CN109370985A - A kind of human umbilical cord mesenchymal stem cells large-scale culture serum free medium - Google Patents

A kind of human umbilical cord mesenchymal stem cells large-scale culture serum free medium Download PDF

Info

Publication number
CN109370985A
CN109370985A CN201811341164.5A CN201811341164A CN109370985A CN 109370985 A CN109370985 A CN 109370985A CN 201811341164 A CN201811341164 A CN 201811341164A CN 109370985 A CN109370985 A CN 109370985A
Authority
CN
China
Prior art keywords
human
umbilical cord
stem cells
mesenchymal stem
cord mesenchymal
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN201811341164.5A
Other languages
Chinese (zh)
Inventor
张昕
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
YOCON BIOLOGY TECHNOLOGY Co.
Original Assignee
Individual
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Individual filed Critical Individual
Priority to CN201811341164.5A priority Critical patent/CN109370985A/en
Publication of CN109370985A publication Critical patent/CN109370985A/en
Pending legal-status Critical Current

Links

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
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0668—Mesenchymal stem cells from other natural sources
    • 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
    • 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
    • C12N2500/05—Inorganic components
    • C12N2500/10—Metals; Metal chelators
    • C12N2500/20—Transition metals
    • 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
    • C12N2500/20—Transition metals
    • C12N2500/22—Zinc; Zn chelators
    • 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
    • C12N2500/20—Transition metals
    • C12N2500/24—Iron; Fe chelators; Transferrin
    • C12N2500/25—Insulin-transferrin; Insulin-transferrin-selenium
    • 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
    • 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
    • C12N2500/36—Lipids
    • 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
    • C12N2500/44—Thiols, e.g. mercaptoethanol
    • 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
    • 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
    • C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
    • 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
    • C12N2501/15—Transforming growth factor beta (TGF-β)
    • 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
    • C12N2501/30—Hormones
    • C12N2501/38—Hormones with nuclear receptors
    • C12N2501/39—Steroid hormones
    • 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
    • C12N2501/70—Enzymes
    • C12N2501/71—Oxidoreductases (EC 1.)
    • 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
    • C12N2501/998—Proteins not provided for elsewhere
    • 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
    • C12N2511/00—Cells for large scale production

Abstract

The invention discloses a kind of human umbilical cord mesenchymal stem cells large-scale culture serum free medium, main component includes amino acid, inorganic salts ingredients, growth factor and albumin ingredient.Human umbilical cord mesenchymal stem cells large-scale culture serum free medium of the invention is free of any serum component, and definite ingredients, overcomes the danger of foreign protei pollution and pathogenic microorganisms, solve the biosafety issues of MSCs clinical application.Using serum free medium of the invention can external large-scale culture human umbilical cord mesenchymal stem cells, can get the umbilical cord mesenchymal stem cells of high quantity, high-purity and safe and high quality, while cell survival rate can be improved, maintain the characteristic of stem cell.The present invention can solve the problems, such as that cell quantity is inadequate, and the cell quantity of every batch of, survival rate and immunophenotype are more stable, meet index of correlation requirement.

