An FDA-Validated, Self-Cleaning Liquid Chromatography-Mass Spectrometry System for Determining Small-Molecule Drugs and Metabolites in Organoid/Organ-on-Chip Medium.

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.

The effects of TGF-ß-induced activation and starvation of vitamin A and palmitic acid on human stem cell-derived hepatic stellate cells.

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-ß. 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-ß-induced activation-related transcriptome. However, TGF-ß-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-ß. 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-ß-induced activation response. Collectively, our work provides an extensive framework for studying human HSCs in healthy and diseased conditions.

Cell identity dynamics and insight into insulin secretagogues when employing stem cell-derived islets for disease modeling.

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.

Glucose Concentration in Regulating Induced Pluripotent Stem Cells Differentiation Toward Insulin-Producing Cells.

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.

Development of a Smart Wireless Multisensor Platform for an Optogenetic Brain Implant.

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.

Pump-Less, Recirculating Organ-on-Chip (rOoC) Platform to Model the Metabolic Crosstalk between Islets and Liver.

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.

Preparative agarose gel electrophoresis for reducing matrix interferences of organoid cell medium prior to LC-MS analysis of insulin.

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.

Simultaneous LC-MS determination of glucose regulatory peptides secreted by stem cell-derived islet organoids.

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.

Characterization of human stem cell-derived hepatic stellate cells and liver sinusoidal endothelial cells during extended in vitro culture.

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-ß. 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.

Mass spectrometry reveals that oxysterols are secreted from non-alcoholic fatty liver disease induced organoids.

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.

Quantitative chemometric phenotyping of three-dimensional liver organoids by Raman spectral imaging.

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.

Development of three-dimensional primary human myospheres as culture model of skeletal muscle cells for metabolic studies.

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.

Pump-less, recirculating organ-on-a-chip (rOoC) platform.

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.

"Organ-in-a-Column" 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 "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.

Academic User View: Organ-on-a-Chip Technology.

Organ-on-a-Chip (OoC) systems bring together cell biology, engineering, and material science for creating systems that recapitulate the in vivo microenvironment of tissues and organs. The versatility of OoC systems enables in vitro models for studying physiological processes, drug development, and testing in both academia and industry. This paper evaluates current platforms from the academic end-user perspective, elaborating on usability, complexity, and robustness. We surveyed 187 peers in 35 countries and grouped the responses according to preliminary knowledge and the source of the OoC systems that are used. The survey clearly shows that current commercial OoC platforms provide a substantial level of robustness and usability-which is also indicated by an increasing adaptation of the pharmaceutical industry-but a lack of complexity can challenge their use as a predictive platform. Self-made systems, on the other hand, are less robust and standardized but provide the opportunity to develop customized and more complex models, which are often needed for human disease modeling. This perspective serves as a guide for researchers in the OoC field and encourages the development of next-generation OoCs.

The Tankyrase Inhibitor OM-153 Demonstrates Antitumor Efficacy and a Therapeutic Window in Mouse Models.

The catalytic enzymes tankyrase 1 and 2 (TNKS1/2) alter protein turnover by poly-ADP-ribosylating target proteins, which earmark them for degradation by the ubiquitin-proteasomal system. Prominent targets of the catalytic activity of TNKS1/2 include AXIN proteins, resulting in TNKS1/2 being attractive biotargets for addressing of oncogenic WNT/ß-catenin signaling. Although several potent small molecules have been developed to inhibit TNKS1/2, there are currently no TNKS1/2 inhibitors available in clinical practice. The development of tankyrase inhibitors has mainly been disadvantaged by concerns over biotarget-dependent intestinal toxicity and a deficient therapeutic window. Here we show that the novel, potent, and selective 1,2,4-triazole-based TNKS1/2 inhibitor OM-153 reduces WNT/ß-catenin signaling and tumor progression in COLO 320DM colon carcinoma xenografts upon oral administration of 0.33-10 mg/kg twice daily. In addition, OM-153 potentiates anti-programmed cell death protein 1 (anti-PD-1) immune checkpoint inhibition and antitumor effect in a B16-F10 mouse melanoma model. A 28-day repeated dose mouse toxicity study documents body weight loss, intestinal damage, and tubular damage in the kidney after oral-twice daily administration of 100 mg/kg. In contrast, mice treated oral-twice daily with 10 mg/kg show an intact intestinal architecture and no atypical histopathologic changes in other organs. In addition, clinical biochemistry and hematologic analyses do not identify changes indicating substantial toxicity. The results demonstrate OM-153-mediated antitumor effects and a therapeutic window in a colon carcinoma mouse model ranging from 0.33 to at least 10 mg/kg, and provide a framework for using OM-153 for further preclinical evaluations. Significance: This study uncovers the effectiveness and therapeutic window for a novel tankyrase inhibitor in mouse tumor models.

Identification of response signatures for tankyrase inhibitor treatment in tumor cell lines.

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/ß-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 ß-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.

Thermoplastic Elastomer (TPE)-Poly(Methyl Methacrylate) (PMMA) Hybrid Devices for Active Pumping PDMS-Free Organ-on-a-Chip Systems.

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.

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 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.

Pancreas-on-a-Chip Technology for Transplantation Applications.

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. 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.