Evaluation of islet function

The dGSIS of the islets, rodent and human, was conducted by connecting MPS to a PERI-4.2 perifusion system (Biorep Technologies, Miami Lakes, FL). Control islets in static culture were transferred into a fresh Acry-Chip, at appropriate time points, for dGSIS assessments to ensure consistency in stimulation profiles. Standard perifusion buffer containing KRBB buffer [115 mM NaCl₂, 4.7 mM KCl, 1.2 mM KH₂PO₄, 1.2 mM MgSO₄, 2.5 mM CaCl₂, 26 mM NaHCO₃, 0.2% (w/v) BSA, and 25 mM HEPES (pH 7.4)] with a selected glucose concentration (low, 3 mM; high, 11 mM) and/or KCl (25 mM) was perifused at a constant flow rate of 30 μl/min. Specifically, islets were first stabilized using a low glucose solution for 60 min and then stimulated with a sequence of 20-min low glucose, 30-min high glucose, 30-min low glucose, 10-min KCl, and 80-min low glucose media. Analytes were collected every 2 min from the outflow tubing and immediately stored at −80°C. Insulin concentrations were quantified using commercially available ELISA kits (Mercodia, Sweden). The values were first converted to micrograms per liter units. They were then converted to picograms per minute using the constant flow rate as listed above, normalized to the number of handpicked islets, and plotted as a function of time. The dead volume within the circulation tubing and islet MPS channels induced a lag of ~8 min. Hence, all dGSIS plots were denoted with 3G, 11G, and/or KCl to represent the stimulation times executed by the perifusion system with an inclusion of the calculated dead volume delay. AUC of an entire region of high glucose, beyond the denoted regions illustrated in the dGSIS plots, was calculated. The peak values were normalized to the corresponding average glucose baseline levels for AUC calculations. Notably, during serial assessments of the islet functionality, the dGSIS performed at earlier time points (i.e., 24, 48, and/or 72 hours) did not include the KCl stimulation step to preserve islet health.

Additional pancreatic hormone secretions were measured using a MILLIPLEX MAP rat metabolic hormone magnetic bead panel–metabolism multiplex assay (MilliporeSigma, Burlington, MA). The assay was performed by following the manufacturers’ recommended protocol with inclusion of proper standards and quality control samples. The antibody-immobilized bead cocktail contained analytes to capture glucagon, active GLP-1, C-peptide, and active amylin. The samples were ran on Luminex MAGPIX system (Luminex Corp., Austin, TX) and the median fluorescent intensity data were analyzed using a five-parameter logistic curve-fitting method to calculate analyte concentration in samples.