Aleksandra Aizenshtadt

• Kømurcu, Kristina Sæterdal; Zawadzka, Malgorzata Elzbieta; Meszka, Igor; Aizenshtadt, Aleksandra; Hrušková, Helena & Aakervik, Lydia Emilie [Show all 10 contributors for this article] (2026). A Validated Mass Spectrometry Platform for Oxysterol Analysis of Single Human Gastruloids and Liver Organoids. Analytical Chemistry. ISSN 0003-2700. 98(9), p. 6792–6804. doi: 10.1021/acs.analchem.5c07140. Full text in Research Archive
• Martinez, Mikel Amirola; Combriat, Thomas Michel Daniel; Ferencevic, Katharina; Wilhelmsen, Ingrid; Dalmao-Fernandez, Andrea & Olsen, Petter Angell [Show all 9 contributors for this article] (2025). Aspects of zone-like identity and holotomographic tracking of human stem cell-derived liver sinusoidal endothelial cells. Frontiers in Cell and Developmental Biology. 13. doi: 10.3389/fcell.2025.1528991. Full text in Research Archive Show summary

  Introduction: Liver sinusoidal endothelial cells (LSEC) are specialized endothelial cells with unique metabolic and barrier functions adapted to the needs of the liver sinusoid. LSECs are highly sensitive to their environment, and this fragile nature causes challenges in analyzing their phenotype under in vitro conditions. Methods: In this work, we first differentiated LSEC-like cells (scLSECs) from two human pluripotent stem cell lines and characterized them by a panel of qPCR markers, immunohistochemistry, substrate oxidation for energy metabolism, scavenger function, and nitric oxide secretion. We then introduced holotomography, a technique that allows to recover quantitative and three-dimensional information about the refractive indexes of cell components, as a tool to image and track scLSEC in vitro in a minimally intrusive, label-free manner. Results and discussion: Holotomography and developed machine learning-based algorithm for image processing allowed us to describe and monitor changes in intracellular pore-like structures over time. Finally, we tested the possibility of inducing aspects of zone-specific LSEC phenotype and metabolism using culture-based treatments, which resulted in modest shifts in marker expression and metabolic activity. The presented strategy provides an advanced tool kit for investigating liver endothelial cells.

• Delon, Ludivine; Busek, Mathias; Menezes, Pedro Duarte; Gadegaard, Nikolaj; Stokowiec, Justyna & Golovin, Alexey [Show all 10 contributors for this article] (2025). Placental Syncytium-on-Chip (PSoC)–Comparison of Forskolin or Mechanical Induced-Syncytialization. Advanced Healthcare Materials. ISSN 2192-2640. doi: 10.1002/adhm.202404462. Full text in Research Archive Show summary

  The placenta is a key embryonic structure that separates maternal and fetal blood systems. The barrier function of the human placenta is performed by villous trophoblasts, i.e. undifferentiated cytotrophoblasts and differentiated syncytiotrophoblats, whose maturation and function are influenced by wall shear stress (WSS) from the maternal blood circulation. Most in vitro placenta models rely on cyclic adenosine monophosphate inducer forskolin (FSK) to establish a placental syncytium. Here a trophoblastic BeWo cell line is used to systematically compare the effect of FSK treatment in static culture with WSS stimulation in a pumpless, recirculating organ-on-chip. It is shown that BeWo cells undergo a similar differentiation under WSS exposure to FSK treatment. A WSS of 0.1 dyn cm−2 leads to cell fusion, polarization, barrier functions, human chorionic gonadotropin (β-hCG) secretion, and increased expression of key transporters. Moreover, WSS induces favorable changes in the levels of FMS-like tyrosine kinase-1 (FLT-1) and Placental Growth Factor (PlGF) suggesting the development of a physiologically relevant placental syncytium-on-chip (PSoC) without the need for FSK. The platform is further expanded to a syncytiotrophoblast/endothelial co-culture showing physiological vascular functions under WSS. The forskolin-free PSoC presented here represents the first pumpless recirculating and scalable platform for physiological placental studies and drug testing.

• Kogler, Stian; Skottvoll, Frøydis Sved; Hruskova, Helena; Rise, Frode; Aizenshtadt, Aleksandra & Krauss, Stefan Johannes Karl [Show all 9 contributors for this article] (2025). Electromembrane Extraction Provides Unprecedented Selectivity for Drugs in Cell Culture Media Used in Organoid and Organ-on-Chip Systems. Analytical Chemistry. ISSN 0003-2700. 97(9), p. 4923–4931. doi: 10.1021/acs.analchem.4c04994. Full text in Research Archive Show summary

