We thank Dr. Davide Selvestrel and prof. Giovanni Sorrentino of the SorrentinoLab at the University of Trieste for helping us perform the EdU proliferation assay. The work was supported by a Banca d&#39;Italia ad hoc grant and intramural FIF grants.

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&#176; 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 Tabl...

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All authors have disclosed any and all conflicts of interest.

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...

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 &#34;the sandwich-culture method,&#34; 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 &#34;domes&#34; 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 groups9,10 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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>A83-01- ALK5 Inhibitor IV</td>
			<td>Twin Helix</td>
			<td>&#160;T3031</td>
			<td></td>
		</tr>
		<tr>
			<td>B27</td>
			<td>Thermofisher Scientific</td>
			<td>0080085SA</td>
			<td></td>
		</tr>
		<tr>
			<td>CFX Connect Real-Time PCR Detection System&#160;</td>
			<td>&#160;Bio-Rad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CHIR99021</td>
			<td>Twin Helix</td>
			<td>T2310</td>
			<td></td>
		</tr>
		<tr>
			<td>Click EdU Alexa 488 imaging kit</td>
			<td>Thermofisher Scientific</td>
			<td>C10499</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen, Type I, solution from rat tail</td>
			<td>Merck</td>
			<td>C3867-1VL</td>
			<td></td>
		</tr>
		<tr>
			<td>Dexamethasone&#160;</td>
			<td>Merck</td>
			<td>D4902</td>
			<td></td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>Merck</td>
			<td>&#160;E9644</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Euroclone</td>
			<td>ECS0180L</td>
			<td></td>
		</tr>
		<tr>
			<td>GELTREX LDEV FREE RGF BME</td>
			<td>Thermofisher Scientific</td>
			<td>A1413202</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin Sodium 25000 IU/5 ml</td>
			<td>B. Braun Melsungen AG</td>
			<td>B01AB01</td>
			<td></td>
		</tr>
		<tr>
			<td>HGF</td>
			<td>Peprotech</td>
			<td>&#160;100-39H&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin-Transferrin-Selenium solution 100x&#160;</td>
			<td>Thermofisher Scientific</td>
			<td>41400045</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine solution</td>
			<td>Euroclone</td>
			<td>ECB3000D</td>
			<td></td>
		</tr>
		<tr>
			<td>Liver Digest Medium&#160;</td>
			<td>Thermofisher Scientific</td>
			<td>&#160;17703-034</td>
			<td></td>
		</tr>
		<tr>
			<td>Liver Perfusion Medium</td>
			<td>Thermofisher Scientific</td>
			<td>17701038</td>
			<td></td>
		</tr>
		<tr>
			<td>N2 supplement</td>
			<td>Thermofisher Scientific</td>
			<td>17502048</td>
			<td></td>
		</tr>
		<tr>
			<td>N-acetylcysteine</td>
			<td>Merck</td>
			<td>A9165</td>
			<td></td>
		</tr>
		<tr>
			<td>Nicotinamide</td>
			<td>Merck</td>
			<td>&#160;N-0636</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-Essential Amino Acids</td>
			<td>Merck</td>
			<td>M7145</td>
			<td></td>
		</tr>
		<tr>
			<td>Normocin</td>
			<td>Aurogene</td>
			<td>ant-nr-1</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS buffer 1X</td>
			<td>PanReac AppliChem</td>
			<td>A0964,9050</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin solution 100x</td>
			<td>Euroclone</td>
			<td>ECB3001D</td>
			<td></td>
		</tr>
		<tr>
			<td>Percoll</td>
			<td>Santa Cruz</td>
			<td>sc-296039A</td>
			<td></td>
		</tr>
		<tr>
			<td>Peristaltic pump</td>
			<td>Ismatec&#8482;&#160;</td>
			<td>MS-4/12 Reglo Digital Pump</td>
			<td></td>
		</tr>
		<tr>
			<td>TNFa</td>
			<td>Peprotech</td>
			<td>&#160;300-01A</td>
			<td></td>
		</tr>
		<tr>
			<td>TRI Reagent</td>
			<td>Merck</td>
			<td>T9424</td>
			<td></td>
		</tr>
		<tr>
			<td>Tubing&#160;</td>
			<td>Ismatec&#8482;&#160;</td>
			<td>&#160;ID.2,79mm</td>
			<td></td>
		</tr>
		<tr>
			<td>Williams&#39; E Medium, no glutamine</td>
			<td>Thermofisher Scientific</td>
			<td>31415029</td>
			<td></td>
		</tr>
		<tr>
			<td>Y27632</td>
			<td>Twin Helix</td>
			<td>T1725</td>
			<td></td>
		</tr>
	</tbody>
</table>

rat liver perfusion and primary hepatocytes isolation: an old procedure crucial for cutting-edge 3d organoids culture

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.

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 studies1,18,19, primary hepatocytes in culture lose their morphology, liver-specific functions, and die within a few days.
<p cla...

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.

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

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 ...

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.

rat-liver-perfusion-primary-hepatocytes-isolation-an-old-procedure

Research

JoVE Journal

Biology

432 Views.  Italian Liver Foundation. 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 ...