11 research outputs found
Determination of insulin secretion from stem cell-derived islet organoids with liquid chromatography-tandem mass spectrometry
Organoids are laboratory-grown 3D organ models, mimicking human organs for e.g. drug development and personalized therapy. Islet organoids (typically 100â200 Âľm), which can be grown from the patientĚs own cells, are emerging as prototypes for transplantation-based therapy of diabetes. Selective methods for quantifying insulin production from islet organoids are needed, but sensitivity and carry-over have been major bottlenecks in previous efforts. We have developed a reverse phase liquid chromatography-tandem mass spectrometry (RPLC-MS/MS) method for studying the insulin secretion of islet organoids. In contrast to our previous attempts using nano-scale LC columns, conventional 2.1 mm inner diameter LC column (combined with triple quadrupole mass spectrometry) was well suited for sensitive and selective measurements of insulin secreted from islet organoids with low microliter-scale samples. Insulin is highly prone to carry-over, so standard tubings and injector parts were replaced with shielded fused silica connectors. As samples were expected to be very limited, an extended Box-Behnken experimental design for the MS settings was conducted to maximize performance. The finale method has excellent sensitivity, accuracy and precision (limit of detection: â¤0.2 pg/ÂľL, relative error: â¤Âą10%, relative standard deviation: <10%), and was well suited for measuring 20 ÂľL amounts of Krebs buffer containing insulin secreted from islet organoids.publishedVersio
Critical evaluation of isolation and characterization techniques of breast cancer exosomes
Exosomes represent a distinct class of extracellular vesicles of endocytic origin secreted by multiple cell types, including tumour cells. An increased release of exosomes, which appears to be a rich source of biomarkers, has been reported from tumour cells. However, current strategies concerning the isolation (and characterization) of exosomes from fluids differ significantly and no consensus method is established. The goal of this work was to evaluate two different exosome isolation methods with two different breast cancer cell lines in culture: differential ultracentrifugation and commercial isolation kit. Evaluation was done using a bicinchoninic acid assay (protein concentration), transmission electron microscopy (morphology), dynamic light scattering (hydrodynamic size), western blotting (targeted protein exosome markers) and nano liquid chromatography tandem mass spectrometry (comprehensive protein identification). The characterization techniques confirmed the isolation of exosomes with both isolation kit and ultracentrifugation. However, the isolated samples did contain contaminations, and there was a clear difference in the protein amount, particle size and populations identified with the two isolation methods. In addition, the majority of the characterization techniques provided poor repeatability, reproducibility and/or demanded extensive optimization. The evaluation showed that the exosome isolation procedures used in this thesis appear to be far from mature. Additionally, the majority of the characterization techniques used in this study provided challenges
3D cell culture models and organ-on-a-chip: Meet separation science and mass spectrometry
In vitro derived simplified 3D representations of human organs or organ functionalities are predicted to play a major role in disease modeling, drug development, and personalized medicine, as they complement traditional cell line approaches and animal models. The cells for 3D organ representations may be derived from primary tissues, embryonic stem cells or induced pluripotent stem cells and come in a variety of formats from aggregates of individual or mixed cell types, selfâorganizing in vitro developed âorganoidsâ and tissue mimicking chips. Microfluidic devices that allow longâterm maintenance and combination with other tissues, cells or organoids are commonly referred to as âmicrophysiologicalâ or âorganâonâaâchipâ systems. Organâonâaâchip technology allows a broad range of âonâchipâ and âoffâchipâ analytical techniques, whereby âonâchipâ techniques offer the possibility of real time tracking and analysis. In the rapidly expanding tool kit for real time analytical assays, mass spectrometry, combined with âonâchipâ electrophoresis, and other separation approaches offer attractive emerging tools. In this review, we provide an overview of current 3D cell culture models, a compendium of current analytical strategies, and we make a case for new approaches for integrating separation science and mass spectrometry in this rapidly expanding research field
Characterization of a selective, iron-chelating antifungal compound that disrupts fungal metabolism and synergizes with fluconazole
The RNAseq data set has been deposited in the ENA database under accession number PRJEB63373 (https://www.ebi.ac.uk/ena/browser/view/PRJEB63373).Fungal infections are a growing global health concern due to the limited number of available antifungal therapies as well as the emergence of fungi that are resistant to first-line antimicrobials, particularly azoles and echinocandins. Development of novel, selective antifungal therapies is challenging due to similarities between fungal and mammalian cells. An attractive source of potential antifungal treatments is provided by ecological niches co-inhabited by bacteria, fungi, and multicellular organisms, where complex relationships between multiple organisms have resulted in evolution of a wide variety of selective antimicrobials. Here, we characterized several analogs of one such natural compound, collismycin A. We show that NR-6226C has antifungal activity against several pathogenic Candida species, including C. albicans and C. glabrata, whereas it only has little toxicity against mammalian cells. Mechanistically, NR-6226C selectively chelates iron, which is a limiting factor for pathogenic fungi during infection. As a result, NR-6226C treatment causes severe mitochondrial dysfunction, leading to formation of reactive oxygen species, metabolic reprogramming, and a severe reduction in ATP levels. Using an in vivo model for fungal infections, we show that NR-6226C significantly increases survival of Candida-infected Galleria mellonella larvae. Finally, our data indicate that NR-6226C synergizes strongly with fluconazole in inhibition of C. albicans. Taken together, NR-6226C is a promising antifungal compound that acts by chelating iron and disrupting mitochondrial functions. IMPORTANCE: Drug-resistant fungal infections are