Use of simulated weightlessness culture method and / or mechanically sensitive ion channel inhibitor for in vitro culture of motor neurons

Abstract

The present application relates to the use of a simulated weightlessness culture method and / or a mechanically sensitive ion channel inhibitor for in vitro culture of motor neurons; in addition, the invention also relates to a method for culturing motor neurons in vitro. The method comprises the step of culturing cells by using a simulated weightlessness culture method and / or a mechanical sensitive ion channel inhibitor as an auxiliary means.

Description

Technical Field

[0001] The present application relates to the field of cell culture, in particular, to the use of simulated weightlessness culture method and / or mechanosensitive ion channel inhibitors for in vitro culture of motor neurons; and also to a method for in vitro culture of motor neurons, comprising: using simulated weightlessness culture method and / or mechanosensitive ion channel inhibitors as auxiliary means for cell culture. Background Art

[0002] In the field of neuroscience, in vitro culture of motor neurons is a key technology that provides an important tool for studying neural development, physiology, drug screening, and neurological diseases. Motor neurons are part of the central nervous system and are responsible for controlling the movement of skeletal muscles. They are essential for understanding motor coordination and the study of neurological diseases.

[0003] Traditionally, the study of motor neurons has relied on animal models, such as mice or rats, and human anatomical tissue. However, these approaches have some challenges: there are ethical and regulatory restrictions on using animal models for research, and animal models cannot fully represent humans due to species differences. Access to human anatomical tissue is restricted, and the number of samples is usually limited, making it difficult to support large-scale studies or drug screening.

[0004] In recent years, with the progress of cell biology, bioengineering and materials science, the technology of culturing motor neurons in vitro has made significant progress. Embryonic stem cells and induced pluripotent stem cells (iPSC) are one of the most common in vitro culture methods. Currently, a variety of technologies and protocols have been developed to direct the differentiation of stem cells into neural precursor cells and then further differentiate them into motor neurons. The continuous innovation of these technologies has made it more feasible to culture motor neurons in vitro, and the obtained motor neurons have been widely used in research in various fields, enabling researchers to better understand the development, function and mechanisms of motor neurons and related diseases.

[0005] Although the technology of in vitro motor neuron differentiation has made significant progress, there are still some limitations and challenges. It is not easy to generate a large number of highly pure cell populations quickly, which is usually required for biochemical analysis, disease modeling and clinical applications. This demand poses a severe challenge to the stem cell field. For this, please refer to: (1) (Du and Chen et al., 2015) This article provides an in vitro culture scheme for gradually differentiating motor neurons from human pluripotent stem cells. This technology can be used to obtain highly pure human motor neurons in vitro, but it takes a long time. (2) (Cui and Yin et al., 2021) This article studies the effect of simulated microgravity on the fate determination of rat neural stem cells: under microgravity conditions, rat neural stem cells are more inclined to neuronal differentiation, but not conducive to glial differentiation. This article reveals the effect of microgravity on in vitro cell culture, but only studies its effect on neural stem cell differentiation. Rats are used instead of humans, and motor neurons and cell maturation are not involved. (3) (Suchyna and Tape et al., 2004) This article describes GsMTx4, a selective inhibitor of mechanosensitive ion channels, a pharmacological tool that can be used for mechanosensitive ion channel inhibition and its mechanism of inhibiting SACs. Summary of the invention

[0006] The present application provides an in vitro culture method for promoting the maturation of motor neurons. The applicant found that when MNP cells were differentiated under simulated microgravity conditions, the motor neuron-specific markers of the obtained cells were expressed earlier and faster, and developed a method for promoting the maturation of motor neuron precursor cells using simulated microgravity. This method is not only efficient and simple, but also meets the requirements of economic efficiency. Relatively speaking, the prior art has completed (1) (Du and Chen et al., 2015) This article provides an in vitro culture scheme for gradually differentiating motor neurons from human pluripotent stem cells. This technology can be used to obtain high-purity human motor neurons in vitro, but it takes a long time. The present invention adds microgravity intervention to this technology, and more mature motor neurons can be obtained in the same time. (2) (Cui and Yin et al., 2021) This article studies the effect of simulated microgravity on the fate determination of rat neural stem cells: rat neural stem cells under microgravity conditions are more inclined to neuronal differentiation, but not conducive to glial differentiation. This article reveals the effect of microgravity on in vitro cell culture, but only studies its effect on neural stem cell differentiation. Rats are used instead of humans, and motor neurons and cell maturation are not involved. This application uses motor neuron precursor cells derived from human embryonic stem cells, and discovered the role of microgravity in promoting the maturation of motor neurons. This application provides a method for culturing motor neurons in vitro using small molecules to simulate microgravity to promote the maturation of motor neurons. The applicant found that when adding a mechanosensitive ion channel inhibitor (GsMTx4, 5 μM) to the culture medium to differentiate MNP cells, the motor neuron-specific markers of the obtained cells were expressed earlier and faster, and developed a method for promoting the maturation of motor neuron precursor cells using small molecule GsMTx4 to simulate microgravity. This method is not only efficient and simple, but also meets economic requirements.

[0007] This application provides:

[0008] 1. Use of simulated weightlessness culture method and / or mechanosensitive ion channel inhibitor for in vitro culture of motor neurons.

[0009] 2. The use according to item 1, wherein the mechanosensitive ion channel inhibitor is GsMTx4.

[0010] 3. The use according to item 1, wherein when the mechanosensitive ion channel inhibitor is used alone, the culture medium comprises: a basal medium consisting of DMEM / F12 and Neurobasal, N2, B27, Glutamax, ascorbic acid, RA, SAG and GsMTx4;

[0011] Preferably, the composition of the culture medium is: DMEM / F12:Neurobasal=1:0.5-2 basal medium, (0.05-5)×N2, (0.05-5)×B27, (0.5-2)×Glutamax, 0.05-0.2 mM ascorbic acid, 0.005-0.02 μM RA, 0.05-0.2 μM SAG and 3-7 μM GsMTx4;

[0012] It is particularly preferred that the composition of the culture medium is: DMEM / F12:Neurobasal=1:1 basal medium, 0.5×N2, 0.5×B27, 1×Glutamax, 0.1 mM ascorbic acid, 0.01 μM RA, 0.1 μM SAG and 5 μM GsMTx4.

[0013] 4. The use according to item 1, wherein the culture conditions used when the simulated weightlessness culture method is used alone are: the culture medium is composed of a basal medium consisting of DMEM / F12 and Neurobasal, N2, B27, Glutamax, ascorbic acid, RA, SAG, a two-dimensional (2D) gyroscope is used to simulate microgravity, which rotates around a horizontal axis to form microgravity, and the culture is carried out under simulated microgravity for more than 3 days;

[0014] Preferably, the culture conditions are: a culture medium composed of: DMEM / F12:Neurobasal=1:0.5-2 basal culture medium, (0.05-5)×N2, (0.05-5)×B27, (0.5-2)×Glutamax, 0.05-0.2 mM ascorbic acid, 0.005-0.02 μM RA, 0.05-0.2 μM SAG, using a two-dimensional (2D) gyroscope to simulate microgravity, rotating around a horizontal axis at a speed of 20-40 rpm to form microgravity, culturing for 4-6 days under simulated microgravity, and changing the medium every other day;

[0015] Particularly preferred culture conditions are: the culture medium composition is: DMEM / F12:Neurobasal=1:1 basal culture medium, 0.5×N2, 0.5×B27, 1×Glutamax, 0.1mM ascorbic acid, 0.01μM RA, 0.1μM SAG, using a two-dimensional (2D) gyroscope to simulate microgravity, rotating around a horizontal axis at a speed of 30rpm to form microgravity, culturing under simulated microgravity for 5 days, and changing the medium every other day.

