Neural stem cell culture medium and application thereof in passively controlling size of neural stem cell aggregate

Abstract

The invention discloses a neural stem cell culture medium which is composed of an Rho kinase inhibitor Y-27632, an HDAC inhibitor sodium valproate, an E-cadherin regulator 8-bromocyclic adenylate, a Wnt signal channel regulator CHIR99021, an anti-aggregation agent polyether F-68, heparin and an NSC complete culture medium. The neural stem cell culture medium is applied to passive control of the size of the neural stem cell aggregate. The invention further discloses a passive control method for the size of the neural stem cell aggregate. The neural stem cell culture medium provided by the invention can effectively and accurately control the size of the cell aggregate, improves the consistency and the cell viability of the cell aggregate under the condition of not influencing the physiological characteristics of cells, avoids the center necrosis problem of the aggregate, provides a more economical and efficient scheme for large-scale culture of neural stem cells, and has wide application prospects. And the application prospects in the fields of cell therapy, drug screening and the like are expanded.

Description

Technical Field

[0001] The invention relates to a neural stem cell culture medium and application thereof in passively controlling the size of neural stem cell aggregates, belonging to the technical field of neural stem cell culture. Background Art

[0002] Neural stem cells tend to form oversized aggregates in suspension culture, leading to central hypoxia and cell necrosis, which in turn affects cell activity and proliferation. Existing technologies primarily rely on mechanical stirring, microcarriers, or microfluidics to control aggregate size. Mechanical stirring, due to the shear force generated during operation, can damage cells, making it difficult to precisely control the microenvironment in which cells reside, making it difficult to achieve the desired effect and posing a risk of contamination. Microcarrier or microfluidics require microcarriers or microfluidic chips to control aggregate size, which has drawbacks such as high cost, high technical difficulty, complex cell harvesting, lack of technical standards and specifications, poor reproducibility, and potential adverse effects on cell physiological properties. Furthermore, additional mechanical and physical methods increase risks, and their safety remains to be investigated.

[0003] Previous studies have shown that culture medium additives can affect cell adhesion and aggregation. However, in practice, the types, concentrations, addition times, and durations of various regulators vary due to differences in cell sources and cell culture environments. Currently, there is a lack of systematic optimization methods for precisely controlling aggregate size, particularly for passively controlling the size of neural stem cell aggregates. Therefore, a simpler, more efficient method for controlling the size of neural stem cell aggregates suitable for large-scale automated production is urgently needed. Summary of the Invention

[0004] In view of the above-mentioned prior art, the present invention provides a neural stem cell culture medium and its application in passively controlling the size of neural stem cell aggregates.

[0005] The present invention is achieved through the following technical solutions:

[0006] A neural stem cell culture medium is composed of a Rho kinase inhibitor Y-27632, an HDAC inhibitor sodium valproate, an E-cadherin regulator 8-bromocyclic adenosine monophosphate (8-bromo-cAMP), a Wnt signaling pathway regulator CHIR99021, an anti-aggregation agent pluronic F-68, heparin, and a complete NSC culture medium. The concentrations of the components are as follows: Y-27632, 5-10 μM; sodium valproate, 0.5-5 mM; 8-bromocyclic adenosine monophosphate, 0.001-1.0 mM; CHIR99021, 1-3 μM; pluronic F-68, 0.1%-1% (w / v, g / mL); and heparin, 1-10 U / mL. The remainder is the complete NSC culture medium.

[0007] The standard name of the Rho kinase inhibitor Y-27632 is: (+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride.

[0008] The standard name of the Wnt signaling pathway regulator CHIR99021 is: 6-[[2-[[4-(2,4-dichlorophenyl)-5-(4-methyl-1H-imidazol-2-yl)pyrimidin-2-yl]amino]ethyl]amino]nicotinonitrile.

[0009] Furthermore, the concentrations of the components are as follows: Y-27632, 10 μM; sodium valproate, 3 mM; 8-bromocyclic adenosine monophosphate, 0.2 mM; CHIR99021, 2 μM; polyether F-68, 0.5%; heparin, 5 U / mL; and the remainder is NSC complete culture medium.

[0010] Furthermore, the NSC complete culture medium is selected from DMEM / F12 complete culture medium.

[0011] The neural stem cell culture medium is used in passively controlling the size of neural stem cell aggregates, and is used as or in preparing a preparation for passively controlling the size of neural stem cell aggregates.

[0012] A method for passively controlling the size of neural stem cell aggregates is as follows: human neural stem cell culture medium is taken and the initial cell density is adjusted to 0.5×10 6 ~2×10 6 cells / mL, add the above neural stem cell culture medium, and culture at 37°C and 5% CO2.

