A Multi-Parametric Islet Perifusion System within a Microfluidic Perifusion Device

Summary

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.

Abstract

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. The device consists of three layers: first layer contains an array of microscale wells (500 μm diameter and 150 μm depth) that help to immobilize the islets while exposed to flow and maximize the exposed surface area of the islets; the second layer contains a circular perifusion chamber (3 mm deep, 7 mm diameter); and the third layer contains an inlet-mixing channel that fans out before injection into the perifusion chamber (2 mm in width, 19 mm in length, and 500 μm in height) for optimizing the mixing efficiency prior to entering the perifusion chamber. The creation of various glucose gradients including a linear, bell shape, and square shapes also can be created in the microfluidic perifusion network and is demonstrated.

Protocol

A. Microfabrication of 3-layer microfluidic perifusion device Bottom Well Master Protocol (150 μm deep wells) Clean the wafer using a razor blade if needed. Clean with Acetone, Methanol, and IPA. Perform plasma treatment at 50 Watts for 30 s. Spin SU8-100 @ 2000 rpm. [Step 1: 500 rpm, 10 s, 100 rpm/s, Step 2: 2000 rpm, 30 s, 300 rpm/s].[NOTE: do not hold the wafer with tweezers after spinning SU8] Soft bake the wafer at 65 °C for 20 min and at…

Discussion

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 hig…

Materials

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