CN104962519B - A kind of preparation method and culture medium of people source astroglia precursor - Google Patents
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Abstract
The invention discloses a kind of preparation methods and culture medium of people source astroglia precursor.Method through the invention, a large amount of astroglia precursor can be obtained in vitro, compared with the conventional method for directly breaking up astroglia by neural stem cell, the ability that the precursor that the present invention obtains has strong proliferative capacity and directed differentiation is astroglia, short-term interior energy obtain rapidly the astroglia of a large amount of high-purities；For cultivating the culture medium of star precursor with ingredient determination, the available feature of commodity；Means for largely generating the astrocyte for scientific research or clinic have the characteristics that of low cost, safe, production efficiency is high, operability is strong.
Description
Technical field
The invention belongs to biomedical sectors, are related to a kind of application induced multi-potent stem cell technology preparation people source astroglia
The method and culture medium of precursor.More particularly to induction people source induced multi-potent stem cell (induced pluripotent
Stem cell, iPSC) method that is divided into brain astroglia precursor (Astrocyte progenitor cell).
Background technology
Astroglia (Astrocyte) is the most a kind of cell of mammal intracerebral distribution, such spongiocyte
In star, the protrusion of many length is sent out from cell space, protrusion is in contact with nerve cell, acts the work(supported and separate nerve cell
Energy.The technology for breaking up acquisition astroglia using induced multi-potent stem cell not only promotes the progress of Neuroscience Research, and
And primary condition is provided applied to clinic for a large amount of astroglia sources that obtain.Astroglia is lured by people source earliest
Multipotential stem cell is led, is obtained by the differentiation step of neural precursor (Fig. 1).
The maintenance culture of people source astroglia precursor is needed using specific culture medium to maintain its precursor category
Property.The current report in relation to people source astroglia precursor is fewer and fewer, most of report in relation to astroglia precursor
Road is all derived from rat or mouse, and scientist can only be obtained, group by detaching human brain tissue by original cuiture and identification
Limited source is knitted, it is few to obtain cell number.The related people source astroglia precursor mentioned in the article that can be found is this
What sample obtained：Purchaser's neural precursor system (cell line is commodity), by culture (culture medium behaviour neural precursor
Culture medium ScienCell Research Laboratories) 1% fetal calf serum of mixing, after 20 days, you can it is positive to obtain CD44
Cell, CD44 is the marker of astroglia precursor, thus the cell be astroglia precursor.Utilize fluidic cell
Instrument can recycle these CD44 positive cells, purifying and culture (people's neural precursor culture medium ScienCell
Research Laboratories mix 1% fetal calf serum).The differentiation product of these precursors is astroglia,
It is that nutrition is provided and is supported for neuron, if astroglia function is impaired, also results in major disease such as A Erci
The silent disease in sea.
Invention content
The present invention is directed to overcome the deficiencies of the prior art and provide a kind of preparation method of people source astroglia precursor and
Culture medium.
