A neural stem cell induction differentiation medium, a preparation method and application thereof

Abstract

The application belongs to the technical field of culture medium, and particularly relates to a neural stem cell induction and differentiation culture medium, a preparation method and application thereof. The neural stem cell induction and differentiation culture medium comprises a basic culture medium and the following ingredients added in the basic culture medium: N-2 additive, transferrin, sodium selenite, iron ilex acid and fluridone. The neural stem cell induction and differentiation culture medium can significantly improve the effect of differentiating pluripotent stem cells into neural stem cells, and the culture medium does not add fetal bovine serum, thereby avoiding the risk of introducing heterologous serum carrying viruses and improving the safety of clinical application, and has a wide application prospect.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of culture medium, and particularly relates to a neural stem cell induction and differentiation culture medium and a preparation method and application thereof. BACKGROUND

[0002] Neural stem cells are a kind of stem cells with self-renewal ability and multi-directional differentiation potential, and exhibit a broad application prospect in the field of regenerative medicine. Neural stem cells can differentiate into neurons, astrocytes and oligodendrocytes, and exhibit great potential in treating neurodegenerative diseases and central nervous injury. With the acceleration of the global aging process and the increase in neuron-damaged diseases such as Alzheimer's disease, the technology of inducing neural stem cells to differentiate into functional neurons provides a possibility for the treatment of such diseases.

[0003] At present, the access to neural stem cells mainly includes directional transdifferentiation of embryonic stem cells and in-vitro differentiation of induced pluripotent stem cells. The directional transdifferentiation of embryonic stem cells involves a complex multi-stage induction process, and is controversial in ethics. However, the existing in-vitro differentiation technology of induced pluripotent stem cells has problems such as low induction efficiency, long cycle, and unstable differentiation results. The traditional neural stem cell induction and differentiation culture system usually adopts a culture medium containing fetal bovine serum, which contains a large number of growth factors and hormones with definite components. This not only has the risk of animal source pollution, but also may carry viruses, increasing the safety hidden danger of clinical application, resulting in large batch difference, uncontrollable differentiation and other problems, which seriously restricts the clinical application of neural stem cells, and makes the large-scale production of standard neural stem cell products face great challenges. Therefore, the optimization of the culture medium for inducing pluripotent stem cells to differentiate into neural cells has important clinical significance.

[0004] Based on the above purpose, the application provides a neural stem cell induction and differentiation culture medium and a preparation method and application thereof. SUMMARY

[0005] The first purpose of the application is to provide a neural stem cell induction and differentiation culture medium.

[0006] The first purpose of the application is achieved by the following technical scheme:

[0007] The neural stem cell induction and differentiation culture medium comprises a basic culture medium and the following components added in the basic culture medium: N-2 additive, transferrin, sodium selenite, iron ilex acid and fluridone.

[0008] Further, the concentration of the N-2 additive in the neural stem cell induction differentiation medium is 0.5-2 v / v%, the concentration of transferrin is 3-10 mg / L, the concentration of sodium selenite is 12-25 μg / L, the concentration of iron icilin is 8-20 μg / L, and the concentration of fluorofenamine is 25-40 μg / L.

[0009] Further, the concentration of the N-2 additive in the neural stem cell induction differentiation medium is 1.2 v / v%, the concentration of transferrin is 6 mg / L, the concentration of sodium selenite is 18 μg / L, the concentration of iron icilin is 14 μg / L, and the concentration of fluorofenamine is 32 μg / L.

[0010] Further, the base medium is DMEM / F12.

[0011] The second object of the present application is to provide a preparation method of the neural stem cell induction differentiation medium.

[0012] The second object of the present application is achieved by the following technical scheme:

[0013] According to the preparation method of the neural stem cell induction differentiation medium, the N-2 additive, transferrin, sodium selenite, iron icilin and fluorofenamine are added into the base medium according to the respective usage amount corresponding to the respective concentration, and then are dissolved and mixed uniformly, and then are filtered and sterilized, so as to obtain the neural stem cell induction differentiation medium.

[0014] The third object of the present application is to provide the application of the neural stem cell induction differentiation medium in the induction differentiation of neural stem cells.