Description

A kind of human umbilical cord mesenchymal stem cells large-scale culture serum free medium
Technical field
The present invention relates to a kind of serum free mediums of human umbilical cord mesenchymal stem cells large-scale culture, belong to cell engineering Technical field.
Background technique
Interstital stem cell (mesenchymal stem cells, MSCs) derives from mesoderm, also referred to as multipotency mesenchyma base Cell plastid (multipotent mesenchymal stromal cells) is one kind of multipotential stem cell, it is found in earliest In marrow, there is the multi-lineage potential broken up to three kinds of endoderm cells, there is low immunogenicity, do not stimulate generation allogeneic It is immunoreacted, not by cytotoxic T cell and NK cell killing and immunoregulation effect.MSCs is in corresponding inductive condition Under can be divided into the different types cell such as bone, fat, cartilage, muscle and liver cell.Umbilical cord mesenchymal stem cells are a kind of presence In people's umbilical cord, the cell mass with multi-lineage potential and self-renewal capacity.It is convenient that it draws materials, from a wealth of sources, uses Belong to " waste utilization ", do not limited by ethics, morals, sufficient cell origin can be provided with clinical to test, especially can be refreshing Treatment zone through systemic disease carrys out new hope.Verified its can be induced to differentiate into osteoblast, insulin secreting cells and Quasi-liver cell etc., and in terms of immunosuppressive therapy have positive potential applicability in clinical practice, however its clinical application must with Based on the external umbilical cord mesenchymal stem cells for obtaining high quantity and high-purity.
Cell culture medium is to determine that cell in-vitro growth is metabolized most important, most direct environmental factor.Current most of conjunctions The growth in vitro of supplement animal blood serum ability sertoli cell is required at cell culture medium, is proliferated, and the use band of animal blood serum Carry out the possibility of animality microbiological contamination culture, therefore pushes the development of Methods of Serum-Free Medium for Animal Cells.Together When, China manages blood product stringent, and human serum AB is not easy to obtain and expensive.Animal blood serum such as fetal calf serum, to people For class, there are foreign protei antigen and carry it is dangerous in the unknown virus of human disease etc., exist to limit umbilical cord MSCs Clinical application.
The microcarrier culture (microcarrier culture) of cell is a kind of for high yield culture attached cell Practical technique, volume of culture is from several milliliters to 6000 liter or more.Using Microcarrier Culture Techniques, suspends and cultivate in ordinary cells In system, every milliliter of culture solution can cultivate millions of cells.
According to the design of system, culture solution usually contains 1-5 grams per liter microcarrier, inoculation 5 × 104- 2 × 105Cell/milli It rises, is stirred by 20-60rpm speed.Perfusion microcarrier culture solution (Perfused microcarrier cultures) can contain height Up to 20g/ liter.When carrying out suspension microcarrier culture for the first time, a kind of method introduced below can refer to.It the volume of culture solution and connects The size of kind amount should do corresponding adjustment according to the difference of culture amount.The culture solution for preparing 100 milliliters, should be by 0.3 gram of micro- load Body is dissolved in 30ml culture medium, is added to rotary container.Inoculate 107Cell mixes gently in the culture solution, and 37 DEG C incubate It educates.Once cell is firmly adhered to the surface of microcarrier, that is, start continuously to stir.Time required for cell adherence mainly takes Certainly in the adhesive efficiency of such cell (attachment efficiency).If the overlong time of cell adherence needs to be interrupted Property (every 30 minutes stir 2 minutes) agitation culture solution to guarantee being uniformly distributed for cell and microcarrier.Then, by the appearance of culture solution Product increases to 50 milliliters.The speed of stirring depends on culture vessel, and wants sufficiently fast to prevent microcarrier from precipitating.1 to 2 day Afterwards, nutrient solution volume increases to 100 milliliters, after 3 to 5 days, and partial medium needs replacing, 7 days or so harvest cells.
