WO2019174535A1 - 3d大脑类器官的制备方法 - Google Patents

3d大脑类器官的制备方法 Download PDF

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WO2019174535A1
WO2019174535A1 PCT/CN2019/077595 CN2019077595W WO2019174535A1 WO 2019174535 A1 WO2019174535 A1 WO 2019174535A1 CN 2019077595 W CN2019077595 W CN 2019077595W WO 2019174535 A1 WO2019174535 A1 WO 2019174535A1
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medium
culture
wrapped
brain
neurobasal
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范靖
王安欣
邹潭
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浙江霍德生物工程有限公司
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Priority to JP2020572596A priority Critical patent/JP2021516997A/ja
Priority to EP19768309.7A priority patent/EP3766965A4/en
Priority to US16/979,981 priority patent/US20210355438A1/en
Publication of WO2019174535A1 publication Critical patent/WO2019174535A1/zh

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    • C12N2501/10—Growth factors
    • C12N2501/13—Nerve growth factor [NGF]; Brain-derived neurotrophic factor [BDNF]; Cilliary neurotrophic factor [CNTF]; Glial-derived neurotrophic factor [GDNF]; Neurotrophins [NT]; Neuregulins
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    • C12N2533/50—Proteins
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Definitions

  • the invention belongs to the technical field of 3D brain organ, and particularly relates to a method for preparing a 3D brain organ from a human neurosphere.
  • Human nerve cells or brain tissue are difficult to obtain and can hardly be cultured in vitro, making many neurological research and drug developments stagnant without a good human model.
  • the differentiation of embryonic stem cells or induced pluripotent stem cells into human neural cells as a neurological disease model is an international innovation and hotspot in recent years.
  • 3D culture can simulate the brain tissue environment and cell communication, it plays an important role in promoting the maturation and function of nerve cells, and the 3D brain organs have an irreplaceable role in the study of human brain development and related diseases. Recently, In vitro culture of various human 3D organs has become a new hot spot.
  • the 3D brain organs obtained by these two methods are difficult to stably control the reproducibility and structural similarity of the products due to the cells and structures that may contain other germ layers at the same time, and are applied in the field of neurological disease models and drug screening. Restricted, but also affects the representativeness and reliability of the data obtained with it.
  • an object of the present invention is to provide a method for preparing a 3D brain organ, and the method provided by the invention can obtain a 3D brain organ with uniform size and structure, and the method is simple and suitable for industrialization.
  • the invention provides a method for preparing a 3D brain organ, comprising:
  • the neurospheres obtained by the RONA method were digested into single cells by accutase, counted, and then inoculated on a cell culture plate in a medium A, cultured until the seventh day, and cultured in the medium B, and cultured until the 25th to 35th days. After the nerve ball is wrapped, the culture is continued for 55 to 65 days, and the culture is continued after the second nerve ball is wrapped;
  • the medium A includes: retinoic acid, BDNF, GDNF, ascorbic acid, cAMP, Neurobasal medium, and B27 additive (without Vitamine A);
  • the medium B includes: BDNF, GDNF, ascorbic acid, cAMP, Neurobasal medium, and B27 additive (without Vitamine A).
  • the invention starts from the highly purified neurosphere obtained by the RONA method, and ensures that more than 99% are neural stem cells, thereby solving the problem that other methods contain non-neuronal stem cells and non-brain tissue in subsequent culture.
  • the invention breaks up the neurospheres into single cells and counts them in a fixed amount, which can ensure the uniformity of the size and composition of the cell clusters, and the size and structure of the 3D brain organs will not be significantly different even after 90 days of culture. .
  • the medium A and the medium B used in the culture process of the present invention ensure that the cultured 3D brain function is induced into the brain tissue of the forebrain, the midbrain and the hindbrain, and the cells and structures of the six layers of the brain cortex. .
  • the present invention is based on the neurosphere (neural sphere) purified by the RONA method, and more than 99% of the human neural precursor cells obtained by the method are neural stem cells.
  • the neurosphere (neurosphere) purified by the RONA method is first digested into single cells by accutase, and counted and seeded on a cell culture plate. Specifically, the present invention counts and implants an equal amount of 1000 to 50,000 cells onto a multi-well cell culture plate to ensure uniform cell cluster size. In one embodiment, an equal number of 1000-10000 cells are seeded into the wells of a round bottom ultra-low attached 96-well cell culture plate.
