CN102268403A - Serum-free culture medium applicable to large-scale single-cell suspension culture of baby hamster kidney cell - Google Patents
Serum-free culture medium applicable to large-scale single-cell suspension culture of baby hamster kidney cell Download PDFInfo
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Abstract
The invention discloses a serum-free culture medium applicable to large-scale single-cell suspension culture of a baby hamster kidney cell (BHK cell). The serum-free culture medium comprises 21 amino acids, 11 inorganic salts, 12 vitamins, one protein hydrolysate, two lipids, two buffer components and six additives. Through the BHK cell serum-free culture medium, a biological product is produced through single-cell suspension culture, so that various adverse factors of serum culture can be avoided, and the problem of difficulty in scaling roller bottle adherent culture can also be eliminated; and thus, the production efficiency of BHK cell culture is increased, the production cost is reduced, meanwhile, the quality of the product is guaranteed, and the medium has a high application value and a great market prospect.
Description
Technical field
The present invention relates to the cell culture medium field, relate in particular to a kind of serum free medium that is applicable to that the extensive single-cell suspension of baby hamster kidney cell is cultivated.
Background technology
Rabies are by natural epidemic disease source property due to the rabies virus or animal derived infecting both domestic animals and human acute infectious disease, and popular wide, case fatality rate is high, and dog is carried out stringent regulations, dog immunity or the dog of going out, and are the positive effective ways of control human rabies.At present this disease still there is not special effective methods of treatment, case fatality rate almost 100%.Foot and mouth disease is a kind of acute, hot, height contagious disease artiodactylous, its virus variation and popular velocity of propagation all are exceedingly fast, can infect the domestic and wild artiodactyl of kind more than 70, worldwide livestock industry and related industries are caused considerable influence, OIE classifies it as must declare transmissible disease, and China is listed in the zoonotic first place of a class with it.Vaccine inoculation is the effective means of successfully preventing, control and even finally eliminate foot and mouth disease.Pseudoabies has been brought disadvantageous effect to the development of domestic livestock breeding industry.Except that the pig at various ages, ox all the susceptible, make zoogenetic infections morbidities such as sheep, dog, cat, rabbit, mouse, mink, fox under field conditions (factors).Rabbit, cavy, mouse susceptible all in the laboratory animal.Wherein the most responsive with rabbit.The rabies vaccine system that China makes with baby hamster kidney cell (bhk cell) now, aftosa vaccine system and pseudo-rabies vaccine are the production of all or part of employing bhk cell, more domestic production technique adopts MEM to add 10% serum and rolls a bottle adherent culture now, and external serum-free bio-reactor high-density suspension culture technology falls behind a lot of.Except above-mentioned animal vaccine product, the recent advances of many biotechnologys aspect as recombinant protein, antibody, produce via bhk cell, all needs large-scale cell in vitro to cultivate, and this just needs considerable cell culture medium.Yet serum and serum protein in the conventional cell culture medium are problematic, and for being unallowed on the extensive bioreactor culture cost, other blood serum substituting methods described in the prior also are very expensive when a large amount of the use.The subject matter of most of commercially available substratum is to provide a large amount of serum compositions (being generally 5%～20%) to substratum, because the high price of serum and limited availability have caused a significant limiting factor.In addition, from the angle of producing, in substratum, add animal serum and/or serum albumin and can make the derived product purifying complicated more, and the virus that wherein may pollute also is serious safety problem.Further, there is batch stability problem in serum.
Therefore, needed for the fermentation of extensive bio-reactor is a kind ofly to provide the cell cell culture medium required basic nutrition, biology and biophysics requisite of growing with quite low cost, the performance of growth of fabulous cell and maximum cell density can be provided, and also be easy to prepare and get from the low-cost composition of relative lesser amt.Preferably, this substratum should not contain serum.
All lack the serum free medium of cultivating at bhk cell specially both at home and abroad, as document
[1]Report uses the HyQ PF CHO of the commercialization substratum Hyclone of main flow in the world, HyQ PF CHO MPS substratum, the ExCell302 of JRH, the Rencyte BHK substratum of ExCell520 substratum and Merck comes the suspension culture bhk cell, and maximum cell density has only reached 2.2 * 10
6Cell count/milliliter is not as good as the low blood serum medium suspension culture maximum cell density 8.0 * 10 of the BHK that has developed
630% of cell count/milliliter.The maximum cell density of the BHK-21 serum-free culture of other bibliographical information is also all 2.0 * 10
6About cell count/milliliter
[2,3]Clear big day one product MD611 substratum of domestic substratum leading enterprise can be effectively as the low blood serum medium of bhk cell adherent culture, in addition, developed in clear big day one can the suspension culture bhk cell low blood serum medium, do not apply for a patent, with the unexposed sale of fashion.Even clear big day one low blood serum medium, the density of suspension culture bhk cell has also only reached 4 * 10
6Cell count/milliliter
[4]In sum, the bhk cell cost effectiveness serum free medium that can be used for large-scale production and not contain serum of developing low-cost significant to the development of domestic animal vaccine industry.
