WO2024207802A1 - 一种基于低强度超声刺激的三维脑类器官的培养方法 - Google Patents

一种基于低强度超声刺激的三维脑类器官的培养方法 Download PDF

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WO2024207802A1
WO2024207802A1 PCT/CN2023/139394 CN2023139394W WO2024207802A1 WO 2024207802 A1 WO2024207802 A1 WO 2024207802A1 CN 2023139394 W CN2023139394 W CN 2023139394W WO 2024207802 A1 WO2024207802 A1 WO 2024207802A1
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brain
intensity ultrasound
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李晓红
陈力群
郭迪
樊秀
常哲瀚
史建新
胡楠
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Tianjin University
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  • Microcephaly is a neurodevelopmental disorder in which the child's head becomes significantly smaller due to abnormal brain development during the fetal or infant period. The patient's brain is underdeveloped and he cannot take care of himself for life. At present, treatment options are limited and the efficacy is not ideal.
  • ASPM is the most common autosomal recessive microcephaly mutation gene.
  • ASPM gene mutates the size of the human brain will be reduced by 50%.
  • ASPM gene knockout mice can only provide very limited reference in the study of human cerebral cortex development (Nature. 2018; 556: 370-375). Studies have shown that the human brain organoid model with ASPM knockout can better reproduce the phenotype of microcephaly (Protein Cell. 2017; 8(11): 823-833).
  • an object of the present invention is to provide a method for culturing three-dimensional brain organoids based on low-intensity ultrasound stimulation.
  • the object of the present invention is to achieve the following:
  • the present invention provides a method for culturing three-dimensional brain organoids based on low-intensity ultrasound stimulation, comprising the following steps:
  • Embryonic stem cells or induced pluripotent stem cells are cultured and differentiated into three-dimensional brain organoids;
  • Low-intensity ultrasound optimizes brain organoid development: Low-intensity ultrasound is used to stimulate and intervene in the development of brain organoids, and the structural changes of three-dimensional brain organoids are recorded at different time points.
  • the specific process of constructing three-dimensional brain organoids in step (1) is as follows: a single cell suspension of embryonic stem cells or induced pluripotent stem cells is inoculated in an ultra-low adhesion U-bottom well plate, neural induction medium is added for culture to form embryoid bodies, and the culture is continued for 8-12 days, and then the embryoid bodies are transferred to a low adhesion well plate, neural differentiation medium is added, and the culture is rotated on a shaker. On the 16th to 20th day of culture, the culture medium is replaced with a mature medium and the culture is continued with rotation.
  • the neural induction medium is DMEM/F-12 medium, which contains 15% serum replacement, 1% minimum essential medium containing non-essential amino acids, 1% L-glutamine substitute, 100 micromoles/liter ⁇ -mercaptoethanol, 100 nanomoles/liter 4-[6-[4-(1-piperazinyl)phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]-quinoline hydrochloride, 10 ⁇ M SB431542, and 2 ⁇ M XAV939.
  • the neural differentiation medium is 50% DMEM/F-12 medium, which contains 50% Neurobasal medium, 0.025% insulin, 0.5% minimum essential medium containing non-essential amino acids, 1% L-glutamine substitute, 1% penicillin/streptomycin, 0.5% N2 supplement, 1% B-27 serum-free additive, vitamin A removed, and 50 micromoles/liter ⁇ -mercaptoethanol.
  • the maturation culture medium is 50% DMEM/F-12 culture medium, which contains 50% Neurobasal culture medium, 0.025% insulin, 0.5% minimum essential culture medium containing non-essential amino acids, 1% L-glutamine substitute, 1% penicillin/streptomycin, 0.5% N-2 additive, 1% B-27 serum-free additive, 50 micromoles/liter ⁇ -mercaptoethanol, 200 micromoles/liter ascorbic acid, and 20 ng/ml brain-derived neurotrophic factor.
