CN107488625B - A kind of fetal bovine serum replacement and its preparation method and application - Google Patents
A kind of fetal bovine serum replacement and its preparation method and application Download PDFInfo
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- CN107488625B CN107488625B CN201710771221.2A CN201710771221A CN107488625B CN 107488625 B CN107488625 B CN 107488625B CN 201710771221 A CN201710771221 A CN 201710771221A CN 107488625 B CN107488625 B CN 107488625B
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- 108091003079 Bovine Serum Albumin Proteins 0.000 title claims abstract description 50
- 239000012091 fetal bovine serum Substances 0.000 title claims abstract description 34
- 238000002360 preparation method Methods 0.000 title claims abstract description 18
- 239000012888 bovine serum Substances 0.000 claims abstract description 18
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 13
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 13
- 239000006228 supernatant Substances 0.000 claims description 37
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 28
- 239000007788 liquid Substances 0.000 claims description 21
- KWGKDLIKAYFUFQ-UHFFFAOYSA-M lithium chloride Chemical compound [Li+].[Cl-] KWGKDLIKAYFUFQ-UHFFFAOYSA-M 0.000 claims description 14
- 239000011780 sodium chloride Substances 0.000 claims description 14
- 230000001376 precipitating effect Effects 0.000 claims description 13
- 238000005119 centrifugation Methods 0.000 claims description 11
- 210000004369 blood Anatomy 0.000 claims description 9
- 239000008280 blood Substances 0.000 claims description 9
- XJLSEXAGTJCILF-RXMQYKEDSA-N (R)-nipecotic acid zwitterion Chemical compound OC(=O)[C@@H]1CCCNC1 XJLSEXAGTJCILF-RXMQYKEDSA-N 0.000 claims description 7
- 239000003146 anticoagulant agent Substances 0.000 claims description 7
- 229940127219 anticoagulant drug Drugs 0.000 claims description 7
- 235000016709 nutrition Nutrition 0.000 claims description 7
- 230000035764 nutrition Effects 0.000 claims description 7
- 210000002381 plasma Anatomy 0.000 claims description 7
- 238000001556 precipitation Methods 0.000 claims description 7
- 239000011781 sodium selenite Substances 0.000 claims description 7
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims description 6
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 6
- XLYOFNOQVPJJNP-ZSJDYOACSA-N heavy water Substances [2H]O[2H] XLYOFNOQVPJJNP-ZSJDYOACSA-N 0.000 claims description 6
- JYGXADMDTFJGBT-VWUMJDOOSA-N hydrocortisone Chemical compound O=C1CC[C@]2(C)[C@H]3[C@@H](O)C[C@](C)([C@@](CC4)(O)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 JYGXADMDTFJGBT-VWUMJDOOSA-N 0.000 claims description 6
- 238000000034 method Methods 0.000 claims description 6
- DAEPDZWVDSPTHF-UHFFFAOYSA-M sodium pyruvate Chemical compound [Na+].CC(=O)C([O-])=O DAEPDZWVDSPTHF-UHFFFAOYSA-M 0.000 claims description 6
- 230000001954 sterilising effect Effects 0.000 claims description 6
- 241000283690 Bos taurus Species 0.000 claims description 5
- 229910003424 Na2SeO3 Inorganic materials 0.000 claims description 5
- 239000012141 concentrate Substances 0.000 claims description 5
- 238000010612 desalination reaction Methods 0.000 claims description 5
- 229960003692 gamma aminobutyric acid Drugs 0.000 claims description 5
- BTCSSZJGUNDROE-UHFFFAOYSA-N gamma-aminobutyric acid Chemical compound NCCCC(O)=O BTCSSZJGUNDROE-UHFFFAOYSA-N 0.000 claims description 5
- 239000000203 mixture Substances 0.000 claims description 5
- 235000015097 nutrients Nutrition 0.000 claims description 4
- 238000004659 sterilization and disinfection Methods 0.000 claims description 4
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 3
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 3
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 claims description 3
- 239000001110 calcium chloride Substances 0.000 claims description 3
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 3
- 229910052927 chalcanthite Inorganic materials 0.000 claims description 3
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims description 3
- 229910000397 disodium phosphate Inorganic materials 0.000 claims description 3
- 229910052564 epsomite Inorganic materials 0.000 claims description 3
- 239000011640 ferrous citrate Substances 0.000 claims description 3
