A microfluidic islet perifusion device was developed for the assessment of dynamic insulin secretion of multiple islets and simultaneous fluorescence imaging of calcium influx and mitochondrial potential changes.
A. Microfabrication of 3-layer microfluidic perifusion device
Bottom Well Master Protocol (150 μm deep wells)
Microchannel Master Protocol (500 μm deep wells)
PDMS solution preparation
B. Experimental setup
Representative Results
Mouse islets were perifused with a linear gradient of 2-25 mM glucose. As shown in Figure 1, calcium influx and insulin secretion are triggered after about 13 minutes of perifusion, corresponding to 6 mM glucose. Changes in Mitochondrial potentials are seen earlier as expected, at about 11 minutes. This data demonstrates the advantage of using this microfluidic network to characterize islet physiology.
Figure 1. Physiological responses to islet cell stimulation.
Traditional islet perifusion systems (macroscale and microscale) have some limitations including complex of setup and design, high technical requirements, and difficulty to create user-prescribed chemical gradients in the system. The microfluidic perifusion system described here overcomes these limitations with simple geometry of design and fabrication. More important, this system can be integrated with fluorescence imaging approach that provide as a unique tool to study islet physiology. The system demonstrated with high signal-noise ratio and spatial-temporal resolution of these fluorescence signals. The current prototype device also can hold multiple perifusion setups in one chip and provide different types of microenvironments for widespread application purposes.
This work was supported by AAUW international fellowship to Adeola Adewola, NIH/NCRR (U42RR023245) to Jose Oberholzer, and The Chicago Diabetes Project.
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
60ml Syringes | BD | |||
Fura-2 fluorescence dye | Molecular Probes Inc. | |||
Rhodamine123 Fluorescence dye | Molecular Probes Inc. | |||
Glucose | Sigma | |||
Bovine Serum Albumin | Sigma | |||
30″ Silicone tubings | Cole Palmer | 1/16 x 1/8in | ||
1.5ml Eppendorf tubes | Fisher | |||
Y-connectors | Cole Palmer | 1/16” & 4mm | ||
Syringe connectors | Cole Palmer | female luer plug 1/16” | ||
Straight connectors | Cole Palmer | 1/16” | ||
Elbow connector | Cole Palmer | 1/16” | ||
Havard syringe pump | Harvard Apparatus | |||
Perifusion device | ||||
Hot plate | PMC | |||
Thermometer | Omega Engineering Inc. | |||
Fraction collector | Gibson | |||
Pippettor | Fisher | |||
Inverted epiflorescence microscope | Olympus | IX71 | ||
Charge-coupled device | QImaging | Retiga-SRV, Fast 1394 |