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 Table of Materials for commercial details.
<ol>
	<li>Warm the water bath at 42 &#176;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.</li>
	<li>Place William&#39;s complete medium and PBS + 3% P/S on ice.</li>
	<li>Prepare the peristaltic pump and connect the tubing with the glass bottle and the bubble trap, both filled with Perfusion buffer.</li>
	<li>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.</li>
</ol>
2. Preparation for hepatocyte isolation
<ol>
	<li>During the animal procedure, expose the following instruments to UV light: Petri dish 100 mm, cell strainer 100 &#181;m, scraper size M, large tweezers, beaker for ice, tray for ice.</li>
</ol>
3. Initial animal procedure and anesthesia
<ol>
	<li>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).</li>
	<li>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.</li>
	<li>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.</li>
	<li>Place the rat in the middle of the dissection tray and secure the limbs using needles.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Inject 100-150 IU of heparin dissolved in 1x PBS (total volume 300 &#181;L) into the vena cava to prevent blood coagulation.</li>
</ol>
4. Cannulation and liver perfusion
<ol>
	<li>Turn on the pump at a low-speed rate (less than 1 mL/min flow).</li>
	<li>Insert the 18 G angiocath in the portal vein at the level of the thread at a flat angle relative to the vein.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
</ol>
5. Liver digestion
<ol>
	<li>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.</li>
	<li>Increase the pump speed to 20 mL/min and periodically press with the swab for liver swelling and relaxation.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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&#39;s complete medium-containing tube. 
	NOTE: Exsanguination and death ensure perfusion via the vena cava. The confirmation of the animal&#39;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.</li>
</ol>
6. Hepatocyte purification
<ol>
	<li>Under the biological hood, transfer the liver into the Petri dish 100 mm with the 15 mL of cold William&#39;s complete medium over a tray full of ice.</li>
	<li>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.</li>
	<li>Collect the medium-containing cells and filter them with the 100 &#181;m filter while transferring them to a sterile conical tube. The filtration allows the removal of connective tissues and undigested tissue fragments.</li>
	<li>Pour about 15 mL of cold William&#39;s complete medium over the smashed liver in the Petri dish to help release more cells. Repeat steps 6.2 and 6.3.</li>
	<li>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 &#181;m filters. 
	NOTE: After the hepatocytes are released and before filtering, the medium should look opaque because of the hepatocytes&#39; presence. The remaining liver should look fibrous and will be discarded.</li>
	<li>Centrifuge the filtered medium containing hepatic cells at 50 x g for 3 min at 4 &#176;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.</li>
	<li>Discard the supernatant without disturbing the cell pellet. Gently add about 40 mL of ice-cold William&#39;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.</li>
	<li>Centrifuge at 50 x g for 3 min at 4 &#176;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.</li>
	<li>Discard the supernatant and resuspend the cell pellet in 25 mL of ice-cold William&#39;s complete medium.</li>
	<li>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.</li>
	<li>Centrifuge at 200 x g for 10 min at 4 &#176;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.</li>
	<li>Aspirate the dead cell layer from the top of the gradient and leave 1-2 mL of the medium with the pellet.</li>
	<li>Resuspend the pellet in 30-40 mL of William&#39;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&#176;C.</li>
	<li>Centrifuge at 200 x g for 10 min at 4&#176;C, then aspirate the supernatant.</li>
</ol>
7. Hepatocyte culture and RNA collection
<ol>
	<li>Add an appropriate amount of medium for cell counting. Count cell viability with 1:10 Trypan blue.</li>
	<li>2D hepatocyte culture
	<ol>
		<li>Plate cells at the concentration of 500,000 cells/well of 6-well dish on a collagen-coated cell plate with pre-warmed William&#39;s complete medium.</li>
		<li>Place the cell culture in a humidified incubator with 95% air, 5% CO2 at 37 &#176;C.</li>
		<li>After 4 h, change the medium to the new pre-warmed William&#39;s complete medium.</li>
		<li>Keep cells in the incubator. Change the medium daily.</li>
		<li>Collect primary hepatocytes in a reagent for RNA extraction before plating (day 0) and at Days 1 to 3 of culture.</li>
		<li>Extract RNA according to the manufacturer&#39;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.</li>
	</ol>
	</li>
	<li>For 3D long-term hepatocyte culture:
	<ol>
		<li>Resuspend the cells in the cold pure matrix at the concentration of 1,000-10,000 cells in 20 &#181;L. 
		NOTE: Try to avoid making matrix bubbles .</li>
		<li>Using pre-chilled pipette tips, plate matrix-containing cell droplets into pre-warmed cell-culture plate.</li>
		<li>Turn the plate upside down and leave it in the cell culture incubator for 20-30 min.</li>
		<li>Turn it up and add the pre-warmed 3D long-term hepatocyte medium and specific culture medium according to Peng&#39;s work22. Keep cells in the incubator.</li>
		<li>Change the 3D long-term hepatocyte medium every 2-3 days.</li>
		<li>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&#39;s work.</li>
	</ol>
	</li>
</ol>

