Three-Dimensional Motor Nerve Organoid Generation

Tatsuya Osaki; Siu Yu  A. Chow; Yui Nakanishi; Joel Hern&#225;ndez; Jiro Kawada; Teruo Fujii; Yoshiho Ikeuchi

doi:10.3791/61544

JoVE Journal
Neuroscience

This content is Free Access.

JoVE Journal Neuroscience

Three-Dimensional Motor Nerve Organoid Generation

三维运动神经器官生成

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09:57 min

September 24, 2020

DOI: 10.3791/61544-v

Tatsuya Osaki^1,2, Siu Yu A. Chow^1,2, Yui Nakanishi^1,2, Joel Hernández^1,3, Jiro Kawada⁴, Teruo Fujii¹, Yoshiho Ikeuchi^1,2

¹Institute of Industrial Science,The University of Tokyo, ²Department of Chemistry and Biotechnology, School of Engineering,The University of Tokyo, ³Faculty of Science and Engineering,Tecnologico de Monterrey, ⁴Jiksak Bioengineering, Inc.

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Overview

This study presents a protocol for generating human iPS cell-derived motor nerve organoids through the spontaneous assembly of axons from a neuronal spheroid in a custom PDMS microculture chip. The platform allows for the investigation of axon bundle development and motor neuron diseases, facilitating efficient drug screening and testing.

Key Study Components

Area of Science

Neuroscience
Stem Cell Biology
Organoid Technology

Background

Motor nerve organoids provide a more physiological model compared to traditional in vitro systems.
Understanding motor neuron diseases like Amyotrophic Lateral Sclerosis (ALS) is crucial for developing effective therapies.
This protocol utilizes human iPS cells to generate a relevant biological model for research.

Purpose of Study

To fabricate motor nerve organoids for studying axon development.
To enhance drug screening for motor neuron diseases using a more accurate cellular environment.
To provide a detailed methodology for generating and utilizing these models in research.

Methods Used

The main platform used is a PDMS microfluidic chip designed for tissue culture.
The biological model consists of motor neurons differentiated from human iPS cells.
The protocol involves several key steps over a period of approximately two to three weeks.
Cultures are maintained in a CO2 incubator, with specific media changes outlined for differentiation.
Motor neuron spheroids are introduced into the microchannel to facilitate axon bundle formation.

Main Results

Axons grow from motor neuron spheroids and assemble into organized bundles through axo-axonal interactions.
Over 60% of differentiated cells express the motor neuron marker HB9, indicating successful differentiation.
The model achieves functional maturation within 12 to 14 days post-differentiation, with key cellular changes noted over time.
Motor nerve organoids show promise for biological analysis relevant to motor neuron diseases.

Conclusions

This study demonstrates a reliable method for generating motor nerve organoids for research and pharmaceutical applications.
It highlights the significance of using human iPS cells in disease modeling and drug screening.
The findings contribute to a deeper understanding of neuronal mechanisms and the development of therapeutics.

Frequently Asked Questions

What are the advantages of using motor nerve organoids?

Motor nerve organoids provide a more physiologically relevant environment for studying motor neuron diseases, enhancing drug screening and testing efficacy.

How is the motor neuron differentiation achieved?

Differentiation is achieved by seeding iPS cells in specific culture media and following a detailed timeline of media changes and supplements over several days.

What types of outcomes are assessed with this method?

The method enables evaluation of cell differentiation, axon growth, and molecular markers of motor neurons, aiding in the study of neuronal development and diseases.

Can this method be adapted for different applications?

Yes, this protocol can be tailored to explore various aspects of neuronal biology and drug testing, making it versatile for research applications.

What are the key limitations of this protocol?

While this method provides significant insights, limitations include the complexity of maintaining culture conditions and the potential for variability in organoid formation.

How does the PDMS microfluidic chip contribute to the study?

The PDMS microfluidic chip facilitates the controlled environment for 3D cell culture, promoting the spontaneous assembly of axon bundles.

该协议提供了一个全面的程序，通过自发组装从组织培养芯片中的球形延伸的强健的轴突束来制造人类iPS细胞衍生的运动神经器官。

该协议有助于使用源自体外人 iPS 细胞的运动神经类器官研究轴突束发展和疾病的潜在机制。通过简单地培养神经元球体和定制的微培养芯片，可以自发产生单向轴突束并用于各种下游实验。运动神经类器官可以通过提供以前的体外系统更具生理性的模型来促进运动神经元疾病（包括 LS）药物的筛选和测试。

在通风橱中，穿戴适当的 PPE，将 3 毫升 SU-8 2100 分配到丙酮清洁的硅晶片上，并将晶片放在旋涂机的中心。通过真空固定晶圆，以每分钟 500 转的速度旋转晶圆 10 秒，使 SU-8 均匀地涂覆在晶圆表面，然后以每分钟 1， 500 转的速度旋转晶圆 30 秒，以每秒 300 转的加速度旋转晶圆，在晶圆上获得一层 150 微米厚的光刻胶。软烘烤后，将光掩模对准光罩对准器，并将晶圆暴露在紫外线下 60 秒。

