WO2019085993A1 - Human neural stem cell culture medium and application - Google Patents

Human neural stem cell culture medium and application Download PDF

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WO2019085993A1
WO2019085993A1 PCT/CN2018/113675 CN2018113675W WO2019085993A1 WO 2019085993 A1 WO2019085993 A1 WO 2019085993A1 CN 2018113675 W CN2018113675 W CN 2018113675W WO 2019085993 A1 WO2019085993 A1 WO 2019085993A1
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neural stem
culture medium
stem cell
cell culture
human neural
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王娟
马静
辛文
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北京全式金生物技术有限公司
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0618—Cells of the nervous system
    • C12N5/0623—Stem cells
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/11—Epidermal growth factor [EGF]
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/40—Regulators of development
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/40—Regulators of development
    • C12N2501/415—Wnt; Frizzeled
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/40—Regulators of development
    • C12N2501/48—Regulators of apoptosis

Definitions

  • the invention relates to the field of cell culture technology. More specifically, it relates to a human neural stem cell culture medium and application.
  • Neural stem cells have the ability to self-replicate and differentiate into cell types such as neural precursor cells, neurons, astrocytes and oligodendrocytes. They are important tools for disease pathogenesis research, drug screening, etc., and are also neurodegenerative. Ideal seed resource cells for alternative treatment of cells with injury-like diseases. At present, there are many ways to obtain neural stem cells in vitro, such as primary isolation and culture from brain tissue, pluripotent stem cells to induce differentiation, and somatic transdifferentiation.
  • the neural stem cell culture solution generally consists of a basic medium, a nutritional additive, and a growth factor, such as the patent CN103045538A, the patent CN105296429A, and the patent CN1536073A.
  • the basic medium is responsible for providing inorganic salts, vitamins, amino acids, glucose, organic substances and the like necessary for cell growth.
  • the basic medium commonly used for culturing neural stem cells is generally commercial DMEM/F12 medium and Neurobasal medium.
  • Nutritional additives are responsible for providing proteins, hormones, trace elements and other components necessary for cell growth.
  • Commercially available N2 and B27 are the more commonly used neural stem cell culture additions.
  • the N2 component is simple and contains only five components of insulin, transferrin, progesterone, putrescine and sodium selenate. The homogeneity between batches is better controlled, but due to the lack of some cell growth requirements, it can not support neural stem cells for a long time.
  • Growing. B27 is effective in supporting the growth of neural stem cells, but its composition is complex. It contains animal extract protein-bovine serum albumin. It is not only difficult to control the stability between batches, but also has the potential to introduce animal-derived pathogenic microorganisms. Cell replacement therapy poses a potential safety hazard. Growth factors are responsible for regulating the relevant signaling pathways to maintain the ability of cells to self-renew. Currently, the growth factors commonly used in cultured neural stem cells are basic fibroblast growth factor (bFGF) and epithelial growth factor (EGF). However, human neural stem cells have limited proliferation ability in the currently available medium, and spontaneous differentiation is more serious.
  • bFGF basic fibroblast growth factor
  • EGF epithelial growth factor
  • One of the objects of the present invention is to provide a human neural stem cell culture medium which is simple in formulation, low in cost, clear in chemical composition, free of animal-derived components, and free of protein extracts and hydrolyzates.
  • Another object of the present invention is to provide the use of the above human neural stem cell culture medium for culturing human neural stem cells.
  • the invention provides a human neural stem cell culture medium, the culture medium comprising the following components: a basic medium, a nutritional additive, a growth factor and a signal pathway regulating small molecule;
  • the growth factor comprises basic fibroblast growth factor (bFGF) and epithelial cell growth factor (EGF), both of which are used at a final concentration of 5-100 ng/mL, preferably 6-50 ng/mL, most It is preferably 10-30 ng/mL.
  • bFGF basic fibroblast growth factor
  • EGF epithelial cell growth factor
  • the signaling pathway regulates a small molecule as one or a combination of a Wnt signaling pathway activator, an apoptosis inhibitory small molecule, and a p53 signaling pathway inhibitor; preferably, a Wnt signaling pathway activator and a p53 signaling pathway inhibitor kind or two combinations.
  • the Wnt signaling pathway activator and final use concentration may be WNT3a, 1-100 ng/mL; 6-BIO, 1-20 ⁇ M; CHIR-98014, 0.1-20 ⁇ M; TDZD-8, 10-100 ⁇ M; AZD1080, 0.1- One or a combination of 50 ⁇ M; CHIR99021, 0.1-20 ⁇ M.
  • it is WNT3a, 1-100 ng/mL or 6-BIO, 1-20 ⁇ M; most preferably, 6-BIO, 1-20 ⁇ M.
  • the apoptosis inhibiting small molecule and the final use concentration may be one or a combination of Bax inhibitor peptide V5, 0.1-100 ⁇ M; iMAC2, 0.01-100 ⁇ M; Embelin, 0.5-500 ⁇ M.
  • it is a combination of one or both of Bax inhibitor peptide V5, 0.1-100 ⁇ M and iMAC2, 0.01-100 ⁇ M; most preferably, Bax inhibitor peptide V5, 0.1-100 ⁇ M.
  • the p53 signaling pathway inhibitor and the final use concentration may be Pifithrin- ⁇ hydrobromide, 0.1-100 ⁇ M; Cyclic Pifithrin- ⁇ hydrobromide, 0.1-100 ⁇ M; Pifithrin- ⁇ , 0.1-100 ⁇ M; SJ 172550, 0.5-500 ⁇ M Combination of species or several.
  • it is Cyclic Pifithrin- ⁇ hydrobromide, 0.1-100 ⁇ M or Pifithrin- ⁇ hydrobromide, 0.1-100 ⁇ M; most preferably, Pifithrin- ⁇ hydrobromide, 0.1-100 ⁇ M.
  • the basal medium is one or two of DMEM/F12 medium and Neurobasal medium; preferably, a DMEM/F12 medium and a Neurobasal medium mixed medium having a volume ratio of 1:1;
  • composition and final concentration of the nutritional additive are: human insulin 0.1-20 mg / L, vitamin C 10-200 mg / L, glutathione 10-100 mg / L, linolenic acid 0.05-5 mg / L, meat
  • the muscarin is 0.2-20 mg/L
  • the N-acetylcysteine is 5-500 ⁇ M
  • the ethanolamine is 0.01-10 mg/L
  • the linoleic acid is 0.05-5 mg/L.
  • the composition and final concentration of the nutritional additive are: human insulin 0.5-15 mg/L, vitamin C 20-100 mg/L, glutathione 20-90 mg/L, linolenic acid 0.1-4 mg/L, respectively.
