As organoids and organ-on-chip (OoC) systems move toward preclinical and clinical applications, there is an increased need for method validation. Using a liquid chromatography-mass spectrometry (LC-MS)-based approach, we developed a method for measuring small-molecule drugs and metabolites in the cell medium directly sampled from liver organoids/OoC systems. The LC-MS setup was coupled to an automatic filtration and filter flush system with online solid-phase extraction (SPE), allowing for robust and automated sample cleanup/analysis. For the matrix, rich in, e.g., protein, salts, and amino acids, no preinjection sample preparation steps (protein precipitation, SPE, etc.) were necessary. The approach was demonstrated with tolbutamide and its liver metabolite, 4-hydroxytolbutamide (4HT). The method was validated for analysis of cell media of human stem cell-derived liver organoids cultured in static conditions and on a microfluidic platform according to Food and Drug Administration (FDA) guidelines with regards to selectivity, matrix effects, accuracy, precision, etc. The system allows for hundreds of injections without replacing chromatography hardware. In summary, drug/metabolite analysis of organoids/OoCs can be performed robustly with minimal sample preparation.

• MeSH
• Chromatography, Liquid methods   MeSH
• Solid Phase Extraction MeSH
• Mass Spectrometry methods   MeSH
• Liver * metabolism   MeSH
• Liquid Chromatography-Mass Spectrometry MeSH
• Small Molecule Libraries analysis   metabolism   chemistry   MeSH
• Lab-On-A-Chip Devices MeSH
• Pharmaceutical Preparations metabolism   analysis   MeSH
• Humans MeSH
• Organoids * metabolism   cytology   MeSH
• Tolbutamide metabolism   analysis   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Validation Study MeSH

Steroid hormone receptors represent a major target in drug discovery. As ligand inducible transcription factors, their activity can be modulated by small lipophilic molecules. Here we describe two panels of potent and selective luciferase reporter cell lines based on cells with low endogenous steroid receptor activity (U2OS). The panels contain reporter cell lines for estrogen receptors α and β, androgen, glucocorticoid, mineralocorticoid, and progesterone receptors. In the first panel, the activation of either synthetic, steroid response elements containing promoter or viral promoter is mediated by full-length steroid receptors. The second panel is based on the expression of the chimeric receptor, which was created by the replacement of the N-terminal part of the molecule by Gal4 DBD and that binds to multiple UAS sites in the reporter promoter. Both panels were extensively characterized by profiling 28 ligands in dose response manner in agonist and antagonist mode. We have analyzed and compared the responses to tested ligands from both panels and concluded that in general both systems generated similar qualitative response in terms of potency, efficacy, partial agonism/antagonism, mixed agonistic/antagonistic profiles and the rank of potencies was well conserved between both panels. However, we have also identified some artifacts introduced by the Gal4/LBD reporter assays in contrast to their full-length receptor reporter counterparts. Keeping in mind the advantages and drawbacks of each reporter format, these cell lines represent powerful and selective tools for profiling large compound libraries (HTS) and for detailed study of mechanisms by which compounds exert their biological effects.

• MeSH
• Transcriptional Activation drug effects   MeSH
• Genetic Engineering methods   MeSH
• Small Molecule Libraries analysis   MeSH
• Humans MeSH
• Luciferases genetics   metabolism   MeSH
• Cell Line, Tumor MeSH
• Drug Discovery methods   MeSH
• Plasmids genetics   MeSH
• Promoter Regions, Genetic drug effects   MeSH
• Recombinant Proteins genetics   metabolism   MeSH
• Genes, Reporter MeSH
• High-Throughput Screening Assays methods   MeSH
• Sequence Deletion MeSH
• Receptors, Steroid agonists   antagonists & inhibitors   genetics   metabolism   MeSH
• Steroids pharmacology   MeSH
• Transfection MeSH
• Transcription Factors genetics   metabolism   MeSH
• Dose-Response Relationship, Drug MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH