CN107418932A - A kind of cell culture fluid for promoting propagation - Google Patents

A kind of cell culture fluid for promoting propagation Download PDF

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Publication number
CN107418932A
CN107418932A CN201710708955.6A CN201710708955A CN107418932A CN 107418932 A CN107418932 A CN 107418932A CN 201710708955 A CN201710708955 A CN 201710708955A CN 107418932 A CN107418932 A CN 107418932A
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parts
cell culture
culture fluid
cell
streptomysin
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CN201710708955.6A
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Inventor
王健
胡小东
赵翔
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Chongqing Sidemu Biological Technology Co Ltd
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Chongqing Sidemu Biological Technology Co Ltd
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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
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0646—Natural killers cells [NK], NKT 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
    • C12N2500/10—Metals; Metal chelators
    • C12N2500/12—Light metals, i.e. alkali, alkaline earth, Be, Al, Mg
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/11—Epidermal growth factor [EGF]
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/13—Nerve growth factor [NGF]; Brain-derived neurotrophic factor [BDNF]; Cilliary neurotrophic factor [CNTF]; Glial-derived neurotrophic factor [GDNF]; Neurotrophins [NT]; Neuregulins
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/999—Small molecules not provided for elsewhere

Abstract

A kind of cell culture fluid for promoting propagation, it is the nutrient solution based on RPMI1640, while adds disodium hydrogen phosphate, potassium dihydrogen phosphate, hyclone, sodium chloride, glucose, potassium chloride, vitamin B12, streptomysin, glutathione, glycine, alanine, aspartic acid, tryptophan, threonine, isoleucine, sodium carboxymethylcellulose.Each composition of culture medium of the present invention is mutually coordinated, collective effect, sufficient nutrition and good environment needed for growth and proliferation of cell can be provided, cytoactive and multiplication capacity can be improved, culture especially suitable for NK cells, using nutrient solution culture of the present invention NK cells fast and stable can be promoted to breed, after cultivating 2 weeks, proliferation times are up to more than 120 times, and fungi, bacterium, mycoplasma etc. are not detected in the culture medium after cultivating, microorganism detection index meets laws and regulations requirement, and raw material needed for preparation are simple and easy to get, is worth marketing application.

