CN107267457A - NSC adhere-wall culture method - Google Patents
NSC adhere-wall culture method Download PDFInfo
- Publication number
- CN107267457A CN107267457A CN201710505536.2A CN201710505536A CN107267457A CN 107267457 A CN107267457 A CN 107267457A CN 201710505536 A CN201710505536 A CN 201710505536A CN 107267457 A CN107267457 A CN 107267457A
- Authority
- CN
- China
- Prior art keywords
- nsc
- culture
- cell
- adhere
- 60nmol
- 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
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
- C12N5/0618—Cells of the nervous system
- C12N5/0623—Stem 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
- C12N2500/30—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
- 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
- C12N2501/115—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
- C12N2501/30—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
- C12N2501/38—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
- C12N2501/40—Regulators of development
- C12N2501/415—Wnt; Frizzeled
-
- 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
- C12N2501/40—Regulators of development
- C12N2501/42—Notch; Delta; Jagged; Serrate
-
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Wood Science & Technology (AREA)
- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Neurology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Neurosurgery (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Developmental Biology & Embryology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
A kind of NSC adhere-wall culture method, using containing 10~20 u mol/mL nonessential amino acid, 10~20ng/mL EGFs, 5~15ng/mL basic fibroblast growth factors, 10~20 u g/mL insulin, 40~60nmol/L hydrocortisones, 10~20nmol/LWNT3a, 40~60nmol/L Notchl, 40~60 u g/mL bovine serum albumin(BSA)s, 15~35ng/mL fibronectins, the collagen types of 50 1 70ng/mL tetra- nerve stem cell culture medium culture of neural stem cells neural.Cultural method of the present invention enables to NSC adherent growth in the case of without the pre- bed board of gelatin, eliminates cumbersome operating procedure and simplifies production procedure.
Description
Technical field
The present invention relates to technical field of cell culture, more particularly to a kind of Culture of neural stem cells method.
Background technology
Nervous system injury disease includes ischemic cerebral disease, hemorrhagic encephalopathic, motor neuron disease etc., is a kind of tight
The disease of human health is threatened again, the survival rate of this kind of disease is improved, disability rate is reduced, and the life of patient is improved to greatest extent
Quality, is the top priority for preventing and treating such disease.Because the discovery of NSC causes controlling for nervous system injury disease
Treatment has new research direction, and NSC also becomes and planted necessary in biomedical engineering and external model research
Daughter cell.
NSC (neural stem cells, NSCs) is that have to be divided into neural neuron, astroglia
With the ability of oligodendroglia, can self-renewing, and be enough to provide the cell mass of a large amount of brain tissue cells, be that a class has point
The mother cell of potential and self refresh ability is split, it can produce the various types of cells of nerve fiber by not reciprocity divisional mode,
Including neuron, oligodendroglia and astroglia.NSC in 1992 is from adults brain striatum
Once it is found, the unrenewable traditional theory of nerve cell is thought so as to break.According to differentiation potential and generation daughter cell
Species NSC can be divided into following several classes：
(1) medullary epithelium cell：Splitting ability is most strong, only deposits Chi embryonic stage, can produce radial neuroglia neuron and
Neuroblast.
(2) radial neuroglia neuron：Generation can be divided in itself and while neuronal precursor or colloid is produced
Cell, main function is to produce projection neuron during infancy neurodevelopment to complete cortex and nerve nucleus etc. in brain
Basic neural tissue cell.
(3) neuroblast：The NSC being primarily present in adult human body, splitting ability can produce neural precursor
Cell and neuron and all kinds of Deiter's cells.
(4) neural precursor：The precursor of all kinds of nerve cells, such as microglia are by Deiter's cells
What precursor was produced.
NSC is as the seed cell in biomedical engineering and external model research, it is necessary to by it in vitro
Culture is enlarged, and how in vitro large-scale culture NSC becomes the emphasis for medical research.Existing training
The Neurobasal culture medium that contains 1 ~ 6 827 of the conventional culture medium of NSC for GIBCO is supported, it is pretreated using gelatin
Blake bottle carry out adhere-wall culture.But the nerve stem cell proliferation ability Jing Guo the medium culture is weaker.Therefore it is badly in need of providing
It is a kind of to strengthen the cultural method of nerve stem cell proliferation ability.
The content of the invention
In view of this, the invention provides a kind of NSC adhere-wall culture method.The Culture of neural stem cells method
The multiplication capacity of NSC can be greatly enhanced, while enabling to NSC in the lazy condition without the pre- bed board of gelatin
Lower adherent growth, eliminates cumbersome operating procedure and simplifies production procedure.
