Single cell glucose-stimulated insulin secretion assay using nanowell-in-microwell plates.

Abstract

Pancreatic β cells secrete insulin in response to elevated levels of glucose. Stem cell derived β (SCβ) cells aim to replicate this glucose-stimulated insulin secretion (GSIS) function, but current preparations cannot provide the same level of insulin as natural β cells. Here, we develop an assay to measure GSIS at the single cell level to investigate the functional heterogeneity of SCβ cells and donor-derived islet cells. Our assay involves randomly depositing single cells and insulin capture microbeads in open-top nanowells (40 × 40 × 55 μm3) fabricated on glass-bottom imaging microwell plates. Insulin secreted from single cells is captured on microbeads and then stained using a detection antibody. The nanowell microstructure limits diffusion of secreted insulin. The glass substrate provides an optically flat surface for quantitative microscopy to measure the concentration of secreted insulin. We used this approach to measure GSIS from SCβ cells and donor-derived islet cells after 15 minutes exposure to 3.3 mM and 16.7 mM glucose. Both cell types exhibited significant GSIS heterogeneity, where elite cells (<20%) produced the majority of the secreted insulin (55-78%). This assay provides an immediate readout of single cell glucose-stimulated insulin secretion in a flexible well plate-based format.