CN108486058A - A method of promotion human pluripotent stem cells directed differentiation is mature neuron - Google Patents
A method of promotion human pluripotent stem cells directed differentiation is mature neuron Download PDFInfo
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Abstract
The invention discloses a kind of methods that promotion human pluripotent stem cells directed differentiation is mature neuron, pass through nerve-inducing and Neural Differentiation culture medium, so that human pluripotent stem cells break up to the method for neuron, this method can be obviously shortened the time needed for differentiation acquisition mature neuron.Compared with prior art, the present invention can induce human pluripotent stem cells to mature neuron in vitro within a short period of time, it substantial saved Neural Differentiation to take, be the cellular transplantation therapy of neurodegenerative disease, drug screening and pathomechanism research provide cell model.
Description
Technical field
The invention belongs to biomedical sectors, and in particular to a kind of promotion human pluripotent stem cells directed differentiation is neural for maturation
The method of member.
Background technology
Central nervous system disease is the current principal disease for threatening the mankind, drastically influences people’s lives quality, such as
Parkinson's formula disease, epilepsy, lateral sclerosis of spinal cord etc..These central system disorders are because of its high rate, high mortality and high cause
The features such as residual rate, brings white elephant to society and family.
The main pathological change of Parkinson formula disease be intracerebral dopaminergic neuron because being denaturalized extensively, it is dead due to lose, especially with
Substantia nigra compacta and corpus straitum are the most notable；Epilepsy is the electric discharge of neuron paroxysmal abnormality, leads to the one of the of short duration obstacle of brain function
Kind chronic recurrent disease；Amyotrophic lateral sclerosis is that a kind of fatal nerve characterized by motor nerve degeneration moves back
Row disease.These neurological diseases do not cure means effectively still so far, can not derived from lesion or the neuron of damage
Regeneration, human pluripotent stem cells have infinite multiplication and vitro directed differentiation to the potential of any one body cell, therefore, using people
The neuron in multipotential stem cell source carries out transplantation treatment and provides possibility for the healing of central nervous system disease.
Human pluripotent stem cells include human embryo stem cell and people's induced multi-potent stem cell, can self-renewing and in specific item
Differentiation becomes a kind of cell of internal any tissue under part.In recent years, research is it has been found that the neural stem cell of transplanting can be whole
The brain tissue into host is closed, and secretory nerve mediator promotes the reparation of nervous system to generate beneficial effect.However,
The research of cellular replacement therapy neurodegenerative disease is primarily present following three problems at present：1. human pluripotent stem cells break up
And the neuronal cell ratio come is limited, cannot provide the neuron of 100% purity, then it plants the presence of cell just with latent
Tumour generate or toxic side effect risk；2. it is very long that people's pluripotent cell is divided into the time required for the process of mature neuron
(in terms of the several years).Therefore after transplanting, cell will pass through to be broken up, is ripe, integrating and can function for a long time, this is greatly
Limit the potential of cellular transplantation therapy；3. if can unpredictably increase immature neural precursor does not enter differential period
It grows, not only has tumor formation risk, also result in neural undue growth, oppress surrounding host cell.
The prior art, which breaks up human pluripotent stem cells, about needs 16 days to neural precursor, however neural precursor
It is ripe then need for quite a long time.In vitro in culture systems, the neuron that human pluripotent stem cells break up was at 30 days
Start express mature neuron marker MAP2, by 50 days after just start express synaptophysin Synapsin, really express
It will be to after 70 days if the mature neuron marker such as glutamate transporter vGlut1 of specificity.If these neurons will be
The functional attributes that mature neuron is embodied in electro physiology experiment then need the longer time.In vivo, the maturation of human neure
Time is even more then number in terms of year.
