CN104962519A - Preparation method and culture medium of human-derived astrocyte precursor cells - Google Patents
Preparation method and culture medium of human-derived astrocyte precursor cells Download PDFInfo
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Abstract
The invention discloses a preparation method and culture medium of human-derived astrocyte precursor cells. The method can be utilized to obtain abundant astrocyte precursor cells in vitro. Compared with the traditional method of directly differentiating astrocyte cells from nerve stem cells, the precursor cells obtained by the method have high multiplication capacity and capacity for directional differentiation into astrocyte cells, and the method can quickly obtain abundant high-purity astrocyte cells in a short time. The culture medium for culturing astrocyte precursor cells has the characteristics of definite components and accessible commercial products. The method for generating abundant astrocyte cells for scientific research or clinic use has the characteristics of low cost, high safety, high production efficiency and high operability.
Description
Technical field
The invention belongs to biomedical sector, relate to a kind of method and the substratum applying induced multi-potent stem cells technology and prepare people source astroglia precursor cell.Be specifically related to induce people source induced multi-potent stem cells (induced pluripotent stemcell, iPSC) to be divided into the method for brain astroglia precursor cell (Astrocyte progenitor cell).
Background technology
Astroglia cell (Astrocyte) is the class cell that in mammal brain, distribution is maximum, and this type of spongiocyte is star, and send the projection of much length from cell space, projection contacts with neurocyte, works the function supported and separate neurocyte.The differentiation of application induced multi-potent stem cells obtains the progress that the technology of astroglia cell not only advances Neuroscience Research, and is applied to clinically provides primary condition for obtaining astroglia cell source in a large number.Astroglia cell is by people source induced multi-potent stem cells the earliest, through neural precursor differentiation step and obtain (Fig. 1).
The maintain of people source astroglia precursor cell, needs to apply specific substratum to maintain its precursor cell attribute.At present about the report of people source astroglia precursor cell is few, the relevant report of astroglia precursor cell of major part is all derive from rat or mouse, and scientist can only by being separated human brain tissue, by original cuiture with identify acquisition, tissue-derived limited, obtain cell number few.The relevant people source astroglia precursor cell mentioned in the article that can find obtains like this: purchaser's neural precursor system (this clone is commodity), 1% foetal calf serum is mixed through cultivating (substratum behaviour neural precursor substratum ScienCell Research Laboratories), after 20 days, the cell of the CD44 positive can be obtained, CD44 is the mark of astroglia precursor cell, therefore this cell is astroglia precursor cell.Utilize flow cytometer, these CD44 positive cells can be reclaimed, purifying and cultivation (people's neural precursor substratum ScienCell Research Laboratories mixes 1% foetal calf serum).The differentiation product of these precursor cells is astroglia cells, for neurone provides nutrition and support, if astroglia cell function is impaired, major disease also can be caused as alzheimer's disease.
Summary of the invention
The present invention is intended to overcome the deficiencies in the prior art, provides preparation method and the substratum of a kind of people source astroglia precursor cell.
In order to achieve the above object, technical scheme provided by the invention is:
The preparation method of described people source astroglia precursor cell comprises the steps:
(1) induced multi-potent stem cells is inoculated in the special culture plate of cell, adopt mTeSR stem cell media, under 37 DEG C of conditions, induced multi-potent stem cells cultivated, every 6-8 days, preferably within 7 days, go down to posterity once, change fresh mTeSR stem cell media every day;
(2) after induced multi-potent stem cells goes down to posterity the 5th day, with aseptic PBS damping fluid rinse cell surface, then add a small amount of (being equivalent to the fresh mTeSR stem cell media half volume changed) embryoid body division culture medium (this embryoid body division culture medium is prior art); Induced multi-potent stem cells cell mass edge is gently scraped with steril cell sleaker, induced multi-potent stem cells cell mass is made to depart from culture plate and be in suspended state, then piping and druming makes induced multi-potent stem cells cell mass disperse repeatedly, induced multi-potent stem cells cell mass after dispersion is transferred to Tissue Culture Dish, under 37 DEG C of conditions, shake cultivation more than 5 days after adding embryoid body division culture medium, within every 2 days, change once fresh embryoid body division culture medium;
(3) induced multi-potent stem cells cell mass suspended concussion cultivation after 5 days, formed the embryoid body cell mass that size is homogeneous, construct similar embryoid body ball-like structure; Embryoid body division culture medium is replaced by the mixed culture medium A be mixed into by N2 substratum, B27 substratum and DMEM/F12 serum free medium, be added with somatomedin bFGF and somatomedin EGF in this mixed culture medium A, somatomedin bFGF and somatomedin EGF in mixed culture medium A concentration be 10 μ g/L; Embryoid body cell mass is moved to together with mixed culture medium A by the culture dish of matrigel Matrigel bag quilt, jog culture dish makes embryoid body cell mass on average be scattered in culture dish bottom surface, quiescent culture 5-7 days under 37 DEG C of conditions, preferably 6 days, changes once fresh mixed culture medium A in every two days; Embryoid body cell mass central authorities directed differentiation is the neural precursor group of rosettes structure; 1L step (3) described mixed culture medium A is mixed into (mixed culture medium not adding aftermentioned somatomedin is commercially available) by 10mL N2 substratum, 20mL B27 substratum and 970mL DMEM/F12 serum free medium, be added with somatomedin bFGF and somatomedin EGF in this mixed culture medium A, somatomedin bFGF and somatomedin EGF in mixed culture medium A concentration be 10 μ g/L.