Reference:
1.?H?Kallel,?Jouini,?S?Majoul,?S?Rourou.?Evaluation?of?various?serum?and?animal?protein?free?media?for?the?production?of?a?veterinary?rabies?vaccine?in?BHK-21?cells.?Biotechnol.?
95(2002)195-204.
2.?The?new?medium?MDSS2N,?free?of?any?animal?protein?supports?cell?growth?and?production?of?various?viruses.?OW?Merten,?H?Kallel,?JC?Manuguerra,?M?Tardy-Panit,?R?Crainic,?F?Delpeyroux,?S?Van?der?Werf,?P?Perrin.?Celltechnol.?
30(1999)191-201.
3.?H?Kallel,?S?Rourou,?S?Majoul,?H?Loukil.?A?novel?process?for?the?production?of?a?veterinary?rabies?vaccine?in?BHK-21?cells?grown?on?microcarriers?in?a?20-l?bioreactor.?Appl?Microbiol?Biotechnol.?
61(2003)441-446.
4. the cell cultures handbook is 2011 editions, clear big day one, 2011.
Summary of the invention
Defective in view of above-mentioned prior art existence, the objective of the invention is to propose a kind of serum free medium that is applicable to that the extensive single-cell suspension of baby hamster kidney cell is cultivated, both can avoid all unfavorable factors of blood serum medium, can eliminate the problem that bottle adherent culture is difficult to the scale amplification of rolling again.
Purpose of the present invention will be achieved by the following technical programs:
A kind of serum free medium that is suitable for the extensive single-cell suspension cultivation of baby hamster kidney cell is characterized in that: comprise amino acid, inorganic salt, VITAMIN, proteolysate, lipid, buffer composition and additive; Described amino acid is the L-tryptophane, L-halfcystine, L-Isoleucine, L-leucine, the L-Xie Ansuan, L-arginine monohydrochloride, L-L-Histidine hydrochloride, L-Methionin, the L-methionine(Met), L-L-glutamic acid, L-phenylalanine, L-asparagus fern acyl, glycine, L-proline(Pro), the L-L-Ala, L-aspartic acid, L-Serine, the L-Threonine, L-glutaminate, L-cystine hydrochloride and L-tyrosine disodium salt; Described inorganic salt are magnesium chloride hexahydrate, anhydrous magnesium sulfate, Calcium Chloride Powder Anhydrous, Repone K, sodium-chlor, sodium dihydrogen phosphate-water, Sodium phosphate dibasic, Zinc vitriol, nine nitric hydrate iron, Sodium Selenite and cupric sulfate pentahydrate; Described VITAMIN is an inositol, vitamins C, pyridoxine phosphate, pyridoxal phosphate, cobalamin, niacinamide, riboflavin, vitamin, D-calcium pantothenate, folic acid, vitamin H (Biotin) and choline chloride 60; Described proteolysate is the rice hydrolyzate; Described lipid is linolic acid and blocked polyethers F-68(Pluronic F-68); Described buffer composition is sodium bicarbonate and hydroxyethyl piperazine ethanesulfonic acid (HEPES); Described additive is a D-glucose, Sodium.alpha.-ketopropionate, reduced glutathion, xanthoglobulin sodium salt, putrescine and Thioctic Acid.
Preferably, the above-mentioned serum free medium that is suitable for the extensive single-cell suspension cultivation of baby hamster kidney cell, wherein: described amino acid whose content is counted with mg/litre:
L-tryptophane 15～750
L-halfcystine 5～204
L-Isoleucine 27～480
L-leucine 30～840
L-Xie Ansuan 20～620
L-arginine monohydrochloride 48～698
L-L-Histidine hydrochloride 30～780
L-Methionin 30～480
L-methionine(Met) 22～190
L-L-glutamic acid 4～30
L-phenylalanine 25～96
Altheine 47～600
Glycine 3～24
L-proline(Pro) 17.25～500
L-L-Ala 3～25
L-aspartic acid 3～20.5
L-Serine 27～670
L-Threonine 50～500
L-glutaminate 365～584
L-cystine hydrochloride 32～64
L-tyrosine disodium salt 56～100.
Preferably, the above-mentioned serum free medium that is suitable for the extensive single-cell suspension cultivation of baby hamster kidney cell, wherein: the content of described VITAMIN is counted with mg/litre:
Vitamins C 0.1～15
Pyridoxine phosphate 0.2～2
Pyridoxal phosphate 0.001～0.1
Cobalamin 0.03～3
Niacinamide 0.1～5
Riboflavin 0.1～0.8
Vitamin 0.2～5
Choline chloride 60 1～8
D-calcium pantothenate 0.1～4
Folic acid 0.1～4
Vitamin H 0.003～0.1
Inositol 2～10.
Preferably, the above-mentioned serum free medium that is suitable for the extensive single-cell suspension cultivation of baby hamster kidney cell, wherein: the content of described inorganic salt is counted with mg/litre:
Magnesium chloride hexahydrate 1～60
Anhydrous magnesium sulfate 1～60
Calcium Chloride Powder Anhydrous 5～160
Repone K 50～400
Sodium-chlor 3000～7000
Sodium dihydrogen phosphate-water 40～200
Sodium phosphate dibasic 10～180
Zinc vitriol 0.1～0.5
Nine nitric hydrate iron 0.01～0.5
Sodium Selenite 0.001～0.02
Cupric sulfate pentahydrate 0.0001～0.002.