In order to realize foregoing invention purpose, the present invention provides following technical scheme：
NSC adhere-wall culture method, it is characterised in that use and contain 10~20 u mol/mL nonessential amino acid, 10
~20ng/mL EGFs, 5~15ng/mL basic fibroblast growth factors, 10~20 u g/mL insulin, 40
~60nmol/L hydrocortisones, 10~20nmol/LWNT3a, 40~60nmol/L Notchl, 40~60 u g/mL ox bloods
Pure albumen, 15~35ng/mL fibronectins, the nerve stem cell culture medium culture god of the collagen types of 50 1 70ng/mL tetra-
Through stem cell.
Preferably, the condition of the culture is 5 ~ 6 CO：、37.C.Further it is preferably：The culture it is thin
Born of the same parents' density is the 5/mL of 5 × l04~1 × 10.
Wherein nonessential amino acid is glutamic acid, alanine, glycine, asparatate, cystine, proline, large bamboo hat with a conical crown and broad brim ammonia
Mixture more than one or both of acid or tyrosine.
The present invention at least has the following advantages that：
The Culture of neural stem cells method that the present invention is provided can greatly enhance the multiplication capacity of NSC.Flow cytometer detection knot
Fruit shows that culture medium of the present invention can enable the surface marker Nestin+CD133+ of the NSC in P6 generations ratio
33.3% is reached, and conventional medium is only 9.8% in the Nestin+CD133+ in P6 generations ratio；The nerve cord that the present invention is cultivated
Cell had obvious exponential phase at 3~6 days, and cell propagation is very fast, and uses the nerve of conventional medium culture method
The growth curve of stem cell shows that cell growth is slow, without obvious logarithmic proliferation.Culture medium of the present invention adds fibronectin and four
Collagen type, enables to NSC adherent growth in the case of without the pre- bed board of gelatin, eliminates cumbersome behaviour
Production procedure is simplified as step.
Embodiment
Agents useful for same in nerve stem cell culture medium and NSC adhere-wall culture method that the present invention is provided, instrument,
Experimental animal etc. can be bought by market.Such as WNT3a therein thinks wound purchased from the magnificent piebald horse in Shanghai, and Notchl is purchased from Wuhan Halothane
Thing；Nonessential amino acid is purchased from Gibco, and article No. is 11140, MEM Non-Essentia Amino Acids
SolutionlOmM (100X).With reference to embodiment, the present invention is expanded on further.
Embodiment
The preparation of culture medium
Nutrient solution formula components are：DMEM/F12,10 u mol/mL nonessential amino acid, 15ng/mL EGFs,
15ng/mL basic fibroblast growth factors, 10 u g/mL insulin, 50nmol/L hydrocortisones, 20nmol/
LWNT3a, 40nmol/L Notchl, 50 u g/mL bovine serum albumin(BSA)s, 35ng/mL fibronectins, the Collagen Type VIs of 50ng/mL tetra-
Albumen.Culture medium preparation method is：Measured according to the rules when using and be well mixed all the components.
Medium culture nerve cell example
The newborn mice of birth 1~4 day, de- neck takes newborn mice brain after putting to death, and peels off after meninx with 4.C PBSs 2
It is secondary, add 2mL Culture of neural stem cells liquid（Test group 1 uses the culture medium of embodiment 1, and test group 2 uses the culture medium of embodiment 2,
Test group 3 uses the culture medium of embodiment 3）, and shredded brain with operating scissors, then use ImL Manual liquid transfering devices
Fragment is dispelled into suspension, cell density is then adjusted to 5 × l04/mL.Suspension is added into 6 orifice plates, per hole 2mL
In 5% CO, 37.The supernatant in 6 orifice plates is drawn after being placed 2 hours, 2 hours in C incubators, another 6 orifice plate relaying is added
Continuous culture, former 6 orifice plate is discarded should not.Culture discards the supernatant in 6 orifice plates after 24 hours, 2mL NSCs are added per hole
Nutrient solution, changes liquid once in every 2~3 days afterwards.Fall culture medium and with containing 0. 04v/v% when cell fusion degree reaches 80%
EDTA concentration is 0.25v/v% trypsin digestion and cell 1 minute, it is come off, then with each group used medium according to
Add pancreatin volume 5 times terminate digestion；Supernatant is removed after centrifugation, sedimentation cell is resuspended with each group used medium, pressed
Added according to 5 × l04/mL sweet degree in blake bottle, labeled as Pl for cell.Repetition is passaged to P6 generations.By above-mentioned each group culture
NSC carries out flow cytometer detection, and surface marker Nestin+, CD133+ of NSC ratio can reach
33.3%th, 29.5%, 24.3%, and conventional medium is only 9. 8% in Nestin+, the CD133+ in P6 generations ratio.
