CN106479978A - A kind of special culture media of neural stem cell and its cultural method - Google Patents
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Abstract
The present invention relates to the special culture media for culture of neural stem cells neural and its cultural method, this neural stem cell special culture media, based on DMEM/F12 culture medium, comprise following density component：300-450 μ g/ml 2 ', 3 '-DIDEOXYADENOSINE -5- triguaiacyl phosphate, 5-15 μ g/ml N2Additive, 15-30ng/ml stem cell factor, 5-7mg/ml thioglycerol, 50-70ng/ml nerve growth factor, 0.05-0.07mmol/ml acetylcholine, 10-25mg/ml sodium carboxymethyl cellulose, 0.005-0.01mmol/ml retinoic acid and 0.110-0.20mmol/ml Folic Acid.Described culture medium prescription is rationally effective, keeps the undifferentiated state of neural stem cell while effectively improving cell amplification speed, does not affect it and breaks up performance, extends passage number.
Description
Technical field
The invention belongs to field of cell culture, particularly to a kind of special culture of neural stem cell
Base and its cultural method.
Background technology
It is to be present in embryo and adult in neural stem cell (Neural Stem Cell, NSC)
One of the nervous tissues such as brain, spinal cord stem cell, is that a class has division potential and from more
The blast cell of new ability, can be updated by not reciprocity divisional mode self replication or be divided into
The various types of cells of the nervous tissues such as neuron, astrocyte, oligodendrocyte,
Can transdifferentiation protometrocyte and Skeletal Muscle Cell.Therefore culture of neural stem cells neural is intended to obtain foot
Enough amounts have propagation and differentiation potential neural stem cell, for transplantation treatment, carry out
Transgeneic procedure is attacked the growth of tumor or is carried out autologous feedback, treatment central nervous system
Functional lesion etc..
Because nerve is the most complicated in body and the tissue of topnotch differentiation, neural stem cell
Culture difficulty is high, and extremely easy Spontaneous Differentiation in incubation, thus lose soon
The characteristic of stem cell and function.So there being the research of a large amount of neural stem cell In vitro culture at present,
It is intended to cultivate the neural stem cell of high-quality and quantity.For example《Early stage people's embryo nerve trunk is thin
Born of the same parents' distribution and separation and Culture》, Lv Haixia, Zhai Wei, Liu Yong etc., XI AN JIAOTONG UNIVERSITY Subject Index (doctor
Learn version), the 2nd phase of volume 24 in April, 2003：97-100；《Human embryo hepatocytes
Separation and Culture and identification》, Luo Shuwei, thank to evergreen, Lu Guang, Central South University's journal (medicine),
2004,29 (2)：129-131；, the people of offer in Chinese patent application CN02134313.6
The cultural method of neural stem cell；The one kind providing in Chinese patent application CN104694475A
New type nerve stem cell cultivates additive and screening technique, application and the training using this additive
Foster base etc..Because serum has the shortcomings that complicated component, quality are unstable, expensive,
Although having culture amplification efficiency during serum higher, but serum-free culture still becoming development and becomes
Gesture, at present typically using interpolation basic fibroblast growth factor (bFGF), epidermal growth
The factor (EGF), antibiotic, N2And/or B24DMEM or DMEM/F12 of the compositions such as additive
Culture fluid as the serum-free medium of neural stem cell, although this culture fluid solve latent
In the problem of virus, pollution, there is culture fluid composition and become apparent from, the advantages of repeated high,
But neural stem cell survival rate is low, propagation slow, pass on and yield poorly, break up serious etc. asking
Topic is still relatively difficult to resolve certainly.
Content of the invention
It is a feature of the present invention that on the basis of forefathers' research, there is provided a kind of nerve trunk is thin
Born of the same parents' special culture media and the method applying described culture medium culturing neural stem cell.Through excessive
Amount test determines the rational formula of culture medium, and the actual conditions of culture of neural stem cells neural,
Effectively In vitro culture and amplification can be carried out to neural stem cell.