  The use of organoids and organ-on-chip technologies as nonanimal methodologies in drug discovery and personalized medicine is rapidly expanding. However, the complexity and small volumes of organoid culture samples present significant analytical challenges, e.g., in drug analysis using liquid chromatography–mass spectrometry (LC–MS). Essentially an electrophoresis across an oil membrane, electromembrane extraction (EME) offers a promising approach for measuring drugs, as it is, for example, compatible with small samples such as organoid and organ-on-chip formats. Given the potential of the technology, there is a need to assess the extraction purity of EME extracts to ensure EME’s compatibility with high-throughput, downstream analysis. This study evaluates the effectiveness of EME for sample cleanup in various common cell culture media used for organoids and organs-on-chips. The media were spiked with 90 small-molecule drugs. Using gel electrophoresis (sodium dodecyl sulfate polyacrylamide gel electrophoresis), high-resolution nuclear magnetic resonance spectroscopy, and LC–MS, we demonstrate that EME provides exhaustive removal of unwanted medium components (proteins, polar molecules, and apolar/neutral molecules) while selectively extracting the spiked small-molecule drugs. The approach was demonstrated with human stem-cell-derived liver organoids, allowing simple detection and monitoring of telltale cytochrome P450 metabolism. Taken together, our observations highlight an unprecedented ability of EME to provide sample cleanup for drug analysis in matrixes compatible with organoids and organ-on-chip technology.

• Cunha, André; Schülke, Christin; Mesri, Alireza; Ruud, Simen Kristoffer; Aizenshtadt, Aleksandra & Ferrari, Giorgio [Show all 16 contributors for this article] (2024). Development of a Smart Wireless Multisensor Platform for an Optogenetic Brain Implant. Sensors. 24(2). doi: 10.3390/s24020575. Full text in Research Archive Show summary

  Implantable cell replacement therapies promise to completely restore the function of neural structures, possibly changing how we currently perceive the onset of neurodegenerative diseases. One of the major clinical hurdles for the routine implementation of stem cell therapies is poor cell retention and survival, demanding the need to better understand these mechanisms while providing precise and scalable approaches to monitor these cell-based therapies in both pre-clinical and clinical scenarios. This poses significant multidisciplinary challenges regarding planning, defining the methodology and requirements, prototyping and different stages of testing. Aiming toward an optogenetic neural stem cell implant controlled by a smart wireless electronic frontend, we show how an iterative development methodology coupled with a modular design philosophy can mitigate some of these challenges. In this study, we present a miniaturized, wireless-controlled, modular multisensor platform with fully interfaced electronics featuring three different modules: an impedance analyzer, a potentiostat and an optical stimulator. We show the application of the platform for electrical impedance spectroscopy-based cell monitoring, optical stimulation to induce dopamine release from optogenetically modified neurons and a potentiostat for cyclic voltammetry and amperometric detection of dopamine release. The multisensor platform is designed to be used as an opto-electric headstage for future in vivo animal experiments.

• Kogler, Stian; Pedersen, Gustav Mathingsdal; Martínez-Ramírez, Felipe; Aizenshtadt, Aleksandra; Busek, Mathias & Krauss, Stefan Johannes Karl [Show all 8 contributors for this article] (2024). An FDA-Validated, Self-Cleaning Liquid Chromatography–Mass Spectrometry System for Determining Small-Molecule Drugs and Metabolites in Organoid/Organ-on-Chip Medium. Analytical Chemistry. ISSN 0003-2700. 96(29), p. 12129–12138. doi: 10.1021/acs.analchem.4c02246. Full text in Research Archive Show summary

  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.

• Hrušková, Helena; Olsen, Christine; Řemínek, Roman; Wang, Chencheng; Aizenshtadt, Aleksandra & Krauss, Stefan Johannes Karl [Show all 10 contributors for this article] (2024). Preparative agarose gel electrophoresis for reducing matrix interferences of organoid cell medium prior to LC-MS analysis of insulin. Journal of Chromatography A. ISSN 0021-9673. 1717. doi: 10.1016/j.chroma.2024.464669. Full text in Research Archive Show summary

  Organoids are 3D cell cultures with microanatomies mimicking aspects of real organs, useful for e.g. animal-free studies of development, disease, and drug discovery. The cell medium of organoid models of Langerhans islets, regulating blood glucose levels by insulin secretion, can be analyzed by liquid chromatography-mass spectrometry (LC-MS). However, organoid medium complexity is a major challenge, as matrix interferences can reduce sensitivity and selectivity, even with optimized LC-MS conditions. By applying preparative agarose gel electrophoresis-electrodialysis (PGE-ED), we were able to decrease the cell medium background signal, allowing for reduced interferences affecting LC-MS analysis of human insulin.

• Wilhelmsen, Ingrid; Combriat, Thomas Michel Daniel; Dalmao-Fernandez, Andrea; Stokowiec, Justyna; Wang, Chencheng & Olsen, Petter Angell [Show all 10 contributors for this article] (2024). The effects of TGF-β-induced activation and starvation of vitamin A and palmitic acid on human stem cell-derived hepatic stellate cells. Stem Cell Research & Therapy. 15(223). doi: 10.1186/s13287-024-03852-8. Full text in Research Archive Show summary