  • 50% Neurobasal culture medium 0.025% insulin, 0.5% minimum essential culture medium containing non-essential amino acids, 1% L-glutamine substitute, 1% penicillin/streptomycin, 0.5% N-2 additive, 1% B-27 serum-free additive, 50 micromoles/liter ⁇ -mercaptoethanol, 200 micromoles/liter ascorbic acid, and 20 ng/ml brain-derived neurotrophic factor.
  • the parameters of the low-intensity ultrasound in step (2) are pulse intensity of 30-50 mW/cm 2 , rated frequency of 1-5 MHz, 5-15% duty cycle, and treatment for 1-5 minutes per day.
  • the starting time point of the stimulation intervention in step (2) is the 15th to 20th day of brain organoid development.
  • the present invention also provides the application of the low-intensity ultrasound stimulation in constructing a microcephaly brain organoid disease model.
  • the human abnormal spindle-like microcephaly-associated protein gene (ASPM) is knocked out in the microcephaly brain organoid disease model.
  • the present invention discovered for the first time that low-intensity ultrasound stimulation optimizes the development of brain organoids, and provided a method for culturing three-dimensional brain organoids based on low-intensity ultrasound stimulation, which can provide a reliable and effective in vitro research model for studying the development process of the nervous system, and provide a new approach for studying the treatment of neurodevelopmental diseases such as microcephaly.
  • Figure 1 is a timeline of the low-intensity ultrasound intervention protocol for brain organoid development
  • FIG2 is a light microscopic comparison of brain organoids treated with low-intensity ultrasound stimulation for different time periods
  • Figure 3 is a comparison of immunofluorescence of brain organoids treated with low-intensity ultrasound stimulation for different time periods, among which: (A): the proportion of proliferating cells (ki67) and neural progenitor cells (SOX2) in the ultrasound group increased significantly; (B): the proportion of cortical neurons (TBR1) in the ultrasound group increased significantly; (C): the proportion of apoptotic cells (TUNEL) in the ultrasound group decreased significantly.
  • FIG4 is an integrated diagram of brain organoids treated with low-intensity ultrasound stimulation at different time points after transplantation into the primary sensory cortex of the host;
  • Figure 5 is a light microscopic image of ASPM knockout brain organoids and ASPM brain organoids after ultrasonic treatment, wherein, (A): light microscopic comparison image of brain organoids between the ASPM knockout group and the control group; (B): light microscopic comparison image of brain organoids between the ASPM knockout group, the control group and the ASPM knockout group after ultrasonic treatment.
  • H9 human embryonic stem cells When H9 human embryonic stem cells grew to 80% confluency, they were digested with accutase for 10 min to form a single cell suspension, and the cells were cultured in a neural induction medium (DMEM/F-12, 15% serum replacement, 1% minimum essential medium containing non-essential amino acids, 1% L-glutamine substitute, 100 ⁇ mol/L ⁇ -mercaptoethanol, 100 nmol/L 4-[6-[4-(1-piperazinyl)phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]-quinoline hydrochloride, 10 ⁇ M SB431542, 2 ⁇ mol/L After resuspending, the cells were seeded in an ultra-low adhesion U-bottom 96-well plate at a ratio of 9000 cells/well and 150 ⁇ L per well to form embryoid bodies.
  • a neural induction medium DMEM/F-12, 15% serum replacement, 1% minimum essential medium containing
  • the medium was replaced by half of the neural induction medium without Y27632 every 2 days.
  • 3mL neural differentiation medium 50% Neurobasal medium, 0.025% insulin, 0.5% minimum essential medium containing non-essential amino acids, 1% L-glutamine substitute, 1% penicillin/streptomycin, 0.5% N-2 supplement, 1% B-27 serum-free supplement, vitamin A removed, 50
  • the medium was replaced with maturation medium (50% DMEM/F-12, 50% Neurobasal medium, 0.025% insulin, 0.5% minimum essential medium containing non-essential amino acids, 1% L-glutamine substitute, 1% penicillin/streptomycin, 0.5% N-2 supplement, 1% B-27 serum-free supplement, 50 ⁇ mol/L ⁇ -mercaptoethanol, 200 ⁇ mol/L ascorbic acid, 20 ng/mL brain-derived neurotrophic factor), and the medium was changed every 3-4 days.
