Rat Liver Perfusion and Primary Hepatocytes Isolation: An Old Procedure Crucial for Cutting-Edge 3D Organoids Culture

Podsumowanie

This protocol presents an optimized two-step collagenase liver perfusion technique in a rat model and shows the use of isolated hepatocytes for in vitro long-term culture of 3D organoids.

Streszczenie

Primary hepatocytes are a commonly used tool for in vitro liver-related studies. However, the maintenance of these cells has always been a challenge due to the rapid loss of morphology, viability, and functionality in culture. A recent approach to long-term culture is the generation of three-dimensional (3D) organoids, an in vitro tool that can recapitulate tissues in a dish based on the marvelous ability of the liver to regenerate itself. Published protocols have been designed to obtain long-term functional 3D organoids from primary adult hepatocytes (Hep-Orgs). The 3D organoid cutting-edge tool requires the ability to isolate cells from adult tissue, and this initial step is crucial for a high-quality final result. The two-step collagenase perfusion, introduced in the 1970s, is still a valid procedure to obtain single hepatocytes. The present article aims to describe all the crucial steps of the surgical procedure, thereby optimizing the primary hepatocytes isolation procedure in the rat model. Moreover, particular attention is paid to the PREPARE guidelines to increase the likelihood of successful procedures and ensure high-quality results. A detailed protocol allows researchers to speed up and optimize the downstream work to establish 3D organoids from primary adult rat hepatocytes. Compared to 2D hepatocytes, Hep-Orgs were still viable and in active proliferation at Day 15, demonstrating a long-term potential.

Wprowadzenie

Primary hepatocytes are an important and widely used tool for in vitro liver-related studies. However, their expansion and maintenance have been historically challenging, as they lose morphology and functionality after a few days in the culture¹. 2D culture is a limiting condition, in particular, for hepatocytes that have a polygonal shape and polarized structure with differentiated apical and basolateral membranes. In fact, hepatocyte adhesion to the plate interferes with their normal activity because it leads to a flat cytoskeleton with limited interaction among cells and between cells and extracellular matrix (ECM), reducing the polarization and the involved signaling pathways².

To bypass the limitations of this method, Dunn and colleagues³ first used the collagen double layer. This culture method, known as "the sandwich-culture method," is based on seeding primary hepatocytes between the two layers of the matrix. This method has many advantages, including long-term culture, maintenance of polygonal morphology, and transcriptional activities comparable to that of freshly isolated hepatocytes⁴. A similar method, based on the same principle, where the underlay is composed of Matrigel while the overlay of collagen, has evidenced the presence of a well-established canalicular network⁵.

Despite the reliability of the sandwich culture method for hepatocyte growth, the present work looks forward to setting up a 3D-organoid culture, an in vitro tool used for recapitulating tissues in a dish and forming a potential bridge toward personalized medicine, allowing the generation of disease-specific biological insights, identifying molecular targets, testing drugs, establishing biobanks, and opening up new horizons for innovative technologies like organ-on-chip⁶. Primary hepatocytes were embedded in hemispherical matrix droplets known as "domes" to allow a robust growth of organoids inside the domes and to guarantee the flow of the soluble factors, including hepatocyte growth factor (HGF) and epidermal growth factor (EGF), from the culture medium. These growth factors directly activate signaling pathways to ensure the survival of the hepatocytes⁷. Liver organoids are usually obtained from stem/progenitor cells isolated from embryonic stages or adult rats, mice, human (and also dogs and cats) liver⁸. Even if the 3D conformation improves the differentiation of stem cells to adult hepatocytes, this tool still lacks the maturity of the original primary tissue⁹. To overcome this problem, in 2018, two different groups^9,10 simultaneously published a protocol to obtain a long-term culture of 3D liver organoids starting from adult primary mouse hepatocytes (referred to as Hep-Orgs). Their protocols recapitulate the proliferative damage response of liver regeneration, growing hepatocytes with a high level of inflammatory cytokines as it occurs after partial hepatectomy. Indeed, the above studies demonstrate that hepatocytes can switch to a ductal state following injury¹² or when cholangiocyte proliferation is suppressed¹³. Their bipotent capacity allows cellular plasticity, which is important for complex structures such as organoids. Therefore, these protocols overcome the problem of the inability to expand primary hepatocytes in vitro.

