The overall goal of this high content beta cell replication screening platform, is to facilitate the investigation of the molecular pathways that restrict beta cell growth. This method can help answer important questions in the beta cell biology field. Such as one of the key signaling molecules and pathways that control beta cell regeneration.
The main advantages of this technique are that it enables increasing the throughput cell lining to a specific assessment and the automated data analyst of a primary islet cell replication. The employment of this technique has the potential to extend toward the development of the regenerative therapy for diabetes. A disease with major health and economic cost to our society.
Begin by using a pancreatic digestive solution loaded 10 milliliter syringe equipped with a 22 gauge needle to chemulate the common bile duct at or just distal to the junction of the cystic and common hepatic ducts. Supporting the common bile duct with a scalple handle, slowly inject 10 milliliters of the solution. After all of the collagenase has been administered, used curved forceps and scissors to separate the inflated pancreas from the descending colon, intestines, stomach, and spline.
Then place up to two pancreata in a 50 milliliter tube on ice containing five milliliters of pancreatic digestion solution. When all of the pancreata have been collected, place the digestion tubes in a 37 degree Celsius water bath for 15 minutes with gentle swirling every three minutes. At the end of the digestion, vigorously shake the tubes vertically 10 times.
And add 10 milliliters of cold wash buffers to the specimens. Vigorously shake the tubes five more times and place them on ice. Next fill the tubes to a final volume of 50 milliliters with cold wash buffer and invert the tubes five times.
Then spin down the tissues and pour off the supernatants, taking care not to dislodge the pellets. Re-suspend the tissue slurry in 25 milliliters of wash buffer followed by gentle vortexing. Repeat the spinning and re-suspension steps two more times.
The pour the tissue suspensions through a 30 mesh tissue sieve into a sterile 250 milliliter beaker. Rinse the digestion tubes with an additional 20 milliliters of wash buffer and pour the washes onto the mesh to remove the undigested pancreatic and fat tissues. Divide the filtered material between two new 50 milliliter tubes and pellet the tissue by centrifugation.
Then decant the supernatant and invert the tubes to drain the excess buffer. To purify the islets, add 20 milliliters of cold density gradient to the pancreatic tissue pellets and gently pipet up and down five times to homogeneously re-suspend the contents. Next slowly overlay 10 milliliters of HBSS down the wall of each tube of gradient taking care to maintain a sharp liquid interface and separate the pancreatic cells by centrifugation.
Use a 10 milliliter pipet to transfer the islet layer from the interface into new 50 milliliter conical tubes. Then wash the islets in 40 to 50 milliliters of wash buffer three times. Inverting their tubes to re-suspend the pellets between each centrifugation.
After the final wash, bring the tubes into a tissue culture hood. Aspirate the supernatant. And re-suspend the pellets in six milliliters of islet medium.
Sieve the islets from each tube in individual wells of six well tissue culture plate. And swirl the plate to collect the islets in the middles of the wells. Evaluate islet purity under the microscope.
To further purify the islets, collect them using a one milliliter pipet and transfer to the adjacent well using six milliliters of islet medium. Repeat islet swirling, collection, transfer and microscopic evaluation until greater than 90 percent purity is achieved. Next, transfer the islets to a single 10 centimeter tissue culture dish containing 20 milliliters of islet medium.
After all of the islets have been harvested, place the culture dish in a tissue culture incubator overnight. Then coat the wells of a 384 well plate with 40 microliters of 804G, conditioned medium per well and place the plate in the incubator overnight. The next day, use a cell scraper to gently detach the islets.
Then transfer the islets into a 50 milliliter tube. Collect the islets by centrifugation. Then wash them with 20 milliliters of warm p, b, s.
Centrifuge for one minute. Aspirate the supernatant. And re-suspend the pellet in zero point two, five percent Trypsin.
Incubate the islets for 10 minutes at 37 degrees Celsius using a one milliliter pipet to aspirate and dispense the islets 10 times at five and 10 minutes. After the second triteration, count a 20 microliter sample of the trypsinized islet cells. If the tissues are fully digested, suspend the dissociative islet cells at a four point five times ten to the four cell per milliliter concentration in islet function medium.
Then use a 12 well manifold aspirator to remove the 804G condition medium from the previously prepared 384 well plate. And sieve 70 microliters of islets per well into rows c to n in columns four to 21. Allow the islets to adhere in the tissue culture incubator for 48 hours.
After two day, use the 12 well manifold aspirator to carefully remove the islet medium and use a 12 channel pipet to add 35 microliters of islet function medium to each well. Then transfer 35 microliters of the freshly prepared two, x compound solutions of interest to each well. And return the islets to the incubator for another 48 hours.
After 48 hours, fix, stain, and analyze the compound treated islets cultures using an automated high content imaging platform. In a typical experiment, the replication rate of the DAPI, PD-1 and insulin positive beta cells is determined by the percentage of beta cells co-expressing Ki-67. Alpha cell replication is measured as the percentage of DAPI positive, PD negative, Glucagon positive, Ki-67 co-expressing cells.
For example in this representative experiment, Dipryridamole was observed to promote the replication of beta but not alpha cells. This technique can be completed in seven days. Following this procedure compounds that in the beta cell replication can be studied further to reveal the mechanisms of action.
This technique has enabled researches in the field of beta cell biology to identify new signaling factors and pathways that regulate beta cell growth. After watching this video, you should have a good understanding of how to test the ability of small molecules to selective stimulate beta cell regeneration.
