CN109337861A - A kind of highly expressed Chinese hamster ovary celI serum free medium of support product - Google Patents
A kind of highly expressed Chinese hamster ovary celI serum free medium of support product Download PDFInfo
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- CN109337861A CN109337861A CN201811341163.0A CN201811341163A CN109337861A CN 109337861 A CN109337861 A CN 109337861A CN 201811341163 A CN201811341163 A CN 201811341163A CN 109337861 A CN109337861 A CN 109337861A
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Abstract
The invention discloses a kind of Chinese hamster ovary celI serum free medium, main component includes amino acid, inorganic salts ingredients, the ingredients such as vitamin, microelement, yeast hydrolyate and albumin.Chinese hamster ovary celI serum free medium of the invention, is free of any serum component, and non-animal derived ingredient compares other serum free mediums, increases its compatibility.Cell can be directly inoculated into this serum free medium from blood serum medium or other serum free mediums, normal growth, eliminate cumbersome domestication process.The present invention improves the stand density and its motility rate of Chinese hamster ovary celI, increases cell in tolerance performance wherein, extends growth platform and hold time, it is most important that improves CHO engineering cell in the ability for wherein expressing foreign protein, greatly promote the yield of product.
Description
Technical field
The present invention relates to a kind of serum free mediums for Chinese hamster ovary celI in vitro culture, belong to technical field of cell culture.
Background technique
Cell culture technology is also cell clone technology, refers under in vitro conditions, by the way that specific nutrition substance is added, together
When environment temperature, pH value osmotic pressure etc. are controlled, make cell kept in suitable vessel activity, normal growth.According to
Training method can be divided into adhere-wall culture and the culture that suspends by the difference of cell adherent manner in vessel.Cell culture medium is
Cell draws the source of nutriment, is the successful key point of cell culture.According to source difference, cell culture medium can divide
For natural medium and artificial synthetic medium.Natural medium is only made of the biofluid naturally occurred.Natural medium
It is highly useful and conveniently, suitable for a variety of different animal cell cultures.But it is definite to these natural mediums due to lacking
The understanding of ingredient, the major defect using natural medium are that repeatability is poor.Artificial synthetic medium is artificially added special
Determine component nutriment, carries out the culture medium for the suitable cell growth that appropriate proportion is formed.As synthetic media is constantly sent out
Exhibition, classification also gradually refine, and can be divided into serum-containing media, culture medium containing serum substitute, arrive serum free medium.Nothing
The use of blood serum medium improves the repeatability of cell culture, avoids the influence due to difference between serum batch, subtracts
The danger that the microbial contaminations such as virus, fungi and mycoplasma are brought by serum is lacked.Meanwhile obtained cell products be also easy to it is pure
Change.The above many advantages have been more and more widely used serum free medium.
Chinese hamster ovary (CHO) cell is a kind of important engineering cell, and foreign gene is easy to express wherein, product
For secreting type, it is easily isolated purifying.So being a kind of important host cell in cell engineering.Currently on the market, including
There are CHO serum-free production medium in import and domestic each famous brand culture medium company, but price is relatively expensive, using rise
Carry out higher cost, so that serum free medium hardly results in large-scale application.Engineered CHO cells in production application are come
It says, under the premise of normal growth, obtains high expression quantity and be only key.Invent it is a collect cell growth with product expression performance in
The serum free medium of one is the actual requirement in market.
Summary of the invention
For the above-mentioned prior art, the present invention provides a kind of highly expressed Chinese hamster ovary celI serum free medium of support product,
The free serum culture of cell can be fully achieved.Using serum free medium culture of the invention, density height, growth can get fastly
And the Chinese hamster ovary celI that expression quantity is high.The present invention can be such that exponential phase cells shorten, plateau extends, and be conducive to the generation of product,
To obtain more target products, production efficiency is improved.
