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CN109609444A - A kind of BHK21Cell non-serum culture medium - Google Patents

A kind of BHK21Cell non-serum culture medium Download PDF

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Publication number
CN109609444A
CN109609444A CN201910090354.2A CN201910090354A CN109609444A CN 109609444 A CN109609444 A CN 109609444A CN 201910090354 A CN201910090354 A CN 201910090354A CN 109609444 A CN109609444 A CN 109609444A
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Prior art keywords
culture medium
cell
bhk
serum culture
cell non
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Pending
Application number
CN201910090354.2A
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Chinese (zh)
Inventor
徐斌
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SUZHOU INDUSTRY PARK SUNRISE BIOTECH ENGINEERING Co Ltd
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SUZHOU INDUSTRY PARK SUNRISE BIOTECH ENGINEERING Co Ltd
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Priority to CN201910090354.2A priority Critical patent/CN109609444A/en
Publication of CN109609444A publication Critical patent/CN109609444A/en
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    • 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
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0684—Cells of the urinary tract or kidneys
    • C12N5/0686—Kidney cells
    • 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
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/05—Inorganic components
    • 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
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/32—Amino acids
    • 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
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/34—Sugars
    • 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
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/38—Vitamins
    • 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
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/46—Amines, e.g. putrescine
    • 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
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/90—Serum-free medium, which may still contain naturally-sourced components

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  • Life Sciences & Earth Sciences (AREA)
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  • Bioinformatics & Cheminformatics (AREA)
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  • General Engineering & Computer Science (AREA)
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  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

The invention discloses a kind of BHK21Cell non-serum culture medium, including glucose, galactolipin, sericin hydrolysate, corn meal agar, amino acids, minerals class, nonionic surfactant, fatty acid ester compounded dose, vitamin, ethanol amine, antioxidant and pH buffer solution, cell in the culture medium when stationary culture with half adherent half suspended state growth, cell survival rate is up to 95% or more；Animal origin-free ingredient improves the biological safety of final products；The virus propagation process that late stage of culture is also supported while sertoli cell is grown, generates higher virus titer；Preparation method is simple, and price is lower, has the prospect of amplification scale production application.