[0016] 5. The use as described in item 1, wherein the culture conditions when the simulated weightlessness culture method and the mechanosensitive ion channel inhibitor are used together are: the culture medium is composed of: a basal medium composed of DMEM / F12 and Neurobasal, N2, B27, Glutamax, ascorbic acid, RA, SAG and GsMTx4, a two-dimensional (2D) gyroscope is used to simulate microgravity, which rotates around a horizontal axis to form microgravity, and the culture is carried out under simulated microgravity for more than 3 days;

[0017] Preferably, the culture conditions are: a culture medium composed of: DMEM / F12:Neurobasal=1:0.5-2 basal culture medium, (0.05-5)×N2, (0.05-5)×B27, (0.5-2)×Glutamax, 0.05-0.2 mM ascorbic acid, 0.005-0.02 μM RA, 0.05-0.2 μM SAG and 3-7 μM GsMTx4, using a two-dimensional (2D) gyroscope to simulate microgravity, rotating around a horizontal axis at a speed of 20-40 rpm to form microgravity, culturing for 4-6 days under simulated microgravity, and changing the medium every other day;

[0018] Particularly preferred culture conditions are: the culture medium composition is: DMEM / F12:Neurobasal=1:1 basal culture medium, 0.5×N2, 0.5×B27, 1×Glutamax, 0.1mM ascorbic acid, 0.01μM RA, 0.1μM SAG and 5μM GsMTx4, using a two-dimensional (2D) gyroscope to simulate microgravity, rotating around a horizontal axis at a speed of 30rpm to form microgravity, culturing under simulated microgravity for 5 days, and changing the medium every other day.

[0019] 6. A method for culturing motor neurons in vitro, comprising the following steps:

[0020] - Use culture medium and suspension culture bottles suitable for suspension culture to culture neurospheres formed by motor neuron precursor cells;

[0021] - Use simulated weightlessness and / or mechanosensitive ion channel inhibitors during the above culture process.

[0022] 7. The method of item 6, wherein the mechanosensitive ion channel inhibitor is GsMTx4.

[0023] 8. The method of item 6, wherein when the mechanosensitive ion channel inhibitor is used alone, the culture medium comprises: a basal medium consisting of DMEM / F12 and Neurobasal, N2, B27, Glutamax, ascorbic acid, RA, SAG and GsMTx4;

[0024] Preferably, the composition of the culture medium is: DMEM / F12:Neurobasal=1:0.5-2 basal medium, (0.05-5)×N2, (0.05-5)×B27, (0.5-2)×Glutamax, 0.05-0.2 mM ascorbic acid, 0.005-0.02 μM RA, 0.05-0.2 μM SAG and 3-7 μM GsMTx4;

[0025] It is particularly preferred that the composition of the culture medium is: DMEM / F12:Neurobasal=1:1 basal medium, 0.5×N2, 0.5×B27, 1×Glutamax, 0.1 mM ascorbic acid, 0.01 μM RA, 0.1 μM SAG and 5 μM GsMTx4.

[0026] 9. The method of item 6, wherein the culture conditions used when the simulated weightlessness culture method is used alone are: the culture medium is composed of a basal medium consisting of DMEM / F12 and Neurobasal, N2, B27, Glutamax, ascorbic acid, RA, SAG, microgravity is simulated using a two-dimensional (2D) gyroscope that rotates around a horizontal axis to form microgravity, and the culture is carried out under simulated microgravity for more than 3 days;

[0027] Preferably, the culture conditions are: a culture medium composed of: DMEM / F12:Neurobasal=1:0.5-2 basal culture medium, (0.05-5)×N2, (0.05-5)×B27, (0.5-2)×Glutamax, 0.05-0.2 mM ascorbic acid, 0.005-0.02 μM RA, 0.05-0.2 μM SAG, using a two-dimensional (2D) gyroscope to simulate microgravity, rotating around a horizontal axis at a speed of 20-40 rpm to form microgravity, culturing for 4-6 days under simulated microgravity, and changing the medium every other day;

[0028] Particularly preferred culture conditions are: the culture medium composition is: DMEM / F12:Neurobasal=1:1 basal culture medium, 0.5×N2, 0.5×B27, 1×Glutamax, 0.1mM ascorbic acid, 0.01μM RA, 0.1μM SAG, using a two-dimensional (2D) gyroscope to simulate microgravity, rotating around a horizontal axis at a speed of 30rpm to form microgravity, culturing under simulated microgravity for 5 days, and changing the medium every other day.

[0029] 10. The method of claim 6, wherein the culture conditions when the simulated weightlessness culture method and the mechanosensitive ion channel inhibitor are used together are: the culture medium composition is: a basal culture medium composed of DMEM / F12 and Neurobasal, N2, B27, Glutamax, ascorbic acid, RA, SAG and GsMTx4, microgravity is simulated using a two-dimensional (2D) gyroscope, which rotates around a horizontal axis to form microgravity, and culture is carried out under simulated microgravity for more than 3 days; preferably, the culture conditions are: the culture medium composition is: DMEM / F12:Neurobasal=1:0.5-2 basal culture medium, (0.05-5)×N2, (0.05-5)×B27, (0.5-2)×Glutamax, 0.05-0.2mM ascorbic acid, 0.005-0.02μM RA, 0.05-0.2μM SAG and 3-7 μM GsMTx4 were cultured in a two-dimensional (2D) gyroscope to simulate microgravity, rotating around the horizontal axis at a speed of 20-40 rpm for 4-6 days under simulated microgravity, with the medium changed every other day;

[0030] Particularly preferred culture conditions are: the culture medium composition is: DMEM / F12:Neurobasal=1:1 basal culture medium, 0.5×N2, 0.5×B27, 1×Glutamax, 0.1mM ascorbic acid, 0.01μM RA, 0.1μM SAG and 5μM GsMTx4, using a two-dimensional (2D) gyroscope to simulate microgravity, rotating around a horizontal axis at a speed of 30rpm to form microgravity, culturing under simulated microgravity for 5 days, and changing the medium every other day.

[0031] Beneficial technical effects achieved by the technical solution of this application

[0032] By adopting the cell culture intervention scheme of the present application (combination or replacement of weightlessness culture method and mechanosensitive ion channel inhibitor), more mature motor neurons can be obtained in the same period of time. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is the technical roadmap for the technical solution of this application.