  Background Hepatic stellate cells (HSC) have numerous critical roles in liver function and homeostasis, while they are also known for their importance during liver injury and fibrosis. There is therefore a need for relevant in vitro human HSC models to fill current knowledge gaps. In particular, the roles of vitamin A (VA), lipid droplets (LDs), and energy metabolism in human HSC activation are poorly understood. Methods In this study, human pluripotent stem cell-derived HSCs (scHSCs), benchmarked to human primary HSC, were exposed to 48-hour starvation of retinol (ROL) and palmitic acid (PA) in the presence or absence of the potent HSC activator TGF-β. The interventions were studied by an extensive set of phenotypic and functional analyses, including transcriptomic analysis, measurement of activation-related proteins and cytokines, VA- and LD storage, and cell energy metabolism. Results The results show that though the starvation of ROL and PA alone did not induce scHSC activation, the starvation amplified the TGF-β-induced activation-related transcriptome. However, TGF-β-induced activation alone did not lead to a reduction in VA or LD stores. Additionally, reduced glycolysis and increased mitochondrial fission were observed in response to TGF-β. Conclusions scHSCs are robust models for activation studies. The loss of VA and LDs is not sufficient for scHSC activation in vitro, but may amplify the TGF-β-induced activation response. Collectively, our work provides an extensive framework for studying human HSCs in healthy and diseased conditions.

• Aizenshtadt, Aleksandra; Wang, Chencheng; Abadpour, Shadab; Menezes, Pedro Duarte; Wilhelmsen, Ingrid & Dalmao-Fernandez, Andrea [Show all 15 contributors for this article] (2024). Pump-Less, Recirculating Organ-on-Chip (rOoC) Platform to Model the Metabolic Crosstalk between Islets and Liver. Advanced Healthcare Materials. ISSN 2192-2640. 13(13). doi: 10.1002/adhm.202303785. Full text in Research Archive Show summary

  Type 2 diabetes mellitus (T2DM), obesity, and metabolic dysfunction-associated steatotic liver disease (MASLD) are epidemiologically correlated disorders with a worldwide growing prevalence. While the mechanisms leading to the onset and development of these conditions are not fully understood, predictive tissue representations for studying the coordinated interactions between central organs that regulate energy metabolism, particularly the liver and pancreatic islets, are needed. Here, a dual pump-less recirculating organ-on-chip platform that combines human pluripotent stem cell (sc)-derived sc-liver and sc-islet organoids is presented. The platform reproduces key aspects of the metabolic cross-talk between both organs, including glucose levels and selected hormones, and supports the viability and functionality of both sc-islet and sc-liver organoids while preserving a reduced release of pro-inflammatory cytokines. In a model of metabolic disruption in response to treatment with high lipids and fructose, sc-liver organoids exhibit hallmarks of steatosis and insulin resistance, while sc-islets produce pro-inflammatory cytokines on-chip. Finally, the platform reproduces known effects of anti-diabetic drugs on-chip. Taken together, the platform provides a basis for functional studies of obesity, T2DM, and MASLD on-chip, as well as for testing potential therapeutic interventions.

• Wang, Chencheng; Abadpour, Shadab; Aizenshtadt, Aleksandra; Dalmao-Fernandez, Andrea; Høyem, Merete & Wilhelmsen, Ingrid [Show all 13 contributors for this article] (2024). Cell identity dynamics and insight into insulin secretagogues when employing stem cell-derived islets for disease modeling. Frontiers in Bioengineering and Biotechnology. 12. doi: 10.3389/fbioe.2024.1392575. Full text in Research Archive Show summary

  Stem cell-derived islets (SC-islets) are not only an unlimited source for cell-based therapy of type 1 diabetes but have also emerged as an attractive material for modeling diabetes and conducting screening for treatment options. Prior to SC-islets becoming the established standard for disease modeling and drug development, it is essential to understand their response to various nutrient sources in vitro. This study demonstrates an enhanced efficiency of pancreatic endocrine cell differentiation through the incorporation of WNT signaling inhibition following the definitive endoderm stage. We have identified a tri-hormonal cell population within SC-islets, which undergoes reduction concurrent with the emergence of elevated numbers of glucagon-positive cells during extended in vitro culture. Over a 6-week period of in vitro culture, the SC-islets consistently demonstrated robust insulin secretion in response to glucose stimulation. Moreover, they manifested diverse reactivity patterns when exposed to distinct nutrient sources and exhibited deviant glycolytic metabolic characteristics in comparison to human primary islets. Although the SC-islets demonstrated an aberrant glucose metabolism trafficking, the evaluation of a potential antidiabetic drug, pyruvate kinase agonist known as TEPP46, significantly improved in vitro insulin secretion of SC-islets. Overall, this study provided cell identity dynamics investigation of SC-islets during prolonged culturing in vitro, and insights into insulin secretagogues. Associated advantages and limitations were discussed when employing SC-islets for disease modeling.

