This protocol facilitates the isolation and reproducible long-term culture of mouse hepatocytes in a 3D collagen sandwich environment to study canonical structure formation and its response to treatment in vitro. Once the technique has been mastered, it's a quick method for obtaining large volumes of highly viable and pure mouse hepatocyte population for in vitro studies. Demonstrating the procedure will be Lenka Sarnova, a technician from our laboratory.
The day before primary hepatocyte isolation, add 100 microliters of 10X DMEM, 485 microliters of distilled water, and 15 microliters of one molar sodium hydroxide in 500 microliters to rat-tail collagen one. Check the pH of the neutralized collagen. It should be about 7.5.
Using a pre-chilled 200 microliter pipette tip, spread 100 microliters of the neutralized collagen solution over each of plastic 3.5 centimeter diameter culture dishes on ice and place the dishes under standard culture conditions overnight. The next morning, rehydrate the collagen layers with one milliliter of 37 degree Celsius PBS per dish for at least three hours at 37 degrees Celsius. To isolate the liver, after confirming a lack of response to toe pinch, place the anesthetized mouse on a dissection mat in the supine position and tape the lower and upper extremities to the mat.
Swab the abdomen with 70%ethanol and make a V-shaped incision from the pubic area to each forelimb. Fold the skin over the chest to uncover the abdominal cavity and place the mat under a dissecting microscope. Move the small intestine and colon in the caudal direction to expose the IVC and load a two milliliter syringe with 2.5 milliliters of 37 degree Celsius solution C.Connect the syringe to the cannula and use a PBS soaked cotton swab to press the liver up to the diaphragm.
Place a soaked suture around the IVC just below the liver. Then using an insulin syringe equipped with a 30 gauge needle bent to a 45 degree angle, inject 10 microliters of 5, 000 units per milliliter of heparin into the portal vein. To cannulate the liver, use microsurgical scissors to make a small incision in the IVC directly next to the liver and insert the cannula into the incision.
Secure the cannula in place with sutures and two surgical knots and cut the portal vein to allow the perfusion buffers to flow out from the liver. Then slowly depress the syringe plunger to perfuse the liver with two milliliters of the solution over a period of about 15 seconds. When all of the solution has been delivered, pre-fill a peristaltic pump with fresh 37 degree Celsius solution C and check the perfusion apparatus to ensure that there are no air bubbles in the system.
Carefully disconnect the cannula from the syringe and quickly but carefully connect the tubing of the running peristaltic pump to the cannula. Immediately start the perfusion at a 2.5 milliliters per minute flow rate. After two minutes, switch to solution D and continue the perfusion for an additional 10 minutes.
At the end of the perfusion, carefully harvest the liver into a 50 milliliter conical tube containing 20 milliliters of 37 degree Celsius solution E.To isolate the primary hepatocytes, holding the liver by the cannula, rub the tissue around the wall of the tube to knock the hepatocytes into the buffer. When all of the tissue has been mashed, transfer the tissue slurry into a 70 micrometer pore nylon strainer to filter the isolated cells into a new 50 milliliter tube. Sediment the liver cells by centrifugation and resuspend the pellet in 20 milliliters of 40%Percoll in DMEM.
Separate the live and dead cells by centrifugation and resuspend the pellet in 20 milliliters of solution E.After centrifuging the cells again, resuspend the pellet in 10 milliliters of fresh solution E for counting. After counting, adjust the primary hepatocyte count to 3.75 times 10 to the five viable cells per milliliter of hepatocyte culture medium. To culture the isolated primary hepatocytes in 3D collagen sandwiches, use a one milliliter pipette to evenly distribute two milliliters of cells onto each collagen-coated 3.5 centimeter diameter dish.
Place the cells in the cell culture incubator for three hours. During the incubation, prepare 100 microliters of neutralized rat-tail collagen one solution per dish as demonstrated. At the end of the incubation, carefully replace the medium and unattached cells with 100 microliters of neutralized collagen per dish and return the plates to the cell culture incubator for an hour.
When the collagen has solidified, carefully add two milliliters of fresh hepatocyte culture medium to each dish and return the cultures to the incubator for three to eight days checking the cultures daily under the microscope. For immunolabeling of the primary hepatocytes within the 3D collagen sandwiches, wash each sandwich carefully with 37 degree Celsius PBS and fix the cultures with one milliliter of 4%paraformaldehyde in PBS per dish for 30 minutes at room temperature. At the end of the fixation, wash the dishes three times for 10 minutes in two milliliters of PBS supplemented with 0.1%Tween 20 per wash.
After the last wash, permeabilize the cells with one milliliter of 0.1 molar glycine and 0.2%Triton X-100 in PBS for one hour at room temperature followed by three washes in PBS plus Tween 20 as just demonstrated. Next, use a 10 microliter load tip connected to a vacuum to gently disturb the top layer of collagen and block any nonspecific binding with one milliliter of 5%bovine serum albumin in PBS Tween for two hours. At the end of the incubation, add the primary antibodies of interest diluted in blocking solution to each dish and incubate overnight at 37 degrees Celsius.
The next morning, wash the dishes with three 15-minute washes in PBS Tween followed by labeling with the appropriate secondary antibodies at 37 degrees Celsius for five hours. At the end of the incubation, wash the cultures two times with PBS Tween and one time with distilled water as demonstrated before mounting the dish with anti-fade mounting medium for microscopy. Within one day of seeding in 3D collagen sandwiches, mouse primary hepatocytes formed clusters and self-organized in an approximately regular network of bile canaliculi.
Within three to six days, clusters of five to 10 cells are usually observed with fully polarized hepatocytes forming a canalicular network. Treatment with either toxin or cytoskeleton-altering drugs results in changes in the hepatocyte cytoskeleton and bile canaliculi width, shape, and number. Although ethanol treatment exhibits only a mild effect on the organization of keratin 8, it increases the tortuosity and distribution of the bile canalicular widths.
The signal intensity of ZO-1 staining is decreased in ethanol-treated bile canaliculi compared to untreated controls suggesting a loss of type junctions after ethanol treatment. The inhibition of actomyosin contractility with Blebbistatin induces the formation of disorderly shaped, thick, rounded bile canaliculi. Additionally, treatment with okadaic acid inhibits phosphatases strongly affecting the physical properties of keratins and resulting in bile canaliculi that are significantly narrowed compared to untreated controls.
Further, ethanol treatment significantly elevates the levels of both alanine and aspartate amino transferases suggesting severe hepatocellular injury while Blebbistatin treatment does not lead to any consideration changes in transaminase levels and okadaic acid treatment triggers mild biochemical changes in alanine aminotransferase but no change in aspartate aminotransferase expression. It is important to keep working relatively quickly during the cannulation and at the beginning of perfusion and to avoid bubbles entering the perfusion system. Cell lysates can be collected in duplicate wells for of proteins of interest to determine whether changes in protein expression and/or activation stages occur during treatment.