Description

A kind of BHK21Cell non-serum culture medium
Technical field
The present invention relates to culture medium preparation technical field more particularly to a kind of BHK21Cell non-serum culture medium.
Background technique
It is thin applied to host used in recombinant viral vector amplification, recombinant protein expression or vaccine prepare with scale at present Born of the same parents' basic source is in mammalian cell, such as baby hamster nephrocyte (BHK21), these cells are usual in traditional incubation It needs to add the cow's serum of a certain amount of (5-10%), contains growth factor, hormone, carrier egg needed for cell growth in serum White, anchoring factor, microelement and other nutriments, can promote cell to grow.But in large-scale production and reality In, the addition of serum has the following disadvantages: the full chemistry sintetics not being commercialized, and the lactoalbumin hydrolysate of animal Chemical component is simultaneously not fixed, and therefore, the quality of culture medium cannot be guaranteed, so that BHK21Vaccine synthesized by cell Quality cannot be guaranteed；Moreover, the shelf-life of the lactoalbumin hydrolysate of animal origin is generally shorter, the ring for needing strict control to store Otherwise border is easy to rotten；Meanwhile the technique of three-in-one preparation method used by the culture medium is relatively complicated, production efficiency It is lower.
In order to overcome serum to add bring drawbacks described above, many R&D institutions or biology doctor from last century the eighties Medicine company just has developed several serum free mediums in succession, but there is also shortcomings simultaneously: in 1. serum free mediums according to So contain animal protein sources ingredient, such as bovine serum albumin(BSA) (BSA), insulin, transferrins, still leaves and apply blood The problem of being likely to occur clearly, furthermore it is expensive, is generally only suitable for laboratory and cultivates use on a small scale；2. the nothing developed at present Blood serum medium can only be suitble to a kind of growth of cell strain to cell specificity with higher, usually a kind of culture medium, without It is suitble to other cell strains；
So being badly in need of developing serum free medium at present, while requiring animal origin-free in the addO-on therapy of the culture medium Ingredient, nutritional ingredient needed for meeting cell growth.
Summary of the invention
The purpose of the present invention is to provide a kind of BHK21Cell non-serum culture medium, the culture medium without animal origin at Point, to improve biological safety, and cell survival rate is high.
In order to achieve the above objectives, the technical solution adopted by the present invention is that: a kind of BHK21Cell non-serum culture medium, including such as Lower component,
Amino acids:
Minerals class:
Further, BHK21Cell non-serum culture medium, including following component,
Amino acids:
Minerals class:
Further, described fatty acid ester compounded dose is one in linolenic acid, linolenic acid, myristic acid and oleic acid Kind is several.
Further, the nonionic surfactant be fatty alcohol polyoxyethylene ether or isomeric alcohol polyethenoxy ether, such as Fatty alcohol polyoxyethylene ether AEO-9 or isomeric alcohol polyethenoxy ether TO-13, nonionic surfactant can prevent cell from existing Serum-free domestication during agglomerate grow, and be conducive to cell oscillation suspend culture when can adapt to suspension growth environment rapidly, Reduce shear action to injure caused by cell, and fatty alcohol polyoxyethylene ether and isomeric alcohol polyethenoxy ether all have environmental protection Property, without taboo regulated substance.
Further, the vitamin is in folic acid, niacinamide, pyridoxol, thiamine, choline chloride and vitamin C It is one or more of.
Further, the auxiliary additive is one of riboflavin or adenine sulfate or two kinds.
Further, the pH buffer solution is acetic acid, succinic acid or combinations thereof.
Culture medium preparation method of the invention is simple, using conventional method by said components according to its respective dissolution characteristics Classification dissolution, is then mixed in apyrogenic ultrapure water, and the pH value that buffer solution adjusts solution is 7.2, is settled to 1000ml It is prepared by i.e. completion after the filtering of 0.22um aseptic filter membrane.
It is as follows the effect of each component in the present invention, amino acid, short needed for sericin hydrolysate provides cell growth Peptide, vitamin and letones etc.；Its nutrient of corn meal agar is less, general cell grown on this culture medium it is poor, But it is suitable for cell to save, improves cell survival rate；Amino acid can supplement Lys, Val, Leu, Ile, Try, Pro-OH etc.；Fat Sour complexing agent: the fatty acid ester compounded dose of component as cell membrane, organelle film, it is complete to be conducive to eucaryotic cell structure, maintains Normal physiological metabolism；Anti-oxidant reagent prevents cell from peroxidation occurs in serum-free domestication incubation；Ethanol amine is made For the precursor substance that phosphatide and cell membrane synthesize, there is facilitation to the growth of cell.
Compared with prior art, the beneficial effects of the present invention are: cell is adherent with half when stationary culture in the culture medium The growth of half suspended state, cell survival rate is up to 95% or more；Animal origin-free ingredient improves the biological safety of final products； The virus propagation process that late stage of culture is also supported while sertoli cell is grown, generates higher virus titer；Preparation method letter Single, price is lower, has the prospect of amplification scale production application.
Specific embodiment
The following is specific embodiments of the present invention, and technical scheme of the present invention will be further described, but the present invention is simultaneously It is not limited to these embodiments.
Embodiment 1
Amino acids:
Minerals class:
Embodiment 2
Amino acids:
Minerals class:
Embodiment 3
Amino acids:
Minerals class:
It is removed from liquid nitrogen BHK21Cell, centrifugation discard supernatant liquid, and it is to be determined to embodiment 3 that a small amount of embodiment 1 is added The complete training liquid that is made into of culture medium cell is resuspended, and be inoculated in sterile culture flask, number of cells 1*104/ mL, adds For the training liquid of above-mentioned same configuration in cultivating three days in 37 DEG C, 5% carbon dioxide incubator, micro- sem observation, cell covers with bottle wall, Form is good；Culture solution is replaced, then carries out sub-bottle culture and continues to cultivate three generations, micro- sem observation, cell covers with bottle wall, and form is good It is good, it is qualified.
Cell in the culture medium when stationary culture with half adherent half suspended state growth, cell survival rate up to 95% with On；Animal origin-free ingredient improves the biological safety of final products；Late stage of culture is also supported while sertoli cell is grown Virus propagation process generates higher virus titer；Preparation method is simple, and price is lower, has amplification scale production application Prospect.
Specific embodiment described herein is only an example for the spirit of the invention.The neck of technology belonging to the present invention The technical staff in domain can make various modifications or additions to the described embodiments or replace by a similar method In generation, however, it does not deviate from the spirit of the invention or beyond the scope of the appended claims.