For achieving the above object, concrete technical scheme of the present invention is as follows：
A kind of neural stem cell special culture media, described culture medium is by DMEM/F12 culture fluid and molten
Matter forms, and described solute and its concentration in DMEM/F12 culture fluid are：300-450μg/ml
2 ', 3 '-DIDEOXYADENOSINE -5- triguaiacyl phosphate, 5-15 μ g/ml N2Additive, 15-30ng/ml do
Cell growth factor, 5-7mg/ml thioglycerol, 50-70ng/ml nerve growth factor,
0.05-0.07mmolol/ml acetylcholine, 10-25mg/ml sodium carboxymethyl cellulose,
0.005-0.01mmolol/ml retinoic acid and 0.110-0.20mmolol/ml Folic Acid.
Preferably, described neural stem cell special culture media, described culture medium is trained by DMEM/F12
Nutrient solution and solute composition, described solute and its concentration in DMEM/F12 culture fluid are：
350 μ g/ml 2 ', 3 '-DIDEOXYADENOSINE -5- triguaiacyl phosphates, 8 μ g/ml N2Additive,
22ng/ml stem cell factor, 6mg/ml thioglycerol, 63ng/ml nerve growth factor,
0.06mmolol/ml acetylcholine, 18mg/ml sodium carboxymethyl cellulose, 0.008mmolol/ml
Retinoic acid and 0.15mmolol/ml Folic Acid.
Neural stem cell special culture media provided by the present invention, formula is rationally effective, keeps
The high motility rate of neural stem cell, keeps nerve while effectively improving cell amplification speed
The undifferentiated state of stem cell, does not affect it and breaks up performance, extend passage number.
On the other hand, the invention provides a kind of apply described neural stem cell special culture media
The method of culture of neural stem cells neural is it is characterised in that methods described comprises the steps：
1) obtain in vitro cranial nerve tissue in aseptic condition, with the PBS dual anti-containing 5%
Rinsed；
2) by step 1) cranial nerve of gained tissue is cut into 1-2mmol3Fragment, add containing by
0.5mg/ml type i collagen enzyme, 0.5mg/ml IV Collagenase Type and 0.1mg/ml deoxidation core
The DMEM/F12 culture medium of ribosomal ribonucleic acid I, in 4-8 DEG C of CO2Digest and decompose in incubator
20-24h, obtains tissue suspension；
3) by step 2) centrifugation of the tissue suspension of gained, go the supernatant, add DMEM/F12
Culture medium is cleaned 3 times, adds cell dissociation buffer, digests 42-57min in 35-38 DEG C, gently
Piping and druming terminates digestion after mixing, and rinses in DMEM/F12, crosses 200-400 mesh cell sieve,
Collect filtrate, centrifugation is abandoned supernatant, obtained single cell suspension；
4) to step 3) add described neural stem cell special culture media in the single cell suspension of gained,
Adjustment cell concentration is 1 × 107Individual/ml, is inoculated in culture dish, cultivates 10-12 days, obtains
To described P0 for neural stem cell；
5) taking step 4) P0 that obtains, for neural stem cell, adds cell dissociation buffer to disappear in 37 DEG C
Change 40s-85s, after piping and druming mixes, be centrifuged 6-10min, abandon the supernatant, add described god
Through stem cell special culture media, cell concentration is made to be 3 × 105Individual/ml, in inoculated and cultured bottle,
And add the diaminoethanes polyacrylamide of 1g/L in culture dish, slowly supply gas mixing all
Even, mixing of slowly supplying gas while often changing a fresh culture every other day, cultivate 3-5 days,
Obtain passing on neural stem cell.
Wherein, dual anti-for cell culture additive：Penicillin and Streptomycin Solution, are a kind of
Containing penicillin (10000IU) and streptomycin (10000 μ g/mL) in 100 times of working concentrations
Mixed liquor.