Because neural precursor, which must exit mitosis, can just be divided into neuronal cell, so, it is effective to inhibit
The proliferation of neural precursor is to promote its one of strategy to terminal nerve cell differentiation.NeuronM disclosed by the invention promotees
In neural maturation culture solution, contain a kind of chain termination thimidine analogue (Azidothymidine, AZT), brain-derived neurotrophy
The factor (brain derived neurotrophic factor, BDNF) and cyclic adenosine monophosphate (Cyclic Adenosine
Monophosphate, cAMP), it can be promoted to mature neuron point in the proliferation for effectively inhibiting neural precursor
Change, increases the differentiation ratio of mature neuron cell and shorten neuronal maturation process, can be carried for cellular transplantation therapy
For more safe and efficient neuronal origin.
Invention content
It is mature neuron the technical problem to be solved in the present invention is to provide a kind of promotion human pluripotent stem cells directed differentiation
Method, to solve the problems such as of the existing technology ineffective.
It is an object of the invention in view of the drawbacks of the prior art, provide a kind of promotion human pluripotent stem cells directed differentiation to be
The method of mature neuron.Pluripotent stem cell differentiation is made one to nerve more particularly to by nerve-inducing and Neural Differentiation culture medium
The method of member.This method can be obviously shortened the time needed for differentiation acquisition mature neuron.
The further object of the present invention is to induce human pluripotent stem cells to obtain mature neuron in vitro in aforementioned manners, is
The cellular transplantation therapy of neurodegenerative disease, drug screening and pathomechanism research provide cell model.
The present invention cultivates human pluripotent stem cells in the system without feeder layer of our independent researches, with
Vitronectin uses Matrigel as matrigel developing approach as matrigel culture human pluripotent stem cells.Pass through addition
A kind of rush nerve maturation culture solution NeuronM containing chain termination compound AZT and BDNF, cAMP promotes human pluripotent stem cells point
Change the time shortened to mature neuron needed for differentiation acquisition mature neuron.
Specific technical solution is as follows：
A method of promotion human pluripotent stem cells directed differentiation is mature neuron, it includes the following steps：
(1) density that E8 culture solution culture human pluripotent stem cells are 60~80% to cell Proliferation is used；
(2) by after the human pluripotent stem cells enzymic digestion obtained in step (1), it is NIM culture solutions that the same day, which replaces culture solution,
It is the 0th day of cell differentiation at this time；
(3) fetal calf serum is added into the middle gained mixed system of step (2) in the 7th day of cell differentiation, and cell is adherent
Culture；NIM culture solutions are replaced after 4~12 hours；
(4) the NIM culture solutions in step (3) in gained system are replaced in the 7th~15 day of cell differentiation every 12~72h；
Break up the 16th day, the class nerve channel structure of formation is blown down, continues to be suspended with NIM culture solutions and cultivate；
(5) the 18th day of cell differentiation is replaced in the mixed system of gained with the neural maturation culture solution of rush in step (4)
NIM culture solutions；The 18th~23 day of cell differentiation is replaced every 12~72h and promotees neural maturation culture solution；
(6) gained cell adhere-wall culture, culture solution in step (5) are replaced with cell containing B27 by the 23rd day of cell differentiation
Cultivate the rush nerve maturation culture solution of additive；
(7) the 26th day of cell differentiation, the culture medium for replacing gained mixture in step (6) are NDM culture mediums, the 26th day
The formation of mature neuron can be observed afterwards.
In step (1), the E8 culture solutions are by FGF2, insulin, vitamin are added in DMEMF12 basal mediums
C, the nutritional ingredients such as selenium and transferrins are prepared.
In step (1), the method for proliferation is：By human pluripotent stem cells culture in the 6 orifice plates that Vitronectin is laid,
Vitronectin is a kind of matrigel of people source, can avoid animal derived materials interference.Used Vitronectin is
Life Technologies companies buy (article No.：A14700), usage is by being added in every milliliter of DMEM/F12
10ulVitronectin lays in 6 orifice plates, is placed at room temperature for 1-4h or 4 DEG C of refrigerator overnight.
In step (2), the enzyme is Dispase enzymes, a concentration of 0.5~5Uml-1；Wherein, the definition of enzyme activity is：It is most suitable
Under temperature condition, enzyme amount needed for conversion 1umol substrates is 1U in 1min.
In step (2), the digestion refers to 1~2 minute in 37 DEG C, the incubator of 5v/v%.