Description

A kind of cell culture fluid for promoting propagation
Technical field
The invention belongs to biological technical field, and in particular to a kind of cell culture fluid for promoting propagation.
Background technology
The growth of cell needs nutrient environment, for maintaining the nutrient matrix of cell growth to be referred to as culture medium.Culture medium is pressed Its physical state can be divided into fluid nutrient medium and solid medium.Fluid nutrient medium is used for large-scale industrial production and physiology The research work of the basic theories such as metabolism.Certain coagulator (such as agar) or solid culture are added in fluid nutrient medium (such as Wheat bran, rice etc.) become as solid medium.Solid medium provides the table of a nutrition and ventilation for the growth of cell Face, the cell produced on such a nutrition surface can form single bacterium colony.Therefore, solid medium cell separation, Identification, counting etc. play considerable effect.Cell required for being separated from multicellular organism and amplification obtain thin Born of the same parents and to cell carry out engineered ex vivo, observation, it is necessary to solve the problems, such as colony-formation assays, with the difficulty or ease of microbial cell culture Compare, relatively difficult is the culture of unicellular culture, particularly zooblast from multicellular organism.Cell culture refers to All in vitro cultures, its implication refers to take out tissue in driven thing live body body, inside physiological environment in analogue body etc. is specific Under the conditions of, incubation culture is carried out, is allowed to survive and grows.Cell culture work has been widely used in biology, medical science, new drug The every field such as research and development, turn into one of most important basic science.
At present, the cell culture medium materials of in the market are relatively complicated, and cost is higher, promote the ability of propagation poor, The cell quantity finally obtained is not ideal enough.
The content of the invention
It is an object of the invention to provide a kind of cell culture fluid for promoting propagation.
The purpose of the present invention is realized by following technical measures：
A kind of cell culture fluid for promoting propagation, it is characterised in that it is the nutrient solution based on RPMI1640, while again Add additives；The additives include disodium hydrogen phosphate, potassium dihydrogen phosphate, hyclone, sodium chloride, glucose, potassium chloride, Vitamin B12, streptomysin, glutathione, glycine, alanine, aspartic acid, tryptophan, threonine, isoleucine, carboxylic first Base sodium cellulosate.
Further, a kind of cell culture fluid for promoting propagation, it is characterised in that it include getting off weight proportion into packet Into：12~18 parts of 1640 culture mediums of RPMI, 0.03~0.08 part of disodium hydrogen phosphate, 0.10~0.18 part of potassium dihydrogen phosphate, tire ox 6~11 parts of serum, 15~23 parts of sodium chloride, 0.03~0.09 part of glucose, 0.02~0.05 part of potassium chloride, vitamin B12 0.001~0.007 part, 0.5~1.1 part of streptomysin, 0.03~0.07 part of glutathione, 0.02~0.06 part of glycine, the third ammonia It is 0.02~0.06 part of acid, 0.03~0.08 part of aspartic acid, 0.02~0.06 part of tryptophan, 0.02~0.06 part of threonine, different 0.03~0.08 part of leucine, 0.5~1.2 part of sodium carboxymethylcellulose.
Further, a kind of cell culture fluid for promoting propagation, it is characterised in that the cell culture fluid is also needed to add and sterilized Water for injection, sterilized water for injection addition are 0.9% to cause the quality volume fraction of sodium chloride in nutrient solution.
In order that the cultivation effect for obtaining cell culture fluid is more obvious, a kind of cell culture fluid for promoting propagation, it includes The composition composition of following weight proportion：13~16 parts of 1640 culture mediums of RPMI, 0.04~0.07 part of disodium hydrogen phosphate, di(2-ethylhexyl)phosphate 0.12~0.15 part of hydrogen potassium, 7~10 parts of hyclone, 17~21 parts of sodium chloride, 0.05~0.08 part of glucose, potassium chloride 0.03 ~0.05 part, 0.002~0.006 part of vitamin B12,0.8~1.0 part of streptomysin, 0.05~0.07 part of glutathione, sweet ammonia 0.04~0.06 part of acid, 0.03~0.05 part of alanine, 0.05~0.07 part of aspartic acid, 0.04~0.06 part of tryptophan, Soviet Union 0.04~0.06 part of propylhomoserin, 0.05~0.07 part of isoleucine, 0.9~1.1 part of sodium carboxymethylcellulose, sterilized water for injection 1800~2350 parts, 0.001~0.003 part of nerve growth factor, 0.001~0.003 part of TGF, epidermal cell life Long 0.001~0.003 part of the factor.
Optimal, a kind of cell culture fluid for promoting propagation, the composition that it includes following weight proportion forms：RPMI 1640 15 parts of culture mediums, 0.05 part of disodium hydrogen phosphate, 0.15 part of potassium dihydrogen phosphate, 8 parts of hyclone, 20 parts of sodium chloride, grape 0.07 part of sugar, 0.05 part of potassium chloride, 0.005 part of vitamin B12,1.0 parts of streptomysin, 0.06 part of glutathione, glycine 0.05 Part, 0.04 part of alanine, 0.06 part of aspartic acid, 0.06 part of tryptophan, 0.06 part of threonine, 0.06 part of isoleucine, carboxylic first 1.0 parts of base sodium cellulosate, 2223 parts of sterilized water for injection, 0.002 part of nerve growth factor, 0.002 part of TGF, table 0.002 part of skin cell growth factor.
