KR101705698B1 - Composition of medium for glial cell cultivation of mouse and method of mouse glial cell differentiation - Google Patents

Abstract

The present invention relates to a medium composition for culturing a mouse glioma cell line and a method for inducing differentiation of mouse glioma cells. The method comprises administering 20 to 80 ug / mL of transferrin, 1 to 10 ng / mL of progesterone, 1 to 10 ng / mL of transferrin to Dulbecco's Modified Eagle's Medium 1 to 10 μg / mL of insulin, 0.1 to 1.5 μg / mL of thyroxine, and 1 to 10 μg / mL of glucose. , It is possible to induce differentiation of glial cells, for example, glial cells, from mouse cortical precursor cells.

Description

[0001] The present invention relates to a medium composition for culturing a mouse glial cell and a method for inducing the differentiation of a mouse glial cell,

The present invention relates to a medium composition for culturing a mouse glial cell capable of inducing differentiation of glial cells from mouse cortical precursor cells and a method for inducing differentiation of mouse glial glial cells.

Neuroglial cells (neuroglial cells) with the cells (ependymal cells) to make the glial tissue. The glial cells fill the spaces between the nerve cells and support them, especially connect them with the capillaries to participate in the metabolism of nerve cells and make water plants of water fibers. Furthermore, they are formed by proliferation of injury or leafing of nerve cells. The glial cells are classified into astrocytes, oligodendrocytes, and microglia in shape, size, and function. The microglial cells are derived from the mesoderm along with the blood vessels. Like nerve cells, it is ectodermal (ectodermal) star.

The oligodendrocytes are small compared to astrocytes and are characterized by a few protrusions with few branches. In addition, it is dark in the former phenomenon (electroencephalogram) because there are a lot of the surface endoplasmic reticulum (vacuole vesicle) and the cytoplasmic tube and the electron density is high. The proliferating ganglion cells adhere closely to the nerve cell body and lie along the nerve fibers. The former corresponds to the lateral cell of the spinal ganglion, and the latter is similar to that of the peripheral nerve. However, it does not participate in the regeneration of nerve fibers, like Shihan cells.

In addition, the proliferating giant cells concentrically surround the axon to form a few seconds.

In order to obtain such a proliferating cell, conventionally, there has been known a technique of extracting proliferating progenitor precursor cells from a rat brain and extracellularly differentiating the cells. However, the above technique requires long-term in vitro culture for one month or more, It is difficult to develop the model and it is difficult to utilize it in animal models of planting diseases ( Neuron 2004 Jul 22; 43 (2): 183-91).

In addition, although techniques for separating cells using an antibody against a protein specifically expressed on the surface of proliferating progenitor cells or differentiated progenitor cells have been known, it is known to utilize the adsorption between a specific antibody and the protein expressed on the cell surface ( Cold Spring Hard Protoc 2013 Sep 1; 2013 (9): 854-68. Doi: 10.1101 / pdb.prot073973.).

Therefore, a mouse which can be easily used as an animal model is used instead of a rat which is difficult to use in an animal model of a planting disease, and an antibody is not required, and therefore, there is a demand for an economical method for differentiating glial cells.

Korean Patent No. 1409336 Korean Patent No. 1323652

It is an object of the present invention to provide a medium composition for culturing a glial cell capable of inducing the differentiation of glial cells from mouse cortical precursor cells.

It is another object of the present invention to provide a method for inducing differentiation of the mouse glioma cells.

In order to accomplish the above object, the present invention provides a medium composition for culturing mouse glioma cells, which comprises 20 to 80 ug / mL of transferrin, 1 to 10 ng / mL of progesterone, 10 to 18 ug / mL of putrescine in DMEM (Dulbecco's Modified Eagle's Medium) mL, sodium ascelenate 1 to 8 ng / mL, glutamine 1 to 5 mM, insulin 1 to 10 ug / mL, thyroxine 0.1 to 1.5 ug / mL, and glucose 1 to 10 ug / mL.

The glial cells may be glial cells.

According to another aspect of the present invention, there is provided a method for inducing differentiation of mouse glioma cells, comprising: (A) contacting cortex precursor cells with a culture medium containing DMEM and 10% FBS, culturing for 7 to 10 days ;

(B) shaking the container in which the cortical precursor cells are cultured at 100 to 300 rpm for 0.5 to 2 hours, discarding the supernatant, adding the medium composition and shaking at 200 to 300 rpm for 12 to 20 hours, Separating the supernatant containing the glial precursor precursor from the bottom of the container through a shaking process except for the cell precursor attached to the bottom;

(C) inducing differentiation by contacting the supernatant separated in step (B) with the culture medium for culturing mouse glioma cells for 5 to 10 days.

