Molecular Imaging of Human Brain Organoids Using Mass Spectrometry

We studied the metabolomics of the developing human brain. A challenge to investigate in vivo. To address this, we use in vitro brain organoids models, and advanced special metabolomics techniques.

Advanced master technology-based technologies have rapidly been applied in recent years for organoid characterization, a trend that is expected to continue. Recent metabolomic studies using mass spectometry imaging or MSI demonstrate the advantages of organoid models in understanding molecular mechanisms of human development and disease, including rare conditions, and applications in personalized medicine. Maintaining the molecular integrity, and morphology of organoids during molecular imaging is challenging, requiring precise sample handling, and preparation. To address this, a specialized technique for high resolution multi MALDI MSI analysis of brain organoids has been developed. It optimizes mass spectrum between imaging conditions, matriculation, and tissue preservation to ensure reliable high quality data. This comprehensive protocol demonstrates high resolution MSI potential, and gives researchers the resources they need to fully utilize these technology to investigate the metabolomic topography of organoids in detail.

Our research will announce our understanding of neurogenesis, metabolite associated with specific cell types in brain organoids. This detailed metabolic profiling will not only illuminate the rule of these metabolites in brain development, but also identify potential target for therapeutic interventions imitate neurodevelopmental disorders.

[Narrator] To begin, remove the flask containing the brain organoids derived from the induced pluripotent stem cells from the incubator. Using wide bore tips, transfer the organoids from the flask to the dish. Wash the organoids three times with DPBS without calcium chloride, and magnesium chloride to rinse the media. Then rinse quickly with distilled water to remove any salts from the DPBS. Add 10 milligrams of gelatin obtained from cold fish skin in a flask containing 100 milliliters of DPBS. Heat and stir the mixture at 70 to 80 degrees Celsius for two hours. Then move the solution to a 37 degrees Celsius incubator for 30 minutes to remove bubbles. Using a small pipette tip, pin down the organoid in the center of a plastic mold. Gently pour the 10% gelatin embedding solution into the mold until the organoid is fully immersed. Place the mold into a Petri dish containing cold 100% ethanol on dry ice. When completely frozen, evidenced by the change of color to solid white, remove the organoid gelatin block from the Petri dish. Wrap the block in aluminum foil or place it in a tin cup. Seal and store the block at minus 80 degrees Celsius until ready for cryo section. For cryo sectioning, place the plastic blocks containing organoids within the cryo chamber set at minus 20 degrees Celsius for 10 to 15 minutes. Then on cryotome, section the organoids into 14 micrometer sections, and mount on indium tin oxide coated glass slides for Mass Spectrometry Imaging or MSI. Store the slides at minus 80 degrees Celsius until imaging. To begin, remove the indium tin oxide coated slide mounted with brain organoid sections from minus 80 degrees Celsius. Place the slide in a desiccate for 20 minutes to minimize condensation of atmospheric water on the surface. After desiccation, using a heated pneumatic sprayer, spray 10 milligrams per milliliter NEDC in 70% methanol onto the organoid sections. To achieve a high resolution MALDI MSI platform for visualizing brain organoid metabolites mount ion source with a dual ion funnel interface Onto a mass spectrometer. Use a connected Q switch frequency tripled ND laser with a 349 nanometer wavelength, a repetition rate of one kilohertz, and a pulse energy of approximately 1.3 to 1.4 micro joules. To avoid oversampling, focus the laser to a spot size of approximately 15 micrometers in diameter. Attach the sample to the MALDI injector stage. Operate and maintain the high pressure ion funnel at 7.4 to 7.5 Torr, and the low pressure ion funnel at 1.6 to 1.8 Torr. Apply radio frequency voltages of 780 kilohertz at 191 volts peak to peak to the low, and 604 kilohertz at 80 volts peak to peak to the high pressure ion funnels. To improve the sensitivity for small metabolites in the low mass range, reduce the RF amplitudes in the low and high pressure funnel of the MALDIDMSI source to approximately 20 and 15% respectively. Set the mass resolution to 70,000. Then select the area and the pixel size of 25 micrometers per pixel. Set the master charge range between 80 to 900 in both negative, and positive ion modes. Keep the automatic gain control turned off, then set the injection time to 250 milliseconds, and acquire furier transform mass spectra in the profile mode. Import the mass spectrometry spectral data directly into the compatible software. Perform baseline correction using the convolution algorithm, and normalize the data using total ion count. Generate the feature list of ion images from the raw data files using a bin width of delta master charge equals 0.01 or 5:00 PPM to distinguish master charge ratio images based on mass defect and pixel coverage. Generate false color or RGB images from individual metabolite ion species. Upload the master charge ratio list of raw data files obtained from MALDI MSI to the Human Metabolome Database to identify metabolites. Krebs cycle related metabolites in 60 day human brain organoids were spatially mapped using MSI with fish gelatin embedding.