- 235000019850 ferrous citrate Nutrition 0.000 claims description 3
- 238000001914 filtration Methods 0.000 claims description 3
- 239000008103 glucose Substances 0.000 claims description 3
- 229960000890 hydrocortisone Drugs 0.000 claims description 3
- APVZWAOKZPNDNR-UHFFFAOYSA-L iron(ii) citrate Chemical compound [Fe+2].OC(=O)CC(O)(C([O-])=O)CC([O-])=O APVZWAOKZPNDNR-UHFFFAOYSA-L 0.000 claims description 3
- 229910001629 magnesium chloride Inorganic materials 0.000 claims description 3
- 229910052603 melanterite Inorganic materials 0.000 claims description 3
- 239000011734 sodium Substances 0.000 claims description 3
- 229910052708 sodium Inorganic materials 0.000 claims description 3
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 claims description 3
- 229910000162 sodium phosphate Inorganic materials 0.000 claims description 3
- 229940054269 sodium pyruvate Drugs 0.000 claims description 3
- 238000010257 thawing Methods 0.000 claims description 3
- 239000013049 sediment Substances 0.000 claims 1
- 239000012894 fetal calf serum Substances 0.000 abstract description 16
- 239000001963 growth medium Substances 0.000 abstract description 16
- 238000004113 cell culture Methods 0.000 abstract description 9
- 230000012010 growth Effects 0.000 abstract description 6
- 238000004519 manufacturing process Methods 0.000 abstract description 4
- 150000001875 compounds Chemical class 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 45
- 210000002966 serum Anatomy 0.000 description 38
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 12
- 230000006872 improvement Effects 0.000 description 7
- 244000309466 calf Species 0.000 description 6
- 230000001413 cellular effect Effects 0.000 description 6
- 210000001519 tissue Anatomy 0.000 description 6
- 229910021642 ultra pure water Inorganic materials 0.000 description 6
- 239000012498 ultrapure water Substances 0.000 description 6
- 230000001605 fetal effect Effects 0.000 description 5
- 241001465754 Metazoa Species 0.000 description 4
- 230000010261 cell growth Effects 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- 206010021703 Indifference Diseases 0.000 description 3
- 108010019160 Pancreatin Proteins 0.000 description 3
- 230000004663 cell proliferation Effects 0.000 description 3
- 230000029087 digestion Effects 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 239000004615 ingredient Substances 0.000 description 3
- 229940055695 pancreatin Drugs 0.000 description 3
- 108010024636 Glutathione Proteins 0.000 description 2
- 239000006143 cell culture medium Substances 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 230000000295 complement effect Effects 0.000 description 2
- 239000000306 component Substances 0.000 description 2
- BVTBRVFYZUCAKH-UHFFFAOYSA-L disodium selenite Chemical compound [Na+].[Na+].[O-][Se]([O-])=O BVTBRVFYZUCAKH-UHFFFAOYSA-L 0.000 description 2
- 210000002950 fibroblast Anatomy 0.000 description 2
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 2
- 230000008676 import Effects 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 230000001737 promoting effect Effects 0.000 description 2
- 229960001471 sodium selenite Drugs 0.000 description 2
- 235000015921 sodium selenite Nutrition 0.000 description 2
- 229960000344 thiamine hydrochloride Drugs 0.000 description 2
- 235000019190 thiamine hydrochloride Nutrition 0.000 description 2
- 239000011747 thiamine hydrochloride Substances 0.000 description 2
- DPJRMOMPQZCRJU-UHFFFAOYSA-M thiamine hydrochloride Chemical compound Cl.[Cl-].CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N DPJRMOMPQZCRJU-UHFFFAOYSA-M 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 1
- YASYEJJMZJALEJ-UHFFFAOYSA-N Citric acid monohydrate Chemical class O.OC(=O)CC(O)(C(O)=O)CC(O)=O YASYEJJMZJALEJ-UHFFFAOYSA-N 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 241000287828 Gallus gallus Species 0.000 description 1
- 206010018910 Haemolysis Diseases 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 241000204031 Mycoplasma Species 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 230000001464 adherent effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 239000012534 cell culture medium component Substances 0.000 description 1
- 230000030833 cell death Effects 0.000 description 1