[0034] Figure 2 The axon growth of neurospheres was observed under a microscope 1 day (D1) and 10 days (D10) after attachment. NG is the culture condition of normal gravity, and SMG is the culture condition of simulated microgravity. Figure 2 It can be seen that after simulated microgravity treatment, more primary neurites grew after the neurospheres were cultured on the wall, which shows that microgravity treatment can promote the maturation of motor neuron precursor cells.

[0035] Figure 3Immunofluorescence staining of neurospheres at 2, 4, and 6 days of differentiation (D2, D4, and D6). NG is the culture condition of normal gravity, and SMG is the culture condition of simulated microgravity. Figure 3 It can be seen that on days 2, 4, and 6, more cells in the neurospheres cultured under simulated microgravity conditions expressed neuronal maturation markers (NEUN) and motor neuron-specific markers (ISL1, MNX1, FOXP1), while fewer cells expressed motor neuron precursor cell markers (OLIG2). Therefore, it can be concluded that simulated microgravity promotes the maturation of motor neuron precursor cells into motor neurons.

[0036] Figure 4 Immunofluorescence staining was performed on neurospheres differentiated for 2, 4, and 6 days. GsMTx4- refers to the culture condition without GsMTx4, and GsMTx4+ refers to the culture condition with GsMTx4. Figure 4 It can be seen that on days 2, 4, and 6, more cells in the neurospheres cultured under simulated microgravity conditions expressed motor neuron-specific markers (ISL1, FOXP1) and fewer motor neuron precursor cell markers (OLIG2). Therefore, it can be concluded that 5 μM GsMTx4 promotes the maturation of motor neuron precursor cells into motor neurons.

[0037] Figure 5 It is a comparison of microgravity and GsMTx4 schemes. The percentage change of positive cells is the absolute value of the change in the proportion of positive cells expressing the corresponding markers (ISL1, FOXP1, OLIG2) compared with the intervention condition (microgravity or GsMTx4 culture) and the normal culture (normal gravity, no addition of GsMTx4). Specifically, the percentage change of OLIG2-positive cells in the microgravity group is the absolute value of the reduction in the percentage of OLIG2-positive cells caused by microgravity compared with the normal culture (normal gravity, no addition of GsMTx4). From Figure 5 It can be seen that on days 2, 4, and 6, compared with microgravity culture, the percentage changes in ISL1-positive cells, FOXP1-positive cells, and OLIG2-positive cells in neurospheres cultured under the condition of GsMTx4 addition were more obvious. Therefore, it can be concluded that 5 μM GsMTx4 has a more obvious effect in promoting the maturation of motor neuron precursor cells into motor neurons. DETAILED DESCRIPTION

[0038] The specific embodiments of the present application will be described in more detail below with reference to the accompanying drawings. Although the specific embodiments of the present application are shown in the accompanying drawings, it should be understood that the present application can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided in order to enable a more thorough understanding of the present application and to fully convey the scope of the present application to those skilled in the art.

[0039] It should be noted that certain words are used in the specification and claims to refer to specific components. Those skilled in the art should understand that technicians may use different nouns to refer to the same component. This specification and claims do not use the difference in nouns as a way to distinguish components, but use the difference in the functions of the components as the criterion for distinction. As mentioned throughout the specification and claims, "including" or "comprising" is an open term, so it should be interpreted as "including but not limited to". The subsequent description of the specification is a preferred embodiment of the implementation of the present application, but the description is based on the general principles of the specification and is not used to limit the scope of the present application. The scope of protection of the present application shall be determined by the attached claims.

[0040] As used herein, "substantially free" with respect to a particular component is used herein to mean that the particular component is not purposefully formulated into the composition and / or is present only as a contaminant or in trace amounts. Thus, the total amount of the particular component resulting from any accidental contamination of the composition is less than 0.05%, preferably less than 0.01%. Most preferred are compositions in which the amount of the particular component is undetectable by standard analytical methods.

[0041] As used in this specification, "a" or "an" may mean one or more. As used in the claims, when used with the word "comprising", the word "a" or "an" may mean one or more than one.

[0042] The term "or" is used in the claims to mean "and / or" unless explicitly stated to refer only to alternatives or the alternatives are mutually exclusive, although the content of this application supports a definition referring only to alternatives and "and / or." As used herein, "another" may mean at least a second or more.

[0043] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the device, the method used to determine the value, or the variation that exists between study subjects.

[0044] The methods for obtaining various biological materials described in the examples are only provided as an experimental method to achieve the specific disclosed purpose, and should not be construed as limiting the sources of biological materials in this application. In fact, the sources of biological materials used are extensive, and any biological material that can be obtained without violating laws and moral ethics can be replaced and used according to the instructions in the examples.

[0045] In a first aspect, the present application provides a simulated weightlessness culture method and / or use of a mechanosensitive ion channel inhibitor.

[0046] In a specific embodiment, provided is the use of simulated weightlessness culture method and / or mechanosensitive ion channel inhibitor for culturing motor neurons in vitro.

[0047] In the context of this specification, simulated weightlessness conditions can be achieved by a 2D gyroscope. In the context of this specification, mechanosensitive (MS) ion channels are multimeric proteins embedded in the membrane that mediate transmembrane ion transport in response to lateral membrane tension, and therefore can play the role of cell morphology receptors. Studies have shown that rapid cell morphology changes can change membrane tension, thereby inducing the opening of MS ion channels and leading to the efflux of permeate and the entry of calcium ions. "Mechanosensitive ion channel inhibitors" can inhibit the above-mentioned functions of the protein. In the context of this specification, "cell morphology changes" can be the effect of gravity.

[0048] In another embodiment, the above use is provided, wherein the mechanosensitive ion channel inhibitor is GsMTx4.

[0049] In yet another embodiment, the above use is provided, wherein, when the mechanosensitive ion channel inhibitor is used alone, the culture medium is composed of: a basal medium composed of DMEM / F12 and Neurobasal, N2, B27, Glutamax, ascorbic acid, RA, SAG, and GsMTx4;

[0050] Preferably, the composition of the culture medium is: DMEM / F12:Neurobasal=1:0.5-2 basic culture medium, specifically, the ratio can be 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1.0, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.5, 1:1.7, 1:1.8, 1:1.9, 1:2.0; (0.05-5)×N2. Specifically, the relative concentration can be 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 3.0, 4.0, 5.0; when the relative concentration is expressed as 0.5×N2, its specific meaning is “this is the actual working concentration of N2; due to the market demand for N2, the relative concentration of N2 is 0.5×N2, which is the actual working concentration of N2. The concentration of the stock solution of the product on sale is 100x, 0.5×N2 means that the final concentration of N2 in the culture solution is 1 / 200 of the concentration of the stock solution"; (0.05-5)×B27, specifically, the relative concentration can be 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2.0, 3.0, 4.0, 5.0; ( 0.5-2)×Glutamax, specifically, the relative concentration can be 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0; 0.05-0.2mM ascorbic acid, specifically, the concentration can be 0.05mM, 0.06mM, 0.07mM, 0.08mM, 0.09mM, 0.10mM, 0.15mM, 0.20mM; 0.005-0.02μM RA, specifically, the concentration can be 0.005 μM, 0.006 μM, 0.007 μM, 0.008 μM, 0.009 μM, 0.01 μM, 0.015 μM, 0.02 μM; 0.05-0.2 μM SAG, specifically, the concentration can be 0.05 μM, 0.06 μM, 0.07 μM, 0.08 μM, 0.09 μM, 0.10 μM, 0.15 μM, 0.2 μM; and 3-7 μM GsMTx4, specifically, the concentration can be 3 μM, 4 μM, 5 μM, 6 μM, 7 μM;

