A Validated Mass Spectrometry Platform for Oxysterol Analysis of Single Human Gastruloids and Liver Organoids

Oxysterols, i.e., hydroxylated cholesterol metabolites, are associated with various signaling pathways and diseases. Their low abundance and structural complexity create analytical challenges, particularly in small sample sizes. We here present an optimized and validated miniaturized sample preparation method that enables oxysterol detection and quantification in single stem cell-derived 3D cell aggregates, as exemplified in human liver organoids (stem cell-based 3D liver models) and human gastruloids (stem cell-based embryo models) using liquid chromatography-mass spectrometry (LC-MS). The method, utilizing enzyme-assisted derivatization with Girard-T reagent, allowed a 10-fold decrease in starting material compared to conventional methodology while maintaining sensitivity and precision. A validation based on Eurachem guidelines confirmed quantitative performance and reproducibility across days and operators. In addition, we introduce a tailored normalization method, allowing same-sample measurements of oxysterols and the total protein content. The miniaturized method enabled successful detection and quantification of oxysterols of expected presence (e.g., 26-hydroxycholesterol), as well as unexpected (24S-hydroxycholesterol) and unknown oxysterols. Using our updated method, we could reveal significant heterogeneity among individual organoids and gastruloids, both between and within cell sources/protocols. Overall, we provide a reliable and high-sensitivity method for analyzing oxysterols in limited biological samples, opening opportunities for further insights into their roles in, e.g., liver function and early embryogenesis.