- 239000013553 cell monolayer Substances 0.000 description 1
- 238000005352 clarification Methods 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000000994 depressogenic effect Effects 0.000 description 1
- 229940042399 direct acting antivirals protease inhibitors Drugs 0.000 description 1
- 239000002158 endotoxin Substances 0.000 description 1
- 210000001339 epidermal cell Anatomy 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008588 hemolysis Effects 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 210000004408 hybridoma Anatomy 0.000 description 1
- 230000003463 hyperproliferative effect Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000008595 infiltration Effects 0.000 description 1
- 238000001764 infiltration Methods 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 206010025482 malaise Diseases 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 210000000130 stem cell Anatomy 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 238000012549 training Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 229960005486 vaccine Drugs 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/0018—Culture media for cell or tissue culture
- C12N5/0037—Serum-free medium, which may still contain naturally-sourced components
Abstract
The invention discloses a kind of fetal bovine serum replacements and its preparation method and application to have obtained good fetal calf serum by the way that specific compound is added in the bovine serum for removing removing protein for sub.When fetal bovine serum replacement of the invention is used for cell culture, the growth conditions of cell are good, and use fetal calf serum almost the same.Stability between fetal bovine serum replacement has preferable batch, the culture medium of the fetal bovine serum replacement of different batches do not make significant difference to the culture of cell.Fetal bovine serum replacement of the invention, production and application cost is relatively low, and price fluctuation is smaller.
Description
Technical field
The present invention relates to a kind of biological products, in particular to a kind of fetal bovine serum replacement and its preparation method and application.
Background technique
Animal blood serum is the maximum natural medium of dosage in cell culture, and cell rich in grows necessary nutrition
Ingredient is usually used in the in vitro culture of zooblast.
Animal blood serum mainly includes fetal calf serum, calf serum, bovine serum, horse serum, sheep blood serum, chicken serum, rabbit blood
It is clear etc., wherein with the most commonly used of fetal calf serum.Growth needed for animal blood serum provides cell Proliferation in cell culture
It is the factor, hormone, transfer protein, adherent and spread over the factor, protease inhibitors and other nutriments on culture substrate.Make
It is most of factor that serum contains promotion cell Proliferation and maintenance with the advantages of serum, it is almost general growth addition
Object, the cell culture suitable for animal, people and insect cell etc..It thus does not need to optimize for each cell line using serum and train
Base is supported, plenty of time and energy are saved.
Protein content is big in serum and complicated component (being greater than 100 kinds), this makes the standardization tested and produced all become tired
Difficulty also makes the downstream separation of biological product of culture cell production and pure such as vaccine, monoclonal antibody and biological activity protein
Change becomes more complicated.Stem cell fostering requirement keeps the developmental characteristic of cell again, does not send out while promoting cell Proliferation
Estrangedization, this requirement to cell culture medium component are harsher.And it may then change cell in vivo normal using serum
State while certain cell (fibroblast) may be promoted to grow, inhibits another kind of cell growth (epidermal cell).It is training
Possibly fibroblast hyper-proliferative can not be prevented using the culture medium containing serum when supporting primary cell, and influence primary cell
Growth.
Although serum contains the ingredient for promoting cell growth, simultaneously also containing endotoxin, hemochrome, complement, antibody etc.