Preferably, the above-mentioned serum free medium that is suitable for the extensive single-cell suspension cultivation of baby hamster kidney cell, wherein: the content of described lipid is counted with mg/litre:
Linolic acid 0.01～0.1
Blocked polyethers F-68 500～1000.
Preferably, the above-mentioned serum free medium that is suitable for the extensive single-cell suspension cultivation of baby hamster kidney cell, wherein: the content of described buffer reagent is counted with mg/litre:
Sodium bicarbonate 2400～2800
Hydroxyethyl piperazine ethanesulfonic acid 2000～3800.
Preferably, the above-mentioned serum free medium that is suitable for the extensive single-cell suspension cultivation of baby hamster kidney cell, wherein: the content of described additive is counted with mg/litre:
D-glucose 1000～5000
Sodium.alpha.-ketopropionate 30～165
Reduced glutathion 0.01～0.9
Xanthoglobulin sodium salt 0.1～5
Putrescine 0.08～0.4
Thioctic Acid 0.1～0.4.
Preferably, the above-mentioned serum free medium that is suitable for the extensive single-cell suspension cultivation of baby hamster kidney cell, wherein: the content of described proteolysate is counted with mg/litre:
Rice hydrolyzate 500～6000.
Preferably, the above-mentioned serum free medium that is suitable for the extensive single-cell suspension cultivation of baby hamster kidney cell, wherein: its each components contents is counted with mg/litre:
L-tryptophane 15～750
L-halfcystine 5～204
L-Isoleucine 27～480
L-leucine 30～840
L-Xie Ansuan 20～620
L-arginine monohydrochloride 48～698
L-L-Histidine hydrochloride 30～780
L-Methionin 30～480
L-methionine(Met) 22～190
L-L-glutamic acid 4～30
L-phenylalanine 25～96
Altheine 47～600
Glycine 3～24
L-proline(Pro) 17.25～500
L-L-Ala 3～25
L-aspartic acid 3～20.5
L-Serine 27～670
L-Threonine 50～500
L-glutaminate 365～584
L-cystine hydrochloride 32～64
L-tyrosine disodium salt 56～100
Vitamins C 0.1～15
Inositol 2～10
Pyridoxine phosphate 0.2～2
Pyridoxal phosphate 0.001～0.1
Cobalamin 0.03～3
Niacinamide 0.1～5
Riboflavin 0.1～0.8
Vitamin 0.2～5
Choline chloride 60 1～8
D-calcium pantothenate 0.1～4
Folic acid 0.1～4
Vitamin H 0.003～0.1
Rice hydrolyzate 500～6000
Magnesium chloride hexahydrate 1～60
Anhydrous magnesium sulfate 1～60
Calcium Chloride Powder Anhydrous 5～160
Repone K 50～400
Sodium-chlor 3000～7000
Sodium dihydrogen phosphate-water 40～200
Sodium phosphate dibasic 10～180
Zinc vitriol 0.1～0.5
Nine nitric hydrate iron 0.01～0.5
Sodium Selenite 0.001～0.02
Cupric sulfate pentahydrate 0.0001～0.002
Linolic acid 0.01～0.1
Blocked polyethers F-68 500～1000
Sodium bicarbonate 2400～2800
Hydroxyethyl piperazine ethanesulfonic acid 2000～3800
D-glucose 1000～5000
Sodium.alpha.-ketopropionate 30～165
Reduced glutathion 0.01～0.9
Xanthoglobulin sodium salt 0.1～5
Putrescine 0.08～0.4
Thioctic Acid 0.1～0.4.
The starting point of most conventional cell culture medium is a kind of basic basic medium, generally comprises amino acid, carbohydrate, VITAMIN, inorganic salt, trace element, damping fluid.For the substratum of most conventional, suitable animal serum or serum albumin and lipid composition joined in this basic basic medium and make and be used for sustenticular cell growth and breeding, expression of recombinant proteins and virus replication or viral product and express necessary perfect medium.Consider the cost and the problem that are associated with serum or sero-abluminous use, aspect other egg white substitutes replacement serum compositions, doing some trials in the prior art.For example, Chinese patent 02110644.4 has been announced a kind of serum free medium, and it is by 1) conventional basic medium, 2) lipid mixtures, 3) peptone is formed, and wherein the protein purpose of hydrolysis is to substitute the protein that generally is found in serum or the albumin in the peptone composition.Bhk cell grows into and contain the equal density of blood serum medium although can support to recombinate, and contains more expensive composition in this substratum, and can not sustenticular cell grow to sufficiently high level to be used for economic production.Therefore, improve still needing aspect the performance of substratum and the total cost two.By adjusting some amino acid whose content, removed the protein content in the substratum, and do not influenced the performance of substratum in the present invention.On serum free medium of the present invention, cultivate bhk cell and obtained 4 times of maximum cell density of being reported to Chinese patent 02110644.4, and not only can support recombinant cell strain expressing protein or antibody, also can support the infection and the propagation of virus.Therefore serum-free perfect medium of the present invention provides and the compare saving of additional cost of serum-free composition described in the prior, and fabulous effect is provided simultaneously.
