CN108865989A - A kind of culture medium of umbilical cord mesenchymal stem cells - Google Patents

A kind of culture medium of umbilical cord mesenchymal stem cells Download PDF

Info

Publication number
CN108865989A
CN108865989A CN201810813395.5A CN201810813395A CN108865989A CN 108865989 A CN108865989 A CN 108865989A CN 201810813395 A CN201810813395 A CN 201810813395A CN 108865989 A CN108865989 A CN 108865989A
Authority
CN
China
Prior art keywords
culture medium
growth factor
umbilical cord
stem cells
mesenchymal stem
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
CN201810813395.5A
Other languages
Chinese (zh)
Inventor
宫丽
董振磊
张炳强
戚凤春
章宏
金铎
于淼
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.)
QINGDAO RE-STORE BIOTECHNOLOGY Co.,Ltd.
Original Assignee
Jilin Jihui Biotechnology Co Ltd
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 Jilin Jihui Biotechnology Co Ltd filed Critical Jilin Jihui Biotechnology Co Ltd
Priority to CN201810813395.5A priority Critical patent/CN108865989A/en
Publication of CN108865989A publication Critical patent/CN108865989A/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
    • C12N5/0665—Blood-borne mesenchymal stem cells, e.g. from umbilical cord blood
    • 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
    • C12N2500/24—Iron; Fe chelators; Transferrin
    • 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
    • C12N2500/38—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
    • C12N2500/70—Undefined extracts
    • C12N2500/76—Undefined extracts from plants
    • 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
    • C12N2501/125—Stem cell factor [SCF], c-kit ligand [KL]
    • 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
    • C12N2501/145—Thrombopoietin [TPO]
    • 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
    • C12N2501/165—Vascular endothelial growth factor [VEGF]
    • 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
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • 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
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/25—Tumour necrosing factors [TNF]
    • 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
    • C12N2501/33—Insulin
    • 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
    • C12N2501/335—Glucagon; Glucagon-like peptide [GLP]; Exendin
    • 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
    • C12N2501/73—Hydrolases (EC 3.)
    • 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

Abstract

A kind of culture medium of umbilical cord mesenchymal stem cells, including DMEM/F12 culture medium, addition in the adding ingredient and ivy extract of DMEM/F12 culture medium, adding ingredient includes following component：Human serum albumins, transferrins, vascular endothelial growth factor, basic fibroblast growth factor, arginine, LIF ELISA, stem cell factor, β cytokine, parathyroid hormone, platelet derived growth factor, tumor necrosis factor α, interleukins, hematopoietin, thrombopoietin, alpha-D-glucose, vitamin C, resveratrol, NaHCO3, gangliosides；Ivy extract is liquid, is extracted from the leaf of ivy by conventional method, final concentration of 20~50mg/L of addition.The present invention solves problems of the prior art, and cell is made to be able to maintain good state and growth rate.