The 3D organoid cutting-edge tool requires the ability to isolate cells from adult tissue, and this initial step is crucial for a high-quality final result. While the 3D organoid preparation could be considered a recent and developing technique (because the organoid definition was coined by Lancaster¹⁴ and Huch¹⁵ only ten years ago), the two-step collagenase perfusion is an old procedure introduced by Seglen in the 1970s¹⁶. Focusing on the most recent publications in the field, the protocols of Ng¹⁷ and Shen¹⁸ could be considered the gold standard for performing the procedure in rats, while the ones of Cabral¹⁹ and Charni-Natan²⁰ for mice. It is not unusual for researchers to focus on choosing the best extracellular matrix (ECM) and growth factors to develop 3D organoids yet bump into failures such as improper surgical procedure, low hepatocytes viability and yield, or high levels of bacterial contamination. These problems lengthen the downstream experiments and increase the number of animals needed for the following experiments. Conversely, if the surgical and isolation procedures are well set up, the high number of viable hepatocytes obtained allows for preparing a huge number of organoids, thus limiting the use of animals. The present protocol mainly addresses these issues using commercial solutions and by optimizing a precise portal vein cannulation that ensures optimal perfusion and digestion steps with the liver in situ.

In order to improve the quality, reproducibility, and translatability of our animal research, we carefully considered the PREPARE guidelines: Planning Research and Experimental Procedures on Animals: Recommendations for Excellence²¹. These reporting guidelines try to increase the likelihood of success through planning and represent an important step in the implementation of the 3Rs of Russel and Burch (replacement, reduction, and refinement). The PREPARE guidelines cover 15 main topics that should be addressed according to each individual research project. We will describe the topics we focused on during the planning and preparation of the project.

The present work aims to describe, in an exhaustive, detailed, and consequential protocol, the crucial steps of the surgery in the rat model to allow researchers to speed up and optimize the downstream work. When we approached these protocols at first, we experienced problems of bacterial contamination, low liver digestion efficiency, low primary hepatocyte yield, and low hepatocyte viability that can easily affect the success of the technique. Showing how to address and solve critical features, this protocol optimized the procedure for primary rat hepatocyte isolation. The rat liver perfusion and the subsequent primary adult hepatocyte isolation are the main preliminary steps for different applications. In particular, this protocol is suitable for all procedures requiring a good yield of high-quality and high-viability adult hepatocytes. The protocol results are appropriate to establish in vitro models to study liver physiology and pathology.

Protokół

All procedures and animal housing were conducted according to the guidelines of the Italian Law and European Community directive. The experimental protocol was approved by the local Animal Care Committee and by the Italian Health Ministry (permit n° 321/2022-PR) according to art.31 of decree 26/2014.

1. Preparation for the animal procedure

NOTE: Please refer to Table 1 for the medium and buffer composition and to the Table of Materials for commercial details.

1. Warm the water bath at 42 °C and then place the Perfusion buffer and 1x PBS in the water bath for about 20 min.
   NOTE: Due to enzyme sensitivity, the Digestion buffer is warmed only when the Perfusion procedure has been successfully started. During the perfusion procedure, maintain the solutions in the water bath.
2. Place William's complete medium and PBS + 3% P/S on ice.
3. Prepare the peristaltic pump and connect the tubing with the glass bottle and the bubble trap, both filled with Perfusion buffer.
4. While priming the pump, fill the tubing with warm Perfusion buffer in order to remove air and wash residual ethanol (low pump speed). This step is also important to check for the correct pump operation and possible tube leakage.