The preferred version of the serum free medium among the present invention comprises amino acid, inorganic salt, VITAMIN, carbohydrate etc., has wherein added proteolysate composition, lipid composition and protection composition.These materials provide sustenticular cell life, growth and breeding necessary basic nutrition composition together.
Serum-free perfect medium of the present invention further comprises the proteolysate composition, and this proteolysate has not only compensated amino acid whose content in the substratum, also provides serum or albuminous surrogate.Term " proteolysate " is meant the protein product of hydrolysis, refers generally to natural protein through the molecular-weight average of acid or the enzyme partial hydrolysis gained protein cleavage product mixtures at about 5 to 30 KD.Selectively, being used for the independent peptone component part of combined preparation proteolysate composition will be through ultrafiltration or similar step purifying in advance, and any residual proteolytic enzyme, intracellular toxin or other potential interfered recombinant protein product that the bhk cell of growing in the substratum expresses or virus and the product of the high component that uses but remove.Therefore, the high-molecular weight protein that expection is used for peptone of the present invention and can be easily provides with serum, serum albumin etc. makes a distinction, and the latter has removed from substratum of the present invention.The concentration of proteolysate relies on several factors in the substratum, for example employed specific proteolysate, the feature of wanting cultured cells system, given peptone cell growth toxigenicity or inhibiting concentration etc., the optimum concn of proteolysate is all determined with experience.General, the total amount of proteolysate is that 0.1 grams per liter arrives in the scope of about 15 grams per liters in serum-free perfect medium of the present invention, is that 0.5 grams per liter is to 10 grams per liters more unobstructedly.Be applicable to that proteolysate of the present invention is commercially available, be mainly the rice hydrolyzate, also can be lactalbumin hydrolysate, casein hydrolysate, Tryptones phosphoric acid meat soup and other plant hydrolyzed thing.
Substratum of the present invention also can contain other water soluble components; Regular Insulin (can strengthen the absorption of carbohydrate) for example; transferrin (Transshipment Permitted iron); trace elements such as selenium; catalase (as the peroxidation protective material); thanomin (can be used as lipid precursor); steroid hormone such as Testosterone; Triiodothyronine such as triiodothyronine; nucleic acid precursor such as xanthoglobulin; Thymine deoxyriboside; Desoxyadenosine and Deoxyribose cytidine, and be used for replenishing of the cell cultures substratum of serum in routine or other nutritive ingredients that serum free medium contains.
The method for preparing substratum is not critical.For example, substratum can make by method as described below, is about to all components and annexation and is dissolved in water with separately suitable concn earlier, and pressurization makes solution filter by a membrane filter and obtains aseptic substratum then.As mentioned above, when cultivating bhk cell for express recombinant protein, antibody or virus, peptone composition in the substratum preferentially prepurification so that later purifying is easier, for example by filtering, carry out ultrafiltration with film subsequently with the little molecular weight boundary of any albumen collected than need or viral product.
Method with culture medium culturing cell of the present invention neither be critical.With the condition approximately identical with conventional substratum with culture medium culturing cell of the present invention.Usually, the cell of growing in serum free medium of the present invention is to cultivate under the appropriate condition at certain temperature range and to selected specific cells.For example, the BHK-21 cell is cultivated in about 36～37.5 ℃ of temperature ranges, and wherein the pH value of substratum preferably is maintained at about in 7.0～7.5 the scope, and is preferred in 7.0～7.2 scopes.Cell cultures is favourable in the good environment of ventilation.
Usually, if can successfully having used in the substratum of serum, albumin or other protein lipid carriers therein, bhk cell grows, this cell can be grown in substratum of the present invention so, serum wherein or protein lipid carrier all are substituted, and optionally add other essential hormone and somatomedins.For example, the commercially available substratum that is used for the bhk cell cultivation of many kinds is arranged, they comprise following commercially available basic medium, Glasgow MEM (BHK-21) [Macpherson et al. Virology. 16:47 (1962)] for example, the Minimum Essential Media (Science of Eagle, 1959,30:432) etc.Substratum of the present invention is not only applicable to the growth of cell, and is applicable to material such as recombinant protein, the factor or the antibody of producing useful physiologically active, can also be used to produce virus vaccines.The U.S. the 7th, 465 uses BHK-21 cell expressing FC-EPO in No. 447 patents.Report such as Kallel uses BHK-21 cell amplification rabies virus (Kallel et al. Jour Biotechnol. 95 (2002) 195-204).Yet benefiting from one of ordinary skill in the art will recognize that according to many other recombinant protein or the viruses of the present invention of present disclosure can be made by this cell strain.Passable recombinant protein includes but not limited to tissue plasminogen activator (TPA), Fc-EPO, the blood coagulation VII factor etc.Passable virus includes but not limited to rabies virus, Pseudorabies virus etc.Further, the bhk cell that can grow in substratum of the present invention can be different subclone cell strains, but BHK-21 cell preferably.The preferred BHK-21 cells of cultivating in substratum of the present invention is suitable for many recombinant proteins, antibody expression, and is suitable for pseudo-rabies and multiple virus amplification such as mad dog, foot and mouth disease.
