The overall goal of this procedure is to isolate and culture neonatal neuron extrahepatic cholangiocytes. This is accomplished by first surgically exposing the abdominal organs to allow access to the liver and bile ducts. The second step of the procedure is to meticulously remove the connective tissue and fats surrounding the bile ducts and gallbladder.
The cleaned bile ducts and gallbladder are then placed on thick collagen gels and cultured for three weeks. The cholangiocytes will migrate from the ducts and are split to thin collagen gels for further culture. Ultimately, results show monolayers of pure cholangiocytes through immunofluorescence microscopy.
Generally, individuals new to this technique generally struggle because of technical difficulties with dissection technique, as well as cleaning of small bile ducts. For this procedure, set up a mouse surgical table close to the tissue culture hood and incubator. Place all the sterilized surgical tools in a beaker containing 70%ethanol.
Put three media loaded, 60 millimeter dishes on ice. Two dishes are needed at the surgical table, but keep the other materials in the hood. The collagen should be on ice.
Begin by placing a euthanized zero to three day old neonatal mouse in a supine position on the dissection board. Start with a small horizontal incision along the midline near the pelvis. Extend the incision roly on both sides, making a U-shaped flap and expose the organs.
Now carefully displace the intestines to the right of the abdomen. To view the liver and bile duct, extend the skin incisions to enlarge the flap if needed. Under the dissection scope, carefully identify and clean the common bile duct.
Pick off connective tissue and surrounding fat. Next, do the same. To clean the gallbladder, remove the bile duct and gallbladder, and place them in one of the prepared dishes there.
Massage the structures using non-rated forceps to remove more connective tissue and eject bile. Then transfer the clean tissues to the second dish. Keep repeating this procedure until tissue is collected from exactly five neonates.
Start by preparing a thick collagen gel. Always make this fresh first. Get all the sterilized solutions together on ice.
Calculate the volumes needed for two milligrams of collagen per milliliter of one XPBS. Add 0.023 parts of sodium hydroxide to each part.Collagen. Mix the aliquots together at room temperature by Pipetting.
It is essential to mix the PBS sodium hydroxide and distilled water before adding the collagen. The collagen stock must be the last component added to the gel, and the final solution needs to be pipetted. Well, this prevents the collagen from clumping.
When the solution begins to take on a gel-like consistency, immediately embed the isolated tissues, then transfer the embedded tissues to the incubator for 30 minutes to solidify. Once solidified, overlay the gel with 3.5 milliliters of BEC media and continue the incubation thereafter. Change the BEC media three times per week Within three weeks, sheets of cells with large nuclei extending from the embedded ducts will form.
These are the cholangiocytes. If the dish has a significant population of fibroblasts, unless they can be cut away, the dish should be discarded. Now, prepare thin collagen gels with one milligram of collagen per milliliter of PBS coat one milliliter on a 100 millimeter plate and spread out the collagen evenly and wait 10 minutes.
After 10 minutes, cover the plate with seven milliliters of DM EMF 12 and incubate it for 10 minutes. After the brief incubation, quickly wash the collagen with one XPBS. Remove all the PBS by aspiration and then add a second layer of collagen just as done to build up the first layer.
Once solidified, overlay the plate with DMF 12 and incubate it for 30 minutes. Now split the cells from the thick gels. First using a spatula or scraper, gently detach the collagen from the plate so it is floating.
Next, prepare filtered collagenase solution and add one milliliter to each 60 millimeter dish of thick collagen. Then transfer the plates to the incubator. After a 30 minute incubation, remove all the solution which contains the cells and transfer it to a 15 milliliter tube.
Spin the tube down and remove the supernatant by aspiration. Resuspend the pellet in 4.5 milliliters of DM F 12 media and spin it down again. Resuspend the pellet in three milliliters of 0.5%Trypsin incubate the cells at 37 degrees Celsius for five minutes during which time the sheets of cells will loosen up.
After five minutes, flow the clumps through a five or 10 milliliter pipette until they break up. Then add five milliliters of BEC media and spin the cells down. A third time resus.
Suspend the pellet in DMM F 12 media as a wash step and pellet the cells again. Now resuspend the cells in BEC media and plate them on the prepared thin layer. Collagen gels grow up the cells and split them on collagen gels as needed.
Using the described protocol, a population of neonatal mouse extrahepatic cholangiocytes with excellent purity, was isolated and stained with K 19 immunofluorescence. After watching this video, you should have a good understanding of how to isolate a pure population of neonatal mouse extrahepatic cholangiocytes by a combination of meticulous duct dissection and outgrowth on thick collagen gels.
