In this video, pancreas tissue that has been isolated and digested previously is subjected to fial gradient purification, which will yield a purified fraction of pancreatic eyelets that can ultimately be cultured in tissue culture flasks until transplantation. Hi, I'm American Chief from the Lab of Human Eyelets Program in Department of Surgery 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.
To start the eyelet purification procedure load the COBE bag clamp all tubes except the green one and set the speed to 1500 and the super out to zero in the hood. Set up the pump and gradient beakers and connect the collection tubing to the green tube. Now the loading of the FI call can begin push spin.
Then add 110 milliliters of fi. Call to the front beaker and start the pump in order to load the COBE bag. As the FI call is loaded, press super out.
Stop the pump, unclamp the pump head and set the super out speed to 100. When the FI call reaches the beaker and all air is expelled from the tubing rec clamp at the pump head, set the spin speed to 3000 and the super out speed to zero. Now prepare to load the gradients by pouring the heavy gradient into the front beaker slightly.
Release the clamp between the two beakers to remove any air. Then pour the light gradient into the back beaker. Turn on the magnetic stir.
Press spin on the COBE machine. Remove the clamp from the tube connecting the two beakers and verify that the heavy and light gradients are mixing. Once the gradients start mixing, turn on the centrifuge.
Wait one minute, then turn on the pump to load the mixing gradients to load the tissue. Let the gradient run low in the front beaker, but not low enough to allow air to enter the loading tube. Then amp the tube connecting the beakers and load the tissue.
Continue adding the rest of the tissue. Then wash the tube that held the tissue with 50 milliliters of wash solution. And use it to wash the front beaker as the last of the tissue enters the bag.
Simultaneously stop the pump UNC clamp at the pump head and clamp tubing above the bag. Continue to centrifuge for five minutes. While centrifugation continues, bring the 12 collection tubes to the hood and loosen their caps.
Attach the collection tubing to the yellow tube and place the sterile collection tip into conical tube number one. Then un clamp the yellow tube and clamp the green tube. When the spin ends, slowly raise super out to 100.
Collect 150 milliliters in tube one. Then collect 30 milliliters in tubes two through 12. Once collection is complete, push stop on the COBE.
Now that the purification has ended, the eyelets can be assessed and cultured. After collecting the purified eyelet fractions, remove a sample from each of the 12 collection tubes and stain with dione. The staining will show witcher layers containing high purity eyelets versus layers with low purity.
Eyelets collect the high and low purity tissue and pool each together, spin and wash twice After the second wash, bring the volume of each to 250 milliliters with final wash buffer. To assess the pooled fractions, start by taking two one milliliter samples from the high of the pooled fractions and one one milliliter sample from the low transfer each one milliliter sample into a conical tube containing nine milliliters of wash solution. Then transfer one milliliter from each 15 milliliter tube into a counting dish.
Examine and count the cells under the microscope. After counting the cells, calculate the number of flasks needed to culture the eyelets using the following formula total, EIN divided by purity of eyelets in decimal form divided by 30, 000 EIN per flask. For example, 100, 000 eyelets with 90%purity are cultured in four flasks.
Transfer the eyelets to T 1 75 flasks and culture in a 37 degrees Celsius and 5%CO2 incubator. After standing with dione, eyelets appear red. This allows for the evaluation of the purity of the eyelets.
Those eyelets are collected in T 1 75 culture flasks and put into the incubator. 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 inner tissue, we need to stop the, the, the digestion as soon as we can by diluting and also cooling down the digestive mixture.
It's also important to handle the tissue gently during collection, washing and mixing. We recommend to use the your University Illinois 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.