In conjunction with Neural Stem Cells ' Growth characteristic, above-mentioned nerve trunk is summed up by lot of experiments thin
The cultural method of born of the same parents and condition, can effectively maintain the optimum state of neural stem cell, and guiding is thin
Born of the same parents breed；Diaminoethanes polyacrylamide is added in passage cell incubation, as
The microcarrier of neural stem cell adhere-wall culture, makes neural stem cell adhere to and micro-carrier surface life
Long, combine the advantage of adhere-wall culture and suspension culture, substantially increase nerve trunk
The yield of cell and quality, the present invention selected diaminoethanes polyacrylamide, with institute
The one-tenth split-phase stated in culture medium is coordinated, in the application of culture of neural stem cells neural, not only permissible
The adhesion of promotion cell and propagation are moreover it is possible to effectively transmit bioactive molecule and nutrient, simultaneously
Keep the activity of neural stem cell.
Preferably, described centrifugal rotational speed is 1300rmp.
Preferably, step 4) P0 for the condition of culture of stem cell is：37 DEG C, 5%CO2's
Humid air, pH value keep 7.3-7.4；Change half amount culture medium, and add 50ug/ within 3rd day
The serum substitute of ml；Fresh culture is changed every three days after 6th day；Preferably, described
The NaOH of value 2M of PH is adjusted it is preferred that described serum substitute is KnockoutTMBlood
Clear substitute.
Find through test, add the serum of 50ug/ml in the culture interstage is to culture medium
Substitute, and keep culture of neural stem cells neural under conditions of above-mentioned pH value, can effectively facilitate thin
Intracellular growth and propagation, yield can increase further.
Preferably, described step 5) condition of culture that passes on neural stem cell is：37℃、5
%CO2Humid air, the 1-2 days pH values keep 7.05-7.15, and PH protects within the 2-3 days
Hold 7.15-7.25, it is 5%CO that described step of supplying gas sends into gas2Humid air, preferably
, the NaOH of value 2M of described PH is adjusted.
Supply gas every other day, make microcarrier persistently keep suspended state, and ensure that O2 and CO2 supplies
Stablizing and fresh of amount, is conducive to adhesion and the growth of neural stem cell, and with nerve trunk
The metabolic process of cell adjusts optimal pH value at any time, be to ensure that cell survival, good growth,
Activity and the essential condition of function maintenance, through many experiments, summarize under above-mentioned pH condition,
The growth of neural stem cell and survival condition are optimal.
Preferably, described cell dissociation buffer be containing 1.5mg/ml trypsin and
The PBS of 0.1mg/ml EDTA.
Preferably, described step 4) described in culture dish pan coating rat-tail glue.
Further preferred, the method that described culture dish is coated rat-tail glue is：Using 0.5mg/ml
Rat-tail glue deionized water solution, be spread evenly across culture dish surface, put into 37 DEG C, 5%
CO2Incubator in be coated 50min, inhale and after abandoning solution, add aseptic water washing 3 times, then plus
Enter DMEM/F12 culture fluid and rinse to dry up for one time and obtain final product.
Neural stem cell special culture media of the present invention, formula is rationally effective, can meet
The nutritional need of Neural Stem Cells ' Growth, effectively facilitate neural stem cell breeds the same of growth
When, keep the stem cell properties of neural stem cell, divide during reducing Culture of neural stem cells
Change behavior probability of happening.
The present invention through lot of experiments, the growth characteristics in conjunction with neural stem cell and demand, always
Bear the method applying above-mentioned neural stem cell special culture media culture of neural stem cells neural, can have
Effect maintains the optimum state of neural stem cell, guides cell proliferation, reduces cell mortality；
Diaminoethanes polyacrylamide is added in passage cell incubation, thin as nerve trunk
The microcarrier of born of the same parents' adhere-wall culture, makes neural stem cell adhere to and micro-carrier surface growth, in conjunction with
The advantage of adhere-wall culture and suspension culture, substantially increases the product of neural stem cell
Amount and quality.
Specific embodiment
With reference to embodiment, the present invention is further described；Following embodiments are illustrative,
It is not determinate it is impossible to limit protection scope of the present invention with following embodiments；The present invention
Used in equipment, if no special requirements, be in the art commonly use equipment；The present invention
Used in method, if no special requirements, be in the art commonly use method.