Other harmful components influence cell growth and even result in cell death.Low-quality serum is often by virus, fungi and mycoplasma etc.
Microorganism infection.Serum origin is unstable, may be caused by area of source environment and cows sickness influence in short supply, causes valence
Lattice wave is dynamic.
Fetal calf serum is a kind of character, the light yellow clarification of appearance, without haemolysis, the slightly sticky thick liquid of foreign, is derived from caesarean birth
Tire ox.Because tire ox is also not in contact with the external world, the quality minimum to the harmful ingredient of cell such as antibody, complement contained in serum
Highest, correspondingly, its production and application cost is also extremely high.In addition, the collection process of fetal calf serum also always by
The query of ethics.Therefore, more meet human serum-free cell culture medium or fetal bovine serum replacement value to be supported energetically
And popularization.
Summary of the invention
It is an object of the present invention to overcome the deficiencies of the prior art and provide a kind of tire ox blood suitable for cell culture
Clear substitute.
It is another object of the present invention to provide a kind of preparation methods of fetal bovine serum replacement.
It is yet a further object of the present invention to provide the culture mediums for having used above-mentioned fetal bovine serum replacement.
The technical solution used in the present invention is:
A kind of fetal bovine serum replacement is made of the bovine serum and nutrition composition of removing removing protein, and each nutrition composition is being gone
Whole content in the bovine serum of removing protein precipitating are as follows:
| CaCl2·2H2O | 154.4mg/L ± 10% |
| CuSO4·5H2O | 0.0013mg/L ± 10% |
| 1% ferrous citrate | 2.46ml/L (0.246%v/v) ± 10% |
| Hydrocortisone | 0.5mg/L ± 10% |
| FeSO4·7H2O | 0.417mg/L ± 10% |
| KCl | 311.8mg/L ± 10% |
| MgCl2·6H2O | 61mg/L ± 10% |
| MgSO4·7H2O | 99.88mg/L ± 10% |
| NaCl | 6.9g/L ± 10% |
| NaH2PO4·2H2O | 70.68mg/L ± 10% |
| Na2HPO4·12H2O | 71.02mg/L ± 10% |
| Glucose | 1.25g/L ± 10% |
| Sodium Pyruvate | 55mg/L ± 10% |
| GABA storing liquid | 2ml/L ± 10% |
| LiCl storing liquid | 2ml ± 10% |
| Nipecotic acid concentrate | 129.7ul/L ± 10% |
| Stock A storing liquid | 20ml/L ± 10% |
| Na2SeO3Storing liquid | 0.2ml/L ± 10% |
Wherein, GABA storing liquid is dissolved in 100ml ultrapure water by 5155mgGABA and is obtained；LiCl storing liquid presses lithium chloride
847.2mg, which is dissolved in 40ml ultrapure water, to be obtained；In nipecotic acid concentrate, the concentration of nipecotic acid is 1mg/ml；Stock A storing liquid
By 165mg thiamine hydrochloride, 50mg reduced glutathione, it is super that 1650mg L-AA -2- phosphoric acid magnesium salts is dissolved in 500ml
It is obtained in pure water；Na2SeO3Storing liquid is dissolved in 100ml ultrapure water by 7mg sodium selenite to be obtained.