• Wang, Chencheng; Abadpour, Shadab; Olsen, Petter Angell; Wang, Daxin; Stokowiec, Justyna & Chera, Simona [Show all 11 contributors for this article] (2024). Glucose Concentration in Regulating Induced Pluripotent Stem Cells Differentiation Toward Insulin-Producing Cells. Transplant International. ISSN 0934-0874. 37. doi: 10.3389/ti.2024.11900. Full text in Research Archive Show summary

  The generation of insulin-producing cells from human-induced pluripotent stem cells holds great potential for diabetes modeling and treatment. However, existing protocols typically involve incubating cells with un-physiologically high concentrations of glucose, which often fail to generate fully functional IPCs. Here, we investigated the influence of high (20 mM) versus low (5.5 mM) glucose concentrations on IPCs differentiation in three hiPSC lines. In two hiPSC lines that were unable to differentiate to IPCs sufficiently, we found that high glucose during differentiation leads to a shortage of NKX6.1+ cells that have co-expression with PDX1 due to insufficient NKX6.1 gene activation, thus further reducing differentiation efficiency. Furthermore, high glucose during differentiation weakened mitochondrial respiration ability. In the third iPSC line, which is IPC differentiation amenable, glucose concentrations did not affect the PDX1/NKX6.1 expression and differentiation efficiency. In addition, glucose-stimulated insulin secretion was only seen in the differentiation under a high glucose condition. These IPCs have higher KATP channel activity and were linked to sufficient ABCC8 gene expression under a high glucose condition. These data suggest high glucose concentration during IPC differentiation is necessary to generate functional IPCs. However, in cell lines that were IPC differentiation unamenable, high glucose could worsen the situation.

• LaLone, Vernon; Aizenshtadt, Aleksandra; Goertz, John; Skottvoll, Frøydis Sved; Mota, Marco Barbero & You, Junji [Show all 15 contributors for this article] (2023). Quantitative chemometric phenotyping of three-dimensional liver organoids by Raman spectral imaging. Cell Reports Methods. 3(4). doi: 10.1016/j.crmeth.2023.100440. Full text in Research Archive Show summary

  Confocal Raman spectral imaging (RSI) enables high-content, label-free visualization of a wide range of molecules in biological specimens without sample preparation. However, reliable quantification of the deconvoluted spectra is needed. Here we develop an integrated bioanalytical methodology, qRamanomics, to qualify RSI as a tissue phantom calibrated tool for quantitative spatial chemotyping of major classes of biomolecules. Next, we apply qRamanomics to fixed 3D liver organoids generated from stem-cell-derived or primary hepatocytes to assess specimen variation and maturity. We then demonstrate the utility of qRamanomics for identifying biomolecular response signatures from a panel of liver-altering drugs, probing drug-induced compositional changes in 3D organoids followed by in situ monitoring of drug metabolism and accumulation. Quantitative chemometric phenotyping constitutes an important step in developing quantitative label-free interrogation of 3D biological specimens.

• Olsen, Christine; Wang, Chencheng; Aizenshtadt, Aleksandra; Abadpour, Shadab; Lundanes, Elsa & Skottvoll, Frøydis Sved [Show all 11 contributors for this article] (2023). Simultaneous LC–MS determination of glucose regulatory peptides secreted by stem cell–derived islet organoids. Electrophoresis. ISSN 0173-0835. 44(21-22), p. 1682–1697. doi: 10.1002/elps.202300095. Full text in Research Archive Show summary

  Abstract For studying stem cell–derived islet organoids (SC‐islets) in an organ‐on‐chip (OoC) platform, we have developed a reversed‐phase liquid chromatography–tandem mass spectrometry (RPLC–MS/MS) method allowing for simultaneous determination of insulin, somatostatin‐14, and glucagon, with improved matrix robustness compared to earlier methodology. Combining phenyl/hexyl‐C18 separations using 2.1 mm inner diameter LC columns and triple quadrupole mass spectrometry, identification and quantification were secured with negligible variance in retention time and quantifier/qualifier ratios, negligible levels of carryover (<2%), and sufficient precision (±10% RSD) and accuracy (±15% relative error) with and without use of an internal standard. The obtained lower limits of quantification were 0.2 µg/L for human insulin, 0.1 µg/L for somatostatin‐14, and 0.05 µg/L for glucagon. The here‐developed RPLC–MS/MS method showed that the SC‐islets have an insulin response dependent on glucose concentration, and the SC‐islets produce and release somatostatin‐14 and glucagon. The RPLC–MS/MS method for these peptide hormones was compatible with an unfiltered offline sample collection from SC‐islets cultivated on a pumpless, recirculating OoC (rOoC) platform. The SC‐islets background secretion of insulin was not significantly different on the rOoC device compared to a standard cell culture well‐plate. Taken together, RPLC–MS/MS method is well suited for multi‐hormone measurements of SC‐islets on an OoC platform.