Description

A kind of culture medium of umbilical cord mesenchymal stem cells
Technical field
The present invention relates to technical field of stem cell culture, more particularly to a kind of culture medium of umbilical cord mesenchymal stem cells.
Background technique
Umbilical cord mesenchymal stem cells, which refer to, is present in one of neonatal umbilical cord tissue versatile stem cell, it can break up At many kinds of histocytes, there is wide potential applicability in clinical practice.It can Successful amplification people using inactivation cord serum cultivating system Umbilical cord mesenchymal stem cells, the cell of culture have the fundamental characteristics of mescenchymal stem cell, for establish mesenchyma stem cell and Clinical application provides theoretical foundation.Mescenchymal stem cell has stronger immunoregulation effect, can promote Radiation in jury function, The histoorgan of damage or lesion can be repaired.Currently, very more mescenchymal stem cell culture base product in the market, disadvantage：1, Using animal-derived sera, while it can also introduce pathogen and heterogenetic antigen.U.S. FDA just considers to be forbidden to use fetal calf serum For cell therapy；2, stem cell is easy differentiation, cannot keep stemness for a long time, and the form of cell is easy to change.
Summary of the invention
The purpose of the present invention is to provide a kind of culture mediums of umbilical cord mesenchymal stem cells, solve and exist in the prior art Animal-derived sera the problem of introducing pathogen and heterogenetic antigen, and cell is made to be able to maintain good state and proliferation Speed, culture medium antioxidant effect are good.
To achieve the above object, the present invention is achieved through the following technical solutions：
A kind of culture medium of umbilical cord mesenchymal stem cells, including DMEM/F12 culture medium, addition are trained in the DMEM/F12 The adding ingredient and ivy extract of base are supported, the adding ingredient includes the ingredient of following final concentration：Human serum albumins 1~ 5g/L, lysozyme 10mg/mL, 5~10mg/L of transferrins, 2~8mg/L of fibronectin, laminin 1~4mg/L, turn 5~10mg/L of ferritin, Fe (NO3)3·9H2O50g/L、FeSO4·7H2O417g/L, 1~3 μ g/L of estradiol, 2~5 μ of testosterone G/L, 1~2 μ g/L of progesterone, 39.25~117.74 μ g/L of dexamethasone, 5~10mg/L of insulin, glucagon-like peptide 12 μ g/L, riboflavin 376.36mg/L, 80.96~242.87mg/L of coacetylase, 4.41~6.17mg/L of butanediamine, taurine 1 ~2mg/L, 0.61~1.85mg/L of ethylaminoethanol, 8.81~26.42mg/L of pyruvic acid, 3.78~7.56 μ g/L of sodium selenate, blood Endothelial tube growth factor-2~8 μ g/L, 4~10 μ g/L of basic fibroblast growth factor, 10~20mg/L of arginine, white blood Sick 1~5 μ g/L of inhibiting factor, 2~8 μ g/L of stem cell factor, 5~10 μ g/mL of β cytokine, parathyroid hormone 35.74~ 71.48ng/L, μ g/L of platelet derived growth factor AB0.5~2.5,0.5~2.5 μ g/L of tumor necrosis factor α, leucocyte 20.5~2.5 μ g/L of interleukin, 0.36~1.09mg/L of hydrocortisone, 1~2mg/L of soybean pancreatin inhibitor, promoting erythrocyte are raw At 1 μ g/L of element, 1 μ g/L of thrombopoietin, alpha-D-glucose 3135mg/L, vitamin C 176.13mg/L, resveratrol 4 μg/L、NaHCO32.2g/L, 5 μ g/L of gangliosides；The ivy extract is liquid, by conventional method from ivy Leaf in extract, final concentration of 20~50mg/L of addition.The blade of ivy is first crushed, then is fermented, pre-swollen is simultaneously It is extracted in 30% ethanol solution, obtains the ivy extract.
Preferably, the adding ingredient further includes the transforming growth factor α and/or 1~4 μ g/ of final concentration of 1~4 μ g/L L transforming growth factor β.
Preferably, the adding ingredient further includes the anthocyanin of final concentration of 1~2 μ g/L.
Beneficial effects of the present invention, first is that the present invention is free of animal blood serum, it is (such as albumin, thin that all the components are all from people Intracellular cytokine etc.), potential animal derived endotoxin or virus in animal blood serum are eliminated, and joined lysozyme in culture medium, It is highly-safe, it is convenient to be applied to clinic；Second is that oxidation resistance is improved present invention adds ivy extract, anthocyanin, Promote cell growth, there is certain repairing effect.
Detailed description of the invention
Fig. 1 is the umbilical cord mesenchymal stem cells form photo that the embodiment of the present invention one is turned out.