In the prior art, there is not yet about human umbilical cord mesenchymal stem cells large-scale culture serum free medium Report.
Summary of the invention
For the above-mentioned prior art, the present invention provides a kind of human umbilical cord mesenchymal stem cells large-scale cultures without blood Clear culture medium is free of animal source component, can fully achieve free serum culture stem cell.Using free serum culture of the invention Base culture human umbilical cord mesenchymal stem cells, the umbilical cord mesenchyma that can obtain high quantity, high-purity and safe and high quality in vitro are dry Cell, while cell survival rate can be improved, maintain the characteristic of stem cell.The present invention can solve the problems, such as that cell quantity is inadequate, and The cell quantity of every batch of, survival rate and immunophenotype are more stable.
The present invention is achieved by the following technical solutions:
A kind of human umbilical cord mesenchymal stem cells large-scale culture serum free medium is to be grouped as by the group of following concentration, respectively Concentration unit is mg/L unless otherwise noted:
L-arginine 100~300；
CuSO4·5H2O 0.0005~0.005；
L- asparagine 25~75；
ASPARTIC ACID 10~30；
Pidolidone 10~30；
Ni(NO3)2·6H2O 0.00002~0.0002；
L-Glutamine 0~500；
ZnSO4·7H2O 0.06~0.6；
Glycine 5~15；
CoCl2·6H2O 0.001~0.008；
L-Histidine 10~30；
NaSiO3·9H2O 0.001~0.01；
L-Isoleucine 25~75；
Na3VO4·12H2O 0.0005~0.005；
L-lysine hydrochloric acid 20~60；
SnCl2·2H2O 0.00001~0.0001；
L-Methionine 10~30；
Na2SeO30.002~0.01；
L-phenylalanine 10~30；
FeSO4·7H2O 0.2~1.6；
L-PROLINE 10~30；
Glucose 1000~4000；
Serine 15~45；
Vitamin C 0.176~0.704；
L-threonine 10~30；
P-hydroxybenzoic acid 0.5~1.5；
L-Trp 5~15；
Sodium Pyruvate 55~550；
Valine 10~30；
Linoleic acid 0.01~0.05；
L-Leu 25~75；
Beta -mercaptoethanol 0.8~4.0；
Two water l-tyrosine disodiums 144~432；
Ethanol amine 1~5；
Two hydrochloric acid 25~75 of l-cysteine；
Dexamethasone 0.001~0.005；
Human transferrin 5~50；
Human serum albumins 1000~5000；
Fibronectin 0.5~5；
Recombinant human fibroblast growth factor 0.1~10；
Recombined human transforming factor β 0.1~10；
Recombinant human epidermal growth factor 0.1~10；
Insulin: 8~20；
Cholesterol: 20~60；
Catalase: 20~50；
Sodium selenite: 17.3~35.0；
1%(mass percent) 3-mercaptoethanol solution: 0.35~1.0ml/L；
Surplus is water.
The recombinant human fibroblast growth factor, recombined human transforming factor, the city recombinant human epidermal growth factor Jun Ke Field is commercially available, and the present invention used is imported product, is purchased from peprotech, product name are as follows: EGF (recombinant human epidermal growth The factor), article number AF-100-15-50；Product name is TGF-b(recombined human transforming factor), article No.: 100-21-100；Quotient The name of an article is (FGF) fibroblast growth factor, commodity article No.: 100-18B-50.
Serum free medium of the invention the preparation method comprises the following steps: take above-mentioned component in addition to water, spy is respectively dissolved according to it Property classification dissolution, then mix, be added water make each component final concentration as described above, adjust pH value to 7.1~7.4 to get work Industry filter element filtering, while nitrogen protection is dispensed and (unstability ingredient is avoided to be oxidized).
Human umbilical cord mesenchymal stem cells large-scale culture serum free medium of the invention is free of any serum component, And definite ingredients, the danger of foreign protei pollution and pathogenic microorganisms is overcome, solves the bio-safety of MSCs clinical application Property problem.
Large-scale culture human umbilical cord mesenchymal stem cells can be used for using serum free medium of the invention, can obtain in vitro The umbilical cord mesenchymal stem cells of high quantity, high-purity and safe and high quality are obtained, while cell survival rate can be improved, maintain stem cell Characteristic.The present invention can solve the problems, such as that cell quantity is inadequate, and the cell quantity of every batch of, survival rate and immunophenotype are more Stablize, meets index of correlation requirement.