  • the medium A was cultured, and the medium A included: retinoic acid, BDNF, GDNF, ascorbic acid, cAMP, Neurobasal medium, and B27 additive (without Vitamine A).
  • the medium A comprises: 1-5 ⁇ M retinoic acid; 10-30 ng/ml BDNF; 10-30 ng/ml GDNF; 0.1-0.5 mM ascorbic acid; 5-15 ⁇ M cAMP; Neurobasal and B27 additives (not Contains Vitamine A).
  • the medium A comprises: 2 ⁇ M retinoic acid; 20 ng/ml BDNF; 20 ng/ml GDNF; 0.2 mM ascorbic acid; 10 ⁇ M cAMP; Neurobasal and B27 additives (without Vitamine A), wherein Neurobasal and B27 The additive (without Vitamine A) is used in a ratio of 50:1.
  • the medium B includes: BDNF, GDNF, ascorbic acid, cAMP, Neurobasal medium, and B27 additive (without Vitamine A).
  • the medium B comprises: 10-30 ng/ml BDNF; 10-30 ng/ml GDNF; 0.1-0.5 mM ascorbic acid; 5-15 ⁇ M cAMP; Neurobasal and B27 additives (without Vitamine A).
  • the medium B comprises: 20 ng/ml BDNF; 20 ng/ml GDNF; 0.2 mM ascorbic acid; 10 ⁇ M cAMP; Neurobasal and B27 additives (without Vitamine A), wherein the volume ratio of Neurobasal and B27 additives It is 50:1.
  • culture medium B When culture medium B continues to culture until the 25th to 35th day, the neurosphere is wrapped and cultured for 55-65 days. After the second nerve ball is wrapped, the culture is continued until 85-100 days, and a uniform simulated human brain can be obtained. It consists of 3D brain organs and can be wrapped and cultured as needed.
  • the neurospheres are wrapped with Matrigel and cultured for 55 to 65 days, and the neurospheres are wrapped for the second time with Matrigel, and the culture is continued.
  • the present invention can also continue to culture after the third time the nerve ball is wrapped until the 85th to 100th day of culture, and preferably, the neurosphere is also wrapped with Matrigel for the third time.
  • the culture is continued until the 30th day, after the nerve ball is wrapped, the culture is continued for 60 days, and after the second nerve ball is wrapped, the culture is continued until 90 days after the third nerve ball is wrapped.
  • the experimental results show that the 3D-type brain corpuscles prepared by the RONA method are relatively uniform in size and shape, and can reach a diameter of 4 mm at 88 days and still continue to grow; It can express markers such as Nestin, Tuj1, Foxg1, TBR2 and NKX2.1, and has the ability to develop forebrain, midbrain and hindbrain; it can also express BRN2, SATB2, CTIP2, TBR1 and other markers in distribution and proportion. Very similar to the brain, with the ability to stabilize the structure of the cerebral cortex.
  • the invention starts from the highly purified neurosphere obtained by the RONA method, and can control and culture a true 3D brain organ with uniform size and structure by a relatively simple method, which has a six-layer cortical structure of the brain and an inhibitory interneuron cell.
  • Each subtype is suitable for in vitro disease research, drug screening, etc., and has significant industrial significance.
  • FIG. 1 is a flow chart showing a process for preparing a 3D brain organ according to Embodiment 1 of the present invention
  • Figure 2 is a photograph of a 3D brain corpuscle cultured to the 17th day in the first embodiment of the present invention
  • Figure 3 is a photograph of a 3D brain corpuscle cultured on day 50 of Example 1 of the present invention.
  • Figure 4 is a photograph of a 3D brain corpuscle cultured to the 50th day in Example 1 of the present invention.
  • Fig. 5 is a photograph of a 3D-type brain corpuscle cultured on day 88 of Example 1 of the present invention.