Fig. 2 is another photo of umbilical cord mesenchymal stem cells form that the embodiment of the present invention one is turned out.
Specific embodiment
It in order to make the object, technical scheme and advantages of the embodiment of the invention clearer, below will be in the embodiment of the present invention Technical solution be clearly and completely described.The person that is not specified actual conditions in embodiment, according to normal conditions or manufacturer builds The condition of view carries out.Production firm person is not specified in component, reagent or instrument used, and being can be by the normal of commercially available purchase acquisition Advise product.
Component, reagent used in experiment and instrument are as follows below：
It is as follows that the invention will be further described below：
Preparing for umbilical cord mesenchymal stem cells is as follows：
(1) umbilical cord is washed：Sterile pincers transfer umbilical cord is added sample cleaning solution and rinses into 10cm sterile petri dish, Repeat 2~3 times, as far as possible removal bloodstain.
(2) sterile tissue, which is cut, is cut into about 2~3cm number section for umbilical cord, and 10~20ml sample cleaning solution is added and washs blood clot, Repeated washing is until basic removal bloodstain, cleaning solution are limpider.Reject blood vessel：Two for pressing blood vessel spiral tendency rejecting umbilical cord are dynamic Arteries and veins and a vein.
(3) umbilical cord tissue for rejecting blood vessel is washed into bloodstain with appropriate sample cleaning solution, repeated washing is until cleaning solution is clear It is clear.
(4) umbilical cord after washing is put into 10cm sterile petri dish, with sterile three tissue shears by umbilical cord scissors at tissue Equal lumps.
(5) homogenate block is averagely seeded in 15cm culture dish, tissue block is uniformly distributed, and appropriate culture completely is added Base.
(6) just setting culture dish is evenly distributed as much as possible tissue block in entire bottom surface, and it is permanent that culture dish is placed in CO2 constant temperature It is cultivated in wet incubator.Condition of culture：37 ± 0.5 DEG C, CO2Volume fraction is：5.0 ± 0.2%.
Embodiment one
A kind of culture medium of umbilical cord mesenchymal stem cells, including DMEM/F12 culture medium, addition are trained in the DMEM/F12 The adding ingredient and ivy extract of base are supported, the adding ingredient includes the ingredient of following final concentration：Human serum albumins 1.25g/L, lysozyme 10mg/mL, transferrins 7.5mg/L, fibronectin 8mg/L, laminin 4mg/L, turn iron egg White 10mg/L, Fe (NO3)3·9H2O 50g/L、FeSO4·7H2O 417g/L, 1.3 μ g/L of estradiol, 2 μ g/L of testosterone, progesterone 1.3 μ g/L, 78.49 μ g/L of dexamethasone, insulin 7mg/L, 2 μ g/L of glucagon-like peptide 1, riboflavin 376.36mg/L, Coacetylase 161.91mg/L, butanediamine 5.30mg/L, taurine 1.25mg/L, ethylaminoethanol 1.22mg/L, pyruvic acid 22.42mg/ L, 5.67 μ g/L of sodium selenate, 6 μ g/L of vascular endothelial growth factor, 10 μ g/L of basic fibroblast growth factor, arginine 20mg/L, 1 μ g/L of LIF ELISA, 8 μ g/L of stem cell factor, 10 μ g/mL of β cytokine, parathyroid hormone 35.74ng/L, platelet derived growth factor AB2.2 μ g/L, 2.5 μ g/L of tumor necrosis factor α, 2.5 μ g/ of interleukin 2 L, hydrocortisone 0.36mg/L, soybean pancreatin inhibitor 1mg/L, 1 μ g/L of hematopoietin, thrombopoietin 1 μ g/L, alpha-D-glucose 3135mg/L, vitamin C 176.13mg/L, 4 μ g/L of resveratrol, NaHCO32.2g/L, neuromere 5 μ g/L of glycosides rouge, 2 μ g/L of transforming growth factor α, 2 μ g/L of anthocyanin；The ivy extract is liquid, by conventional method It is extracted from the leaf of ivy, the final concentration of 50mg/L of addition.
By each ingredient, by its, respectively characteristic dissolves, and mixing, HEPES constant volume, 0.22 μm of filter membrane filtration sterilization obtains Culture medium I, 4 DEG C of preservations.
Embodiment two
