Islets perifusion with an automated multi-channel fraction collector OMNICOLL
Pancreatic islets are thought to play a key role in the pathophysiology of Type 1 and Type 2 diabetes through the failure of islet β-cells to secrete sufficient quantities of insulin to regulate blood glucose.
The definitive measure of β-cell quality in an islet is the measurement of β-cell function, i.e., the ability of the islets to release insulin in a controlled manner in response to minute changes in ambient glucose levels.
The use of perifusion systems to understand β-cell kinetics in vitro has been a key factor in the elucidation of islet physiology and pathophysiology. Islet perifusion involves the exposure of islets to continuous flow with nutrients, gas, or reagents. Several researchers published perifusion systems with the control of temperature, pH and flow rate of the perifusate.
Currently there is no standardized, inexpensive apparatus and protocol that can be replicated easily in any laboratory.
The islet perifusion set-up consists of four perifusion columns connected to four channel isamtec pumps, to pump 4 different perifusion solution into 4 parallel islets-containing columns simultaneously with the flow rate of 100 µL/min. Continuous flow or dynamic perifusion of the solution containing glucose and secretagogues through the islets is the most accurate assessment of regulated insulin release in vitro.
It is possible to use any third-party peristaltic pumps by the OMNICOLL fraction collector, if the pump can be controlled remotely by the analog signals.
The columns harboring the islets and the perifusion medium were kept at 37°C in a temperature controlled chamber. The islets were perifused for 10 – 15 min per stimulus to allow proper exposure of the islets and collection of their secretion product.
Perifusate (100 μL/min) from 4 columns were collected simultaneously every minute by an automated 4-channel fraction collector OMNICOLL into 96-well plates. The OMNICOLL 4-channel sample collector collects top 4 wells of the 96 well plate every minute (A, B, C & D - each representing a different treatment condition) to collect 12 minutes of fractions and then move to the bottom four rows (E, F, G & H rows) of the 96-well plate.
Other multi-channel customization of OMNICOLL fraction collector for 96-well plates (or other recipients) can be done.
The perifusate in the collecting 96-well plates were kept at <4°C to preserve the integrity of the analytes.
OMNICOLL fraction collector base (containing the fractions/samples) can be placed in a cold bath or any other thermo-stabilized container.
The collected samples were assayed for insulin, glucagon, or somatostatin concentration. Measurement of the different hormones can be done in different wells of the same 96 well plate.
In principle, 96 samples from multiple islet preparations with different experimental conditions can be measured simultaneously allowing for high throughput screening.
OTHER MULTI-CHANNEL CONFIGURATION WITH 96-WELL PLATES
LAMBDA Laboratory Instruments. April, 2017