In step (2)~(5), the NIM culture solutions are by DMEM/F12, NEAA and N2 with 90~99：0.5~5：0.5
~5 volume ratio is formulated.
In step (5)~(6), the rush nerve culture solution in the NIM culture solutions described in step (2)~(5) by being added
Chain termination thimidine analogue AZT, BDNF and cAMP are prepared；Wherein, a concentration of 1~500 μm of ol/L of AZT, BDNF's is dense
Degree is 1~100ng/mL, a concentration of 0.5~10 μm of ol/L of cAMP.In step (6), the cell culture containing B27 additive
Rush nerve maturation culture solution in, B27 cell culture additive and the volume ratio for promoting neural maturation culture solution are 0.5~5：95~
99.5。
In step (7), the NDM culture mediums configure by the following method to be obtained：By Neurobasal medium, NEAA
With N2 with 97~99：0.5~2：After 0.5~2 volume ratio mixing, B27, BDNF and cAMP are added；Wherein, NDM culture mediums
In, the volume ratio of B27 and NDM culture mediums is 1：A concentration of 1~100ng ml of 10~100, BDNF-1, a concentration of the 0.5 of cAMP
~10 μm of ol/L.
Advantageous effect：
1, at the 4th week of differentiation, compared with default method, the neural bulb diameter obtained through this method differentiation significantly reduces about
27%；After adherent 2 days, the percentage of Ki67 positive cells participates in experiment than control group significant decrease about 56.5%, EdU and shows
After the cell of this method differentiation acquisition is adherent, compared with original differentiation method, EdU positive cell percentages have been remarkably decreased about
41.5%.Therefore, illustrate that this method effectively inhibits the proliferative capacity of neural stem cell.
2, technical scheme of the present invention is inducing the 4th week broken up, the neural precursor mark in the neuron of differentiation
Object PAX6 positive rates are reduced to 71.63 ± 4.2%, are better than conventional method 90.34 ± 1.7%；It is neural in the neuron of differentiation
Precursor marker SOX2 positive rates are reduced to 7.63 ± 1.2%, are better than the 41.34 ± 1.7% of default method；35th day,
Neural precursor marker SOX2 positive rates are reduced to 0 in the neuron of differentiation, better than default method 20.34 ±
1.8%.Therefore, illustrate that this method effectively inhibits the proliferative capacity of neural precursor.
3, technical scheme of the present invention is at the 5th week of induction differentiation, it can obtain 85.71 in the cell of differentiation ±
4.6% neuron (expression neuronal marker Tuj-1), better than acquiescence differentiation method 72.42 ± 2.5%；In the god of differentiation
Through there is 82.92 ± 1.7% maturing rate (expression maturation neuronal marker MAP2) in member, be better than conventional method 73.95 ±
2.2%.Therefore, this method has been effectively promoted the maturation of neuron.
4, for technical scheme of the present invention at the 5th week of induction differentiation, the cellular morphology complexity of differentiation was higher than conventional method,
The length of mature neuron longest protrusion is 1.42 times of conventional method group, and the protrusion number of terminals of each ripe nerve is default method
1.58 times of group, the level-one bump count of each ripe nerve are 1.52 times of conventional method group.Therefore, illustrate that this method is effective
Promote the maturation of neuron.
5, technical scheme of the present invention broke up marker after the neuronal synapse of acquisition at the 11st week of induction differentiation
VGlut expression density is 1.77 times of conventional method group, and presynaptic markers Synaptophysin expression density is conventional method
1.5 times of group, vGlut and Synaptophysin coexpression density are 1.97 times of conventional method groups.Therefore, illustrate this method
It has been effectively promoted the maturation of neuron.
6, method of inducing differentiation of the invention is that efficiently and directionally breaks up the human pluripotent stem cells without feeder layer to Glutamatergic
Neuron, existing technology culture people pluripotent stem cell Dependent Animals feeder layer or differentiation efficiency are relatively low.
7, easy to operate without heterologous factor in addition, this method is compared with existing technologies, repeatability is high.