A kind of culture medium of umbilical cord mesenchymal stem cells, including DMEM/F12 culture medium, addition are trained in the DMEM/F12 The adding ingredient and ivy extract of base are supported, the adding ingredient includes the ingredient of following final concentration：Human serum albumins 5g/ L, lysozyme 10mg/mL, transferrins 5mg/L, fibronectin 2mg/L, laminin 4mg/L, transferrins 5mg/L, Fe(NO3)3·9H2O 50g/L、FeSO4·7H2O 417g/L, 3 μ g/L of estradiol, 5 μ g/L of testosterone, 1 μ g/L of progesterone, fill in 39.25 μ g/L of rice pine, insulin 10mg/L, 2 μ g/L of glucagon-like peptide 1, riboflavin 376.36mg/L, coenzyme A161.91mg/L, butanediamine 5.30mg/L, taurine 1.25mg/L, ethylaminoethanol 0.61mg/L, pyruvic acid 22.42mg/L, selenium Sour 3.78 μ g/L of sodium, 2 μ g/L of vascular endothelial growth factor, 4 μ g/L of basic fibroblast growth factor, arginine 10mg/L, 1 μ g/L of LIF ELISA, 8 μ g/L of stem cell factor, 5 μ g/mL of β cytokine, parathyroid hormone 71.48ng/L, blood are small Plate source property growth factor AB0.5 μ g/L, 0.5 μ g/L of tumor necrosis factor α, 0.5 μ g/L of interleukin 2, hydrocortisone 1.09mg/L, soybean pancreatin inhibitor 2mg/L, 1 μ g/L of hematopoietin, 1 μ g/L of thrombopoietin, the Portugal α-D- Grape sugar 3135mg/L, vitamin C 176.13mg/L, 4 μ g/L of resveratrol, NaHCO32.2g/L, 5 μ g/L of gangliosides, turn Change grouth factor beta 2 μ g/L, 2 μ g/L of anthocyanin；The ivy extract is liquid, by conventional method from the leaf of ivy Middle extraction, the final concentration of 20mg/L of addition.
By each ingredient, by its, respectively characteristic dissolves, and mixing, HEPES constant volume, 0.22 μm of filter membrane filtration sterilization obtains Culture medium II, 4 DEG C of preservations.
Comparative example one
Growth of mesenchymal stem cells serum free medium, source LONZA, article No. 00190632, be labeled as culture medium III, 4 DEG C save.
Cell proliferation velocity contrast experiment
The good umbilical cord mesenchymal stem cells of growth conditions are taken, with 0.05% pancreas enzyme -EDTA vitellophag, digestion is to It sets microscopically observation to be become round and fallen off by shuttle shape to cell is most of, culture medium I, culture medium II and culture medium is added III, by 5 × 104/cm2It is inoculated in 96 well culture plates, every hole adds respectively states two culture mediums, 100 μ l.Culture plate one is taken simultaneously Column are added growth medium but cell are not added, and every hole culture medium is 100 μ l, as blank control group.Every 12h takes 4 holes, MTT ratio Color method surveys 570nm wavelength absorbance value (D570 value), and continuous to survey 3 days, the result tested is as shown in table 1, table 2, table 3.
1 umbilical cord mesenchymal stem cells of table cultivate value-added speed table in culture medium I
Time 12h 24h 36h 48h 60h 72h 84h
D570 value 0.12 0.25 0.39 0.4 0.4 0.4 0.4
2 umbilical cord mesenchymal stem cells of table cultivate value-added speed table in culture medium II
Time 12h 24h 36h 48h 60h 72h 84h
D570 value 0.11 0.22 0.35 0.4 0.4 0.4 0.4
3 umbilical cord mesenchymal stem cells of table cultivate value-added speed table in culture medium III
Time 12h 24h 36h 48h 60h 72h 84h
D570 value 0.08 0.12 0.22 0.32 0.4 0.4 0.4
Culture medium I and culture medium II are all made of component and method of the invention and prepare, and difference is that the end of adding ingredient is dense Degree is different.Culture medium III is to use more growth of mesenchymal stem cells serum free medium on the market.It can from table 1, table 2, table 3 To obtain, using the umbilical cord mesenchymal stem cells of culture medium I of the invention and the culture of culture medium II, cell proliferation speed is obvious The umbilical cord mesenchymal stem cells cultivated better than culture medium III.
The stem cell that the culture medium I of embodiment one is cultivated is in kilter, as depicted in figs. 1 and 2.
The present invention is free of animal blood serum, and all the components are all from people (such as albumin, cell factor), eliminate animal blood Potential animal derived endotoxin or virus in clear, and joined lysozyme in culture medium, it is highly-safe, it is convenient to be applied to face Bed.Present invention adds ivy extract, resveratrol, anthocyanin, improve oxidation resistance, promote cell growth, have Certain repairing effect, the cellular morphology turned out are good.
Although the embodiments of the present invention have been disclosed as above, but its is not only in the description and the implementation listed With.It can be applied to various suitable the field of the invention completely.It for those skilled in the art, can be easily Realize other modification.Therefore without departing from the general concept defined in the claims and the equivalent scope, the present invention is simultaneously unlimited In specific details and figure shown and described herein.