  • Carnitine 0.5-15mg/L, N-acetylcysteine 10-400 ⁇ M, ethanolamine 0.05-8mg/L, linoleic acid 0.1-4mg/L.
  • the composition and final concentration of the nutritional additive are: human insulin 1-10 mg / L, vitamin C 30-80 mg / L, glutathione 30-80 mg / L, linolenic acid 1-3 mg / L, carnitine 1-10 mg / L, N-acetylcysteine 50-100 ⁇ M, ethanolamine 0.1-5 mg / L, linoleic acid 1-3 mg / L.
  • the invention also provides the use of the above human neural stem cell culture medium for culturing human neural stem cells.
  • any range recited in the present invention includes any value between the end value and the end value, and Any subrange of arbitrary values between end values or end values.
  • the medium of the invention has simple formula, low cost, clear chemical composition, no animal-derived components, no protein extract and hydrolyzate, so the batch is stable and there is no potential introduction of animal origin and humanity.
  • the risk of pathogenic microorganisms is safer and can be applied to the clinical research and clinical experiments of human neural stem cell culture.
  • the present invention firstly applies a small signal-modulating small molecule to the culture of human neural stem cells, and can support the rapid proliferation and dry maintenance of human neural stem cells of various sources under the action of signaling pathway regulating small molecules.
  • the technical problem that human neural stem cells are easily differentiated in vitro and difficult to be amplified in a long-term manner is solved.
  • Fig. 1 is a view showing the morphology of human neural stem cells after culture for 10 passages in the experimental group and the control medium.
  • Fig. 2 shows the expression ratio of the neural stem cell marker gene Nestin after 10 generations of human neural stem cells cultured in the experimental group and the control group, respectively, by flow cytometry.
  • Figure 3 shows a photograph of Nestin immunofluorescence staining of human neural stem cells after 10 passages in an experimental group culture medium.
  • Figure 4 shows the expression of the neuronal marker gene TUJ1 and the glial marker gene GFAP by immunofluorescence after 10 days of random differentiation of human neural stem cells.
  • the basal medium DMEM/F12 medium and Neurobasal medium used in the examples of the present invention were purchased from Life Technologies; the growth factors bFGF and EGF were purchased from Peprotech; other materials were commonly used in the art.
  • a human neural stem cell culture medium comprising the following components:
  • Base medium 50% DMEM/F12 medium and 50% Neurobasal medium.
  • Nutritional additive the composition and final concentration of the nutritional additive are: human insulin 10mg / L, vitamin C 50mg / L, glutathione 50mg / L, linolenic acid 1mg / L, carnitine 20mg / L, N-acetylcysteine 5 ⁇ M, ethanolamine 5 mg/L, and linoleic acid 1 mg/L.
  • bFGF 20 ng/mL
  • EGF 20 ng/mL
  • the signaling pathway regulates the small molecule component and its final use concentration: Pifithrin- ⁇ hydrobromide, 5 ⁇ M and 6-BIO, 2 ⁇ M.
  • a human neural stem cell culture medium comprising the following components:
  • Base medium 50% DMEM/F12 medium and 50% Neurobasal medium.
  • Nutritional additive The composition and final concentration of the nutritional additive are: human insulin 0.1 mg / L, vitamin C 10 mg / L, glutathione 10 mg / L, linolenic acid 0.05 mg / L, carnitine 0.2 mg /L, N-acetylcysteine 5 ⁇ M, ethanolamine 0.01 mg/L, linoleic acid 0.05 mg/L.
  • bFGF 5 ng/mL
  • EGF 5 ng/mL
  • the signaling pathway regulates the small molecule component and its final use concentration: WNT3a, 1 ng/mL and Bax inhibitor peptide V5, 0.1 ⁇ M and Cyclic Pifithrin- ⁇ hydrobromide, 0.1 ⁇ M.
  • a human neural stem cell culture medium comprising the following components:
  • Base medium 50% DMEM/F12 medium and 50% Neurobasal medium.
  • Nutritional additive the composition and final concentration of the nutritional additive are: human insulin 20mg / L, vitamin C 200mg / L, glutathione 100mg / L, linolenic acid 5mg / L, carnitine 20mg / L, N-acetylcysteine 500 ⁇ M, ethanolamine 10 mg/L, and linoleic acid 5 mg/L.
  • bFGF 100 ng/mL
  • EGF 100 ng/mL
  • the signaling pathway regulates the small molecule component and its final use concentration: WNT3a, 1 ng/mL and Bax inhibitor peptide V5, 100 ⁇ M and Cyclic Pifithrin- ⁇ hydrobromide, 100 ⁇ M.
  • the formulation of the human neural stem cell culture medium used in the experimental group was the same as in Example 1;
  • the human neural stem cell culture medium used in the control group was the medium of Example 1 minus the signaling pathway regulating small molecules.
  • the polyornithine (PLO) was diluted to 15 ⁇ g/mL with PBS and added to the Petri dish to be coated.
  • PLO polyornithine
  • Table 1 Incubate at 37 °C for 2 h or 4 °C overnight, not to let the incubation process The bottom of the dish is dried. Discard the PLO, rinse it twice with PBS, and rinse it with DMEM/F12. Laminin was diluted to 5 ⁇ g/mL with DMEM/F12 and added to the PLO-coated dish. See Table 1 for the amount of addition, incubate at 37 ° C for 2 h or 4 ° C overnight, pay attention to the incubation process. Do not let the bottom of the dish dry.
  • a method of obtaining human neural stem cells by inducing differentiation of human pluripotent stem cells is provided.
  • the medium in which human pluripotent stem cells differentiate into neural stem cells is formulated as follows:
  • Base medium 50% DMEM/F12 medium and 50% Neurobasal medium.
  • Nutritional Additive Formulation of the nutritional additive in the same manner as in Example 1.
  • SB431542 10 ⁇ M and LDN193189 100 nM (a commercially available small molecule that is commonly used in the art to induce differentiation of human pluripotent stem cells into neural stem cells).
  • Methods for inducing differentiation of human pluripotent stem cells into neural stem cells are as follows:
  • Human pluripotent stem cells with good growth condition were digested into single cells by Accutase, inoculated into a petri dish previously coated with PLO-Laminin at a density of 10 5 cells/mL, and differentiated into neural stem cells in human pluripotent stem cells.
  • Nestin-positive neural stem cells can be obtained by culturing for 4 days in the medium.
  • Human neural stem cells were cultured in the culture medium of the experimental group and the control group, respectively, and the cultivation steps were as follows:
  • Figure 1 shows the morphological map of human neural stem cells cultured in the experimental group and the control group for 10 generations respectively. It can be seen that human neural stem cells are in the experimental group of human neural stem cells. The growth state in the medium was better than that of the control human neural stem cell culture medium.