[0015] According to the application of the neural stem cell induction differentiation medium, the induced pluripotent stem cells are differentiated into neural stem cells.

[0016] Further, the specific steps of the induction of the pluripotent stem cells into neural stem cells are as follows:

[0017] (1) the pluripotent stem cells are cultured by using the mTeSR-plus medium, and the single cells are obtained by cell digestion when the cell fusion degree reaches 70-80%;

[0018] (2) the single cells obtained in step (1) are washed and resuspended in the mTeSR-plus medium containing the ROCK inhibitor, and then are inoculated in the cell culture plate and cultured for 20-30 h;

[0019] (3) then, the cell culture medium of step (2) is replaced by the neural stem cell induction differentiation medium, the medium is replaced every 2 days, and the continuous culture is performed for 4-6 days.

[0020] Furthermore, in step (2), the ROCK inhibitor is Y-27632 or Thiazovivin.

[0021] Furthermore, in step (2), the concentration of the ROCK inhibitor in the mTeSR-plus medium is 8-10 μM.

[0022] Furthermore, in step (2), the inoculation density is 7-8 × 10⁻⁸. 3 / cm 2 .

[0023] Compared with the prior art, the main advantages of the present invention are:

[0024] This invention prepares a neural stem cell induction differentiation medium by adding N-2 additive, transferrin, sodium selenite, ferromalonic acid, and flufenidone to a basal culture medium. This medium significantly enhances the differentiation of pluripotent stem cells into neural stem cells. Furthermore, this medium does not contain fetal bovine serum, avoiding the risk of introducing viruses from heterologous serum and eliminating the need for antibiotics. Simultaneously, the raw materials for this medium are readily available, highly stable, and the feeder-free induction method avoids contamination from other cells, improving the safety of clinical applications and demonstrating broad application prospects. Attached Figure Description

[0025] Figure 1 Morphological image of neural stem cells obtained by culturing in the neural stem cell induction differentiation medium prepared in Example 1 of the present invention (magnification of 100x).

[0026] Figure 2 Morphological image of neural stem cells obtained by culturing in the neural stem cell induction differentiation medium prepared in Example 2 of the present invention (magnification of 100x).

[0027] Figure 3 Morphological image of neural stem cells obtained by culturing in the neural stem cell induction differentiation medium prepared in Example 3 of the present invention (magnification of 100x).

[0028] Figure 4 The image shows the morphology of neural stem cells obtained by culturing in the neural stem cell induction differentiation medium prepared in Comparative Example 1 of this invention (magnification of 100x).

[0029] Figure 5 The image shows the morphology of neural stem cells obtained by culturing in the neural stem cell induction differentiation medium prepared in Comparative Example 2 of this invention (magnification is 100x).

[0030] Figure 6 The image shows the morphology of neural stem cells obtained by culturing in the neural stem cell induction differentiation medium prepared in Comparative Example 3 of this invention (magnification is 100x).

[0031] Figure 7 The morphology of the neural stem cells obtained by culturing the neural stem cell induction differentiation medium prepared for the present application comparative example 4 (magnification 100 times) ;

[0032] Figure 8 The morphology of the neural stem cells obtained by culturing the neural stem cell induction differentiation medium prepared for the present application comparative example 5 (magnification 100 times) ;

[0033] Figure 9 The influence of different neural stem cell induction differentiation media on the expression level of neural stem cell marker SOX2;

[0034] Figure 10 The influence of different neural stem cell induction differentiation media on the expression level of neural stem cell marker Nestin. DETAILED DESCRIPTION

[0035] The following is a further detailed description of the present application in conjunction with specific preferred embodiments, which cannot be deemed to limit the specific implementation of the present application to these descriptions. For those of ordinary skill in the art to which the present application belongs, without departing from the concept of the present application, a number of simple deductions or substitutions can also be made, which should be deemed to fall within the protection scope of the present application. The specific conditions not mentioned in the examples are carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments used, if not specifically mentioned, are conventional products obtained through market channels.