As the further improvement of above-mentioned fetal bovine serum replacement, bovine serum of each nutrition composition in removal albumen precipitation
In whole content are as follows:
| CaCl2·2H2O | 154.4mg/L ± 5% |
| CuSO4·5H2O | 0.0013mg/L ± 5% |
| 1% ferrous citrate | 2.46ml/L (0.246%v/v) ± 5% |
| Hydrocortisone | 0.5mg/L ± 5% |
| FeSO4·7H2O | 0.417mg/L ± 5% |
| KCl | 311.8mg/L ± 5% |
| MgCl2·6H2O | 61mg/L ± 5% |
| MgSO4·7H2O | 99.88mg/L ± 5% |
| NaCl | 6.9g/L ± 5% |
| NaH2PO4·2H2O | 70.68mg/L ± 5% |
| Na2HPO4·12H2O | 71.02mg/L ± 5% |
| Glucose | 1.25g/L ± 5% |
| Sodium Pyruvate | 55mg/L ± 5% |
| GABA storing liquid | 2ml/L ± 5% |
| LiCl storing liquid | 2ml ± 5% |
| Nipecotic acid concentrate | 129.7ul/L ± 5% |
| Stock A storing liquid | 20ml/L ± 5% |
| Na2SeO3Storing liquid | 0.2ml/L ± 5% |
More preferably, the further improvement as above-mentioned fetal bovine serum replacement, each nutrition composition is in removal albumen precipitation
Whole content in bovine serum are as follows:
As the further improvement of above-mentioned fetal bovine serum replacement, go the bovine serum of removing protein the preparation method is as follows:
1) it takes stalwartness at bovine blood, anti-coagulants is added, extracting yellow blood plasma under aseptic condition after standing, centrifugation removal precipitating,
Take supernatant；
2) CaCl of final concentration of 20mM is added in supernatant2, 37 DEG C of water-bath 2h；
3) supernatant after water-bath is freezed, places melt later, remove albumen precipitation and collect supernatant；
4) NaCl is added into supernatant to saltout, separates supernatant, centrifugation removal precipitating collects supernatant；
5) NaCl mass concentration is transferred to 0.7%, obtains the bovine serum of removing protein by desalination.
A kind of preparation method of fetal bovine serum replacement, includes the following steps:
1) it takes stalwartness at bovine blood, anti-coagulants is added, extracting yellow blood plasma under aseptic condition after standing, centrifugation removal precipitating,
Take supernatant；
2) CaCl of final concentration of 20mM is added in supernatant2, 37 DEG C of water-bath 2h；
3) supernatant after water-bath is freezed, places melt later, remove albumen precipitation and collect supernatant；
4) NaCl of final concentration of 4.79M is added into supernatant, places precipitating, separates supernatant, centrifugation removal precipitating is collected
Supernatant；
5) by supernatant desalination, adjusting NaCl mass concentration is 0.7%, obtains the bovine serum of removing protein；
6) nutrient is added in bovine serum, each nutrient is as described above in the final concentration of bovine serum；
7) it sterilizes, be packaged to be fetal bovine serum replacement.
As the further improvement of above-mentioned preparation method, anti-coagulants is citric acid three sodium solution.
As the further improvement of above-mentioned preparation method, 4 DEG C stand 4~for 24 hours after extracting yellow blood plasma under aseptic condition, 4 DEG C,
3000rpm centrifugation removal precipitating.
It, will freezing 4 at -20 DEG C~-40 DEG C of supernatant after water-bath~for 24 hours as the further improvement of above-mentioned preparation method
Afterwards, it is placed at room temperature for thawing.
As the further improvement of above-mentioned preparation method, the method for sterilizing is filtration sterilization or irradiation sterilization.
A kind of cell culture medium, added with above-mentioned fetal bovine serum replacement.
The beneficial effects of the present invention are:
Fetal bovine serum replacement of the invention has replaces fetal calf serum well, when being used for cell culture, the life of cell
Long status is good, and uses fetal calf serum almost the same.
Stability between fetal bovine serum replacement of the invention has preferable batch, the fetal bovine serum replacement of different batches
Culture medium does not make significant difference to the culture of cell.
Fetal bovine serum replacement of the invention, production and application cost are relatively low.
Detailed description of the invention
Fig. 1 is the cellular morphology photo of different fetal calf serums He fetal bovine serum replacement culture SP2/0 cell of the present invention；
Fig. 2 is the cellular morphology photo of different fetal calf serums He fetal bovine serum replacement culture 293T cell of the present invention；
Fig. 3 is the cellular morphology photo of different fetal calf serum culture A549 cells；
Fig. 4 is the cellular morphology photo of different fetal calf serum culture A549 cells；
Fig. 5 is the cellular morphology photo of different fetal calf serum culture A549 cells.
