CN101974481A - Serum free culture medium for growing various cells derived from kidney tissue - Google Patents
Serum free culture medium for growing various cells derived from kidney tissue Download PDFInfo
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Abstract
The invention relates to a serum free culture medium for growing various cells derived from the kidney tissue. In the serum free culture medium, dulbecco's modified eagle medium (DMEM)/ nutrient mixture F-12(F12)(1:1) is used as a basic culture medium and comprises the components including yeast autolysate, soy protein hydrolysate, amino acids, a fatty acid combining agent, an antioxidant reagent, reduced glutathione, a polyamine mixture, ethanolamine, beta-mercaptoethanol, glycerol, minerals, Pluronic F-68 and the like, and the pH value is 7.2. The culture medium of the invention can be used for culturing various cell lines derived from the kidney tissue, such as HEK293 cells, Marc145 cells, Vero cells and BHK21 cells and for propagating viruses.
Description
Technical field
The present invention relates to a kind of serum free medium that is used for multiple renal tissue derived cell growth, can be used to support the cultivation of HEK293 cell, Vero cell, Marc145 cell or BHK21 cell especially, belong to the cell engineering field.
Background technology
Being applied to the used host cell of recombinant viral vector amplification, expression of recombinant proteins or vaccine scale preparation at present is mammiferous renal tissue derived cell substantially, as human embryonic kidney cell (HEK293), African green ape nephrocyte (Vero), RhMK (Marc145) and immature hamster nephrocyte (BHK21) etc.These cells need to add the bovine serum of a certain amount of (5～10%) usually in traditional culturing process, contain required somatomedin, hormone, carrier proteins, anchoring factor, trace element and other nutritive substances of cell growth in the serum, can promote the cell growth.But in large-scale production and practical application, there is following shortcoming in the interpolation of serum:
1) pollution of serum susceptible viral, mycoplasma or other pathogenic agent;
2) mass discrepancy between the serum of different batches causes the mass discrepancy between the finished product batch;
3) existence of a large amount of serum increases the difficulty of producing the later stage purifying, increases technical process, improves production cost, and the rate of recovery reduces;
4) the part serum composition is difficult to thorough removal, has a strong impact on end product quality, has potential safety hazard.
Add the above-mentioned defective of bringing in order to overcome serum, some serum free mediums have just been developed in succession from many R﹠D institutions of the eighties in last century or biological medicine company, as the Excell series serum free medium that JRH company under the Sigma house flag produces, VP-AGT series serum free medium that Gibco company produces under the Invitrogen house flag or the like.
Above-mentioned serum free medium all has characteristics separately, can grow by sustenticular cell greatly, but also has weak point simultaneously:
1) still contains animal proteinum in the serum free medium and come derived components, as bovine serum albumin (BSA), Regular Insulin, Transferrins,iron complexes etc., still leaving over and using the problem that serum may occur, it costs an arm and a leg in addition, and use is cultivated in generally only suitable laboratory on a small scale;
2) the serum free medium pair cell of exploitation has higher specificity at present, and common a kind of substratum can only be fit to a kind of growth of cell strain, and is not suitable for other cell strains;
So at present be badly in need of developing being applicable to the suitable type serum free medium that is all the animal kidney tissue-derived cell, require animal origin-free composition in the interpolation component of this substratum simultaneously, satisfy the required nutritive ingredient of cell growth.
Summary of the invention
The object of the present invention is to provide a kind of serum free medium that is used for multiple renal tissue derived cell growth, this substratum does not contain animal derived components, improving biological safety, and can be applicable to the cultivation of multiple renal tissue derived cell, cell survival rate height.
The invention provides a kind of no animal component source, serum free medium that is used for multiple renal tissue derived cell growth, comprise following component:
The pH value of described substratum is 7.2.
Above component is the chemistry or the biochemical reagents of Sigma company.