Claims (7)

1. a kind of BHK21Cell non-serum culture medium, which is characterized in that including following component,
Amino acids:
Minerals class:
。
2. BHK according to claim 121Cell non-serum culture medium, which is characterized in that including following component,
Amino acids:
Minerals class:
。
3. BHK according to claim 121Cell non-serum culture medium, which is characterized in that described fatty acid ester compounded dose is One or more of linolenic acid, linolenic acid, myristic acid and oleic acid.
4. BHK according to claim 121Cell non-serum culture medium, which is characterized in that the nonionic surfactant For fatty alcohol polyoxyethylene ether or isomeric alcohol polyethenoxy ether.
5. BHK according to claim 121Cell non-serum culture medium, which is characterized in that the vitamin is folic acid, cigarette One or more of amide, pyridoxol, thiamine, choline chloride and vitamin C.
6. BHK according to claim 121Cell non-serum culture medium, which is characterized in that the auxiliary additive is core yellow Element or one of adenine sulfate or two kinds.
7. BHK according to claim 121Cell non-serum culture medium, which is characterized in that the pH buffer solution is acetic acid Or one of succinic acid or two kinds.
CN201910090354.2A 2019-01-30 2019-01-30 A kind of BHK21Cell non-serum culture medium Pending CN109609444A (en)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110592000A (en) * 2019-08-13 2019-12-20 苏州易迈吉生物医药科技有限公司 Serum-free medium supporting high-density suspension culture of BHK (baby hamster kidney) cells
CN112322577A (en) * 2020-11-05 2021-02-05 上海奥浦迈生物科技股份有限公司 Serum-free medium for large-scale culture of CHO cells and application thereof
CN112442486A (en) * 2020-11-05 2021-03-05 上海奥浦迈生物科技股份有限公司 Culture medium for maintaining in vitro culture CHO DG44 cell later-stage viability rate and application thereof
CN116042506A (en) * 2022-10-31 2023-05-02 洛阳赛奥生物工程技术有限公司 A preparation method and application of a serum-free medium for cultivating BHK21 cells

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CN105018416A (en) * 2014-07-15 2015-11-04 内蒙古金源康生物工程有限公司 Serum-free animal origin-free culture medium for suspension culture of BHK-21 cells and preparation method of serum-free animal origin-free culture medium
CN105255812A (en) * 2014-07-15 2016-01-20 内蒙古金源康生物工程有限公司 Low-serum culture medium suitable for BHK-21 cell spinner flask culture and preparation method thereof
CN107435037A (en) * 2016-05-27 2017-12-05 上海倍谙基生物科技有限公司 A kind of serum free medium for bhk cell

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110592000A (en) * 2019-08-13 2019-12-20 苏州易迈吉生物医药科技有限公司 Serum-free medium supporting high-density suspension culture of BHK (baby hamster kidney) cells
CN112322577A (en) * 2020-11-05 2021-02-05 上海奥浦迈生物科技股份有限公司 Serum-free medium for large-scale culture of CHO cells and application thereof
CN112442486A (en) * 2020-11-05 2021-03-05 上海奥浦迈生物科技股份有限公司 Culture medium for maintaining in vitro culture CHO DG44 cell later-stage viability rate and application thereof
CN112442486B (en) * 2020-11-05 2023-02-10 上海奥浦迈生物科技股份有限公司 Culture medium for maintaining late-stage viability of CHO DG44 cells cultured in vitro and application thereof
CN116042506A (en) * 2022-10-31 2023-05-02 洛阳赛奥生物工程技术有限公司 A preparation method and application of a serum-free medium for cultivating BHK21 cells

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