在热板上烘烤后，在光刻胶显影剂中显影晶片 10 至 20 分钟，并在轨道振荡器上搅拌，在此过程中更换显影溶液一次。为了准备用于组织培养的芯片底层，将新鲜制备的 PDMS 倒入晶圆上至所需厚度，并在真空室中对混合物进行脱气。将 PDMS 在 60 摄氏度的烘箱中固化至少三个小时。

冷却后，用刀片从晶片上切下固化的 PDMS，然后用未固化的 PDMS 烘烤将培养基储液槽粘合到 PDMS 底层，得到组装好的 PDMS 组织培养芯片。为了制备用于运动神经类器官形成的 PDMS 芯片，请在含有 70% 乙醇的培养皿中对芯片和 76 x 52 毫米的显微镜玻璃消毒至少一小时。孵育结束时，将芯片放在湿显微镜玻璃上。

干燥一夜后，芯片应粘附在玻璃上。将 30 微升稀释的基底膜基质液滴在 DMEM/F12 中，放在通道入口的一侧，然后从入口的另一侧吸出溶液，使基质涂在微通道表面。然后将 PDMS 芯片与涂层溶液在培养皿中在室温下孵育 1 小时。

为了诱导 3D iPS 细胞分化为运动神经元，将每孔 4 次 10 至 4 个 iPS 细胞接种到 96 孔 U 底板的适当孔中，溶于 100 微升饲养层和补充有 10 微摩尔 Y-27632 的无血清细胞培养基中。第二天，用 100 微升补充有 10 微摩尔 SB431542 和 100 纳摩尔 LDN-193189 的 KSR 培养基替换每个孔中的上清液。在第 2 天和第 3 天，用 100 微升 KSR 培养基替换上清液，补充有 10 微摩尔SB431542、100 纳摩尔 LDN-193189、5 微摩尔 DAPT、5 微摩尔 SU5402、1 微摩尔视黄酸和 1 微摩尔平滑激动剂。

在第 4 天和第 5 天，用 75% KSR 培养基和 25% N 2 培养基的混合物替换上清液，并补充有 10 微摩尔SB431542、100 纳摩尔 LDN193189、5 微摩尔 DAPT、5 微摩尔 SU5402、1 微摩尔视黄酸和 1 微摩尔平滑激动剂。在第 6 天和第 7 天，用 50% KSR 培养基和 50% N2 培养基的混合物替换上清液，并补充有 5 微摩尔 DAPT、5 微摩尔 SU5402、1 微摩尔视黄酸和 1 微摩尔平滑激动剂。在第 8 天和第 9 天，用 25% KSR 培养基和 75% N2 培养基补充 5 微摩尔 DAPT、5 微摩尔 SU5402、1 微摩尔视黄酸和 1 微摩尔平滑激动剂的混合物替换上清液。

在第 10 天和第 11 天，用补充有 5 微摩尔 DAPT、5 微摩尔 SU5402、1 微摩尔维甲酸和 1 微摩尔平滑激动剂的 N2 培养基替换上清液。在第 12 天，用 100 微升成熟培养基替换每个孔中的上清液，每孔补充 20 ng/毫升脑源性神经营养因子。为了诱导运动神经类器官的形成，用 150 微升成熟培养基代替 PDMS 芯片中的涂层溶液，每毫升补充 20 纳克脑源性神经营养因子，并使用宽口径微量移液器尖端轻轻地将运动神经元球体从 96 孔板的一个孔中加入芯片微通道的入口。

在

培养皿中的组织培养芯片附近放置一个小的无菌水库，以防止介质蒸发，并将培养皿置于 37 摄氏度和 5% 二氧化碳的培养箱中。每两到三天，用补充有 20 ng/mL 脑源性神经营养因子的新鲜成熟培养基替换培养基储液库中心的一半用尽培养基。轴突将从运动神经元球体生长到通道中，在两到三周的时间内自发组装成一个束，形成运动神经类器官。

运动神经元在 3D 分化程序中在 12 到 14 天内分化。重要的是，超过 60% 的细胞在分化过程中表达运动神经元标志物 HB9，运动神经元球体中大约 80% 的细胞是 SMI32 阳性。在微通道作为物理导向下，轴突在引入球体后 24 小时内从运动神经元球体伸长，形成轴突-轴突相互作用的束缚。

轴突在接下来的 3 到 4 天内到达微通道的中心，又过了 10 天后延伸到微通道的另一端。通过将 PDMS 从显微镜玻璃上拆下，可以从芯片中收集运动神经类器官进行生物分析。然后可以使用手术刀或镊子在显微镜下解剖和分离轴突束和细胞体。

在运动神经类器官的轴突束中，树突状标志物蛋白不能通过 Western blotting 检测到。确保球体完全落到培养芯片内的底部非常重要。我们展示了对位点和底部的检查，可以帮助确定球体在芯片中的位置。

分离的轴突束可以检查各种其他方法。可以获得大量的轴突并用于需要大量材料的生化测定。

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