[0051] It is particularly preferred that the composition of the culture medium is: DMEM / F12:Neurobasal=1:1 basal culture medium, 0.5×N2, 0.5×B27, 1×Glutamax, 0.1mM ascorbic acid, 0.01μM RA, 0.1μM SAG and 5μM GsMTx4. In the context of this specification, 0.5×N2 represents the actual working concentration of N2; since the concentration of the stock solution of the commercial N2 product is 100x, 0.5×N2 represents that the final concentration of N2 in the culture solution is 1 / 200 of the concentration of the stock solution. In the context of this specification, 0.5×B27 represents the actual working concentration of B27; since the concentration of the stock solution of the commercial B27 product is 50x, 0.5×B27 represents that the final concentration of B27 in the culture solution is 1 / 100 of the concentration of the stock solution. In the context of this specification, 1×Glutamax represents the actual working concentration of Glutamax; since the concentration of the stock solution of commercially available Glutamax is 100x, 1×Glutamax means that the final concentration of Glutamax in the culture medium is 1 / 100 of the concentration of the stock solution.

[0052] In yet another specific embodiment, the above-mentioned use is used, wherein the culture conditions used when the simulated weightlessness culture method is used alone are: the culture medium is composed of: a basal medium composed of DMEM / F12 and Neurobasal, N2, B27, Glutamax, ascorbic acid, RA, SAG, a two-dimensional (2D) gyroscope is used to simulate microgravity, which rotates around a horizontal axis to form microgravity, and the culture is carried out under simulated microgravity for more than 3 days;

[0053] Preferably, the culture conditions are: the culture medium composition is: DMEM / F12:Neurobasal=1:0.5-2 basic culture medium, specifically, the ratio can be 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1.0, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.5, 1:1.7, 1:1.8, 1:1.9, 1:2 .0; (0.05-5)×N2. Specifically, the relative concentration can be 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 3.0, 4.0, 5.0; when the relative concentration is expressed as 0.5×N2, its specific meaning here is "this is the actual working concentration of N2; due to N 2 The concentration of the stock solution of the commercial product is 100x, 0.5×N2 means that the final concentration of N2 in the culture solution is 1 / 200 of the concentration of the stock solution”; (0.05-5)×B27. Specifically, the relative concentration can be 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2.0, 3.0, 4.0, 5.0; (0.5-2)×Glutamax, specifically, the relative concentration can be 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0; 0.05-0.2mM ascorbic acid, specifically, the concentration can be 0.05mM, 0.06mM, 0.07mM, 0.08mM, 0.09mM, 0.10mM, 0.15mM, 0.20mM; 0.005-0.02μM RA, specifically, the concentration can be 0.005μM, 0.006μM, 0.007μM, 0.008μM, 0.009μM, 0.01μM, 0.015μM, 0.02μM; 0.05-0.2μM SAG, specifically, the concentration can be 0.05μM, 0.06μM, 0.07μM, 0.08μM, 0.09μM, 0.10μM, 0.15μM, 0.2μM; using a two-dimensional (2D) gyroscope to simulate microgravity, rotating around a horizontal axis at a speed of 20-40rpm to form microgravity, specifically, it can be 20rpm, 25rpm, 30rpm, 35rpm, 40rpm; culturing under simulated microgravity for 4-6 days, specifically, it can be 4 days, 5 days or 6 days, and changing the medium every other day;

[0054] Particularly preferred culture conditions are: the culture medium composition is: DMEM / F12:Neurobasal=1:1 basal culture medium, 0.5×N2, 0.5×B27, 1×Glutamax, 0.1mM ascorbic acid, 0.01μM RA, 0.1μM SAG, using a two-dimensional (2D) gyroscope to simulate microgravity, rotating around a horizontal axis at a speed of 30rpm to form microgravity, culturing under simulated microgravity for 5 days, and changing the medium every other day.

[0055] In another embodiment, the above-mentioned use is provided, wherein the culture conditions when the simulated weightlessness culture method and the mechanosensitive ion channel inhibitor are used together are: the culture medium is composed of: a basal medium composed of DMEM / F12 and Neurobasal, N2, B27, Glutamax, ascorbic acid, RA, SAG and GsMTx4, microgravity is simulated using a two-dimensional (2D) gyroscope, which rotates around a horizontal axis to form microgravity, and culture is carried out for more than 3 days under simulated microgravity;

[0056] Preferably, the culture conditions are: the culture medium composition is: DMEM / F12:Neurobasal=1:0.5-2 basic culture medium, specifically, the ratio can be 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1.0, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.5, 1:1.7, 1:1.8, 1:1.9, 1:2 .0; (0.05-5)×N2. Specifically, the relative concentration can be 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 3.0, 4.0, 5.0; when the relative concentration is expressed as 0.5×N2, its specific meaning here is "this is the actual working concentration of N2; due to N 2 The concentration of the stock solution of the commercial product is 100x, 0.5×N2 means that the final concentration of N2 in the culture solution is 1 / 200 of the concentration of the stock solution”; (0.05-5)×B27. Specifically, the relative concentration can be 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2.0, 3.0, 4.0, 5.0; (0.5-2)×Glutamax, specifically, the relative concentration can be 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0; 0.05-0.2mM ascorbic acid, specifically, the concentration can be 0.05mM, 0.06mM, 0.07mM, 0.08mM, 0.09mM, 0.10mM, 0.15mM, 0.20mM; 0.005-0.02μM RA, specifically, the concentration can be 0.005μM, 0.006μM, 0.007μM, 0.008μM, 0.009μM, 0.01μM, 0.015μM, 0.02μM; 0.05-0.2μM SAG, specifically, the concentration can be 0.05μM, 0.06μM, 0.07μM, 0.08μM, 0.09μM, 0.10μM, 0.15μM, 0.2μM; and 3-7μM GsMTx4, specifically, the concentration can be 3μM, 4μM, 5μM, 6μM, 7μM; using a two-dimensional (2D) gyroscope to simulate microgravity, rotating around a horizontal axis at a speed of 20-40rpm to form microgravity, specifically, the speed can be 20rpm, 25rpm, 30rpm, 35rpm, 40rpm; culturing under simulated microgravity for 4-6 days, specifically, it can be 4 days, 5 days or 6 days, and changing the medium every other day;

[0057] Particularly preferred culture conditions are: the culture medium composition is: DMEM / F12:Neurobasal=1:1 basal culture medium, 0.5×N2, 0.5×B27, 1×Glutamax, 0.1mM ascorbic acid, 0.01μM RA, 0.1μM SAG and 5μM GsMTx4, using a two-dimensional (2D) gyroscope to simulate microgravity, rotating around a horizontal axis at a speed of 30rpm to form microgravity, culturing under simulated microgravity for 5 days, and changing the medium every other day.