The glial cells may be glial cells.

In the step (B), the medium composition may include DMEM and 10% FBS.

The present invention provides a culture medium suitable for obtaining glomeruli glioma cells differentiated by using a mouse, and when differentiated using the same, it is possible to smoothly differentiate into normal glial cell glia But also the degree of differentiation is excellent.

In addition, when the method of inducing differentiation of mouse glioma cells of the present invention is used, it is possible to study the mechanism of regulating the herbalization process using various mouse models, and to provide drug screening for the discovery of therapeutic agents for hereditary diseases, And a system capable of monitoring the recovery process.

FIG. 1 is a photograph of the mouse cortical precursor cells cultured according to the production example of the present invention, taken according to the flow of the culture time.
FIG. 2 is a photograph of the cultured mouse cortical precursor cells of FIG. 1 (a) photographed after prolonged shaking at 37 ° C, (b) removing microgranular progenitor cells, (C) photographs of progenitor cell precursor cells in supernatant after prolonged shaking.
FIG. 3 is a photograph showing the process of inducing the differentiation of glomeruli precursor cells into glomeruli cells according to the present invention.
Fig. 4 is a photograph showing the differentiation pattern of the differentiated glial cells according to the example of the present invention and the comparative example.
FIG. 5 is a graph showing the degree of differentiation of glomeruli ganglia differentiated according to Examples and Comparative Examples of the present invention.
Fig. 6 is a photograph showing the differentiation pattern of the differentiated glial cells according to the example of the present invention and the comparative example.

The present invention relates to a medium composition for culturing a mouse glial cell capable of inducing differentiation of glial cells from mouse cortical precursor cells and a method for inducing differentiation of mouse glial glial cells.

Hereinafter, the present invention will be described in detail.

The medium composition for culturing mouse glioma cells according to the present invention is prepared by adding 20 to 80 ug / mL of transferrin, 1 to 10 ng / mL of progesterone, 10 to 18 ng / mL of putrescine to DMEM (Dulbecco's Modified Eagle's Medium) mL, 1 to 8 ng / mL of sodium selenite, 1 to 5 mM of glutamine, 1 to 10 ug / mL of insulin, 0.1 to 1.5 ug / mL of thyroxine, 1 to 10 ug / mL of glucose, and a remaining amount of distilled water (or B27 solution).

In the case of using a culture medium having a different composition without using the culture medium having the above composition as the culture medium composition, it is impossible to smoothly differentiate into the glioma cells of the mouse normally.

The glial cells can be classified into astrocytes, glial cells, and microglia according to their morphology, size, and function. In the present invention, the glial cells are divided into a myelin sheath (membrane wrapping the branches of nerve cells) It means the glial cells involved.

DL-alpha-tocopherol acetate, DL-alpha-tocopherol, vitamin A, BSA (Life Technologies), and the like are used as the DMEM for DEMO , Catalase, insulin, superoxide dismutase, corticosterone, D-galactose, ethanolamine HCl, glutathione ion, L-carnitine HCl, linoleic acid, linoleic acid, progesterone, putrescine 2HCl, sodium selenite) and triiodo-1-thyronine.

In addition, the present invention provides a method for inducing differentiation of mouse glioma cells from mouse cortical precursor cells.

The method for inducing the differentiation of mouse glioma cells of the present invention comprises the steps of (A) contacting mouse cortical precursor cells to a medium composition comprising DMEM and 10% FBS for 7 to 10 days; (B) shaking the container in which the cortical precursor cells are cultured at 100 to 300 rpm for 0.5 to 2 hours, discarding the supernatant, adding the medium composition and shaking at 200 to 300 rpm for 12 to 20 hours, Separating the supernatant containing the glial precursor precursor from the bottom of the container through a shaking process except for the cell precursor attached to the bottom; And (C) inducing differentiation by contacting the supernatant separated in step (B) with the culture medium for 5 to 10 days.

First, in step (A), the mouse cortical precursor cells are contacted with a culture composition containing DMEM and 10% FBS commonly used and cultured for 7 to 10 days.

