Microfluidic System for Generation of Sinusoidal Glucose Waveforms for Entrainment of Islets of Langerhans

Abstract

A microfluidic system was developed to produce sinusoidal waveforms of glucose to entrain oscillations of intracellular [Ca2+] in islets of Langerhans. The work described is an improvement to a previously reported device where two pneumatic pumps delivered pulses of glucose and buffer to a mixing channel. The mixing channel acted as a low pass filter to attenuate these pulses to produce the desired final concentration. Improvements to the current device included increasing the average pumping frequency from 0.83 Hz to 3.33 Hz by modifying the on-chip valves to minimize adhesion between the PDMS and glass within the valve. The cutoff frequency of the device was increased from 0.026 Hz to 0.061 Hz for sinusoidal fluorescein waves by shortening the length of the mixing channel to 3.3 cm. The value of the cutoff frequency was chosen between the average pumping frequency and the low frequency (∼0.0056 Hz) glucose waves that were needed to entrain islets of Langerhans. In this way, the pulses from the pumps were attenuated greatly, but the low frequency glucose waves were not. Using this microfluidic system, a total flow rate of 1.5 ± 0.1 μL min-1 was generated and used to deliver sinusoidal waves of glucose concentration with a median value of 11 mM and amplitude of 1 mM to a chamber that contained an islet of Langerhans loaded with the Ca2+-sensitive fluorophore, indo-1. Entrainment of the islets was demonstrated by pacing the rhythm of intracellular [Ca2+] oscillations to oscillatory glucose levels produced by the device. The system should be applicable to a wide range of cell types to aid in understanding cellular responses to dynamically changing stimuli.

Synchronization of mouse islets of Langerhans by glucose waveforms

“…Because glucose acts as a global signal to all islets within the pancreas, islets become entrained to the oscillatory glucose levels, resulting in synchronized insulin oscillations. We (27) and others (3,9,18,24) have demonstrated entrainment of islet responses to oscillatory glucose levels. However, there is a lack of quantitative evidence of groups of islets synchronizing based on this mechanism.…”

“…1. As described elsewhere (27,28), two pneumatic pumps were used to create temporal glucose oscillations in the mixing channel, which were delivered to an 860-nl chamber that held ϳ20 islets. The benefits of using this device for testing islet synchronization are that it is highly automated, allows complex waveforms to be generated, and allows facile imaging of [Ca 2ϩ ] i .…”

“…Islets of Langerhans were collected as described previously (27). For [Ca 2ϩ ]i monitoring, 1.0 l of 5.0 mM fura 2-AM in DMSO and 1.0 l Pluronic F-127 in DMSO were mixed and transferred into 2 ml of RPMI to form a final fura 2-AM concentration of 2.5 M. Each batch of islets was incubated in this solution at 37°C and 5% CO2 for 45 min.…”

“…The differences in the results may be due to the glucose profile applied (10-min glucose waveforms vs. 5-min waveforms) or islet preparation (whole islets vs. cell clusters). We have developed microfluidic devices that produce glucose profiles mimicking those found in vivo to explore islet synchronization (27,28). We have found that sinusoidal glucose waveforms can entrain [Ca 2ϩ ] i within single islets, even to complex gradient profiles that included phase shifts in the glucose waves (27).…”

Microfluidic platform for assessing pancreatic islet functionality through dielectric spectroscopy

“…Although mature and specific, these techniques lack dynamic assessment, are costly and time-consuming, and they involve multistep protocols that often result in error. 10 To remedy these limitations, many microfluidic platforms have been developed to assess islet functionality by coupling with techniques including zinc sampling, 11 insulin ELISA, 12 Ca 2þ , 13,14 and NADPH imaging. 15 Many of the challenges with dynamic islet functionality analysis using insulin ELISA still remain when coupling with microfluidic systems.…”