In order to achieve the above object, technical solution provided by the invention is：
The preparation method of the people source astroglia precursor includes the following steps：
(1) induced multi-potent stem cell is inoculated in the special culture plate of cell, using mTeSR stem cell medias, in 37
Induced multi-potent stem cell is cultivated under the conditions of DEG C, every 6-8 days, passage in preferably 7 days was primary, replaces daily fresh
MTeSR stem cell medias；
(2) it after induced multi-potent stem cell passes on the 5th day, with sterile PBS buffer solution rinse cell surface, is then added few
Measuring (the fresh mTeSR stem cell media half volume for being equivalent to replacement) embryoid body differential medium, (embryoid body breaks up
Culture medium is the prior art)；Induced multi-potent stem cell cell mass edge is gently scraped with steril cell sleaker, makes induced multi-potent stem cell
Cell mass is detached from culture plate and is in suspended state, and then piping and druming makes induced multi-potent stem cell cell mass disperse repeatedly, will disperse
Induced multi-potent stem cell cell mass afterwards is transferred to Tissue Culture Dish, is shaken under the conditions of 37 DEG C after adding embryoid body differential medium
Culture 5 days or more is swung, primary fresh embryoid body differential medium is replaced within every 2 days；
(3) induced multi-potent stem cell cell mass suspension shake culture forms the embryoid that size is uniform, construction is similar after 5 days
The embryoid body cell mass of body chondritic；Embryoid body differential medium is changed to by N2 culture mediums, B27 culture mediums and DMEM/
In the mixed culture medium A that F12 serum free mediums are mixed into, mixed culture medium A added with growth factor bFGF and growth because
The concentration of sub- EGF, growth factor bFGF and growth factor EGF in mixed culture medium A is 10 μ g/L；By embryoid body cell
Group is moved to together with mixed culture medium A by the coated culture dishes of matrigel Matrigel, jog culture dish makes embryoid body cell
It rolls into a ball and is averagely scattered in culture dish bottom surface, stationary culture 5-7 days under the conditions of 37 DEG C preferably 6 days, is replaced primary fresh for every two days
Mixed culture medium A；Embryoid body cell mass is centrally oriented the neural precursor group for being divided into rosettes structure；1L steps (3)
The mixed culture medium A is mixed by 10mL N2 culture mediums, 20mL B27 culture mediums and 970mL DMEM/F12 serum free mediums
At (mixed culture medium for being not added with aftermentioned growth factor is commercially available), in mixed culture medium A added with growth factor bFGF and
The concentration of growth factor EGF, growth factor bFGF and growth factor EGF in mixed culture medium A is 10 μ g/L.
(4) choose neural precursor group with sterile needle to be placed in sterile centrifugation tube, addition is equivalent to 2/3 body of centrifuge tube
Long-pending DMEM/F12 serum free mediums, centrifuge under the conditions of 1000rpm, collect cell, then replace and fresh are cultivated by N2
Added with life in the mixed culture medium B that base, B27 culture mediums and DMEM/F12 serum free mediums are mixed into, mixed culture medium B
The culture medium of long factor bFGF, growth factor B MP-4 and the KSR of alternative serum, growth factor bFGF is in mixed culture medium B
A concentration of 20 μ g/L of a concentration of 10 μ g/L, growth factor B MP-4 in mixed culture medium B, the culture medium of the KSR of alternative serum
Additive amount be mixed culture medium B total volumes 10%；The cell precipitation collected is blown and beaten, cell precipitation is made to be resuspended in fresh cultured
In base, cell liquid is formed, cell liquid is laid in the coated Tissue Culture Dish of matrigel, the stationary culture 3 under the conditions of 37 DEG C
After it, the cell volume in cell liquid expands, and begins to differentiate into astroglia precursor, and the cell of proliferation starts to express low water
Flat GFAP marker proteins are to get people source astroglia precursor.1L steps (4) the mixed culture medium B is trained by 10mL N2
Foster base, 20mL B27 culture mediums and 970mL DMEM/F12 serum free mediums, which are mixed into, (is not added with the mixed of aftermentioned growth factor
It is commercially available to close culture medium), added with growth factor bFGF, growth factor B MP-4 and alternative serum in mixed culture medium B
The culture medium of KSR, a concentration of 10 μ g/Ls of the growth factor bFGF in mixed culture medium B, growth factor B MP-4 are being mixed
A concentration of 20 μ g/L in base B, the additive amount of the culture medium of the KSR of alternative serum are the 10% of mixed culture medium B total volumes.
Preferably, DMEM/F12 serum free medium 990mL are contained in 1L steps (2) the embryoid body differential medium,
N2 culture medium 10mL also contain A-8301 and Dorsomorphin, A-8301 and Dorsomorphin and break up culture in embryoid body
A concentration of 1 μm of ol/L in base.
Preferably, the method further includes following steps：LIF ELISA, white blood are added into mixed culture medium B
A concentration of 20 μ g/L of the sick inhibiting factor in mixed culture medium B sets the people source astroglia precursor that step (4) obtains
The directed differentiation of astroglia is carried out in the mixed culture medium B added with LIF ELISA.