Detailed description of the invention
Fig. 1: microcarrier culture apparatus.
Fig. 2: microcarrier culture cell microphoto 100 ×.
Specific embodiment
Below with reference to embodiment, the present invention is further illustrated.
Instrument involved in following embodiments, reagent, material etc. are unless otherwise noted existing in the prior art Conventional instrument, reagent, material etc., can be obtained by regular commercial sources.Experimental method involved in following embodiments, inspection Survey method etc. is unless otherwise noted existing routine experiment method in the prior art, detection method etc..
Embodiment 1 prepares human umbilical cord mesenchymal stem cells large-scale culture serum free medium
The concentration of each raw material is as follows, and each concentration unit is mg/L unless otherwise noted:
L-arginine 200；
CuSO4·5H2O 0.002；
L- asparagine 50；
ASPARTIC ACID 20；
Pidolidone 20；
Ni(NO3)2·6H2O 0.0001；
L-Glutamine 300；
ZnSO4·7H2O 0.3；
Glycine 10；
CoCl2·6H2O 0.005；
L-Histidine 20；
NaSiO3·9H2O 0.005；
L-Isoleucine 50；
Na3VO4·12H2O 0.002；
L-lysine hydrochloric acid 40；
SnCl2·2H2O 0.00005；
L-Methionine 20；
Na2SeO30.005；
L-phenylalanine 20；
FeSO4·7H2O 1.0；
L-PROLINE 20；
Glucose 3000；
Serine 30；
Vitamin C 0.500；
L-threonine 20；
P-hydroxybenzoic acid 1.0；
L-Trp 10；
Sodium Pyruvate 300；
Valine 20；
Linoleic acid 0.03；
L-Leu 50；
Beta -mercaptoethanol 2.0；
Two water l-tyrosine disodiums 300；
Ethanol amine 3；
Two hydrochloric acid 50 of l-cysteine；
Dexamethasone 0.003；
Human transferrin 30；
Human serum albumins 2500；
Fibronectin 3；
Recombinant human fibroblast growth factor 5；
Recombined human transforming factor β 5；
Recombinant human epidermal growth factor 5；
Insulin: 10；
Cholesterol: 40；
Catalase: 30；
Sodium selenite: 25.0；
1% 3-mercaptoethanol solution: 0.8ml/L；
Surplus is water.
The preparation method comprises the following steps: taking above-mentioned component in addition to water, according to its respectively dissolution characteristics classification dissolution, then mixes, add Enter water and make each component final concentration as described above, adjust pH value to 7.1~7.4 to get, industrial filter element filtering, while nitrogen protection It is dispensed and (unstability ingredient is avoided to be oxidized).
Embodiment 2 prepares human umbilical cord mesenchymal stem cells large-scale culture serum free medium
The concentration of each raw material is as follows, and each concentration unit is mg/L unless otherwise noted:
L-arginine 100；
CuSO4·5H2O 0.0005；
L- asparagine 75；
ASPARTIC ACID 30；
Pidolidone 10；
Ni(NO3)2·6H2O 0.00002；
L-Glutamine 500；
ZnSO4·7H2O 0.6；
Glycine 5；
CoCl2·6H2O 0.001；
L-Histidine 30；
NaSiO3·9H2O 0.01；
L-Isoleucine 25；
Na3VO4·12H2O 0.0005；
L-lysine hydrochloric acid 60；
SnCl2·2H2O 0.0001；
L-Methionine 10；
Na2SeO30.002；
L-phenylalanine 30；
FeSO4·7H2O 1.6；
L-PROLINE 10；
Glucose 1000；
Serine 45；
Vitamin C 0.704；
L-threonine 10；
P-hydroxybenzoic acid 0.5；
L-Trp 15；
Sodium Pyruvate 550；
Valine 10；
Linoleic acid 0.01；
L-Leu 75；
Beta -mercaptoethanol 4.0；
Two water l-tyrosine disodiums 144；
Ethanol amine 1；
Two hydrochloric acid 75 of l-cysteine；
Dexamethasone 0.005；
Human transferrin 5；
Human serum albumins 1000；
Fibronectin 5；
Recombinant human fibroblast growth factor 10；
Recombined human transforming factor β 0.1；
Recombinant human epidermal growth factor 0.1；
Insulin: 20；
Cholesterol: 20；
Catalase: 20；
Sodium selenite: 35.0；
1% 3-mercaptoethanol solution: 1.0ml/L；
Surplus is water.
The preparation method comprises the following steps: taking above-mentioned component in addition to water, according to its respectively dissolution characteristics classification dissolution, then mixes, add Enter water and make each component final concentration as described above, adjust pH value to 7.1~7.4 to get, industrial filter element filtering, while nitrogen protection It is dispensed and (unstability ingredient is avoided to be oxidized).
Embodiment 3 prepares human umbilical cord mesenchymal stem cells large-scale culture serum free medium
Each material concentration is as follows, and each concentration unit is mg/L unless otherwise noted:
