A Simple and Rapid Method for Simultaneous Isolation of Primary Islets and Primary Pancreatic Acinar Cells from Mice

This study developed a simplified method to efficiently extract functional primary islets and acinar cells from the mouse pancreas offering a valuable tool for studying intracellular communication in the pathogenesis of acute pancreatitis induced diabetes PPDMA. Current methods for isolating primary islets from mice mainly rely on bile duct cannulation. This technique requires precise localization and cannulation of the bile duct, followed by in vivo perfusion of the pancreatic parenchyma collagenase P solution.

It is quite difficult to perform without specialized training, achieving effective and efficient pancreatic perfusion is challenging. This video prevents a simple and rapid method for isolating primary mount islets suitable for researchers with no perfusion experience. The method also enables the simultaneous acquisition of primary pancreatic acinar cells, yielding sufficient quantity of high quality islets and acinar cells.

It allows researchers to conduct experiment within the same pathologic and physiologic context, thereby facilitating in depth analysis of the interaction mechanism between islets and the acinar cells. Isolation of pancreatic islets and pancreatic acinar cells PACs. Add Hank's balanced salt solution and collagenase P solution to the 12-well plate and add three milliliter of collagenase P solution to a 50 milliliter centrifuge tube for subsequent digestion.

Anesthetize the mouse with 1.25%tribromoethanol before surgery followed by euthanasia. Make a V-shaped abdominal incision to open the mouse's abdominal cavity. The dark red lymphoid organ in the left hypochondriac region is the spleen, and the white organ attached to the spleen is the pancreas.

Carefully perform blunt dissection of the pancreas along the lower edge of the stomach and the pancreaticoduodenal junction. Wash the isolated pancreatic tissue in Hank's balanced salt solution and remove residual mesenteric attachments, the spleen, and peripancreatic adipose tissue. Move the pre-processed pancreas to a 12-well plate pre added with two milliliter of collagenase P solution.

Hold the pancreas in place with tweezers using the left hand and use a one milliliter syringe with the right hand to inject collagenase P solution into the pancreatic parenchyma until the pancreatic tissue appears translucent and edematous. Cut the pancreas into one to two millimeters cubed off tissue pieces quickly with surgical scissors. Cut off the distal one to 1.5 centimeters of a one milliliter pipette tip to enlarge the opening, and use this modified tip to transfer the pancreatic tissue pieces together with two milliliter of collagenase P solution into a 50 milliliter centrifuge tube preloaded with three milliliter of collagenase P solution.

Incubate the centrifuge tube in a 37 degrees Celsius water bath for 12 minutes, gently shaking the tube every five to six minutes during this period. Add 10 milliliter of RPMI 1640 complete medium to terminate the digestive effect of collagenase P solution. Pipette the pellet repeatedly with a five milliliters Pasteur pipette, 15 to 20 up and down strokes, until no obvious large tissue clumps remain.

Add 10 milliliters of RPMI 1640 complete medium. Then centrifuge at 180 times G for two minutes at four degrees Celsius. And discard the supernatant Resuspend the pellet with 20 milliliters of RPMI 1640 complete medium.

Filter the suspension through a 40-mesh sieve. Then centrifuge at 180 times G for two minutes at four degrees Celsius. Discard the supernatant gently.

Add 20 milliliters of Ficoll solution to resuspend the pellet. Then slowly add 15 milliliters of RPMI 1640 complete medium. At this point, a clear liquid interface can be observed.

Centrifuge gently at 640 times G for 20 minutes at 25 degrees Celsius. Carefully remove the centrifuge tube, and aspirate the upper red medium layer using a five milliliters Pasteur pipette. Then, carefully aspirate the liquid containing islets at the liquid layer interface and transfer it to a new 50 milliliters centrifuge tube.

Add 20 milliliters of RPMI 1640 complete medium. Centrifuge at 180 times G for two minutes at four degrees Celsius, and discard the supernatant. Resuspend the pellet with 20 milliliters of RPMI 1640 complete medium.

Centrifuge at 180 times G for two minutes at four degrees Celsius, and discard the supernatant. Resuspend the pellet with 10 milliliters of RPMI 1640 complete medium. And transfer it to a 60 millimeter cell culture dish.

Under an inverted biological microscope 100 times magnification, manually pick islets using a 20 microliter pipette. Transfer the selected islets to a 24-well cell culture plate preloaded with 500 microliters of RPMI 1640 complete medium per well. Discard the Ficoll solution layer.

Resuspend the pellet with 10 milliliters of DMEM complete medium. Filter through a 100 micrometer cell strainer. Centrifuge at 180 times G for two minutes at four degrees Celsius.

And discard the supernatant. Resuspend the pellet with 10 milliliters of DMEM complete medium. Centrifuge at 180 times G for two minutes at four degrees Celsius and discard the supernatant.

Then, resuspend the pellet with five milliliters of DMEM complete medium for subsequent experiments. After Ficoll solution density gradient centrifugation, islets were observed near the interface between the transparent colorless liquid layer and the medium with packs present as sediment at the bottom of the tube. Islets were usually round oval, golden brown, with a stable yield of 120 plus or minus five per mouse, Figure A and Figure B.Freshly isolated PACs were spherical and clustered.

Acinar cells had darker apical ends with visible zymogen granules and the yield was 1.6 to 1.95 times 10 to the seventh power cells per mouse, Figure C and Figure D.Islet and acinar cell clacein PI staining show most cells stained green calcein, live, and few red PI, dead, Figure A.Image J-based quantitative analysis reveal isolated islets and acinar cells have viability rates of 97.52 plus or minus 0.16%and 96.55 plus or minus 0.95%respectively, Figure B.Isolated pancreatic acinar cells, a basal amylase activity of 0.79 plus or minus 0.01 units per milliliter. After stimulation with 10 nano molar, 20 nano molar and 50 nano molar caerulein, their amylase activities were 1.45 plus or minus 0.03 units per milliliter, 1.65 plus or minus 0.05 units per milliliter, and 1.39 plus or minus 0.02 units per milliliter respectively. One-way ANOVA showed all caerulein groups had significantly different amylase activity versus the control group, all P value less than 0.001.

Additionally, the 20 nano molar group differed significantly from the 10 nano molar group, P value less than 0.001, figure A.Isolated islets at insulin secretion of 0.27 plus or minus 0.04 nanograms per milliliter per islet per hour when stimulated with 5.6 millimolar glucose and 0.94 plus or minus 0.04 nanograms per milliliter per islet per hour with 22 millimolar glucose, GSI equals 3.44, Figure B.This study established a method for simultaneous isolation of primary mouse islets and pancreatic acinar cells without complex in vivo perfusion. With a long technical barrier, the method is easy to operate, yielding 120 plus or minus 5 islets and 1.6 to 1.95 times 10 to the seventh power acinar cells per mouse with both cell types showing a viability rate of over 96%The isolated islets exhibit normal glucose-stimulated insulin secretion and the acinar cells are sensitive to caerulein stimulation. This confirms that the cells obtained via this method have intact functions, making them suitable for studies on pancreatic exocrine and endocrine interactions.

Although, the method is limited by the need for basic mouse anatomy knowledge, the requirement for reagent dosage adjustments, restrictions on the number of mice processed simultaneously, and the unvalidated application risk. It still provides a reliable cell isolation protocol for researchers lacking perfusion experiences.