The present invention is achieved by the following technical solutions:
A kind of highly expressed Chinese hamster ovary celI serum free medium of support product, is grouped as by the group of following concentration:
Substance title adds concentration (mg/L)
Amino acid
Glycine 150-750
L-Alanine 5-50
L-arginine hydrochloride 200-630
Altheine 300-1500
L-Aspartic acid 110-320
L-cysteine hydrochloride 50-200
Pidolidone 135-250
L-Histidine hydrochloride 160-500
L-Isoleucine 125-525
L-Leu 50-450
L lysine HCL 55-280
L-Methionine 45-375
L-phenylalanine 30-160
L-PROLINE 56-90
Serine 220-355
L-threonine 120-350
L-Trp 110-230
L-tyrosine disodium 250-450
Valine 250-460
L-cysteine 100-300
Hydroxyproline 2.4-3.5
Vitamin
Choline chloride 130-250
D-VB5 calcium 5-8
Folic acid 1.7-3.5
Niacinamide 3.7-6.6
Puridoxine hydrochloride 6.5-9.5
Riboflavin 0.5-2.3
Thiamine salt hydrochlorate 3.8-5.6
Vitamin B12 1-10
Inositol 10-100
Lipoic acid 5-10
Biotin 0.003-0.03
Retinol 0.005-0.5
P-aminobenzoic acid 0.008-0.015
Inorganic salts
Calcium chloride dihydrate 100-200
Sodium bicarbonate 1000-4000
Epsom salt 50-150
Potassium chloride 500-950
Sodium chloride 2500-5000
Seven hypophosphite monohydrate disodium hydrogen 100-300
Microelement
Inclined alum acid ammonium 0.00015-0.00036
Sodium metasilicate 0.002-0.05
Tetrahydrate manganese chloride 0.00002-0.0001
CoCL2 6H2O 0.001-0.002
Aluminum Chloride Hexahydrate 0.0001-0.0005
Chromium chloride hexahydrate 0.00001-0.0001
Six water nickel chloride 0.00001-0.0001
Eight water oxygen zirconium chloride 0.00001-0.0001
Germanium dioxide 0.00003-0.0003
White vitriol 0.1-1
Ferrous sulfate heptahydrate 0.25-1
Cupric sulfate pentahydrate 0.002-0.02
Sodium selenite 0.0005-0.005
Carbohydrate and other compositions
Linoleic acid 0.02-0.2
Linolenic acid 0.005-0.05
Lecithin 0.5-5
Stearic acid 0.01-0.025
Palmitoleic acid 0.003-0.009
Glucose 3000-6000
Sodium Pyruvate 50-200
Yeast hydrolyate 200-1000
Putrescine hydrochloride 0.2-2
Rh-insulin 1-10
68 500-2000 of pluronic
Taurine 100-250
Ascorbic acid 50-200
Dextran sulfate 10-50
Hydrocortisone 0.1-0.5
Ethanol amine 0.25-0.75
Thymidine 0.08-0.8
Ironic citrate 2000-10000
Recombination human serum albumin 0.2-0.5
Tween-80 1-5
Surplus is water.
Serum free medium of the invention the preparation method comprises the following steps: take above-mentioned component in addition to water, spy is respectively dissolved according to it
Property classification dissolution, then mix, be added water make each component final concentration as described above, adjust pH value to 7.0~7.4, infiltration is depressed into
300~350 mOsm/kg are to get, industrial filter element filtering, while nitrogen protection is dispensed and (avoids unstability ingredient by oxygen
Change).
The highly expressed Chinese hamster ovary celI serum free medium of support product of the invention is free of any serum component, avoids latent
Pollution and risk, solve biosafety issues, while providing convenience for downstream purification.
Detailed description of the invention
Fig. 1: Chinese hamster ovary celI from be transferred in commercially available culture medium continuous passage culture in 3 culture medium of embodiment as a result, inoculation
Afterwards respectively on day 4, cell was passed in the 6th day and the 8th day, continuous passage 4 times, cells show growth stablize.
