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

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.

An LC-MS/MS Method for the Simultaneous Quantification of Insulin, Cortisol, Glucagon-like Peptide 1, Ghrelin, and Osteocalcin

“…Regardless, across replicates of all three QC levels with seven hormones, more than 76.2% of the measured concentrations had a % error within 20% of the nominal values, and more than 85.7% of the QC samples had a % error within 25% of the nominal values, which is within common LC-MS protein assay accuracy standards [18]. The challenges of achieving high accuracy for multiple peptide hormones using MS have been previously reported [19]. The lower accuracy in low-concentration QC samples (near the lower limit of quantification) compared to medium and high QC samples has also been reported in MS peptide and protein quantification [20].…”

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

“…[104] Nevertheless, rOoC chips show, for example, some insulin absorption (29%) as recently confirmed by mass spectroscopic measurements. [49] Possible causes for this unspecific binding might be the employed membrane and the used ECM material in the OCs rather than the chip surface itself. However, adsorption to the chip should be further investigated.…”

“…Although tissue models based on human donor material still represent the gold standard for functional and/or metabolic studies, hPSC-derived organoids start to resemble adult functionality and metabolic features thus rendering them suitable for disease modeling while offering better scalability and long-term stability in culture. [105] Throughout a series of improvements, we and others have obtained pluripotent stem cell-derived sc-islets [49,106,107] and sc-liver organoids [54,62,[108][109][110] that approach adult metabolic Figure 7. Anti-diabetic drug testing on-chip.…”

“…To establish an in vitro model of the islets-liver metabolic axis on the dual-rOoC chip platform, human embryonic stem cells (hESC; line H1) were differentiated to sc-islet and sc-liver organoids using modified lineage-specific differentiation protocols (Figures 3a and 4a). Sc-islet organoids were generated according to a previously published 6-stage differentiation protocol [41,49] (Figure 3a) as described in the Experimental Section. Gene expression analysis of the sc-islets (Figure 3b) demonstrated the expression of key signature genes for 𝛼-𝛽and 𝛾-cells in the organoids.…”

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

“…A Page Ruler protein prestained ladder (Invitrogen, Waltham, PA, USA) was used to identify the molecular weight of proteins and to track the progress of the separation. The full SDS-PAGE procedure was performed according to Olsen et al 8 Liver Organoids and Organ-on-Chips. Liver organoids were generated from human-induced pluripotent stem cells (H1 ESC cell line, WiCell, HMGUi001-A, HMGU, and WTC-11, Coriell Institute for Medical Research) as previously described.…”