Description of the drawings
Fig. 1 is the neural stem cell light microscopic figure below of differentiation to the 28th day suspension growth, visible apparent nerve ball in figure
Structure, scale=250 μm；Default is acquiescence differentiation group, and what NeuronM groups used is the promotion nerve of this patent introduction
First ripe inductive differentiation medium；
Fig. 2 is to breaking up to the diameter of the 28th day suspension growth neural stem cell the statistics knot after measuring and analyzing
Fruit is schemed；Compared with giving tacit consent to differentiation group, the neural bulb diameter obtained through this method differentiation reduces about 27%, and two groups have significant system
Meter learns difference；
Fig. 3 is to carrying out EdU (the 5- Brdurds of acetenyl -2 ' after differentiation to the 29th day nerve cell adhere-wall culture
Nucleosides) incorporation experiment, and carry out immunofluorescence dyeing；EdU is inserted into cell Proliferation in the DNA molecular replicated,
The cell of cell division S phases can be marked, the effective percentage for reflecting cell Proliferation；Fig. 3 be Hoechst (blue) and
The positive cell of EdU (red)；Scale=100 μm；
Fig. 4 carries out immunofluorescence dye to carrying out EdU incorporation experiments after differentiation to the 29th day nerve cell adhere-wall culture
Color, and to statistical results chart that EdU positive cells are counted；Compared with giving tacit consent to differentiation group, the god through this method differentiation acquisition
About 41.5% is had dropped through EdU positive cell percentages in cell；Two groups have significant significant difference；
Fig. 5 is to carry out Ki67 immunofluorescence dyeings to breaking up to the 29th day laggard row of nerve cell adhere-wall culture；Ki67
It is the marker for marking proliferative activity, Fig. 5 is the positive cell of Hoechst (blue) and Ki67 (red)；Scale=100
μm；
Fig. 6 to differentiation to carrying out Ki67 immunofluorescence dyeings after the 29th day nerve cell adhere-wall culture, and to Ki67 sun
The statistical results chart that property cell is counted；Compared with giving tacit consent to differentiation group, Ki67 in the nerve cell obtained through this method differentiation
Positive cell percentage has dropped about 56.5%；
Fig. 7 is to carry out PAX6 and Tuj-1 immunofluorescence dyeings to breaking up to the 30th day neuron；PAX6 is neural precursor
The marker of cell, Tuj-1 are the markers of neuron；Fig. 7 is Hoechst (blue) and PAX6 (red), Tuj-1 (green)
Positive cell；Scale=100 μm；
Fig. 8 is counted to breaking up to the 30th day neuron into PAX6 immunofluorescence dyeings, and to PAX6 positive cells
Statistical results chart；Compared with giving tacit consent to differentiation group, PAX6 positive cell percentages in the nerve cell obtained through this method differentiation
Have dropped about 18.71%；Two groups have significant significant difference；
Fig. 9 is to carry out SOX2 immunofluorescence dyeings to breaking up to the 30th day neuron；SOX2 is neural precursor
Marker；Fig. 9 is the positive cell of Hoechst (blue) and SOX2 (green)；Scale=100 μm；
Figure 10 is to breaking up the neuron to the 30th day and the 35th day into SOX2 immunofluorescence dyeings, and to SOX2 positive cells
The statistical results chart counted；Compared with giving tacit consent to differentiation group, SOX2 in the 30th day neuron obtained through this method differentiation
Positive cell percentage has dropped about 35%, and SOX2 positive cell percentages have dropped about 20% in the 35th day neuron, sun
Property cell number be 0；Two groups have significant significant difference；
Figure 11 is to carry out MAP2 and Tuj-1 immunofluorescence dyeings to breaking up to the 35th day neuron；Tuj-1 is neuron
Marker, MAP2 is the marker of mature neuron；Figure 11 is Hoechst (blue) and MAP2 (red), Tuj-1 (green)
Positive cell；Scale=100 μm；
Figure 12 be carry out MAP2 and Tuj-1 immunofluorescence dyeings to breaking up to the 35th day neuron, and to MAP2 and
The statistical results chart that Tuj-1 positive cells are counted；Compared with giving tacit consent to differentiation group, in the neuron obtained through this method differentiation