Fig. 2: growth figure of the Chinese hamster ovary celI that Chinese hamster ovary celI is observed under the microscope in 3 culture medium of embodiment.
Fig. 3: the Chinese hamster ovary celI growth curve of batch cultivation and corresponding Cell viability in 3 culture medium of embodiment.
Fig. 4: Chinese hamster ovary celI batch cultivation in 3 culture medium of embodiment collects the testing result of antibody expression amount.
Specific embodiment
Below with reference to embodiment, the present invention is further illustrated.
Heretofore described raw material is import cell culture grade reagent, and is stored according to related request.Following experiments
Process is routine experiment method unless otherwise instructed.Feature of the invention is described below in conjunction with attached drawing.It should be understood that
Following embodiment is served only for explaining the present invention, rather than limits the scope of the invention.
Embodiment 1 prepares Chinese hamster ovary celI serum free medium
The concentration of each raw material is as follows:
Substance title adds concentration (mg/L)
Amino acid
Glycine 650
L-Alanine 46
L-arginine hydrochloride 324
Altheine 850
L-Aspartic acid 240
L-cysteine hydrochloride 118
Pidolidone 187
L-Histidine hydrochloride 235
L-Isoleucine 220
L-Leu 430
L lysine HCL 166
L-Methionine 207
L-phenylalanine 122
L-PROLINE 74.5
Serine 256
L-threonine 156
L-Trp 153.3
L-tyrosine disodium 306.8
Valine 323
L-cysteine 185
Hydroxyproline 2.79
Vitamin
Choline chloride 155.5
D-VB5 calcium 6.21
Folic acid 1.97
Niacinamide 4.57
Puridoxine hydrochloride 9.12
Riboflavin 1.07
Thiamine salt hydrochlorate 4.22
Vitamin B12 3.6
Inositol 79
Lipoic acid 8.83
Biotin 0.011
Retinol 0.026
P-aminobenzoic acid 0.01
Inorganic salts
Calcium chloride dihydrate 150
Sodium bicarbonate 3700
Epsom salt 77
Potassium chloride 500
Sodium chloride 4500
Seven hypophosphite monohydrate disodium hydrogens 250
Microelement
Inclined alum acid ammonium 0.00022
Sodium metasilicate 0.0025
Tetrahydrate manganese chloride 0.00008
CoCL2 6H2O 0.0011
Aluminum Chloride Hexahydrate 0.0003
Chromium chloride hexahydrate 0.00005
Six water nickel chlorides 0.00005
Eight water oxygen zirconium chlorides 0.00005
Germanium dioxide 0.00005
White vitriol 0.9
Ferrous sulfate heptahydrate 0.95
Cupric sulfate pentahydrate 0.0135
Sodium selenite 0.00179
Carbohydrate and other compositions
Linoleic acid 0.098
Linolenic acid 0.015
Lecithin 3.4
Stearic acid 0.0183
Palmitoleic acid 0.007
Glucose 5000
Sodium Pyruvate 170
Yeast hydrolyate 550
Putrescine hydrochloride 0.49
Rh-insulin 5
Pluronic 68 1000
Taurine 200
Ascorbic acid 110
Dextran sulfate 25
Hydrocortisone 0.4
Ethanol amine 0.5
Thymidine 0.54
Ironic citrate 8000
Recombination human serum albumin 0.3
Tween-80 1.5
Surplus is water.
The preparation method comprises the following steps: taking above-mentioned component in addition to water, according to its respectively dissolution characteristics classification dissolution, then mixes, add
Enter water and make each component final concentration as described above, adjust pH value to 7.0~7.4, infiltration be depressed into 300~350 mOsm/kg to get,
Industrial filter element filtering, while nitrogen protection is dispensed and (unstability ingredient is avoided to be oxidized).