  • Example 6 is a tissue section and staining photograph of precursor cells of different brain regions of 3D type cerebral bodies cultured in the first week from Example 1;
  • Example 7 is a tissue section and staining photograph of different cerebral cortical neurons of 3D type brain bodies cultured on the 88th day in Example 1;
  • Example 8 is a tissue section and staining photograph of 3D type cerebral glial cells and nerve cells cultured to the 63rd day in Example 1;
  • Fig. 9 is a tissue section and staining photograph of 3D-type brain glial cells and nerve cells cultured on the 63rd day in Example 1.
  • FIG. 1 is a flow chart of a process for preparing a 3D brain organ according to Embodiment 1 of the present invention.
  • Step 1 After obtaining the neurosphere (neural sphere) from the RONA method (see the article "Cultured networks of excitatory projection neurons and inhibitory interneurons for studying human cortical neurotoxicity” published by Xu JC and Fan J in Science Translational Medicine in April 2016) After being digested into three cells by accutase, the same number of 5000 cells were inoculated into the wells of a 96-well cell culture plate with a low-bottomed round bottom, and cultured on a circular shaker of a carbon dioxide incubator at 37 ° C using medium A.
  • medium A is a final concentration of 2 ⁇ M retinoic acid, 20 ng / ml BDNF and GDNF, 0.2 mM ascorbic acid, 10 ⁇ M cAMP Neurobasal and B27 additives (without Vitamine A), Wherein, the ratio of the amount of Neurobasal and B27 additive is 50:1;
  • Step 2 On the second day, a uniform-sized neurosphere was observed in each well.
  • the medium was changed and the medium B was used to continue the culture; the medium B was at a final concentration of 20 ng/ Ml BDNF and GDNF, 0.2 mM ascorbic acid, 10 ⁇ M cAMP Neurobasal and B27 additive (without Vitamine A), wherein the ratio of Neurobasal and B27 additives is 50:1;
  • Step 3 When cultured until the 30th day, the neurospheres were wrapped with Matrigel on the surface of the non-hydrophilic sterile material, and the cells were further cultured in 96 wells until the 60th day. The Matrigel was further wrapped and cultured until the 90th day, and a uniform simulation was obtained.
  • a 3D brain organ composed of the human brain and can be wrapped and cultured as needed.
  • FIG. 2 is a photograph of a 3D-type brain corpuscle cultured to the 17th day in the first embodiment of the present invention
  • FIG. 3 is a 3D class cultured to the 50th day in the first embodiment of the present invention
  • Fig. 4 is a photograph of a 3D-type brain corpuscle cultured to the 50th day in the first embodiment of the present invention
  • Fig. 5 is a photograph of a 3D-type brain corpuscle cultured on the 88th day in the first embodiment of the present invention.
  • the size and shape of the 3D brain corpuscles are relatively uniform, and the diameter can reach 4 mm at 88 days and can continue to grow, while the other 3D brain corpuscles obtained by most methods are the same. Time is hard to grow to such a size and stay healthy.
  • Fig. 6 is a tissue section and staining photograph of precursor cells of different brain regions of 3D type cerebral bodies cultured in the first week from Example 1, wherein Nestin is a common marker protein expressed by neural precursor cells. Tuj1 is a protein marker commonly expressed by nerve cells, Foxg1 is a marker of forebrain precursor cells, TBR2 is a marker of the neural progenitor cells of the midbrain Subventricalzone and hippocampus, and NKX2.1 is a marker of hindbrain precursor cells. DAPI is a DNA dye. As can be seen from Fig. 6, the 3D-type brain corpuscle obtained by the method of the present invention has the ability to develop the forebrain, midbrain and hindbrain.
  • Fig. 7 is a tissue section and staining photograph of different cerebral cortical neurons of 3D type corpuscles cultured in the first day from the first day to the 88th day, wherein REELIN, BRN2, SATB2, CTIP2, and TBR1 are cerebral cortex I, respectively. /II, III, IV, V, VI markers of various layers of nerve cells. As can be seen from Fig. 7, the 3D-type brain corpuscle obtained by the production method of the present invention expresses markers of the above different cortex.
  • FIG. 8 is a tissue section and staining photograph of 3D type cerebral glial cells and nerve cells cultured on the 63rd day of Example 1, wherein nNOS, PV and SST are inhibitory brain neurons, respectively. Markers, MAP2 is a marker of relatively mature nerve cells, and DAPI is a DNA dye.
