoic acid 0.03-6.5mg/L
D calcium pantothenate 1-9mg/L
Pyridoxal hydrochloride 1.5-10mg/L
Puridoxine hydrochloride 0.1-3mg/L
Riboflavin 0.001-0.5mg/L
Vitamin B12 0.5-5mg/L
Sodium carboxymethylcellulose 0.1-2g/L
Glucose 1-20g/L
Phenol red 5-50mg/L
Ethanol amine 0.5-5mg/L
L glutamine 50-400mg/L
Sodium bicarbonate 1-3.95g/L
Fructose 0-6g/L
Zinc sulfate 0.05-0.5mg/L
Copper sulphate 0.01-0.05mg/L
Ferric nitrate 0.05-1mg/L
Ammonium metavanadate 0.001-0.02mg/L
Potassium iodide 0.00001-0.01mg/L
Stannic chloride 0.000018-0.00018mg/L
Nickel sulfate 0.0002-0.001mg/L
Sodium metasilicate 0.005-0.018mg/L
Lipoic acid 0.8-50mg/L
Vitamin E 0.2-2mg/L
Sodium selenite 0.001-0.1mg/L
Cholesterol 0.00002-0.006mg/L
Linoleic acid 0.0005-0.15mg/L
Linolenic acid 0.0005-0.85mg/L
Arachidonic acid 0.0001-0.03mg/L
Progesterone 0.0003-0.002mg/L
Tween 80 1-10mg/L
Thymidine 3-6mg/L
Myristic acid 0.2-0.8 mg/L
Stearic acid 0.18-0.5mg/L
L-cysteine 16.5-70.8 mg/L
Magnesium chloride 30-300mg/L
Sodium butyrate 27-429mg/L
Hypoxanthine 0.5-5mg/L
Adenine 2-8mg/L
Ferrous sulfate 3-10mg/L
Putrescine 0.02-1.6mg/L
Taurine 5-60mg/L
Citric acid 60-120mg/L
Beta -mercaptoethanol 0.1-9.9mg/L
Dexamethasone 0.001-0.1mg/L
Bismuth ammonium citrate 0.05-0.2mg/L
HEPES 2-6mg/L
Caffeic acid 0.05-0.5mg/L
The third dipeptides of paddy 20-200mg/L
Cobalt nitrate 0.0248mg/L
Boratex 0.0177mg/L
Ammonium molybdate 0.00925mg/L.
2. the preparation method of culture medium described in claim 1, which is characterized in that 1-10mg Tween 80 is dissolved in 50ml injection
With in water, 0.0005-0.15mg linoleic acid, 0.0005-0.85mg linolenic acid, 0.0001-0.03mg arachidonic is then added
Acid, 0.2-2mg vitamin E, 0.2-0.8mg myristic acid, 0.18-0.5mg stearic acid sufficiently to its dissolution are mended with water for injection
Liquid adds 4.5-10g sodium chloride, 1-20g glucose, 0-6g fructose, stirs to clarify, with water for injection fluid infusion to 500ml
To 900ml, finally add:
Calcium chloride 100-300mg/L
Magnesium sulfate 50-150mg/L
Potassium chloride 300-800mg/L
Disodium hydrogen phosphate 100-200mg/L
Potassium dihydrogen phosphate 20-150mg/L
L R-gene 100-600mg/L
L-Asp 20-100mg/L
L-Asn 200-600mg/L
L-Cys-Cys dihydrochloride 100-300mg/L
L histidine monohydrochloride 10-200mg/L
L-Leu 100-200mg/L
L-Ile 20-200mg/L
Glycine 0-80mg/L
L lysine hydrochloride 60-200mg/L
L-Met 5-200mg/L
L-Phe 15-150mg/L
L-Pro 0-50mg/L
L-Ser 80-200mg/L
L-Thr 30-100mg/L
L-Trp 8-45mg/L
L-Tyr disodium salt 10-130mg/L
L-Val 100-200mg/L
L ornithine hydrochloride 5-25mg/L
L citrulling 10-50mg/L
L-Glu 0-80 mg/L
Oxaloacetic acid 10-80mg/L
Biotin 0.004-2mg/L
Choline chloride 5-80mg/L
Vitamin C 0.3-15mg/L
Folic acid 2.5-13mg/L
Inositol 2-30mg/L
