CN107201339A - Nerve stem cell culture medium - Google Patents
Nerve stem cell culture medium Download PDFInfo
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- CN107201339A CN107201339A CN201710505538.1A CN201710505538A CN107201339A CN 107201339 A CN107201339 A CN 107201339A CN 201710505538 A CN201710505538 A CN 201710505538A CN 107201339 A CN107201339 A CN 107201339A
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Abstract
A kind of nerve stem cell culture medium, contains 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.The NSC of medium culture of the present invention had obvious exponential phase at 3~6 days, and cell propagation is very fast, and uses the growth curve of the NSC of conventional medium culture to show that cell growth is slow, without obvious logarithmic proliferation.
Description
Technical field
The present invention relates to technical field of cell culture, more particularly to a kind of nerve stem cell culture medium.
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 culture medium of nerve stem cell proliferation ability.
The content of the invention
In view of this, the invention provides a kind of nerve stem cell culture medium, the nerve stem cell culture medium can be significantly
Strengthen the multiplication capacity of NSC, while enabling to NSC adherent life under the lazy condition without the pre- bed board of gelatin
It is long, eliminate cumbersome operating procedure and simplify production procedure.
In order to realize foregoing invention purpose, the present invention provides following technical scheme：
A kind of nerve stem cell culture medium, including DMEM/F12, nonessential amino acid, EGF, basic fibroblast are thin
The intracellular growth factor, insulin, hydrocortisone, WNT3a, Notchl and bovine serum albumin(BSA).Wherein 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/L WNT3a, 40~60nmol/L
Notchl, 40~60 u g/mL bovine serum albumin(BSA)s.
Preferably, nerve stem cell culture medium also includes fibronectin and four collagen types.
Further it is preferably：Contain 10~20 u mol/mL nonessential amino acid, 10 in nerve stem cell culture medium
~20ng/mL EGFs, 5~15ng/mL basic fibroblast growth factors, 10~20 u g/mL insulin, 40
~60nmol/L hydrocortisones, 10~20nmol/L WNT3a, 40~60nmol/L Notchl, 40~60 u g/mL ox bloods
Pure albumen, 15~35ng/mL fibronectins, the collagen types of 50~70ng/mL tetra-.
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 nerve stem cell culture medium that the present invention is provided can greatly enhance the multiplication capacity of NSC.Flow cytometer detection result
It has been shown that, culture medium of the present invention can enable the surface marker Nestin+CD133+ of the NSC in P6 generations ratio reach
To 33.3%, and conventional medium is only 9.8% in the Nestin+CD133+ in P6 generations ratio；The god of medium culture of the present invention
There is obvious exponential phase through stem cell, cell propagation is very fast, and uses the god of conventional medium culture at 3~6 days
Growth curve through stem cell shows that cell growth is slow, without obvious logarithmic proliferation；
Embodiment
The invention discloses the Culture of neural stem cells that the preparation of a kind of nerve stem cell culture medium and culture medium, the present invention are provided
Base agents useful for same, instrument, experimental animal etc. can be bought by market.Wherein, WNT3a thinks wound, Notchl purchases purchased from the magnificent piebald horse in Shanghai
From Wuhan Halothane thing；Nonessential amino acid is purchased from Gibco, and article No. is 11140, MEM Non-Essentia Amino Acids
SolutionlOmM (100X) etc..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 times,
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, examination
Test group 3 and use 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.
The NSC of above-mentioned each group culture is subjected to flow cytometer detection, the surface marker Nestin of NSC
+, CD133+ ratio can reach 33.3%, 29.5%, 24.3%, and conventional medium is in Nestin+, the CD133+ in P6 generations
Ratio is only 9. 8%.
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 adherent growth in the case of without the pre- bed board of gelatin simultaneously,
Eliminate cumbersome operating procedure and simplify production procedure.
Claims (3)
1. a kind of nerve stem cell culture medium, it is characterised in that：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 cow's serums
Albumin.
2. nerve stem cell culture medium according to claim 1, it is characterised in that：The nerve stem cell culture medium is also wrapped
Include fibronectin and four collagen types.
3. nerve stem cell culture medium according to claim 2, it is characterised in that：Containing 15~35ng/mL fibronectins,
The collagen types of 50 1 70ng/mL tetra-.
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| CN201710505538.1A CN107201339A (en) | 2017-06-28 | 2017-06-28 | Nerve stem cell culture medium |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107663516A (en) * | 2017-11-02 | 2018-02-06 | 北京全式金生物技术有限公司 | A kind of human nerve stem cell culture medium and application |
| CN110684735A (en) * | 2019-11-13 | 2020-01-14 | 安徽科门生物科技有限公司 | Culture medium for rapidly proliferating neural stem cells |
| CN111254115A (en) * | 2018-12-03 | 2020-06-09 | 北京银丰鼎诚生物工程技术有限公司 | Preparation method of culture medium suitable for large-scale culture of clinical-grade neural stem cells |
| CN116144598A (en) * | 2023-04-06 | 2023-05-23 | 广州准优生物科技有限公司 | Culture medium for promoting proliferation and differentiation of neural stem cells and application of culture medium |
-
2017
- 2017-06-28 CN CN201710505538.1A patent/CN107201339A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107663516A (en) * | 2017-11-02 | 2018-02-06 | 北京全式金生物技术有限公司 | A kind of human nerve stem cell culture medium and application |
| WO2019085993A1 (en) * | 2017-11-02 | 2019-05-09 | 北京全式金生物技术有限公司 | Human neural stem cell culture medium and application |
| CN111254115A (en) * | 2018-12-03 | 2020-06-09 | 北京银丰鼎诚生物工程技术有限公司 | Preparation method of culture medium suitable for large-scale culture of clinical-grade neural stem cells |
| CN110684735A (en) * | 2019-11-13 | 2020-01-14 | 安徽科门生物科技有限公司 | Culture medium for rapidly proliferating neural stem cells |
| CN116144598A (en) * | 2023-04-06 | 2023-05-23 | 广州准优生物科技有限公司 | Culture medium for promoting proliferation and differentiation of neural stem cells and application of culture medium |
| CN116144598B (en) * | 2023-04-06 | 2023-09-01 | 中科中銮生物科技(广东)有限公司 | Culture medium for promoting proliferation and differentiation of neural stem cells and application of culture medium |
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Application publication date: 20170926 |