• Busek, Mathias; Aizenshtadt, Aleksandra; Koch, Timo; Frank, Anna; Delon, Ludivine Sylvie Claude & Martinez, Mikel Amirola [Show all 12 contributors for this article] (2023). Pump-less, recirculating organ-on-a-chip (rOoC) platform. Lab on a Chip. ISSN 1473-0197. doi: 10.1039/D2LC00919F. Full text in Research Archive
• Kømurcu, Kristina Sæterdal; Wilhelmsen, Ingrid; Thorne, James L.; Krauss, Stefan Johannes Karl; Wilson, Steven Ray Haakon & Aizenshtadt, Aleksandra [Show all 7 contributors for this article] (2023). Mass spectrometry reveals that oxysterols are secreted from non-alcoholic fatty liver disease induced organoids. Journal of Steroid Biochemistry and Molecular Biology. ISSN 0960-0760. 232. doi: 10.1016/j.jsbmb.2023.106355. Full text in Research Archive Show summary

  Oxysterols are potential biomarkers for liver metabolism that are altered under disease conditions such as non-alcoholic fatty liver disease (NAFLD). We here apply sterolomics to organoids used for disease modeling of NAFLD. Using liquid chromatography-mass spectrometry with on-line sample clean-up and enrichment, we establish that liver organoids produce and secrete oxysterols. We find elevated levels of 26-hydroxycholesterol, an LXR agonist and the first oxysterol in the acidic bile acid synthesis, in medium from steatotic liver organoids compared to untreated organoids. Other upregulated sterols in medium from steatotic liver organoids are dihydroxycholesterols, such as 7α,26–dihydroxycholesterol, and 7α,25-dihydroxycholesterol. Through 26-hydroxycholesterol exposure to human stem cell-derived hepatic stellate cells, we observe a trend of expressional downregulation of the pro-inflammatory cytokine CCL2, suggesting a protective role of 26-hydroxycholesterol during early-phased NAFLD disease development. Our findings support the possibility of oxysterols serving as NAFLD indicators, demonstrating the usefulness of combining organoids and mass spectrometry for disease modeling and biomarker studies.

• Wilhelmsen, Ingrid; Martinez, Mikel Amirola; Stokowiec, Justyna; Wang, Chencheng; Aizenshtadt, Aleksandra & Krauss, Stefan Johannes Karl (2023). Characterization of human stem cell-derived hepatic stellate cells and liver sinusoidal endothelial cells during extended in vitro culture. Frontiers in Bioengineering and Biotechnology. 11. doi: 10.3389/fbioe.2023.1223737. Full text in Research Archive Show summary

  Background: There is a significant need for predictive and stable in vitro human liver representations for disease modeling and drug testing. Hepatic stellate cells (HSCs) and liver sinusoidal endothelial cells (LSECs) are important non-parenchymal cell components of the liver and are hence of relevance in a variety of disease models, including hepatic fibrosis. Pluripotent stem cell- (PSC-) derived HSCs (scHSCs) and LSECs (scLSECs) offer an attractive alternative to primary human material; yet, the suitability of scHSCs and scLSECs for extended in vitro modeling has not been characterized. Methods: In this study, we describe the phenotypic and functional development of scHSCs and scLSECs during 14 days of 2D in vitro culture. Cell-specific phenotypes were evaluated by cell morphology, immunofluorescence, and gene- and protein expression. Functionality was assessed in scHSCs by their capacity for intracellular storage of vitamin A and response to pro-fibrotic stimuli induced by TGF-β. scLSECs were evaluated by nitric oxide- and factor VIII secretion as well as endocytic uptake of bioparticles and acetylated low-density lipoprotein. Notch pathway inhibition and co-culturing scHSCs and scLSECs were separately tested as options for enhancing long-term stability and maturation of the cells. Results and Conclusion: Both scHSCs and scLSECs exhibited a post-differentiation cell type-specific phenotype and functionality but deteriorated during extended culture with PSC line-dependent variability. Therefore, the choice of PSC line and experimental timeframe is crucial when designing in vitro platforms involving scHSCs and scLSECs. Notch inhibition modestly improved long-term monoculture in a cell line-dependent manner, while co-culturing scHSCs and scLSECs provides a strategy to enhance phenotypic and functional stability.

• Dalmao-Fernandez, Andrea; Aizenshtadt, Aleksandra; Bakke, Hege Gilbø; Krauss, Stefan Johannes Karl; Rustan, Arild Christian & Thoresen, G. Hege [Show all 7 contributors for this article] (2023). Development of three-dimensional primary human myospheres as culture model of skeletal muscle cells for metabolic studies. Frontiers in Bioengineering and Biotechnology. 11, p. 1–13. doi: 10.3389/fbioe.2023.1130693. Full text in Research Archive Show summary

  Introduction: Skeletal muscle is a major contributor to whole-body energy homeostasis and the utilization of fatty acids and glucose. At present, 2D cell models have been the most used cellular models to study skeletal muscle energy metabolism. However, the transferability of the results to in vivo might be limited. This project aimed to develop and characterize a skeletal muscle 3D cell model (myospheres) as an easy and low-cost tool to study molecular mechanisms of energy metabolism. Methods and results: We demonstrated that human primary myoblasts form myospheres without external matrix support and carry structural and molecular characteristics of mature skeletal muscle after 10 days of differentiation. We found significant metabolic differences between the 2D myotubes model and myospheres. In particular, myospheres showed increased lipid oxidative metabolism than the 2D myotubes model, which oxidized relatively more glucose and accumulated more oleic acid. Discussion and conclusion: These analyses demonstrate model differences that can have an impact and should be taken into consideration for studying energy metabolism and metabolic disorders in skeletal muscle.