Embodiment 1
A kind of be used for neural stem cell special culture media, described culture medium is by DMEM/F12 culture fluid and molten
Matter forms, and described solute and its concentration in DMEM/F12 culture fluid are：300μg/ml 2′,3
'-DIDEOXYADENOSINE -5- triguaiacyl phosphate, 5 μ g/ml N2Additive, 15ng/ml stem cell growth
The factor, 5mg/ml thioglycerol, 50ng/ml nerve growth factor, 0.05mmol/ml acetyl
Choline, 10mg/ml sodium carboxymethyl cellulose, 0.005mmol/ml retinoic acid and 0.110mmol/ml
Folic Acid.
Embodiment 2
A kind of be used for neural stem cell special culture media, described culture medium is by DMEM/F12 culture fluid and molten
Matter forms, and described solute and its concentration in DMEM/F12 culture fluid are：350μg/ml 2′,3
'-DIDEOXYADENOSINE -5- triguaiacyl phosphate, 8 μ g/ml N2Additive, 22ng/ml stem cell growth
The factor, 6mg/ml thioglycerol, 63ng/ml nerve growth factor, 0.06mmol/ml acetyl
Choline, 18mg/ml sodium carboxymethyl cellulose and 0.15mmol/ml Folic Acid.
Embodiment 3
A kind of cultural method of neural stem cell, comprises the steps：
1) obtain in vitro cranial nerve tissue in aseptic condition, with the PBS dual anti-containing 5%
Rinsed；
2) by step 1) cranial nerve of gained tissue is cut into 1mmol3Fragment, adds containing by 0.5mg/ml
Type i collagen enzyme, 0.5mg/ml IV Collagenase Type and 0.1mg/ml DNA (deoxyribonucleic acid) I
DMEM/F12 culture medium, in 8 DEG C of CO2Digest and decompose 20h in incubator, obtains tissue and hangs
Liquid；
3) by step 2) centrifugation of the tissue suspension of gained, go the supernatant, add DMEM/F12
Culture medium is cleaned 3 times, adds cell dissociation buffer, digests 57min in 35 DEG C, gently blows and beats mixed
Terminate digestion after even, rinse in DMEM/F12, cross 200-400 mesh cell sieve, collect filter
Liquid, supernatant is abandoned in centrifugation, obtains single cell suspension；
4) to step 3) add described neural stem cell special culture media in the single cell suspension of gained,
Adjustment cell concentration is 1 × 105Individual/ml, is inoculated in culture dish, cultivates 12 days, obtains
Described P0 is for neural stem cell；
5) taking step 4) P0 that obtains, for neural stem cell, adds cell dissociation buffer to disappear in 37 DEG C
Change 85s, after piping and druming mixes, be centrifuged 10min, abandon supernatant, add described neural stem cell special
With culture medium, cell concentration is made to be 3 × 106Individual/ml, in inoculated and cultured bottle, and to culture dish
The diaminoethanes polyacrylamide of middle addition 1g/L, mix homogeneously of slowly supplying gas, often every other day
Change mixing of slowly supplying gas while a fresh culture, cultivate 3-5 days, obtain passing on god
Through stem cell.
Wherein, neural stem cell special culture media is made up of DMEM/F12 culture fluid and solute, described
Solute and its concentration in DMEM/F12 culture fluid are：450 μ g/ml 2 ', 3 '-dideoxies
Adenosine -5- triguaiacyl phosphate, 15 μ g/ml N2Culture additive, 30ng/ml stem cell growth
The factor, 7mg/ml thioglycerol, 70ng/ml nerve growth factor, 0.07mmol/ml second
Phatidylcholine, 25mg/ml sodium carboxymethyl cellulose, 0.01mmol/ml retinoic acid and
0.20mmol/ml Folic Acid.