Tuj-1 positive cell percentages rise about 13.3%, MAP2 positive cell percentages and rise about 17.08%, the MAP2 positives
Ratio of the cell in Tuj-1 positive cells rises 8.97%；Two groups have significant significant difference；
Figure 13 is to carry out MAP2 immunofluorescence dyeings to breaking up to the 40th day neuron；Figure 11 is Hoechst (blue)
With the positive cell of MAP2 (reflecting the degree that MAP2 expression grows from weak to strong to white by red)；Scale=100 μm；
Figure 14 is the neuron progress MAP2 immunofluorescence dyeings to differentiation to the 40th day, and identical to MAP2 expression intensities
Cell carry out morphological analysis after statistical results chart；Compared with giving tacit consent to differentiation group, the maturation obtained through this method differentiation is neural
The length of first longest protrusion is its 1.42 times, and the protrusion number of terminals of each ripe nerve is its 1.58 times, each ripe nerve
Level-one bump count is its 1.52 times；
Figure 15 is to carry out vGlut and Tuj-1 immunofluorescence dyeings to breaking up to the 80th day neuron；Tuj-1 is nerve
The marker of member, vGlut is vesica glutamate transporter, is the mark that glutamate neuron maturation is just expressed to certain phase
Object；Figure 15 is the positive cell of Hoechst (blue) and Tuj-1 (green), and the expression presentation of vGlut (red) is dotted, such as white
Color arrow is signified；Scale=100 μm；
Figure 16 is to carry out vGlut and Synaptophysin immunofluorescence dyeings to breaking up to the 80th day neuron；
VGlut is vesica glutamate transporter, is the postsynaptic marker that glutamate neuron maturation is just expressed to certain phase；
Synaptophysin is Synaptophysin, is the presynaptic marker that neuronal maturation is just expressed to certain phase；Figure 16
Dotted, vGlut is presented in expression for Hoechst (blue) positive cell, vGlut (red) and Synaptophysin (green)
Mark (yellow) is total to Synaptophysin to show to form Synaptic junction, such as white arrow meaning；Scale=100 μm；
Figure 17 is the neuron progress vGlut and Synaptophysin immunofluorescence dyeings to differentiation to the 80th day, and right
The statistical results chart that vGlut and Synaptophysin positive punctate appearances are counted；Compared with giving tacit consent to differentiation group, through we
The neuron vGlut expression density that method differentiation obtains is its 1.77 times, and it is its 1.5 times that Synaptophysin, which expresses density,
VGlut and Synaptophysin coexpression density is its 1.97 times.
Specific implementation mode
Embodiment 1
A method of promoting human pluripotent stem cells Differentiating Into Neurons, includes the following steps：
(1) using the method culture human pluripotent stem cells of no feeder layer, until cell Proliferation is the density that can be passed on.
(2) the human pluripotent stem cells enzymic digestion that will be obtained in step (1), it is NIM that then the same day, which replaces culture solution, at this time
It is the 0th day of cell differentiation.
Human pluripotent stem cells can be observed within (3) the 1st days and form embryoid body cell ball (Embryonic bodies, EBs).
(4) the 7th days adherent by EB.
The class nerve channel structure that cell forms rose style (Rossete) can be observed from (5) the 10th days.
The class nerve channel structure of formation is blown down at (6) the 16th days, next day can be observed neural precursor and be formed.
It is the NeuronM containing AZT, BDNF and cAMP to replace within (7) the 18th days culture solution.Wherein, a concentration of the 1~500 of AZT
μm ol/L, a concentration of 0.5~10 μm of ol/L of a concentration of 1~100ng/mL of BDNF, cAMP.
(8) the 23rd days adherent by neural precursor, and used medium is still NeuronM.
NDM is replaced medium within (9) the 26th days, the inside includes B27, cAMP and BDNF, gradual visible neuronal cell shape
State occurs, and has a large amount of mature neurons to be formed after the 26th, and can be identified in different time points.Wherein, B27 uses volume ratio
It is 1:10~1:A concentration of 0.5~10 μm of ol/L of 100, BDNF a concentration of 1~100ng/mL, cAMP..