The present invention optimizes and filters out the method (Fig. 2) for preparing astroglia precursor using induced multi-potent stem cell.
Compared with the astroglia of differentiated, which has proliferation and directed differentiation ability, a large amount of offer high-purities
Astroglia provides source.The technology is for the pathogenesis of brain diseases, the regeneration of brain and repair mechanism and then applies
There is very important application prospect in clinical treatment.
The present invention will be further described below：
Induced multi-potent stem cell is inoculated in the 6 special culture plates of hole cell by the present invention, keep induced multi-potent stem cell with
The state of cell mass is passed on, and cell mass size is uniform (Fig. 3).Need to keep in incubation highdensity cell mass into
Row culture, it is generally primary every passage in 7 days.The cell dedifferented is chosen in passage using sterile needle.For cultivating induction in this stage
The culture medium of multipotential stem cell be commercially available special mTeSR stem cell medias (Stemcell Technologies,
Vancouver,Canada).2 milliliters of fresh mediums are replaced daily.After stem cell passes on the 5th day, buffered with sterile PBS
Then liquid (pH7.4) rinse cell surface replaces 1 milliliter embryoid body (Embryonic Body, EB) differential medium, the training
Foster base contains DMEM/F12 serum free mediums (Thermo Fisher Scientific), N2 culture mediums (Thermo Fisher
Scientific, Inc), the Dorsomorphin of A-8301 (Stemgent, the San Diego, CA) and 1 μm of ol/L of 1 μm of ol/L
(Chemdea, Ridgewood, NJ, USA) rolls into a ball edge with the light cell scraping of steril cell sleaker, cell mass is gradually made to be detached from culture
Ware is at suspended state, and then blowing and beating 5 times repeatedly makes cell mass disperse.All cell masses are transferred to the 6 of low adhesion
Orifice plate Tissue Culture Dish, and add 2 milliliters of embryoid differential medium.Shake culture five days or more in 37 DEG C of incubators, often
Replace a fresh medium within two days.
Suspension shake culture can form the embryoid body chondritic (Fig. 4) that size is uniform, construction is similar after five days.Gently move
Original culture solution is removed, the mixed culture being mixed by N2 culture mediums, B27 culture mediums and DMEM/F12 serum free mediums is changed to
Base (Thermo Fisher Scientific), addition basic fibroblast growth factor bFGF (R＆D Systems,
Minneapolis, MN), recombinant human epidermal growth factor EGF (R＆D Systems, Minneapolis, MN).Embryoid body is thin
It is moved to together with born of the same parents group and fresh medium by matrigel Matrigel (BD Bioscience) coated culture dish, gently shaking
Shaking culture dish makes embryoid averagely be scattered in culture dish bottom surface, stationary culture 6 days or so in 37 DEG C of incubators, replaces one within every two days
Secondary fresh cell medium.Embryoid body cell China Youth Communist League Center Committee meeting directed differentiation is rosettes structure (Neuralrosettes) (figure
5).Show that these cells express the marker PAX6 and Nestin (Fig. 6) of neural precursor using immunofluorescence dyeing identification.
Choose neural precursor group with sterile needle to be placed in 15 milliliters of sterile centrifugation tube, adds 10 milliliters of serum-frees
DMED/F12 culture mediums centrifuge 5 minutes, cell is collected by centrifugation in 1000 rpms of speed, replace it is fresh by N2 culture mediums,
The mixed culture medium (Thermo Fisher Scientific) that B27 culture mediums and DMEM/F12 serum free mediums are mixed into
(commercially available N2 culture mediums are concentrate, and 100x (100 times of dilutions when use), B27 culture mediums are commercially available concentrate, and 50X (makes
50 times of dilutions of used time)), culture medium is interior to add two kinds of growth factors of bFGF and BMP-4, adds alternative serum
The culture medium (Thermo Fisher Scientific) of KnockoutSerum Replacer (KSR).And then it firmly blows and beats thin
Born of the same parents are precipitated, and are allowed to be resuspended in fresh medium, cell liquid are laid in the coated Tissue Culture Dish of matrigel, stationary culture 3
After it, cell volume expands, and begins to differentiate into astroglia precursor, and the cell of proliferation starts to express low-level GFAP marks
Will albumen, thus obtain at high proportion (>95%) astroglia precursor.There is the identified cell very high BrdU to mix
Activity shows the ability (Fig. 6) of its high proliferation.