  • Figure 2 shows the expression ratio of human neural stem cell marker gene Nestin after human cultured neural stem cells culture in experimental and control human neural stem cell culture medium for 10 generations. After culture for 10 generations, in the experimental group medium, human nerve The expression ratio of stem cells in Nestin was still above 90%, while in the control medium, the expression ratio of Nestin was less than 50%.
  • Figure 3 shows the immunofluorescence staining of the human neural stem cell marker gene Nestin after culture of human neural stem cells in the experimental group for 10 generations.
  • neural stem cells cultured in the present invention still have the potential to differentiate into neurons and glial cells after 10 generations of culture.
  • the formula of the neural stem cell random differentiation culture solution is as follows:
  • Neurobasal medium 49%.
  • Nutritional Additive The formulation is the same as the nutritional additive formulation of Example 1.
  • the neural stem cells were digested and inoculated according to the method described in Example 4.
  • the cells were replaced with neural stem cells randomly differentiated culture solution on the 2nd day after inoculation, and then changed every other day until the 10th day of culture, and the results were confirmed by immunofluorescence staining.
  • Fig. 4 it can be seen from Fig. 4 that human neural stem cells still have the potential to differentiate into neurons and glial cells after 10 days of random differentiation in the culture of the neural stem cell culture medium for 10 days.

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Abstract

Provided is a human neural stem cell culture medium. The culture medium comprises the following components: a basal culture medium, a nutritional additive, a growth factor, and a signaling pathway-regulating small molecule. Also provided is an application of the culture medium in culturing human neural stem cells.

Description

一种人神经干细胞培养基及应用Human neural stem cell culture medium and application 技术领域Technical field
本发明涉及细胞培养技术领域。更具体地，涉及一种人神经干细胞培养基及应用。The invention relates to the field of cell culture technology. More specifically, it relates to a human neural stem cell culture medium and application.
背景技术Background technique
各种神经损伤和退行性的神经系统疾病的发病率呈逐年上升趋势，传统的医学方法很难彻底治愈这类疾病，细胞替代性治疗是彻底治愈这类疾病潜在的最有效的手段。The incidence of various neurological and degenerative neurological diseases is increasing year by year. It is difficult to completely cure these diseases by traditional medical methods. Cell replacement therapy is the most effective means to completely cure such diseases.
神经干细胞具有自我复制能力和分化为神经前体细胞、神经元、星形胶质细胞和少突角质细胞等细胞类型的潜能，是疾病发病机制研究、药物筛选等的重要工具，也是神经退行性和损伤类疾病细胞替代性治疗的理想种子资源细胞。目前，已经有许多途径可以在体外获得神经干细胞，如从脑组织内原代分离培养获得，多能性干细胞诱导分化获得，体细胞转分化获得。Neural stem cells have the ability to self-replicate and differentiate into cell types such as neural precursor cells, neurons, astrocytes and oligodendrocytes. They are important tools for disease pathogenesis research, drug screening, etc., and are also neurodegenerative. Ideal seed resource cells for alternative treatment of cells with injury-like diseases. At present, there are many ways to obtain neural stem cells in vitro, such as primary isolation and culture from brain tissue, pluripotent stem cells to induce differentiation, and somatic transdifferentiation.
神经干细胞培养液一般由基础培养基、营养添加剂和生长因子组成，如专利CN103045538A、专利CN105296429A和专利CN1536073A。基础培养基负责提供细胞生长必须的无机盐、维生素、氨基酸、葡萄糖、有机物等物质，目前培养神经干细胞常用的基础培养基一般是商品化的DMEM/F12 medium和Neurobasal medium。营养添加剂负责提供细胞生长必须的蛋白、激素、微量元素等成分，商售的N2和B27是比较常用的神经干细胞培养添。N2成分简单，只含有胰岛素、转铁蛋白、孕酮、腐胺和硒酸钠五种成分，批次之间均一性比较好控制，但是由于缺乏一些细胞生长需求物，不能长期支持神经干细胞的生长。B27在支持神经干细胞生长方面效果较好，但是其成分复杂，含有动物提取蛋白-牛血清白蛋白，不仅批次之间稳定性难以控制，还会有潜在引入动物源性病原微生物的风险，为细胞替代性治疗带来了潜在的安全隐患。生长因子负责调控相关的信号通路来维持细胞的自我更新能力，目前培养神经干细胞常用的生长因子是碱性成纤维细胞生长因子(bFGF)和上皮细胞生长因子(EGF)。但是人神经干细胞在目前已有的培养基中增殖能力有限，自发分化比较严重。The neural stem cell culture solution generally consists of a basic medium, a nutritional additive, and a growth factor, such as the patent CN103045538A, the patent CN105296429A, and the patent CN1536073A. The basic medium is responsible for providing inorganic salts, vitamins, amino acids, glucose, organic substances and the like necessary for cell growth. Currently, the basic medium commonly used for culturing neural stem cells is generally commercial DMEM/F12 medium and Neurobasal medium. Nutritional additives are responsible for providing proteins, hormones, trace elements and other components necessary for cell growth. Commercially available N2 and B27 are the more commonly used neural stem cell culture additions. The N2 component is simple and contains only five components of insulin, transferrin, progesterone, putrescine and sodium selenate. The homogeneity between batches is better controlled, but due to the lack of some cell growth requirements, it can not support neural stem cells for a long time. Growing. B27 is effective in supporting the growth of neural stem cells, but its composition is complex. It contains animal extract protein-bovine serum albumin. It is not only difficult to control the stability between batches, but also has the potential to introduce animal-derived pathogenic microorganisms. Cell replacement therapy poses a potential safety hazard. Growth factors are responsible for regulating the relevant signaling pathways to maintain the ability of cells to self-renew. Currently, the growth factors commonly used in cultured neural stem cells are basic fibroblast growth factor (bFGF) and epithelial growth factor (EGF). However, human neural stem cells have limited proliferation ability in the currently available medium, and spontaneous differentiation is more serious.
因此，需要提供一种新型的人神经干细胞培养基，以解决上述问题。Therefore, there is a need to provide a novel human neural stem cell culture medium to solve the above problems.
发明内容Summary of the invention
本发明的目的之一在于提供一种人神经干细胞培养基，该培养基配方简单、成本低廉、化学成分明确，不含动物源性成分，不含蛋白提取物和水解物。One of the objects of the present invention is to provide a human neural stem cell culture medium which is simple in formulation, low in cost, clear in chemical composition, free of animal-derived components, and free of protein extracts and hydrolyzates.
本发明的目的之二在于提供上述人神经干细胞培养基在培养人神经干细胞的应用。Another object of the present invention is to provide the use of the above human neural stem cell culture medium for culturing human neural stem cells.