Embodiment 4
A kind of cultural method of neural stem cell, comprises the steps：
1) obtain in vitro cranial nerve tissue in aseptic condition, with the PBS dual anti-containing 5%
Rinsed；
2) by step 1) cranial nerve of gained tissue is cut into 2mmol3Fragment, adds containing by 0.5mg/ml
Type i collagen enzyme, 0.5mg/ml IV Collagenase Type and 0.1mg/ml DNA (deoxyribonucleic acid) I
DMEM/F12 culture medium, in 4 DEG C of CO2Digest and decompose 24h in incubator, obtains tissue and hangs
Liquid；
3) by step 2) centrifugation of the tissue suspension of gained, go the supernatant, add DMEM/F12
Culture medium is cleaned 3 times, adds cell dissociation buffer, digests 42min in 38 DEG C, gently blows and beats mixed
Terminate digestion after even, rinse in DMEM/F12, cross 200-400 mesh cell sieve, collect filter
Liquid, supernatant is abandoned in centrifugation, obtains single cell suspension；
4) to step 3) add described neural stem cell special culture media in the single cell suspension of gained,
Adjustment cell concentration is 1 × 105Individual/ml, is inoculated in culture dish, cultivates 10 days, obtains
Described P0 is for neural stem cell；
5) taking step 4) P0 that obtains, for neural stem cell, adds cell dissociation buffer to disappear in 37 DEG C
Change 40s, after piping and druming mixes, be centrifuged 6min, abandon supernatant, add described neural stem cell special
With culture medium, cell concentration is made to be 3 × 106Individual/ml, in inoculated and cultured bottle, and to culture dish
The diaminoethanes polyacrylamide of middle addition 1g/L, mix homogeneously of slowly supplying gas, often every other day
Change mixing of slowly supplying gas while a fresh culture, cultivate 3-5 days, obtain passing on god
Through stem cell.
Wherein, neural stem cell special culture media is made up of DMEM/F12 culture fluid and solute, described
Solute and its concentration in DMEM/F12 culture fluid are：350 μ g/ml 2 ', 3 '-two take off
Oxygen adenosine -5- triguaiacyl phosphate, 8 μ g/ml N2Additive, 22ng/ml stem cell factor,
6mg/ml thioglycerol, 63ng/ml nerve growth factor, 0.06mmol/ml acetylcholine,
18mg/ml sodium carboxymethyl cellulose and 0.15mmol/ml Folic Acid.
Embodiment 5
A kind of cultural method of neural stem cell, comprises the steps：
1) obtain in vitro cranial nerve tissue in aseptic condition, with the PBS dual anti-containing 5%
Rinsed；
2) by step 1) cranial nerve of gained tissue is cut into 2mmol3Fragment, adds containing by 0.5mg/ml
Type i collagen enzyme, 0.5mg/ml IV Collagenase Type and 0.1mg/ml DNA (deoxyribonucleic acid) I
DMEM/F12 culture medium, in 6 DEG C of CO2Digest and decompose 24h in incubator, obtains tissue and hangs
Liquid；
3) by step 2) centrifugation of the tissue suspension of gained, go the supernatant, add DMEM/F12
Culture medium is cleaned 3 times, adds containing 1.5mg/ml trypsin and 0.1mg/ml EDTA
PBS digests 50min in 36 DEG C, terminates digestion, after gently piping and druming mixes in DMEM/F12
Rinsing, crosses 200-400 mesh cell sieve, collects filtrate, and supernatant is abandoned in centrifugation, obtains unicellular outstanding
Liquid；Wherein, described centrifugal rotational speed is 1300rpm；
4) to step 3) add described neural stem cell special culture media in the single cell suspension of gained,
Adjustment cell concentration is 1 × 105Individual/ml, is inoculated in culture dish, and in 37 DEG C, 5%CO2
Humid air, pH value keep 7.3-7.4 under conditions of cultivated；Change half amount within 3rd day
Culture medium, and add the Knockout of 50ug/mlTMSerum substitute；After 6th day every
Change fresh culture within two days；Culture obtained described P0 for neural stem cell after 11 days；
5) taking step 4) P0 that obtains, for neural stem cell, adds and contains 1.5mg/ml Trypsin
The PBS of enzyme and 0.1mg/ml EDTA digests 40s in 37 DEG C, after piping and druming mixes, 1300rpm
It is centrifuged 8min under rotating speed, abandons supernatant, add described neural stem cell special culture media, make
Cell concentration is 3 × 106Individual/ml, in inoculated and cultured bottle, and adds 1g/L in culture dish
Diaminoethanes polyacrylamide, mix homogeneously of slowly supplying gas, in 37 DEG C, 5%CO2's
Cultivated under conditions of humid air；Often change slow while a fresh culture every other day
Send into 5%CO2Humid air mixed, cultivate 3-5 days, obtain passing on nerve trunk thin
Born of the same parents.