[0058] In a second aspect, the present application provides a method for culturing motor neurons in vitro.

[0059] In one embodiment, a method for culturing motor neurons in vitro is provided, comprising: culturing neurospheres formed by motor neuron precursor cells using a culture medium suitable for suspension culture and a suspension culture bottle; - the culture temperature is 37 degrees Celsius, 5% CO 2 concentration, incubator oxygen supply; - simulated weightlessness and / or mechanosensitive ion channel inhibitors were used during the above culture process.

[0060] In another specific embodiment, the above method is provided, wherein the mechanosensitive ion channel inhibitor is GsMTx4.

[0061] In yet another specific embodiment, the above method is provided, wherein, when the mechanosensitive ion channel inhibitor is used alone, the culture medium is composed of: a basal medium composed of DMEM / F12 and Neurobasal, N2, B27, Glutamax, ascorbic acid, RA, SAG, and GsMTx4;

[0062] Preferably, the composition of the culture medium is: DMEM / F12:Neurobasal=1:0.5-2 basic culture medium, specifically, the ratio can be 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1.0, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.5, 1:1.7, 1:1.8, 1:1.9, 1:2.0; (0.05-5)×N2. Specifically, the relative concentration can be 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 3.0, 4.0, 5.0; when the relative concentration is expressed as 0.5×N2, its specific meaning is “this is the actual working concentration of N2; due to the market demand for N2, the relative concentration of N2 is 0.5×N2, which is the actual working concentration of N2. The concentration of the stock solution of the product on sale is 100x, 0.5×N2 means that the final concentration of N2 in the culture solution is 1 / 200 of the concentration of the stock solution"; (0.05-5)×B27, specifically, the relative concentration can be 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2.0, 3.0, 4.0, 5.0; ( 0.5-2)×Glutamax, specifically, the relative concentration can be 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0; 0.05-0.2mM ascorbic acid, specifically, the concentration can be 0.05mM, 0.06mM, 0.07mM, 0.08mM, 0.09mM, 0.10mM, 0.15mM, 0.20mM; 0.005-0.02μM RA, specifically, the concentration can be 0.005 μM, 0.006 μM, 0.007 μM, 0.008 μM, 0.009 μM, 0.01 μM, 0.015 μM, 0.02 μM; 0.05-0.2 μM SAG, specifically, the concentration can be 0.05 μM, 0.06 μM, 0.07 μM, 0.08 μM, 0.09 μM, 0.10 μM, 0.15 μM, 0.2 μM; and 3-7 μM GsMTx4, specifically, the concentration can be 3 μM, 4 μM, 5 μM, 6 μM, 7 μM;

[0063] It is particularly preferred that the composition of the culture medium is: DMEM / F12:Neurobasal=1:1 basal culture medium, 0.5×N2, 0.5×B27, 1×Glutamax, 0.1mM ascorbic acid, 0.01μM RA, 0.1μM SAG and 5μM GsMTx4. In one embodiment, the above-mentioned use is provided, wherein the culture conditions used when the simulated weightlessness culture method is used alone are: the culture medium composition is DMEM / F12:Neurobasal=1:1 basal culture medium, 0.5×N2, 0.5×B27, 1×Glutamax, 0.1mM ascorbic acid, 0.01μM RA, 0.1μM SAG, 5μM GsMTx4.

[0064] In the context of this specification, 0.5×N2 represents the actual working concentration of N2; since the concentration of the commercial stock solution of N2 is 100x, 0.5×N2 represents that the final concentration of N2 in the culture medium is 1 / 200 of the concentration of the stock solution. In the context of this specification, 0.5×B27 represents the actual working concentration of B27; since the concentration of the commercial stock solution of B27 is 50x, 0.5×B27 represents that the final concentration of B27 in the culture medium is 1 / 100 of the concentration of the stock solution. In the context of this specification, 1×Glutamax represents the actual working concentration of Glutamax; since the concentration of the commercial stock solution of Glutamax is 100x, 1×Glutamax represents that the final concentration of Glutamax in the culture medium is 1 / 100 of the concentration of the stock solution.

[0065] In a specific embodiment, the above method is provided, wherein the culture conditions used when the simulated weightlessness culture method is used alone are: the culture medium is composed of: a basal medium composed of DMEM / F12 and Neurobasal, N2, B27, Glutamax, ascorbic acid, RA, SAG, microgravity is simulated using a two-dimensional (2D) gyroscope, which rotates around a horizontal axis to form microgravity, and culture is carried out under simulated microgravity for more than 3 days;

[0066] Preferably, the culture conditions are: the culture medium composition is: DMEM / F12:Neurobasal=1:0.5-2 basic culture medium, specifically, the ratio can be 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1.0, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.5, 1:1.7, 1:1.8, 1:1.9, 1:2 .0; (0.05-5)×N2. Specifically, the relative concentration can be 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 3.0, 4.0, 5.0; when the relative concentration is expressed as 0.5×N2, its specific meaning here is "this is the actual working concentration of N2; due to N 2 The concentration of the stock solution of the commercial product is 100x, 0.5×N2 means that the final concentration of N2 in the culture solution is 1 / 200 of the concentration of the stock solution”; (0.05-5)×B27. Specifically, the relative concentration can be 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2.0, 3.0, 4.0, 5.0; (0.5-2)×Glutamax, specifically, the relative concentration can be 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0; 0.05-0.2mM ascorbic acid, specifically, the concentration can be 0.05mM, 0.06mM, 0.07mM, 0.08mM, 0.09mM, 0.10mM, 0.15mM, 0.20mM; 0.005-0.02μM RA, specifically, the concentration can be 0.005μM, 0.006μM, 0.007μM, 0.008μM, 0.009μM, 0.01μM, 0.015μM, 0.02μM; 0.05-0.2μM SAG, specifically, the concentration can be 0.05μM, 0.06μM, 0.07μM, 0.08μM, 0.09μM, 0.10μM, 0.15μM, 0.2μM; using a two-dimensional (2D) gyroscope to simulate microgravity, rotating around a horizontal axis at a speed of 20-40rpm to form microgravity, specifically, it can be 20rpm, 25rpm, 30rpm, 35rpm, 40rpm; culturing under simulated microgravity for 4-6 days, specifically, it can be 4 days, 5 days or 6 days, and changing the medium every other day;

[0067] Particularly preferred culture conditions are: the culture medium composition is: DMEM / F12:Neurobasal=1:1 basal culture medium, 0.5×N2, 0.5×B27, 1×Glutamax, 0.1mM ascorbic acid, 0.01μM RA, 0.1μM SAG, using a two-dimensional (2D) gyroscope to simulate microgravity, rotating around a horizontal axis at a speed of 30rpm to form microgravity, culturing under simulated microgravity for 5 days, and changing the medium every other day.