[0036] Example 1

[0037] A neural stem cell induction differentiation medium, in terms of the final concentration of the neural stem cell induction differentiation medium, comprises DMEM / F12 basic medium and N-2 additive 1.2 v / v%, transferrin 6 mg / L, sodium selenite 18 μg / L, iron quebracho acid 14 μg / L, fluridone 32 μg / L added in the basic medium.

[0038] The specific preparation method of the neural stem cell induction differentiation medium is as follows:

[0039] N-2 additive, transferrin, sodium selenite, iron quebracho acid and fluridone are taken and added into the basic medium according to the use amount corresponding to the respective concentration, fully dissolved and mixed, and then filtered with a filter membrane to remove bacteria, to obtain the neural stem cell induction differentiation medium.

[0040] Example 2

[0041] A neural stem cell induction differentiation medium, based on the final concentration of the neural stem cell induction differentiation medium, includes DMEM / F12 basal medium and N-2 additives added to the basal medium at 0.5 v / v%, transferrin at 3 mg / L, sodium selenite at 12 μg / L, iron malonic acid at 8 μg / L, and flufenidone at 25 μg / L.

[0042] The specific preparation method of the neural stem cell differentiation induction culture medium is as follows:

[0043] Take N-2 additive, transferrin, sodium selenite, iron malonic acid, and flufenidone, and add them to the basal culture medium according to their respective concentrations and dosages. Dissolve and mix thoroughly, then filter through a filter membrane to remove bacteria, thus obtaining the neural stem cell induction differentiation culture medium.

[0044] Example 3

[0045] A neural stem cell induction differentiation medium, based on the final concentration of the neural stem cell induction differentiation medium, includes DMEM / F12 basal medium and N-2 additive 2v / v%, transferrin 10 mg / L, sodium selenite 25 μg / L, iron malonic acid 20 μg / L, and flufenidone 40 μg / L added to the basal medium.

[0046] The specific preparation method of the neural stem cell differentiation induction culture medium is as follows:

[0047] Take N-2 additive, transferrin, sodium selenite, iron malonic acid, and flufenidone, and add them to the basal culture medium according to their respective concentrations and dosages. Dissolve and mix thoroughly, then filter through a filter membrane to remove bacteria, thus obtaining the neural stem cell induction differentiation culture medium.

[0048] Comparative Example 1

[0049] The difference between Comparative Example 1 and Example 1 is that iron-malolactic acid is omitted from the composition of the neural stem cell induction differentiation culture medium.

[0050] Comparative Example 2

[0051] The difference between Comparative Example 2 and Example 1 is that flufenidone is omitted from the composition of the neural stem cell induction differentiation culture medium.

[0052] Comparative Example 3

[0053] The difference between Comparative Example 3 and Example 1 is that the amount of iron-containing malolactic acid in the neural stem cell induction differentiation culture medium is 40 μg / L, while the rest are the same as in Example 1.

[0054] Comparative Example 4

[0055] The difference between Comparative Example 4 and Example 1 is that the amount of firogptifimbine in the component of the neural stem cell induction and differentiation medium is 5 μg / L, and the others are the same as those in Example 1.

[0056] Comparative Example 5

[0057] The difference between Comparative Example 5 and Example 1 is that the amount of firogptifimbine in the component of the neural stem cell induction and differentiation medium is 60 μg / L, and the others are the same as those in Example 1.

[0058] Test Example 1

[0059] (1) The pluripotent stem cells were cultured using mTeSR-plus medium, and when the cell confluence reached 75%, the cells were digested to obtain single cells.

[0060] (2) The single cells obtained in step (1) were washed and resuspended in mTeSR-plus medium containing 10 μM ROCK inhibitor (Y-27632), and then inoculated in Matrigel-coated cell culture plates at a density of 8 x 10 3 / cm 2 for 24 h.

[0061] (3) Then, the medium of step (2) was replaced with the neural stem cell induction and differentiation medium prepared in Examples 1-3 and Comparative Examples 1-5, and the cells were further cultured in a cell incubator at 37°C and 5% CO2, with medium replacement every 2 days. After 6 days of continuous culture, the differentiated neural stem cells were obtained. The morphologies of the neural stem cells cultured in the neural stem cell induction and differentiation medium of Examples 1-3 and Comparative Examples 1-5 are shown in Figures 1-8 , where the magnification is 100 times.