Wherein, neural stem cell special culture media is made up of DMEM/F12 culture fluid and solute, described
Solute and its concentration in DMEM/F12 culture fluid are：420 μ g/ml 2 ', 3 '-dideoxies
Adenosine -5- triguaiacyl phosphate, 10 μ g/ml N2Culture additive, 18ng/ml stem cell growth
The factor, 6mg/ml thioglycerol, 60ng/ml nerve growth factor, 0.06mmol/ml second
Phatidylcholine, 20mg/ml sodium carboxymethyl cellulose and 0.13mmol/ml Folic Acid.
Preferably, step 4) in the 1-2 days pH values keep 7.05-7.15, PH protects within the 2-3 days
Hold 7.15-7.25；
It is further preferred that described pH value is adjusted by the NaOH of 2M.
Embodiment 6
A kind of cultural method of neural stem cell, the difference with embodiment 5 is,
Further define, step 4) in culture dish pan coating rat-tail glue, wherein, rat-tail glue
Method for coating is：Using the rat-tail glue deionized water solution of 0.5mg/ml, it is spread evenly across culture
Ware surface, puts into 37 DEG C, 5%CO2Incubator in be coated 50min, inhale and add after abandoning solution
Aseptic water washing 3 times, adds DMEM/F12 culture fluid and rinses and dry up for one time and obtain final product.
Test 1：Neurocyte rises in value situation in vitro
1) it is grouped
Test group：Liver stem cells culture medium of the present invention；
Matched group：Neuronal cell cultures base provided in Chinese patent application CN104818245A；
2) test method
Cultured and amplified in vitro method, selects the method described in embodiment 4.
Using trypan blue classics staining, cell is counted, according to the method training of embodiment 4
Foster amplifying liver stem cells, count primary stem cell, the 3rd generation, the 6th generation and the work of the 9th generation respectively
The quantity of neural stem cell, the results are shown in Table 1.
Table 1 neural stem cell cultured and amplified in vitro cell quantity
As can be seen from Table 1, the cultural method of neural stem cell of the present invention can be effective
In vitro culture neural stem cell, and liver stem cells special culture media provided by the present invention is to liver
The amplification cultivation of stem cell is very efficient, has the difference of highly significant compared with matched group, in addition through platform
Expect that the blue classics measurable test group of staining passes on rear Cell viability every time all more than 86%, more right
Variant according to group 63%.
Test 2：Neural stem cell differentiating state-detection
1) it is grouped
Test group：Liver stem cells culture medium of the present invention；
Matched group：Neuronal cell cultures base provided in Chinese patent application CN02134313.6；
2) test method
Cultured and amplified in vitro method, selects the method described in embodiment 4.
Take according to embodiment 4 method culture P10 for neural stem cell, wherein, nerve trunk
During passage, add Brdu (5 μm of ol/L) in the medium.With 4% paraformaldehyde in room
The fixing cell of temperature, PBS washs three times.For the dyeing of intracellular antigen, 0.1%
Broken cell film 30 minutes in Triton X-100, are then incubated 30 in 5% Normal Goat Serum
Minute to block non-specific binding, an anti-incubation 2 hours, wherein one anti-use main
Antibody is：Mouse monoclonal anti-tubulin TuJ1 (1:750 dilutions), for contaminating neuron；
The anti-GFAP of rabbit polyclonal (1:400 dilutions), for contaminating astrocyte；Mouse monoclonal resists
O4(1:10 dilutions), for contaminating oligodendrocyte and its precursor.Wash after three times with PBS,
Incubation 1 hour in the sheep anti-mouse igg two of biotin labeling resists；Washed after three times with PBS,
Streptavidin alkaline phosphatase enzymatic solution is incubated 30 minutes；Finally shown with substrate B CIP/NBT
Color, observes under inverted microscope and counts.
