Introduction.Primary hepatocytes are an important tool for in vitro liver related studies. However, the expansion and maintenance of these cells have been historically challenging as they lose morphology and functionality after a few days in culture. 3D organoids are a cutting-edge tool able to recapitulate tissues in a dish for a long-term culture, overcoming the problem of the inability to expand to the primary hepatocytes in vitro.
The overall goal of the present work is to describe in an exhaustive, detailed, and consequential protocol, the crucial steps of rat liver perfusion, and primary hepatocytes isolation, in order to allow researchers to speed and optimize the 3D organoid culture, Warm water bath at 42 degrees Celsius, and then place perfusion buffer and PBS one pair in the water bath for 20 minutes. Place primary hepatocytes complete medium and PBS, added with 3%pen-strepped on ice. Prepare the peristaltic pump and connect the tubing with the glass bottle and the bubble trap, both filled with perfusion buffer.
Fill the tubing with warm perfusion buffer in order to remove air bubbles and to wash residual ethanol. Anesthetized the adult rat by intraperitoneal injection of anesthetic mix. Shave the abdomen and clean it with 70%ethanol to reduce bacterial contamination during the surgery.
Place the rat in the middle of the dissection tray and secure the limbs using needles. Make a U-shaped incision through the skin and muscle, from the center of the lower abdomen, to the rib cage. Clamp the skin and folded up to the head, exposing the intestines.
Carefully move the intestine to the left side of the animal, out of the abdominal cavity, and expose the hepatic portal vein and vena cava. Using pliers, run a suture under the portal vein and prepare two knots, one centimeter, one from the other, that surround the vein itself. Inject 100 to 150 units of heparin dissolved in PBS into the vena cover to prevent blood coagulation.
Turn on the pump with a low speed rate. Insert the 18-gauge angiocath in the portal vein, at the level of the thread in a flat angle relative to the vein. Remove the inner needle to leave the cannula in the vein.
Connect to the outlet end of it using a luer lock connector. Close the knot, and stabilize the tube in the right position. Then cut the vena cava to let the blood exit.
The liver should quickly start swelling and bleaching in two to three seconds. This confirms perfusion buffer is flowing through the liver. Slowly increase the pump speed at 10 milliliter per minute, and maintain at least 10 minutes to allow the cleaning of the liver from blood.
During the perfusion, apply pressure with swab to the vena cava for 10 seconds intervals. The liver should swell during clamp, and relax upon vein release, leading to enhanced cell dissociation. Repeat the pressure periodically.
After the perfusion, switch the perfusion solution to the pre-warmed digestion solution without interrupting the flow and avoiding any air bubble in the tubing. Increase pump speed at 20 milliliter per minute, and periodically continue to press with swab for liver swelling and relaxation. After digestion buffer, liver should look mushy.
At this point, the needle can be removed and the pump stopped. For the liver dissection, gently grab the central connective tissue, between the lobes with forceps. Cut all the connection to other organs, and lift the liver.
Wash the liver, just immersing in the pre-chilled PBS, plus 3%pen-strep solution for few seconds. Then place in the pre-chilled William's medium-containing tube. Under biological hood, transfer the liver into the Petri dish with 15 milliliters cold William's complete medium, over a tray full of ice.
Disturb the tissue vigorously with the scraper to release the cells in the medium. If the liver digestion is correctly executed, the release should be easy. Collect the medium containing cells and filter it with the filter while transferring in a sterile Falcon tube.
Pour about 15 milliliters of cold William's complete medium, over the smashed liver in the Petri dish to help release more cells and repeat the filtration. At the end of the release phase, the filter medium should opaque after freeing hepatic cells. The remaining liver should look fibrous and would be discarded.
Centrifuge the filtered medium containing hepatic cells with low speed break. Discard the supernatant and resuspend the cell pellet in 40 milliliters ice-cold William's complete medium, then repeat the centrifugation. Discard the supernatant, and then resuspend the cell palette in 25 milliliters ice-cold William's complete medium, Add 25 milliliters, 90%Percoll solution into the tube and gently mix.
You should obtain a visible palette on the bottom of the tube corresponding to the viable hepatocytes, and the layer on the top of the gradient composed by dead hepatocytes. Aspirate the dead cell layer from the top of the gradient and leave one, two milliliters of medium with a pellet. Resuspend the pellet in 30, 40 milliliters of medium.
Centrifuge at 200g for 10 minutes at four degrees Celsius, then aspirate supernatant. Add an appropriate amount of medium for the counting. Count cell viability with one to 10 trypan blue.
For 2D culture, primary hepatocytes were plated at a concentration of 500, 000 cells per well of six multi-well on collagen-coated cell plates with pre-warmed William's complete medium, and placed in the incubator. After three to four hours, medium was changed to new pre-warmed William's complete medium. For 3D hepatocyte culture, primary hepatocytes were suspended in cold pure Geltrex matrix at the concentration of 1, 000 cells in 20 microliters of Geltrex.
The plate was turned upside down and left in the incubator for 20 to 30 minutes to allow matrix solidification. Then the plate was turned up, and 3D-specific culture medium was added. The medium was changed every two to three days.
Representative results. At the end of the setting up procedures, we obtained a cell yield of up to one per 10 to the eight cells per isolation from the liver of about 300 grams rat. The cell viability between 78%and 97%has been established by trypan blue, counting after Percoll density gradient.
At four hours after plating, the hepatocytes showed a cuboidal morphology. On day one, primary hepatocytes acquired their typical hexagonal shape, and lipid droplets start to be visible. On day two to three, the lipid droplets increase, while on day four to five, the cells accumulate actin stress fibers.
A drastic reduction of viability occurred already in the first days of culture reaching 49.5%on day one. that lows at 33.5%on day three, and reaches 9.1%on day five. On day zero of plating hepatic organoids, we're measuring 30 micrometers.
On day two of culture, hepatic organoids nearly doubled their dimensions, underlining size growth that is maintained also at day five. Hepatic organoid shape on day one was round shape, while they showed the bunch of grape shape on day two. On day 15, a proliferative activity of hepatocytes, green cells, was present even if the EdU incorporation was low, 9%Conclusion.
3D organoids are considered a frontier for personalized medicine, and allow a long-term hepatocytes culture. Compared with 2D hepatocytes, hepatic organoids were still viable and in active proliferation at day 15, demonstrating a longer term potential. The quality of cutting-edge 3D technique, requires a good yield of viable hepatocytes and a well-performed liver perfusion and isolation.
We clarify steps that could appear as more critical in primary rat hepatocyte isolation, and summarize the scattered tips that can easily affect the success of the technique in the rat model.