Claims (3)

1. a kind of culture medium of umbilical cord mesenchymal stem cells, which is characterized in that including DMEM/F12 culture medium, addition described The adding ingredient and ivy extract of DMEM/F12 culture medium, the adding ingredient include the ingredient of following final concentration：People's blood Pure 1~5g/L of albumen, lysozyme 10mg/mL, 5~10mg/L of transferrins, 2~8mg/L of fibronectin, laminin 1~4mg/L, 5~10mg/L of transferrins, Fe (NO3)3·9H2O50g/L、FeSO4·7H2O417g/L, 1~3 μ g/ of estradiol L, 2~5 μ g/L of testosterone, 1~2 μ g/L of progesterone, 39.25~117.74 μ g/L of dexamethasone, 5~10mg/L of insulin, pancreas are high 12 μ g/L of blood glucose element sample peptide, riboflavin 376.36mg/L, 80.96~242.87mg/L of coacetylase, 4.41~6.17mg/ of butanediamine L, 1~2mg/L of taurine, 0.61~1.85mg/L of ethylaminoethanol, 8.81~26.42mg/L of pyruvic acid, sodium selenate 3.78~ 7.56 μ g/L, 2~8 μ g/L of vascular endothelial growth factor, 4~10 μ g/L of basic fibroblast growth factor, arginine 10~ 20mg/L, 1~5 μ g/L of LIF ELISA, 2~8 μ g/L of stem cell factor, 5~10 μ g/mL of β cytokine, parathyroid hormone 35.74~71.48ng/L of element, μ g/L of platelet derived growth factor AB0.5~2.5,0.5~2.5 μ g/ of tumor necrosis factor α L, 0.5~2.5 μ g/L of interleukin 2,0.36~1.09mg/L of hydrocortisone, 1~2mg/L of soybean pancreatin inhibitor, rush 1 μ g/L of erythropoietin(EPO), 1 μ g/L of thrombopoietin, alpha-D-glucose 3135mg/L, vitamin C 176.13mg/L, 4 μ g/L of resveratrol, NaHCO32.2g/L, 5 μ g/L of gangliosides；The ivy extract is liquid, passes through conventional side Method is extracted from the leaf of ivy, final concentration of 20~50mg/L of addition.
2. a kind of culture medium of umbilical cord mesenchymal stem cells according to claim 1, which is characterized in that the adding ingredient It further include the transforming growth factor α and/or 1~4 μ g/L transforming growth factor β of final concentration of 1~4 μ g/L.
3. a kind of culture medium of umbilical cord mesenchymal stem cells according to claim 1, which is characterized in that the adding ingredient It further include the anthocyanin of final concentration of 1~2 μ g/L.
CN201810813395.5A 2018-07-23 2018-07-23 A kind of culture medium of umbilical cord mesenchymal stem cells Pending CN108865989A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201810813395.5A CN108865989A (en) 2018-07-23 2018-07-23 A kind of culture medium of umbilical cord mesenchymal stem cells

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201810813395.5A CN108865989A (en) 2018-07-23 2018-07-23 A kind of culture medium of umbilical cord mesenchymal stem cells