Outstanding effect of the present invention is: the present invention can adopt conventional preparation method to produce, and its using method also is an ordinary method; Bhk cell serum-free suspension culture base single-cell suspension of the present invention is cultivated and is produced biological products, do not contain serum, component is clear and definite, help the separation and purification of product, improve product quality, the all unfavorable factors that both can avoid serum to cultivate can be eliminated the problem that bottle adherent culture is difficult to the scale amplification of rolling again; Support the bhk cell cultivation of going down to posterity for a long time, need not the adaptive process of long-term and complex; Support bhk cell single-cell suspension growth that can be good, preparation is used easily, and with low cost; be suitable for the powder culture medium preparation; and be used for the large-scale production utilization of biological products, and improve the production efficiency that bhk cell is cultivated, have excellent application value and huge market outlook.
Following constipation closes the embodiment accompanying drawing, the specific embodiment of the present invention is described in further detail, so that technical solution of the present invention is easier to understand, grasp.
Description of drawings
Fig. 1 is the BHK-21 cell growth curve in the bhk cell serum free medium (hyclone CHO-PF and EX 302) of serum free medium of the present invention (BS-SFM) and prior art respectively; Cells/ml: cell count/milliliter;
Fig. 2 is the BHK-21 cell is cultivated the production rabies vaccine respectively in the MEM substratum that is adding 10% calf serum (CS) of serum free medium of the present invention (BS-SFM) and prior art and commercialization serum free medium (CHO-PF and EX 302) a output, wherein, planting malicious titre is 10
5.0LD
50/ 0.03 milliliter.Infection proportion is 1%(v/v); LD
50/ 0.03ml:LD
50/ 0.03 milliliter; Time(h): the time (hour).
Embodiment
Embodiment 1
A kind of serum free medium that is suitable for the extensive single-cell suspension cultivation of baby hamster kidney cell of present embodiment, it comprises 21 seed amino acids, 11 kinds of inorganic salt, 12 kinds of VITAMIN, a kind of proteolysate, 2 kinds of lipids, 2 kinds of buffer compositions, 6 kinds of additives; The material that describes below is all available from Sigma.
Utilization listed composition and amount in table 1 have prepared the particular of present embodiment serum free medium, are labeled as BS-SFM.
Table 1:
| Composition | Concentration |
| Calcium chloride | 116mg/L |
| 50 * main salt | 20 ml/L |
| 10000 * less important salt | 0.1 ml/L |
| 200 * VITAMIN and |
5 ml/L |
| Glucose | 4500 mg/L |
| Glutamine | 484 mg/ |
| 10 * amino acid | 100 ml/ |
| 10% Pluronic F-68 | 10 ml/L |
| Sodium bicarbonate | 2600 mg/L |
| The rice hydrolyzate | 2000 mg/L |
Table 2:50 * main salt
| Composition | Concentration (mg/litre) |
| Magnesium chloride hexahydrate | 1700 |
| Repone K | 15500 |
| Sal epsom | 2450 |
| Sodium-chlor | 225000 |
| Sodium dihydrogen phosphate-water | 6500 |
| Sodium phosphate dibasic | 3900 |
Table 3:10000 * less important salt
| Composition | Concentration (mg/litre) |
| Zinc vitriol | 4320 |
| Nine nitric hydrate iron | 1000 |
| Sodium Selenite | 100 |
| Cupric sulfate pentahydrate | 1.3 |
Table 4:200 * VITAMIN and other
| Composition | Concentration (mg/litre) |
| Vitamins C | 400 |
| Pyridoxine phosphate | 200 |
| Pyridoxal phosphate | 20 |
| Cobalamin | 200 |
| Niacinamide | 200 |
| Riboflavin | 100 |
| Vitamin | 200 |
| Choline chloride 60 | 600 |
| Sodium.alpha.-ketopropionate | 11000 |
| Reduced glutathion | 60 |
| Inositol | 800 |
| The xanthoglobulin sodium salt | 478 |
| Putrescine | 20 |
| Thioctic Acid | 30 |
| Linolic acid | 10.4 |
| The D-calcium pantothenate | 400 |
| Folic acid | 400 |
| Vitamin H | 16 |
Table 5:10 * amino acid
| Composition | Concentration (mg/litre) |
| The L-tryptophane | 200 |
| The L-halfcystine | 1800 |
| The L-leucine | 1050 |
| The L-Isoleucine | 1050 |
| The L- |
96 |
| The L-arginine monohydrochloride | 14750 |
| The L-L-Histidine hydrochloride | 3600 |
| L-Methionin | 38200 |
| The L-methionine(Met) | 16700 |
| L-L-glutamic acid | 1400 |
| The L-phenylalanine | 4200 |
| Altheine | 48600 |
| Glycine | 600 |
| The L-proline(Pro) | 32700 |
| The L-L-Ala | 400 |
| The L-aspartic acid | 1400 |
| The L-Serine | 62400 |
| The L-Threonine | 24700 |
| The L-cystine hydrochloride | 400 |
| L-tyrosine disodium salt | 6800 |
Table 6
| Cultivate batch (repetition) | Maximum cell density (10 6Cell count/milliliter) |
| Repeat 1 | 7.0 |
| Repeat 2 | 7.2 |
| Repeat 3 | 7.3 |
Liquid concentrate with table 2 to the listed composition of table 5 and amount and make.Powdered enriched material makes according to last table 1 listed composition and amount.Enriched material and powder add in 800 ml waters by the listed order of table 1.The pH value of final mixture is transferred to 7.2～7.3 and volume added to 1 liter.Amino acid except that glutamine makes according to table 5.