L-arginine 300；
CuSO4·5H2O 0.005；
L- asparagine 25；
ASPARTIC ACID 10；
Pidolidone 30；
Ni(NO3)2·6H2O 0.0002；
L-Glutamine 0；
ZnSO4·7H2O 0.06；
Glycine 15；
CoCl2·6H2O 0.008；
L-Histidine 10；
NaSiO3·9H2O 0.001；
L-Isoleucine 75；
Na3VO4·12H2O 0.005；
L-lysine hydrochloric acid 20；
SnCl2·2H2O 0.00001；
L-Methionine 30；
Na2SeO30.01；
L-phenylalanine 10；
FeSO4·7H2O 0.2；
L-PROLINE 30；
Glucose 4000；
Serine 15；
Vitamin C 0.176；
L-threonine 30；
P-hydroxybenzoic acid 1.5；
L-Trp 5；
Sodium Pyruvate 55；
Valine 30；
Linoleic acid 0.05；
L-Leu 25；
Beta -mercaptoethanol 0.8；
Two water l-tyrosine disodiums 432；
Ethanol amine 5；
Two hydrochloric acid 25 of l-cysteine；
Dexamethasone 0.001；
Human transferrin 50；
Human serum albumins 5000；
Fibronectin 0.5；
Recombinant human fibroblast growth factor 0.1；
Recombined human transforming factor β 10；
Recombinant human epidermal growth factor 10；
Insulin: 8；
Cholesterol: 60；
Catalase: 50；
Sodium selenite: 17.3；
1% 3-mercaptoethanol solution: 0.35ml/L；
Surplus is water.
The preparation method comprises the following steps: taking above-mentioned component in addition to water, according to its respectively dissolution characteristics classification dissolution, then mixes, add Enter water and make each component final concentration as described above, adjust pH value to 7.1~7.4 to get, industrial filter element filtering, while nitrogen protection It is dispensed and (unstability ingredient is avoided to be oxidized).
Experiment
1, culture medium prepared by embodiment 2 is placed in T25 Tissue Culture Flask, by umbilical cord mesenchymal stem cells with 1.0 × 104cells/cm2Inoculation, cultivates at 37 DEG C of temperature, counts the time that climbs out of of primary cell, the growth time in per generation.
2, culture medium prepared by embodiment 2 is placed in micro-carriers cell culture rolling bottle, by umbilical cord mesenchymal stem cells with 2.0×105Cells/mL inoculation, using the magnetic stirring apparatus of certain brand, is cultivated at 37 DEG C of temperature, the stirring of 45 rpm speed.
Meanwhile umbilical cord mesenchymal stem cells are cultivated for control with lonza serum free medium (market is commercially available).
Conclusion:
Primary cell climbs out of the time: common cell culture medium carries out umbilical cord plant block method and climbs cell, and visible cell is climbed within 8~10 days Out, serum free medium of the invention climbs out of cell stage and foreshortens to 5~7 days, improves cell separative efficiency.
The per generation growth time of cell is monitored by continuous 10 generation, average per generation growth time at 2~3 days, than lonza without Blood serum medium improves one day, has saved incubation time, has improved work efficiency.
Continuous passage number: traditional MSC serum free medium was passaged to after 6 generations, and cell growth is slowed by, the present invention Culture medium can continuous passage to 10 generations, cell still keeps its differentiation potential, and lonza culture medium was passaged to for the 8th generation, and cell is raw Length obviously slows down, and will appear adherent difficulty, and cell is easily fallen off, and causes the later period that can not pass on.
It can carry out the culture of extensive human umbilical cord mesenchymal stem cells, training using bioreactor in vitro using the present invention It supports 7 day time, cell quantity can expand 15-20 times, and the umbilical cord mesenchyma for obtaining high quantity, high-purity and safe and high quality is dry thin Born of the same parents, while improving cell survival rate, maintain the characteristic of stem cell, and the culture of lonza culture medium 7 days, it is only capable of 10 times of amplification, is made It can get more cells with serum free medium same time of the invention.
Above-mentioned, although specific embodiments of the present invention have been described in conjunction with the embodiments, not protects to the present invention The limitation of range, those skilled in the art should understand that, based on the technical solutions of the present invention, those skilled in the art The various modifications or changes that can be made are not needed to make the creative labor still within protection scope of the present invention.