[0062] The results are shown in Figures 1-8 , which are the morphologies of the neural stem cells cultured in the neural stem cell induction and differentiation medium prepared in Examples 1-3 and Comparative Examples 1-5. As can be seen from the figure, compared with Comparative Examples 1-5, the neural stem cell induction and differentiation medium prepared in Examples 1-3 has better induction and differentiation effect. It is shown that by adding ilexgenin A and firogptifimbine to the basic medium, the effect of differentiating pluripotent stem cells into neural stem cells can be significantly improved.

[0063] Test Example 2

[0064] The neural stem cells obtained by culturing the neural stem cell induction medium of Example 1-3 and Comparative Example 1-5 of Test Example 1 were collected, and the expression levels of neural stem cell markers were detected by using fluorescence quantitative PCR. The total RNA of the neural stem cells was extracted by using an RNA extraction kit, and the cDNA was obtained by reverse transcription of the obtained RNA by using a PrimeScript RT Master Mix kit. The specific operation method is described in the kit instruction manual. The cDNA was used as a template, and the expression levels of neural stem cell markers SOX2 and Nestin in different groups were detected by using real-time fluorescence quantitative PCR technology. The specific operation steps are as follows:

[0065] (1) Design of primers of neural stem cell markers SOX2, Nestin and internal reference gene β-actin

[0066] The primers were designed according to the sequences of SOX2 and Nestin genes, and β-actin gene was used as an internal reference gene. The upstream primer sequence of the SOX2 gene is shown in SEQ ID NO. 1, and the downstream primer sequence is shown in SEQ ID NO. 2; the upstream primer sequence of the Nestin gene is shown in SEQ ID NO. 3, and the downstream primer sequence is shown in SEQ ID NO. 4; and the upstream primer sequence of the internal reference gene β-actin is shown in SEQ ID NO. 5, and the downstream primer sequence is shown in SEQ ID NO. 6. The quantitative PCR primer sequences are shown in Table 1. Each PCR reaction was set in triplicate.

[0067] Table 1 Sequence table

[0068]

[0069] (2) Fluorescence quantitative PCR reaction program

[0070] Real-time fluorescence quantitative PCR detection was performed by using a 2 × SYBR Green qPCR Master Mix kit, and the primers of neural stem cell markers SOX2 and Nestin and the primers of β-actin internal reference gene were synthesized in advance. The qPCR reaction was performed on a fluorescence quantitative PCR instrument, and the cDNA sequence corresponding to the stem cell markers SOX2 and Nestin was amplified. The fluorescence quantitative PCR reaction system is shown in Table 2. The cDNA was used as a template, and the upstream and downstream primer sequences of the neural stem cell markers SOX2 and Nestin were added, respectively, and the reaction system was mixed and configured with SYBR dye and sterile water. The fluorescence quantitative PCR reaction program is shown in Table 3. Each sample was set in triplicate, β-actin was used as an internal reference, and the relative expression amount of the neural stem cell marker was calculated by using the Ct method, and the formula was Ct=Ct 目的基因 -Ctβ-actin Each group of tests was performed in triplicate. The relative expression of neural stem cell markers SOX2 and Nestin was achieved by drawing a column chart, and the results are shown in FIGS. Figure 9 、 Figure 10

[0071] Table 2 Fluorescent quantitative PCR system

[0072]

[0073] Table 3 Fluorescent quantitative PCR reaction program

[0074]

[0075] The results are shown in FIGS. Figure 9 、 Figure 10 , respectively, the effects of different groups of neural stem cell induction and differentiation medium on the expression levels of neural stem cell markers SOX2 and Nestin. As can be seen from the figures, compared with the comparative examples 1-5, the expression levels of neural stem cell markers SOX2 and Nestin in the neural stem cells obtained by the medium induction and differentiation of the inventive examples 1-3 were significantly increased, which proved that the neural stem cell induction and differentiation medium could successfully induce pluripotent stem cells into neural stem cells. It is shown that after adding ilegans acid and fluridone to the basic medium, the effect of differentiating pluripotent stem cells into neural stem cells can be significantly improved.