为达到上述目的，本发明采用下述技术方案：In order to achieve the above object, the present invention adopts the following technical solutions:
本发明提供了一种人神经干细胞培养基，所述培养基包括以下组分：基础培养基、营养添加剂、生长因子和信号通路调控小分子；The invention provides a human neural stem cell culture medium, the culture medium comprising the following components: a basic medium, a nutritional additive, a growth factor and a signal pathway regulating small molecule;
进一步，所述生长因子包括碱性成纤维细胞生长因子(bFGF)和上皮细胞生长因子(EGF)，二者的最终使用浓度均为5-100ng/mL，优选的为6-50ng/mL，最优选的为10-30ng/mL。Further, the growth factor comprises basic fibroblast growth factor (bFGF) and epithelial cell growth factor (EGF), both of which are used at a final concentration of 5-100 ng/mL, preferably 6-50 ng/mL, most It is preferably 10-30 ng/mL.
所述信号通路调控小分子为Wnt信号通路激活剂、凋亡抑制小分子、p53信号通路抑制剂中的一种或几种组合；优选的，为Wnt信号通路激活剂和p53信号通路抑制剂一种或两种组合。The signaling pathway regulates a small molecule as one or a combination of a Wnt signaling pathway activator, an apoptosis inhibitory small molecule, and a p53 signaling pathway inhibitor; preferably, a Wnt signaling pathway activator and a p53 signaling pathway inhibitor Kind or two combinations.
其中，among them,
所述Wnt信号通路激活剂及最终使用浓度，可以是WNT3a，1-100ng/mL；6-BIO，1-20μM；CHIR-98014，0.1-20μM；TDZD-8，10-100μM；AZD1080，0.1-50μM；CHIR99021，0.1-20μM中的一种或者几种的组合。优选的，为WNT3a，1-100ng/mL或6-BIO，1-20μM；最优选的，为6-BIO，1-20μM。The Wnt signaling pathway activator and final use concentration may be WNT3a, 1-100 ng/mL; 6-BIO, 1-20 μM; CHIR-98014, 0.1-20 μM; TDZD-8, 10-100 μM; AZD1080, 0.1- One or a combination of 50 μM; CHIR99021, 0.1-20 μM. Preferably, it is WNT3a, 1-100 ng/mL or 6-BIO, 1-20 μM; most preferably, 6-BIO, 1-20 μM.
所述凋亡抑制小分子及最终使用浓度，可以是Bax inhibitor peptide V5，0.1-100μM；iMAC2，0.01-100μM；Embelin，0.5-500μM中的一种或者几种的组合。优选的，为Bax inhibitor peptide V5,0.1-100μM和iMAC2，0.01-100μM中的一种或两种的组合；最优选的，为Bax inhibitor peptide V5,0.1-100μM。The apoptosis inhibiting small molecule and the final use concentration may be one or a combination of Bax inhibitor peptide V5, 0.1-100 μM; iMAC2, 0.01-100 μM; Embelin, 0.5-500 μM. Preferably, it is a combination of one or both of Bax inhibitor peptide V5, 0.1-100 μM and iMAC2, 0.01-100 μM; most preferably, Bax inhibitor peptide V5, 0.1-100 μM.
所述p53信号通路抑制剂及最终使用浓度，可以是Pifithrin-α hydrobromide，0.1-100μM；Cyclic Pifithrin-α hydrobromide，0.1-100μM；Pifithrin-μ，0.1-100μM；SJ 172550，0.5-500μM中的一种或几种的组合。优选的，为Cyclic Pifithrin-α hydrobromide，0.1-100μM或Pifithrin-α hydrobromide，0.1-100μM；最优选的，为Pifithrin-α hydrobromide，0.1-100μM。The p53 signaling pathway inhibitor and the final use concentration may be Pifithrin-α hydrobromide, 0.1-100 μM; Cyclic Pifithrin-α hydrobromide, 0.1-100 μM; Pifithrin-μ, 0.1-100 μM; SJ 172550, 0.5-500 μM Combination of species or several. Preferably, it is Cyclic Pifithrin-α hydrobromide, 0.1-100 μM or Pifithrin-α hydrobromide, 0.1-100 μM; most preferably, Pifithrin-α hydrobromide, 0.1-100 μM.
进一步，所述基础培养基为DMEM/F12 medium、Neurobasal medium中的一种或两种；优选的，为体积比为1:1的DMEM/F12 medium和Neurobasal  medium混合培养基；Further, the basal medium is one or two of DMEM/F12 medium and Neurobasal medium; preferably, a DMEM/F12 medium and a Neurobasal medium mixed medium having a volume ratio of 1:1;
进一步，所述营养添加剂组成成分和最终使用浓度分别为：人胰岛素0.1-20mg/L、维生素C 10-200mg/L、谷胱甘肽10-100mg/L、亚麻酸0.05-5mg/L、肉毒碱0.2-20mg/L、N-乙酰半胱氨酸5-500μM、乙醇胺0.01-10mg/L、亚油酸0.05-5mg/L。Further, the composition and final concentration of the nutritional additive are: human insulin 0.1-20 mg / L, vitamin C 10-200 mg / L, glutathione 10-100 mg / L, linolenic acid 0.05-5 mg / L, meat The muscarin is 0.2-20 mg/L, the N-acetylcysteine is 5-500 μM, the ethanolamine is 0.01-10 mg/L, and the linoleic acid is 0.05-5 mg/L.
优选的，所述营养添加剂的组成成分和最终使用浓度分别为：人胰岛素0.5-15mg/L、维生素C 20-100mg/L、谷胱甘肽20-90mg/L、亚麻酸0.1-4mg/L、肉毒碱0.5-15mg/L、N-乙酰半胱氨酸10-400μM、乙醇胺0.05-8mg/L、亚油酸0.1-4mg/L。Preferably, the composition and final concentration of the nutritional additive are: human insulin 0.5-15 mg/L, vitamin C 20-100 mg/L, glutathione 20-90 mg/L, linolenic acid 0.1-4 mg/L, respectively. Carnitine 0.5-15mg/L, N-acetylcysteine 10-400μM, ethanolamine 0.05-8mg/L, linoleic acid 0.1-4mg/L.
最优选的，所述营养添加剂的组成成分和最终使用浓度分别为：人胰岛素1-10mg/L、维生素C 30-80mg/L、谷胱甘肽30-80mg/L、亚麻酸1-3mg/L、肉毒碱1-10mg/L、N-乙酰半胱氨酸50-100μM、乙醇胺0.1-5mg/L、亚油酸1-3mg/L。Most preferably, the composition and final concentration of the nutritional additive are: human insulin 1-10 mg / L, vitamin C 30-80 mg / L, glutathione 30-80 mg / L, linolenic acid 1-3 mg / L, carnitine 1-10 mg / L, N-acetylcysteine 50-100 μM, ethanolamine 0.1-5 mg / L, linoleic acid 1-3 mg / L.
