Molecular Imaging of Human Brain Organoids Using Mass Spectrometry

Saleh  M. Khalil; Gerarda Cappuccio; Feng Li; Mirjana Maletic-Savatic

doi:10.3791/66997

JoVE Journal
Neuroscience

This content is Free Access.

JoVE Journal Neuroscience

Molecular Imaging of Human Brain Organoids Using Mass Spectrometry

使用质谱法对人脑类器官进行分子成像

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08:04 min

September 27, 2024

DOI: 10.3791/66997-v

Saleh M. Khalil*^1,2, Gerarda Cappuccio*^1,2, Feng Li^3,4, Mirjana Maletic-Savatic^1,2,4,5

¹Department of Pediatrics -Neurology,Baylor College of Medicine, ²Jan and Dan Duncan Neurological Research Institute,Texas Children's Hospital, ³Department of Pathology & Immunology,Baylor College of Medicine, ⁴Center for Drug Discovery,Baylor College of Medicine, ⁵Department of Neuroscience,Baylor College of Medicine

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Overview

This study presents an advanced method for mass spectrometry imaging (MSI) that allows for the detailed mapping of metabolite distributions within brain organoids. Utilizing in vitro models derived from induced pluripotent stem cells, the research investigates human brain metabolomics, especially during development and in relation to neurodevelopmental disorders.

Key Study Components

Area of Science

Neuroscience
Metabolomics
Mass Spectrometry Imaging

Background

Investigating metabolomics in vivo is challenging; thus, in vitro brain organoids are leveraged.
Recent advances in MSI offer new insights into the molecular mechanisms of brain development and disease.
Maintaining organoid morphology and integrity during imaging is critical for accurate data.

Purpose of Study

To develop a protocol for high-resolution MSI of brain organoids.
To explore metabolite signatures associated with specific cell types and neurodevelopment.
To identify potential therapeutic targets for neurodevelopmental disorders.

Methods Used

The study employs organoid models derived from induced pluripotent stem cells.
Brain organoids are prepared using specialized tissue handling and preservation techniques for MSI analysis.
The high-resolution MSI workflow includes detailed cryo-sectioning and optimization of mass spectrometry conditions.
Specific embedding protocols and settings for the mass spectrometer are outlined for reliable imaging results.

Main Results

Molecular mapping of the Krebs cycle metabolites was achieved in brain organoids.
High-resolution imaging revealed localized distributions of metabolites associated with neurogenesis.
The findings provide insights into the role of metabolites in brain development and potential therapeutic avenues.

Conclusions

The study demonstrates a robust approach for detailed metabolomic profiling in brain organoids.
The findings enhance understanding of neurodevelopment and could inform treatment strategies for developmental disorders.
This method holds promise for future research into metabolic processes underlying brain function.

Frequently Asked Questions

What are the advantages of using brain organoids in this study?

Brain organoids provide a relevant model for studying human brain development and disease due to their ability to mimic key aspects of brain tissue.

How is the biological model implemented in this research?

The study utilizes organoids derived from induced pluripotent stem cells, allowing for the investigation of human-specific neurodevelopmental processes.

What types of data are obtained from mass spectrometry imaging?

MSI provides spatially resolved metabolite distributions, enabling insights into metabolic pathways and their alterations during brain development.

How can this method be applied or adapted for future research?

The outlined MSI protocol can be adapted to investigate other organoid models or conditions, enhancing the study of metabolism in various diseases.

What are the key limitations or considerations for this study?

Maintaining the integrity and morphology of organoids during preparation and imaging is critical and can pose technical challenges.

开发了一种先进的脑类器官质谱成像（MSI）方法，该方法允许在这些模型中绘制代谢物分布图。这项技术提供了对早期发育和疾病期间大脑代谢途径和代谢物特征的见解，有望更深入地了解人脑功能。

我们研究了发育中的人脑的代谢组学。在体内研究的挑战。为了解决这个问题，我们使用体外脑类器官模型和先进的特殊代谢组学技术。

近年来，基于先进主技术的技术已迅速应用于类器官表征，这一趋势预计将持续下去。最近使用质谱成像或 MSI 的代谢组学研究证明了类器官模型在理解人类发育和疾病的分子机制（包括罕见疾病）以及在个性化医疗中的应用方面的优势。在分子成像过程中保持类器官的分子完整性和形态具有挑战性，需要精确的样品处理和制备。为了解决这个问题，已经开发了一种用于脑类器官高分辨率多 MALDI MSI 分析的专门技术。它优化了成像条件、培养和组织保存之间的质谱，以确保可靠的高质量数据。该综合协议展示了高分辨率 MSI 的潜力，并为研究人员提供了充分利用这些技术详细研究类器官代谢组学形貌所需的资源。