  • maturation medium 50% DMEM/F-12, 50% Neurobasal medium, 0.025% insulin, 0.5% minimum essential medium containing non-essential amino acids, 1% L-glutamine substitute, 1% penicillin/streptomycin, 0.5% N-2 supplement, 1% B-27 serum-free supplement, 50 ⁇ mol/L ⁇ -mercaptoethanol, 200 ⁇ mol/L ascorbic acid, 20 ng/mL brain-derived neurotrophic factor
  • Low-intensity ultrasound stimulation to intervene in the development of brain organoids Treatment began on the 18th day of brain organoid development. About 6-8 brain organoids were cultured in each well of a low-adhesion 6-well plate containing 3 mL of maturation culture medium. Sterile coupling agent ( ⁇ 1 mm thick) was applied to the bottom of each well of the low-adhesion 6-well plate. The ultrasound probe (ME Sonicator 740, 5 cm2) was placed under the plate in close contact without gaps. The parameters of the ultrasound therapy instrument were adjusted: the pulse intensity was 40 mW/cm2, the rated frequency was 3 MHz, the duty cycle was 10%, and the treatment was 2 min per day.
  • the coupling agent on the bottom plate was wiped off and the plate was returned to the 80 rpm shaker for continuous culture.
  • the proportions of neural progenitor cells (SOX2), cortical neurons (TBR1), proliferating cells (ki67), and apoptotic cells (TUNEL) were detected on the 35th, 55th, and 75th days of brain organoid development.
  • the results are shown in Figure 3. It can be seen that the brain organoids after low-intensity ultrasound stimulation showed an increase in size, an increase in the proportion of neural progenitor cells and cortical neurons, a significant increase in the proportion of proliferating cells, and a significant decrease in the proportion of apoptotic cells, which effectively promoted the structural development of brain organoids.
  • Detection of brain organoid integration ability after low-intensity ultrasound stimulation intervention The GFP-carrying brain organoids treated with ultrasound on the 40th day were transplanted into the primary sensory cortex of Nod-SCID mice, and the survival ability and integration ability of the brain organoids in the host were tested at the 2nd and 5th months of transplantation. The results are shown in Figure 4. The survival ability of brain organoids treated with low-intensity ultrasound stimulation in the host after transplantation was significantly increased, and the integration ability with the host was significantly improved.
  • Embryonic stem cells were infected with Crispr-cas9-mediated ASPM knockout lentivirus, and single cell clones were selected after two weeks of drug screening for gene fragment verification and knockout effect identification to construct an ASPM knockout embryonic stem cell line.
  • the human brain organoids were cultured and identified according to the method for constructing human brain organoids in Example 1.
  • the low-intensity ultrasound intervention parameters of the common brain organoids in Example 2 were used for treatment and detection.
  • the specific results are shown in Figure 5. It can be seen that the microcephalic brain organoids after low-intensity ultrasound treatment showed a significant increase in size.