It is 24 hours or so that the astroglia precursor of acquisition, which has self duplication ability, proliferating cycle, can be passed
Generation and freezen protective are in liquid nitrogen.For the astroglia precursor of acquisition, 20 μ g/L can be added in above-mentioned culture solution
LIF ELISA (Leukemia inhibition factor, LIF) carry out astroglia directed differentiation, 48
Hour after can be obtained a large amount of high-purities (>95%) astroglia (Fig. 7), is directly used in scientific research and clinic.
The present invention illustrates the key component applied to culture astroglia precursor defined medium, avoids general
Culture based articles in cause cell differentiation the unknown factor.The medium component is stablized, and preparation method is simple.Use the culture
The astroglia precursor of base culture can maintain proliferation and differentiation capability for a long time, and repeatability is high, and different batches have been effectively ensured
Stability between product, is conducive to the standardization of products, has higher commercial application value.
By means of the invention it is also possible to obtain a large amount of astroglia precursor in vitro, and by neural stem cell
Directly differentiation astroglia conventional method compare (table 1), the precursor that the present invention obtains have strong proliferative capacity with
And the ability that directed differentiation is astroglia, short-term interior energy obtain rapidly the astroglia of a large amount of high-purities；For
The culture medium of star precursor is cultivated with ingredient determination, the available feature of commodity；For largely generating for scientific research or
The means of clinical astrocyte have the characteristics that of low cost, safe, production efficiency is high, operability is strong.
Description of the drawings
Fig. 1 is the conventional method schematic diagram that astroglia obtains；From neural stem cell to Astrocyte differentiation
When, it will produce the pollution of neuronal cell and oligodendroglia；
Fig. 2 is the method for the present invention schematic diagram；Astroglia precursor has proliferative capacity, and is specifically divided into a large amount of
Astroglia；
Fig. 3 behaviours source induced multi-potent stem cell figure；
Fig. 4 is that embryoid body cell is cliqued graph；
The neural precursor of Fig. 5 rosettes structures is cliqued graph；
Fig. 6 is neural precursor figure；It is identified by immunofluorescence dyeing, most cells express neural precursor
Marker Nestin and Pax6；
Fig. 7 is astroglia precursor；The BrdU positives show that the cell is the cell of proliferation；
Fig. 8 is astroglia；It is identified by immunofluorescence dyeing, astroglia precursor inhibits by leukaemia
After the differentiation of factor induced orientation, most cells express the marker GFAP of astroglia.