As shown in table 6, with the BHK-21 cell with 0.8 * 10
6Cell count/milliliter density is inoculated in Corning Incorporated's 500 ml shake flasks, and the maximum cell density that cell can reach has provided the resulting maximum cell density of BHK-21 on the BS-SFM substratum that is grown in three different batches.The results are shown in accompanying drawing 1 with prior art serum free medium (hyclone CHO-PF and EX 302) contrast is cultivated.Learn obviously from The above results, be grown in the passable cell density preferably of cell in the serum free medium cheaply of the present invention.Duplicate on the BHK-21 cell that this serum free medium also can support rabies virus to cultivate in this substratum in addition.
Although aforesaid invention is quite detailed the describing of purpose of illustrating and understanding by explanation and embodiment, in appended claim scope, still have some change and change obviously.For example, the relative quantity of the separate constituent that proposes in table 1～5 can be made an amendment according to the specific needs of interested specific cells system by those skilled in the art, and this need be known to those skilled in the art and be easy to and utilizes.Usually, the specified quantitative of listing in table 1～5 can change in about 50% scope.
Embodiment 2
A kind of serum free medium that is suitable for the extensive single-cell suspension cultivation of baby hamster kidney cell of present embodiment, it comprises 21 seed amino acids, 11 kinds of inorganic salt, 12 kinds of VITAMIN, a kind of proteolysate, 2 kinds of lipids, 2 kinds of buffer compositions, 6 kinds of additives; The material that describes below is all available from Sigma.
Composition is as follows, counts with mg/litre:
L-tryptophane 350
L-halfcystine 150
L-Isoleucine 200
L-leucine 300
L-Xie Ansuan 220
L-arginine monohydrochloride 258
L-L-Histidine hydrochloride 500
L-Methionin 300
L-methionine(Met) 50
L-L-glutamic acid 25
L-phenylalanine 25
Altheine 420
Glycine 20
L-proline(Pro) 19
L-L-Ala 20
L-aspartic acid 20
L-Serine 300
L-Threonine 50
L-glutaminate 390
L-cystine hydrochloride 45
L-tyrosine disodium salt 80
D-glucose 4600
Rice hydrolyzate 2500
HEPES 2000
Pluronic?F-68 800
Yeast hydrolyate 1000
Sodium.alpha.-ketopropionate 105
Reduced glutathion 0.05
Xanthoglobulin sodium salt 0.5
Putrescine 0.1
Thioctic Acid 0.1
Linolic acid 0.01
Vitamins C 0.1
Pyridoxine phosphate 0.2
Pyridoxal phosphate 0.01
Cobalamin 0.02
Niacinamide 2
Riboflavin 0.7
Vitamin 2
Choline chloride 60 7
D-calcium pantothenate 2
Vitamin H 0.1
Magnesium chloride hexahydrate 60
Anhydrous magnesium sulfate 60
Calcium Chloride Powder Anhydrous 100
Repone K 141
Sodium-chlor 6300
Sodium dihydrogen phosphate-water 40
Sodium phosphate dibasic 20
Zinc vitriol 0.1
Nine nitric hydrate iron 0.1
Sodium Selenite 0.01
Cupric sulfate pentahydrate 0.0003
Said components is dissolved in the no thermal source ultrapure water prepares, can be fit to the extensive single-cell suspension serum free medium of bhk cell.Use above-mentioned BS-SFM serum free medium in 500 ml shake flasks of Corning Incorporated, to cultivate the BHK-21 cell, reach 1.5 * 10 at cell density
6Behind cell count/milliliter, infect rabies virus, maximum virus titer is 10
7LD
50/ 0.03 milliliter, reach leading domestic level.That uses the commercialization culture medium culturing under the same terms the results are shown in Figure 2.The training method that the traditional basic medium that uses the BS-SFM substratum to use with respect to state intradermal vaccine enterprise adds 10% serum improves maximum viral yield above 70%, compare with external similar main flow commercialization substratum, BS-SFM substratum viral yield is slightly excellent, but cost but has only every liter of dozens of yuan, import commercialization substratum well below every liter in hundreds of unit can be applied in domestic animal vaccine manufacturing enterprise or people's medication manufacturing enterprise.
The present invention still has numerous embodiments, and all employing equivalents or equivalent transformation and all technical schemes of forming all drop within protection scope of the present invention.