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Abstract

一种基于低强度超声刺激的三维脑类器官的培养方法，该方法首先培养脑类器官，然后通过低强度超声干预脑类器官的发育，通过分析全发育周期低强度超声对脑类器官结构的优化作用，并且研究了低强度超声刺激对人类异常纺锤体样小头畸形相关蛋白基因(ASPM)敲除的小头畸形脑类器官培养中的作用。首次发现低强度超声刺激优化了脑类器官发育，可为研究神经系统发育过程提供可靠的、有效的体外研究模型，并为研究小头畸形等神经发育相关疾病的治疗提供新途径。

Description

一种基于低强度超声刺激的三维脑类器官的培养方法 背景技术
神经系统发育经历神经诱导、图式形成、细胞命运决定及特化、神经细胞极性建立与迁移、神经环路形成等过程。深入开展神经发育的机制研究以指导预防及治疗这类重大神经发育缺陷疾病具有重要意义。小头畸形是胎儿期或婴儿期大脑发育异常导致的患儿头部明显变小的一种神经系统发育障碍性疾病。患者脑部发育不全，终身不能自理生活。目前，治疗手段有限，疗效不理想。
脑类器官凭借细胞组成多样性高、结构组织复杂度高、电生理功能相对成熟、基因表达模式与人类相近等优点，是模拟婴儿脑发育过程的良好三维模型。ASPM是最常见的常染色体隐性小头畸形突变基因。当ASPM基因发生突变时，人脑尺寸将减少50％。而ASPM基因灭活仅能使小鼠的大脑萎缩10％，ASPM基因敲除小鼠在人类脑皮层发育的研究中只能提供十分有限的参考(Nature.2018；556:370-375)。而研究表明，ASPM敲除的人源性脑类器官模型可较好地重现小头畸形的表型(Protein Cell.2017；8(11):823-833)。
然而，将低强度超声应用于三维脑类器官发育的相关研究还未见报道。
 发明内容
鉴于此，本发明的目的是提供一种基于低强度超声刺激的三维脑类器官的培养方法。
本发明目的是通过以下方式实现：
本发明提供一种基于低强度超声刺激的三维脑类器官的培养方法，包括以下步骤：
(1)构建三维脑类器官：将胚胎干细胞或诱导性多能干细胞进行培养，分化为三维脑类器官；
(2)低强度超声优化脑类器官发育：利用低强度超声在脑类器官发育过程中进行刺激干预，在不同时间点记录三维脑类器官的结构变化。
基于上述技术方案，进一步地，步骤(1)中构建三维脑类器官的具体过程为：将胚胎干细胞或诱导性多能干细胞的单细胞悬液接种在超低黏附U型底孔板中，加入神经诱导培养基培养，形成拟胚体，培养至8-12天，转移到低黏附孔板中，加入神经分化培养基后摇床旋转培养，培养第16-20天，更换为成熟培养基继续旋转培养。
基于上述技术方案，进一步地，所述的神经诱导培养基为DMEM/F-12培养基，培养基中含有15％ 血清替代物，1％ 含非必须氨基酸的最低必需培养基，1％ L-谷氨酰胺的替代品，100微摩尔/升β-巯基乙醇，100纳摩尔/升 4-[6-[4-(1-哌嗪基)苯基]吡唑并[1,5-a]嘧啶-3-基]-喹啉盐酸盐，10μM SB431542，2μMXAV939。
基于上述技术方案，进一步地，所述的神经分化培养基为50％ DMEM/F-12培养基，培养基中含有50％ Neurobasal 培养基，0.025％ 胰岛素，0.5％ 含非必须氨基酸的最低必需培养基，1％L-谷氨酰胺的替代品，1％ 青霉素/链霉素，0.5％ N2 supplement，1％ B-27无血清添加剂，去除维生素A，50微摩尔/升β-巯基乙醇。
基于上述技术方案，进一步地，所述的成熟培养基为50％ DMEM/F-12培养基，培养基中含有50％ Neurobasal 培养基，0.025％ 胰岛素，0.5％ 含非必须氨基酸的最低必需培养基，1％L-谷氨酰胺的替代品，1％ 青霉素/链霉素，0.5％ N-2添加剂，1％ B-27无血清添加剂，50微摩尔/升β-巯基乙醇，200微摩尔/升 抗坏血酸，20纳克/毫升 脑源性神经营养因子。
基于上述技术方案，进一步地，步骤(2)中所述的低强度超声的参数为脉冲强度为30-50mW/cm 2 ，额定频率为1-5MHz，5-15％占空比，每天处理1-5min。
基于上述技术方案，进一步地，步骤(2)中刺激干预开始时间点为脑类器官发育的第15-20天。
本发明还提供所述的低强度超声刺激在构建小头畸形脑类器官疾病模型中的应用。
基于上述技术方案，进一步地，所述的小头畸形脑类器官疾病模型中敲除了人类异常纺锤体样小头畸形相关蛋白基因(ASPM)。
