Kristina Sæterdal Kømurcua,b, Malgorzata Elzbieta Zawadzkaa,b, Igor Meszkab, Aleksandra Aizenshtadtb,
Helena Hruškováa, Lydia Emilie Aakervika, James L Thornec, Stefan Johannes Karl Kraussb, Steven Ray Wilsona,b,
and Hanne Røberg-Larsena,b
a Section of Chemical Life Science, Department of Chemistry, University of Oslo, Norway
b Hybrid Technology Hub, Faculty of Medicine, University of Oslo, Norway
c School of Food Science and Nutrition, University of Leeds, Leeds LS2 9JT, United Kingdom
Email: k.s.komurcu@kjemi.uio.no
Oxysterols, hydroxylated forms of cholesterol, are associated with a plethora of diseases, including liver disorders. Liver organoids, i.e. laboratory-grown miniaturized liver models, are alternatives to animal models in liver disease research. In our previous work, we demonstrated the upregulation of oxysterols in liver organoid disease models using liquid chromatography – mass spectrometry (LC-MS) [1]. However, conventional LC-MS analysis of oxysterols typically requires a derivatization step to enhance the ionization efficiency of these neutral compounds, which results in a significant dilution of the sample. This poses a challenge when targeting low-abundance oxysterols in limited sample sizes such as organoids. In addition, being able to assess oxysterols at the single-organoid levels could give valuable insights into organoid heterogeneity.
We here present a miniaturized sample preparation method that enables the quantification of oxysterols in a single liver organoid using LC-MS [2]. Applying experimental design to optimize all reagents involved in the derivatization reaction, we achieved a 10-fold reduction in final sample volume without compromising reaction efficiency. Methanol volume had the most significant impact on yield (p<0.0001), while cholesterol oxidase and isopropanol amounts showed a modest effect (p=0.03-0.04).
The method was validated with regard to linearity (25-500 pM, R2 > 0.99), intra-day precision (<15% RSD), inter-day precision (<20% RSD) and relative error (<10%). Additionally, our method allowed for the measurement of oxysterols and total protein content in the same sample, enabling normalization of oxysterols between experiments. With a final volume of 75 µL after derivatization, we successfully quantified 26-hydroxycholesterol in a single organoid. Interestingly, we observed a high degree of heterogeneity between single organoids (>40%), even after normalization.
Overall, we present a highly sensitive method enabling single organoid sterolomics, which could further advance studies of oxysterols in liver diseases using single organoids. Future work aims to further increase sensitivity by downscaling the chromatographic system to capillary LC, where preliminary results suggest a further 10-fold increase in sensitivity. This could allow quantification of the less-abundant dihydroxycholesterols in single organoids.
Figure 1 Our original sample preparation procedure for oxysterols is downscaled by a 10-fold using experimental design, allowing us to now measure oxysterols and total protein content in a single liver organoid.
References
Kømurcu et al, J Steroid Biochem Mol Biol, 2023
Kømurcu and Zawadzka et al, manuscript in progress, 2025