Specific embodiment
Below with reference to embodiment, technical solution of the present invention is further illustrated.
Embodiment 1
A kind of fetal bovine serum replacement, preparation method are as follows:
1) selection figure stalwartness takes at ox and is upgraded to unit when blood with 5, anti-coagulants (4% two citric acid monohydrates three are added
Sodium) it measures as 500ml；4 DEG C of refrigerator overnights are placed, the blood plasma of extracting yellow under aseptic condition, are taken after 4 DEG C of 3000rpm centrifugation 10min
Clearly；
2) CaCl of final concentration of 20mM is added into supernatant2, 37 DEG C of water-bath 2h；
3) -20 DEG C place 12 hours after, be placed at room temperature for thawings, removal albumen precipitation collection supernatant；
4) NaCl of final concentration of 4.79M, 4 DEG C of precipitating 12h are added into supernatant；
5) 5000rpm is centrifuged 15min, collects supernatant；
6) NaCl concentration is transferred to 0.7% by desalination；
7) cell is added and grows required ion, is added final concentration of:
8) pH to 7.4 is adjusted；290,0.22 μm of filter filtering is depressed into once with NaCl adjusting infiltration or direct irradiate is gone out
Bacterium；
Wherein, GABA storing liquid is dissolved in 100ml ultrapure water by 5155mgGABA and is obtained；LiCl storing liquid presses lithium chloride
847.2mg, which is dissolved in 40ml ultrapure water, to be obtained；In nipecotic acid concentrate, the concentration of nipecotic acid is 1mg/ml；Stock A storing liquid
By 165mg thiamine hydrochloride, 50mg reduced glutathione, it is super that 1650mg L-AA -2- phosphoric acid magnesium salts is dissolved in 500ml
It is obtained in pure water；Na2SeO3Storing liquid is dissolved in 100ml ultrapure water by 7mg sodium selenite to be obtained.
The performance of fetal bovine serum replacement and fetal calf serum compares:
SP2/0 cell culture
The fetal bovine serum replacement for using different fetal calf serums and embodiment 1 respectively is configured to 20%FBS-DMEM culture
Base cultivates SP2/0 cell as follows:
1) 80%~90% SP2/0 Tissue Culture Flask will have been grown to from 37 DEG C, 5% CO2It takes out, uses in incubator
75% alcohol is sprayed on bottle surface, and Tissue Culture Flask rotation is put in Biohazard Safety Equipment, is exhausted using Sterile pipette former
Some culture mediums are added 1ml PBS solution and wash away remaining culture medium, and exhaust PBS；
2) to the 25cm2The pancreatin of 1ml 0.25% is added in culture bottle, digests 30~60s；
3) when gap occurs in microscopic observation cell monolayer, DMEM culture medium of isometric 3 times containing serum is added and terminates
Digestion；
4) cell is moved in 15ml centrifuge tube, is centrifuged 800rpm/3min；
5) supernatant is removed, adds 5ml's cell is resuspended containing serum DMEM culture medium；
6) cell count, the density of passage are 2~5 × 104A cell/cm2Carry out secondary culture, each 25cm2Culture
Bottle plus 5ml contain the DMEM culture medium of serum, are put in 37 DEG C, 5%CO2It is cultivated in incubator.
The growth conditions and related biochemical indicator of cell are compared, as a result as follows:
Illustrate: the universal 01UC of company A, company A hybridoma O2HO, B company, import famous brand name serum be tire ox
Serum.
From the data in table and scheme to be obtained with the culture medium culture of different serum thin as can be seen that for SP2/0 cell
Basic indifference in born of the same parents' form, fetal bovine serum replacement of the invention and commercially available fetal calf serum effect without significant difference, meanwhile, blood
Clear substitute batch between stability it is preferable, performance is close.