本发明相对于现有技术具有的有益效果如下：
本发明首次发现低强度超声刺激优化了脑类器官发育，提供一种基于低强度超声刺激的三维脑类器官的培养方法，可为研究神经系统发育过程提供可靠的、有效的体外研究模型，并为研究小头畸形等神经发育相关疾病的治疗提供了新途径。
 附图说明
为了更清楚地说明本发明实施例，下面将对实施例涉及的附图进行简单地介绍。
图1为低强度超声干预脑类器官发育方案的时间轴；
图2为低强度超声刺激处理不同时间的脑类器官光镜对比图；
图3为低强度超声刺激处理不同时间的脑类器官免疫荧光对比图，其中，(A)：超声组增殖细胞(ki67)和神经祖细胞(SOX2)比例明显上升；(B)：超声组皮质神经元(TBR1)比例明显上升；(C)：超声组凋亡细胞(TUNEL)比例明显下降。
图4为低强度超声刺激处理的脑类器官移植入宿主初级感觉皮层不同时间点的整合图；
图5为ASPM敲除脑类器官及超声处理后的ASPM脑类器官光镜图，其中，(A)：ASPM敲除组和对照组的脑类器官光镜对比图；(B)：ASPM敲除组、对照组和超声处理后的ASPM敲除组的脑类器官光镜对比图。
 具体实施方式
下面结合实施例对本发明进行详细的说明，但本发明的实施方式不限于此，显而易见地，下面描述中的实施例仅是本发明的部分实施例，对于本领域技术人员来讲，在不付出创造性劳动性的前提下，获得其他的类似的实施例均落入本发明的保护范围。
实施例1
脑类器官构建：待H9人胚胎干细胞生长融合度高达80％时，使用accutase消化10min形成单细胞悬液，使用含50μM Y27632神经诱导培养基(DMEM/F-12，15％血清替代物，1％ 含非必须氨基酸的最低必需培养基，1％ L-谷氨酰胺的替代品，100微摩尔/升β-巯基乙醇，100纳摩尔/升 4-[6-[4-(1-哌嗪基)苯基]吡唑并[1,5-a]嘧啶-3-基]-喹啉盐酸盐，10μM SB431542，2微摩尔/升 3,5,7,8-四氢-2-[4-(三氟甲基)苯基]-4H-噻喃并[4,3-D]嘧啶-4-酮)重悬后，以9000个细胞/孔，每孔150μL的比例将细胞接种在超低黏附U型底96孔板中，形成拟胚体。每2天使用不含Y27632的神经诱导培养基半量换液一次。培养第10天时，使用5mL巴士吸管将其转移至低黏附6孔板中，每孔6-8个拟胚体，每孔3mL神经分化培养基(50％ DMEM/F-12，50％Neurobasal 培养基，0.025％ 胰岛素，0.5％含非必须氨基酸的最低必需培养基，1％ L-谷氨酰胺的替代品，1％青霉素/链霉素，0.5％ N-2添加剂，1％ B-27无血清添加剂，去除维生素A，50微摩尔/升β-巯基乙醇)，80rpm摇床旋转培养，每隔2天全量换液一次。培养第18天，更换为成熟培养基(50％DMEM/F-12，50％ Neurobasal 培养基，0.025％ 胰岛素，0.5％含非必须氨基酸的最低必需培养基，1％ L-谷氨酰胺的替代品，1％ 青霉素/链霉素，0.5％ N-2添加剂，1％ B-27无血清添加剂，50微摩尔/升β-巯基乙醇，200微摩尔/升 抗坏血酸，20纳克/毫升 脑源性神经营养因子)，每隔3-4天全量换液一次。
实施例2
低强度超声刺激干预脑类器官发育：从脑类器官发育第18天开始处理，约6-8个脑类器官培养在含3mL成熟培养基的低黏附6孔板每孔中，在低黏附6孔板每孔底部涂抹无菌耦合剂(＜1mm厚)，超声探头(ME Sonicator 740,5平方厘米)放置在孔板下方，紧密接触，无空隙产生。调整超声波治疗仪的参数：脉冲强度为40mW/cm 2 ，额定频率为3MHz，10％占空比，每天处理2min。处理后擦拭底板耦合剂，将孔板放回80rpm摇床持续培养。在脑类器官发育的第35天、第55天、第75天进行神经祖细胞(SOX2)、皮质神经元(TBR1)、增殖细胞(ki67)、凋亡细胞(TUNEL)比例检测。结果如图3所示，可以看出低强度超声刺激处理后的脑类器官表现为尺寸增大，神经祖细胞，皮质神经元比例增加，增殖细胞比例明显增加，凋亡细胞比例显著减少，有效促进脑类器官的结构发育。
实施例3
低强度超声刺激干预后的脑类器官整合能力检测：将第40天的超声处理的携带GFP的脑类器官移植入Nod-SCID小鼠初级感觉皮层中，在移植第2个月和移植第5个月检测脑类器官在宿主中的存活能力和与宿主的整合能力。结果见图4，低强度超声刺激处理后的脑类器官移植后在宿主中存活能力明显增高，与宿主整合能力明显提高。