[0068] In another specific embodiment, the above method is provided, wherein the culture conditions when the simulated weightlessness culture method and the mechanosensitive ion channel inhibitor are used together are: the culture medium is composed of: a basal medium composed of DMEM / F12 and Neurobasal, N2, B27, Glutamax, ascorbic acid, RA, SAG and GsMTx4, microgravity is simulated using a two-dimensional (2D) gyroscope, which rotates around a horizontal axis to form microgravity, and culture is carried out under simulated microgravity for more than 3 days;

[0069] Preferably, the culture conditions are: the culture medium composition is: DMEM / F12:Neurobasal=1:0.5-2 basic culture medium, specifically, the ratio can be 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1.0, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.5, 1:1.7, 1:1.8, 1:1.9, 1:2 .0; (0.05-5)×N2. Specifically, the relative concentration can be 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 3.0, 4.0, 5.0; when the relative concentration is expressed as 0.5×N2, its specific meaning here is "this is the actual working concentration of N2; due to N 2 The concentration of the stock solution of the commercial product is 100x, 0.5×N2 means that the final concentration of N2 in the culture solution is 1 / 200 of the concentration of the stock solution”; (0.05-5)×B27. Specifically, the relative concentration can be 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2.0, 3.0, 4.0, 5.0; (0.5-2)×Glutamax, specifically, the relative concentration can be 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0; 0.05-0.2mM ascorbic acid, specifically, the concentration can be 0.05mM, 0.06mM, 0.07mM, 0.08mM, 0.09mM, 0.10mM, 0.15mM, 0.20mM; 0.005-0.02μM RA, specifically, the concentration can be 0.005μM, 0.006μM, 0.007μM, 0.008μM, 0.009μM, 0.01μM, 0.015μM, 0.02μM; 0.05-0.2μM SAG, specifically, the concentration can be 0.05μM, 0.06μM, 0.07μM, 0.08μM, 0.09μM, 0.10μM, 0.15μM, 0.2μM; and 3-7μM GsMTx4, specifically, the concentration can be 3μM, 4μM, 5μM, 6μM, 7μM; using a two-dimensional (2D) gyroscope to simulate microgravity, rotating around a horizontal axis at a speed of 20-40rpm to form microgravity, specifically, the speed can be 20rpm, 25rpm, 30rpm, 35rpm, 40rpm; culturing under simulated microgravity for 4-6 days, specifically, it can be 4 days, 5 days or 6 days, and changing the medium every other day; the culture conditions are particularly preferred: the culture medium composition is: DMEM / F12:Neurobasal=1:1 basic culture medium, 0.5×N2, 0.5×B27, 1×Glutamax, 0.1mM ascorbic acid, 0.01μMRA, 0.1μM SAG and 5μM GsMTx4, using a two-dimensional (2D) gyroscope to simulate microgravity, rotating around the horizontal axis at a speed of 30rpm to form microgravity, cultured under microgravity for 5 days, and changing the medium every other day.

[0070] Examples

[0071] Experimental methods and conditions:

[0072] (1) Embryonic stem cell maintenance and culture

[0073] 1) Evenly spread 1 ml of 0.1% gelatin solution per well in a six-well plate and incubate in a 37°C, 5% CO2 incubator for at least 30 min.

[0074] 2) Aspirate the gelatin solution, add MEF culture medium, and revive the irradiated MEF cells at a rate of 2.5×10 per well. 5 The cells were inoculated into a six-well plate, mixed and cultured overnight in a 37°C, 5% CO2 incubator. The MEF culture medium was configured as follows:

[0075] 45ml DMEM

[0076] 5ml FBS

[0077] 3) The next day, remove the MEF culture medium and replace it with maintenance medium. The maintenance medium is prepared as follows:

[0078] 39ml DMEM / F12

[0079] 10ml KSR

[0080] 0.5ml Glutamax

[0081] 0.5ml NEAA

[0082] 20ul BFGF (final concentration 20ng / ml)

[0083] 0.35ul β-mercaptoethanol

[0084] 4) H9 cells were digested with dispase solution and then inoculated onto MEF cells at a ratio of 1:4-1:6.

[0085] (2) Directed differentiation of motor neurons

[0086] 1) Human pluripotent stem cells (hPSCs) were differentiated into neuroepithelial cells (NEPs) expressing SOX1 with a positive rate of >95% using chemically defined neural culture medium (DMEM / F12:Neurobasal=1:1 culture medium containing 0.5×N2, 0.5×B27, 1×Glutamax, 0.1mM ascorbic acid, 2μM DMH1, 2μM SB, 3μM CHIR) for 6 days with medium replacement every other day.

[0087] 2) NEP cells were passaged at a ratio of 1:6, and the culture medium was changed (DMEM / F12:Neurobasal=1:1 culture medium, containing 0.5×N2, 0.5×B27, 1×Glutamax, 0.1mM ascorbic acid, 2μM DMH1, 2μM SB, 1μM CHIR, 0.1μM RA, 1μM SAG) to further differentiate into motor neuron precursor cells (MNPs) expressing olig2 positivity >90%.

[0088] 3) The adherent MNPs were collected and centrifuged (1000 rpm, 3.5 min) and suspended in culture. The culture medium was DMEM / F12:Neurobasal=1:1 culture medium containing 0.5×N2, 0.5×B27, 1×Glutamax, 0.1 mM ascorbic acid, 0.01 μM RA, and 0.1 μM SAG.

[0089] (3) Microgravity intervention (see Figure 1 (a))

[0090] 1) On the second day of suspension, the motor neuron precursor cells grew into free-floating spheres. The neurospheres were collected and suspended in a 25 cm2 cell culture flask at a density of 5×10 6 cells / flask, the culture medium was DMEM / F12:Neurobasal=1:1 culture medium containing 0.5×N2, 0.5×B27, 1×Glutamax, 0.1mM ascorbic acid, 0.01μM RA, 0.1μM SAG, the flask was filled with fresh culture medium to avoid the influence of shear stress, and then randomly divided into normal gravity group and simulated microgravity group.

[0091] 2) Use a two-dimensional (2D) cyclotron to simulate microgravity. The simulated microgravity group was incubated in a cyclotron and rotated around the horizontal axis at a speed of 30 rpm to form microgravity. The neurospheres were cultured under simulated microgravity for 5 days, and the medium was changed every other day to finally obtain motor neurons (MN).

[0092] (4) GsMTx4 intervention (see Figure 1 (b))

[0093] 1) On the second day of suspension, the motor neuron precursor cells grew into free-floating spheres. The neurospheres were collected and suspended in a 25 cm2 cell culture flask at a density of 5×10 6 cells / bottle, the culture medium was DMEM / F12:Neurobasal=1:1 culture medium containing 0.5×N2, 0.5×B27, 1×Glutamax, 0.1mM ascorbic acid, 0.01μM RA, 0.1μM SAG, and then randomly divided into GsMTx4-group and GsMTx4+group.

[0094] 2) The GsMTx4+ group was treated with 5 μM GsMTx4, while the GsMTx4- group was not treated. The cells were cultured for 5 days, and the medium was changed every other day to finally obtain motor neurons (MN).

