The overall goal of this procedure is to isolate intact islets of Langerhans from the neonatal mouse pancreas. This method can help answer the key questions in adding development and function. Such as insulin excretion, gene expression, para cell survival and functional maintenance.
The main advantage of this method is that it's simple and straight forward while still allowing new researchers to isolate a substantial number of intact islet for down strain studies. Begin by spraying a neonate in the supine position with 70%ethanol. Then, use tweezers to lift the skin over the abdomen and cut the skin and muscle layers longitudinally along the midline from the genitals to the ribcage.
Next, transfer all of the internal organs into a 100 centimeter dish and locate the pancreas, which is a white scattered organ with the ventral portion that clings to the duodenum and the dorsal portion that clings to the stomach and spleen. Add HBSS to dish to submerge the organs so that the pancreatic tissues no longer cling to the duodenum, stomach, or spleen. Then use a pair of tweezers to peel the pancreas away from surrounding tissues.
Transfer the pancreas into a new 60 millimeter dish containing HBSS and use scissors to cut one to five pancreata into five millimeter pieces or smaller. When all of the tissue has been minced, transfer the pieces into one point five milliliter microcentrifuge tubes and add collagenase working solutions to the samples. Then incubate the tissue fragments at 37 degrees celsius, inverting the tubes two times every minute.
After five minutes, tap the tube to monitor the digestion status of the pieces. Continue the digestion for another ten minutes, until the lysate is cloudy and most of the pancreata appear as fragmented cell clusters less than two millimeters in diameter. At the end of the digestion, pellet the lysate by microcentrifugation and use a p1000 pipette to carefully remove the supernatent, which should appear turbid but free of cell clusters.
Next, add one millileter of fresh RPMI 16/40 complete medium to the pellet. Then gently tap to resuspend the fragmented lysate pieces and invert the tube ten times. After centrifuging, invert the resuspended lysate at least two more times as just demonstrated until the supernatent is clear.
Then resuspend the final pellet in one milliliter of fresh complete medium and add two milliliters of polysucrose and sodium diatrazoate at a 1.077 g/ml density to a 15 milliliter conical tube on ice. Using a Pasteur pipette, carefully layer the lysate on top of the gradient solution without mixing the layers and separate the cells by centrifugation. Transfer the medium and the islet containing interphase layers into a new 15 milliliter tube and gently but thoroughly mix the islets with ten milliliters of fresh medium for centrifugation.
After a second wash and fresh complete medium, resuspend the islet and duct containing pellet in four milliliters of fresh complete medium. Now seed the islets onto a 60 millimeter dish and add five milliliters of complete medium to the cells. Transfer the dish under a dissecting microscope and turn on the bright field illumination adjusting the light intensity to approximately 50%of the maximum power.
At a 50x magnification, the islets will appear as slightly pink clusters and the acinar cells and dark, irregularly shaped clusters. Using a p20 pipette tip, harvest the individual islets, taking care to avoid the acinar clusters, dispensing the islet enriched fractions in a new 60 millimeter dish containing complete medium. When all of the islets have been picked from the original dish, harvest the islets from the collection dish into a new 60 millimeter dish containing four milliliters of fresh complete medium two more times, until only pure islets are present in the final collection dish.
A proper digestion and isolation will yield islets with smooth outer surfaces. Insufficient collagenase digestion can result in large, easily visualized islets that can not be completely separated from the acinar tissues, reducing the overall islet yield even though larger islets are produced. In the case of over digestion, although acinar tissues can be completely separated from the islets, islet structure is prone to be compromised resulting in rough surface islets observed under the microscope.
Neonatal islets isolated by this method display high basal insulin secretion levels with glucose stimulated insulin secretion profiles typical of immature islet beta cells, suggesting that this islet isolation procedure largely conserves the functional properties of neonatal islets. Once mastered, this technique can be finished within just two hours if performed properly. When attempting the procedure, it is important to remember to precentrifuge the collagenase in order to remove any insoluble debris and to calcium and magnesium supplemented washing buffer and to use pretreated washing buffer in order to stop the tissue dissociation quickly.
For in this procedure, our experiment, such as glucose stimulated insulin secretion can be performed to answer additional questions such as insulin secretion dynamics in neonatal beta cells. Also, this technique pave ways for researchers in the field of beta cell biology to do additional essays, such as gene expression or other metabolic studies. After watching this video, you should have a good understanding of how to isolate intact islets from neonatal mouse pancreas for your down strain studies.
Don't forget, working with collagenase powder will be extremely hazardous, so precautions such as wearing gloves and face mask should be always taken when using this material.
