Biology subjects

Aizenshtadt, A.

Publications and source records attributed to Aizenshtadt, A..

7 recordsLinked to original sources

bioRxiv2026

Modelling mechanisms and treatment of cholangiopathies with a bile duct on a chip

Background and aims Model systems for bile duct disorders are needed for testing therapeutic interventions. Current models have poor human relevance or limited potential for recreating the complex bile duct microenvironment at scale. We aimed to generate a humanized microphysiological system to model and treat cholangiopathies. Methods An in vitro bile duct was created using 3D printed microfluidic chips containing a collagen-embedded canal seeded with patient-derived primary human cholangiocytes. Barrier permeability and compound transport across the epithelium was measured, and disruption of the barrier was performed with lipopolysaccharide treatment. The duct was challenged with the known hepatotoxicant Chlorpromazine. Biliatresone was used to model biliary-atresia and treated using N-acetyl-L-cysteine. Results Cholangiocytes in the bile duct chip established a tight, polarized epithelial barrier. Verapamil and Linerixibat inhibited transport of rhodamine 123 and cholyl-lys-fluorescein respectively with 66 % (p = 0.0004) and 57 % (p = 0.03) reduction. 10 g/mL lipopolysaccharide led to a loss of epithelial barrier integrity, measured by an increase of over 1000 % in leakage of both 3 kDa (p = 0.0002) and 10 kDa dextran (p = 0.0001) along with upregulation of cytokines. Chlorpromazine displayed dose-dependent toxicity with EC50 values of 84, 140 and 96 M for three patient lines. Biliatresone induced a dose-dependent abnormal phenotype with loss of viability. The induced phenotype could be treated with N-acetyl-L-cysteine, improving viability from 23 % to 59 % (p < 0.0001) with treatment of 2 g/mL Biliatresone. Conclusions Our novel platform allows complex studies of bile duct biology, testing of off-target effects from drugs and treatment of a disease phenotype.

cell biology↗

bioRxiv2025

Uncovering the transcriptional hallmarks of endothelial cell aging via integrated single-cell analysis

Endothelial cells (ECs) are critical regulators of vascular function and exhibit specialized, organ-specific roles across tissues. During aging, these cells become dysfunctional, resulting in increased susceptibility to cardiovascular disease and its associated mortality. While single-cell transcriptomics studies have revealed extensive endothelial heterogeneity across tissues and conditions, a comprehensive atlas of human EC transcriptomes over the course of the adult human lifespan is still lacking. Here, we present the Human Aging Endothelial Cell Atlas (HAECA), a harmonized single-cell transcriptomic compendium of over 375,000 ECs from 12 human tissues throughout adulthood. Using HAECA, we identified age-associated transcriptional shifts, including a decline in angiogenic gene expression in venous ECs and widespread alterations in extracellular matrix (ECM)- and mechanotransduction-associated pathways. We validated these findings in aging human skin and further uncovered a p21-linked transcriptional program in ECs, confirmed in both in vitro and in vivo models and linked to cellular senescence. Together, our study provides a high-resolution transcriptome reference across spatial as well as temporal axes of the human endothelium.

cell biology↗

bioRxiv2023

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 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 carry-over (< 2%), and sufficient precision ({+/-} 10% RSD) and accuracy ({+/-} 15% relative error) with and without use of internal standard. 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 off-line sample collection from SC-islets cultivated on a pump-less, recirculating organ-on-chip (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 is well suited for multi-hormone measurements of SC-islets on an organ-on-chip platform.

biochemistry↗

bioRxiv2023

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 7,26-dihydroxycholesterol, and 7,25-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. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=118 SRC="FIGDIR/small/529551v2_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@9de7fdorg.highwire.dtl.DTLVardef@e9b328org.highwire.dtl.DTLVardef@f14101org.highwire.dtl.DTLVardef@e2e9c3_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2022

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

Current organ-on-a-chip (OoC) systems mimic important aspects of specific organ and tissue functions, however, many commercial and academic devices are either too simple for advanced assays or require a complicated support set-up including external driving systems such as pumps and tubing that hamper scalability and robustness. We have developed a novel, pump-less directional flow recirculating organ-on-a-chip (rOoC) platform that creates continuous or pulsed directional gravity-driven flow by a combination of a 3D-tilting system and an optimized microfluidic layout. The rOoC platform allows growing and connecting tissue or organ representations on-chip with the possibility of incorporating barrier functions, gradients, and circulating cells. Using the rOoC platform we demonstrate simple and reproducible endothelialisation, hepatic organoid integration, and the first steps of vascularization of 3D organ representations on-chip.

bioengineering↗

bioRxiv2020

Organoid-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 co-loaded 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 drugs 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.

bioengineering↗

bioRxiv2020

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. Multi-well EME (Parallel-EME) holding 100 L 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.

biochemistry↗