Embodiment 2 prepares Chinese hamster ovary celI serum free medium
The concentration of each raw material is as follows:
Substance title adds concentration (mg/L)
Amino acid
Glycine 500
L-Alanine 30
L-arginine hydrochloride 500
Altheine 505
L-Aspartic acid 240
L-cysteine hydrochloride 130
Pidolidone 155
L-Histidine hydrochloride 290
L-Isoleucine 450
L-Leu 125
L lysine HCL 250
L-Methionine 125
L-phenylalanine 60
L-PROLINE 60
Serine 350
L-threonine 300
L-Trp 215
L-tyrosine disodium 400
Valine 400
L-cysteine 252
Hydroxyproline 3.3
Vitamin
Choline chloride 215
D-VB5 calcium 7.5
Folic acid 3.2
Niacinamide 5.8
Puridoxine hydrochloride 7.5
Riboflavin Tetrabutyrate .1
Thiamine salt hydrochlorate 5.0
Vitamin B12 8.2
Inositol 50
Lipoic acid 6.2
Biotin 0.02
Retinol 0.3
P-aminobenzoic acid 0.013
Inorganic salts
Calcium chloride dihydrate 180
Sodium bicarbonate 3700
Epsom salt 120
Potassium chloride 500
Sodium chloride 4500
Seven hypophosphite monohydrate disodium hydrogens 250
Microelement
Inclined alum acid ammonium 0.0003
Sodium metasilicate 0.03
Tetrahydrate manganese chloride 0.00005
CoCL2 6H2O 0.0015
Aluminum Chloride Hexahydrate 0.0003
Chromium chloride hexahydrate 0.00005
Six water nickel chlorides 0.00005
Eight water oxygen zirconium chlorides 0.00005
Germanium dioxide 0.00005
White vitriol 0.2
Ferrous sulfate heptahydrate 0.5
Cupric sulfate pentahydrate 0.01
Sodium selenite 0.003
Carbohydrate and other compositions
Linoleic acid 0.15
Linolenic acid 0.03
Lecithin 1.5
Stearic acid 0.012
Palmitoleic acid 0.004
Glucose 4500
Sodium Pyruvate 100
Yeast hydrolyate 850
Putrescine hydrochloride 1.5
Rh-insulin 8
Pluronic 68 1500
Taurine 150
Ascorbic acid 150
Dextran sulfate 50
Hydrocortisone 0.2
Ethanol amine 0.6
Thymidine 0.2
Ironic citrate 5000
Recombination human serum albumin 0.4
Tween-80 1.5
Surplus is water.
The preparation method comprises the following steps: taking above-mentioned component in addition to water, according to its respectively dissolution characteristics classification dissolution, then mixes, add
Enter water and make each component final concentration as described above, adjust pH value to 7.0~7.4, infiltration be depressed into 300~350 mOsm/kg to get,
Industrial filter element filtering, while nitrogen protection is dispensed and (unstability ingredient is avoided to be oxidized).
Embodiment 3 prepares Chinese hamster ovary celI serum free medium
Each material concentration is as follows:
Substance title adds concentration (mg/L)
Amino acid
Glycine 650
L-Alanine 30
L-arginine hydrochloride 300
Altheine 1000
L-Aspartic acid 150
L-cysteine hydrochloride 150
Pidolidone 200
L-Histidine hydrochloride 200
L-Isoleucine 320
L-Leu 430
L lysine HCL 185
L-Methionine 200
L-phenylalanine 150
L-PROLINE 60
Serine 285
L-threonine 250
L-Trp 200
L-tyrosine disodium 423
Valine 405
L-cysteine 155
Hydroxyproline 3.0
Vitamin
Choline chloride 150
D-VB5 calcium 5.8
Folic acid 3.3
Niacinamide 4.2
Puridoxine hydrochloride 8.5
Riboflavin 1.8
Thiamine salt hydrochlorate 4.5
Vitamin B12 5.0
Inositol 25
Lipoic acid 8.5
Biotin 0.025
Retinol 0.3
P-aminobenzoic acid 0.01
Inorganic salts
Calcium chloride dihydrate 150
Sodium bicarbonate 3700
Epsom salt 77
Potassium chloride 900
Sodium chloride 4500
Seven hypophosphite monohydrate disodium hydrogens 250
Microelement
Inclined alum acid ammonium 0.00031
Sodium metasilicate 0.045
Tetrahydrate manganese chloride 0.00008
CoCL2 6H2O 0.0015
Aluminum Chloride Hexahydrate 0.0004
Chromium chloride hexahydrate 0.00003
Six water nickel chlorides 0.00003
Eight water oxygen zirconium chlorides 0.00003
Germanium dioxide 0.00003
White vitriol 0.5
Ferrous sulfate heptahydrate 0.8
Cupric sulfate pentahydrate 0.015
Sodium selenite 0.004
Carbohydrate and other compositions
Linoleic acid 0.15
Linolenic acid 0.035
Lecithin 1.5
Stearic acid 0.021
Palmitoleic acid 0.005
Glucose 5000
Sodium Pyruvate 100
Yeast hydrolyate 800
Putrescine hydrochloride 1.5
Rh-insulin 8
Pluronic 68 1500
Taurine 180
Ascorbic acid 150
Dextran sulfate 30
Hydrocortisone 0.3
Ethanol amine 0.55
Thymidine 0.3
Ironic citrate 3000
Recombination human serum albumin 0.4
Tween-80 3.5
Surplus is water.
The preparation method comprises the following steps: taking above-mentioned component in addition to water, according to its respectively dissolution characteristics classification dissolution, then mixes, add
Enter water and make each component final concentration as described above, adjust pH value to 7.0~7.4, infiltration be depressed into 300~350 mOsm/kg to get,
Industrial filter element filtering, while nitrogen protection is dispensed and (unstability ingredient is avoided to be oxidized).
Experiment
Chinese hamster ovary celI serum free medium prepared by embodiment 3, for doing cell culture.Specific method is will be in commercially available culture
Stablize the Chinese hamster ovary celI of culture in base with 0.5e6 The density of cells/ml is seeded in the finished product culture medium of the invention.In 125 mL
It is cultivated in shaking flask, inoculation volume is 30 mL.Condition of culture is 5% CO2, and temperature is 37 DEG C, 110 r/min of shaking speed.
Daily continuous sampling carries out cell count after inoculation, while taking the motility rate of trypan blue staining calculating cell.
As a result
Suspension Chinese hamster ovary celI growth conditions in the serum free medium are preferable, and cell individually disperses to suspend, without obvious cell knot
Group.Cell viability is high, batch cultivation, and motility rate can maintain 90% or more before plateau, and continuous culture performance is stablized.
Therefore, in this culture medium, cell expresses exogenous albumen ability is also remarkably reinforced.Batch cultivation can harvest in every liter of culture
Protein product reaches 466mg/L.
Above-mentioned, although specific embodiments of the present invention have been described in conjunction with the embodiments, not protects to the present invention
The limitation of range, those skilled in the art should understand that, based on the technical solutions of the present invention, those skilled in the art
The various modifications or changes that can be made are not needed to make the creative labor still within protection scope of the present invention.