[0076] Further analysis shows that, compared with example 1, the composition of the neural stem cell induction and differentiation medium of comparative example 1 omits ilegans acid; the composition of the neural stem cell induction and differentiation medium of comparative example 2 omits fluridone; the composition of the neural stem cell induction and differentiation medium of comparative example 3 uses 40 μg / L of ilegans acid; the composition of the neural stem cell induction and differentiation medium of comparative example 4 uses 5 μg / L of fluridone; and the composition of the neural stem cell induction and differentiation medium of comparative example 5 uses 60 μg / L of fluridone, and the induction effects of the five groups of neural stem cell induction and differentiation medium are significantly decreased. It can be seen that when the concentration of fluridone in the neural stem cell induction and differentiation medium is 25-40 μg / L and the concentration of ilegans acid is 8-20 μg / L, the effect of promoting pluripotent stem cells to differentiate into neural stem cells is the best.

[0077] Finally, it should be pointed out that the above examples are only used to illustrate the technical solutions of the present application, but not to limit it. The basic principles and main features of the present application have been described above with specific embodiments, and some modifications or substitutions can be made on the basis of the present application, but these modifications or substitutions do not make the corresponding technical solutions deviate from the scope of the present application.​

Claims

1. A neural stem cell induction differentiation medium, characterized by, The neural stem cell induction differentiation medium comprises a basic medium and the following components added in the basic medium: N-2 supplement, transferrin, sodium selenite, iron tannic acid and fluorofenamine; the concentration of N-2 supplement in the neural stem cell induction differentiation medium is 0.5-2 v / v %, the concentration of transferrin is 3-10 mg / L, the concentration of sodium selenite is 12-25 μg / L, the concentration of iron tannic acid is 8-20 μg / L, and the concentration of fluorofenamine is 25-40 μg / L.

2. The neural stem cell induction differentiation medium according to claim 1, wherein, The concentration of N-2 supplement in the neural stem cell induction differentiation medium is 1.2 v / v %, the concentration of transferrin is 6 mg / L, the concentration of sodium selenite is 18 μg / L, the concentration of iron tannic acid is 14 μg / L, and the concentration of fluorofenamine is 32 μg / L.

3. The neural stem cell induction differentiation medium of claim 1, wherein, The basic medium is DMEM / F12.

4. The method for preparing the neural stem cell induction differentiation culture medium according to any one of claims 1-3, characterized in that, N-2 supplement, transferrin, sodium selenite, iron tannic acid and fluorofenamine are added into the basic medium according to the respective usage amount corresponding to the respective concentration, dissolved and mixed thoroughly, then filtered and sterilized to obtain the neural stem cell induction differentiation medium.

5. Use of a neural stem cell induction differentiation medium according to any one of claims 1 to 3, characterized in that, The neural stem cell induction differentiation medium is used for inducing pluripotent stem cells to differentiate into neural stem cells.

6. The use of the neural stem cell induction differentiation medium according to claim 5, characterized in that, The specific steps for inducing pluripotent stem cells to differentiate into neural stem cells are as follows: (1) pluripotent stem cells are cultured using mTeSR-plus medium, and when the cell confluence reaches 70-80 %, the cells are digested to obtain single cells; (2) the single cells obtained in step (1) are washed and resuspended in mTeSR-plus medium containing a ROCK inhibitor, and then inoculated in a cell culture plate for culture for 20-30 h; (3) then the medium of step (2) is replaced with the neural stem cell induction differentiation medium, and the medium is replaced every 2 days, and the cells are continuously cultured for 4-6 days to obtain the neural stem cells.

7. The use of the neural stem cell induction differentiation medium according to claim 6, characterized in that, In step (2), the ROCK inhibitor is Y-27632 or Thiazovivin.

8. The use of the neural stem cell induction differentiation medium according to claim 6, characterized in that, In step (2), the concentration of the ROCK inhibitor in the mTeSR-plus medium is 8-10 μM.

9. The use of the neural stem cell induction differentiation medium according to claim 6, characterized in that, The inoculation density in step (2) is 7-8 x 10 3 / cm 2 .

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