293T cell culture:
The fetal bovine serum replacement for using different fetal calf serums and embodiment 1 respectively is configured to 10%FBS-DMEM culture
Base cultivates 293T cell as follows:
1) 80%~90% 293T Tissue Culture Flask will have been grown to from 37 DEG C, 5% CO2It takes out, uses in incubator
75% alcohol is sprayed on bottle surface, and Tissue Culture Flask rotation is put in Biohazard Safety Equipment, is exhausted using Sterile pipette former
Some culture mediums are added 1ml PBS solution and wash away remaining culture medium, and exhaust PBS；
2) to the 25cm2The pancreatin of 1ml 0.25% is added in culture bottle, digests 3~5min；
3) when intercellular gap increases, DMEM culture of isometric 3 times containing serum is added in microscopic observation cell rounding
Base terminates digestion；
4) cell is moved in 15ml centrifuge tube, is centrifuged 1000rpm/3min；
5) supernatant is removed, adds 5ml's cell is resuspended containing serum DMEM culture medium；
6) cell count, the density of passage are 2~5 × 104A cell/cm2Carry out secondary culture, each 25cm2Culture
Bottle plus 5ml contain the DMEM culture medium of serum, are put in 37 DEG C, 5%CO2It is cultivated in incubator.
By disclosed method culture 293T cell is cultivated, observation compares the growth conditions of cell.Experimental result is as follows:
From the data in table and scheme to be obtained with the culture medium culture of different serum thin as can be seen that for 293T cell
Basic indifference in born of the same parents' form, fetal bovine serum replacement of the invention and commercially available fetal calf serum effect without significant difference, meanwhile, blood
Clear substitute batch between stability it is preferable, performance is close.
A549 cell culture
The fetal bovine serum replacement for using different fetal calf serums and embodiment 1 respectively is configured to 10%FBS-DMEM culture
Base cultivates A549 cell as follows:
1) 80%~90% A549 Tissue Culture Flask will have been grown to from 37 DEG C, 5% CO2It takes out, uses in incubator
75% alcohol is sprayed on bottle surface, and Tissue Culture Flask rotation is put in Biohazard Safety Equipment, is exhausted using Sterile pipette former
Some culture mediums are added 1ml PBS solution and wash away remaining culture medium, and exhaust PBS；
2) to the 25cm2The pancreatin of 1ml 0.25% is added in culture bottle, digests 1~3min；
3) when intercellular gap increases, DMEM culture of isometric 3 times containing serum is added in microscopic observation cell rounding
Base terminates digestion；
4) cell is moved in 15ml centrifuge tube, is centrifuged 1000rpm/3min；
5) supernatant is removed, adds 5ml's cell is resuspended containing serum DMEM culture medium；
6) cell count, the density of passage are 2~5 × 104A cell/cm2Carry out secondary culture, each 25cm2Culture
Bottle plus 5ml contain the DMEM culture medium of serum, are put in 37 DEG C, 5%CO2It is cultivated in incubator.
By disclosed method culture A549 cell is cultivated, in 5 generations of continuous culture, observe the growth conditions of comparison cell.Experiment
As a result as follows:
From the data and figure in table as can be seen that passing through the culture in five generations, the form and cell of A549 cell, which double, to be kept
Normally.For A549 cell, basic indifference in the cellular morphology that is obtained with the culture medium culture of different serum, company A Front
(general) supports the culture of A549 cell completely, and it is good compared with South America import brand serum to show.Fetal calf serum of the invention replaces
For object and commercially available fetal calf serum effect without significant difference, meanwhile, serum substitute batch between stability it is preferable, performance is close.