• Brinch, Shoshy Alam; Amundsen-Isaksen, Enya; Espada, Sandra; Hammarström, Clara Louise; Aizenshtadt, Aleksandra & Olsen, Petter Angell [Show all 18 contributors for this article] (2022). The Tankyrase Inhibitor OM-153 Demonstrates Antitumor Efficacy and a Therapeutic Window in Mouse Models. Cancer Research Communications. 2(4), p. 233–245. doi: 10.1158/2767-9764.CRC-22-0027. Full text in Research Archive
• Kogler, Stian; Aizenshtadt, Aleksandra; Harrison, Sean; Skottvoll, Frøydis Sved; Berg, Henriette Engen & Abadpour, Shadab [Show all 14 contributors for this article] (2022). "Organ-in-a-Column" Coupled On-line with Liquid Chromatography-Mass Spectrometry. Analytical Chemistry. ISSN 0003-2700. 94(50), p. 17677–17684. doi: 10.1021/acs.analchem.2c04530. Full text in Research Archive Show summary

  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 “organ-in-a-column” 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 “on column” with heroin, followed by on-line monitoring of the drug’s phase 1 metabolism. Enzymatic metabolism of heroin produced in the “organ-in-a-column” 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.

• Busek, Mathias; Aizenshtadt, Aleksandra; Martinez, Mikel Amirola; Delon, Ludivine Sylvie Claude & Krauss, Stefan Johannes Karl (2022). Academic User View: Organ-on-a-Chip Technology. Biosensors. ISSN 2079-6374. 12(2). doi: 10.3390/bios12020126. Full text in Research Archive Show summary

  The pleasantness of gentle stroking (CT-targeted touch) varies highly between individuals and studies, indicating that relevant factors may not be accounted for. We propose that the affective value of a touch event is determined by how well its perceived purpose matches the goals of the touch receiver. The perceived purpose or meaning of touch is in turn informed by the sensory characteristics together with the setting, person factors, and the touchee’s expectations. Affective touch is often a sign of affection, intended to soothe or show support. In a typical lab study however, the toucher is a stranger and its purpose is research. The purpose of laboratory touch is nevertheless compatible with the goal of participants, namely to contribute to research. To fully understand how the perception of affective touch emerges, more studies should directly manipulate participants’ beliefs about the purpose of touch.

• Skottvoll, Frøydis Sved; Aizenshtadt, Aleksandra; Hansen, Frederik Andre; Martinez, Mikel Amirola; Andersen, Jannike Mørch & Bogen, Inger Lise [Show all 11 contributors for this article] (2022). Direct Electromembrane Extraction-Based Mass Spectrometry: A Tool for Studying Drug Metabolism Properties of Liver Organoids. Analysis & Sensing. 2(2). doi: 10.1002/anse.202100051. Full text in Research Archive Show summary

  This work introduces a strategy for organoid analysis - direct Electromembrane Extraction based Mass Spectrometry (dEME-MS) – 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 1 μM methadone, methadone metabolism was monitored from 0 to 24 hours (11 time points). All analytes had <0.4 % variance in retention times with >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.

• Busek, Mathias; Aizenshtadt, Aleksandra; Koch, Timo; Frank, Anna; Delon, Ludivine Sylvie Claude & Martinez, Mikel Amirola [Show all 12 contributors for this article] (2022). Pump-less, recirculating organ-on-a-chip (rOoC) platform. Lab on a Chip. ISSN 1473-0197. p. 345–361. doi: 10.1039/d2lc00919f. Full text in Research Archive Show summary

  We developed a novel, pump-less directional flow recirculating organ-on-a-chip (rOoC) platform that creates controlled unidirectional gravity-driven flow by a combination of a 3D-tilting system and an optimized microfluidic layout. The rOoC platform was assembled utilizing a layer-to-layer fabrication technology based on thermoplastic materials. It features two organoid compartments supported by two independent perfusion channels and separated by a hydrogel barrier. We developed a computational model to predict wall shear stress values and then measured the flow rate in the microfluidic channels with micro-Particle-Image-Velocimetry (μPIV). The suitability of the rOoC for functional culture of endothelial cells was tested using HUVECs seeded in the perfusion channels. HUVECs aligned in response to the directional flow, formed a barrier and were able to sprout into the organoid compartments. Next, we demonstrated the viability of human stem-cell derived liver organoids in the organoid compartments. Finally, we show the possibility to circulate immune cells in the microfluidic channels that retain viability without being trapped or activated. The rOoC platform allows growing and connecting of two or more tissue or organ representations on-chip with the possibility of applying gradients, endothelial barriers, microvasculature and circulating cells independent of external tubing and support systems.

• Skottvoll, Frøydis Sved; Hansen, Frederik; Harrison, Sean; Boger, Ida Caroline Sneis; Mrsa, Ago & Restan, Magnus Saed [Show all 14 contributors for this article] (2021). Electromembrane extraction and mass spectrometry for liver organoid drug metabolism studies. Analytical Chemistry. ISSN 0003-2700. 93, p. 3576–3585. doi: 10.1021/acs.analchem.0c05082. Full text in Research Archive Show summary

  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 μL 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.