本发明还提供了上述人神经干细胞培养基在培养人神经干细胞中的应用。The invention also provides the use of the above human neural stem cell culture medium for culturing human neural stem cells.
另外，如无特殊说明，本发明所用到的试剂、组分均可通过市售商购获得，如果没有特别说明，本发明所记载的任何范围包括端值以及端值之间的任何数值以及以端值或者端值之间的任意数值所构成的任意子范围。In addition, the reagents and components used in the present invention are commercially available, unless otherwise specified, and any range recited in the present invention includes any value between the end value and the end value, and Any subrange of arbitrary values between end values or end values.
本发明的有益效果如下：The beneficial effects of the present invention are as follows:
1、本发明培养基配方简单，成本低廉，化学成分明确，不含动物源性成分，不含蛋白提取物和水解物，因此批次之间均一稳定，没有潜在引入动物源性和人源性病原微生物的风险，更加安全，可以应用在临床研究和临床实验人神经干细胞的培养中。1. The medium of the invention has simple formula, low cost, clear chemical composition, no animal-derived components, no protein extract and hydrolyzate, so the batch is stable and there is no potential introduction of animal origin and humanity. The risk of pathogenic microorganisms is safer and can be applied to the clinical research and clinical experiments of human neural stem cell culture.
2、本发明首次将信号通路调控小分子应用到了人神经干细胞的培养中，在信号通路调控小分子的作用下，可以长期支持各种来源的人神经干细胞的快速增殖和干性的维持，成功解决了人神经干细胞在体外培养易分化和难以长期扩增的技术问题。2. The present invention firstly applies a small signal-modulating small molecule to the culture of human neural stem cells, and can support the rapid proliferation and dry maintenance of human neural stem cells of various sources under the action of signaling pathway regulating small molecules. The technical problem that human neural stem cells are easily differentiated in vitro and difficult to be amplified in a long-term manner is solved.
附图说明DRAWINGS
下面结合附图对本发明的具体实施方式作进一步详细的说明。The specific embodiments of the present invention will be further described in detail below with reference to the accompanying drawings.
图1示出人神经干细胞在实验组和对照组培养基中培养10代后的形态图。Fig. 1 is a view showing the morphology of human neural stem cells after culture for 10 passages in the experimental group and the control medium.
图2示出流式细胞术检测人神经干细胞分别在实验组和对照组培养基中培养10代后神经干细胞标志基因Nestin的表达比例。Fig. 2 shows the expression ratio of the neural stem cell marker gene Nestin after 10 generations of human neural stem cells cultured in the experimental group and the control group, respectively, by flow cytometry.
图3示出人神经干细胞在实验组培养基中培养10代后Nestin免疫荧光染色图片。Figure 3 shows a photograph of Nestin immunofluorescence staining of human neural stem cells after 10 passages in an experimental group culture medium.
图4示出人神经干细胞随机分化10天后免疫荧光检测神经元标志基因TUJ1和胶质细胞标志基因GFAP的表达情况。Figure 4 shows the expression of the neuronal marker gene TUJ1 and the glial marker gene GFAP by immunofluorescence after 10 days of random differentiation of human neural stem cells.
具体实施方式Detailed ways
为了更清楚地说明本发明，下面结合优选实施例和附图对本发明做进一步的说明。附图中相似的部件以相同的附图标记进行表示。本领域技术人员应当理解，下面所具体描述的内容是说明性的而非限制性的，不应以此限制本发明的保护范围。In order to explain the present invention more clearly, the present invention will be further described in conjunction with the preferred embodiments and the accompanying drawings. Similar components in the drawings are denoted by the same reference numerals. It should be understood by those skilled in the art that the following detailed description is intended to be illustrative and not restrictive.
本发明中实施例中所使用的基础培养基DMEM/F12 medium和Neurobasal medium购自Life Technologies；生长因子bFGF和EGF购自Peprotech；其他材料均为本领域常用材料。The basal medium DMEM/F12 medium and Neurobasal medium used in the examples of the present invention were purchased from Life Technologies; the growth factors bFGF and EGF were purchased from Peprotech; other materials were commonly used in the art.
实施例1 人神经干细胞培养基Example 1 Human neural stem cell culture medium
一种人神经干细胞培养基，所述培养基包括以下组分：A human neural stem cell culture medium comprising the following components:
基础培养基(体积比)：50％DMEM/F12 medium和50％Neurobasal medium。Base medium (volume ratio): 50% DMEM/F12 medium and 50% Neurobasal medium.
营养添加剂：所述营养添加剂的组成成分和最终使用浓度分别为：人胰岛素10mg/L、维生素C 50mg/L、谷胱甘肽50mg/L、亚麻酸1mg/L、肉毒碱20mg/L、N-乙酰半胱氨酸5μM、乙醇胺5mg/L、亚油酸1mg/L。Nutritional additive: the composition and final concentration of the nutritional additive are: human insulin 10mg / L, vitamin C 50mg / L, glutathione 50mg / L, linolenic acid 1mg / L, carnitine 20mg / L, N-acetylcysteine 5 μM, ethanolamine 5 mg/L, and linoleic acid 1 mg/L.
生长因子组成组分和其最终使用浓度：bFGF：20ng/mL；EGF：20ng/mL。Growth factor component and its final use concentration: bFGF: 20 ng/mL; EGF: 20 ng/mL.
信号通路调控小分子组成组分和和其最终使用浓度：Pifithrin-α hydrobromide，5μM和6-BIO，2μM。The signaling pathway regulates the small molecule component and its final use concentration: Pifithrin-α hydrobromide, 5 μM and 6-BIO, 2 μM.
实施例2 人神经干细胞培养基Example 2 Human neural stem cell culture medium
一种人神经干细胞培养基，所述培养基包括以下组分：A human neural stem cell culture medium comprising the following components:
基础培养基(体积比)：50％DMEM/F12 medium和50％Neurobasal medium。Base medium (volume ratio): 50% DMEM/F12 medium and 50% Neurobasal medium.
营养添加剂：所述营养添加剂的组成成分和最终使用浓度分别为：人胰岛素0.1mg/L、维生素C 10mg/L、谷胱甘肽10mg/L、亚麻酸0.05mg/L、 肉毒碱0.2mg/L、N-乙酰半胱氨酸5μM、乙醇胺0.01mg/L、亚油酸0.05mg/L。Nutritional additive: The composition and final concentration of the nutritional additive are: human insulin 0.1 mg / L, vitamin C 10 mg / L, glutathione 10 mg / L, linolenic acid 0.05 mg / L, carnitine 0.2 mg /L, N-acetylcysteine 5 μM, ethanolamine 0.01 mg/L, linoleic acid 0.05 mg/L.