Claims (6)
1. a kind of fetal bovine serum replacement is made of, it is characterised in that: each nutrition the bovine serum and nutrition composition of removing removing protein
Whole content of the component in the bovine serum for removing removing protein are as follows:
Wherein, go the bovine serum of removing protein the preparation method is as follows:
1) it takes stalwartness at bovine blood, anti-coagulants is added, extracting yellow blood plasma under aseptic condition after standing, centrifugation removal precipitating takes
Clearly；
2) CaCl of final concentration of 20mM is added in the supernatant that step 1) obtains2, 37 DEG C of water-bath 2h；
3) supernatant after step 2 water-bath is freezed, places melt later, remove albumen precipitation and collect supernatant；
4) NaCl is added into the supernatant of step 3) to saltout, separates supernatant, centrifugation removal precipitating collects supernatant；
5) NaCl mass concentration is transferred to 0.7%, obtains the bovine serum of removing protein by desalination.
2. a kind of preparation method of fetal bovine serum replacement, includes the following steps:
1) it takes stalwartness at bovine blood, anti-coagulants is added, extracting yellow blood plasma under aseptic condition after standing, centrifugation removal precipitating takes
Clearly；
2) CaCl of final concentration of 20mM is added in the supernatant that step 1) obtains2, 37 DEG C of water-bath 2h；
3) supernatant after step 2 water-bath is freezed, places melt later, remove albumen precipitation and collect supernatant；
4) NaCl of final concentration of 4.79 M is added into the supernatant of step 3), places precipitating, separates supernatant, centrifugation removal is heavy
It forms sediment, collects supernatant；
5) by the supernatant desalination of step 4), adjusting NaCl mass concentration is 0.7%, obtains the bovine serum of removing protein；
6) nutrient is added in bovine serum, each nutrient is as described in claim 1 in the final concentration of bovine serum；
7) it sterilizes, be packaged to be fetal bovine serum replacement.
3. preparation method according to claim 2, it is characterised in that: anti-coagulants is citric acid three sodium solution.
4. preparation method according to claim 2, it is characterised in that: 4 DEG C stand 4~for 24 hours after extracting yellow under aseptic condition
Blood plasma, 4 DEG C, 3000rpm centrifugation removal precipitating.
5. preparation method according to claim 2, it is characterised in that: will be freezed at -20 DEG C~-40 DEG C of supernatant after water-bath
4~for 24 hours after, be placed at room temperature for thawing.
6. preparation method according to claim 2, it is characterised in that: the method for sterilizing is filtration sterilization or irradiation sterilization.
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| CN201710771221.2A CN107488625B (en) | 2017-08-31 | 2017-08-31 | A kind of fetal bovine serum replacement and its preparation method and application |
| PCT/CN2017/101552 WO2019041378A1 (en) | 2017-08-31 | 2017-09-13 | Fetal bovine serum substitute, preparation method therefor, and application thereof |
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| CN115197894A (en) * | 2022-07-08 | 2022-10-18 | 江苏丰华生物制药有限公司 | Method for culturing CHO (Chinese hamster ovary) cells by adopting serum substitute |
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| CN1800367A (en) * | 2005-01-05 | 2006-07-12 | 上海席诺生物技术有限公司 | Embryo cattle serum substitute without serum or animal protein for animal cell culture |
| CN101760442B (en) * | 2010-01-15 | 2012-06-27 | 华东理工大学 | Serum-free medium for MDCK cell large-scale adherent culture and single-cell suspension culture |
| SG11201402108YA (en) * | 2011-11-11 | 2014-06-27 | Essential Pharmaceuticals Llc | Kit comprising serum replacement and labile factors |
| MX2016008251A (en) * | 2013-12-20 | 2016-10-13 | Essential Pharmaceuticals Llc | Media for cell culture. |
| CN104830765A (en) * | 2015-04-30 | 2015-08-12 | 上海泰坦科技股份有限公司 | High efficiency fetal calf serum substitute |
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Denomination of invention: A fetal bovine serum substitute and its preparation method and application Effective date of registration: 20231227 Granted publication date: 20181221 Pledgee: Industrial and Commercial Bank of China Limited Guangzhou tianpingjia sub branch Pledgor: GUANGZHOU RUITE BIOTECHNOLOGY Co.,Ltd. Registration number: Y2023980074564 |