Claims (5)

1. a kind of human umbilical cord mesenchymal stem cells large-scale culture serum free medium, it is characterised in that: be by following concentration What group was grouped as, each concentration unit is mg/L unless otherwise noted:
L-arginine 100~300；
CuSO4·5H2O 0.0005~0.005；
L- asparagine 25~75；
ASPARTIC ACID 10~30；
Pidolidone 10~30；
Ni(NO3)2·6H2O 0.00002~0.0002；
L-Glutamine 0~500；
ZnSO4·7H2O 0.06~0.6；
Glycine 5~15；
CoCl2·6H2O 0.001~0.008；
L-Histidine 10~30；
NaSiO3·9H2O 0.001~0.01；
L-Isoleucine 25~75；
Na3VO4·12H2O 0.0005~0.005；
L-lysine hydrochloric acid 20~60；
SnCl2·2H2O 0.00001~0.0001；
L-Methionine 10~30；
Na2SeO30.002~0.01；
L-phenylalanine 10~30；
FeSO4·7H2O 0.2~1.6；
L-PROLINE 10~30；
Glucose 1000~4000；
Serine 15~45；
Vitamin C 0.176~0.704；
L-threonine 10~30；
P-hydroxybenzoic acid 0.5~1.5；
L-Trp 5~15；
Sodium Pyruvate 55~550；
Valine 10~30；
Linoleic acid 0.01~0.05；
L-Leu 25~75；
Beta -mercaptoethanol 0.8~4.0；
Two water l-tyrosine disodiums 144~432；
Ethanol amine 1~5；
Two hydrochloric acid 25~75 of l-cysteine；
Dexamethasone 0.001~0.005；
Human transferrin 5~50；
Human serum albumins 1000~5000；
Fibronectin 0.5~5；
Recombinant human fibroblast growth factor 0.1~10；
Recombined human transforming factor β 0.1~10；
Recombinant human epidermal growth factor 0.1~10；
Insulin: 8~20；
Cholesterol: 20~60；
Catalase: 20~50；
Sodium selenite: 17.3~35.0；
1% 3-mercaptoethanol solution: 0.35~1.0ml/L；
Surplus is water.
2. human umbilical cord mesenchymal stem cells large-scale culture serum free medium according to claim 1, it is characterised in that: It is to be grouped as by the group of following concentration, each concentration unit is mg/L:
L-arginine 200；
CuSO4·5H2O 0.002；
L- asparagine 50；
ASPARTIC ACID 20；
Pidolidone 20；
Ni(NO3)2·6H2O 0.0001；
L-Glutamine 300；
ZnSO4·7H2O 0.3；
Glycine 10；
CoCl2·6H2O 0.005；
L-Histidine 20；
NaSiO3·9H2O 0.005；
L-Isoleucine 50；
Na3VO4·12H2O 0.002；
L-lysine hydrochloric acid 40；
SnCl2·2H2O 0.00005；
L-Methionine 20；
Na2SeO30.005；
L-phenylalanine 20；
FeSO4·7H2O 1.0；
L-PROLINE 20；
Glucose 3000；
Serine 30；
Vitamin C 0.500；
L-threonine 20；
P-hydroxybenzoic acid 1.0；
L-Trp 10；
Sodium Pyruvate 300；
Valine 20；
Linoleic acid 0.03；
L-Leu 50；
Beta -mercaptoethanol 2.0；
Two water l-tyrosine disodiums 300；
Ethanol amine 3；
Two hydrochloric acid 50 of l-cysteine；
Dexamethasone 0.003；
Human transferrin 30；
Human serum albumins 2500；
Fibronectin 3；
Recombinant human fibroblast growth factor 5；
Recombined human transforming factor β 5；
Recombinant human epidermal growth factor 5；
Insulin: 10；
Cholesterol: 40；
Catalase: 30；
Sodium selenite: 25.0；
1% 3-mercaptoethanol solution: 0.8；
Surplus is water.
3. human umbilical cord mesenchymal stem cells large-scale culture serum free medium according to claim 1, it is characterised in that: It is to be grouped as by the group of following concentration, each concentration unit is mg/L:
L-arginine 100；
CuSO4·5H2O 0.0005；
L- asparagine 75；
ASPARTIC ACID 30；
Pidolidone 10；
Ni(NO3)2·6H2O 0.00002；
L-Glutamine 500；
ZnSO4·7H2O 0.6；
Glycine 5；
CoCl2·6H2O 0.001；
L-Histidine 30；
NaSiO3·9H2O 0.01；
L-Isoleucine 25；
Na3VO4·12H2O 0.0005；
L-lysine hydrochloric acid 60；
SnCl2·2H2O 0.0001；
L-Methionine 10；
Na2SeO30.002；
L-phenylalanine 30；
FeSO4·7H2O 1.6；
L-PROLINE 10；
Glucose 1000；
Serine 45；
Vitamin C 0.704；
L-threonine 10；
P-hydroxybenzoic acid 0.5；
L-Trp 15；
Sodium Pyruvate 550；
Valine 10；
Linoleic acid 0.01；
L-Leu 75；
Beta -mercaptoethanol 4.0；
Two water l-tyrosine disodiums 144；
Ethanol amine 1；
Two hydrochloric acid 75 of l-cysteine；
Dexamethasone 0.005；
Human transferrin 5；
Human serum albumins 1000；
Fibronectin 5；
Recombinant human fibroblast growth factor 10；
Recombined human transforming factor β 0.1；
Recombinant human epidermal growth factor 0.1；
Insulin: 20；
Cholesterol: 20；
Catalase: 20；
Sodium selenite: 35.0；
1% 3-mercaptoethanol solution: 1.0；
Surplus is water.
4. the system of human umbilical cord mesenchymal stem cells large-scale culture serum free medium according to any one of claims 1 to 3 Preparation Method, it is characterised in that: take component in addition to water, according to its respectively dissolution characteristics classification dissolution, then mix, water, which is added, to be made Each component final concentration as described in any one of claims 1 to 3, adjust pH value to 7.1~7.4 to get.
5. human umbilical cord mesenchymal stem cells large-scale culture serum free medium according to any one of claims 1 to 3 is being trained Support the application in human umbilical cord mesenchymal stem cells.
CN201811341164.5A 2018-11-12 2018-11-12 A kind of human umbilical cord mesenchymal stem cells large-scale culture serum free medium Pending CN109370985A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201811341164.5A CN109370985A (en) 2018-11-12 2018-11-12 A kind of human umbilical cord mesenchymal stem cells large-scale culture serum free medium

