Our focus is on type 1 diabetes research, emphasizing therapies that regulate the immune system and restore beta alpha cell functions. We have developed a unique study platform, using living human pancreas slices from organ donations, complementing isolated islet research. This approach enables detailed analysis of value cellular interactions and functions.
Our collaboration with a network for pancreatic organ donors offers community investigators access to pancreatic tissue sizes. However, culturing these sizes presents challenges as digestive enzymes can degrade the tissue and reduce viability. Therefore, there's a pressing need to establish culture conditions that maintain both, viability and a physiological environment for accurate functional studies.
Our protocol enables the collection of functional data on all cells within the pancreas, so investigators can use it for a variety of studies. Our own focus is on modeling type 1 diabetes and evaluating mechanisms of actions for drugs. A key question for me is to address alpha cell function and their role in type 1 diabetes.
Our protocol not only overcomes the survivor bias associated with isolated islets, but also uniquely facilitates the study of immune cells crucial in the context of type 1 diabetes. By studying whole pancreas tissue, we can assess function and responses that more closely reflect the in vivo environment. We're committed to sharing advancements in the pancreas slice platform with others.
Through Ampod, our goal is to encourage collaboration, enabling more effective addressing of critical questions by modeling disease mechanisms and testing potential therapeutic drugs, we aim to contribute significantly to the advancement on novel treatments. To begin, place the human pancreas tissue and baseline buffer under a stereo microscope, use forceps and scissors to gently remove the connective, fibrotic and adipose tissues. With the scissors, cut the tissue into multiple pieces with an area of approximately 0.5 cubic centimeters.
Then blot the pieces dry on tissue paper. Now transfer four pieces into a 35 millimeter Petri dish. Fill the dish with 3.8%agarose solution until all the pieces are fully submerged.
Once the agarose solution has solidified, cut out the tissue pieces. Run the scalpel along the edge of the dish to remove the agarose and carefully separate the tissue blocks. To slice the tissue pieces, glue the tissue blocks onto the metal plate of a vibratome.
Mount the plate onto the tray of the instrument, then fill the tray with baseline buffer. Set the vibratome to automated slicing at 120 micrometers and adjust the start and end positions. Now move the blade shortly above the tissue.
Start slicing at slow speeds with an amplitude of 0.8 millimeters. Use a small brush to collect the slices in baseline buffer containing aprotinin for one hour. Place the slices on an orbital shaker at slow speed to flush out the enzymes released during slicing.
To test the tissue viability, transfer a single tissue slice into a well of a 12 well plate filled with 990 microliters of baseline buffer. Next pipette fluorescein diacetate into the well. Add 10 microliters of propidium iodide diluted in 990 microliters of PBS to the tissue slices.
Then immerse the slices in PBS in another well for one minute. Finally, mount the slices on a glass slide with a cover slip for imaging. A viability of 80 to 90%was observed in the freshly sliced pancreatic tissue.
Lowered viability was observed at the cutting surface due to cell damage. Delayed sectioning resulted in decreased viability. To begin switch on the perfusion machine, launch the perfusion software, input the chamber count, then label the input solutions.
Now connect the inflow and outflow tubing of the perfusion machine. Next, place three agarose embedded human pancreatic tissue slices under a stereo microscope. Use a brush and scalpel to trim the agarose of the tissues down to a minimum.
Add a drop of baseline buffer onto the grid of a slice chamber. Gently transfer each slice onto each grid. Assemble the individual slice chamber parts from top to bottom.
To study isolated islets, use a minimum of 30 islets per column for insulin secretion, or 100 islets for glucagon detection and add islets to the chamber filled with baseline buffer. Screw the lid on top to close the islet chamber. Next, click on the Steps tab, input the number of rows, set the step time to 90 minutes and click Apply.
After adding the steps for all solutions, press the Overview tab to see the overview of the protocol. Press the Plate Map tab to view the visual plate representation. Begin priming the cells with the loading solutions.
Use the chamber heating and tray cooling pump during perfusion. Monitor the chambers for accurate flow and bubbles. Change the collection plates during protocol.
Store the trays at 4 degrees Celsius for same day quantification. After perfusion is complete, disassemble the chambers and gently invert each part into a dish containing baseline buffer to collect the slices. Transfer the slices into a tube for lysis or fixation.
Flush the machine with water, 10%bleach and a combination of water and air to clean it. Dilute the prepared calcium dye in 3 milliliters of baseline HEPES buffer solution in a 3 centimeter wide Petri dish, add aprotinin in the diluted dye solution. Then transfer a single slice into the solution.
Incubate the slice in the dark for 30 minutes to 1 hour on an orbital shaker at room temperature. In the meantime, connect the filled perfusion syringes and tubing. Switch on the instruments heater and adjust the pump flow at a flow rate of 0.5 milliliters per minute.
Now, gently place a single slice into the imaging chamber of the confocal microscope. Secure the slice with a harp. Connect the in and outflow tubing to the chamber, then start the pump.
Identify the imaging area under low magnification using Reflection, then switch to a higher magnification objective and adjust the position accordingly. Set the resolution to a minimum of 256 by 256, the imaging interval between 5-10 seconds and the Z-Stack range to 30-60 micrometers with an interval of 10 micrometers between planes. Begin imaging with solution switch as per the experiment.
Dynamic hormone secretion was observed in the viable slice resulting in robust insulin secretion. Delayed sectioning of poor tissue quality resulted in diminished or absent response to high glucose and membrane depolarization. A heat map generated by intensity changes during calcium imaging showed responding cells after switching to increased glucose concentration.
