Type of Document Dissertation Author Zhang, Xinyu Author's Email Address email@example.com URN etd-08042011-064658 Title Synchronization Of Islets Of Langerhans By Glucose Waveforms In Microfluidic Devices Degree Doctor of Philosophy Department Chemistry and Biochemistry, Department of Advisory Committee
Advisor Name Title Michael G. Roper Committee Chair André M. Striegel Committee Member John G. Dorsey Committee Member Qing-Xiang Sang Committee Member Richard Bertram University Representative Keywords
- Calcium Oscillations
- Islets Of Langerhans
Date of Defense 2011-06-29 Availability unrestricted AbstractThe work in this dissertation presents development of microfluidic devices for cellular perfusion with temporal gradients of glucose. Initially, a microfluidic perfusion system was developed for automated delivery of glucose to single islets. The 3-layer glass/polymer device contained two diaphragm pumps, a 12 cm mixing channel and a 0.2 ěL cell chamber. By altering the flow rate ratio of a stimulus pump and a diluent pump, a series of output concentrations were produced while a constant 1.5 + 0.1 ěL/min flow rate was maintained. The output concentrations were varied in time producing step gradients and other waveforms, such as sine and triangle waves, at different amplitudes and frequencies. Glucose in step gradients applied to islets between 3 and 20 mM brought fluorescence oscillations of [Ca2+] ([Ca2+]i) indicators. This application displayed that the system could be useful in future studies of cellular physiology.
The microfluidic system was improved to decrease broadening of sinusoidal glucose waveforms to entrain [Ca2+]i oscillations of single islets. Improvements to the microfluidic device raised the average pumping frequency of the on-chip diaphragm pumps from 0.83 Hz to 3.33 Hz by modifying the pneumatic valves to minimize adhesion between the polydimethylsiloxane (PDMS) and glass within the valve. This pumping frequency at 3.33 Hz allowed the cutoff frequency of the mixing channel, analyzed as a low pass filter, to increase from 0.026 to 0.061 Hz. Sinusoidal waves generated at 0.0033 Hz had low broadening due to the high cutoff frequency. Glucose in sinusoidal waves of 11 mM (median) ± 1 mM (amplitude) were delivered to single islets loaded with the Ca2+-sensitive fluorophore, indo-1. Entrainment of the islets was demonstrated by pacing the rhythm of indo-1 fluorescence 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 dynamic concentrations of stimuli.
To apply glucose waves to dynamic stimulation of multiple islets simultaneously, a cylindical chamber of 1 mm x 1 mm (diameter x height) was used to contain these islets. When a glucose wave traveled in the chamber, islets distant from the chamber inlet sensed the glucose waveform with considerable delay and amplitude attenuation compared to islets close to the inlet. To decrease the delay and amplitude attenuation of glucose waves, finite element analysis was employed to geometrically optimize the chamber. In a chamber optimized with 4 inlets, ~ 20 islets were simultaneously simulated by glucose waveforms and [Ca2+]i was imaged using Fura-2 fluorescence. All islets were entrained to a sinusoidal waveform of glucose (11 mM median, 1 mM amplitude, and a 5 min period) producing synchronized oscillations of Fura-2 fluorescence. The results were analyzed by a synchronization index and spectral analysis. These results indicated that an oscillatory glucose level synchronized the activity of a heterogeneous islet population, serving as preliminary evidence that islets could be synchronized in vivo through oscillatory glucose levels produced by a liver-pancreas feedback loop.
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