The strong cell of general multiplication capacity, growth curve can be in " S " type, can be divided into 3 growth periods：First stage is suitable
Ying Qi, cell propagation is slower：Second stage is exponential phase, and cell proliferation rate accelerates, in logarithmic growth；Phase III is
Plateau, cells proliferation slowed down.According to the cell growth curve figure in seven days generations of NSC P6, it can be seen that 0~2 day is suitable
Ying Qi, 3~6 days is exponential phase, and cell propagation is very fast, and cell propagation is slack-off within the 7th day, enters plateau.Cell growth
Curve map understands that the multiplication capacity of the cell is stronger.And use the growth curve of the NSC of conventional medium culture to show
Show that cell growth is slow, without obvious logarithmic proliferation.It can be seen that, the nerve stem cell culture medium that the present invention is provided can greatly enhance god
Multiplication capacity through stem cell.The culture medium of the present invention enables to NSC in the situation without the pre- bed board of gelatin simultaneously
Lower adherent growth, eliminates cumbersome operating procedure and simplifies production procedure.
Claims (3)
1. a kind of NSC adhere-wall culture method, it is characterised in that use and contain 10~20 u mol/mL non-essential aminos
Acid, 10~20ng/mL EGFs, 5~15ng/mL basic fibroblast growth factors, 10~20 u g/mL pancreas islet
Element, 40~60nmol/L hydrocortisones, 10~20nmol/LWNT3a, 40~60nmol/L Notchl, 40~60 u g/mL
Bovine serum albumin(BSA), 15~35ng/mL fibronectins, the nerve stem cell culture medium training of the collagen types of 50 1 70ng/mL tetra-
Support NSC.
2. NSC adhere-wall culture method according to claim 1, it is characterised in that the condition of the culture is 5 ~
6 CO：、37C.
3. NSC adhere-wall culture method according to claim 1 or 2, it is characterised in that the cell of the culture
Density is the 5/mL of 5 × l04~1 × 10.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710505536.2A CN107267457A (en) | 2017-06-28 | 2017-06-28 | NSC adhere-wall culture method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710505536.2A CN107267457A (en) | 2017-06-28 | 2017-06-28 | NSC adhere-wall culture method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN107267457A true CN107267457A (en) | 2017-10-20 |
Family
ID=60070964
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710505536.2A Pending CN107267457A (en) | 2017-06-28 | 2017-06-28 | NSC adhere-wall culture method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107267457A (en) |
-
2017
- 2017-06-28 CN CN201710505536.2A patent/CN107267457A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104928247B (en) | A kind of nerve stem cell culture medium and NSC adhere-wall culture method | |
| CN106479971A (en) | A kind of serum-free medium for cultivating mescenchymal stem cell and method | |
| CN107201339A (en) | Nerve stem cell culture medium | |
| CN101748096A (en) | Sub totipotential stem cell and preparation method and application thereof | |
| CN101203601A (en) | Transplantation of differentiated immature adipocytes and biodegradable scaffold for tissue augmentation | |
| CN106399230A (en) | Method for culturing skin-origin fibroblasts | |
| Deng et al. | Stage-specific differentiation of iPSCs toward retinal ganglion cell lineage | |
| CN106916787A (en) | A kind of limbal stem cell culture medium and its cultural method | |
| CN109749997A (en) | A kind of Limbus corneae stem cell serum-free culture medium and its cultural method | |
| CN111560344A (en) | Method for constructing brain-like tissue by using umbilical cord mesenchymal stem cells | |
| CN102899285A (en) | Method for differentiation of embryonic stem cells into nerve cells through in vitro induction | |
| CN105106240B (en) | A kind of stem cell medicine and its purposes in vascular interventional treatment cerebral apoplexy | |
| CN117778313B (en) | Differentiation method and application of mesenchymal stem cells obtained from brain organoids | |
| CN105754943B (en) | A kind of naked mole cultured hippocampal neuron method | |
| CN108865997B (en) | Culture medium and culture method for in vitro culture of astrocytes | |
| Zhang et al. | Schwann cells originating from skin-derived precursors promote peripheral nerve regeneration in rats | |
| CN103060265A (en) | Primary culture method of elderly rat brain vascular endothelial cell | |
| CN108070558A (en) | A kind of preparation method of clinical grade neural stem cell | |
| CN105087475B (en) | A kind of method that cell culture fluid and its application and induction dental pulp stem cell break up to neural-like cells | |
| CN102703380B (en) | Sub-totipotent stem cell, preparation method and application thereof | |
| CN107267457A (en) | NSC adhere-wall culture method | |
| CN107164325B (en) | The preparation method and kit of the oligodendroglia in the source MSCs | |
| CN110229860A (en) | A kind of Animal Skin small-molecular peptides preparation method promoting Marrow Stromal Cells in Proliferation | |
| CN111733133B (en) | Method for promoting differentiation and growth of epidermal stem cells | |
| CN105441381B (en) | Method of the external evoked human adipose mesenchymal stem cells to functional hepatocyte differentiation |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20171020 |
|
| WD01 | Invention patent application deemed withdrawn after publication |