Integrated perfusion and separation systems for entrainment of insulin secretion from islets of Langerhans

Abstract

A microfluidic system was developed to investigate the entrainment of insulin secretion from islets of Langerhans to oscillatory glucose levels. A gravity-driven perfusion system was integrated with a microfluidic system to deliver sinusoidal glucose waveforms to the islet chamber. Automated manipulation of the height of the perfusion syringes allowed precise control of the ratio of two perfusion solutions into a chamber containing 1 – 10 islets. Insulin levels in the perfusate were measured using an online competitive electrophoretic immunoassay with a sampling period of 10 s. The insulin immunoassay had a detection limit of 3 nM with RSDs of calibration points ranging from 2 – 8%. At 11 mM glucose, insulin secretion from single islets was oscillatory with a period ranging from 3 – 6 min. Application of a small amplitude sinusoidal wave of glucose with a period of 5 or 10 min, shifted the period of the insulin oscillations to this forcing period. Exposing groups of 6 – 10 islets to a sinusoidal glucose wave synchronized their behavior, producing a coherent pulsatile insulin response from the population. These results demonstrate the feasibility of the developed system for the study of oscillatory insulin secretion and can be easily modified for investigating the dynamic nature of other hormones released from different cell types.

The Pancreatic β-Cell: A Bioenergetic Perspective

“…As discussed above, single islet insulin secretion monitored in a microfluidic device showed high-amplitude oscillations with a period of ϳ5 min in the presence of constant elevated glucose (550). Remarkably, the periodicity could be entrained by imposing small sinusoidal oscillations in applied glucose with a period different from the "natural" cycle time in constant glucose (FIGURE 9B) (124,550).…”

“…The pulsatility of insulin secretion can thus be accounted for by the periodic activation of VACCs. However, one puzzling feature, at least to the author, is an apparent disconnect between the periodicity of insulin secretion from single islets, which at roughly 5 min is reported to be rather insensitive to glucose concentration (43,107,550), and that of bursting, the frequency of which can be glucose-dependent and continuous in the presence of very high glucose (47, 160,555). Burst termination has been ascribed to the activation of slow Ca 2ϩ -activated Ca 2ϩ channels as [Ca 2ϩ ] c accumulated during a burst (18,131,552).…”

“…In response to elevated glucose, insulin release detected in the portal vein oscillates with a period of 5-6 min (488). While no single mechanism appears to account for all aspects of the oscillatory entrainment of the entire population of islets in the pancreas in the absence of physical contact, a plausible mechanism involves a negative-feedback loop involving the liver, with some combination of insulin-mediated hepatic glucose uptake and/or glucose release during peaks and troughs, respectively, of insulin secretion (516,550). The model is not without problems, notably in the ability of the perfused pancreas to maintain insulin secretory oscillations, suggesting the existence of a hypothetical intrapancreatic pacemaker (494).…”

“…In the presence of constant elevated glucose, islets retain an autonomous oscillator controlling insulin secretion with a typical periodicity of 2-6 min (43, 107,550). A recent representative study employed a microfluidic device to monitor insulin secretion from single isolated mouse islets following a 3-11 mM step increase in glucose (FIGURE 9A) (550). The study was interesting in that insulin secretion followed the classic time course of first-and second-phase release by varying the amplitude, rather than the frequency, of the individual pulses (FIGURE 2B) (see also Ref.…”

The quest to make fully functional human pancreatic beta cells from embryonic stem cells: climbing a mountain in the clouds

“…Tools are available for optical imaging of voltage responses, which enables high-throughput analyses of thousands of cells and will therefore likely supplant traditional electrophysiology for the characterisation of in vitro derived cells. Highthroughput electrophysiology and lab-on-a-chip technologies will be particularly useful in both understanding the phenotypic range of human islet cells and assessing their in vitro differentiated counterparts side by side [58][59][60]. The islet biologists and mathematical modellers who are most familiar with these aspects of beta cell behaviour should be engaged in studies of in vitro differentiated cells to dissect the properties of all of the major ion currents, the shape of the action potentials and the distinctive bursting patterns that are normally seen in healthy beta cells (Fig.…”