生长因子组成组分和其最终使用浓度：bFGF：5ng/mL；EGF：5ng/mL。Growth factor component and its final use concentration: bFGF: 5 ng/mL; EGF: 5 ng/mL.
信号通路调控小分子组成组分和和其最终使用浓度：WNT3a，1ng/mL和Bax inhibitor peptide V5,0.1μM和Cyclic Pifithrin-α hydrobromide，0.1μM。The signaling pathway regulates the small molecule component and its final use concentration: WNT3a, 1 ng/mL and Bax inhibitor peptide V5, 0.1 μM and Cyclic Pifithrin-α hydrobromide, 0.1 μM.
实施例3 人神经干细胞培养基Example 3 Human neural stem cell culture medium
一种人神经干细胞培养基，所述培养基包括以下组分：A human neural stem cell culture medium comprising the following components:
基础培养基(体积比)：50％DMEM/F12 medium和50％Neurobasal medium。Base medium (volume ratio): 50% DMEM/F12 medium and 50% Neurobasal medium.
营养添加剂：所述营养添加剂的组成成分和最终使用浓度分别为：人胰岛素20mg/L、维生素C 200mg/L、谷胱甘肽100mg/L、亚麻酸5mg/L、肉毒碱20mg/L、N-乙酰半胱氨酸500μM、乙醇胺10mg/L、亚油酸5mg/L。Nutritional additive: the composition and final concentration of the nutritional additive are: human insulin 20mg / L, vitamin C 200mg / L, glutathione 100mg / L, linolenic acid 5mg / L, carnitine 20mg / L, N-acetylcysteine 500 μM, ethanolamine 10 mg/L, and linoleic acid 5 mg/L.
生长因子组成组分和其最终使用浓度：bFGF：100ng/mL；EGF：100ng/mL。Growth factor component and its final use concentration: bFGF: 100 ng/mL; EGF: 100 ng/mL.
信号通路调控小分子组成组分和和其最终使用浓度：WNT3a，1ng/mL和Bax inhibitor peptide V5,100μM和Cyclic Pifithrin-α hydrobromide，100μM。The signaling pathway regulates the small molecule component and its final use concentration: WNT3a, 1 ng/mL and Bax inhibitor peptide V5, 100 μM and Cyclic Pifithrin-α hydrobromide, 100 μM.
实施例4 人神经干细胞生物培养Example 4 Human neural stem cell biological culture
1、人神经干细胞培养基1. Human neural stem cell culture medium
实验组所用的人神经干细胞培养基的配方同实施例1；The formulation of the human neural stem cell culture medium used in the experimental group was the same as in Example 1;
对照组所用的人神经干细胞培养基为实施例1的培养基减去信号通路调控小分子。The human neural stem cell culture medium used in the control group was the medium of Example 1 minus the signaling pathway regulating small molecules.
2、人神经干细胞的培养2. Culture of human neural stem cells
2.1PLO-Laminin培养皿的包被2.1PLO-Laminin Petri Dish
将多聚鸟氨酸(PLO)用PBS稀释至15μg/mL，加入到待包被的培养皿中，加入的量请参考表1，37℃孵育2h或者4℃过夜，注意孵育过程中不要让培养皿的底部干掉。弃掉PLO，用PBS润洗两遍，DMEM/F12润洗一遍。将层黏连蛋白(Laminin)用DMEM/F12稀释至5μg/mL，加入到PLO包被过的培养皿中，加入的量请见表1，37℃孵育2h或者4℃过夜，注意孵育过程中不要让培养皿的底部干掉。The polyornithine (PLO) was diluted to 15 μg/mL with PBS and added to the Petri dish to be coated. For the amount of addition, please refer to Table 1. Incubate at 37 °C for 2 h or 4 °C overnight, not to let the incubation process The bottom of the dish is dried. Discard the PLO, rinse it twice with PBS, and rinse it with DMEM/F12. Laminin was diluted to 5 μg/mL with DMEM/F12 and added to the PLO-coated dish. See Table 1 for the amount of addition, incubate at 37 ° C for 2 h or 4 ° C overnight, pay attention to the incubation process. Do not let the bottom of the dish dry.
表1 包被培养皿所需的PLO和Laminin的量Table 1 Amount of PLO and Laminin required to coat the dish
培养皿规格Petri dish specification 生长面积(cm 2) Growing area (cm 2 ) 加入的量(mL)Amount added (mL)
6孔板6-well plate 10cm 2/well 10cm 2 /well 1mL/well1mL/well
12孔板12-well plate 4cm 2/well 4cm 2 /well 0.4mL/well0.4mL/well
24孔板24-well plate 2cm 2/well 2cm 2 /well 0.2mL/well0.2mL/well
35mm培养皿35mm culture dish 10cm 2 10cm 2 1mL1mL
60mm培养皿60mm culture dish 20cm 2 20cm 2 2mL2mL
100mm培养皿100mm culture dish 60cm 2 60cm 2 6mL6mL
2.2人神经干细胞的获得2.2 Human neural stem cell acquisition
采用将人多能性干细胞诱导分化获得人神经干细胞的方法。A method of obtaining human neural stem cells by inducing differentiation of human pluripotent stem cells.
其中人多能性干细胞分化为神经干细胞的培养基，配方如下：The medium in which human pluripotent stem cells differentiate into neural stem cells is formulated as follows:
基础培养基(体积比)：50％DMEM/F12 medium和50％Neurobasal medium。Base medium (volume ratio): 50% DMEM/F12 medium and 50% Neurobasal medium.
营养添加剂：同实施例1中营养添加剂的配方。Nutritional Additive: Formulation of the nutritional additive in the same manner as in Example 1.
诱导分化的小分子：SB431542 10μM和LDN193189 100nM(为本领域内常用的诱导人多能性干细胞分化为神经干细胞的商售小分子)。Small molecules that induce differentiation: SB431542 10 μM and LDN193189 100 nM (a commercially available small molecule that is commonly used in the art to induce differentiation of human pluripotent stem cells into neural stem cells).
将人多能性干细胞诱导分化为神经干细胞的方法如下：Methods for inducing differentiation of human pluripotent stem cells into neural stem cells are as follows:
将生长状况良好的人多能性干细胞用Accutase消化成单细胞，按照10 5cells/mL的密度接种至事先包被好PLO-Laminin的培养皿中，并在人多能性干细胞分化为神经干细胞的培养基中培养4天，即可以获得Nestin阳性的神经干细胞。 Human pluripotent stem cells with good growth condition were digested into single cells by Accutase, inoculated into a petri dish previously coated with PLO-Laminin at a density of 10 5 cells/mL, and differentiated into neural stem cells in human pluripotent stem cells. Nestin-positive neural stem cells can be obtained by culturing for 4 days in the medium.