Specifically, the brain is removed from the mouse after 1 day of birth, and the cortex is collected by removing the midbrain, the male membrane, and the olfactory precursor from the brain. 20% FBS, 1% Pen / Strep, and DNase I (0.003%) were added to the cortex after adding an enzyme digestion solution (trypsin, final 0.0125%) and left in a 25-38 캜 wheel incubator for 5-20 minutes. Is added, and then the mixture is shaken 1 to 3 times and incubated at room temperature for 5 to 20 minutes. After the incubation, the supernatant was subjected to primary centrifugation, and the supernatant was removed. Then, the supernatant was firstly resuspended in 5 to 20 mL of a mixed solution of DMEM and 10% FBS, filtered using a strainer of 80 to 150 μm and centrifuged again. And then resuspended in 10 to 30 mL of a mixed solution of DMEM and 10% FBS. The secondary resuspension was filtered using a 50-90 μm strainer and further centrifuged again. The supernatant was then removed, and the solution was subjected to tertiary resuspension in 10 to 30 mL of a mixed solution of DMEM and 10% FBS. And then cultured for 7 to 10 days to cultivate the mouse cortical precursor cells.

Next, in step (B), the container in which the mouse cortical progenitor cells are cultured is shaken at 100 to 300 rpm for 0.5 to 2 hours, then the supernatant is discarded, the medium composition is added, and the mixture is incubated at 200 to 300 rpm , The cell precursor attached to the bottom of the container is shaken. After shaking, only the supernatant containing the glial precursor precursor remote from the bottom of the container is taken, and the glial cell precursor cells attached to the bottom of the container are separated. The supernatant recovery can be performed 2-3 times to isolate pure glial cells, for example, glial cells.

The neural progenitor cells contained in the mouse cortical precursor cells are composed of astrocytic progenitor cells, progenitor precursor cells, and small precursor cells. Each of the neuron precursor cells is adsorbed on the bottom of the flask according to the kind of cells The degree is different. The glial progenitor precursor cell to be obtained in the present invention is a cell that is adsorbed firmly to the flask bottom than the astrocytes, but adsorbed firmly to the bottom of the flask. Therefore, After the cells are stabilized to a certain extent, they are shaken again. Only the progenitor cells isolated from the bottom are extracted. After stabilizing and shaking again, astrocytes are still attached to the floor.

 Specifically, the culture container in which the cultured mouse cortical precursor cells grow is shaken at a temperature of 30 to 38 ° C for 0.5 to 2 hours at a rate of 100 to 300 rpm, and then the supernatant is removed to remove the microbial cells. The culture medium was filled with the culture medium containing DMEM and 10% FBS, the cells adsorbed on the bottom in an incubator were stabilized for 3 to 7 hours, and then shaken again at a speed of 200 to 400 rpm for 12 to 20 hours. Except for the cells (astrocytes, etc.) adsorbed on the bottom, only the supernatant is taken. The supernatant is subjected to quaternary centrifugation, followed by washing with PBS, followed by fifth centrifugation, followed by removing PBS, adding trypsin-EDTA, and stirring at room temperature for 5 minutes to 20 minutes. After the above stirring, trypsin-EDTA is removed, and 10 to 30 mL of a mixed solution of DMEM and 20% FBS can be added to the culture.

The cultured culture was centrifuged at 6 times, and the supernatant was removed. 10 to 30 mL of a mixture of DMEM and 10% FBS was added to the suspension, and the suspension was applied to collect cell precursor cells (primary) And then incubated in a culture vessel for 0.5 to 2 hours in an incubator. After the incubation, remove the supernatant by removing the cells attached to the bottom (removal of astrocytic precursor cells) and centrifuging the supernatant containing cells that are still not adsorbed. The above procedure is repeated two more times to collect pure neuron precursor cells and apply them. The applied suspension solution is filtered to extract and apply spindle cell precursor cells. After 15-30 hours, the solution mixed with DMEM and 10% FBS is replaced.

Next, in step (C), the proliferating progenitor precursor cells extracted in step (B) are contacted with the medium composition for culturing a glial cell of the present invention prepared by the method of the present invention and cultured for 5 to 10 days, To induce the growth of the glial cells of the mouse. It is preferable that the medium composition for culturing the glial cells of the mouse is changed every 2 to 3 days during the culture.

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the present invention. Such variations and modifications are intended to be within the scope of the appended claims.

