This procedure begins by obtaining normal donor pancreas tissue. After a brief trimming step, the distended pancreas is cut into even pieces and moved into the digestion chamber. The pancreas is cannulated and collagenase solution is infused through the cannula by an automated perfusion machine.
Finally, pancreatic tissue is collected in large volume, conical tubes, and ultimately purified further for eyelet transplantation, which will be shown in a subsequent video. Hi, I'm American Chief from the Lab of Human Eyelet Program in the Department of Survey at the University of Illinois at Chicago. Hi, my name is Barbara Barbero and I'm also for the Human Eyelet Program.
Today we'll show you a procedure for human pancreatic eyelet isolation. We use this procedure in our facility in order to harvest high quality human eyelets to for a clinical usage. Let's get started.
Human eyelet isolation is a time sensitive procedure that involves teamwork and hence four individuals are needed to conduct this procedure. The team is led by the surgeon who dissects and cannulate the pancreas. The eyelet isolation begins the minute the team enters the UIC eyelid isolation facility.
Team members ensure that all necessary instrumentation like the centrifuge is checked and turned on. They also make sure to sanitize all hoods and surfaces. Two people will set up the cannulation table, slush machine digestion, circuit perfusion, unit cope purification machine, and surgical tables.
Simultaneously, two other team members will prepare the media that is needed during isolation. Next, bacteriology tubes, sampling tubes and assessment tubes and plates are prepared. Prepare the purification tube set as follows, the first tube empty and marked at 150 milliliters.
Tubes two through 12 filled with 200 milliliters of M1 99. Media supplemented with human albumin or wash solution and marked at 230 milliliters. And finally, one 50 milliliter conical tube filled with 50 milliliters wash solution.
Once these preparation steps are complete, we are ready to begin the preparation and perfusion of the pancreas. To begin preparation and perfusion of the pancreas, an operator removes the organ from packaging. Using sterile technique, the surgeon will transfer the pancreas to the decontamination table.
The surgeon will fill one 60 cc syringe with the original pancreas preservation solution and hand it to an operator who will use it to fill prepared procurement bacteriology bottles. After a visual inspection to determine whether the organ is suitable for eyelet isolation, the surgeon will ask an operator to prepare collagenase today. Prepare is a choate.
If the pancreas is damaged during procurement or anatomically abnormal, it cannot be used for eyelet isolation. Remember that Resus suspension time varies between different enzyme brands and lots. When the surgeon is ready, bring the decontamination solutions to the decontamination table and pour them into the appropriate containers.
The surgeon will decontaminate the pancreas by washing the pancreas for one minute. In each of the decontamination solutions, the pancreas is then placed in a small sterile jar in which it will be weighed. After the weight is entered into the batch record, the pancreas is placed in the cannulation pan.
With some preservation solution, the surgeon will expose and partially incise the main duct and cannulate each half of the pancreas with a cannula secured by, suture your to perfuse the pancreas. First, move it to the perfusion unit and pour the enzyme solution into the basin. Total perfusion time is about 10 minutes at 80 millimeters of mercury for five minutes.
Then at 180 millimeters of mercury for five minutes, the pancreas will appear distended. After perfusion, move the perfused pancreas to the cannulation pan where the fatty tissue is carefully trimmed from the pancreas. Then cut the pancreas into 10 to 12 pieces and transfer it to the recording chamber for digestion.
To begin the digestion step, transfer the pancreas to the empty record chamber. Add one, two and a half milliliter vial of pulmozyme put on the screen being careful to avoid blocking the chamber, close the chamber and fill up the system. Start pumping the solution for five minutes.
Gently rock the chamber to evenly disperse warm solution throughout. After five minutes, gently shake the chamber for the whole digestion phase. Maintain the temperature at 37 degrees Celsius.
Take a sample every two minutes and detect if free eyelets are found under the scope record the eyelet evaluation information in the digestion table of the batch record. Continue to monitor the percentage of eyelets until there are 50%of them floating free and isolated in the solution. Once 50%of the eyelets are found free in the samples, digestion is stopped by adding cold RPMI dilution solution.
The circuit tissue is collected in 500 milliliter conical tubes prefilled with 30 milliliters. Human albumin. Spin the conical tubes at 1100 RPM for one minute at four degrees Celsius.
Once the tubes are spun, suction off the supernatant and wash the pellet. With cold wash solution, Combine all collection tubes suspended with digested pancreatic tissue into a single 500 milliliter conical. When a tube is full of washed tissue spin and repeat the washing process until all tissue has been washed multiple times.
Spin this tube and transfer the tissue into a single 250 milliliter conical tube. Bring the volume up to 250 milliliters with M1 99 washing solution, supplemented with human albumin and prepare for sampling. Take a one milliliter sample with a pipette and add to a 15 milliliter conical tube prefilled with nine milliliters of wash solution.
Then mixed by gently inverting the 15 milliliter conical tube. And from this sample out one milliliter directly into a grid counting dish. Count the cells and calculate the cell number.
Using a dilution factor of 2, 500, spin the 250 milliliter conical tube and add 150 milliliters of uw. University of Wisconsin Solution. When UW is added, makes the cell suspension thoroughly using the pipette record and communicate to the purification operator.
The start time in uw. Leave the tubes on ice and swirl occasionally. Now the eyelets can be purified further at this step, a sample from un purified pancreatic tissue is taken for counting.
When dione solution is added to the sample, only the eyelid population will be stained red. Then their quality and quantity can be evaluated. We've just demonstrated a procedure for isolating high number of quality human pancreatic eyelet, which is important for successful eyelet transplantation.
Based on our experience, as soon as 40 to 50%of human eyelets are free from the surrounding as inner tissue, we need to stop the the digestion as soon as we can by diluting and also cooling down the digestion mixture. It's also important to handle the tissue gently during collection, washing and mixing. We recommend to use the U University really nice Chicago purification method, which results in a superior recovery of a highly pure human eyelets.
This method also significantly shortened the isolation time, which will minimize the ischemia associated injury to the eyelet. So that's it. Thanks for watching and good luck with your experiments.