Claims (9)
1. one kind is suitable for the serum free medium that the extensive single-cell suspension of baby hamster kidney cell is cultivated, and it is characterized in that: comprise amino acid, inorganic salt, VITAMIN, proteolysate, lipid, buffer composition and additive;
Described amino acid is the L-tryptophane, L-halfcystine, L-Isoleucine, L-leucine, the L-Xie Ansuan, L-arginine monohydrochloride, L-L-Histidine hydrochloride, L-Methionin, the L-methionine(Met), L-L-glutamic acid, L-phenylalanine, L-asparagus fern acyl, glycine, L-proline(Pro), the L-L-Ala, L-aspartic acid, L-Serine, the L-Threonine, L-glutaminate, L-cystine hydrochloride and L-tyrosine disodium salt;
Described inorganic salt are magnesium chloride hexahydrate, anhydrous magnesium sulfate, Calcium Chloride Powder Anhydrous, Repone K, sodium-chlor, sodium dihydrogen phosphate-water, Sodium phosphate dibasic, Zinc vitriol, nine nitric hydrate iron, Sodium Selenite and cupric sulfate pentahydrate;
Described VITAMIN is an inositol, vitamins C, pyridoxine phosphate, pyridoxal phosphate, cobalamin, niacinamide, riboflavin, vitamin, D-calcium pantothenate, folic acid, vitamin H and choline chloride 60;
Described proteolysate is the rice hydrolyzate;
Described lipid is linolic acid and blocked polyethers F-68;
Described buffer composition is sodium bicarbonate and hydroxyethyl piperazine ethanesulfonic acid;
Described additive is a D-glucose, Sodium.alpha.-ketopropionate, reduced glutathion, xanthoglobulin sodium salt, putrescine and Thioctic Acid.
2. the serum free medium that is suitable for the extensive single-cell suspension cultivation of baby hamster kidney cell according to claim 1, it is characterized in that: described amino acid whose content is counted with mg/litre:
L-tryptophane 15～750
L-halfcystine 5～204
L-Isoleucine 27～480
L-leucine 30～840
L-Xie Ansuan 20～620
L-arginine monohydrochloride 48～698
L-L-Histidine hydrochloride 30～780
L-Methionin 30～480
L-methionine(Met) 22～190
L-L-glutamic acid 4～30
L-phenylalanine 25～96
Altheine 47～600
Glycine 3～24
L-proline(Pro) 17.25～500
L-L-Ala 3～25
L-aspartic acid 3～20.5
L-Serine 27～670
L-Threonine 50～500
L-glutaminate 365～584
L-cystine hydrochloride 32～64
L-tyrosine disodium salt 56～100.
3. the serum free medium that is suitable for the extensive single-cell suspension cultivation of baby hamster kidney cell according to claim 1, it is characterized in that: the content of described VITAMIN is counted with mg/litre:
Vitamins C 0.1～15
Pyridoxine phosphate 0.2～2
Pyridoxal phosphate 0.001～0.1
Cobalamin 0.03～3
Niacinamide 0.1～5
Riboflavin 0.1～0.8
Vitamin 0.2～5
Choline chloride 60 1～8
D-calcium pantothenate 0.1～4
Folic acid 0.1～4
Vitamin H 0.003～0.1
Inositol 2～10.
4. the serum free medium that is suitable for the extensive single-cell suspension cultivation of baby hamster kidney cell according to claim 1, it is characterized in that: the content of described inorganic salt is counted with mg/litre:
Magnesium chloride hexahydrate 1～60
Anhydrous magnesium sulfate 1～60
Calcium Chloride Powder Anhydrous 5～160
Repone K 50～400
Sodium-chlor 3000～7000
Sodium dihydrogen phosphate-water 40～200
Sodium phosphate dibasic 10～180
Zinc vitriol 0.1～0.5
Nine nitric hydrate iron 0.01～0.5
Sodium Selenite 0.001～0.02
Cupric sulfate pentahydrate 0.0001～0.002.
5. the serum free medium that is suitable for the extensive single-cell suspension cultivation of baby hamster kidney cell according to claim 1, it is characterized in that: the content of described lipid is counted with mg/litre:
Linolic acid 0.01～0.1
Blocked polyethers F-68 500～1000.
6. the serum free medium that is suitable for the extensive single-cell suspension cultivation of baby hamster kidney cell according to claim 1, it is characterized in that: the content of described buffer reagent is counted with mg/litre:
Sodium bicarbonate 2400～2800
Hydroxyethyl piperazine ethanesulfonic acid 2000～3800.
7. the serum free medium that is suitable for the extensive single-cell suspension cultivation of baby hamster kidney cell according to claim 1, it is characterized in that: the content of described additive is counted with mg/litre:
D-glucose 1000～5000
Sodium.alpha.-ketopropionate 30～165
Reduced glutathion 0.01～0.9
Xanthoglobulin sodium salt 0.1～5
Putrescine 0.08～0.4
Thioctic Acid 0.1～0.4.
8. the serum free medium that is suitable for the extensive single-cell suspension cultivation of baby hamster kidney cell according to claim 1, it is characterized in that: the content of described proteolysate is counted with mg/litre:
Rice hydrolyzate 500～6000.