In the present invention, the culture dish used in human pluripotent stem cells needs small with Vitronectin matrigel room temperatures coating 2
When, culture solution used is E8, partly changes liquid daily.
In the present invention, human pluripotent stem cells digestion the specific steps are：E8 culture solutions are siphoned away first, are added
Dispase(1Uml-1), volume is the half of E8 culture solutions.Cell is then positioned over 37 DEG C, 5%CO2Incubator in be incubated
2 minutes, can be observed under the microscope clone edge it is shinny, then Dispase is siphoned away, be added DMEM/F12 wash once, then
New DMEM/F12 is added to blow down clone, is placed in centrifuge tube and centrifuges, 800rpm, 5 minutes.Upper layer DMEM/F12 is siphoned away,
Cell induction differentiation liquid is added, cell is transferred to suspend in culture bottle and is cultivated, this is differentiation the 0th day.
In the present invention, cell will form EBs in the 1st of differentiation the day, and will be constantly proliferated in 5 days thereafter, form
It is larger and rounded.At 1-4 days of differentiation, liquid is partly changed daily, after breaking up the 4th day, partly changes liquid every other day, culture solution is NIM.
In the present invention, when breaking up the 7th day, 10% FBS is added in NIM, by cell adhere-wall culture in culture dish,
Completely new NIM is replaced after 10 hours.
In the present invention, from breaking up the 10th day, the cell that adhere-wall culture can be observed forms rose style (Rossete)
Class nerve channel structure indicates that human pluripotent stem cells are divided into neuro-epithelial cell at this time.The 7-15 days of differentiation, are partly changed every other day
Liquid, culture solution are NIM.
In the present invention, the cell of adhere-wall culture is blown down when breaking up the 16th day, the specific steps are：Directly use the rifle of 1ml
Head is blown and beaten against cell, and the cell blown down is transferred in centrifuge tube, is stood 5 minutes, is made its natural subsidence to tube bottom.Then will
Culture solution siphons away, and completely new NIM is added, cell is transferred in culture bottle, adds the B27 that volume ratio is 2%, reduces piping and druming
When to the mechanical damage of cell.
In the present invention, when breaking up the 17th day, neural precursor will be obtained.When breaking up the 18th day, it is added containing special
The NeuronM culture solutions of component partly changed liquid every other day to differentiation the 23rd day.
In the present invention, when breaking up the 23rd day, by the adherent differentiation of neural precursor, the specific steps are：It first will be adherent
Slide used is coated with Matrigel, is placed 2 hours at room temperature.Then neural precursor is sucked out, is placed in centrifuge tube
In, TrypLE is added and places incubator digestion 2-3 minutes.TrypLE is siphoned away later, DMEM/F12 is added and washes once, adds
New DMEM/F12 blows and beats cell with the pipette tips of 200ul, every time after piping and druming 5 time suction top layer containing individual cells
DMFM/12 is placed in another centrifuge tube, reduces the piping and druming repeatedly to cell.Then 10ul is sucked out to be positioned in cell counting board
It is counted, is finally diluted to the density kind of 2x10^4/ml on slide with culture solution, each slide 100ul.After 2 hours
Whether adherent observe neural precursor, if adherent, fluid infusion to 500ul.Culture solution used in the process of this is still
NeuronM, and the B27 that volume ratio is 2% is added wherein, liquid is partly changed every other day.Culture solution is replaced within 26th day to be NDM and add
Enter B27 (1:50), BDNF (10ng ml-1) and cAMP (1 μM), hereafter changes liquid at an interval of three and half days.
