Busek, M. | Search bioRxiv

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↗

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↗