Production Example 1. Culture of mouse cortical precursor cells

The brain was removed from the mouse (ICR species) 1 day after birth and placed in a Petri dish containing HBSS (Hanks' Balanced Salt Solution), then the midbrain, membrane and olfactory precursors were removed from the brain and placed in a 15 mL conical tube containing HBSS Collect the cortex. The HBSS is removed and the cortex is crushed by pipetting the solution once or twice with a 10 mL pipette with DMEM and 10% FBS (Fetal Bovine Serum). After incubation for 15 min (150 rpm) in a 37 ° C wheel incubator, the cells were mixed with DMEM, 20% FBS, 1% Pen / Strep and DNase I (0.003%) in an enzyme digestion solution (final 0.0125% trypsin) Add the resulting mixture and gently shake the tube 3 times and incubate at room temperature for 10 minutes. After the culture, centrifugation was carried out at a speed of 1000 rpm for 10 minutes, and the supernatant was removed. Then, the supernatant was resuspended in 10 mL of a mixed solution of DMEM and 10% FBS, filtered using a 100-μm strainer, and centrifuged Min). Remove the supernatant and resuspend in 20 mL of a mixture of DMEM and 10% FBS. The resuspension was filtered using a 70 [mu] m strainer and centrifuged again (800 rpm, 10 min), and the supernatant was removed and resuspended in 20 mL of a mixture of DMEM and 10% FBS, After the filter and after 24 hours, the culture composition was changed and the mouse cortex precursor cells were cultured by replacing the new medium composition (DMEM + 10% FBS) every 3 days and culturing for 7 days.

Production Example 2. Culture of rat cortical precursor cells

Brains were removed from Sprague Dawley rats 1 day after birth and placed in a Petri dish containing HBSS (Hanks' Balanced Salt Solution). Then the brain, membrane and olfactory precursors were removed from the brain, and the enzyme digestion solution (final 0.025% Trypsin) and 10% FBS in an Erlenmeyer flask using a 25-mL pipette and shake the mixture in a 37 ° C incubator for 30 minutes. Add a mixture of DMEM, 20% FBS, 1% Pen / Strep and DNase I (0.008%), gently shake the tube 3 times and incubate at room temperature for 10 minutes. After the culture, centrifugation was carried out at a speed of 1000 rpm for 10 minutes, and the supernatant was removed. The supernatant was then resuspended in 10 mL of a mixed solution of DMEM and 10% FBS, filtered using a 210-μm strainer, centrifuged (1000 rpm, Min). Remove the supernatant and resuspend in 20 mL of a mixture of DMEM and 10% FBS. The resuspension was filtered using a 130 [mu] m strainer and centrifuged again (800 rpm, 10 min) and the supernatant was removed and resuspended in 20 mL of a mixture of DMEM and 10% FBS, After 24 hours of filtration, the culture medium was replaced with fresh medium composition (DMEM + 10% FBS) every 3 days and cultured for 7 days to cultivate the cortical precursor cells.

Production Examples 3 to 6. Preparation of Medium Composition

Were mixed according to the composition shown in Table 1 below to prepare respective medium compositions. The following basal medium eagle (BME), F12, DMEM and B27 are materials commonly used in the art.

division Production Example 3 Production Example 4 Production Example 5 Production Example 6 Basic badge DMEM BMEF12 BMEF12 DMEM Transferrin (ug / mL) 50 100 50 - Progesterone (ng / mL) 6.2 12.8 6.2 - Gt; (ug / mL) < / RTI > 16 20 16 - Sodium selenite (ng / mL) 5 10.4 5 - Glutamine (mM) 4 6.6 4 4 Insulin (ug / mL) 5 25 5 - Thyroxine (ug / mL) 0.8 0.8 0.8 - Glucose (ug / mL) 6 6 6 6 B27 Balance Balance Balance Balance

< Example  And Comparative Example >

Example  One. Manufacturing example  1 + Manufacturing example  3

The mouse cortical precursor cells cultured in Preparation Example 1 were placed in a flask and shaken at a speed of 200 rpm for 1 hour at 37 ° C. Subsequently, the supernatant was removed (microsomal cells were removed) and 10 mL of fresh DMEM and 10% Lt; RTI ID = 0.0 &gt; 37 C &lt; / RTI &gt; for 5 hours. After the incubation, the culture was shaken at a temperature of 37 ° C for 15 hours at 250 rpm. The supernatant was then centrifuged (1500 rpm, 10 minutes), washed with PBS and centrifuged at 1500 rpm for 10 minutes. After removing PBS, trypsin-EDTA was added (0.25%, 1.5 mL, 10 minutes) and stirred at room temperature. After the above stirring, trypsin-EDTA was removed and cultured for 10 minutes using a 20 mL medium composition containing DMEM and 20% FBS.