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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><tbody><tr><td>A83-01- ALK5 Inhibitor IV</td><td>Twin Helix</td><td>&amp;nbsp;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&amp;nbsp;</td><td>&amp;nbsp;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&amp;nbsp;</td><td>Merck</td><td>D4902</td><td></td></tr><tr><td>EGF</td><td>Merck</td><td>&amp;nbsp;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>&amp;nbsp;100-39H&amp;nbsp;</td><td></td></tr><tr><td>Insulin-Transferrin-Selenium solution 100x&amp;nbsp;</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&amp;nbsp;</td><td>Thermofisher Scientific</td><td>&amp;nbsp;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>&amp;nbsp;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&amp;trade;&amp;nbsp;</td><td>MS-4/12 Reglo Digital Pump</td><td></td></tr><tr><td>TNFa</td><td>Peprotech</td><td>&amp;nbsp;300-01A</td><td></td></tr><tr><td>TRI Reagent</td><td>Merck</td><td>T9424</td><td></td></tr><tr><td>Tubing&amp;nbsp;</td><td>Ismatec&amp;trade;&amp;nbsp;</td><td>&amp;nbsp;ID.2,79mm</td><td></td></tr><tr><td>Williams&#039; 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.
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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 ...

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

Research

JoVE Journal

Biology

894 Views.  Italian Liver Foundation. Inleiding.Primaire hepatocyten zijn een belangrijk hulpmiddel voor in vitro levergerelateerde studies. De uitbreiding en het onderhoud van deze cellen zijn echter historisch uitdagend geweest, omdat ze na een paar dagen in cultuur morfologie en functionaliteit verliezen. 3D-organoïden zijn een geavanceerd hulpmiddel dat in staat is om weefsels in een schaal te recapituleren voor een langdurige kweek, waardoor het probleem van het onvermogen om in vitro uit t...

Video: Perfusie van rattenlever en isolatie van primaire hepatocyten: een oude procedure die cruciaal is voor geavanceerde 3D-organoïdencultuur

A Simple and Efficient Method to Isolate Macrophages from Mixed Primary Cultures of Adult Liver Cells

