Filtration-Based Rapid ECM-Hydrogel Elimination Enables Robust MALDI MSI of Intestinal Organoids.

The rapid development of matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) offers a powerful approach for the 2D metabolic profiling of intestinal organoids. However, its application remains constrained by challenges in sample preparation. Conventional extracellular matrix (ECM)-based hydrogel removal methods, such as repeated centrifugation or enzymatic digestion, are labor-intensive and time-consuming, often causing structural disruption of organoids and degradation of metabolites, thereby compromising the spectral quality and metabolite coverage. In this study, we established a filtration-based rapid ECM-hydrogel elimination (FREE) method using a 70 μm filter, which reduced treatment time from 75 to 25 min while preserving organoid structural integrity, cell viability, and metabolic stability and, more importantly, improved data quality, with a 26.6% increase in metabolite coverage and enhanced signal intensity. Applying this workflow to an LPS-induced inflammatory intestinal organoid model, we observed spatial metabolic remodeling associated with epithelial polarity disruption. Notably, phosphatidylserine (PS) became enriched at the apical membrane, while phosphatidylethanolamine (PE) exhibited a basolateral side, suggesting a link between loss of polarity and early apoptotic signaling. Overall, FREE provides an efficient pretreatment strategy that enables MSI research on intestinal organoids to dissect the mechanisms of inflammation-associated metabolic remodeling.