我们的研究将宣布我们对神经发生的理解，神经发生是与脑类器官中特定细胞类型相关的代谢物。这种详细的代谢分析不仅将阐明这些代谢物在大脑发育中的规律，还可以确定模仿神经发育障碍的治疗干预的潜在目标。

[旁白]首先，从培养箱中取出装有源自诱导多能干细胞的脑类器官的烧瓶。使用宽口径尖端，将类器官从烧瓶转移到培养皿中。用不含氯化钙的DPBS和氯化镁洗涤类器官三次以冲洗培养基。然后用蒸馏水快速冲洗以去除 DPBS 中的任何盐分。将 10 毫克从冷鱼皮中提取的明胶加入装有 100 毫升 DPBS 的烧瓶中。将混合物在 70 至 80 摄氏度下加热并搅拌两个小时。然后将溶液移至37摄氏度的培养箱中30分钟以去除气泡。使用小移液器吸头，将类器官固定在塑料模具的中心。将 10% 明胶包埋溶液轻轻倒入模具中，直至类器官完全浸入。将模具放入装有冷 100% 乙醇的干冰培养皿中。当完全冷冻时，颜色变为纯白色证明，从培养皿中取出类器官明胶块。用铝箔包裹块或将其放入锡杯中。将块密封并储存在零下 80 摄氏度，直到准备好进行冷冻切片。对于冷冻切片，将含有类器官的塑料块放置在设置为零下20摄氏度的冷冻室中10至15分钟。然后在冷冻过程中，将类器官切成 14 微米的切片，并安装在氧化铟锡涂层载玻片上进行质谱成像或 MSI。将载玻片储存在零下 80 摄氏度直至成像。首先，从零下80摄氏度取出安装有脑类器官切片的氧化铟锡涂层载玻片。将载玻片置于干燥剂中 20 分钟，以尽量减少表面大气中的水凝结。干燥后，使用加热的气动喷雾器，将每毫升10毫克的NEDC在70%甲醇中喷洒到类器官切片上。为了实现用于可视化脑类器官代谢物的高分辨率 MALDI MSI 平台，将具有双离子漏斗接口的离子源安装到质谱仪上。使用连接的 Q 开关频率三倍 ND 激光器，波长为 349 纳米，重复率为 1 kHz，脉冲能量约为 1.3 至 1.4 微焦耳。为避免过度采样，请将激光聚焦到直径约为 15 微米的光斑尺寸。将样品连接到 MALDI 进样器级。作和维护高压离子漏斗在 7.4 至 7.5 Torr，低压离子漏斗在 1.6 至 1.8 Torr。将 780 kHz 的射频电压施加到 191 伏峰值到低点，在 604 伏峰到峰值到 80 伏时施加到高压离子漏斗的峰值。为了提高低质量范围内小代谢物的灵敏度，将MALDIDMSI源的低压和高压漏斗中的射频幅度分别降低到大约20%和15%。将质量分辨率设置为 70,000。然后选择每个像素 25 微米的区域和像素大小。在负离子和正离子模式下将主电荷范围设置为 80 至 900。关闭自动增益控制，然后将进样时间设置为 250 毫秒，并在配置文件模式下获取 furier 变换质谱图。将质谱谱数据直接导入兼容软件。使用卷积算法执行基线校正，并使用总离子计数对数据进行归一化。使用等于 0.01 或 5：00 PPM 的 delta 主电荷的箱宽从原始数据文件中生成离子图像的特征列表，以根据质量缺陷和像素覆盖率区分主电荷比图像。从单个代谢物离子种类生成假彩色或 RGB 图像。将从 MALDI MSI 获得的原始数据文件的主电荷比列表上传到人类代谢组数据库，以识别代谢物。使用 MSI 和鱼明胶包埋对 60 天人脑类器官中的 Krebs 循环相关代谢物进行空间映射。