2. Preparation for hepatocyte isolation

1. During the animal procedure, expose the following instruments to UV light: Petri dish 100 mm, cell strainer 100 µm, scraper size M, large tweezers, beaker for ice, tray for ice.

3. Initial animal procedure and anesthesia

1. Anesthetize the adult rat (250-350 g body weight; 10-12 weeks old) by intraperitoneal injection of anesthetics mix (final concentration for xylazine: 22.5 mg/kg; for ketamine: 112.5 mg/kg; approx. final volume: 0.8-1.0 mL).
2. Monitor the depth of anesthesia by toe pinching until the rat no longer responds to noxious stimuli. It usually takes 5 min. The anesthetic dose ensures deep, surgical anesthesia that minimizes the risk of suffering or distress during the procedure
   NOTE: Exsanguination is the secondary method of rat euthanasia used in this experiment, and it ensures that the animal has been euthanized effectively.
3. Shave the abdomen and clean it with 70% ethanol to reduce bacterial contamination during the surgery.
   NOTE: This is a non-survival surgery, so full asepsis is not maintained.
4. Place the rat in the middle of the dissection tray and secure the limbs using needles.
5. 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 fold it up to the head, exposing the intestines.
6. 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.
7. Using pliers run a cotton thread under the portal vein and prepare 2 knots, 1 cm one from the other, that surround the vein itself.
   NOTE: A black thread is easier to visualize.
8. Inject 100-150 IU of heparin dissolved in 1x PBS (total volume 300 µL) into the vena cava to prevent blood coagulation.

4. Cannulation and liver perfusion

1. Turn on the pump at a low-speed rate (less than 1 mL/min flow).
2. Insert the 18 G angiocath in the portal vein at the level of the thread at a flat angle relative to the vein.
3. Manually remove the inner needle to leave the cannula in the vein. Blood will flow inside it, filling up the plug and avoiding trapped air bubbles in the following step.
4. While the perfusion buffer flows in the tubing, connect the cannula by pressing the C button to the outlet end using a Luer lock connector.
5. At this point, tie the knots, one around the catheter inside the vein and one around the catheter, before entering the vein to stabilize the tube in the right position.
6. Cut the vena cava to let the blood exit. Ensure that the liver starts swelling and bleaching quickly (seconds) in 2-3 s. This confirms that the perfusion buffer is correctly flowing through the liver.
   NOTE: If some lobes remain red, the angiocatheter is placed too deeply. If there are only red spots, lightly tapping on the liver can help the perfusion in the specific points. Pour some 1x PBS over the open abdomen to help move the blood and check for leaks.
7. Slowly increase the pump flow to 10 mL/min and maintain it for at least 10 min to allow the cleaning of the liver from blood.
8. During the perfusion, apply pressure to the vena cava with a cotton bud for 10 s intervals: Ensure that the liver swells upon clamping and relaxes upon release. Repeat the pressure periodically (5-10 times) and check for liver swelling and relaxation.
   NOTE: This step serves as a crucial checkpoint for visibly validating perfusion. It has been essential in ensuring high hepatocyte vitality and maximizing the final yield. By actively verifying this process, the buffer can effectively reach all parts of the liver vasculature, which passive blood clearing alone would not achieve.

5. Liver digestion

1. After the perfusion, switch to the pre-warmed Digestion buffer without interrupting the flow and avoiding air bubbles in the tubing.
   NOTE: Temporarily reducing the flow can help perform this passage. The digestion buffer includes phenol red, which facilitates visualization of switching from the Perfusion buffer to the Digestion buffer.
2. Increase the pump speed to 20 mL/min and periodically press with the swab for liver swelling and relaxation.
3. After this step (lasting about 15 minutes), ensure that the liver becomes increasingly mushy. At this point, the needle can be removed, and the pump stops.
4. As the liver capsule is fragile after digestion, gently and carefully dissect the liver: grab the central connective tissue between the lobes with forceps. Cut all the connections to other organs and lift the liver.
5. Wash the liver, immersing it in the pre-chilled PBS + 3% P/S solution for a few seconds. Then, place it in the pre-chilled William's complete medium-containing tube.
   NOTE: Exsanguination and death ensure perfusion via the vena cava. The confirmation of the animal's death comes from the pneumothorax induced the moment the liver is explanted, and the diaphragm is fully dissected, and upon inspection, breathing and heartbeat are no longer functional.