Niacinamide 1-18mg/L
P-aminobenzoic acid 0.03-6.5mg/L
D calcium pantothenate 1-9mg/L
Pyridoxal hydrochloride 1.5-10mg/L
Puridoxine hydrochloride 0.1-3mg/L
Riboflavin 0.001-0.5mg/L
Vitamin B12 0.5-5mg/L
Sodium carboxymethylcellulose 0.1-2g/L
Phenol red 5-50mg/L
Ethanol amine 0.5-5mg/L
L glutamine 50-400mg/L
Sodium bicarbonate 1-3.95g/L
Zinc sulfate 0.05-0.5mg/L
Copper sulphate 0.01-0.05mg/L
Ferric nitrate 0.05-1mg/L
Ammonium metavanadate 0.001-0.02mg/L
Potassium iodide 0.00001-0.01mg/L
Stannic chloride 0.000018-0.00018mg/L
Nickel sulfate 0.0002-0.001mg/L
Sodium metasilicate 0.005-0.018mg/L
Lipoic acid 0.8-50mg/L
Sodium selenite 0.001-0.1mg/L
Cholesterol 0.00002-0.006mg/L
Progesterone 0.0003-0.002mg/L
Thymidine 3-6mg/L
L-cysteine 16.5-70.8 mg/L
Magnesium chloride 30-300mg/L
Sodium butyrate 27-429mg/L
Hypoxanthine 0.5-5mg/L
Adenine 2-8mg/L
Ferrous sulfate 3-10mg/L
Putrescine 0.02-1.6mg/L
Taurine 5-60mg/L
Citric acid 60-120mg/L
Beta -mercaptoethanol 0.1-9.9mg/L
Dexamethasone 0.001-0.1mg/L
Bismuth ammonium citrate 0.05-0.2mg/L
HEPES 2-6mg/L
Caffeic acid 0.05-0.5mg/L
The third dipeptides of paddy 20-200mg/L
Cobalt nitrate 0.0248mg/L
Boratex 0.0177mg/L
Ammonium molybdate 0.00925mg/L
After addition, continue stirring to being completely dissolved, then with water for injection fluid infusion to 1000ml, 0.22 μm of filter is crossed and filtered out
Bacterium, 4 DEG C are kept in dark place.
3. culture medium described in claim 1 is in diploid cell growth phase or answering as culture medium in the cell maintenance stage
With.
4. the application that culture medium described in claim 1 is produced for people with mad dog, varicella, polio vaccine.
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| CN108103003B (en) * | 2017-12-12 | 2021-03-05 | 四川百诺吉科技有限公司 | Serum-free medium adapting to PK-15 full-suspension growth, preparation method thereof and full-suspension domestication method applied to PK-15 cells |
| CN109106950B (en) * | 2018-09-11 | 2021-12-10 | 中国农业科学院兰州兽医研究所 | Annular theileriosis vaccine protective agent and preparation method thereof |
| CN109536435A (en) * | 2018-12-20 | 2019-03-29 | 镇江桥美科技有限公司 | A kind of Serum-free and protein-free medium |
| CN110257328A (en) * | 2019-08-14 | 2019-09-20 | 广州赛莱拉干细胞科技股份有限公司 | A kind of mesenchymal stem cell serum-free culture medium |
| CN113957042A (en) * | 2020-07-20 | 2022-01-21 | 维他利肤(北京)生物科技有限公司 | Preparation method of stem cell zero-protein culture medium and stem cell growth factor |
| CN115627255B (en) * | 2022-12-01 | 2023-04-07 | 天信和(苏州)生物科技有限公司 | Method for culturing human diploid cells by adopting low-serum culture medium |
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