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although the isolation methods of liver macrophages have been well-described4-6, most of these methods require sophisticated equipment, such as a centrifugal elutriator and technical skills. Here, we provide a novel method to obtain liver macrophages in sufficient number and purity from mixed primary cultures of adult rat liver cells, as schematically illustrated in Figure 1.
After dissociation of the liver cells by two-step perfusion method7,8,a fraction mostly composed of parenchymal hepatocytes is prepared and seeded into T75 tissue culture flasks with culture medium composed of DMEM and 10% FCS.Parenchymal hepatocytes lose the epithelial cell morphology within a few days in culture, degenerate or transform into fibroblast-like cells (Figure 2). As the culture proceeds, around day 6, phase contrast-bright, round macrophage-like cells start to proliferate on the fibroblastic cell sheet (Figure 2). The growth of the macrophage-like cells continue and reach to maximum levels around day 12, covering the cell sheet on the flask surface. By shaking of the culture flasks, macrophages are readily suspended into the culture medium. Subsequent transfer and short incubation in plastic dishes result in selective adhesion of macrophages(Figure 3), where as other contaminating cells remain suspended. After several rinses with PBS, attached macrophages are harvested. More than 106 cells can be harvested repeatedly from the same T75 tissue culture flask at two to three day intervals for more than two weeks(Figure 3).The purities of the isolated macrophages were 95 to 99%, as evaluated by flow cytometry or immunocytochemistry with rat macrophage-specific antibodies (Figure 4).The isolated cells show active phagocytosis of polystylene beads (Figure 5), proliferative response to recombinant GM-CSF, secretion of inflammatory/anti-inflammatory cytokines upon stimulation with LPS, and formation of multinucleated giant cells9.
In conclusion, we provide a simple and efficient method to obtain liver macrophages in sufficient number and purity without complex equipment and skills.This method might be applicable to other mammalian species.

A novel method to obtain macrophages from primary culture of rat liver cells is described. This method utilizes the proliferation of macrophages in the culture, followed by shaking of culture flasks and purification by selective attachment to plastic dishes. This technique efficiently provides liver macrophages without complex equipment and skills.

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although ...

Immunology and Infection

Integrative Protocol for Generation of iPSC-Derived 3D Human Hepatic Organoids

Obtaining stable hepatic cells in culture poses a significant challenge for liver studies. Bearing this in mind, an optimized method is depicted utilizing human induced pluripotent stem cells (hiPSCs) to generate 3D cultures of human hepatic organoids (HHOs). The utilization of HHOs offers a valuable approach to understanding liver development, unraveling liver diseases, conducting high-throughput studies for drug development, and exploring the potential for liver transplantation. In the former investigation, through immunofluorescence and quantitative RT-PCR techniques, the progression was monitored, identifying the presence of various cell populations, such as hepatoblasts and the two types of hepatoblast-derived cells: cholangiocytes or hepatocyte-like cells, across different developmental stages. This report presents a straightforward 3D protocol starting from hiPSC to acquire HHOs that mirror the stages of human embryo development. The protocol, spanning 46-50 days, encompasses several steps: (i) meticulous management of hiPSC culture to generate HHOs, (ii) initiation of cell differentiation in 2D and the subsequent transition to 3D, and (iii) an optimized dissociation strategy to break down HHOs into single cells for single-cell RNA sequencing. As an illustration of the broad applications of this approach, the present protocol was previously applied to unravel the role of thyroid hormone signaling in developing liver cells.

Human iPSC-derived 3D hepatic organoids constitute a potential tool for understanding the thyroid hormone's action on liver development.

Obtaining stable hepatic cells in culture poses a significant challenge for liver studies. Bearing this in mind, an optimized method is depicted utilizing ...

Developmental Biology

"Liver-on-a-Chip" Cultures of Primary Hepatocytes and Kupffer Cells for Hepatitis B Virus Infection

Despite the exceptional infectivity of the hepatitis B virus (HBV) in vivo, where only three viral genomes can result in a chronicity of experimentally infected chimpanzees, most in vitro models require several hundreds to thousands of viral genomes per cell in order to initiate a transient infection. Additionally, static 2D cultures of primary human hepatocytes (PHH) allow only short-term studies due to their rapid dedifferentiation. Here, we describe 3D liver-on-a-chip cultures of PHH, either in monocultures or in cocultures with other nonparenchymal liver-resident cells. These offer a significant improvement to studying long-term HBV infections with physiological host cell responses. In addition to facilitating drug efficacy studies, toxicological analysis, and investigations into pathogenesis, these microfluidic culture systems enable the evaluation of curative therapies for HBV infection aimed at eliminating covalently closed, circular (ccc)DNA. This presented method describes the set-up of PHH monocultures and PHH/Kupffer cell co-cultures, their infection with purified HBV, and the analysis of host responses. This method is particularly applicable to the evaluation of long-term effects of HBV infection, treatment combinations, and pathogenesis.

