Quantitative and Temporal Control of Oxygen Microenvironment at the Single Islet Level

Summary

Microfluidic oxygen control confers more than just convenience and speed over hypoxic chambers for biological experiments. Especially when implemented via diffusion through a membrane, microfluidic oxygen can provide simultaneous liquid and gas phase modulations at the microscale-level. This technique enables dynamic multi-parametric experiments critical for studying islet pathophysiology.

Abstract

Simultaneous oxygenation and monitoring of glucose stimulus-secretion coupling factors in a single technique is critical for modeling pathophysiological states of islet hypoxia, especially in transplant environments. Standard hypoxic chamber techniques cannot modulate both stimulations at the same time nor provide real-time monitoring of glucose stimulus-secretion coupling factors. To address these difficulties, we applied a multilayered microfluidic technique to integrate both aqueous and gas phase modulations via a diffusion membrane. This creates a stimulation sandwich around the microscaled islets within the transparent polydimethylsiloxane (PDMS) device, enabling monitoring of the aforementioned coupling factors via fluorescence microscopy. Additionally, the gas input is controlled by a pair of microdispensers, providing quantitative, sub-minute modulations of oxygen between 0-21%. This intermittent hypoxia is applied to investigate a new phenomenon of islet preconditioning. Moreover, armed with multimodal microscopy, we were able to look at detailed calcium and K_ATP channel dynamics during these hypoxic events. We envision microfluidic hypoxia, especially this simultaneous dual phase technique, as a valuable tool in studying islets as well as many ex vivo tissues.

Introduction

Dynamic hypoxia is important in biology, specifically for islet transplants

Dynamic hypoxia is an important physiological as well as pathophysiological parameter in many biological tissues. Change in oxygen, for example, is a potent developmental signal in angiogenesis. Moreover, spatial and temporal patterns in hypoxia modulate HIF1-alpha and play roles in diseases like pancreatic cancer. Hypoxia is also a confounding factor affecting islet transplant outcomes. Recently, temporally oscillations of hypoxia, or intermittent hypoxia (IH) have demonstrated benefits in "preconditioning" islets¹. However, both static and transien....

Protocol

1. Preparing the Mouse Islets

1. Dissect C57BL/6 mice and isolate islets by collagenase digestion and Ficoll density gradient separation. (Refer to JOVE articles referenced in^2,3).
2. Incubate islets in RPMI-1640 medium containing 10% FBS, 1% penicillin/streptomycin, and 20 mM HEPES in Petri dishes (37 °C, 5% CO₂). Post-isolation, culture islets for 24 hr prior to use in experiments. Use the islets within 1-2 days to ensure consistent results.

2. Making the Microfluidic Platform

1. Generate the microstructure geometries on transparency photomasks for each device layer: 1) inlet....

Results

Central to this islet hypoxia technique is the ability to modulate aqueous and gaseous phase stimulation in the same microfluidic chamber with minute-scale transients. Figure 1 is a representative result of the a) dual stimulations and b) fast modulations measured within the islet chamber. Aqueous modulation, shown by introduction of fluorescein into the chamber, achieves equilibrium in three to four minutes of mixing. Furthermore, oxygen can be stepped from 5-21% with fast transients, enabling cycling o.......

Discussion

The multiple modalities integrated in this islet hypoxia technique present several points noted here for troubleshooting. First the isolated islets continue to degrade and disintegrate in culture due to digestive enzymes from acinar cells. Standardizing experiments to 1-2 days after islet isolation is thus critical in obtaining consistent results. Second, the aqueous flow was stopped during hypoxia and intermittent hypoxia to prevent convective clearance at the boundary between laminar flow and diffusion. This seems to l.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Reagent/Material
Spinner	Laurell	WS-400
SU8	MicroChem	SU8-2150/SU8-2100
Digital Hotplate	PMC Dataplate	722A
UV Curing Lamp	OmniCure	S1000
PMDS	Dow Chemical	Sylgard 184
Corona Wand	ETP	BD-20AC
Vacuum Chamber	Bel-Art	420220000
Microdispensers	The Lee Company	IKTX0322000A
5 V and 20 V DC Power	Radio Shack
NI USB	National Instrument	NI USB-6501
Thermometer	Omega Engineering, Inc.
Peristaltic Pump	Gilson	Minipulse 2
Oxygen Sensor	Ocean Optics	NeoFox
Fraction Collector	Gilson	203
Pippette	Fisher Scientific	Finnpipette II 100μl
Inverted Epifluorescence Microscope	Leica	DMI 4000B
50 ml Conical Tubes	Fisher Scientific
Fura-2 Fluorescence Dye	Molecular Probes, Life Technologies
Rhodamine 123 Fluorescence Dye	Molecular Probes, Life Technologies
Culture Media	Sigma-Aldrich	RPMI-1640
HEPES	Sigma-Aldrich
Glucose	Sigma-Aldrich
Bovine Serum Albumin	Sigma-Aldrich
30 in Silicone Tubings	Cole-Parmer		1/16 in x 1/8 in
1.5 ml Eppendorf Tubes	Fisher Scientific
Y-connectors	Cole-Parmer		1/16 in and 4 mm
Syringe Connectors	Cole-Parmer		female Luer plug 1/16 in
Straight Connectors	Cole-Parmer		1/16 in
Elbow Connector	Cole-Parmer		1/16 in