In the present invention, immunofluorescence technique can be used in the neuron identification of different time points.The specific steps are：It will need to reflect
One piece of the slide of fixed cell and culture is chosen on the culture dish for being placed in and doing used in immunofluorescence.Polyformaldehyde is added
(PFA) fixed half an hour；Then Phosphate Buffered Saline (PBS) are changed to wash 3 times, every time 5 minutes；It is added
0.2% Triton, 10 minutes；10% Donkey-Serum closings 1 hour are added；Primary antibody is added, with 0.1% Triton
Donkey-Serum with 5% is prepared, and is positioned over mistake in 4 DEG C of refrigerators；Primary antibody is siphoned away every other day, is washed 3 times, every time 10 points with PBS
Clock；Secondary antibody is added, is prepared with 5% Donkey-Serum, is incubated half an hour；Then secondary antibody is siphoned away, washes 3 times again with PBS, often
Secondary 10 minutes；It is finally closed on glass slide with mountant, uses fluorescence microscope.
The results show, the neuron percentage that the method for the present invention obtains increase, and mature neuron percentage increases, god
More abundant through first protrusion quantity, projection length dramatically increases, and synapsin expression quantity dramatically increases.
Claims (7)
1. a kind of method promoting human pluripotent stem cells directed differentiation as mature neuron, which is characterized in that it includes following step
Suddenly：
(1) density that E8 culture solution culture human pluripotent stem cells are 60~80% to cell Proliferation is used；
(2) by after the human pluripotent stem cells enzymic digestion obtained in step (1), it is NIM culture solutions that the same day, which replaces culture solution, at this time
It is the 0th day of cell differentiation；
(3) the 7th day of cell differentiation, is added fetal calf serum into gained mixed system in step (2), and by the adherent training of cell
It supports；NIM culture solutions are replaced after 4~12 hours；
(4) the NIM culture solutions in step (3) in gained system are replaced in the 7th~15 day of cell differentiation every 12~72h；Differentiation
16th day, the class nerve channel structure of formation is blown down, continues to be suspended with NIM culture solutions and cultivate；
(5) NIM is replaced in the 18th day of cell differentiation in the mixed system of gained in step (4) with the neural maturation culture solution of rush
Culture solution；The 18th~23 day of cell differentiation is replaced every 12~72h and promotees neural maturation culture solution；
(6) gained cell adhere-wall culture, culture solution in step (5) are replaced with cell culture containing B27 by the 23rd day of cell differentiation
The rush nerve maturation culture solution of additive；
(7) the 26th day of cell differentiation, the culture medium for replacing gained mixture in step (6) are NDM culture mediums, can after the 26th day
Observe the formation of mature neuron.
2. according to the method described in claim 1, it is characterized in that, in step (2), the enzyme is Dispase enzymes, a concentration of
0.5~5Uml-1。
3. according to the method described in claim 1, it is characterized in that, in step (2), the digestion refers to the 5v/ at 37 DEG C
1~2 minute in the incubator of v%.
4. according to the method described in claim 1, it is characterized in that, in step (2)~(5), the NIM culture solutions by
DMEM/F12, NEAA and N2 are with 90~99：0.5~5：0.5~5 volume ratio is formulated.
5. according to the method described in claim 1, it is characterized in that, in step (5)~(6), the rush nerve culture solution by
Chain termination thimidine analogue AZT, BDNF and cAMP are added in NIM culture solutions described in step (2)~(5) to be prepared；Wherein,
A concentration of 1~500 μm of ol/L of AZT, a concentration of 0.5~10 μm of ol/L of a concentration of 1~100ng/mL of BDNF, cAMP.
6. according to the method described in claim 1, it is characterized in that, in step (6), the cell culture containing B27 additive
Rush nerve maturation culture solution in, B27 cell culture additive and the volume ratio for promoting neural maturation culture solution are 0.5~5：95~
99.5。
7. according to the method described in claim 1, it is characterized in that, in step (7), the NDM culture mediums are by the following method
Configuration obtains：By Neurobasal medium, NEAA and N2 with 97~99：0.5~2：After 0.5~2 volume ratio mixing, then
B27, BDNF and cAMP is added；Wherein, in NDM culture mediums, the volume ratio of B27 and NDM culture mediums is 1：10~100, BDNF's
A concentration of 1~100ng ml-1, a concentration of 0.5~10 μm of ol/L of cAMP.
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