  • the 3D brain corpuscle obtained by the manufacturing method of the present invention contains at least three inhibitory brain neurons which play important roles in brain development and function, and are relatively mature.
  • FIG. 9 is a tissue section and staining photograph of 3D type cerebral glial cells and nerve cells cultured on the 63rd day from Example 1, wherein GFAP and MAP2 are glial cells and nerve cells, respectively. Marker, DAPI is a DNA dye. According to statistics, the proportion of glial cells in all cells of 3D type brain bodies is about 50-70%. As can be seen from Fig. 9, the 3D-type brain corpuscle obtained by the production method of the present invention contains glial cells which play an important role in brain development and function, and the approximate proportion and distribution are very close to the composition of the human brain.

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Abstract

提供了一种3D大脑类器官的制备方法，包括：将RONA法获得的神经球经accutase消化为单细胞，计数后接种于细胞培养板上在培养基A中培养，培养至第7天后在培养基B中继续培养，培养至第25～35天时，Matrigel包裹神经球后继续培养至55～65天，第二次Matrigel包裹神经球后继续培养。还提供了一种用于培养3D大脑类器官的培养基。

Description

3D大脑类器官的制备方法 技术领域
本发明属于3D大脑类器官技术领域，尤其涉及一种从人神经球制备3D大脑类器官的方法。
背景技术
人的神经细胞或大脑组织很难获得，在体外几乎无法培养，使得很多神经疾病的研究和药物研发没有很好的人源模型而停滞不前。由胚胎干细胞或诱导多能干细胞诱导分化得到人的神经细胞作为神经疾病模型是这几年国际上的创新技术和热点。同时由于3D培养更能模拟大脑组织环境和细胞交流，对于神经细胞的成熟和功能有着重要的促进作用，并且所得到3D大脑类器官在研究人大脑发育和相关疾病上有不可替代的作用，最近各种人3D器官的体外培养成为新的热点。
传统的人3D大脑类器官的制作方法一般通过从拟胚胎体(Embryonic Body)开始，如2013年及2014年Lancaster等发表在Nature及Nature protocol上的3D大脑类器官的制作方法使得界内在体外使用人多能干细胞(包括胚胎干细胞及诱导多能干细胞)得到3D的模拟人大脑的类器官上前进了一大步。最近Qian等2016年在Cell上发表的也是使用类似的方法从人多能干细胞制作大脑类器官来研究寨卡病毒引发小脑症的机制，并且发明了小的多孔板搅拌装置来降低因子的用量和成本，以及增加均一性。但是，这两种方法所得到的3D大脑类器官由于同时可能含有其他胚层的细胞和结构，难以稳定控制得到产品的可重复性，结构相似性等，在神经系统疾病模型和药物筛选领域的应用受到限制，同时也影响到用其所获得数据的代表性和可靠性。
2017年Lee等在Neuropsychopharmacology上发表的文章中，其手工挑选出大小在50000～200000微米之间的玫瑰花样细胞簇(主要为NPC，神经前体细胞)，对其进行诱导，但是其得到的3D新大脑皮层小体不具有发育出后脑(NKX2.1染色为阴性)的潜能。而且，Lee等的方法较为复杂，无法做到简 易，大批量而且均一。
发明内容
有鉴于此，本发明的目的在于提供一种3D大脑类器官的制备方法，本发明提供的方法可以得到大小、结构均一的3D大脑类器官，且方法简易，适于产业化。
