The overall goal of this procedure is to isolate primary pancreatic ASIN cells and maintain them in vitro. This is accomplished by first extracting the pancreas from the mouse house. Next, pancreatic asinine are dissociated using a moderate enzymatic haase one a digestion.
Then the pancreatic asinine are mechanically dissociated by successive pipetting. Finally, the dispersed asinine are seeded to maintain them in culture. Ultimately, results can be obtained that show the quality of the ASIN R preparation through immunohistochemistry or immunofluorescence experiments.
The main advantage of this technique of our existing methods, like extraction by complete pancreatic tissue digestion and dissociation or fact sorting, is that it permit a rapid insulation of primary pancreatic dispersed asy sustainable for more than one week. In culture, this method can help answer a key question in the exocrine pancreas field, such as better deciphering the physiological mechanisms, as well as all trans differentiation and tumorgenesis processes taking part in the pancreas. This procedure is performed in a level two sterile microbiological safety cabinet with sterile dissection equipment.
Refer to text protocol for all buffer recipes. After euthanizing a mouse according to the text protocol, secure it and use 70%ethanol to spray the abdomen with dissecting scissors and forceps. Make a V-shape incision at the genital area and extend it up to the diaphragm to completely open the abdominal cavity.
Position the liver lobes against the diaphragm, then exteriorize the gut and colon placing them to the left. After locating the rectum with a pair of curved forceps and dissecting scissors, grab and suction it. Using the same pair of forceps, pull the intestine to carefully unroll the bowel from the rectum to the stomach.
The pancreas can now be located as a small strip between the stomach and the beginning of the bowel with its ligations to the spleen intact. Using Noyes scissors and a pair of forceps, carefully cut the pancreas along the bowel and liberated with the spleen from the rest of the digestive track. While holding the spleen carefully section the pancreas attached to it, ensuring that no mesenteric fat or adjacent tissue is collected.
Next, rinse the pancreas twice in one X Hank's balance salt solution or HBSS white pose. Tissue will float, making it easy to see and should be removed from the pancreas. Rinse the pancreas twice more in HBSS.
If the pancreas needs to be transported to the cell culture facility, keep it in HBSS on ice to dissociate the pancreas. Begin by placing it into a sterile Petri dish containing five milliliters of HBSS using forceps in a scalpel. Mince the tissue into small pieces, approximately one to three millimeters cubed size.
Transfer the tissue into a sterile 50 milliliter polypropylene tube and centrifuge for two minutes at 450 Gs and four degrees Celsius. Aspirate and discard the supernatant to remove cell fragments and blood cells. Next, add 10 milliliters of Kase one a solution and using a 25 milliliter serological pipette, transfer the tissue to a 25 square centimeter flask.
Incubated for 20 to 30 minutes at 37 degrees Celsius and every five minutes using sterile serological pipettes that gradually decrease in size, pipette up and down 10 times to mechanically dissociate the pancreatic tissue. When the tissue fragments are completely dissociated and the solution is turbid. Stop the enzymatic reaction by adding 10 milliliters of cold buffered washing solution.
Then transfer the mixture to a sterile 50 milliliter polypropylene tube. Transfer the sample to a sterile 50 milliliter polypropylene tube and centrifuge for two minutes. Then carefully aspirate and discard the supernatant to remove the collegiate one a solution.
Wash the tissue three times with 10 milliliters each of buffered washing solution. Spinning for three minutes After each rinse After the last wash, resus suspend the cell pellet in seven milliliters of complete waymouth medium. Filter the through a 100 micrometer filter, which will allow the pancreatic ASIN our structures of 10 to 15 cells to pass through.
Then rinse the filter with six milliliters of complete way myths medium. See two milliliters per well of the isolated asinine in a six well cultured dish. And incubate them for 24 hours at 37 degrees Celsius and 5%CO2.
Next, transfer the asinine in suspension into a new six well cultured dish to eliminate the contaminant cells and cellular remnants that have adhered overnight to seed on matrix scaffolds. One day prior coat a six well culture dish with type one collagen by incubating one milliliter of collagen solution in each well for one hour at 37 degrees Celsius. Aspirate the solution and use PBS to rinse the cells twice.
Allow the wells to dry for at least 12 hours in a sterile environment before use. Place the six well culture dish in a level two safety cabinet. Next, transfer the isolated primary asinine into the wells of the collagen encoded dish and incubate them for two days to allow them to adhere to the substrate on day three.
Change the culture medium to eliminate non-viable cells and continue to replace the medium every three days. With time and culture, the cells will progressively spread on the collagen containing support. Shown here is a typical day one representation of pancreatic asinine, isolated.
Using this protocol, transferring the asin our cells into a fresh culture dish eliminates the contaminating cellular fragments and adherent cells as demonstrated in this figure. When cultured on type one collagen, asar cells spread and lose their asar morphology. And 3D organization giving rise to a monolayer of spindle shaped cells Once mastered, this technique can be done in one hour if it's performed properly.
While attempting this procedure, it's important to remember that the rapidity of pancreas extraction and the attention dedicated to the enzymatic and mechanical dissociations are essential for suitable yield and quality of dispersed as in iation.
