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.

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.

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.