Specific implementation mode
The preparation method of the people source astroglia precursor includes the following steps：
(1) induced multi-potent stem cell is inoculated in the 6 special culture plates of hole cell, using mTeSR stem cell medias, in
Induced multi-potent stem cell is cultivated under the conditions of 37 DEG C, it is primary every passage in 7 days, it is dry thin that the fresh mTeSR of 2mL are replaced daily
Born of the same parents' culture medium；
(2) it after induced multi-potent stem cell passes on the 5th day, with sterile PBS buffer solution rinse cell surface, is then added
1ml embryoid body differential mediums gently scrape induced multi-potent stem cell cell mass edge with steril cell sleaker, keep induced multi-potent dry thin
Born of the same parents' cell mass is detached from culture plate and is in suspended state, and then piping and druming makes induced multi-potent stem cell cell mass disperse repeatedly, will divide
Induced multi-potent stem cell cell mass after dissipating is transferred to 6 orifice plates Tissue Culture Dish, adds after embryoid body differential medium at 37 DEG C
Under the conditions of shake culture 5 days or more, replace within every 2 days primary fresh embryoid body differential medium；1L steps (2) described embryoid body
In differential medium contain DMEM/F12 serum free mediums 990mL, N2 culture medium 10mL, also contain A-8301 and
A concentration of 1 μm of ol/L of Dorsomorphin, A-8301 and Dorsomorphin in embryoid body differential medium；
(3) induced multi-potent stem cell cell mass suspension shake culture forms the embryoid that size is uniform, construction is similar after 5 days
The embryoid body cell mass of body chondritic；Embryoid body differential medium is changed to by N2 culture mediums, B27 culture mediums and DMEM/
In the mixed culture medium A that F12 serum free mediums are mixed into, mixed culture medium A added with growth factor bFGF and growth because
The concentration of sub- EGF, growth factor bFGF and growth factor EGF in mixed culture medium A is 10 μ g/L；By embryoid body cell
Group is moved to together with mixed culture medium A by the coated culture dishes of matrigel Matrigel, jog culture dish makes embryoid body cell
It rolls into a ball and is averagely scattered in culture dish bottom surface, stationary culture 5-7 days under the conditions of 37 DEG C replaces primary fresh mixed culture for every two days
Base A；Embryoid body cell mass is centrally oriented the neural precursor group for being divided into rosettes structure；1L steps (3) described mixing
Culture medium A is mixed by 10mL N2 culture mediums, 20mL B27 culture mediums and 970mL DMEM/F12 serum free mediums, this is mixed
It closes in culture medium A and is trained in mixing added with growth factor bFGF and growth factor EGF, growth factor bFGF and growth factor EGF
The concentration supported in base A is 10 μ g/L；
(4) choose neural precursor group with sterile needle to be placed in 15mL sterile centrifugation tubes, add the DMEM/ of 10mL
F12 serum free mediums centrifuge 5min under the conditions of 1000rpm, collect cell, then replace fresh by N2 culture mediums, B27
Growth factor is added in the mixed culture medium B that culture medium and DMEM/F12 serum free mediums are mixed into, mixed culture medium B
The culture medium of the KSR of bFGF, growth factor B MP-4 and alternative serum, growth factor bFGF are a concentration of in mixed culture medium B
A concentration of 20 μ g/L of 10 μ g/L, growth factor B MP-4 in mixed culture medium B, the addition of the culture medium of the KSR of alternative serum
Amount is the 10% of mixed culture medium B total volumes；The cell precipitation collected is blown and beaten, cell precipitation is made to be resuspended in fresh culture,
Cell liquid is formed, cell liquid is laid in the coated Tissue Culture Dish of matrigel, the stationary culture after 3 days under the conditions of 37 DEG C,
Cell volume in cell liquid expands, and begins to differentiate into astroglia precursor, and the cell of proliferation starts to express low-level
GFAP marker proteins are to get people source astroglia precursor；1L steps (4) the mixed culture medium B is cultivated by 10mL N2
Base, 20mL B27 culture mediums and 970mL DMEM/F12 serum free mediums are mixed into, added with growth in mixed culture medium B
The culture medium of the KSR of factor bFGF, growth factor B MP-4 and alternative serum, growth factor bFGF are dense in mixed culture medium B
Degree be 10 μ g/L, a concentration of 20 μ g/Ls of the growth factor B MP-4 in mixed culture medium B, the culture medium of the KSR of alternative serum
Additive amount is the 10% of mixed culture medium B total volumes；
(5) LIF ELISA is added into mixed culture medium B, LIF ELISA is in mixed culture medium B
The people source astroglia precursor that step (4) obtains is placed in this and is added with LIF ELISA by a concentration of 20 μ g/L
The directed differentiation of astroglia is carried out in mixed culture medium B.
By means of the invention it is also possible to obtain a large amount of astroglia precursor in vitro, and by neural stem cell
The conventional method of directly differentiation astroglia compares (table 1), which has strong proliferative capacity and directed differentiation
For the ability of astroglia, short-term interior energy obtains rapidly the astroglia of a large amount of high-purities；Before cultivating star
The culture medium of body cell is with ingredient determination, the available feature of commodity；For largely generating the star for scientific research or clinic
The means of cell have the characteristics that of low cost, safe, production efficiency is high, operability is strong.