The cultured culture was subjected to tertiary centrifugation (1500 rpm, 10 minutes), the supernatant was removed, and 20 mL of a solution obtained by mixing DMEM and 10% FBS was added to the culture solution. The culture solution was first resuspended, After incubation for 1 hour, the supernatant was collected by centrifugation (1500 rpm, 10 minutes), and the supernatant was removed by DMEM And 10% FBS was added thereto, followed by re-suspending, and then the second ash suspension was again applied. After culturing for 1 hour, the preadipocyte precursor cells not adsorbed on the bottom are collected (secondary), and the collected (secondary) preadipocyte precursor cell suspension is applied third. After incubation for 1 hour after the third application, the non-adsorbed suspension is filtered with a 40-μm cell strainer, and after 24 hours, pure pregelatinized progenitor cells are extracted while the solution mixed with DMEM and 10% FBS is replaced.

The extracted dendritic cells precursor cells were contacted with the medium composition prepared according to Preparation Example 3, and cultured for 6 days to induce differentiation, and then the dendritic cells of the mice were cultured.

Comparative Example 1 Production Example 2 + Production Example 4

The rat cortical precursor cells cultured in Preparation Example 2 were shaken at a speed of 200 rpm for 1 hour at 37 ° C. Then, the supernatant was removed, and a 10 mL medium composition containing fresh DMEM and 10% FBS was added thereto. Lt; / RTI &gt; After the incubation, the mixture was shaken at 37 ° C for 12 hours at 250 rpm. The supernatant was then centrifuged at 1500 rpm for 10 minutes, washed with PBS and centrifuged at 1500 rpm for 10 minutes. After removing PBS, trypsin-EDTA was added (0.25%, 5 mL, 10 minutes) and stirred at room temperature. After the agitation, the trypsin-EDTA was removed and the enzyme was inactivated using a 20 mL medium composition containing DMEM and 20% FBS.

After the culture was subjected to tertiary centrifugation (1500 rpm, 10 minutes), the supernatant was removed, and 20 mL of a solution obtained by mixing DMEM and 10% FBS was added thereto. After incubation for 20 min, the unadsorbed cell precursor cells were collected (primary), and the supernatant was removed by centrifugation (1500 rpm, 10 min). 20 mL of DMEM and 10% And then the second ash suspension is applied again. After culturing for 1 hour, the preadipocyte precursor cells not adsorbed on the bottom are collected (secondary), and the collected (secondary) preadipocyte precursor cell suspension is applied third. After 1 hour of the third application, the undifferentiated suspension is filtered with a 40-μm cell strainer, and after 4 hours, the solution containing the mixture of DMEM and 10% FBS is replaced while extracting the proliferating progenitor cells.

The extracted proliferating progenitor precursor cells were contacted with the medium composition prepared according to Production Example 4, and cultured for 10 days to induce differentiation, and the proliferating cells of the rat were cultured.

Comparative Example  2. Manufacturing example  1 + Manufacturing example  5

The same procedure as in Example 1 was carried out except that the culture supernatant of mouse was cultured using the culture composition prepared in Preparation Example 5 instead of Preparation Example 3.

Comparative Example  3. Manufacturing example  1 + Manufacturing example  6

The culture supernatant of the mouse was cultured in the same manner as in Example 1 except that the culture composition prepared in Preparation Example 6 was used instead of the preparation Example 3.

< Test Example >

Test Example  1. Photographed by inducing differentiation of mouse cortical precursor cells into spleen cells of mouse

FIG. 1 is a photograph of the mouse cortical precursor cells cultured according to Production Example 1 of the present invention, according to the culture time (1, 3, 5 and 7 days). The photograph was taken using a Cool snap camera (Roper Scientific, Sarasota) equipped with an Olympus microscope (BX51, O).

2 is a photograph of the mouse cortical precursor cells cultured in Production Example 1 according to Example 1 of the present invention after being shaken (a) for 15 hours, (b) a microgranular progenitor cell was removed, (C) photographs of progenitor cell progenitor cells adsorbed on the floor after removing astrocytic progenitor cells. FIG. The same equipment as in Fig. 1 was used for the photographing.