• Busek, Mathias; Nøvik, Steffen; Aizenshtadt, Aleksandra; Martinez, Mikel Amirola; Combriat, Thomas Michel Daniel & Grünzner, Stefan [Show all 7 contributors for this article] (2021). Thermoplastic elastomer (Tpe)–poly(methyl methacrylate) (pmma) hybrid devices for active pumping pdms-free organ-on-a-chip systems. Biosensors. ISSN 2079-6374. 11(5). doi: 10.3390/bios11050162. Full text in Research Archive Show summary

  Polydimethylsiloxane (PDMS) has been used in microfluidic systems for years, as it can be easily structured and its flexibility makes it easy to integrate actuators including pneumatic pumps. In addition, the good optical properties of the material are well suited for analytical systems. In addition to its positive aspects, PDMS is well known to adsorb small molecules, which limits its usability when it comes to drug testing, e.g., in organ-on-a-chip (OoC) systems. Therefore, alternatives to PDMS are in high demand. In this study, we use thermoplastic elastomer (TPE) films thermally bonded to laser-cut poly(methyl methacrylate) (PMMA) sheets to build up multilayered microfluidic devices with integrated pneumatic micro-pumps. We present a low-cost manufacturing technology based on a conventional CO2 laser cutter for structuring, a spin-coating process for TPE film fabrication, and a thermal bonding process using a pneumatic hot-press. UV treatment with an Excimer lamp prior to bonding drastically improves the bonding process. Optimized bonding parameters were characterized by measuring the burst load upon applying pressure and via profilometer-based measurement of channel deformation. Next, flow and long-term stability of the chip layout were measured using microparticle Image Velocimetry (uPIV). Finally, human endothelial cells were seeded in the microchannels to check biocompatibility and flow-directed cell alignment. The presented device is compatible with a real-time live-cell analysis system.

• Mygland, Line; Mahmuda, Shoshy; Strand, Martin Frank; Olsen, Petter Angell; Aizenshtadt, Aleksandra & Lund, Kaja [Show all 21 contributors for this article] (2021). Identification of response signatures for tankyrase inhibitor treatment in tumor cell lines. iScience. 24(7). doi: 10.1016/j.isci.2021.102807. Full text in Research Archive Show summary

  Small-molecule tankyrase 1 and tankyrase 2 (TNKS1/2) inhibitors are effective antitumor agents in selected tumor cell lines and mouse models. Here, we characterized the response signatures and the in-depth mechanisms for the antiproliferative effect of tankyrase inhibition (TNKSi). The TNKS1/2-specific inhibitor G007-LK was used to screen 537 human tumor cell lines and a panel of particularly TNKSi-sensitive tumor cell lines was identified. Transcriptome, proteome, and bioinformatic analyses revealed the overall TNKSi-induced response signatures in the selected panel. TNKSi-mediated inhibition of wingless-type mammary tumor virus integration site/b-catenin, yes-associated protein 1 (YAP), and phosphatidylinositol-4,5-bisphosphate 3-kinase/AKT signaling was validated and correlated with lost expression of the key oncogene MYC and impaired cell growth. Moreover, we show that TNKSi induces accumulation of TNKS1/2-containing b-catenin degradasomes functioning as core complexes interacting with YAP and angiomotin proteins during attenuation of YAP signaling. These findings provide a contextual and mechanistic framework for using TNKSi in anticancer treatment that warrants further comprehensive preclinical and clinical evaluations.

• Waaler, Jo; Mygland, Line; Tveita, Anders Aune; Strand, Martin Frank; Solberg, Nina & Olsen, Petter Angell [Show all 19 contributors for this article] (2020). Tankyrase inhibition sensitizes melanoma to PD-1 immune checkpoint blockade in syngeneic mouse models. Communications Biology. 3. doi: 10.1038/s42003-020-0916-2. Full text in Research Archive Show summary

  The development of immune checkpoint inhibitors represents a major breakthrough in cancer therapy. Nevertheless, a substantial number of patients fail to respond to checkpoint pathway blockade. Evidence for WNT/β-catenin signaling-mediated immune evasion is found in a subset of cancers including melanoma. Currently, there are no therapeutic strategies available for targeting WNT/β-catenin signaling. Here we show that a specific small-molecule tankyrase inhibitor, G007-LK, decreases WNT/β-catenin and YAP signaling in the syngeneic murine B16-F10 and Clone M-3 melanoma models and sensitizes the tumors to anti-PD-1 immune checkpoint therapy. Mechanistically, we demonstrate that the synergistic effect of tankyrase and checkpoint inhibitor treatment is dependent on loss of β-catenin in the tumor cells, anti-PD-1-stimulated infiltration of T cells into the tumor and induction of an IFNγ- and CD8+ T cell-mediated anti-tumor immune response. Our study uncovers a combinatorial therapeutical strategy using tankyrase inhibition to overcome β-catenin-mediated resistance to immune checkpoint blockade in melanoma.