实施例4
ASPM敲除的小头畸形脑类器官模型构建及超声处理：采用Crispr-cas9介导的ASPM敲除的慢病毒感染胚胎干细胞，药筛两周后挑选单细胞克隆，进行基因片段验证及敲除效果鉴定，构建ASPM敲除的胚胎干细胞系。
随后，按照实施例1中的人脑类器官构建方法进行培养与鉴定。待第30天后，按照实施例2中的低强度超声干预普通脑类器官的参数方案进行处理与检测，具体结果见图5，可以看出，低强度超声处理后的小头畸形脑类器官表现为尺寸明显增大。
最后应说明的是：以上各实施例仅用以说明本发明的技术方案，而非对其限制；尽管参照前述各实施例对本发明进行了详细的说明，本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分或者全部技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本发明各实施例技术方案的范围。

Claims (9)

  1. 一种基于低强度超声刺激的三维脑类器官的培养方法，其特征在于，包括以下步骤：
    (1)构建三维脑类器官：将胚胎干细胞或诱导性多能干细胞进行培养，分化为三维脑类器官；
    (2)低强度超声优化脑类器官发育：利用低强度超声在脑类器官发育过程中进行刺激干预，在不同时间点记录三维脑类器官的结构变化。
  2. 根据权利要求1所述的培养方法，其特征在于，步骤(1)中构建三维脑类器官的具体过程为：将胚胎干细胞或诱导性多能干细胞的单细胞悬液接种在超低黏附U型底孔板中，加入神经诱导培养基培养，形成拟胚体，培养至8-12天，转移到低黏附孔板中，加入神经分化培养基后摇床旋转培养，培养第16-20天，更换为成熟培养基继续旋转培养。
  3. 根据权利要求2所述的培养方法，其特征在于，所述的神经诱导培养基为DMEM/F-12培养基，培养基中含有15％血清替代物，1％含非必须氨基酸的最低必需培养基，1％L-谷氨酰胺的替代品，100微摩尔/升β-巯基乙醇，100纳摩尔/升 4-[6-[4-(1-哌嗪基)苯基]吡唑并[1,5-a]嘧啶-3-基]-喹啉盐酸盐，10微摩尔/升4-[4-(1,3-苯并二唑-5-基)-5-(2-吡啶基)-1H-咪唑-2-基]-苯酰胺水合物，2微摩尔/升 3,5,7,8-四氢-2-[4-(三氟甲基)苯基]-4H-噻喃并[4,3-D]嘧啶-4-酮。
  4. 根据权利要求2所述的培养方法，其特征在于，所述的神经分化培养基为50％DMEM/F-12培养基，培养基中含有50％Neurobasal 培养基，0.025％胰岛素，0.5％含非必须氨基酸的最低必需培养基，1％L-谷氨酰胺的替代品，1％青霉素/链霉素，0.5％N-2添加剂，1％B-27无血清添加剂，去除维生素A，50微摩尔/升β-巯基乙醇。
  5. 根据权利要求2所述的培养方法，其特征在于，所述的成熟培养基为50％DMEM/F-12培养基，培养基中含有50％Neurobasal 培养基，0.025％胰岛素，0.5％含非必须氨基酸的最低必需培养基，1％L-谷氨酰胺的替代品，1％青霉素/链霉素，0.5％N-2添加剂，1％B-27无血清添加剂，50微摩尔/升β-巯基乙醇，200微摩尔/升 抗坏血酸，20纳克/毫升 脑源性神经营养因子。
  6. 根据权利要求1所述的培养方法，其特征在于，步骤(2)中所述的低强度超声的参数为脉冲强度为30-50mW/cm 2 ，额定频率为1-5MHz，5-15％占空比，每天处理1-5min。
  7. 根据权利要求1所述的培养方法，其特征在于，步骤(2)中刺激干预开始时间点为脑类器官发育的第15-20天。
  8. 权利要求1所述的低强度超声刺激在构建小头畸形脑类器官疾病模型中的应用。
  9. 根据权利要求8所述的应用，其特征在于，所述的小头畸形脑类器官疾病模型中敲除了人类异常纺锤体样小头畸形相关蛋白基因(ASPM)。
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Families Citing this family (5)

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Publication number Priority date Publication date Assignee Title