Table 2 test group and the neural stem cell differentiating result of matched group
Understand, the cell of test group more than 95% assumes TuJ1, GFAP through test result analysis
O4 anti-with mouse monoclonal is feminine gender, and Brdu is positive, thus proves using the present invention's
Liver stem cells the 10th generation of the Selective agar medium Secondary Culture also less phenomenon that differentiation degeneration occurs,
Maintain the cell quality of primary liver stem cells, application Brdu labelling can further illustrate nerve
The division growth ability of stem cell；And the cell of matched group only more than 56% assumes TuJ1, GFAP
O4 anti-with mouse monoclonal is feminine gender, and after passing for 10 generations, hepatic stem cell differentiation is relatively serious.
Test 3：Neural stem cell differentiating behavioral value
1) it is grouped
Test group：Liver stem cells culture medium of the present invention；
Matched group：Neuronal cell cultures base provided in Chinese patent application CN02134313.6；
2) test method
Cultured and amplified in vitro method, selects the method described in embodiment 4.
The P10 taking method culture according to embodiment 4 for neural stem cell, with pancreatin digestion and
Hyclone is planted respectively after terminating and is used the coated little training of poly-D-lysine in advance in diameter 315cm
In foster ware, with there being blood serum medium DMEM/F12+5%Fcs to carry out differentiation culture, cultivate 24h
Carry out exempting from Fluorometric assay using the method for test 2 afterwards, testing result is shown in Table 3.
Table 3 test group and the neural stem cell differentiating result of matched group
Can know that test group neural stem cell, can be multidirectional under the conditions of differentiation culture through result of the test
It is divided into neuronal cell, astrocyte and oligodendrocyte, illustrate that the present invention is carried
For neural stem cell special culture media and cultural method same to the effective amplification of neural stem cell
When, maintain it as the characteristic of stem cell, maintain its differentiation potential；Compared with matched group,
Test group neural stem cell is more easy to be divided into neuronal cell, weakens it spontaneous in incubation
It is divided into the tendency of glial cell.
Conclusion
From above-mentioned test, using Culture of neural stem cells method of the present invention and its specially
With culture medium, can effective In vitro culture neural stem cell, and nerve provided by the present invention
Stem cell special culture media is very efficient to the amplification cultivation of neural stem cell, and effectively keeps
The stem cell potential of neural stem cell, the 10th generation also less phenomenon that differentiation degeneration occurs.
Claims (10)
1. a kind of neural stem cell special culture media is it is characterised in that described culture medium is by DMEM/F12
Culture fluid and solute composition, described solute and its concentration in DMEM/F12 culture fluid are：
300-450 μ g/ml 2 ', 3 '-DIDEOXYADENOSINE -5- triguaiacyl phosphate, 5-15 μ g/ml N2Add
Plus agent, 15-30ng/ml stem cell factor, 5-7mg/ml thioglycerol, 50-70ng/ml
Nerve growth factor, 0.05-0.07mmol/ml acetylcholine, 10-25mg/ml carboxymethyl are fine
The plain sodium of dimension, 0.005-0.01mmol/ml retinoic acid and 0.110-0.20mmol/ml leaf
Acid.
2. neural stem cell special culture media as claimed in claim 1 is it is characterised in that described culture
Base is made up of DMEM/F12 culture fluid and solute, described solute and its DMEM/F12 culture
Concentration in liquid is：350 μ g/ml 2 ', 3 '-DIDEOXYADENOSINE -5- triguaiacyl phosphates, 8 μ g/ml
N2Additive, 22ng/ml stem cell factor, 6mg/ml thioglycerol, 63ng/ml god
Through somatomedin, 0.06mmol/ml acetylcholine, 18mg/ml sodium carboxymethyl cellulose,
0.008mmol/ml retinoic acid and 0.15mmol/ml Folic Acid.