6. Hepatocyte purification

1. Under the biological hood, transfer the liver into the Petri dish 100 mm with the 15 mL of cold William's complete medium over a tray full of ice.
2. Press the organ vigorously with the scraper to release the cells in the medium.
   NOTE: If the digestion phase is correctly executed, the cell release should be easy and not require much pressure on the liver. Do not cut the liver in pieces; leave it intact. Keeping the liver submerged in the medium improves cell release.
3. Collect the medium-containing cells and filter them with the 100 µm filter while transferring them to a sterile conical tube. The filtration allows the removal of connective tissues and undigested tissue fragments.
4. Pour about 15 mL of cold William's complete medium over the smashed liver in the Petri dish to help release more cells. Repeat steps 6.2 and 6.3.
5. Repeat point 6.4 at least once, until all cells are released. If necessary, divide the medium into more conical tubes and use multiple 100 µm filters.
   NOTE: After the hepatocytes are released and before filtering, the medium should look opaque because of the hepatocytes' presence. The remaining liver should look fibrous and will be discarded.
6. Centrifuge the filtered medium containing hepatic cells at 50 x g for 3 min at 4 °C with a low-speed brake.
   NOTE: Hepatocytes are denser than other liver cells. Due to low centrifugation force, only hepatocytes will be in the pellet, while other cells will remain in the supernatant.
7. Discard the supernatant without disturbing the cell pellet. Gently add about 40 mL of ice-cold William's complete medium in the tube and slowly pipette up and down 2 to 3 times to resuspend the cell pellet.
   NOTE: The cell pellet must be resuspended carefully in order to avoid damaging the cells.
8. Centrifuge at 50 x g for 3 min at 4 °C.
   NOTE: For the first tries, count the viable cells with a 1:10 trypan blue assay after the second centrifugation before proceeding to the next steps. This can avoid wasting media and time if all the cells are dead.
9. Discard the supernatant and resuspend the cell pellet in 25 mL of ice-cold William's complete medium.
10. Add 25 mL of 90% density gradient solution into the tube and gently mix.
    NOTE: A density gradient solution separates viable hepatocytes from dead hepatocytes and cell debris based on their density.
11. Centrifuge at 200 x g for 10 min at 4 °C. A visible pellet must be obtained on the bottom of the tube, corresponding to the viable hepatocytes, and a clear layer on the top of the gradient composed of dead hepatocytes.
    NOTE: If there is no pellet at all, try to mix the solution again by swirling and repeat the centrifugation.
12. Aspirate the dead cell layer from the top of the gradient and leave 1-2 mL of the medium with the pellet.
13. Resuspend the pellet in 30-40 mL of William's complete medium.
    NOTE: For the entire procedure, use only 25 mL serological pipettes. Smaller bore pipettes may reduce hepatocyte viability. All the steps should be conducted on ice to maintain +4°C.
14. Centrifuge at 200 x g for 10 min at 4°C, then aspirate the supernatant.