The comparison of table 1 the method for the present invention and conventional method
Claims (8)
1. a kind of preparation method of people source astroglia precursor, which is characterized in that described method includes following steps：
（1）Induced multi-potent stem cell is inoculated in the special culture plate of cell, using mTeSR stem cell medias, in 37 DEG C
Under the conditions of induced multi-potent stem cell is cultivated, it is primary every passage in 6-8 days, it is dry thin to replace fresh mTeSR daily
Born of the same parents' culture medium；
（2）After induced multi-potent stem cell passes on the 5th day, with sterile PBS buffer solution rinse cell surfaces, then it is added quasi-
Idiosome differential medium；Induced multi-potent stem cell cell mass edge is gently scraped with steril cell sleaker, keeps induced multi-potent stem cell thin
Born of the same parents group is detached from culture plate and is in suspended state, and then piping and druming makes induced multi-potent stem cell cell mass disperse repeatedly, after dispersion
Induced multi-potent stem cell cell mass be transferred to Tissue Culture Dish, shaken under the conditions of 37 DEG C after adding embryoid body differential medium
Culture 5 days or more is swung, primary fresh embryoid body differential medium is replaced within every 2 days；
（3）Induced multi-potent stem cell cell mass suspension shake culture forms the embryoid body that size is uniform, construction is similar after 5 days
The embryoid body cell mass of chondritic；Embryoid body differential medium is changed to by N2 culture mediums, B27 culture mediums and DMEM/
Growth factor bFGF and life are added in the mixed culture medium A that F12 serum free mediums are mixed into, mixed culture medium A
Long factor EGF, the concentration of growth factor bFGF and growth factor EGF in mixed culture medium A are 10 μ g/L；It will intend
It is moved to together with idiosome cell mass and mixed culture medium A by the coated culture dishes of matrigel Matrigel, jog culture dish
Embryoid body cell mass is set averagely to be scattered in culture dish bottom surface, stationary culture 5-7 days under the conditions of 37 DEG C is replaced primary for every two days
Fresh mixed culture medium A；Embryoid body cell mass is centrally oriented the neural precursor group for being divided into rosettes structure；
（4）Choose neural precursor group with sterile needle to be placed in sterile centrifugation tube, addition is equivalent to 2/3 volume of centrifuge tube
DMEM/F12 serum free mediums, centrifuged under the conditions of 1000rpm, collect cell, then replace fresh by N2
The mixed culture medium B that culture medium, B27 culture mediums and DMEM/F12 serum free mediums are mixed into, mixed culture medium B
In added with growth factor bFGF, growth factor B MP-4 and alternative serum KSR culture medium, growth factor bFGF
A concentration of 20 μ of a concentration of 10 μ g/L in mixed culture medium B, growth factor B MP-4 in mixed culture medium B
G/L, the additive amount of the culture medium of the KSR of alternative serum are the 10% of mixed culture medium B total volumes；Blow and beat the cell collected
Precipitation, makes cell precipitation be resuspended in fresh culture, forms cell liquid, and cell liquid, which is laid in the coated cell of matrigel, to be trained
It supports in ware, after 3 days, the cell volume in cell liquid expands stationary culture, begins to differentiate into astroglia under the conditions of 37 DEG C
Precursor, the cell of proliferation start to express low-level GFAP marker proteins to get people source astroglia precursor；1L
Step（2）Contain DMEM/F12 serum free mediums 990mL, N2 culture medium 10mL in the embryoid body differential medium,
It is dense in embryoid body differential medium also to contain A-8301 and Dorsomorphin, A-8301 and Dorsomorphin
Degree is 1 μm of ol/L.
2. the method as described in claim 1, which is characterized in that step（1）It is that induced multi-potent stem cell is inoculated in 6
In the special culture plate of hole cell, carried out using mTeSR stem cell medias, under the conditions of 37 DEG C to induced multi-potent stem cell
Culture, it is primary every passage in 7 days, the fresh mTeSR stem cell medias of 2mL are replaced daily.