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CN117230146A (zh) * 2023-10-28 2023-12-15 天津大学 一种用于模拟胚胎时期环境污染物对大脑发育损伤研究的脑类器官模型的应用
CN118048308B (zh) * 2024-01-29 2025-03-14 天津大学 基于h9诱导产生含有视泡的类脑器官及眼脑融合培养方法
CN119432724B (zh) * 2024-10-15 2025-11-18 天津大学 一种三维片上脑的培养、光刺激与信号处理方法
CN119464212A (zh) * 2024-11-19 2025-02-18 天津大学 一种pfos皮层脑类器官损伤模型及其构建方法和应用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112226410A (zh) * 2020-07-16 2021-01-15 广州市妇女儿童医疗中心（广州市妇幼保健院、广州市儿童医院、广州市妇婴医院、广州市妇幼保健计划生育服务中心） 一种原发性小头畸形疾病模型及其构建方法和应用
WO2022134229A1 (zh) * 2020-12-23 2022-06-30 武汉睿健医药科技有限公司 一种神经干细胞诱导分化培养基及诱导分化方法
CN116376840A (zh) * 2023-04-03 2023-07-04 天津大学 一种基于低强度超声刺激的三维脑类器官的培养方法

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20190382731A1 (en) * 2016-07-11 2019-12-19 Trustees Of Tufts College Artificial Brain Tissue
CN109136185B (zh) * 2017-06-28 2021-09-21 中国科学院生物物理研究所 一种类脑器官器的制备方法和应用
KR102228400B1 (ko) * 2019-09-24 2021-03-16 고려대학교 산학협력단 뇌 오가노이드 제조 방법
CN113717940B (zh) * 2021-08-27 2024-01-19 深圳康沃先进制造科技有限公司 简化的大脑皮层微组织模型及其构建方法
CN115404218B (zh) * 2022-01-13 2024-09-24 南华大学附属第一医院 一种包含胶质细胞的3d人脑类器官培养方法
CN114657127B (zh) * 2022-02-28 2024-09-13 合肥燃音生物科技有限公司 一种大脑类器官模型及其制备方法与应用
CN114457015A (zh) * 2022-03-11 2022-05-10 中国科学院广州生物医药与健康研究院 纹状体类脑器官及其培养基、培养方法和应用

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112226410A (zh) * 2020-07-16 2021-01-15 广州市妇女儿童医疗中心（广州市妇幼保健院、广州市儿童医院、广州市妇婴医院、广州市妇幼保健计划生育服务中心） 一种原发性小头畸形疾病模型及其构建方法和应用
WO2022134229A1 (zh) * 2020-12-23 2022-06-30 武汉睿健医药科技有限公司 一种神经干细胞诱导分化培养基及诱导分化方法
CN116376840A (zh) * 2023-04-03 2023-07-04 天津大学 一种基于低强度超声刺激的三维脑类器官的培养方法

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
CAKIR BILAL; XIANG YANGFEI; TANAKA YOSHIAKI; KURAL MEHMET H.; PARENT MAXIME; KANG YOUNG-JIN; CHAPETON KAYLEY; PATTERSON BENJAMIN; : "Engineering of human brain organoids with a functional vascular-like system", NATURE METHODS, vol. 16, no. 11, 7 October 2019 (2019-10-07), New York, pages 1169 - 1175, XP036917493, ISSN: 1548-7091, DOI: 10.1038/s41592-019-0586-5 *