The preferred version of serum free medium of the present invention is that each component concentration is as follows:
Component concentration
DMEM/F12(1∶1) 14.8g/L
Yeast is from hydrolyzate 2g/L
Soybean protein hydrolyate 1g/L
Amino acids
L-L-Ala 10mg/L
L-Xie Ansuan 80mg/L
L-leucine 30mg/L
L-Isoleucine 50mg/L
L-methionine(Met) 190mg/L
L-proline(Pro) 100mg/L
L-phenylalanine 100mg/L
L-tryptophane 150mg/L
L-glycine 15mg/L
L-Serine 100mg/L
L-Threonine 120mg/L
L-halfcystine 50mg/L
Altheine 50mg/L
L-glutaminate 400mg/L
L-tyrosinase 15 0mg/L
L-aspartic acid 40mg/L
L-L-glutamic acid 50mg/L
L-Methionin 60mg/L
L-arginine 50mg/L
L-Histidine 50mg/L
Oxyproline 10mg/L
Fatty acid complex mixture 0.26ml/L
Anti-oxidant reagent 0.21ml/L
Polyamine mixture 0.21ml/L
Reduced glutathion 3mg/L
Thanomin 1.5ml/L
Beta-mercaptoethanol 0.5ml/L
Glycerine 8g/L
The mineral substance class
CaCl
2 75mg/L
MgCl
2 60mg/L
MgSO
4 50mg/L
NaHCO
3 1.5g/L
FeSO
4·7H
2O 18mg/L
Fe(NO
3)
3·9H
2O 8mg/L
Ironic citrate 2mg/L
Pluronic?F-68 1g/L
The pH of described substratum is 7.2.
Serum free medium of the present invention is a basic medium with DMEM/F12 (1: 1), and each the composition effect that is added is as follows:
1) yeast is from hydrolyzate: the required vitamin B group of cell growth, nucleosides material etc. are provided.
2) soybean protein hydrolyate: provide the cell growth required amino acid, small peptide, VITAMIN and letones etc.
3) amino acid:, replenish Lys, Val, Leu, Ile, Try, Pro-OH etc. according to the growth velocity of renal tissue derived cell and the characteristic of amino acid metabolism.
4) fatty acid complex mixture: lipid acid is as the component of cytolemma, organoid film, and it is complete to help cellularstructure, keeps the normal physiological metabolism.
5) anti-oxidant reagent: prevent that cell from serum-free domestication culturing process peroxidation taking place.
6) reduced glutathion: reduced glutathion, effectively suppress the cell peroxidation.
7) polyamine mixture: carry the polyamines class material of positive charge, maintain the stable and cell normal physiological metabolism of nucleic acid construct.
8) thanomin: phosphatide and cytolemma synthetic precursor substance, the growth of pair cell has promoter action.
9) beta-mercaptoethanol: prevent the cell peroxidation, and effectively suppress cell clustering phenomena in the domestication process.
10) glycerine: the outside atmosphere of stabilized cell incubation growth, reduce the injury that the outside atmosphere pair cell causes.
11) mineral substance: CaCl
2, MgCl
2, MgSO
4, NaHCO
3, FeSO
47H
2O etc.
12) Pluronic F-68: nonionic surfactant can prevent cell poly-group growth in serum-free domestication process, and helps cell can adapt to the suspension growth environment rapidly when the vibration suspension culture, reduces the injury that the shearing action pair cell causes.
In sum, basal culture medium is according to growth, the metabolic characteristic of renal tissue derived cell, in basic medium, add non-animal-origin nutritive ingredient, satisfy the needs of cell growth, add antioxidant component and cellular metabolism and regulate material, assist the kidney derived cell progressively to adapt to the serum-free culture environment, the intracellular reactive oxygen species generation metabolism disorder that inhibition causes owing to serum-free domestication process, even necrocytosis.Substratum of the present invention can be used for the clone in multiple renal tissue source, as the cultivation and the virus multiplication of HEK293 cell, Marc145 cell, Vero cell and BHK21 cell.It has the following advantages:
1) can support the cell growth in above-mentioned multiple renal tissue source, and be not limited to above-mentioned several renal tissue derived cell;
2) above-mentioned cell leaves standstill in this substratum when cultivating and grows with half adherent half suspended state, and cell survival rate reaches more than 95%;
3) the sustenticular cell cultivation of going down to posterity for a long time;
4) animal origin-free composition, the biological safety of raising the finished product;
Also support the virus multiplication process of late stage of culture when 5) sustenticular cell is grown, produce higher virus titer;
6) price is lower, possesses the prospect of amplification scale production application.
