Organoids and Organ-on-a-Chip: A Need for Separation Science

Organoids are predicted to become important tools for personalized medicine and are alternatives to animal models. Separation science and mass spectrometry (MS) are key approaches for studying organoids and organ‑on-a-chip systems. Applications include the study of organoid drug metabolism and biomarker discovery.

Organoids are three-dimensional (3D) models of organs grown in the laboratory. Organoids can be grown using stem cells from a patient and can be developed into miniatures of organs such as livers, kidneys, and lungs.

The organoid research field is developing fast, and organoids are predicted to become important tools for personalized medicine and alternatives to animal models. When a miniature organ model is grown or placed in microfluidic chips that provide nutrients and connections to other “mini-organs”, we refer to these as organ-on-a-chip (OoC) units.

The Hybrid Technology Hub is located at the University of Oslo and focuses on the development and testing of organoids and OoCs. Here, biologists, physicists, informaticians, and chemists collaborate in a highly multidisciplinary environment. Our role is to develop analytical tools for analyzing organoids and OoCs. We find that research in organoids and OoCs greatly benefits from separation science and mass spectrometry (MS) (1). We present here some examples of our work that we will be presenting at HPLC 2023 in Düsseldorf (along with new results!).

Disease Models

Liver organoids can be exposed to compounds such as lipids to create models of non-alcoholic fatty liver disease (NAFLD). We speculated that oxysterols (hydroxylated metabolites of cholesterol) could serve as markers of disease progression, as there were signs of this being the case when analyzing blood samples from NAFLD patients (2). Using an automated filtration and filter backflush plumbing (AFFL) upstream to solid-phase extraction–liquid chromatography (SPE–LC)–MS, we could observe that several oxysterols were indeed significantly upregulated compared to the controls (2). This pilot serves as an example of how controlled studies of organoids can be used as a relatively simple tool in biomarker discovery.

Drug Metabolism

Liver organoids can be used to predict drug metabolism. We have used liver models to identify drug metabolites (3) and to study pharmacokinetics (4). However, we wish to reduce the number of sample preparation steps applied for extracting and isolating analytes. One approach has been to apply electromembrane extraction (EME), which is electrophoresis across an oil membrane (5). Using EME in a 96-well format, we were able to perform simple extractions of drugs and metabolites prior to LC–MS analysis (5). The approach was also compatible with a simple capillary electrophoresis-ultraviolet (CE-UV) system (5).

Organ-on-a-Chip and LC–MS

Based on the success with the 96-plate format, we explored the use of chips with EME. Liver organoids were placed in the device and fractions were collected using a valve setup connecting to the LC–MS system. Metabolism of methadone was monitored in a fully automated fashion (6). In parallel with this, we have developed a method where liver organoids are placed within LC column housing, which we call “organ in a column”. The columns were again coupled to the LC–MS system via a valve, allowing for fully automated analysis of drug metabolism (7).

Hormones

In addition to liver organoids, we worked with islet organoids, that is, models of the regions of the pancreas that produce hormones such as insulin. Islet organoids are being developed for transplantation purposes for addressing type 1 diabetes, but they can also be coupled with liver organoids in multi-organ chip systems for studying inter-organ interactions.

We have developed methods for measuring insulin secreted from islet organoids (8). A key issue has been to address nonspecific absorption of our analyte and matrix effects from cell medium and other related buffers used for organoids and OoC systems (often containing significant amounts of albumin). We are currently expanding the method to include other hormones for multi-analyte measurements.

Conclusions (And What To Look Forward To)

We have found that separation science and mass spectrometry are highly valuable tools for studying organoids and OoC systems. These organ models can vary in composition and functionality, with a large range of interesting analytes. Therefore, we are dependent on having versatile and selective analytical tools such as liquid chromatography and mass spectrometry to separate isomers or hormones from uninteresting (but interfering) cell medium proteins. We are exploring the possibilities of coupling OoC systems with LC–MS. EME is an exciting tool for selective extraction and provides very clean samples—perhaps so clean that we may be able to employ simpler detectors than mass spectrometry for certain applications. This will then require even more attention to our chromatographic systems, which is a pleasure, especially to us chromatographers.

References

1. Kogler, S.; Kømurcu, K. S.; Olsen, C.; et al. Organoids, Organ-on-a-Chip, Separation Science and Mass Spectrometry: An Update. TrAC, Trends Anal. Chem. 2023, 161, 116996. DOI: 10.1016/j.trac.2023.116996
2. Kømurcu, K. S.; Wilhelmsen, I.; Thorne, J. L.; et al. Mass Spectrometry Reveals that Oxysterols are Secreted from Non-Alcoholic Fatty Liver Disease Induced Organoids. bioRxiv 2023, DOI: 10.1101/2023.02.22.529551
3. LaLone, V.; Aizenshtadt, A.; Goertz, J.; et al. Quantitative Chemometric Phenotyping of Three-Dimensional Liver Organoids by Raman Spectral Imaging (qRamanomics). Cells Rep. Methods 2023, 3 (4), 100440. DOI: 10.1016/j.crmeth.2023.100440
4. Harrison, S.P.; Sillar, R.; Tanaka, Y. et al. Scalable Production of Tissue-Like Vascularised Liver Organoids from Human PSCs. bioRxiv 2020, DOI: 10.1101/2020.12.02.406835
5. Skottvoll, F. S.; Harrison, S.; Boger, I. S. et al. Electromembrane Extraction and Mass Spectrometry for Liver Organoid Drug Metabolism Studies. Anal. Chem. 2021, 93, 3576–3585. DOI: 10.1021/acqas.analchem.0c05082
6. Skottvoll, F. S.; Aizenshtadt, A.; Hansen, F. A. et al. Direct Electromembrane Extraction-Based Mass Spectrometry: A Tool for Studying Drug Metabolism Properties of Liver Organoids. Analysis Sensing 2022, 2 (2), e202100051. DOI: 10.1002/anse.202100051
7. Kogler, S.; Aizenshtadt, A.; Harrison, S.; et al. “Organ-in-a-Column” Coupled Online with Liquid Chromatography–Mass Spectrometry. Anal. Chem. 2022, 94 (50), 17677–17684. DOI: 10.1021/acs.analchem.2c04530
8. Olsen, C.; Wang, C.; Abadour, S. et al. Determination of insulin secretion from stem cell-derived islets with liquid chromatography–tandem mass spectrometry. J. Chromatogr. B Analyst Technol. Biomed. Life Sci. 2023, 1215, 123577. DOI: 10.1016/j.jchromb.2022.123577

Steven Ray Wilson is a professor in analytical chemistry at the University of Oslo, specializing in separation science and mass spectrometry. He is a PI in the Center of Excellence, the Hybrid Technology Hub. Wilson also has several collaborations with clinicians related to diagnostics (proteomics, metabolomics).

Hanne Røberg-Larsen is an associate professor in analytical chemistry at the University of Oslo, and the leader of the bioanalytical chemistry group in the Department of Chemistry. Participating in numerous multidisciplinary collaborations, she specializes in LC–MS of biosamples, with a focus on sterolomics and clinical applications.

Steven Ray Wilson will present his keynote lecture on Wednesday 21 June at 14:00 in Hall Y.

Hanne Røberg-Larsen will present her talk on Thursday 22 June at 10:10 in Hall Y.