9. the serum free medium that is suitable for the extensive single-cell suspension cultivation of baby hamster kidney cell according to claim 1 comprises amino acid, inorganic salt, VITAMIN, proteolysate, lipid, buffer composition and additive is characterized in that, its each components contents is counted with mg/litre:
L-tryptophane 15～750
L-halfcystine 5～204
L-Isoleucine 27～480
L-leucine 30～840
L-Xie Ansuan 20～620
L-arginine monohydrochloride 48～698
L-L-Histidine hydrochloride 30～780
L-Methionin 30～480
L-methionine(Met) 22～190
L-L-glutamic acid 4～30
L-phenylalanine 25～96
Altheine 47～600
Glycine 3～24
L-proline(Pro) 17.25～500
L-L-Ala 3～25
L-aspartic acid 3～20.5
L-Serine 27～670
L-Threonine 50～500
L-glutaminate 365～584
L-cystine hydrochloride 32～64
L-tyrosine disodium salt 56～100
Vitamins C 0.1～15
Inositol 2～10
Pyridoxine phosphate 0.2～2
Pyridoxal phosphate 0.001～0.1
Cobalamin 0.03～3
Niacinamide 0.1～5
Riboflavin 0.1～0.8
Vitamin 0.2～5
Choline chloride 60 1～8
D-calcium pantothenate 0.1～4
Folic acid 0.1～4
Vitamin H 0.003～0.1
Rice hydrolyzate 500～6000
Magnesium chloride hexahydrate 1～60
Anhydrous magnesium sulfate 1～60
Calcium Chloride Powder Anhydrous 5～160
Repone K 50～400
Sodium-chlor 3000～7000
Sodium dihydrogen phosphate-water 40～200
Sodium phosphate dibasic 10～180
Zinc vitriol 0.1～0.5
Nine nitric hydrate iron 0.01～0.5
Sodium Selenite 0.001～0.02
Cupric sulfate pentahydrate 0.0001～0.002
Linolic acid 0.01～0.1
Blocked polyethers F-68 500～1000
Sodium bicarbonate 2400～2800
Hydroxyethyl piperazine ethanesulfonic acid 2000～3800
D-glucose 1000～5000
Sodium.alpha.-ketopropionate 30～165
Reduced glutathion 0.01～0.9
Xanthoglobulin sodium salt 0.1～5
Putrescine 0.08～0.4
Thioctic Acid 0.1～0.4.
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| CN103184195A (en) * | 2013-03-08 | 2013-07-03 | 苏州市沃美生物技术有限公司 | Powder parvovirus maintenance medium and preparation method thereof |
| CN105255810A (en) * | 2015-11-09 | 2016-01-20 | 内蒙古金源康生物工程有限公司 | Serum-free and animal-source-free culture medium suitable for insect cell Sf-9 |
| CN105505853A (en) * | 2015-12-23 | 2016-04-20 | 中农威特生物科技股份有限公司 | Low-serum culture medium for high-density suspension culture of BHK-21 cells and application of low-serum culture medium in proliferation of FMDVs (foot and mouth disease viruses) |
| CN106244518A (en) * | 2016-08-04 | 2016-12-21 | 天信和(苏州)生物科技有限公司 | A kind of Marc 145 low blood serum medium of cell and preparation method thereof |
| CN108103003A (en) * | 2017-12-12 | 2018-06-01 | 四川百诺吉科技有限公司 | A kind of serum free medium and preparation method thereof for adapting to the full suspension growths of PK-15 and cell suspend acclimation method entirely |
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| CN114107170A (en) * | 2021-11-12 | 2022-03-01 | 广东省华晟生物技术有限公司 | Cat kidney suspension cell line and construction method and application thereof |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0954563B1 (en) * | 1996-10-10 | 2008-07-02 | Invitrogen Corporation | Animal cell culture media comprising plant-derived nutrients |
| CN102021139A (en) * | 2009-09-11 | 2011-04-20 | 华东理工大学 | Chinese hamster ovary culture medium as well as preparation method and application thereof |
| CN102115729A (en) * | 2009-12-31 | 2011-07-06 | 北京清大天一科技有限公司 | Method for culturing baby hamster kidney (BHK) 21 cell in serum-free way, and vaccine preparation method |
-
2011
- 2011-08-01 CN CN2011102179489A patent/CN102268403A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0954563B1 (en) * | 1996-10-10 | 2008-07-02 | Invitrogen Corporation | Animal cell culture media comprising plant-derived nutrients |
| CN102021139A (en) * | 2009-09-11 | 2011-04-20 | 华东理工大学 | Chinese hamster ovary culture medium as well as preparation method and application thereof |
| CN102115729A (en) * | 2009-12-31 | 2011-07-06 | 北京清大天一科技有限公司 | Method for culturing baby hamster kidney (BHK) 21 cell in serum-free way, and vaccine preparation method |
Non-Patent Citations (1)
| Title |
|---|
| 滕小锘: "表达乙型肝炎表面抗原的重组CHO细胞无血清培养基的优化及生物反应器培养", 《中国生物制品学杂志》, vol. 23, no. 10, 31 October 2010 (2010-10-31), pages 1080 - 1086 * |
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