(4) choose neural precursor by sterile needle roll into a ball and be placed in sterile centrifugation tube, add the DMEM/F12 serum free medium being equivalent to centrifuge tube 2/3 volume, centrifugal under 1000rpm condition, collecting cell, then change fresh in N2 substratum, the mixed culture medium B that B27 substratum and DMEM/F12 serum free medium are mixed into, somatomedin bFGF is added with in this mixed culture medium B, the substratum of the KSR of growth factor B MP-4 and alternative serum, the concentration of somatomedin bFGF in mixed culture medium B is 10 μ g/L, the concentration of growth factor B MP-4 in mixed culture medium B is 20 μ g/L, the addition of the substratum of the KSR of alternative serum is 10% of mixed culture medium B cumulative volume, the cell precipitation that piping and druming is collected, cell precipitation is made to be resuspended in fresh culture, form enchylema, enchylema is laid in the Tissue Culture Dish of matrigel bag quilt, under 37 DEG C of conditions, quiescent culture is after 3 days, and the cell volume in enchylema expands, and starts to be divided into astroglia precursor cell, the cell of propagation starts to express low-level GFAP marker protein, obtains people source astroglia precursor cell.The described mixed culture medium B of 1L step (4) is by 10mL N2 substratum, 20mL B27 substratum and 970mL DMEM/F12 serum free medium are mixed into (mixed culture medium not adding aftermentioned somatomedin is commercially available), somatomedin bFGF is added with in this mixed culture medium B, the substratum of the KSR of growth factor B MP-4 and alternative serum, the concentration of somatomedin bFGF in mixed culture medium B is 10 μ g/L, the concentration of growth factor B MP-4 in mixed culture medium B is 20 μ g/L, the addition of the substratum of the KSR of alternative serum is 10% of mixed culture medium B cumulative volume.
Preferably, containing DMEM/F12 serum free medium 990mL in the described embryoid body division culture medium of 1L step (2), N2 substratum 10mL, also containing A-8301 and Dorsomorphin, A-8301 and the Dorsomorphin concentration in embryoid body division culture medium is 1 μm of ol/L.
Preferably, described method also comprises the steps: to add leukaemia inhibitory factor in mixed culture medium B, the concentration of leukaemia inhibitory factor in mixed culture medium B is 20 μ g/L, and people source astroglia precursor cell step (4) obtained is placed in the directed differentiation that this mixed culture medium B being added with leukaemia inhibitory factor carries out astroglia cell.
Optimization of the present invention also filters out the method (Fig. 2) applied induced multi-potent stem cells and prepare astroglia precursor cell.Compared with the astroglia cell of differentiated, this precursor cell has propagation and directed differentiation ability, provides highly purified astroglia cell to provide source in a large number.This technology is for the pathogeny of encephalopathy, the regeneration of brain and repair mechanism and then be applied to clinical treatment and have very important application prospect.
