Pancreas cells are sensitive target for ROS induced damages, which is associated with development of type-2 diabetes. A pancreas cell-based assays will help identify ROS inducing chemicals. The main advantage of this technique is to use isolated pancreas islet cells for simple and effective identification of the ROS inducing chemicals which may have the added diabetogenic potentials.
This method can also be used for an in vitro analysis on the effects of chemicals on a physiological function of the pancreas such as a glucose-stimulated insulin secretion. Use a pair of curved forceps to clamp the duodenum close to the position of the sphincter of Oddi. With another pair of curved forceps, clamp the duodenum wall to block the bile pathway.
Find the common bile duct and with a 30 gauge needle and a five milliliter syringe, slowly inject three milliliters of collagenase solution. Then, reposition the mouse with the head proximal and the feet distal. Ensure that the common bile duct and pancreas are inflated after the injection.
Using the curved forceps and fine scissors, remove the distended pancreas to separate it from the descending colon, intestines, stomach and spleen. Then, place the organ in a 50 milliliter tube containing three milliliters of collagenase solution and keep it on ice. Place each 50 milliliter tube into a 37 degrees Celsius water bath and incubate them for 15 to 20 minutes.
Vortex the tubes, then pool four to five digested organs in a large sterile sink strainer and use a scoop to thoroughly break apart the pancreas. With a 23 gauge needle and ten milliliter syringe containing wash solution, vigorously pipet the digested cells off the screen. Then, collect the wash solution in a 50 milliliter tube.
Centrifuge the tubes at 97g and four degrees Celsius for one minute and decant the supernatant. Add 25 milliliters of wash solution to the tubes, and resuspend the tissue by gently vortexing. Then, spin down the tissue, and decant the supernatant.
Repeat the wash three times, taking care not to dislodge the pelleted tissue. To purify the islets, add ten milliliters of purification solution to the washed tissue pellet and gently resuspend the contents. Gently overlay five milliliters of isolation solution onto the purification solution.
Ensure that a sharp liquid interface is maintained between the two solutions. Centrifuge the tube at 560g at four degrees Celsius for 15 minutes, with slow acceleration and no break. Then, insert the pipet into the interface and collect the islet layer.
Transfer the solution into new 50 milliliter tubes before washing the islet as just demonstrated. After removing the final wash, add ten milliliters of islet culture solution and gently and thoroughly mix to resuspend the islets. Then, using a multi-channel pipet transfer 100 microliters per well to the wells of a 96 well plate.
Incubate the plate in a tissue culture incubator for 12 hours. Then, centrifuge the plate at 112g and remove the culture medium. Dilute the environmental chemicals in islet culture medium.
Add 100 micro liters of each of the chemical diluents into separate wells of the plate. Resuspend the islets in each well and incubate the plate at 37 degrees Celsius for 12 hours. Following chemical treatment of the islets, use 1x PBS to wash the samples.
Then, add five millimolar N-Acetyl Cysteine or NAC to the wells, as an antioxidant to quench ROS and incubate the plates at 37 degrees Celsius for 12 hours. Centrifuge the 96 well plate at 112g then, add five micromolar solution of fluorescent probe and incubate the plate at 37 degrees Celsius for 60 minutes. Use PBS to wash the treated islet cells three times and use a microplate reader at 480 nanometers to measure the fluorescence.
Finally, perform the glucose-stimulated insulin secretion, or GSIS assay, on the ROS inducing xenobiotic treated and untreated islets, to measure the xenobiotic effects on the physiological function of the pancreatic islets. Shown here, are healthy pancreatic islets, isolated from C57 Black/6 mice, that are round or oval in shape and are relatively uniformed in size. In this GSIS assay, using isolated islets stimulated with glucose to secrete insulin, a dose-dependent response was observed.
When isolated islets were treated with the xenobiotics listed here, several chemicals including Atrazine, BaP, BPA and PCB caused a significant induction of reactive oxygen species. However, the addition of NAC was able to quench the production of ROS by the chemical treatments. In addition, pancreatic islets pretreated with PCB126 and BPA showed a significant decrease in insulin secretion compared to the control group, confirming the important role of ROS in causing damage to the physiological function of pancreatic beta cells which lead to T2DM.
Once mastered, the isolation of the pancreas islets can be done in two hours if it is performed properly. While attempting this procedure it's important to use gradient centrifugation to purify the islets away from the other pancreatic debris. Following this procedure, other methods like glucose-stimulated insulin secretion can be performed in order to answer additional questions like effects of the chemicals on the physiological functions of the pancreas islets.
After its development, this technique paved the way for researchers in the field of toxicology to explore diabetogenic potentials of the environmental chemicals in isolated mouse pancreas islet cells. After watching this video, you should have a good understanding of how to isolate the mouse pancreas islets and use these islet cells for identification.