• Kogler, Stian; Harrison, Sean; Aizenshtadt, Aleksandra; Skottvoll, Frøydis Sved; Abadpour, Shadab & Krauss, Stefan [Show all 10 contributors for this article] (2020). “Organoid-in-a-column” coupled on-line with liquid chromatography-mass spectrometry. BioRxiv. ISSN 0362-4331. doi: 10.1101/2020.09.08.282756. Full text in Research Archive
• Abadpour, Shadab; Aizenshtadt, Aleksandra; Olsen, Petter Angell; Shoji, Kayoko; Wilson, Steven Ray Haakon & Krauss, Stefan [Show all 7 contributors for this article] (2020). Pancreas-on-a-Chip Technology for Transplantation Applications. Current Diabetes Reports. ISSN 1534-4827. 20. doi: 10.1007/s11892-020-01357-1. Full text in Research Archive Show summary

  Abstract Purpose of Review Human pancreas-on-a-chip (PoC) technology is quickly advancing as a platform for complex in vitro modeling of islet physiology. This review summarizes the current progress and evaluates the possibility of using this technology for clinical islet transplantation. Recent Findings PoC microfluidic platforms have mainly shown proof of principle for long-term culturing of islets to study islet function in a standardized format. Advancement in microfluidic design by using imaging-compatible biomaterials and biosensor technology might provide a novel future tool for predicting islet transplantation outcome. Progress in combining islets with other tissue types gives a possibility to study diabetic interventions in a minimal equivalent in vitro environment. Summary Although the field of PoC is still in its infancy, considerable progress in the development of functional systems has brought the technology on the verge of a general applicable tool that may be used to study islet quality and to replace animal testing in the development of diabetes interventions.

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• Kogler, Stian; Aizenshtadt, Aleksandra; Harrison, Sean Philip; Sullivan, Gareth John; Lundanes, Elsa & Bogen, Inger Lise [Show all 9 contributors for this article] (2023). Organ-in-a-column – automated on-line incubation, sampling, and LC-MS analysis for measurement of drug metabolites. Full text in Research Archive
• Røberg-Larsen, Hanne; Kømurcu, Kristina Sæterdal; Kogler, Stian; Olsen, Christine; Skottvoll, Frøydis Sved & Aizenshtadt, Aleksandra [Show all 8 contributors for this article] (2023). Organoids, Organ-on-a-chip and Mass Spectrometry. Full text in Research Archive
• Wilson, Steven Ray Haakon; Røberg-Larsen, Hanne; Krauss, Stefan Johannes Karl & Aizenshtadt, Aleksandra (2022). Organ-in-a-column - An automated on-line incubation and analysis system for drug metabolism studies. Full text in Research Archive
• Skottvoll, Frøydis Sved; Aizenshtadt, Aleksandra; Harrison, Sean; Krauss, Stefan Johannes Karl; Sullivan, Gareth & Bogen, Inger Lise [Show all 9 contributors for this article] (2022). Organ-in-a-column - On-line incubation and LC-MS-analysis for drug metabolism studies. Full text in Research Archive
• Kømurcu, Kristina Sæterdal; Aizenshtadt, Aleksandra; Thorne, James L; Krauss, Stefan Johannes Karl; Wilson, Steven Ray Haakon & Røberg-Larsen, Hanne (2022). Oxysterols are secreted from Non-alcoholic Fatty Liver disease induced organoids. Full text in Research Archive
• Kømurcu, Kristina Sæterdal; Aizenshtadt, Aleksandra; Thorne, James L; Wilson, Steven Ray Haakon & Røberg-Larsen, Hanne (2022). Measuring oxysterols using LC-MS for the understanding of non alcoholic fatty liver disease (NAFLD). Full text in Research Archive
• Kømurcu, Kristina Sæterdal; Aizenshtadt, Aleksandra; Thorne, James L; Krauss, Stefan Johannes Karl; Wilson, Steven Ray Haakon & Røberg-Larsen, Hanne (2022). Measuring oxysterols secreted from liver organoids by LC-MS for the understanding of Non-alcoholic fatty liver disease. Full text in Research Archive
• Kømurcu, Kristina Sæterdal; Aizenshtadt, Aleksandra; Thorne, James L.; Krauss, Stefan Johannes Karl; Wilson, Steven Ray Haakon & Røberg-Larsen, Hanne (2022). Oxysterols are secreted from Non-Alcoholic Fatty Liver Disease (NAFLD) induced organoids. Full text in Research Archive
• Kømurcu, Kristina Sæterdal; Aizenshtadt, Aleksandra; Thorne, James L; Krauss, Stefan Johannes Karl; Wilson, Steven Ray Haakon & Røberg-Larsen, Hanne (2022). Measuring oxysterols secreted from liver organoids by LC-MS for the understanding of Non-alcoholic fatty liver disease. Full text in Research Archive
• Nordli, Henriette; Aizenshtadt, Aleksandra; Krauss, Stefan Johannes Karl; Wilson, Steven Ray Haakon & Røberg-Larsen, Hanne (2021). Oxysterols secreted from a Non-Alcoholic Fatty Liver Disease (NAFLD) induced liver organoids. Full text in Research Archive

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