The present invention will be further described below:
Induced multi-potent stem cells is inoculated in the special culture plate of 6 porocyte by the present invention, keeps induced multi-potent stem cells to go down to posterity with the state of cell mass, cell mass size homogeneous (Fig. 3).Need to keep highdensity cell mass to cultivate in culturing process, generally went down to posterity once every 7 days.Go down to posterity and use sterile needle to choose the cell dedifferented.Be commercially available special mTeSR stem cell media (Stemcell Technologies, Vancouver, Canada) for cultivating the substratum of induced multi-potent stem cells in this stage.Change 2 milliliters of fresh mediums every day.After stem cell is gone down to posterity the 5th day, with aseptic PBS damping fluid (pH7.4) rinse cell surface, then embryoid body (the Embryonic Body of 1 milliliter is changed, EB) division culture medium, this substratum contains DMEM/F12 serum free medium (Thermo Fisher Scientific), N2 substratum (Thermo Fisher Scientific, Inc), A-8301 (the Stemgent of 1 μm of ol/L, San Diego, Dorsomorphin (the Chemdea of CA) and 1 μm of ol/L, Ridgewood, NJ, USA), with steril cell sleaker light cell scraping group edge, cell mass is progressively made to depart from culture dish, it is made to be in suspended state, then piping and druming makes cell mass disperse 5 times repeatedly.All cells group is transferred to 6 orifice plate Tissue Culture Dishs of low adhesion, and adds embryoid division culture medium 2 milliliters.In 37 DEG C of incubators, shake cultivation more than five days, within every two days, change a fresh medium.
Suspend concussion cultivation can be formed after five days size homogeneous, construct similar embryoid body ball-like structure (Fig. 4).Remove original nutrient solution gently, be replaced by the mixed culture medium (Thermo Fisher Scientific) be mixed into by N2 substratum, B27 substratum and DMEM/F12 serum free medium, add Prostatropin bFGF (R & D Systems, Minneapolis, MN), recombinant human epidermal growth factor EGF (R & D Systems, Minneapolis, MN).Embryoid body cell mass and fresh medium together being moved to wraps in the culture dish of quilt by matrigel Matrigel (BDBioscience), jiggling culture dish makes embryoid on average be scattered in culture dish bottom surface, quiescent culture about 6 days in 37 DEG C of incubators, changes a fresh cell medium in every two days.Embryoid body cell mass central authorities can directed differentiation be rosettes structure (Neuralrosettes) (Fig. 5).The qualification of application immunofluorescence dyeing shows mark PAX6 and Nestin (Fig. 6) of these cell expressing neural precursors.
Choose neural precursor by sterile needle to roll into a ball and the sterile centrifugation tube being placed in 15 milliliters, add 10 milliliters of serum-free DMED/F12 substratum centrifugal 5 minutes, the centrifugation collecting cell of 1000 rpms, change fresh in N2 substratum, mixed culture medium (Thermo Fisher Scientific) that B27 substratum and DMEM/F12 serum free medium are mixed into (N2 substratum commercially available be concentrated solution, 100x (during use 100 times of dilutions), B27 substratum is commercially available concentrated solution, 50X (during use 50 times of dilutions)), bFGF and BMP-4 two kinds of somatomedins are added in substratum, add the substratum (Thermo Fisher Scientific) of the KnockoutSerum Replacer (KSR) of alternative serum.And then firmly blow and beat cell precipitation, make it to be resuspended in fresh medium, enchylema is laid in the Tissue Culture Dish of matrigel bag quilt, quiescent culture is after 3 days, cell volume expands, start to be divided into astroglia precursor cell, the cell of propagation starts to express low-level GFAP marker protein, obtains at high proportion the astroglia precursor cell of (>95%) thus.Through identifying that this cell has very high BrdU and mixes activity, show the ability (Fig. 6) of its high proliferation.
The astroglia precursor cell obtained has self duplication ability, and proliferating cycle is 24 hours, can carry out going down to posterity with freezen protective in liquid nitrogen.For the astroglia precursor cell obtained, leukaemia inhibitory factor (the Leukemia inhibition factor of 20 μ g/L can be added in above-mentioned nutrient solution, LIF) directed differentiation of astroglia cell is carried out, the astroglia cell (Fig. 7) of a large amount of high purity (>95%) can be obtained after 48 hours, be directly used in scientific research and clinical.
The present invention illustrates the key ingredient being applied to and cultivating astroglia precursor cell defined medium, avoids the unknown factor of trigger cell differentiation in general substratum goods.This medium component is stablized, and compound method is simple.Use astroglia precursor cell energy long term maintenance propagation and the differentiation capability of this culture medium culturing, repeatability is high, effectively ensure that the stability between different batches product, be conducive to the standardization of products to have higher commercial application value.