[0013] The present invention achieves precise control of the size of cell aggregates through culture medium additives (Rho kinase inhibitor Y-27632, HDAC inhibitor sodium valproate, E-cadherin regulator 8-bromo-cAMP, Wnt signaling pathway regulator CHIR99021, anti-aggregation agent polyether F-68 and heparin). Among them, Y-27632 inhibits Rho kinase activity, regulates cytoskeleton-related cell-to-cell connections and cell migration, and thus affects the aggregation state of cell spheres, making the cell spheres more uniform in shape and size; sodium valproate affects the related histone deacetylation process, thereby regulating gene expression and changing the cell Some biological behaviors of the cell-cell interaction affect the adhesion and aggregation characteristics between cells, helping to make the morphology and aggregation of cell spheroids more uniform. CHIR99021 and 8-bromo-cAMP promote cell-cell adhesion by regulating the Wnt signaling pathway and E-cadherin-related signaling pathways, respectively, making the aggregation of cell spheroids more compact and uniform. These culture medium additives can effectively and precisely control the size of cell aggregates without relying on additional mechanical or physical means. At the same time, they improve the consistency and cell viability of cell aggregates without affecting the physiological properties of cells, avoiding the problem of central necrosis of aggregates, making them suitable for large-scale production. All culture medium additives are commercially available products in the prior art.

[0014] The neural stem cell culture medium and the method for passively controlling the size of neural stem cell aggregates of the present invention have the following advantages:

[0015] (1) Only culture medium additives are used to control the size of cell aggregates, without the need for complex mechanical equipment, microcarriers, and microfluidic chips, thus reducing operational difficulty and cost;

[0016] (2) Through the rational and effective combination of culture medium additives, the size of cell aggregates can be more accurately controlled, while at the same time, the consistency and cell viability of cell aggregates can be improved without affecting the physiological characteristics of cells, thus avoiding the problem of central necrosis of aggregates;

[0017] (3) The culture medium additives added are basically drugs that are widely used in clinical practice and new drug research, and have many advantages such as compliance with regulations, clear ingredients, reliable quality, good stability, and high safety;

[0018] (4) Suitable for large-scale cell culture systems and can be integrated into automated production processes;

[0019] (5) It is applicable to the passive control method of the size of neural stem cell aggregates, providing a more economical and efficient solution for the large-scale cultivation of neural stem cells, and also expanding the application prospects in the fields of cell therapy, drug screening, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 : Photographs of cells in Example.

[0021] Figure 2 : Photograph of cells of Comparative Example 1.

[0022] Figure 3 : Photograph of cells of Comparative Example 2.

[0023] Figure 4 : Photograph of cells of Comparative Example 3.

[0024] Figure 5 : Photograph of cells of Comparative Example 4.

[0025] Figure 6 : Photograph of cells of Comparative Example 5.

[0026] Figure 7 : Comparison of cell diameters (μm).

[0027] Figure 8 : Comparison of cell viability (%).

[0028] Figure 9 : Comparison of cell proliferation times.

[0029] Figure 10 : Comparison of stemness markers (%).

[0030] Figure 11 : Comparison of cell apoptosis ratio (%). DETAILED DESCRIPTION

[0031] The present invention will be further described below in conjunction with the embodiments. However, the scope of the present invention is not limited to the following embodiments. Those skilled in the art will appreciate that various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention.

[0032] Unless otherwise specified, the instruments, reagents, and materials involved in the following examples are all conventional instruments, reagents, and materials available in the prior art and can be obtained through regular commercial channels. The experimental methods and detection methods involved in the following examples are all conventional experimental methods and detection methods available in the prior art, unless otherwise specified.

[0033] The NSC complete culture medium used in the present invention is DMEM / F12 complete culture medium (Gibco).

[0034] Example Neural Stem Cell Culture Medium and Passive Control Method for Neural Stem Cell Aggregate Size

[0035] (1) Preparation of neural stem cell culture

[0036] A neural stem cell culture medium is composed of a Rho kinase inhibitor Y-27632, an HDAC inhibitor sodium valproate, an E-cadherin regulator 8-bromo-cAMP, a Wnt signaling pathway regulator CHIR99021, an anti-aggregation agent pluronic F-68, heparin, and a complete NSC culture medium, wherein the concentrations of the components are as follows: Y-27632, 10 μM; sodium valproate, 3 mM; 8-bromo-cAMP, 0.2 mM; CHIR99021, 2 μM; pluronic F-68, 0.5%; heparin, 5 U / mL; and the remainder is a complete NSC culture medium.

[0037] The preparation method of the neural stem cell culture medium is as follows: Y-27632, sodium valproate, 8-bromo-cAMP, CHIR99021, polyether F-68 and heparin are added to the NSC complete culture medium, and mixed evenly to obtain the culture medium.

[0038] (II) Passive control method of neural stem cell aggregate size

[0039] Here are the steps:

[0040] (1) Take human neural stem cell culture medium and adjust the initial cell density to 1×10 6 cells / mL.