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201811341164.5A CN109370985A (en) 2018-11-12 2018-11-12 A kind of human umbilical cord mesenchymal stem cells large-scale culture serum free medium

Publications (1)

Publication Number Publication Date
CN109370985A true CN109370985A (en) 2019-02-22

Family

ID=65384389

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201811341164.5A Pending CN109370985A (en) 2018-11-12 2018-11-12 A kind of human umbilical cord mesenchymal stem cells large-scale culture serum free medium

Country Status (1)

Country Link
CN (1) CN109370985A (en)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111440764A (en) * 2020-02-21 2020-07-24 广东国科细胞科技有限公司 Serum-free culture medium of mesenchymal stem cells and clinical-grade large-scale culture method of mesenchymal stem cells
CN112159795A (en) * 2020-10-13 2021-01-01 肖利霞 Passage amplification method for umbilical cord mesenchymal stem cell working cells in public bank
WO2021120613A1 (en) * 2019-12-16 2021-06-24 江苏艾洛特医药研究院有限公司 Serum-free cell culture medium additive, culture medium, and uses
CN114480272A (en) * 2022-02-22 2022-05-13 张国锋 Additive and culture medium for umbilical cord mesenchymal stem cell culture and preparation method of additive and culture medium
WO2023065384A1 (en) * 2021-10-22 2023-04-27 中国医学科学院生物医学工程研究所 Culture medium for human mesenchymal stem cell

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105886464A (en) * 2016-06-07 2016-08-24 广东万海细胞生物科技有限公司 Serum-free culture medium for umbilical cord blood mesenchymal stem cells
CN106635978A (en) * 2016-12-21 2017-05-10 广东科玮生物技术股份有限公司 Serum-free culture medium for umbilical cord mesenchymal stem cells, as well as preparation method and application thereof
CN107043747A (en) * 2017-06-23 2017-08-15 王晓柯 A kind of human umbilical cord mesenchymal stem cells serum free medium
CN108251359A (en) * 2017-12-20 2018-07-06 上海华新生物高技术有限公司 A kind of mesenchymal stem cell serum-free culture medium and cultural method

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105886464A (en) * 2016-06-07 2016-08-24 广东万海细胞生物科技有限公司 Serum-free culture medium for umbilical cord blood mesenchymal stem cells
CN106635978A (en) * 2016-12-21 2017-05-10 广东科玮生物技术股份有限公司 Serum-free culture medium for umbilical cord mesenchymal stem cells, as well as preparation method and application thereof
CN107043747A (en) * 2017-06-23 2017-08-15 王晓柯 A kind of human umbilical cord mesenchymal stem cells serum free medium
CN108251359A (en) * 2017-12-20 2018-07-06 上海华新生物高技术有限公司 A kind of mesenchymal stem cell serum-free culture medium and cultural method