By method of the present invention, a large amount of astroglia precursor cells can be obtained in vitro, with directly broken up the traditional method of astroglia cell by neural stem cell compared with (table 1), the precursor cell that the present invention obtains has strong multiplication capacity and directed differentiation is the ability of astroglia cell, can obtain rapidly a large amount of highly purified astroglia cell in a short time; Substratum for cultivating star precursor cell has composition and determines, the available feature of commodity; For scientific research or clinical Astrocytic means, there is with low cost, that security is high, production efficiency is high, workable feature for producing in a large number.
Accompanying drawing explanation
Fig. 1 is the traditional method schematic diagram that astroglia cell obtains; During by neural stem cell to Astrocyte differentiation, the pollution of neuronal cell and oligodendrocyte can be produced;
Fig. 2 is the inventive method schematic diagram; Astroglia precursor cell has multiplication capacity, and is specifically divided into a large amount of astroglia cell;
Fig. 3 behaves source induced multi-potent stem cells figure;
Fig. 4 is that embryoid body cell is cliqued graph;
The neural precursor of Fig. 5 rosettes structure is cliqued graph;
Fig. 6 is neural precursor figure; Through immunofluorescence dyeing qualification, most cells expresses mark Nestin and Pax6 of neural precursor;
Fig. 7 is astroglia precursor cell; The BrdU positive shows that this cell is the cell of propagation;
Fig. 8 is astroglia cell; Through immunofluorescence dyeing qualification, astroglia precursor cell is after the differentiation of leukaemia inhibitory factor induced orientation, and most cells expresses the mark GFAP of astroglia cell.
Embodiment
The preparation method of described people source astroglia precursor cell comprises the steps:
(1) induced multi-potent stem cells is inoculated in the special culture plate of 6 porocyte, adopt mTeSR stem cell media, under 37 DEG C of conditions, induced multi-potent stem cells cultivated, went down to posterity once every 7 days, change the mTeSR stem cell media that 2mL is fresh every day;
(2) after induced multi-potent stem cells goes down to posterity the 5th day, with aseptic PBS damping fluid rinse cell surface, then 1ml embryoid body division culture medium is added, induced multi-potent stem cells cell mass edge is gently scraped with steril cell sleaker, induced multi-potent stem cells cell mass is made to depart from culture plate and be in suspended state, then piping and druming makes induced multi-potent stem cells cell mass disperse repeatedly, induced multi-potent stem cells cell mass after dispersion is transferred to 6 orifice plate Tissue Culture Dishs, under 37 DEG C of conditions, cultivation 5 days more than is shaken after adding embryoid body division culture medium, within every 2 days, change once fresh embryoid body division culture medium, containing DMEM/F12 serum free medium 990mL in the described embryoid body division culture medium of 1L step (2), N2 substratum 10mL, also containing A-8301 and Dorsomorphin, A-8301 and the Dorsomorphin concentration in embryoid body division culture medium is 1 μm of ol/L,
(3) induced multi-potent stem cells cell mass suspended concussion cultivation after 5 days, formed the embryoid body cell mass that size is homogeneous, construct similar embryoid body ball-like structure; Embryoid body division culture medium is replaced by the mixed culture medium A be mixed into by N2 substratum, B27 substratum and DMEM/F12 serum free medium, be added with somatomedin bFGF and somatomedin EGF in this mixed culture medium A, somatomedin bFGF and somatomedin EGF in mixed culture medium A concentration be 10 μ g/L; Embryoid body cell mass is moved to together with mixed culture medium A by the culture dish of matrigel Matrigel bag quilt, jog culture dish makes embryoid body cell mass on average be scattered in culture dish bottom surface, quiescent culture 5-7 days under 37 DEG C of conditions, changes once fresh mixed culture medium A in every two days; Embryoid body cell mass central authorities directed differentiation is the neural precursor group of rosettes structure; The described mixed culture medium A of 1L step (3) is mixed into by 10mL N2 substratum, 20mL B27 substratum and 970mLDMEM/F12 serum free medium, be added with somatomedin bFGF and somatomedin EGF in this mixed culture medium A, somatomedin bFGF and somatomedin EGF in mixed culture medium A concentration be 10 μ g/L;
(4) choose neural precursor by sterile needle roll into a ball and be placed in 15mL sterile centrifugation tube, add the DMEM/F12 serum free medium of 10mL, centrifugal 5min under 1000rpm condition, collecting cell, then change fresh in N2 substratum, the mixed culture medium B that B27 substratum and DMEM/F12 serum free medium are mixed into, somatomedin bFGF is added with in this mixed culture medium B, the substratum of the KSR of growth factor B MP-4 and alternative serum, the concentration of somatomedin bFGF in mixed culture medium B is 10 μ g/L, the concentration of growth factor B MP-4 in mixed culture medium B is 20 μ g/L, the addition of the substratum of the KSR of alternative serum is 10% of mixed culture medium B cumulative volume, the cell precipitation that piping and druming is collected, cell precipitation is made to be resuspended in fresh culture, form enchylema, enchylema