Medium preparation method of the present invention is simple, adopt conventional method with said components according to its dissolution characteristics classification dissolving (being undertaken) separately by the operation steps that sigma company product description provides, be mixed in then in the apyrogenic ultrapure water, the pH value of regulator solution is 7.2, is settled to 1000ml after promptly finish preparation behind the 0.22um sterile filtration membrane filtration.
Description of drawings
Fig. 1 (1)～Fig. 1 (4) is the state graph that HEK293 cell, Marc145 cell, Vero cell and BHK21 cell are grown in serum free medium of the present invention under 20 times of object lens; Wherein: Fig. 1 (1) is the HEK293 cell, and Fig. 1 (2) is the Marc145 cell, and Fig. 1 (3) is the Vero cell, and Fig. 1 (4) is the BHK21 cell.
Fig. 2 is the HEK293 cell graphic representation of the growth curve in the DMEM of serum free medium of the present invention and interpolation 10% serum, cell survival rate, specific growth rate respectively; Wherein: legend, △ and zero represent growth curve, cell survival rate and the specific growth rate of HEK293 cell in serum free medium of the present invention respectively, legend ■, ▲, ● represent growth curve, cell survival rate and the specific growth rate of HEK293 cell in the DMEM that contains 10% serum consumption respectively.
Fig. 3 for the marc145 cell respectively at serum free medium of the present invention with contain growth curve, cell survival rate, specific growth rate graphic representation among the DMEM of 10% serum consumption; Wherein, legend, △ and zero represent growth curve, cell survival rate and the specific growth rate of marc145 cell in serum free medium of the present invention respectively, legend ■, ▲, ● represent growth curve, cell survival rate and the specific growth rate of marc145 cell in the DMEM that contains 10% serum consumption respectively.
Fig. 4 for the vero cell respectively at serum free medium of the present invention with contain growth curve, cell survival rate, specific growth rate graphic representation among the DMEM of 10% serum consumption; Wherein, legend, △ and zero represent growth curve, cell survival rate and the specific growth rate of vero cell in serum free medium of the present invention respectively, legend ■, ▲, ● represent growth curve, cell survival rate and the specific growth rate of vero cell in the DMEM that contains 10% serum consumption respectively.
Fig. 5 for the BHK21 cell respectively at serum free medium of the present invention with contain growth curve, cell survival rate, specific growth rate graphic representation among the DMEM of 10% serum consumption; Wherein, legend, △ and zero represent growth curve, cell survival rate and the specific growth rate of BHK21 cell in serum free medium of the present invention respectively, legend ■, ▲, ● represent growth curve, cell survival rate and the specific growth rate of BHK21 cell in the DMEM that contains 10% serum consumption respectively.
Embodiment
Below in conjunction with drawings and Examples, the invention will be further described.
The preparation of embodiment 1 serum free medium of the present invention
Raw material is sigma company product in the table 1, the operation steps that provides by sigma company product description, with each raw material of table 1 according to its dissolution characteristics dissolving of classifying separately, under 25 ℃ of conditions, mix then, the pH value of regulating the gained mixed solution is 7.2, be settled to 1000ml, promptly obtain substratum, the content of each component in substratum is as shown in the table.
When each composition mixes, preferably add an amount of apyrogenic ultra-clean pure water to 800ml, lucifuge stirs 30min, and it is fully dissolved, and adjusting the pH value is 7.2, and then is settled to 1000ml.The target substratum for preparing, after 0.22 μ m membrane filtration degerming, it is stand-by to keep in Dark Place in 4 ℃ of refrigerators.