2.3人神经干细胞的培养2.3 Human neural stem cell culture
将人神经干细胞分别在实验组和对照组的培养液中进行培养，培养的步骤如下：Human neural stem cells were cultured in the culture medium of the experimental group and the control group, respectively, and the cultivation steps were as follows:
弃掉人多能性干细胞分化为神经干细胞的培养基，用PBS清洗一遍，加入适量的Accutase，加入的量以盖过细胞为宜，37℃消化3min，此时细胞呈现松散的贴壁状态，小心弃掉Accutase，用人神经干细胞培养基重新悬浮细胞，并进行细胞计数，按照10 5cells/mL的密度接种至事先包被好的培养皿中。 Discard the medium in which human pluripotent stem cells differentiate into neural stem cells, wash them once with PBS, add appropriate amount of Accutase, and add the amount to cover the cells, and digest at 37 °C for 3 min. At this time, the cells are loosely attached. The Accutase was carefully discarded, the cells were resuspended in human neural stem cell culture medium, and cell counts were performed, and inoculated into a previously coated petri dish at a density of 10 5 cells/mL.
培养结果如图1-3所示，图1给出了人神经干细胞在实验组和对照组培养液中分别培养10代后的形态图，由此可以看出人神经干细胞在实验组人神经干细胞培养基中的生长状态要比对照组人神经干细胞培养基的好。图2给出了人神经干细胞在实验组和对照组人神经干细胞培养基中分别培养10代后人神经干细胞标志性基因Nestin的表达比例，培养10代后，在实验组 培养基中，人神经干细胞Nestin的表达比例仍然在90％以上，而在对照组培养基中，Nestin的表达比例缺不足50％。由此可以看出，我们筛选出的信号通路调控小分子，在维持神经干细胞自我更新和干性维持方面具有重要的作用。图3给出了实验组人神经干细胞在实验组培养液中培养10代后人神经干细胞标志基因Nestin的免疫荧光染色图片。The culture results are shown in Figure 1-3. Figure 1 shows the morphological map of human neural stem cells cultured in the experimental group and the control group for 10 generations respectively. It can be seen that human neural stem cells are in the experimental group of human neural stem cells. The growth state in the medium was better than that of the control human neural stem cell culture medium. Figure 2 shows the expression ratio of human neural stem cell marker gene Nestin after human cultured neural stem cells culture in experimental and control human neural stem cell culture medium for 10 generations. After culture for 10 generations, in the experimental group medium, human nerve The expression ratio of stem cells in Nestin was still above 90%, while in the control medium, the expression ratio of Nestin was less than 50%. It can be seen that the signal pathways we screen out regulate small molecules and play an important role in maintaining the self-renewal and dry maintenance of neural stem cells. Figure 3 shows the immunofluorescence staining of the human neural stem cell marker gene Nestin after culture of human neural stem cells in the experimental group for 10 generations.
实施例5 本发明培养得到的神经干细胞的分化能力试验Example 5 Differentiation ability test of neural stem cells cultured in the present invention
对神经干细胞进行分化，发现本发明培养的神经干细胞培养10代后仍具有分化成神经元和胶质细胞的潜能。Differentiation of neural stem cells revealed that the neural stem cells cultured in the present invention still have the potential to differentiate into neurons and glial cells after 10 generations of culture.
具体的方法如下：The specific method is as follows:
1、神经干细胞随机分化培养液的配方如下：1. The formula of the neural stem cell random differentiation culture solution is as follows:
DMEM/F12 medium：49％；DMEM/F12 medium: 49%;
Neurobasal medium：49％。Neurobasal medium: 49%.
营养添加剂：配方同实施例1中的营养添加剂配方。Nutritional Additive: The formulation is the same as the nutritional additive formulation of Example 1.
血清(Fetal Bovin Serum,FBS)：1％。Serum (Fetal Bovin Serum, FBS): 1%.
2、人神经干细胞在体外随机分化成神经元和胶质细胞的方法如下：2. The method for random differentiation of human neural stem cells into neurons and glial cells in vitro is as follows:
按照实施例4所述的方法将神经干细胞消化接种，接种后第2天给细胞换成神经干细胞随机分化培养液，以后隔日换液，直到培养到第10天，进行免疫荧光染色鉴定，结果如图4所示，从图4中可以看出人神经干细胞在本神经干细胞培养液培养10代后，随机分化10天后，仍然具有分化成神经元和胶质细胞的潜能。The neural stem cells were digested and inoculated according to the method described in Example 4. The cells were replaced with neural stem cells randomly differentiated culture solution on the 2nd day after inoculation, and then changed every other day until the 10th day of culture, and the results were confirmed by immunofluorescence staining. As shown in Fig. 4, it can be seen from Fig. 4 that human neural stem cells still have the potential to differentiate into neurons and glial cells after 10 days of random differentiation in the culture of the neural stem cell culture medium for 10 days.
显然，本发明的上述实施例仅仅是为清楚地说明本发明所作的举例，而并非是对本发明的实施方式的限定，对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式的变化或变动，这里无法对所有的实施方式予以穷举，凡是属于本发明的技术方案所引伸出的显而易见的变化或变动仍处于本发明的保护范围之列。It is apparent that the above-described embodiments of the present invention are merely illustrative of the present invention and are not intended to limit the embodiments of the present invention, and those skilled in the art can also make the above description. It is to be understood that various changes and modifications may be made without departing from the spirit and scope of the invention.

Claims (10)

  1. 一种人神经干细胞培养基，其特征在于，所述培养基包括以下组分：基础培养基、营养添加剂、生长因子和信号通路调控小分子。A human neural stem cell culture medium, characterized in that the culture medium comprises the following components: a basal medium, a nutritional additive, a growth factor, and a signaling pathway regulating small molecule.
  2. 根据权利要求1所述的人神经干细胞培养基，其特征在于，所述生长因子包括碱性成纤维细胞生长因子和上皮细胞生长因子，二者的最终使用浓度均为5-100ng/mL，优选的为6-50ng/mL，最优选的为10-30ng/mL。The human neural stem cell culture medium according to claim 1, wherein the growth factor comprises basic fibroblast growth factor and epithelial cell growth factor, and the final use concentration of both is 5-100 ng/mL, preferably It is 6-50 ng/mL, and most preferably 10-30 ng/mL.