The present invention has following beneficial effect：
Present invention culture medium based on RPMI1640 culture mediums, adds corresponding additives so that present invention culture Each composition of base is mutually coordinated, collective effect, can provide sufficient nutrition and good environment needed for growth and proliferation of cell, can improve thin Cytoactive and multiplication capacity, especially suitable for the culture of NK cells, using nutrient solution culture of the present invention NK cells can be promoted quick Stable propagation, after culture 2 weeks, proliferation times are up to more than 120 times, and do not detected in the culture medium after cultivating fungi, bacterium, Mycoplasma etc., microorganism detection index meet laws and regulations requirement, and raw material needed for preparation are simple and easy to get, are worth marketing application.
Embodiment
The present invention is specifically described below by embodiment, it is necessary to it is pointed out here that be that following examples are only used It is further described in the present invention, it is impossible to limiting the scope of the invention is interpreted as, without departing substantially from spirit of the invention In the case of essence, the modifications or substitutions made to the inventive method, step or condition belong to the scope of the present invention.
Embodiment 1
A kind of cell culture fluid for promoting propagation includes each component of following proportioning：
Title Dosage
RPMI1640 15g
Disodium hydrogen phosphate 0.05g
Dipotassium hydrogen phosphate 0.15g
Hyclone 8g
Sodium chloride 20g
Glucose 0.07g
Potassium chloride 0.05g
Microorganism B12 0.005g
Streptomysin 1.0g
Glutathione 0.06g
Glycine 0.05g
Alanine 0.04g
Aspartic acid 0.06g
Tryptophan 0.06g
Threonine 0.06g
Isoleucine 0.06g
Sodium carboxymethylcellulose 1.0g
Sterilized water for injection 2223ml
Nerve growth factor 0.002g
TGF 0.002g
Epithelical cell growth factor 0.002g
Preparation method：Above-mentioned other materials except sterilized water for injection are weighed, are placed in suitable appropriate vessel, in 15~20 DEG C At a temperature of be stirred 5~10 minutes, it is to be mixed uniformly after, add sterilized water for injection, continue to be uniformly mixed, produce.
NK cell culture experiments：
Cell culture fluid made from Example 1, NK cells are inoculated in nutrient solution, satisfied in 37 DEG C, 5% carbon dioxide With cultivated in the CO2gas incubator of temperature 2 weeks, add within every 2 days above-mentioned cell culture fluid, record growing state, experimental result Show, NK cells breed 150 times.
Embodiment 2
A kind of cell culture fluid for promoting propagation includes each component of following proportioning：
Title Dosage (g)
RPMI1640 13g
Disodium hydrogen phosphate 0.04g
Dipotassium hydrogen phosphate 0.12g
Hyclone 7g
Sodium chloride 17g
Glucose 0.05g
Potassium chloride 0.03g
Microorganism B12 0.002g
Streptomysin 0.8g
Glutathione 0.05g
Glycine 0.04g
Alanine 0.03g
Aspartic acid 0.05g
Tryptophan 0.04g
Threonine 0.04g
Isoleucine 0.05g
Sodium carboxymethylcellulose 0.9g
Sterilized water for injection 1889ml
Nerve growth factor 0.001g
TGF 0.001g
Epithelical cell growth factor 0.001g
Preparation method：Above-mentioned other materials except sterilized water for injection are weighed, are placed in suitable appropriate vessel, in 15~20 DEG C At a temperature of be stirred 5~10 minutes, it is to be mixed uniformly after, add sterilized water for injection, continue to be uniformly mixed, produce.
By the experimental method of embodiment 1, NK cell culture experiments are carried out, after cultivating two weeks, test result indicates that, NK cells More than 130 times of propagation.
Embodiment 3
A kind of cell culture fluid for promoting propagation includes each component of following proportioning：
Title Dosage (g)
RPMI1640 16g
Disodium hydrogen phosphate 0.07g
Dipotassium hydrogen phosphate 0.15g
Hyclone 10g
Sodium chloride 21g
Glucose 0.08g
Potassium chloride 0.05g
Microorganism B12 0.006g
Streptomysin 1.0g
Glutathione 0.07g
Glycine 0.06g
Alanine 0.05g
Aspartic acid 0.07g
Tryptophan 0.06g
Threonine 0.06g
Isoleucine 0.07g
Sodium carboxymethylcellulose 1.1g
Sterilized water for injection 2333ml
Nerve growth factor 0.003g
TGF 0.003g
Epithelical cell growth factor 0.003g
Preparation method：Above-mentioned other materials except sterilized water for injection are weighed, are placed in suitable appropriate vessel, in 15~20 DEG C At a temperature of be stirred 5~10 minutes, it is to be mixed uniformly after, add sterilized water for injection, continue to be uniformly mixed, produce.
By the experimental method of embodiment 1, NK cell culture experiments are carried out, after cultivating two weeks, test result indicates that, NK cells More than 120 times of propagation.