Claims (5)
1. a kind of Chinese hamster ovary celI serum free medium, which is characterized in that be grouped as by the group of following concentration:
Substance title adds concentration (mg/L)
Amino acid
Glycine 150-750
L-Alanine 5-50
L-arginine hydrochloride 200-630
Altheine 300-1500
L-Aspartic acid 110-320
L-cysteine hydrochloride 50-200
Pidolidone 135-250
L-Histidine hydrochloride 160-500
L-Isoleucine 125-525
L-Leu 50-450
L lysine HCL 55-280
L-Methionine 45-375
L-phenylalanine 30-160
L-PROLINE 56-90
Serine 220-355
L-threonine 120-350
L-Trp 110-230
L-tyrosine disodium 250-450
Valine 250-460
L-cysteine 100-300
Hydroxyproline 2.4-3.5
Vitamin
Choline chloride 130-250
D-VB5 calcium 5-8
Folic acid 1.7-3.5
Niacinamide 3.7-6.6
Puridoxine hydrochloride 6.5-9.5
Riboflavin 0.5-2.3
Thiamine salt hydrochlorate 3.8-5.6
Vitamin B12 1-10
Inositol 10-100
Lipoic acid 5-10
Biotin 0.003-0.03
Retinol 0.005-0.5
P-aminobenzoic acid 0.008-0.015
Inorganic salts
Calcium chloride dihydrate 100-200
Sodium bicarbonate 1000-4000
Epsom salt 50-150
Potassium chloride 500-950
Sodium chloride 2500-5000
Seven hypophosphite monohydrate disodium hydrogen 100-300
Microelement
Inclined alum acid ammonium 0.00015-0.00036
Sodium metasilicate 0.002-0.05
Tetrahydrate manganese chloride 0.00002-0.0001
CoCL2 6H2O 0.001-0.002
Aluminum Chloride Hexahydrate 0.0001-0.0005
Chromium chloride hexahydrate 0.00001-0.0001
Six water nickel chloride 0.00001-0.0001
Eight water oxygen zirconium chloride 0.00001-0.0001
Germanium dioxide 0.00003-0.0003
White vitriol 0.1-1
Ferrous sulfate heptahydrate 0.25-1
Cupric sulfate pentahydrate 0.002-0.02
Sodium selenite 0.0005-0.005
Carbohydrate and other compositions
Linoleic acid 0.02-0.2
Linolenic acid 0.005-0.05
Lecithin 0.5-5
Stearic acid 0.01-0.025
Palmitoleic acid 0.003-0.009
Glucose 3000-6000
Sodium Pyruvate 50-200
Yeast hydrolyate 200-1000
Putrescine hydrochloride 0.2-2
Rh-insulin 1-10
68 500-2000 of pluronic
Taurine 100-250
Ascorbic acid 50-200
Dextran sulfate 10-50
Hydrocortisone 0.1-0.5
Ethanol amine 0.25-0.75
Thymidine 0.08-0.8
Ironic citrate 2000-10000
Recombination human serum albumin 0.2-0.5
Tween-80 1-5
Surplus is water.
2. Chinese hamster ovary celI serum free medium according to claim 1, which is characterized in that be grouped as by the group of following concentration:
Substance title adds concentration (mg/L)
Amino acid
Glycine 650
L-Alanine 46
L-arginine hydrochloride 324
Altheine 850
L-Aspartic acid 240
L-cysteine hydrochloride 118
Pidolidone 187
L-Histidine hydrochloride 235
L-Isoleucine 220
L-Leu 430
L lysine HCL 166
L-Methionine 207
L-phenylalanine 122
L-PROLINE 74.5
Serine 256
L-threonine 156
L-Trp 153.3
L-tyrosine disodium 306.8
Valine 323
L-cysteine 185
Hydroxyproline 2.79
Vitamin
Choline chloride 155.5
D-VB5 calcium 6.21
Folic acid 1.97
Niacinamide 4.57
Puridoxine hydrochloride 9.12
Riboflavin 1.07
Thiamine salt hydrochlorate 4.22
Vitamin B12 3.6
Inositol 79
Lipoic acid 8.83
Biotin 0.011
Retinol 0.026
P-aminobenzoic acid 0.01
Inorganic salts
Calcium chloride dihydrate 150
Sodium bicarbonate 3700
Epsom salt 77
Potassium chloride 500
Sodium chloride 4500
Seven hypophosphite monohydrate disodium hydrogens 250
Microelement
Inclined alum acid ammonium 0.00022
Sodium metasilicate 0.0025
Tetrahydrate manganese chloride 0.00008
CoCL2 6H2O 0.0011
Aluminum Chloride Hexahydrate 0.0003
Chromium chloride hexahydrate 0.00005
Six water nickel chlorides 0.00005
Eight water oxygen zirconium chlorides 0.00005
Germanium dioxide 0.00005
White vitriol 0.9
Ferrous sulfate heptahydrate 0.95
Cupric sulfate pentahydrate 0.0135
Sodium selenite 0.00179
Carbohydrate and other compositions
Linoleic acid 0.098
Linolenic acid 0.015
Lecithin 3.4
Stearic acid 0.0183
Palmitoleic acid 0.007
Glucose 5000
Sodium Pyruvate 170
Yeast hydrolyate 550
Putrescine hydrochloride 0.49
Rh-insulin 5
Pluronic 68 1000
Taurine 200
Ascorbic acid 110
Dextran sulfate 25
Hydrocortisone 0.4
Ethanol amine 0.5
Thymidine 0.54
Ironic citrate 8000
Recombination human serum albumin 0.3
Tween-80 1.5
Surplus is water.
