Molecular Imaging of Human Brain Organoids Using Mass Spectrometry

Brain organoid models serve as a powerful tool for studying human brain development and function. Mass spectrometry imaging (MSI), a cutting-edge technology, allows us to map the spatial distribution of diverse molecules such as lipids, sugars, amino acids, drugs, and their metabolites within these organoids, all without the need for specific molecular probes. High-quality MSI data hinge on meticulous sample preparation. Fixatives play a pivotal role, but conventional options such as glutaraldehyde, paraformaldehyde, and cryopreserving such as sucrose may inadvertently impact tissue metabolites. Optimal fixation entails flash freezing in liquid nitrogen. However, for small organoids, a more suitable approach involves transitioning the organoids directly from the incubator into a warmed embedding solution, followed by freezing in dry ice-cooled ethanol. Another critical step is the embedding prior to cryosectioning, which also requires materials compatible with MSI, as traditional options can interfere with matrix deposition and ionization. Here, an optimized protocol for high resolution-MALDI-MSI of human brain organoids is presented, encompassing sample preparation, sectioning, and imaging using mass spectrometry. This method showcases the molecular distribution of small metabolites, such as amino acids, with high mass accuracy and sensitivity. As such, coupled with complementary studies of brain organoids, it can assist in illuminating complex processes governing early brain development, metabolic cell fate trajectories, and distinctive metabolite signatures. Furthermore, it provides insights into the precise locations of molecules within the organoid, enriching our understanding of the spatial organization of 3D brain organoid models. As the field continues to advance, a growing number of studies leveraging MSI to delve into brain organoids and complex biological systems is anticipated, thereby deepening the understanding of the metabolic aspects of human brain function and development.