an emerging global threat, and pan-resistance to current antifungal therapies is an increasing problem. Clearly, there is a need for new antifungal drugs. In this study, we characterized a novel antifungal agent, the collismycin analog NR-6226C. NR-6226C has a favorable toxicity profile for human cells, which is essential for further clinical development. We unraveled the mechanism of action of NR-6226C and found that it disrupts iron homeostasis and thereby depletes fungal cells of energy. Importantly, NR-6226C strongly potentiates the antifungal activity of fluconazole, thereby providing inroads for combination therapy that may reduce or prevent azole resistance. Thus, NR-6226C is a promising compound for further development into antifungal treatment.This work was supported by grants from the Norwegian Cancer Society (project numbers 182524 and 208012), the Norwegian Health Authority South-East (2017064, 2017072, 2018012, and 2019096), the Research Council of Norway (261936, 301268, and 262652). O.Z. is supported by grant PID2020-114546RB by MCIN/AEI/10.13039/501100011033.S
Direct Electromembrane Extraction based Mass Spectrometry: A Tool for Studying Drug Metabolism Properties of Liver Organoids
This work introduces a strategy for organoid analysis - direct Electromembrane Extraction based Mass Spectrometry (dEME-MS) â for coupling liver organoids with mass spectrometry (MS). dEME-MS comprises electrophoresis of selected small molecules from a culture chamber across an oil membrane, and to a MS compatible solution. This enables clean micro-extraction of drugs and their metabolites as produced in the liver organoids to capillary liquid chromatography-mass spectrometry. Applying dEME-MS, proof-of-concept of directly measuring methadone metabolism is demonstrated on adult liver organoids. With 50 liver organoids and 1â
ÎźM methadone, methadone metabolism was monitored from 0 to 24â
hours (11â
time points). All analytes had 100 measurements. dEME-MS is capable of automated and selective monitoring of drug metabolism in liver organoids, and could serve as a valuable tool for automated drug discovery efforts
Ultracentrifugation versus kit exosome isolation: nanoLCâMS and other tools reveal similar performance biomarkers, but also contaminations
Aim: For isolation of exosomes, differential ultracentrifugation and an isolation kit from a major vendor were compared.
Materials & methods: âCase studyâ exosomes isolated from patient-derived cells from glioblastoma multiforme and a breast cancer cell line were analyzed.
Results: Transmission electron microscopy, dynamic light scattering, western blotting, and so forth, revealed comparable performance. Potential protein biomarkers for both diseases were also identified in the isolates using nanoLCâMS. Western blotting and nanoLCâMS also revealed negative exosome markers regarding both isolation approaches.
Conclusion: The two isolation methods had an overall similar performance, but we hesitate to use the term âexosome isolationâ as impurities may be present with both isolation methods. NanoLCâMS can detect disease biomarkers in exosomes and is useful for critical assessment of exosome enrichment procedures
Electromembrane extraction and mass spectrometry for liver organoid drug metabolism studies
Liver organoids are emerging tools for precision drug development and toxicity screening. We demonstrate that electromembrane extraction (EME) based on electrophoresis across an oil membrane is suited for segregating selected organoid-derived drug metabolites prior to mass spectrometry (MS)-based measurements. EME allowed drugs and drug metabolites to be separated from cell medium components (albumin, etc.) that could interfere with subsequent measurements. Multiwell EME (parallel-EME) holding 100 ÎźL solutions allowed for simple and repeatable monitoring of heroin phase I metabolism kinetics. Organoid parallel-EME extracts were compatible with ultrahigh-performance liquid chromatography (UHPLC) used to separate the analytes prior to detection. Taken together, liver organoids are well-matched with EME followed by MS-based measurements
âOrgan-in-a-Columnâ Coupled On-line with Liquid Chromatography-Mass Spectrometry
Organoids, i.e.,
laboratory-grown organ models developed from stem
cells, are emerging tools for studying organ physiology, disease modeling,
and drug development. On-line analysis of organoids with mass spectrometry
would provide analytical versatility and automation. To achieve these
features with robust hardware, we have loaded liquid chromatography
column housings with induced pluripotent stem cell (iPSC) derived
liver organoids and coupled the âorgan-in-a-columnâ
units on-line with liquid chromatography-mass spectrometry (LC-MS).
Liver organoids were coloaded with glass beads to achieve an even
distribution of organoids throughout the column while preventing clogging.
The liver organoids were interrogated âon columnâ with
heroin, followed by on-line monitoring of the drugâs phase
1 metabolism. Enzymatic metabolism of heroin produced in the âorgan-in-a-columnâ
units was detected and monitored using a triple quadrupole MS instrument,
serving as a proof-of-concept for on-line coupling of liver organoids
and mass spectrometry. Taken together, the technology allows direct
integration of liver organoids with LC-MS, allowing selective and
automated tracking of drug metabolism over time
âOrgan-in-a-Columnâ Coupled On-line with Liquid Chromatography-Mass Spectrometry
Organoids, i.e.,
laboratory-grown organ models developed from stem
cells, are emerging tools for studying organ physiology, disease modeling,
and drug development. On-line analysis of organoids with mass spectrometry
would provide analytical versatility and automation. To achieve these
features with robust hardware, we have loaded liquid chromatography
column housings with induced pluripotent stem cell (iPSC) derived
liver organoids and coupled the âorgan-in-a-columnâ
units on-line with liquid chromatography-mass spectrometry (LC-MS).
Liver organoids were coloaded with glass beads to achieve an even
distribution of organoids throughout the column while preventing clogging.
The liver organoids were interrogated âon columnâ with
heroin, followed by on-line monitoring of the drugâs phase
1 metabolism. Enzymatic metabolism of heroin produced in the âorgan-in-a-columnâ
units was detected and monitored using a triple quadrupole MS instrument,
serving as a proof-of-concept for on-line coupling of liver organoids
and mass spectrometry. Taken together, the technology allows direct
integration of liver organoids with LC-MS, allowing selective and
automated tracking of drug metabolism over time