Table 1. is applicable to the serum free medium component and the content in the no animal component source of HEK293, Marc145, Vero, BHK21 cell cultures
Serum free medium is as follows to the domestication culturing step of HEK293 cell, Marc145 cell, Vero cell and BHK21 cell:
1) cultivation that respectively HEK293 cell, Marc145 cell, Vero cell and the BHK21 cell of normal cultivation is reduced the serum consumption adapts to:
The first step: adopt ordinary method (consumption of serum is 10% in the DMEM substratum) to cultivate HEK293 cell, Marc145 cell, Vero cell and BHK21 cell respectively, then above-mentioned cell is seeded to respectively in the DMEM substratum that adds 5% (volumetric concentration) serum and cultivates, the adaptation through 1～2 generation can adapt to the culture condition that DMEM adds 5% serum fully;
Second step: it is to cultivate in the 2%DMEM substratum that each cell that has adapted to the DMEM substratum that adds 5% serum is seeded to the serum consumption respectively, and HEK293 cell and Marc145 cell adapt to through 3 generations through 2 generations, Vero cell and BHK21 cell can adapt to the culture condition that DMEM adds 2% serum fully;
The 3rd step: it is to cultivate in 1% the DMEM substratum that each cell that has adapted to the DMEM substratum that adds 2% serum is seeded to the serum consumption respectively, and HEK293 cell and Marc145 cell adapt to through 5 generations through 3 generations, Vero cell and BHK21 cell can adapt to the culture condition that DMEM adds 1% serum fully;
2) carrying out the serum-free culture domestication adapts to:
Be the cell that sets out with the cell that has adapted to the DMEM culture condition that adds 1% serum, according to 100%, 75%, 50%, the usage ratio of 25% and 0% (volume ratio), progressively reduce the consumption of DMEM+1% blood serum medium (adding 1% serum in the DMEM substratum), simultaneously according to 0%, 25%, 50%, 75% and 100% usage ratio (volume ratio) progressively increases the consumption of serum free medium of the present invention (pressing described in the embodiment one), progressively tame the nutritional condition of above-mentioned 4 kinds of cell adapted serum free mediums with this mixed culture medium, realize that finally 100% uses this serum free medium to cultivate the HEK293 cell fully, the Marc145 cell, Vero cell and BHK21 cell.
HEK293 cell and Marc145 cell through 5 generations tame, domestication just can adapt to serum free medium of the present invention through 8～10 generations for Vero cell and BHK21 cell.As seen, reduce the serum consumption by several times and cultivate adaptation and serum-free culture domestication, can make the cell adapted serum free medium of the present invention of HEK293 cell, Marc145 cell, Vero cell and BHK21, and be half adherent half growth conditions that suspends.
In 37 ℃, 5%CO
2Culture environment in, cultivate in the square vase at T75, leave standstill cultivation HEK293 cell, Marc145 cell, Vero cell, BHK21 cell respectively with serum free medium of the present invention, their cell growthhabit is seen Fig. 1 (1)～Fig. 1 (4) respectively, as shown in the figure, HEK293 cell, Marc145 cell, Vero cell and the BHK21 cell in renal tissue source, part is presented skin sample form in this serum free medium, be attached to the culture surface growth slightly, the part culture surface that is suspended in spherical in shape is grown.Cell growthhabit integral body trends towards sphere, and the scattered length of cell based one's duty has conglomeration slightly.
Cultivate and went down to posterity once in 3～4 days, with 0.05%EDTA solution peptic cell, dispel cell gently when going down to posterity, remove digestion solution through the centrifugal 2min of 1000rpm, re-suspended cell divides bottle can finish the cultivation of normally going down to posterity in fresh serum free medium, and the general bottle ratio of dividing is 1 biography 3.In the whole culturing process, the survival rate of cell generally all reaches more than 95%.As Fig. 2～shown in Figure 5, HEK293 cell, Marc145 cell, Vero cell, the growth curve of BHK21 cell in this serum free medium all are better than the growth curve in DMEM+10% serum, specific growth rate all is higher than the specific growth rate of traditional substratum at the cultivation initial stage, illustrates that this substratum can support above-mentioned cell normal growth well.