[0041] (2) Add 5 mL of human neural stem cell culture medium to a T75 culture flask, and then add 10 mL of neural stem cell culture medium. Culture at 37°C and 5% CO2 for 7 days without changing the medium.

[0042] (3) After 7 days of culture, take photos under a microscope to count their diameters.

[0043] (4) The cultured human neural stem cells were digested with Accutase (cell dissociation reagent) to form a single cell suspension, and the cells were counted using an AOPI cell counter to calculate the cell viability and cell proliferation times.

[0044] (5) Take 4×10 6 cells / group, and flow cytometry was used to detect stemness markers and cell apoptosis.

[0045] (3) Setting of comparison ratio

[0046] The neural stem cells were cultured in the above-prepared neural stem cell culture medium as the experimental group. At the same time, five control groups (numbered as comparative examples 1 to 5) were set up. The culture media used to culture the neural stem cells in the five control groups were as follows:

[0047] Comparative Example 1: NSC complete culture medium.

[0048] Comparative Example 2: Composed of the following components at the following concentrations: sodium valproate, 3 mM; 8-bromo-cAMP, 0.2 mM; CHIR99021, 5 μM; polyether F-68, 0.5%; heparin, 5 U / mL; the remainder being complete NSC culture medium. That is, compared to Example 2, the Rho kinase inhibitor Y-27632 was not included.

[0049] Comparative Example 3: Composed of the following components at the following concentrations: Y-27632, 10 μM; sodium valproate, 8 mM; 8-bromo-cAMP, 0.2 mM; CHIR99021, 2 μM; the remainder being complete NSC culture medium. That is, compared to Example 3, the anti-aggregating agent polyether F-68 and heparin were not included.

[0050] Comparative Example 4: Composed of the following components at the following concentrations: Y-27632, 10 μM; sodium valproate, 3 mM; CHIR99021, 2 μM; pluronic acid F-68, 0.5%; heparin, 5 U / mL; the remainder being complete NSC culture medium. That is, compared to Example 4, the E-cadherin regulator 8-bromo-cAMP was not included.

[0051] Comparative Example 5: Composed of the following components at the following concentrations: Y-27632, 10 μM; sodium valproate, 3 mM; 8-bromo-cAMP, 0.2 mM; CHIR99021, 5 μM; heparin, 5 U / mL; the remainder being complete NSC culture medium. That is, compared to Example 5, the anti-aggregating agent, polyether F-68, was not included.

[0052] The method for culturing neural stem cells is the same as that described in (2) above.

[0053] (IV) Experimental results

[0054] (1) Comparison of cell spheroid morphology and diameter

[0055] The cells of Examples and Comparative Examples 1 to 5 were cultured for 7 days, and white light photos of not less than 100 cell spheres were taken under an inverted microscope (10X) in each group of cells. The photos of the cells cultured in Examples and Comparative Examples 1 to 5 are as follows: Figures 1 to 6 Image pro 6.0 software was used to calculate the diameter of the cell spheres in each group. The results are shown in Table 1. The cell diameter comparison chart is shown in Figure 7 shown.

[0056] Table 1

[0057] Group Cell morphological changes Cell diameter (μm) Example Suspended spheres, uniform refraction, and uniform dispersion of cell spheres 151±20 Comparative Example 1 Suspended spheres, uniform refraction, and uniform dispersion of cell spheres 173±46 Comparative Example 2 Suspended spherical, irregular cell spheres 89±26 Comparative Example 3 Suspended spherical cells, uneven refraction, many dead cells, small cell spheres 110±40 Comparative Example 4 Suspended spheres, loose and irregular cell spheres, and significantly smaller cell spheres 120±68 Comparative Example 5 Suspended spheres, uneven cell size, and adherence to the wall 101±52

[0058] Depend on Figure 1As can be seen from Figure 1 and Table 1, the cell sphere morphology of Example and Comparative Example 1 both showed typical neural cell sphere morphology, but the standard deviation of the diameter of the cell sphere of Example was significantly lower than that of Comparative Example 1, indicating that the uniformity of the cell sphere of Example was better than that of Comparative Example 1; the diameter of the cell sphere of Comparative Examples 2 to 5 was too small, which affected its cell proliferation, biological characteristics and differentiation ability.

[0059] (2) Comparison of cell viability and cell proliferation

[0060] The cells cultured for 7 days in Examples and Comparative Examples 1 to 5 were digested with Accutase to form a single cell suspension, 40 μL of each suspension was taken, and the cell viability was detected by AOPI cell counting method. The results are shown in Table 2. The cell viability comparison is shown in Figure 2. Figure 8 As shown in the figure, the cell proliferation times comparison is shown in Figure 9 shown.