is laid in the Tissue Culture Dish of matrigel bag quilt, under 37 DEG C of conditions, quiescent culture is after 3 days, and the cell volume in enchylema expands, and starts to be divided into astroglia precursor cell, the cell of propagation starts to express low-level GFAP marker protein, obtains people source astroglia precursor cell, the described mixed culture medium B of 1L step (4) is mixed into by 10mL N2 substratum, 20mL B27 substratum and 970mLDMEM/F12 serum free medium, the substratum of the KSR of somatomedin bFGF, growth factor B MP-4 and alternative serum is added with in this mixed culture medium B, the concentration of somatomedin bFGF in mixed culture medium B is 10 μ g/L, the concentration of growth factor B MP-4 in mixed culture medium B is 20 μ g/L, and the addition of the substratum of the KSR of alternative serum is 10% of mixed culture medium B cumulative volume,
(5) in mixed culture medium B, leukaemia inhibitory factor is added, the concentration of leukaemia inhibitory factor in mixed culture medium B is 20 μ g/L, and people source astroglia precursor cell step (4) obtained is placed in the directed differentiation that this mixed culture medium B being added with leukaemia inhibitory factor carries out astroglia cell.
By method of the present invention, a large amount of astroglia precursor cells can be obtained in vitro, with directly broken up the traditional method of astroglia cell by neural stem cell compared with (table 1), this precursor cell has strong multiplication capacity and directed differentiation is the ability of astroglia cell, can obtain rapidly a large amount of highly purified astroglia cell in a short time; Substratum for cultivating star precursor cell has composition and determines, the available feature of commodity; For scientific research or clinical Astrocytic means, there is with low cost, that security is high, production efficiency is high, workable feature for producing in a large number.
The comparison of table 1 the inventive method and traditional method
Claims (9)
1. a preparation method for people source astroglia precursor cell, is characterized in that, described method comprises the steps:
(1) induced multi-potent stem cells is inoculated in the special culture plate of cell, adopts mTeSR stem cell media, under 37 DEG C of conditions, induced multi-potent stem cells cultivated, went down to posterity every 6-8 days once, change fresh mTeSR stem cell media every day;
(2), after induced multi-potent stem cells goes down to posterity the 5th day, with aseptic PBS damping fluid rinse cell surface, embryoid body division culture medium is then added; Induced multi-potent stem cells cell mass edge is gently scraped with steril cell sleaker, induced multi-potent stem cells cell mass is made to depart from culture plate and be in suspended state, then piping and druming makes induced multi-potent stem cells cell mass disperse repeatedly, induced multi-potent stem cells cell mass after dispersion is transferred to Tissue Culture Dish, under 37 DEG C of conditions, shake cultivation more than 5 days after adding embryoid body division culture medium, within every 2 days, change once fresh embryoid body division culture medium;
(3) induced multi-potent stem cells cell mass suspended concussion cultivation after 5 days, formed the embryoid body cell mass that size is homogeneous, construct similar embryoid body ball-like structure; Embryoid body division culture medium is replaced by the mixed culture medium A be mixed into by N2 substratum, B27 substratum and DMEM/F12 serum free medium, be added with somatomedin bFGF and somatomedin EGF in this mixed culture medium A, somatomedin bFGF and somatomedin EGF in mixed culture medium A concentration be 10 μ g/L; Embryoid body cell mass is moved to together with mixed culture medium A by the culture dish of matrigel Matrigel bag quilt, jog culture dish makes embryoid body cell mass on average be scattered in culture dish bottom surface, quiescent culture 5-7 days under 37 DEG C of conditions, changes once fresh mixed culture medium A in every two days; Embryoid body cell mass central authorities directed differentiation is the neural precursor group of rosettes structure;
(4) choose neural precursor by sterile needle roll into a ball and be placed in sterile centrifugation tube, add the DMEM/F12 serum free medium being equivalent to centrifuge tube 2/3 volume, centrifugal under 1000rpm condition, collecting cell, then change fresh in N2 substratum, the mixed culture medium B that B27 substratum and DMEM/F12 serum free medium are mixed into, somatomedin bFGF is added with in this mixed culture medium B, the substratum of the KSR of growth factor B MP-4 and alternative serum, the concentration of somatomedin bFGF in mixed culture medium B is 10 μ g/L, the concentration of growth factor B MP-4 in mixed culture medium B is 20 μ g/L, the addition of the substratum of the KSR of alternative serum is 10% of mixed culture medium B cumulative volume, the cell precipitation that piping and druming is collected, cell precipitation is made to be resuspended in fresh culture, form enchylema, enchylema is laid in the Tissue Culture Dish of matrigel bag quilt, under 37 DEG C of conditions, quiescent culture is after 3 days, and the cell volume in enchylema expands, and starts to be divided into astroglia precursor cell, the cell of propagation starts to express low-level GFAP marker protein, obtains people source astroglia precursor cell.