[0095] (5) Motor neuron adherent culture

[0096] 1) Coating of cell culture dishes:

[0097] Prepare a culture dish and add an appropriate amount of Matrigel under sterile conditions. Evenly coat the surface of the culture dish with Matrigel and then solidify in an incubator at 37°C (about 1 hour).

[0098] 2) Cell seeding: Collect neurospheres, add the cell suspension to a culture dish containing Matrigel, mix the cells and Matrigel evenly, and place the culture dish back into the 37°C incubator for culture.

[0099] 3) Observation of results: Observe the axonal growth of neurospheres in Matrigel. The results are shown in Figure 2 , Figure 2 The axon growth of neurospheres was observed under a microscope 1 day (D1) and 10 days (D10) after attachment. NG is the culture condition of normal gravity, and SMG is the culture condition of simulated microgravity.

[0100] (6) Immunofluorescence staining

[0101] 1) Collect neurospheres, wash once with PBS medium, add 4% paraformaldehyde (precooled at 4°C), and fix at room temperature for 30 minutes.

[0102] 2) Remove the paraformaldehyde and add 30% sucrose to dehydrate at 4°C

[0103] 3) After the neurospheres sink to the bottom, the sucrose solution is removed and OCT is added for frozen sectioning.

[0104] 4) Wash the sections three times with PBS medium, add blocking solution, and incubate at room temperature for 30 minutes.

[0105] 5) Aspirate the blocking solution, add primary antibody solution, and incubate overnight at 4°C.

[0106] 6) The next day, discard the primary antibody solution and wash the cells three times with PBS culture medium, each time for 10 minutes.

[0107] 7) Add the secondary antibody solution and incubate at room temperature in the dark for 50 min.

[0108] 8) Aspirate and discard the secondary antibody solution, add Hoechest and incubate for 5 minutes.

[0109] 9) Wash three times with PBS medium, 10 min each time.

[0110] 10) Seal the slides with sealing medium.

[0111] 11) Observe with a regular fluorescence microscope or take photos under a confocal microscope.

[0112] The above experiments prove that:

[0113] Microgravity is formed by rotating around a horizontal axis at a speed of 20-40 rpm, especially at 30 rpm, using a two-dimensional (2D) gyroscope. Cultivating neurospheres under simulated microgravity can promote the maturation of motor neuron precursor cells into motor neurons. Using 3-7 μM, especially 5 μM of GsMTx4 to inhibit mechanosensitive ion channels can promote the maturation of motor neuron precursor cells into motor neurons.

[0114] The experimental results can be found in Figure 2 , Figure 3 , Figure 4 as well as Figure 5 .

[0115] from Figure 2 It can be seen that after simulated microgravity treatment, more primary neurites grew after the neurospheres were cultured on the wall, which shows that microgravity treatment can promote the maturation of motor neuron precursor cells.

[0116] Figure 3 Immunofluorescence staining of neurospheres at 2, 4, and 6 days of differentiation (D2, D4, and D6). NG is the culture condition of normal gravity, and SMG is the culture condition of simulated microgravity. Figure 3 It can be seen that on days 2, 4, and 6, more cells in the neurospheres cultured under simulated microgravity conditions expressed neuronal maturation markers (NEUN) and motor neuron-specific markers (ISL1, MNX1, FOXP1), while fewer cells expressed motor neuron precursor cell markers (OLIG2). Therefore, it can be concluded that simulated microgravity promotes the maturation of motor neuron precursor cells into motor neurons.

[0117] Figure 4Immunofluorescence staining was performed on neurospheres differentiated for 2, 4, and 6 days. GsMTx4- refers to the culture condition without GsMTx4, and GsMTx4+ refers to the culture condition with GsMTx4. Figure 4 It can be seen that on days 2, 4, and 6, more cells in the neurospheres cultured with the addition of GsMTx4 expressed motor neuron-specific markers (ISL1, FOXP1) and fewer motor neuron precursor cell markers (OLIG2). Therefore, it can be concluded that 5 μM GsMTx4 promotes the maturation of motor neuron precursor cells into motor neurons.

[0118] Figure 5 It is a comparison of microgravity and GsMTx4 schemes. The percentage change of positive cells is the absolute value of the change in the proportion of positive cells expressing the corresponding markers (ISL1, FOXP1, OLIG2) compared with the intervention condition (microgravity or GsMTx4 culture) and the normal culture (normal gravity, no addition of GsMTx4). Specifically, the percentage change of OLIG2-positive cells in the microgravity group is the absolute value of the reduction in the percentage of OLIG2-positive cells caused by microgravity compared with the normal culture (normal gravity, no addition of GsMTx4). From Figure 5 It can be seen that on days 2, 4, and 6, compared with microgravity culture, the percentage changes in ISL1-positive cells, FOXP1-positive cells, and OLIG2-positive cells in neurospheres cultured under the condition of GsMTx4 addition were more obvious. Therefore, it can be concluded that 5 μM GsMTx4 has a more obvious effect in promoting the maturation of motor neuron precursor cells into motor neurons.

[0119] When simulated microgravity and GsMTx4 were used simultaneously as auxiliary culture conditions, a more obvious effect of promoting the maturation of motor neuron precursor cells into motor neurons was observed.

[0120] Although the embodiments of the present application are described above in conjunction with the accompanying drawings, the present application is not limited to the above specific embodiments and application fields, and the above specific embodiments are merely illustrative and instructive, rather than restrictive. A person of ordinary skill in the art can also make many forms under the guidance of this specification and without departing from the scope of protection of the claims of the present application, all of which belong to the scope of protection of the present application.

Claims

1. Use of simulated weightlessness culture method and / or mechanosensitive ion channel inhibitor for in vitro culture of motor neurons.

2. The use according to claim 1, wherein The mechanosensitive ion channel inhibitor is GsMTx4.

3. The use according to claim 1, wherein When the mechanosensitive ion channel inhibitor was used alone, the culture medium composition was: basal medium composed of DMEM / F12 and Neurobasal, N2, B27, Glutamax, ascorbic acid, RA, SAG, and GsMTx4; Preferably, the composition of the culture medium is: DMEM / F12:Neurobasal=1:0.5-2 basal medium, (0.05-5)×N2, (0.05-5)×B27, (0.5-2)×Glutamax, 0.05-0.2 mM ascorbic acid, 0.005-0.02 μM RA, 0.05-0.2 μM SAG and 3-7 μM GsMTx4; It is particularly preferred that the composition of the culture medium is: DMEM / F12:Neurobasal=1:1 basal medium, 0.5×N2, 0.5×B27, 1×Glutamax, 0.1 mM ascorbic acid, 0.01 μM RA, 0.1 μM SAG and 5 μM GsMTx4.