本发明提供了一种3D大脑类器官的制备方法，包括：
将RONA法获得的神经球经accutase消化为单细胞，计数后接种于细胞培养板上在培养基A中培养，培养至第7天后在培养基B中继续培养，培养至第25～35天时，包裹神经球后继续培养至55～65天，第二次包裹神经球后继续培养；
所述培养基A包括：维甲酸、BDNF、GDNF、抗坏血酸、cAMP、Neurobasal培养基和B27添加剂(不含Vitamine A)；
所述培养基B包括：BDNF、GDNF、抗坏血酸、cAMP、Neurobasal培养基和B27添加剂(不含Vitamine A)。
本发明从RONA方法得到的高度纯化的神经球开始，保证了99％以上是神经干细胞，从而解决了其他方法含有非神经命运的干细胞及在后续培养中会出现非大脑组织的问题。本发明将神经球打散成为单细胞后计数并按固定数量接种，可以保证细胞团的大小和组成的均一性，即使培养至90天以后3D类脑器官的大小及结构也不会出现显著差异。同时本发明培养过程中使用的培养基A和培养基B确保所培养的3D大脑类器官能被诱导成为前脑，中脑和后脑三个脑区的组织，以及大脑六层皮层的细胞和结构。
本发明从通过RONA法纯化得到的neurosphere(神经球)出发，该方法获得的人神经前体细胞99％以上是神经干细胞。RONA法纯化得到neurosphere(神经球)的具体过程参见Xu JC和Fan J于2016年4月在Science Translational Medicine上所发表的Cultured networks of excitatory projection neurons and inhibitory interneurons for studying human cortical neurotoxicity一文。
本发明首先将通过RONA法纯化得到的neurosphere(神经球)经accutase消化为单细胞，计数后接种于细胞培养板上。具体而言，本发明计数后种植 同等数量的1000～50000个细胞至多孔细胞培养板上，保证细胞团大小的均一。在一个实施例中，接种同等数量的1000～10000个细胞至圆底超低附的96孔细胞培养板的孔内。
接种后在培养基A中进行培养，所述培养基A包括：维甲酸(retinoic acid)、BDNF、GDNF、抗坏血酸(ascorbic acid)、cAMP、Neurobasal培养基和B27添加剂(不含Vitamine A)。在一个实施例中，所述培养基A包括：1～5μM维甲酸；10～30ng/ml BDNF；10～30ng/ml GDNF；0.1～0.5mM抗坏血酸；5～15μM cAMP；Neurobasal和B27添加剂(不含Vitamine A)。在一个实施例中，所述培养基A包括：2μM维甲酸；20ng/ml BDNF；20ng/ml GDNF；0.2mM抗坏血酸；10μM cAMP；Neurobasal和B27添加剂(不含Vitamine A)，其中，Neurobasal和B27添加剂(不含Vitamine A)的用量比为50:1。
接种后在培养基A中置于37℃，5％CO 2细胞培养箱中的低速圆周摇床上摇动培养，每3～5天半量换液，第二天即可观察到每个孔内都形成一个大小均一的神经球，培养至第7天更换至培养基B中同等条件继续培养。
在本发明中，所述培养基B包括：BDNF、GDNF、抗坏血酸、cAMP、Neurobasal培养基和B27添加剂(不含Vitamine A)。在一个实施例中，所述培养基B包括：10～30ng/ml BDNF；10～30ng/ml GDNF；0.1～0.5mM抗坏血酸；5～15μM cAMP；Neurobasal和B27添加剂(不含Vitamine A)。在一个实施例中，所述培养基B包括：20ng/ml BDNF；20ng/ml GDNF；0.2mM抗坏血酸；10μM cAMP；Neurobasal和B27添加剂(不含Vitamine A)，其中，Neurobasal和B27添加剂的体积比为50:1。
在培养基B继续培养至第25～35天时，包裹神经球后继续培养至55～65天，第二次包裹神经球后继续培养，培养至85-100天左右，可以得到均一的模拟人大脑组成的3D大脑类器官，并可根据需求继续包裹和培养。
具体而言，本发明在培养至第25～35天时，用Matrigel包裹神经球后继续培养至55～65天，用Matrigel第二次包裹神经球后继续培养。
具体而言，本发明还可以在培养至第85～100天第三次包裹神经球后继续培养，优选地，第三次也用Matrigel包裹神经球。
在一个实施例中，本发明培养至第30天时，包裹神经球后继续培养至60天，第二次包裹神经球后继续培养至90天第三次包裹神经球后继续培养。