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021120613A1 (en) * 2019-12-16 2021-06-24 江苏艾洛特医药研究院有限公司 Serum-free cell culture medium additive, culture medium, and uses
CN111440764A (en) * 2020-02-21 2020-07-24 广东国科细胞科技有限公司 Serum-free culture medium of mesenchymal stem cells and clinical-grade large-scale culture method of mesenchymal stem cells
CN112159795A (en) * 2020-10-13 2021-01-01 肖利霞 Passage amplification method for umbilical cord mesenchymal stem cell working cells in public bank
WO2023065384A1 (en) * 2021-10-22 2023-04-27 中国医学科学院生物医学工程研究所 Culture medium for human mesenchymal stem cell
CN114480272A (en) * 2022-02-22 2022-05-13 张国锋 Additive and culture medium for umbilical cord mesenchymal stem cell culture and preparation method of additive and culture medium

Similar Documents

Publication Publication Date Title
CN109370985A (en) A kind of human umbilical cord mesenchymal stem cells large-scale culture serum free medium
CN103857789B (en) Prepare mescenchymal stem cell basal medium and utilize the method that mescenchymal stem cell basal medium prepares cell therapy product and the differentiation product obtained by this culture medium
CN107043747A (en) A kind of human umbilical cord mesenchymal stem cells serum free medium
CN101864393B (en) Serum-free culture medium without animal origin components for culturing Vero cell micro-carrier
CN103583511B (en) A kind of mesenchymal stem cell cryopreserving liquid and parenteral solution
CN101760442B (en) Serum-free medium for MDCK cell large-scale adherent culture and single-cell suspension culture
CN102827807B (en) Serum-free culture medium for mesenchymal stem cells
CN105420182A (en) Serum-free medium for umbilical cord mesenchymal stem cells
CN103153318B (en) For the cell culture system of the Bioreactor scaleup of cell
CN106754670B (en) Mesenchymal stem cell serum-free medium and preparation method and application thereof
CN101984048A (en) Culture medium for culturing mesenchymal stem cells
CN104164405A (en) Serum-free culture system for efficiently culturing human umbilical cord mesenchymal stem cells in vitro
CN101418330B (en) Non protein culture medium adapted to large-scale culture of NSO cell and production of antibody
CN105112363B (en) A kind of serum free medium of human adipose mesenchymal stem cells and preparation method thereof
JP6714932B2 (en) Method for producing vertebrate adipose tissue-derived mesenchymal cell line
JP6990659B2 (en) Chemically defined medium for culturing cancer stem cell (CSC) -containing cell populations
CN103881971B (en) Culture medium and culture method for culturing and/or amplifying mesenchymal stem cells
CN109749997A (en) A kind of Limbus corneae stem cell serum-free culture medium and its cultural method
Carmelo et al. Scalable ex vivo expansion of human mesenchymal stem/stromal cells in microcarrier-based stirred culture systems
CN102191215B (en) Human-derived serum-free culture medium and preparation method thereof
CN108359632A (en) Mdck cell system, the method and its application for replicating virus
CN101831403B (en) Method for amplifying mesenchymal stem cells of human umbilical cord and placenta in vitro
CN102653728A (en) Method for gradually culturing animal cells in scale-up manner by using bioreactor and micro-carrier
CN108795841A (en) A kind of ST culture mediums and its application
CN104195102B (en) The method that inducing human embryo stem cell breaks up to neuroderm

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
TA01 Transfer of patent application right

Effective date of registration: 20200904

Address after: 304, 3rd floor, No.7, Fengxian Middle Road, Haidian District, Beijing 100094

Applicant after: YOCON BIOLOGY TECHNOLOGY Co.

Address before: 100094, Beijing, Haidian District Feng Yin Zhong Road, No. 7, block B

Applicant before: Wang Xiaoke

TA01 Transfer of patent application right
RJ01 Rejection of invention patent application after publication

Application publication date: 20190222

RJ01 Rejection of invention patent application after publication