3 is a graph showing the process of inducing the differentiation of proliferating progenitor precursor cells extracted according to Example 1 of the present invention into proliferating gonads according to the passage of time (1, 2, 3, 4 and 5 days) It is a photograph. The same equipment as in Fig. 1 was used for photographing.

As shown in Fig. 1, it was confirmed that the mouse cortical precursor cells were well cultured with time in a medium in which they were generally used.

As shown in Fig. 2, the progenitor precursor cells, astrocytic precursor cells and small precursor cells are all mixed at first, but (a) the process of shaking the cultured cortex precursor cells and the multiple reproduction (C) only the proliferating progenitor precursor cells were extracted by sequentially removing the small cell precursor cells and the astrocytic precursor cells through the turbid process.

In addition, as shown in FIG. 3, the proliferating progenitor precursor cells were cultured in a culture medium prepared according to the present invention, and the proliferation of the progenitor precursor cells was further differentiated into proliferating glial cells over time.

Test Example  2. Measurement of differentiation pattern_1

Immunocytochemistry was used to measure the pattern and degree of differentiation. The primary antibody is an antibody that recognizes MBP (myelin basic protein) protein expressed in differentiated dendritic cells and an antibody that recognizes olig2 protein expressed in all stages of dendritic cells regardless of the degree of differentiation And an antibody with a fluorescent protein was used as the secondary antibody (FIGS. 4 and 6). The degree of differentiation was measured by calculating the percentage of MBP-positive differentiated from Olig2-positive (Fig. 5), and the pattern of differentiation was determined based on the distribution of MBP (Fig. 6).

FIG. 4 is a photograph (Cell Observer Z1, Zeiss) of the differentiation pattern of the differentiated glial cells according to the example of the present invention and the comparative example, and FIG. 5 is a photograph Fig. 2 is a graph showing the degree of differentiation of protruding cells.

As shown in FIG. 4 and FIG. 5, it was confirmed that the proliferating glioma cells differentiated according to Example 1 were normally differentiated from the progenitor glioblast precursor cells, and the degree of differentiation was as high as about 80% or more. On the other hand, it was confirmed that the epidermal cells according to Comparative Example 1 internal 3 were abnormally differentiated and the degree of differentiation was as low as about 40% or less. In particular, according to Comparative Example 1, it was confirmed that almost all of the glutamatergic cells using the rat were abnormally differentiated and the differentiation correction was less than about 5%, which is very small.

Test Example  3. Measurement of differentiation pattern_2

FIG. 6 is a photograph (Zeiss, Z1) of the differentiation pattern of the differentiated glial cells according to the example of the present invention and the comparative example. The method for confirming the differentiation pattern was carried out using the same antibody staining method as in Test Example 2 above.

In the photograph of FIG. 6, the arrow indicates an epidermal growth factor in which the differentiation does not proceed smoothly, and the arrowhead is an abnormal epidermal growth factor.

As shown in Fig. 6, the glomeruli ganglia differentiated according to Example 1 were normally differentiated from glial glial precursor cells. It was confirmed that the differentiated glial cells differentiated according to Comparative Examples 1 to 3 did not progress smoothly or were abnormally differentiated.

Claims (5)

Ml of progesterone, 10 to 18 ug / ml of putrescine, 1 to 8 ng / ml of sodium ascelenate, 1 to 5 mM of glutamine in DMEM (Dulbecco's Modified Eagle's Medium), 20 to 80 ug / ml of transferrin, , Insulin 1 to 10 ug / mL, thyroxine 0.1 to 1.5 ug / mL, and glucose 1 to 10 ug / mL. delete (A) contacting mouse cortical precursor cells to a medium composition comprising DMEM and 10% FBS for 7 to 10 days;
(B) shaking the container in which the cortical precursor cells are cultured at 100 to 300 rpm for 0.5 to 2 hours, discarding the supernatant, adding the medium composition and shaking at 200 to 300 rpm for 12 to 20 hours, Separating the supernatant containing the glial progenitor precursor from the bottom of the container through a shaking process except for the cell precursor attached to the bottom; And
(C) contacting the supernatant separated in step (B) to the medium composition of claim 1 for 5 to 10 days to induce differentiation, and inducing differentiation of the mouse papillary germ cells How to. delete 4. The method according to claim 3, wherein the culture medium in step (B) comprises DMEM and 10% FBS.

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