Analysis of Non-Human Primate Pancreatic Islet Oxygen Consumption

Summary

This protocol demonstrates the accurate and reproducible measurement of oxygen consumption in non-human primate pancreatic islets. The islet loading techniques and coating of the microplate provide a framework for efficient measurement of respiration in other types of cultured spheroids.

Abstract

The measurement of oxygen consumption in spheroid clusters of cells, such as ex vivo pancreatic islets, has historically been challenging. We demonstrate the measurement of islet oxygen consumption using a 96-well microplate designed for the measurement of oxygen consumption in spheroids. In this assay, spheroid microplates are coated with a cell and tissue adhesive on the day prior to the assay. We utilize a small volume of adhesive solution to encourage islet adherence to only the bottom of the well. On the day of the assay, 15 islets are loaded directly into the base of each well using a technique that ensures optimal positioning of islets and accurate measurement of oxygen consumption. Various aspects of mitochondrial respiration are probed pharmacologically in non-human primate islets, including ATP-dependent respiration, maximal respiration, and proton leak. This method allows for consistent, reproducible results using only a small number of islets per well. It can theoretically be applied to any cultured spheroids of similar size.

Introduction

In order to maintain normal blood glucose levels, the pancreatic β cell must sense elevations in glucose and secrete insulin accordingly. The coupling of insulin secretion with glucose levels is directly linked to glucose metabolism and the production of ATP through mitochondrial oxidative phosphorylation. Thus, mitochondria play a critical role in stimulus-secretion coupling¹. Assessing β-cell mitochondrial function can reveal defects that lead to impaired insulin secretion. The secretion of glucagon by pancreatic α cells is also closely tied to mitochondrial function². Although immortalized islet cell li....

Protocol

1. Preparation of Microplate and Sensor Cartridge on the Day Prior to Running the Assay

Islets were isolated from three year old Japanese macaques as previously described¹³. This method is very similar to that used to isolate human islets from cadaver donors, but differs from mice, in which pancreata are often inflated with collagenase solution while the animal is under sedation and prior to organ removal. Islet retrieval was conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee (IACUC) of the Oregon National Primate Research Center (ONPRC) and Oregon Health and Science Universi....

Results

To load islets into microplate, 15 islets should be aspirated in 15 µL of media, as shown in Figure 1A. Islets will naturally settle toward the bottom of the pipet tip within a few seconds. Then, the pipet tip is lowered to the bottom of the well. The tip is very slightly lifted, and a small volume (about 5 µL) is pipetted out along with the islets. This technique results in consistent placement of islet at the bottom of the micropl.......

Discussion

The study of islet oxygen consumption has previously been hampered by the spherical shape of islets, their lack of adherence to culture surfaces, and the number of islets required per well. In this protocol, we highlight the efficacy of the 96-well spheroid microplate for measuring islet oxygen consumption on a small number of islets and demonstrate a technique for handling and loading islets which is technically feasible and produces consistent results.

In order for islets to adhere to the bo.......

Materials

Name	Company	Catalog Number	Comments
Cell culture dish, 60 mm X 15 mm style	Corning	430166
Cell-Tak Cell and Tissue Adhesive	Corning	354240
Conical tube, 50 mL	Falcon	352070
Dextrose anhydrous	Fisher Scientific	BP350-1	For glucose solution, 200 mg/ml, sterile filetered
Disposable reservoirs (sterile), 25 ML	Vistalab	3054-1033	for loading multichannel pipet
EZFlow Sterile 0.45 μm PES Syringe Filter, 13 mm	Foxx Life Sciences	371-3115-OEM
L-glutamine	Gibco	25030-081	200 mM (100x)
Multichannel pipette tips	ThermoFisher Scientific	94410810
Multichannel pipette, 15-1250 μL	ThermoFisher Scientific	4672100BT	Recommended
P20, P200, and P1000 pipettes	Eppendorf	2231000602
pH Probe	Hanna Instruments	HI2210-01
Pipette tips, 20 μL, 200 μL, 1000 μL	Olympus	24-404, 24-412, 24-430
Seahorse XF Base Media	Agilent	103334-100
Seahorse XF Cell Mito Stress Test Kit	Agilent	103015-100	Includes Oligomycin, FCCP, and Rotenone/Antimycin A
Seahorse XFe96 Analyzer	Agilent	S7800B	Including prep station with 37 °C non-CO2 incubator
Seahorse XFe96 Spheroid Fluxpak Mini	Agilent	102905-100	Includes sensor cartridge, spheroid microplate, and calibrant
Sodium bicarbonate	Fisher Scientific	BP328-500
Sodium pyruvate	Gibco	11360-070	100 mM (100x)
Stereo Microscope	Olympus	SZX9
Syringe (sterile), 5 mL	BD	309603	For sterile filtration
Water (sterile)	Sigma	W3500-500mL