实验结果表明，本发明从RONA方法得到的高度纯化的神经球出发，制备的3D类脑小体的大小和形状都相对非常均一，而且在88天时直径可达到4mm并且仍然能继续生长；且其能够表达Nestin、Tuj1、Foxg1、TBR2、NKX2.1等标志物，具备发育出前脑、中脑和后脑的能力；其也能够表达BRN2，SATB2，CTIP2，TBR1等标志物，且在分布和比例上与大脑非常相似，具备稳定得到具有大脑皮层结构的能力。
本发明从RONA方法得到的高度纯化的神经球开始，用较为简单的方法可以控制并培养得到大小及结构均一的真正的3D大脑类器官，其具有大脑的六层皮层结构和抑制性中间神经细胞各亚型，适用于体外疾病研究、药物筛选等，具有重大的产业化的意义。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据提供的附图获得其他的附图。
图1为本发明实施例1提供的3D大脑类器官的制备工艺流程图；
图2是本发明实施例1培养至第17天的3D类脑小体照片；
图3是本发明实施例1培养至第50天的3D类脑小体照片；
图4是本发明实施例1培养至第50天的3D类脑小体照片；
图5是本发明实施例1培养至第88天的3D类脑小体照片。
图6为实施例1培养至第10周的3D类脑小体不同脑区的前体细胞的组织切片及染色照片；
图7为实施例1培养至第88天的3D类脑小体不同大脑皮层神经细胞的组织切片及染色照片；
图8为实施例1培养至第63天的3D类脑小体神经胶质细胞及神经细胞的组织切片及染色照片；
图9为实施例1培养至第63天的3D类脑小体神经胶质细胞及神经细胞的组织切片及染色照片。
具体实施方式
下面将对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例1
参见图1，图1为本发明实施例1提供的3D大脑类器官的制备工艺流程图。
步骤一、从RONA法(见Xu JC和Fan J于2016年4月在Science Translational Medicine上所发表的Cultured networks of excitatory projection neurons and inhibitory interneurons for studying human cortical neurotoxicity一文)得到neurosphere(神经球)后，经accutase消化为单细胞，计数后分别接种同等数量的5000个细胞至圆底超低附的96孔细胞培养板的孔内，使用培养基A放置在37摄氏度的二氧化碳孵箱的圆周摇床上培养7天，每3～5天半量换液培养；培养基A为含有终浓度2μM retinoic acid，20ng/ml BDNF and GDNF，0.2mM ascorbic acid，10μM cAMP的Neurobasal及B27添加剂(不含Vitamine A)，其中，Neurobasal和B27添加剂的用量比为50:1；
步骤二、第二天即可观察到每个孔内都形成一个大小均一的神经球，在培养至第7天时，更换培养基，使用培养基B继续培养；培养基B为含有终浓度20ng/ml BDNF and GDNF，0.2mM ascorbic acid，10μM cAMP的Neurobasal及B27添加剂(不含Vitamine A)，其中，Neurobasal和B27添加剂的用量比为50:1；
步骤三，培养至第30天时，在非亲水的无菌材料表面用Matrigel包裹神经球，继续在96孔中培养至第60天，继续包裹Matrigel并培养至第90天，可以得到均一的模拟人大脑组成的3D大脑类器官，并可根据需求继续包裹和培养。
参见图2、图3、图4和图5，图2是本发明实施例1培养至第17天的3D类脑小体照片，图3是本发明实施例1培养至第50天的3D类脑小体照片，图4是本发明实施例1培养至第50天的3D类脑小体照片，图5是本发明实施例1培养至第88天的3D类脑小体照片。由图2～图5可知，3D类脑小体的大小和形状都相对非常均一，而且在88天时直径可达到4mm并且仍然能继续生长，而其他大部分 方法得到的3D类脑小体在同样时间很难生长到这样的大小并保持健康。
参见图6，图6为实施例1培养至第10周的3D类脑小体不同脑区的前体细胞的组织切片及染色照片，其中，Nestin是神经前体细胞表达的常见的标志性蛋白，Tuj1是神经细胞普遍表达的蛋白标志物，Foxg1是前脑前体细胞的标志物，TBR2是中脑Subventricalzone以及hippocampus的神经前体细胞的标志物，NKX2.1是后脑前体细胞的标志物，DAPI是DNA染料。由图6可知，本发明的方法得到的3D类脑小体有能发育出前脑、中脑和后脑的能力。