3. one kind application neural stem cell special culture media as claimed in claim 1 or 2 culture nerve
The method of stem cell is it is characterised in that methods described comprises the steps：
1) obtain in vitro cranial nerve tissue in aseptic condition, with the PBS dual anti-containing 5%
Rinsed；
2) by step 1) cranial nerve of gained tissue is cut into 1-2mm3Fragment, adds containing by 0.5mg/ml
Type i collagen enzyme, 0.5mg/ml IV Collagenase Type and 0.1mg/ml DNA (deoxyribonucleic acid) I
DMEM/F12 culture medium, in 4-8 DEG C of CO2Digest and decompose 20-24h in incubator, obtains group
Knit suspension；
3) by step 2) centrifugation of the tissue suspension of gained, abandon the supernatant, add DMEM/F12
Culture medium is cleaned 3 times, adds cell dissociation buffer, digests 42-57min in 35-38 DEG C, gently
Piping and druming terminates digestion after mixing, and rinses in DMEM/F12, crosses 200-400 mesh cell sieve,
Collect filtrate, centrifugation is abandoned supernatant, obtained single cell suspension；
4) to step 3) add described neural stem cell special culture media in the single cell suspension of gained,
Adjustment cell concentration is 1 × 107Individual/ml, is inoculated in culture dish, cultivates 10-12 days, obtains
To described P0 for neural stem cell；
5) taking step 4) P0 that obtains, for neural stem cell, adds cell dissociation buffer to disappear in 37 DEG C
Change 40s-85s, after piping and druming mixes, be centrifuged 6-10min, abandon the supernatant, add described god
Through stem cell special culture media, cell concentration is made to be 3 × 105Individual/ml, in inoculated and cultured bottle,
And add the diaminoethanes polyacrylamide of 1g/L in culture dish, slowly supply gas mixing all
Even, mixing of slowly supplying gas while often changing a fresh culture every other day, cultivate 3-5 days,
Obtain passing on neural stem cell.
4. Culture of neural stem cells method as claimed in claim 3 is it is characterised in that described centrifugation
Rotating speed is 1300rpm.
5. Culture of neural stem cells method as claimed in claim 3 is it is characterised in that step 4)
P0 for the condition of culture of stem cell is：37 DEG C, 5%CO2Humid air, pH value keep
7.3-7.4；Change half amount culture medium, and add the serum substitute of 50ug/ml within 3rd day；
Fresh culture is changed every three days after 6th day.
6. Culture of neural stem cells method as claimed in claim 3 is it is characterised in that step 5)
The condition of culture passing on neural stem cell is：37 DEG C, 5%CO2Humid air, 1-2
Its pH value keeps 7.05-7.15, and PH keeps 7.15-7.25, described step of supplying gas within the 2-3 days
Rapid gas of sending into is 5%CO2Humid air.
7. the Culture of neural stem cells method as described in claim 5 or 6 is it is characterised in that nerve
In stem cell incubation, pH value is adjusted by the NaOH of 2M.
8. the method for Culture of neural stem cells as claimed in claim 3 is it is characterised in that described thin
Born of the same parents' Digestive system is the PBS containing 1.5mg/ml trypsin and 0.1mg/ml EDTA.
9. the cultural method of neural stem cell as claimed in claim 3 is it is characterised in that step 4)
Described in culture dish pan coating rat-tail glue.
10. the cultural method of neural stem cell as claimed in claim 9 is it is characterised in that described
The method that culture dish is coated rat-tail glue is：Rat-tail glue deionization using 0.5mg/ml is water-soluble
Liquid, is spread evenly across culture dish surface, puts into 37 DEG C, 5%CO2Incubator in be coated
50min, inhales to abandon and adds aseptic water washing 3 times after solution, adds DMEM/F12 culture fluid
Rinse to dry up for one time and obtain final product.
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