Preparing ductal epithelial organoids for high-spatial-resolution molecular profiling using mass spectrometry imaging

Area of Science:

• Biotechnology
• Cell Biology
• Analytical Chemistry

Background:

• Organoid cultures are 3D models mimicking human tissues, crucial for disease modeling and drug discovery.
• Mass spectrometry imaging (MSI) offers unparalleled molecular analysis in situ, but sample preparation for organoids is challenging.
• Preserving the intricate 3D structure and molecular localization of organoids for MSI is essential for accurate biological insights.

Purpose of the Study:

• To develop and validate a protocol for preparing hollow organoids for mass spectrometry imaging (MSI).
• To ensure the preservation of 3D morphology and spatiotemporal molecular distribution during sample preparation.
• To enable accurate molecular identification using tandem mass spectrometry on prepared organoid samples.

Main Methods:

• A protocol focusing on organoid collection, embedding in gelatin, and MSI-specific sample preparation was developed.
• Recommendations for data acquisition and molecular identification by tandem mass spectrometry were provided.
• The protocol was designed for compatibility with various organoid types and origins.

Main Results:

• The developed protocol successfully preserves the 3D morphological structure of hollow organoids.
• Spatiotemporal distribution of a wide array of molecules within organoids is retained during sample preparation.
• Accurate molecular identification is achievable through tandem mass spectrometry following the protocol.

Conclusions:

• This protocol provides a robust method for preparing organoids for mass spectrometry imaging.
• The technique allows for comprehensive molecular profiling of organoids while maintaining structural integrity.
• The rapid 1-day protocol facilitates high-throughput analysis of diverse organoid models.