3. the method as described in claim 1, which is characterized in that step（2）It is to wait for that induced multi-potent stem cell passes on the 5th day
Afterwards, with sterile PBS buffer solution rinse cell surfaces, embryoid body differential medium is then added, it is light with steril cell sleaker
Induced multi-potent stem cell cell mass edge is scraped, induced multi-potent stem cell cell mass is made to be detached from culture plate and is in suspended state, so
Piping and druming makes induced multi-potent stem cell cell mass disperse repeatedly afterwards, and the induced multi-potent stem cell cell mass after dispersion is transferred to 6
Orifice plate Tissue Culture Dish adds after embryoid body differential medium under the conditions of 37 DEG C shake culture 5 days or more, and every 2 days more
Change primary fresh embryoid body differential medium.
4. the method as described in claim 1, which is characterized in that 1L steps（3）The mixed culture medium A is by 10mL
N2 culture mediums, 20mL B27 culture mediums and 970mL DMEM/F12 serum free mediums are mixed into, mixed culture medium A
In be added with growth factor bFGF and growth factor EGF, growth factor bFGF and growth factor EGF are being mixed
Concentration in base A is 10 μ g/L.
5. the method as described in claim 1, which is characterized in that 1L steps（4）The mixed culture medium B is by 10mL
N2 culture mediums, 20mL B27 culture mediums and 970mL DMEM/F12 serum free mediums are mixed into, mixed culture medium B
In added with growth factor bFGF, growth factor B MP-4 and alternative serum KSR culture medium, growth factor bFGF
A concentration of 20 μ of a concentration of 10 μ g/L in mixed culture medium B, growth factor B MP-4 in mixed culture medium B
G/L, the additive amount of the culture medium of the KSR of alternative serum are the 10% of mixed culture medium B total volumes.
6. the method as described in claim 1, which is characterized in that step（4）It is to choose neural precursor group with sterile needle
It is placed in 15mL sterile centrifugation tubes, the DMEM/F12 serum free mediums of 10mL is added, under the conditions of 1000rpm
5min is centrifuged, cell is collected.
7. the method as described in claim 1, which is characterized in that the method further includes following steps：To mixed culture medium
LIF ELISA, a concentration of 20 μ g/L of the LIF ELISA in mixed culture medium B, by step are added in B
（4）The people source astroglia precursor of acquisition is placed in the mixed culture medium B added with LIF ELISA and carries out
The directed differentiation of astroglia.
8. a kind of culture medium being used to prepare people source astroglia precursor, which is characterized in that the culture medium is to be mixed with N2
With the DMEM/F12 serum-free mixed culture mediums of B27, added with growth factor bFGF, growth factor in the culture medium
The culture medium of BMP-4 and the KSR of alternative serum, growth factor bFGF a concentration of 10 μ g/L, growth factor B MP-4 are dense
The additive amount for the culture medium that degree is 20 μ g/L, KSR is the 10% of culture medium total volume.
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| CN105695408B (en) * | 2016-03-22 | 2019-09-27 | 中国人民解放军第二军医大学 | A kind of naked mole Astrocytes Primary Cultivation |
| CN107488629A (en) * | 2017-08-14 | 2017-12-19 | 中国科学院广州生物医药与健康研究院 | A kind of directed differentiation method of human pluripotent stem cells |
| US20220119765A1 (en) * | 2019-01-22 | 2022-04-21 | Korea University Research And Business Foundation | Differentiation method of neural stem cells manufactured by direct cell conversion into astrocytes |
| CN114763531B (en) * | 2021-01-12 | 2023-07-21 | 内蒙古大学 | Method for inducing differentiation of neural stem cells into astrocytes, uses and compositions |
| CN116478923B (en) * | 2022-04-26 | 2024-01-02 | 浙江霍德生物工程有限公司 | Preparation method of astrocyte |
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