4. The use according to claim 1, wherein The culture conditions used when the simulated weightlessness culture method was used alone were: the culture medium consisted of: a basal medium consisting of DMEM / F12 and Neurobasal, N2, B27, Glutamax, ascorbic acid, RA, SAG, microgravity was simulated using a two-dimensional (2D) gyroscope, which rotated about a horizontal axis to form microgravity, and cultured in simulated microgravity for more than 3 days; Preferably, the culture conditions are: a culture medium composed of: DMEM / F12:Neurobasal=1:0.5-2 basal culture medium, (0.05-5)×N2, (0.05-5)×B27, (0.5-2)×Glutamax, 0.05-0.2 mM ascorbic acid, 0.005-0.02 μM RA, 0.05-0.2 μM SAG, using a two-dimensional (2D) gyroscope to simulate microgravity, rotating around a horizontal axis at a speed of 20-40 rpm to form microgravity, culturing for 4-6 days under simulated microgravity, and changing the medium every other day; Particularly preferred culture conditions are: the culture medium composition is: DMEM / F12:Neurobasal=1:1 basal culture medium, 0.5×N2, 0.5×B27, 1×Glutamax, 0.1mM ascorbic acid, 0.01μM RA, 0.1μM SAG, using a two-dimensional (2D) gyroscope to simulate microgravity, rotating around a horizontal axis at a speed of 30rpm to form microgravity, culturing under simulated microgravity for 5 days, and changing the medium every other day.

5. The use according to claim 1, wherein The culture conditions when the simulated weightlessness culture method and the mechanosensitive ion channel inhibitor are used together are as follows: the culture medium is composed of a basal medium composed of DMEM / F12 and Neurobasal, N2, B27, Glutamax, ascorbic acid, RA, SAG, and GsMTx4, microgravity is simulated using a two-dimensional (2D) gyroscope, which rotates around a horizontal axis to form microgravity, and culture is carried out under simulated microgravity for more than 3 days; Preferably, the culture conditions are: a culture medium composed of: DMEM / F12:Neurobasal=1:0.5-2 basal culture medium, (0.05-5)×N2, (0.05-5)×B27, (0.5-2)×Glutamax, 0.05-0.2 mM ascorbic acid, 0.005-0.02 μM RA, 0.05-0.2 μM SAG and 3-7 μM GsMTx4, using a two-dimensional (2D) gyroscope to simulate microgravity, rotating around a horizontal axis at a speed of 20-40 rpm to form microgravity, culturing for 4-6 days under simulated microgravity, and changing the medium every other day; Particularly preferred culture conditions are: the culture medium composition is: DMEM / F12:Neurobasal=1:1 basal culture medium, 0.5×N2, 0.5×B27, 1×Glutamax, 0.1mM ascorbic acid, 0.01μM RA, 0.1μM SAG and 5μM GsMTx4, using a two-dimensional (2D) gyroscope to simulate microgravity, rotating around a horizontal axis at a speed of 30rpm to form microgravity, culturing under simulated microgravity for 5 days, and changing the medium every other day.

6. A method for culturing motor neurons in vitro, comprising the following steps: - Use culture medium and suspension culture bottles suitable for suspension culture to culture neurospheres formed by motor neuron precursor cells; - Use simulated weightlessness and / or mechanosensitive ion channel inhibitors during the above culture process.

7. The method of claim 6, wherein: The mechanosensitive ion channel inhibitor is GsMTx4.

8. The method of claim 6, wherein: When the mechanosensitive ion channel inhibitor was used alone, the culture medium composition was: basal medium composed of DMEM / F12 and Neurobasal, N2, B27, Glutamax, ascorbic acid, RA, SAG, and GsMTx4; Preferably, the composition of the culture medium is: DMEM / F12:Neurobasal=1:0.5-2 basal medium, (0.05-5)×N2, (0.05-5)×B27, (0.5-2)×Glutamax, 0.05-0.2 mM ascorbic acid, 0.005-0.02 μM RA, 0.05-0.2 μM SAG and 3-7 μM GsMTx4; It is particularly preferred that the composition of the culture medium is: DMEM / F12:Neurobasal=1:1 basal medium, 0.5×N2, 0.5×B27, 1×Glutamax, 0.1 mM ascorbic acid, 0.01 μM RA, 0.1 μM SAG and 5 μM GsMTx4.

9. The method of claim 6, wherein: The culture conditions used when the simulated weightlessness culture method was used alone were: the culture medium consisted of: a basal medium consisting of DMEM / F12 and Neurobasal, N2, B27, Glutamax, ascorbic acid, RA, SAG, microgravity was simulated using a two-dimensional (2D) gyroscope, which rotated about a horizontal axis to form microgravity, and cultured in simulated microgravity for more than 3 days; Preferably, the culture conditions are: a culture medium composed of: DMEM / F12:Neurobasal=1:0.5-2 basal culture medium, (0.05-5)×N2, (0.05-5)×B27, (0.5-2)×Glutamax, 0.05-0.2 mM ascorbic acid, 0.005-0.02 μM RA, 0.05-0.2 μM SAG, using a two-dimensional (2D) gyroscope to simulate microgravity, rotating around a horizontal axis at a speed of 20-40 rpm to form microgravity, culturing for 4-6 days under simulated microgravity, and changing the medium every other day; Particularly preferred culture conditions are: the culture medium composition is: DMEM / F12:Neurobasal=1:1 basal culture medium, 0.5×N2, 0.5×B27, 1×Glutamax, 0.1mM ascorbic acid, 0.01μM RA, 0.1μM SAG, using a two-dimensional (2D) gyroscope to simulate microgravity, rotating around a horizontal axis at a speed of 30rpm to form microgravity, culturing under simulated microgravity for 5 days, and changing the medium every other day.

10. The method of claim 6, wherein: The culture conditions when the simulated weightlessness culture method and the mechanosensitive ion channel inhibitor are used together are as follows: the culture medium is composed of a basal medium composed of DMEM / F12 and Neurobasal, N2, B27, Glutamax, ascorbic acid, RA, SAG, and GsMTx4, microgravity is simulated using a two-dimensional (2D) gyroscope, which rotates around a horizontal axis to form microgravity, and culture is carried out under simulated microgravity for more than 3 days; Preferably, the culture conditions are: a culture medium composed of: DMEM / F12:Neurobasal=1:0.5-2 basal culture medium, (0.05-5)×N2, (0.05-5)×B27, (0.5-2)×Glutamax, 0.05-0.2 mM ascorbic acid, 0.005-0.02 μM RA, 0.05-0.2 μM SAG and 3-7 μM GsMTx4, using a two-dimensional (2D) gyroscope to simulate microgravity, rotating around a horizontal axis at a speed of 20-40 rpm to form microgravity, culturing for 4-6 days under simulated microgravity, and changing the medium every other day; Particularly preferred culture conditions are: the culture medium composition is: DMEM / F12:Neurobasal=1:1 basal culture medium, 0.5×N2, 0.5×B27, 1×Glutamax, 0.1mM ascorbic acid, 0.01μM RA, 0.1μM SAG and 5μM GsMTx4, using a two-dimensional (2D) gyroscope to simulate microgravity, rotating around a horizontal axis at a speed of 30rpm to form microgravity, culturing under simulated microgravity for 5 days, and changing the medium every other day.

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