Claims (5)

1. a kind of cell culture fluid for promoting propagation, it is characterised in that it is the nutrient solution based on RPMI1640, while is added again Enter additives；The additives include disodium hydrogen phosphate, potassium dihydrogen phosphate, hyclone, sodium chloride, glucose, potassium chloride, dimension Raw plain B12, streptomysin, glutathione, glycine, alanine, aspartic acid, tryptophan, threonine, isoleucine, carboxymethyl Sodium cellulosate.
2. a kind of cell culture fluid for promoting propagation as claimed in claim 1, it is characterised in that it includes getting off weight proportion Composition composition：12 ~ 18 parts of 1640 culture mediums of RPMI, 0.03 ~ 0.08 part of disodium hydrogen phosphate, potassium dihydrogen phosphate 0.10 ~ 0.18 Part, 6 ~ 11 parts of hyclone, 15 ~ 23 parts of sodium chloride, 0.03 ~ 0.09 part of glucose, 0.02 ~ 0.05 part of potassium chloride, vitamin B12 0.001 ~ 0.007 part, 0.5 ~ 1.1 part of streptomysin, 0.03 ~ 0.07 part of glutathione, 0.02 ~ 0.06 part of glycine, alanine 0.02 ~ 0.06 part, 0.03 ~ 0.08 part of aspartic acid, 0.02 ~ 0.06 part of tryptophan, 0.02 ~ 0.06 part of threonine, isoleucine 0.03 ~ 0.08 part, 0.5 ~ 1.2 part of sodium carboxymethylcellulose.
A kind of 3. cell culture fluid for promoting propagation as claimed in claim 1 or 2, it is characterised in that the cell culture fluid Also need to add sterilized water for injection, sterilized water for injection addition is to cause the quality volume fraction of sodium chloride in nutrient solution 0.9%。
4. a kind of cell culture fluid for promoting propagation as claimed in claim 3, it is characterised in that it includes following weight proportion Composition composition：13 ~ 16 parts of 1640 culture mediums of RPMI, 0.04 ~ 0.07 part of disodium hydrogen phosphate, potassium dihydrogen phosphate 0.12 ~ 0.15 Part, 7 ~ 10 parts of hyclone, 17 ~ 21 parts of sodium chloride, 0.05 ~ 0.08 part of glucose, 0.03 ~ 0.05 part of potassium chloride, vitamin B12 0.002 ~ 0.006 part, 0.8 ~ 1.0 part of streptomysin, 0.05 ~ 0.07 part of glutathione, 0.04 ~ 0.06 part of glycine, alanine 0.03 ~ 0.05 part, 0.05 ~ 0.07 part of aspartic acid, 0.04 ~ 0.06 part of tryptophan, 0.04 ~ 0.06 part of threonine, isoleucine 0.05 ~ 0.07 part, 0.9 ~ 1.1 part of sodium carboxymethylcellulose, 1800 ~ 2350 parts of sterilized water for injection, nerve growth factor 0.001 ~ 0.003 part, 0.001 ~ 0.003 part of TGF, 0.001 ~ 0.003 part of epithelical cell growth factor.
5. a kind of cell culture fluid for promoting propagation, it is characterised in that the composition that it includes following weight proportion forms：RPMI 1640 15 parts of culture mediums, 0.05 part of disodium hydrogen phosphate, 0.15 part of potassium dihydrogen phosphate, 8 parts of hyclone, 20 parts of sodium chloride, grape 0.07 part of sugar, 0.05 part of potassium chloride, 0.005 part of vitamin B12,1.0 parts of streptomysin, 0.06 part of glutathione, glycine 0.05 Part, 0.04 part of alanine, 0.06 part of aspartic acid, 0.06 part of tryptophan, 0.06 part of threonine, 0.06 part of isoleucine, carboxylic first 1.0 parts of base sodium cellulosate, 2223 parts of sterilized water for injection, 0.002 part of nerve growth factor, 0.002 part of TGF, table 0.002 part of skin cell growth factor.
CN201710708955.6A 2017-08-17 2017-08-17 A kind of cell culture fluid for promoting propagation Pending CN107418932A (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107964532A (en) * 2017-12-27 2018-04-27 重庆斯德姆生物技术有限公司 Human plactnta Subaerial blue green algae is induced to differentiate into the method and its application of cardiac muscle cell
CN108060127A (en) * 2017-12-12 2018-05-22 成都源泉生物科技有限公司 A kind of immunocyte basal medium

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108060127A (en) * 2017-12-12 2018-05-22 成都源泉生物科技有限公司 A kind of immunocyte basal medium
CN107964532A (en) * 2017-12-27 2018-04-27 重庆斯德姆生物技术有限公司 Human plactnta Subaerial blue green algae is induced to differentiate into the method and its application of cardiac muscle cell

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Application publication date: 20171201