3. Chinese hamster ovary celI serum free medium according to claim 1, which is characterized in that be grouped as by the group of following concentration:
Substance title adds concentration (mg/L)
Amino acid
Glycine 650
L-Alanine 30
L-arginine hydrochloride 300
Altheine 1000
L-Aspartic acid 150
L-cysteine hydrochloride 150
Pidolidone 200
L-Histidine hydrochloride 200
L-Isoleucine 320
L-Leu 430
L lysine HCL 185
L-Methionine 200
L-phenylalanine 150
L-PROLINE 60
Serine 285
L-threonine 250
L-Trp 200
L-tyrosine disodium 423
Valine 405
L-cysteine 155
Hydroxyproline 3.0
Vitamin
Choline chloride 150
D-VB5 calcium 5.8
Folic acid 3.3
Niacinamide 4.2
Puridoxine hydrochloride 8.5
Riboflavin 1.8
Thiamine salt hydrochlorate 4.5
Vitamin B12 5.0
Inositol 25
Lipoic acid 8.5
Biotin 0.025
Retinol 0.3
P-aminobenzoic acid 0.01
Inorganic salts
Calcium chloride dihydrate 150
Sodium bicarbonate 3700
Epsom salt 77
Potassium chloride 900
Sodium chloride 4500
Seven hypophosphite monohydrate disodium hydrogens 250
Microelement
Inclined alum acid ammonium 0.00031
Sodium metasilicate 0.045
Tetrahydrate manganese chloride 0.00008
CoCL2 6H2O 0.0015
Aluminum Chloride Hexahydrate 0.0004
Chromium chloride hexahydrate 0.00003
Six water nickel chlorides 0.00003
Eight water oxygen zirconium chlorides 0.00003
Germanium dioxide 0.00003
White vitriol 0.5
Ferrous sulfate heptahydrate 0.8
Cupric sulfate pentahydrate 0.015
Sodium selenite 0.004
Carbohydrate and other compositions
Linoleic acid 0.15
Linolenic acid 0.035
Lecithin 1.5
Stearic acid 0.021
Palmitoleic acid 0.005
Glucose 5000
Sodium Pyruvate 100
Yeast hydrolyate 800
Putrescine hydrochloride 1.5
Rh-insulin 8
Pluronic 68 1500
Taurine 180
Ascorbic acid 150
Dextran sulfate 30
Hydrocortisone 0.3
Ethanol amine 0.55
Thymidine 0.3
Ironic citrate 3000
Recombination human serum albumin 0.4
Tween-80 3.5
Surplus is water.
4. the preparation method of Chinese hamster ovary celI serum free medium according to any one of claims 1 to 3, it is characterised in that: take
Component is stated, according to its respectively dissolution characteristics classification dissolution, then mixes, is dissolved in deionized water, adjust solution ph and exist
Between 7.0-7.4, osmotic pressure gets product culture medium between 300~350 mOsm/kg after filtration sterilization.
5. application of the Chinese hamster ovary celI serum free medium according to any one of claims 1 to 3 in culture suspension Chinese hamster ovary celI.
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