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

## TL;DR

This paper introduces a new, highly sensitive method for analyzing oxysterols in small biological samples like human liver and embryo models.

## Contribution

A miniaturized, validated LC–MS method for oxysterol analysis in single organoids and gastruloids, with improved sensitivity and reproducibility.

## Key findings

- The method enables oxysterol detection in single organoids with 10-fold less starting material than conventional methods.
- Significant heterogeneity in oxysterol levels was observed among individual organoids and gastruloids.
- The method detected both expected and unexpected oxysterols, including 26-hydroxycholesterol and 24S-hydroxycholesterol.

## Abstract

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.