Publications (1)

Publication Number Publication Date
CN108865989A true CN108865989A (en) 2018-11-23

Family

ID=64304249

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201810813395.5A Pending CN108865989A (en) 2018-07-23 2018-07-23 A kind of culture medium of umbilical cord mesenchymal stem cells

Country Status (1)

Country Link
CN (1) CN108865989A (en)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110317779A (en) * 2019-06-06 2019-10-11 深圳市艾一生命科技有限公司 The umbilical cord mesenchymal stem cells culture medium of serum-free
CN110331130A (en) * 2019-07-03 2019-10-15 依科赛生物科技（太仓）有限公司 Mesenchymal stem cell serum-free medium and application thereof
CN111518761A (en) * 2020-05-25 2020-08-11 青岛瑞思德生物科技有限公司 Method for in vitro screening, activating, amplifying and cryopreserving of mesenchymal stem cells and establishing cell bank of mesenchymal stem cells
CN114591901A (en) * 2022-03-24 2022-06-07 和携科技有限公司 Method for extracting exosomes of animal umbilical cord mesenchymal stem cells
CN116410921A (en) * 2023-02-09 2023-07-11 北京益华生物科技有限公司 Human umbilical cord mesenchymal stem cell induction culture medium, induction method and application
EP4092106A4 (en) * 2020-01-13 2024-03-06 Qingdao Restore Biotechnology Co Ltd Inducer for inducing mesenchymal stem cells to differentiate into islet cells

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101363012A (en) * 2008-09-27 2009-02-11 上海交通大学 Method for amplifying hematopoietic stem cell and progenitor cell using rhDSL recombinant protein
CN103243071A (en) * 2013-05-09 2013-08-14 陈云燕 Clinical-grade human mesenchymal stem cell serum-free complete medium
CN104164405A (en) * 2014-08-12 2014-11-26 赛业（苏州）生物科技有限公司 Serum-free culture system for efficiently culturing human umbilical cord mesenchymal stem cells in vitro
CN105132369A (en) * 2015-08-25 2015-12-09 北京瑞思德生物医学研究院 Induction medium and inducer for transformation of mesenchymal stem cells into testosterone secretory 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

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101363012A (en) * 2008-09-27 2009-02-11 上海交通大学 Method for amplifying hematopoietic stem cell and progenitor cell using rhDSL recombinant protein
CN103243071A (en) * 2013-05-09 2013-08-14 陈云燕 Clinical-grade human mesenchymal stem cell serum-free complete medium
CN104164405A (en) * 2014-08-12 2014-11-26 赛业（苏州）生物科技有限公司 Serum-free culture system for efficiently culturing human umbilical cord mesenchymal stem cells in vitro
CN105132369A (en) * 2015-08-25 2015-12-09 北京瑞思德生物医学研究院 Induction medium and inducer for transformation of mesenchymal stem cells into testosterone secretory 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

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
周婷婷等: "无血清培养体系原代培养脐带间充质干细胞", 《中国组织工程研究》 *
孙亚如等: "培养体系对维持人脐带间充质干细胞特性及其体外扩增效率的影响", 《中国组织工程研究》 *
王振宇等: "《天然产物分离技术》", 31 January 2012, 北京：中国轻工业出版社 *
赵中振等: "《当代药用植物典 第3册》", 29 February 2008, 世界图书出版公司 *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110317779A (en) * 2019-06-06 2019-10-11 深圳市艾一生命科技有限公司 The umbilical cord mesenchymal stem cells culture medium of serum-free
CN110331130A (en) * 2019-07-03 2019-10-15 依科赛生物科技（太仓）有限公司 Mesenchymal stem cell serum-free medium and application thereof
EP4092106A4 (en) * 2020-01-13 2024-03-06 Qingdao Restore Biotechnology Co Ltd Inducer for inducing mesenchymal stem cells to differentiate into islet cells
CN111518761A (en) * 2020-05-25 2020-08-11 青岛瑞思德生物科技有限公司 Method for in vitro screening, activating, amplifying and cryopreserving of mesenchymal stem cells and establishing cell bank of mesenchymal stem cells
CN111518761B (en) * 2020-05-25 2022-04-29 青岛瑞思德生物科技有限公司 Method for in vitro screening, activating, amplifying and cryopreserving of mesenchymal stem cells and establishing cell bank of mesenchymal stem cells
CN114591901A (en) * 2022-03-24 2022-06-07 和携科技有限公司 Method for extracting exosomes of animal umbilical cord mesenchymal stem cells
CN114591901B (en) * 2022-03-24 2023-11-24 和携科技有限公司 Extraction method of animal umbilical cord mesenchymal stem cell exosome
CN116410921A (en) * 2023-02-09 2023-07-11 北京益华生物科技有限公司 Human umbilical cord mesenchymal stem cell induction culture medium, induction method and application
CN116410921B (en) * 2023-02-09 2024-01-23 北京益华生物科技有限公司 Human umbilical cord mesenchymal stem cell induction culture medium, induction method and application