7. Hepatocyte culture and RNA collection

1. Add an appropriate amount of medium for cell counting. Count cell viability with 1:10 Trypan blue.
2. 2D hepatocyte culture
   1. Plate cells at the concentration of 500,000 cells/well of 6-well dish on a collagen-coated cell plate with pre-warmed William's complete medium.
   2. Place the cell culture in a humidified incubator with 95% air, 5% CO₂ at 37 °C.
   3. After 4 h, change the medium to the new pre-warmed William's complete medium.
   4. Keep cells in the incubator. Change the medium daily.
   5. Collect primary hepatocytes in a reagent for RNA extraction before plating (day 0) and at Days 1 to 3 of culture.
   6. Extract RNA according to the manufacturer's protocol and reverse-transcribe it to cDNA for evaluation of hepatic marker genes by RT-qPCR. The primer list is presented in Table 2.
3. For 3D long-term hepatocyte culture:
   1. Resuspend the cells in the cold pure matrix at the concentration of 1,000-10,000 cells in 20 µL.
      NOTE: Try to avoid making matrix bubbles .
   2. Using pre-chilled pipette tips, plate matrix-containing cell droplets into pre-warmed cell-culture plate.
   3. Turn the plate upside down and leave it in the cell culture incubator for 20-30 min.
   4. Turn it up and add the pre-warmed 3D long-term hepatocyte medium and specific culture medium according to Peng's work²². Keep cells in the incubator.
   5. Change the 3D long-term hepatocyte medium every 2-3 days.
   6. At least after 14 days of culture, the Hep-Orgsare separated from the matrix and viable Hep-Orgs are evaluated by Trypan blue and EdU proliferation assay and passaged according to Peng's work.

Wyniki

At the end of the set-up procedures (step 6.13), we obtained a cell yield of up to 1 x 10⁸ cells per isolation from the liver of about 300 g of a rat. Cell viability between 78% and 97% was established by Trypan blue counting.

As already described in previous studies^1,18,19, primary hepatocytes in culture lose their morphology, liver-specific functions, and die within a few days.

Dyskusje

3D-organoids are a frontier for personalized medicine and allow a long-term hepatocyte culture. The quality of this innovative technique requires a good yield of viable primary hepatocytes and well-performed liver perfusion and hepatocytes isolation. This old procedure is still widely used; however, it comprises different steps that can be challenging. Approaching the procedure, we experienced critical issues such as bacterial contamination, low liver digestion efficiency, low primary hepatocyte yield, and low hepatocyte...

Materiały

Name	Company	Catalog Number	Comments
A83-01- ALK5 Inhibitor IV	Twin Helix	T3031
B27	Thermofisher Scientific	0080085SA
CFX Connect Real-Time PCR Detection System	Bio-Rad
CHIR99021	Twin Helix	T2310
Click EdU Alexa 488 imaging kit	Thermofisher Scientific	C10499
Collagen, Type I, solution from rat tail	Merck	C3867-1VL
Dexamethasone	Merck	D4902
EGF	Merck	E9644
Fetal bovine serum (FBS)	Euroclone	ECS0180L
GELTREX LDEV FREE RGF BME	Thermofisher Scientific	A1413202
Heparin Sodium 25000 IU/5 ml	B. Braun Melsungen AG	B01AB01
HGF	Peprotech	100-39H
Insulin-Transferrin-Selenium solution 100x	Thermofisher Scientific	41400045
L-Glutamine solution	Euroclone	ECB3000D
Liver Digest Medium	Thermofisher Scientific	17703-034
Liver Perfusion Medium	Thermofisher Scientific	17701038
N2 supplement	Thermofisher Scientific	17502048
N-acetylcysteine	Merck	A9165
Nicotinamide	Merck	N-0636
Non-Essential Amino Acids	Merck	M7145
Normocin	Aurogene	ant-nr-1
PBS buffer 1X	PanReac AppliChem	A0964,9050
Penicillin-streptomycin solution 100x	Euroclone	ECB3001D
Percoll	Santa Cruz	sc-296039A
Peristaltic pump	Ismatec™	MS-4/12 Reglo Digital Pump
TNFa	Peprotech	300-01A
TRI Reagent	Merck	T9424
Tubing	Ismatec™	ID.2,79mm
Williams' E Medium, no glutamine	Thermofisher Scientific	31415029
Y27632	Twin Helix	T1725