参见图7，图7为实施例1培养至第88天的3D类脑小体不同大脑皮层神经细胞的组织切片及染色照片，其中，REELIN，BRN2，SATB2，CTIP2，TBR1依次分别是大脑皮层I/II，III，IV，V，VI各层神经细胞的标志物。由图7可知，本发明所述的制作方法得到的3D类脑小体表达上述不同皮层的标志物。
参见图8，图8为实施例1培养至第63天的3D类脑小体神经胶质细胞及神经细胞的组织切片及染色照片，其中，nNOS,PV及SST分别是抑制性大脑神经元的标志物，MAP2是相对成熟的神经细胞的标志物，DAPI是DNA染料。由图8可知，本发明所述的制作方法得到的3D类脑小体至少含有在大脑发育和功能上起着十分重要作用的这三种抑制性大脑神经元，并且相对成熟。
参见图9，图9为实施例1培养至第63天的3D类脑小体神经胶质细胞及神经细胞的组织切片及染色照片，其中，GFAP及MAP2分别是神经胶质细胞和神经细胞的标志物，DAPI是DNA染料。经统计，神经胶质细胞在3D类脑小体所有细胞中的比例大约为50-70％。由图9可知，本发明所述的制作方法得到的3D类脑小体含有在大脑发育和功能上起着十分重要作用的神经胶质细胞，并且大致比例和分布非常接近人大脑的组成。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (10)

  1. 一种3D大脑类器官的制备方法，其特征在于，包括：
    将RONA法获得的神经球经accutase消化为单细胞，计数后接种于细胞培养板上在培养基A中培养，培养至第7天后在培养基B中继续培养，培养至第25～35天时，包裹神经球后继续培养至55～65天，第二次包裹神经球后继续培养；
    所述培养基A包括：维甲酸、BDNF、GDNF、抗坏血酸、cAMP、Neurobasal培养基和B27添加剂；
    所述培养基B包括：BDNF、GDNF、抗坏血酸、cAMP、Neurobasal培养基和B27添加剂。
  2. 根据权利要求1所述的制备方法，其特征在于，将RONA法获得的神经球经accutase消化为单细胞，计数后接种同等数量的1000～50000个细胞至多孔细胞培养板上在培养基A中培养。
  3. 根据权利要求2所述的制备方法，其特征在于，在培养基A中培养的条件为：在37℃，5％CO 2细胞培养箱中的低速圆周摇床上摇动培养。
  4. 根据权利要求1～3任意一项所述的制备方法，其特征在于，所述培养基A包括：1～5μM维甲酸；10～30ng/ml BDNF；10～30ng/ml GDNF；0.1～0.5mM抗坏血酸；5～15μM cAMP；Neurobasal和B27添加剂(不含Vitamine A)；
    所述培养基B包括：10～30ng/ml BDNF；10～30ng/ml GDNF；0.1～0.5mM抗坏血酸；5～15μM cAMP；Neurobasal和B27添加剂(不含Vitamine A)。
  5. 根据权利要求4所述的制备方法，其特征在于，所述培养基A包括：2μM维甲酸；20ng/ml BDNF；20ng/ml GDNF；0.2mM抗坏血酸；10μM cAMP；Neurobasal和B27添加剂(不含Vitamine A)；
    所述培养基B包括：20ng/ml BDNF；20ng/ml GDNF；0.2mM抗坏血酸；10μM cAMP；Neurobasal和B27添加剂(不含Vitamine A)。
  6. 根据权利要求1所述的制备方法，其特征在于，培养至第25～35天时，用Matrigel包裹神经球后继续培养至55～65天，用Matrigel第二次包裹神经球后继续培养。
  7. 根据权利要求1所述的制备方法，其特征在于，还包括：培养至第85～100天第三次包裹神经球后继续培养。
  8. 根据权利要求7所述的制备方法，其特征在于，培养至第85～100天用Matrigel第三次包裹神经球后继续培养。
  9. 根据权利要求7所述的制备方法，其特征在于，培养至第30天时，包裹神经球后继续培养至60天，第二次包裹神经球后继续培养至90天第三次包裹神经球后继续培养。
  10. 用于培养3D大脑类器官的培养基，其特征在于，包括培养基A和培养基B；
    所述培养基A包括：维甲酸、BDNF、GDNF、抗坏血酸、cAMP、Neurobasal培养基和B27添加剂；B27添加剂中不含维生素A；
    所述培养基B包括：BDNF、GDNF、抗坏血酸、cAMP、Neurobasal培养基和B27添加剂；B27添加剂中不含维生素A。
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