HOU XUANDI, QIU ZHIHAI, XIAN QUANXIANG, KALA SHASHWATI, JING JIANING, WONG KIN FUNG, ZHU JIEJUN, GUO JINGHUI, ZHU TING, YANG MINYI: "Precise Ultrasound Neuromodulation in a Deep Brain Region Using Nano Gas Vesicles as Actuators", ADVANCED SCIENCE, vol. 8, no. 21, 1 November 2021 (2021-11-01), Germany, pages 1 - 12, XP093057348, ISSN: 2198-3844, DOI: 10.1002/advs.202101934 *
HOU XUANDI, QIU ZHIHAI, XIAN QUANXIANG, KALA SHASHWATI, JING JIANING, WONG KIN FUNG, ZHU JIEJUN, GUO JINGHUI, ZHU TING, YANG MINYI: "Precise Ultrasound Neuromodulation in a Deep Brain Region Using Nano Gas Vesicles as Actuators", ADVANCED SCIENCE, vol. 8, no. 21, 1 November 2021 (2021-11-01), Germany, pages 2101934, XP093057348, ISSN: 2198-3844, DOI: 10.1002/advs.202101934 *
LEI XU, TANG XIAOYAN , LIU YAN: "Research progresses of 3D brain organoids derived from pluripotent stem cells", JOURNAL OF NANJING MEDICAL UNIVERSITY(NATURAL SCIENCES), vol. 40, no. 1, 15 January 2020 (2020-01-15), pages 133 - 140, XP093081136, DOI: 10.7655/NYDXBNS20200128 *
VICTORIA COTERO, FAN YING, TSAAVA TEA, KRESSEL ADAM M., HANCU ILEANA, FITZGERALD PAUL, WALLACE KIRK, KAANUMALLE SIREESHA, GRAF JOH: "Noninvasive sub-organ ultrasound stimulation for targeted neuromodulation", NATURE COMMUNICATIONS, vol. 10, no. 1, pages 1 - 12, XP055719759, DOI: 10.1038/s41467-019-08750-9 *
XIANG YANGFEI; TANAKA YOSHIAKI; PATTERSON BENJAMIN; KANG YOUNG-JIN; GOVINDAIAH GUBBI; ROSELAAR NAOMI; CAKIR BILAL; KIM KUN-YONG; L: "Fusion of Regionally Specified hPSC-Derived Organoids Models Human Brain Development and Interneuron Migration", CELL STEM CELL, vol. 21, no. 3, 27 July 2017 (2017-07-27), AMSTERDAM, NL , pages 383 - 398, XP085189924, ISSN: 1934-5909, DOI: 10.1016/j.stem.2017.07.007 *

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