[0061] Table 2

[0062] Group Cell viability (%) Cell proliferation multiples Example 96.18 6.52 Comparative Example 1 93.57 5.91 Comparative Example 2 85.31 3.25 Comparative Example 3 82.36 3.09 Comparative Example 4 89.29 4.27 Comparative Example 5 87.72 4.06

[0063] From Table 2, Figure 8 、 Figure 9 It can be seen that compared with the comparative examples, the examples performed better in terms of both cell survival rate and cell proliferation multiples, indicating that the neural stem cell culture medium used in the examples has obvious advantages in maintaining cell survival and promoting cell proliferation.

[0064] (3) Comparison of stemness markers and cell apoptosis

[0065] The cells cultured for 7 days in Examples and Comparative Examples 1 to 5 were digested with Accutase to form a single cell suspension. 3×10 6 Cells were used for stemness marker detection, 1×10 6 Cells were used for cell cycle analysis, 1×10 6 The cells were used for apoptosis detection, and the results are shown in Table 3. The comparison of stemness markers is shown in Figure 3. Figure 10 As shown in the figure, the comparison of cell apoptosis ratio is shown in Figure 11 shown.

[0066] Table 3

[0067]

[0068] From Table 3, Figure 10 、 Figure 11It can be seen that compared with the comparative examples, the cell apoptosis rate of the embodiment is significantly lower than that of comparative examples 1 to 5, which indicates that the neural stem cell culture medium of the embodiment can effectively inhibit cell apoptosis and maintain cell activity, possibly by better maintaining the expression of stemness markers and other mechanisms, so that the cells are in a more stable, undifferentiated and well-survived state. This shows that the various additives in the culture medium prepared by the present invention interact and regulate cell aggregation in terms of signal pathway coordination and intercellular connection regulation. For example, sodium valproate and Y-27632 work together to make the interaction between cells in the cell sphere more orderly, which is conducive to maintaining uniformity. In addition, 8-bromo-cAMP ensures that the cells in the cell sphere adhere tightly and orderly from different angles, avoiding local excessive aggregation or looseness, and further optimizing the aggregation state of the cell sphere. Then, under the pathway regulation of CHIR99021, the cells are guided by orderly signals and are in the appropriate position in the cell sphere, ensuring the uniformity of the overall structure of the cell sphere.

[0069] (4) Conclusion

[0070] Comparison of neural stem cells cultured in the neural stem cell culture medium of the present invention in terms of cell morphology, cell diameter, cell viability, cell proliferation ratio, flow cytometry (cycle, apoptosis), etc., demonstrates that the neural stem cell culture medium of the present invention can more accurately control the size of cell aggregates, while improving the consistency and cell viability of cell aggregates without affecting the physiological properties of the cells, thereby avoiding the problem of central necrosis of the aggregates.

[0071] The above examples are provided to those skilled in the art to fully disclose and describe how to implement and use the claimed embodiments, rather than to limit the scope of the disclosure herein. Modifications that are obvious to those skilled in the art will fall within the scope of the appended claims.

Claims

1. A neural stem cell culture medium, characterized in that: The invention is composed of a Rho kinase inhibitor Y-27632, an HDAC inhibitor sodium valproate, an E-cadherin regulator 8-bromocyclic adenosine monophosphate (BAMP), a Wnt signaling pathway regulator CHIR99021, an anti-aggregation agent polyether F-68, heparin, and a complete NSC culture medium. The concentrations of the components are as follows: Y-27632, 5 to 10 μM; sodium valproate, 0.5 to 5 mM; 8-bromocyclic adenosine monophosphate (BAMP), 0.001 to 1.0 mM; CHIR99021, 1 to 3 μM; polyether F-68, 0.1% to 1%; heparin, 1 to 10 U / mL; and the remainder is a complete NSC culture medium.

2. The neural stem cell culture medium according to claim 1, characterized in that The concentrations of the components are as follows: Y-27632, 10 μM; sodium valproate, 3 mM; 8-bromocyclic adenosine monophosphate, 0.2 mM; CHIR99021, 2 μM; polyether F-68, 0.5%; heparin, 5 U / mL; and the remainder is NSC complete culture medium.

3. The neural stem cell culture medium according to claim 1, wherein: The NSC complete culture medium is selected from DMEM / F12 complete culture medium.

4. Use of the neural stem cell culture medium according to claim 1 or 2 in passively controlling the size of neural stem cell aggregates.

5. Use of the neural stem cell culture medium according to claim 1 or 2 as or in the preparation of a preparation for passively controlling the size of neural stem cell aggregates.

6. A method for passively controlling the size of neural stem cell aggregates, characterized in that: Take the culture medium of human neural stem cells and adjust the initial cell density to 0.5×10 6 ~2×10 6 cells / mL, add the neural stem cell culture medium according to claim 1, 2 or 3, and culture at 37°C and 5% CO2.

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