Similar Documents

Publication Publication Date Title
CN108865989A (en) A kind of culture medium of umbilical cord mesenchymal stem cells
CN101412985B (en) Serum-free medium for in vitro cultivation and amplification of mesenchymal stem cells
CN103404509B (en) Cell preserving fluid as well as preparation method and application of cell preserving fluid
CN104805054B (en) Stem cell serum-free culture medium
CN108309822A (en) A kind of preparation method of human umbilical cord mesenchymal stem cells paracrine factor freeze-dried powder
CN110564682B (en) Method for large-scale production of human adipose-derived mesenchymal stem cell exosomes
CN108823156A (en) For the clinical grade human umbilical cord mesenchymal stem cells composite factor of reparation and the preparation method of freeze-dried powder
CN102807965B (en) Method for preparing tissue engineered cornea and device of method
CN103893825B (en) Method for preparing bacterial cellulose compounded amnion extracellular matrix material containing collagen
CN106754639A (en) A kind of mescenchymal stem cell factor large-scale producing method
CN101757691A (en) Preparation method of tissue engineering cornea
CN109022343A (en) A kind of preparation method of ginseng stem cell
CN106038598A (en) Method for preparing human-derived stem cell secretion bioactive factor and lysate
CN107129967A (en) A kind of serum free medium system for primary human tonsillar cell
CN110305839A (en) Mesenchymal stem cell serum-free culture medium
CN107418928A (en) A kind of human mesenchymal stem cell Osteoinductive differentiation culture medium and preparation method
CN107362391A (en) A kind of preparation method of autologous three skin fibroblasts preparation
CN111557893A (en) Preparation method of mask containing stem cell exosome components
WO2021098025A1 (en) Method for in-vitro activation of adipose stem cells to transform into proto-chondrocytes
CA2373391A1 (en) Spheroid preparation
CN112655554B (en) Method for accelerating induction of apple callus and growth based on LED light quality
CN109385397A (en) A kind of serum free medium and preparation method thereof for cultivating mescenchymal stem cell
CN102925407B (en) In-vitro culture method of triploid loach fin cells
CN107312744A (en) A kind of Buccal mucosa cell nutrient solution containing serum
CN107129966A (en) A kind of corneal epithelial cell nutrient solution containing serum

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
TA01 Transfer of patent application right

Effective date of registration: 20200515

Address after: 266000, No. 1, 2, building 7, Qingdao blue bio Pharmaceutical Industrial Park, 368 Hedong Road, Hedong District, Qingdao, Shandong

Applicant after: QINGDAO RE-STORE BIOTECHNOLOGY Co.,Ltd.

Address before: 130000 North Lake Science and Technology Development Zone, Changchun, Jilin, China, 501 A11, North Lake Science and Technology Park, 3333 Shengbei street.

Applicant before: JILIN JIHUI BIOTECHNOLOGY Co.,Ltd.

WD01 Invention patent application deemed withdrawn after publication
WD01 Invention patent application deemed withdrawn after publication

Application publication date: 20181123