2. the method for claim 1, it is characterized in that, step (1) is inoculated in by induced multi-potent stem cells in the special culture plate of 6 porocyte, adopt mTeSR stem cell media, under 37 DEG C of conditions, induced multi-potent stem cells cultivated, went down to posterity once every 7 days, change the mTeSR stem cell media that 2mL is fresh every day.
3. the method for claim 1, it is characterized in that, step (2) is after induced multi-potent stem cells goes down to posterity the 5th day, with aseptic PBS damping fluid rinse cell surface, then embryoid body division culture medium is added, induced multi-potent stem cells cell mass edge is gently scraped with steril cell sleaker, induced multi-potent stem cells cell mass is made to depart from culture plate and be in suspended state, then piping and druming makes induced multi-potent stem cells cell mass disperse repeatedly, induced multi-potent stem cells cell mass after dispersion is transferred to 6 orifice plate Tissue Culture Dishs, under 37 DEG C of conditions, cultivation 5 days more than is shaken after adding embryoid body division culture medium, within every 2 days, change once fresh embryoid body division culture medium.
4. the method for claim 1, it is characterized in that, containing DMEM/F12 serum free medium 990mL in the described embryoid body division culture medium of 1L step (2), N2 substratum 10mL, also containing A-8301 and Dorsomorphin, A-8301 and the Dorsomorphin concentration in embryoid body division culture medium is 1 μm of ol/L.
5. the method for claim 1, it is characterized in that, the described mixed culture medium A of 1L step (3) is mixed into by 10mL N2 substratum, 20mL B27 substratum and 970mL DMEM/F12 serum free medium, be added with somatomedin bFGF and somatomedin EGF in this mixed culture medium A, somatomedin bFGF and somatomedin EGF in mixed culture medium A concentration be 10 μ g/L.
6. the method for claim 1, it is characterized in that, the described mixed culture medium B of 1L step (4) is mixed into by 10mL N2 substratum, 20mL B27 substratum and 970mL DMEM/F12 serum free medium, the substratum of the KSR of somatomedin bFGF, growth factor B MP-4 and alternative serum is added with in this mixed culture medium B, the concentration of somatomedin bFGF in mixed culture medium B is 10 μ g/L, the concentration of growth factor B MP-4 in mixed culture medium B is 20 μ g/L, and the addition of the substratum of the KSR of alternative serum is 10% of mixed culture medium B cumulative volume.
7. the method for claim 1, it is characterized in that, step (4) chooses neural precursor by sterile needle roll into a ball and be placed in 15mL sterile centrifugation tube, adds the DMEM/F12 serum free medium of 10mL, centrifugal 5min under 1000rpm condition, collecting cell.
8. the method for claim 1, it is characterized in that, described method also comprises the steps: to add leukaemia inhibitory factor in mixed culture medium B, the concentration of leukaemia inhibitory factor in mixed culture medium B is 20 μ g/L, and people source astroglia precursor cell step (4) obtained is placed in the directed differentiation that this mixed culture medium B being added with leukaemia inhibitory factor carries out astroglia cell.
9. the substratum for the preparation of people source astroglia precursor cell, it is characterized in that, described substratum is the DMEM/F12 serum-free mixed culture medium being mixed with N2 and B27, the substratum of the KSR of somatomedin bFGF, growth factor B MP-4 and alternative serum is added with in this substratum, somatomedin bFGF concentration is 10 μ g/L, growth factor B MP-4 concentration is 20 μ g/L, and the addition of the substratum of KSR is 10% of substratum cumulative volume.
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