  3. 根据权利要求1所述的人神经干细胞培养基，其特征在于，所述信号通路调控小分子为Wnt信号通路激活剂、凋亡抑制小分子、p53信号通路抑制剂中的一种或几种组合；优选的，为Wnt信号通路激活剂和p53信号通路抑制剂一种或两种组合。The human neural stem cell culture medium according to claim 1, wherein the signaling pathway regulating small molecule is one or a combination of a Wnt signaling pathway activator, an apoptosis inhibitory small molecule, and a p53 signaling pathway inhibitor. Preferably, one or a combination of a Wnt signaling pathway activator and a p53 signaling pathway inhibitor.
  4. 根据权利要求1所述的人神经干细胞培养基，其特征在于，所述Wnt信号通路激活剂及最终使用浓度为WNT3a，1-100ng/mL；6-BIO，1-20μM；CHIR-98014，0.1-20μM；TDZD-8，10-100μM；AZD1080，0.1-50μM；CHIR99021，0.1-20μM中的一种或者几种的组合；优选的，为WNT3a，1-100ng/mL或6-BIO，1-20μM；最优选的，为6-BIO，1-20μM。The human neural stem cell culture medium according to claim 1, wherein the Wnt signaling pathway activator and the final use concentration are WNT3a, 1-100 ng/mL; 6-BIO, 1-20 μM; CHIR-98014, 0.1 -20 μM; TDZD-8, 10-100 μM; AZD1080, 0.1-50 μM; CHIR99021, 0.1-20 μM in combination with one or more; preferably, WNT3a, 1-100 ng/mL or 6-BIO, 1- 20 μM; most preferably, 6-BIO, 1-20 μM.
  5. 根据权利要求1所述的人神经干细胞培养基，其特征在于，所述凋亡抑制小分子及最终使用浓度为Bax inhibitor peptide V5，0.1-100μM；iMAC2，0.01-100μM；Embelin，0.5-500μM中的一种或几种的组合；优选的，为Bax inhibitor peptide V5,0.1-100μM和iMAC2，0.01-100μM中的一种或两种的组合；最优选的，为Bax inhibitor peptide V5,0.1-100μM。The human neural stem cell culture medium according to claim 1, wherein the apoptosis inhibiting small molecule and the final use concentration are Bax inhibitor peptide V5, 0.1-100 μM; iMAC2, 0.01-100 μM; Embelin, 0.5-500 μM One or a combination of several; preferably, one or a combination of Bax inhibitor peptide V5, 0.1-100 μM and iMAC2, 0.01-100 μM; most preferred, Bax inhibitor peptide V5, 0.1-100 μM .
  6. 根据权利要求1所述的人神经干细胞培养基，其特征在于，所述p53信号通路抑制剂及最终使用浓度为Pifithrin-αhydrobromide，0.1-100μM；Cyclic Pifithrin-αhydrobromide，0.1-100μM；Pifithrin-μ，0.1-100μM；SJ172550，0.5-500μM中的一种或几种的组合；优选的，为Cyclic Pifithrin-αhydrobromide，0.1-100μM或Pifithrin-αhydrobromide，0.1-100μM；最优选的，为Pifithrin-αhydrobromide，0.1-100μM。The human neural stem cell culture medium according to claim 1, wherein the p53 signaling pathway inhibitor and the final use concentration are Pifithrin-αhydrobromide, 0.1-100 μM; Cyclic Pifithrin-αhydrobromide, 0.1-100 μM; Pifithrin-μ, 0.1-100 μM; SJ172550, a combination of one or more of 0.5-500 μM; preferably, Cyclic Pifithrin-αhydrobromide, 0.1-100 μM or Pifithrin-αhydrobromide, 0.1-100 μM; most preferably, Pifithrin-αhydrobromide, 0.1 -100 μM.
  7. 根据权利要求1所述的人神经干细胞培养基，其特征在于，所述基础培养基为DMEM/F12 medium、Neurobasal medium中的一种或两种；优选的，为体积比为1:1的DMEM/F12 medium和Neurobasal medium混合培养基。The human neural stem cell culture medium according to claim 1, wherein the basal medium is one or both of DMEM/F12 medium and Neurobasal medium; preferably, DMEM is 1:1 by volume. /F12 medium and Neurobasal medium mixed medium.
  8. 根据权利要求1所述的人神经干细胞培养基，其特征在于，所述营养添加剂组成成分和最终使用浓度分别为：人胰岛素0.1-20mg/L、维生素C 10-200mg/L、谷胱甘肽10-100mg/L、亚麻酸0.05-5mg/L、肉毒碱0.2-20mg/L、N-乙酰半胱氨酸5-500μM、乙醇胺0.01-10mg/L、亚油酸0.05-5mg/L。The human neural stem cell culture medium according to claim 1, wherein the nutritional additive component and the final use concentration are: human insulin 0.1-20 mg/L, vitamin C 10-200 mg/L, glutathione, respectively. 10-100 mg/L, linolenic acid 0.05-5 mg/L, carnitine 0.2-20 mg/L, N-acetylcysteine 5-500 μM, ethanolamine 0.01-10 mg/L, linoleic acid 0.05-5 mg/L.
  9. 根据权利要求8所述的人神经干细胞培养基，其特征在于，所述营养添加剂的组成成分和最终使用浓度分别为：人胰岛素0.5-15mg/L、维生素C 20-100mg/L、谷胱甘肽20-90mg/L、亚麻酸0.1-4mg/L、肉毒碱0.5-15mg/L、N-乙酰半胱氨酸10-400μM、乙醇胺0.05-8mg/L、亚油酸0.1-4mg/L；优选的，所述营养添加剂的组成成分和最终使用浓度分别为：人胰岛素1-10mg/L、维生素C 30-80mg/L、谷胱甘肽30-80mg/L、亚麻酸1-3mg/L、肉毒碱1-10mg/L、N-乙酰半胱氨酸50-100μM、乙醇胺0.1-5mg/L、亚油酸1-3mg/L。The human neural stem cell culture medium according to claim 8, wherein the composition and final use concentration of the nutritional additive are: human insulin 0.5-15 mg/L, vitamin C 20-100 mg/L, glutathione, respectively. Peptide 20-90mg/L, linolenic acid 0.1-4mg/L, carnitine 0.5-15mg/L, N-acetylcysteine 10-400μM, ethanolamine 0.05-8mg/L, linoleic acid 0.1-4mg/L Preferably, the composition and final concentration of the nutritional additive are: human insulin 1-10 mg / L, vitamin C 30-80 mg / L, glutathione 30-80 mg / L, linolenic acid 1-3 mg / L, carnitine 1-10 mg / L, N-acetylcysteine 50-100 μM, ethanolamine 0.1-5 mg / L, linoleic acid 1-3 mg / L.
  10. 一种如权利要求1-9任一所述的人神经干细胞培养基在培养人神经干细胞的应用。Use of a human neural stem cell culture medium according to any one of claims 1-9 for culturing human neural stem cells.
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