Claims (3)
1. one kind is used for the serum free medium that multiple renal tissue derived cell is grown, and comprises following component:
Component concentration
DMEM/F12(1∶1) 14.8g/L
Yeast is from hydrolyzate 1～3g/L
Soybean protein hydrolyate 1～3g/L
Amino acids
L-L-Ala 10～30mg/L
L-Xie Ansuan 50～150mg/L
L-leucine 30～80mg/L
L-Isoleucine 50～180mg/L
L-methionine(Met) 150～300mg/L
L-proline(Pro) 100～250mg/L
L-phenylalanine 100～250mg/L
L-tryptophane 50～300mg/L
L-glycine 0～50mg/L
L-Serine 50～150mg/L
L-Threonine 50～250mg/L
L-halfcystine 50～250mg/L
Altheine 50～200mg/L
L-glutaminate 250～400mg/L
L-tyrosinase 15 0～250mg/L
L-aspartic acid 20～80mg/L
L-L-glutamic acid 50～200mg/L
L-Methionin 60～100mg/L
L-arginine 50～300mg/L
L-Histidine 50～300mg/L
Oxyproline 0～40mg/L
Fatty acid complex mixture 0.1～0.3ml/L
Anti-oxidant reagent 0.1～0.3ml/L
Polyamine mixture 0.1～0.3ml/L
Reduced glutathion 1～3mg/L
Thanomin 1.5～3ml/L
Beta-mercaptoethanol 0.5～2ml/L
Glycerine 5～20g/L
The mineral substance class
CaCl
2 50～154mg/L
MgCl
2 20～60mg/L
MgSO
4 20～50mg/L
NaHCO
3 1～1.5g/L
FeSO
4·7H
2O 5～20mg/L
Fe(NO
3)
3·9H
2O 2～10mg/L
Ironic citrate 2～20mg/L
Pluronic?F-68 0.5～1g/L
The pH of described substratum is 7.2.
2. substratum according to claim 1 is characterized in that described substratum comprises following component:
Component concentration
DMEM/F12(1∶1) 14.8g/L
Yeast is from hydrolyzate 2g/L
Soybean protein hydrolyate 1g/L
Amino acid
L-L-Ala 10mg/L
L-Xie Ansuan 80mg/L
L-leucine 30mg/L
L-Isoleucine 50mg/L
L-methionine(Met) 190mg/L
L-proline(Pro) 100mg/L
L-phenylalanine 100mg/L
L-tryptophane 150mg/L
L-glycine 15mg/L
L-Serine 100mg/L
L-Threonine 120mg/L
L-halfcystine 50mg/L
Altheine 50mg/L
L-glutaminate 400mg/L
L-tyrosinase 15 0mg/L
L-aspartic acid 40mg/L
L-L-glutamic acid 50mg/L
L-Methionin 60mg/L
L-arginine 50mg/L
L-Histidine 50mg/L
Oxyproline 10mg/L
Fatty acid complex mixture 0.26ml/L
Anti-oxidant reagent 0.21ml/L
Polyamine mixture 0.21ml/L
Reduced glutathion 3mg/L
Thanomin 1.5ml/L
Beta-mercaptoethanol 0.5ml/L
Glycerine 8g/L
Mineral substance
CaCl
2 75mg/L
MgCl
2 60mg/L
MgSO
4 50mg/L
NaHCO
3 1.5g/L
FeSO
4·7H
2O 18mg/L
Fe(NO
3)
3·9H
2O 8mg/L
Ironic citrate 2mg/L
Pluronic?F-68 1g/L
The pH of described substratum is 7.2.
3. according to the described substratum of claim 1, it is characterized in that described renal tissue derived cell is HEK293 cell, Vero cell, Marc145 cell or BHK21 cell.
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