The goal of this protocol is to provide a step-by-step guide to perform 3-D &#34;liver-on-a-chip&#34; infection experiments with the hepatitis B virus.

Despite the exceptional infectivity of the hepatitis B virus (HBV) in vivo, where only three viral genomes can result in a chronicity of experimentally ...

Isolation and Primary Culture of Rat Hepatic Cells

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology and other related subjects. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 1 and, since then, has undergone many modifications. The most commonly used technique was described by Seglenin 1976 2. Essentially, hepatocytes are dissociated from anesthetized adult rats by a non-recirculating collagenase perfusion through the portal vein. The isolated cells are then filtered through a 100 &mu;m pore size mesh nylon filter, and cultured onto plates. After 4-hour culture, the medium is replaced with serum-containing or serum-free medium, e.g. HepatoZYME-SFM, for additional time to culture. These procedures require surgical and sterile culture steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol, which allow consistently in the generation of viable hepatocytes in large numbers.

Primary hepatocytes provide a valuable tool to evaluate biochemical, molecular, and metabolic functions in a physiologically relevant experimental system. We describe a reliable protocol for rat in situ liver perfusion, which consistently generates viable hepatocytes up to 1.0 &times; 108 cells per preparation with cell viability between 88 ~ 96%.

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

Human Liver Spheroids from Peripheral Blood for Liver Disease Studies

Human liver cells can form a three-dimensional (3D) structure capable of growing in culture for some weeks, preserving their functional capacity. Due to their nature to cluster in the culture dishes with low or no adhesive characteristics, they form aggregates of multiple liver cells that are called human liver spheroids. The forming of 3D liver spheroids relies on the natural tendency of hepatic cells to aggregate in the absence of an adhesive substrate. These 3D structures possess better physiological responses than cells, which are closer to an in vivo environment. Using 3D hepatocyte cultures has numerous advantages when compared with classical two-dimensional (2D) cultures, including a more biologically relevant microenvironment, architectural morphology that reassembles natural organs as well as a better prediction regarding disease state and in vivo-like responses to drugs. Various sources can be used to generate spheroids, like primary liver tissue or immortalized cell lines. The 3D liver tissue can also be engineered by using human embryonic stem cells (hESCs) or induced pluripotent stem cells (hiPSCs) to derive hepatocytes. We have obtained human liver spheroids using blood-derived pluripotent stem cells (BD-PSCs) generated from unmanipulated peripheral blood by activation of human membrane-bound GPI-linked protein and differentiated to human hepatocytes. The BD-PSCs-derived human liver cells and human liver spheroids were analyzed by light microscopy and immunophenotyping using human hepatocyte markers.

Here we present a non-genetic method to generate human autologous liver spheroids using mononuclear cells isolated from steady-state peripheral blood.

Human liver cells can form a three-dimensional (3D) structure capable of growing in culture for some weeks, preserving their functional capacity. Due to ...

Bioengineering

Isolation of Human Hepatocytes by a Two-step Collagenase Perfusion Procedure

The liver, an organ with an exceptional regeneration capacity, carries out a wide range of functions, such as detoxification, metabolism and homeostasis. As such, hepatocytes are an important model for a large variety of research questions. In particular, the use of human hepatocytes is especially important in the fields of pharmacokinetics, toxicology, liver regeneration and translational research. Thus, this method presents a modified version of a two-step collagenase perfusion procedure to isolate hepatocytes as described by Seglen 1.
Previously, hepatocytes have been isolated by mechanical methods. However, enzymatic methods have been shown to be superior as hepatocytes retain their structural integrity and function after isolation. This method presented here adapts the method designed previously for rat livers to human liver pieces and results in a large yield of hepatocytes with a viability of 77&plusmn;10%. The main difference in this procedure is the process of cannulization of the blood vessels. Further, the method described here can also be applied to livers from other species with comparable liver or blood vessel sizes.

A modified two-step collagenase perfusion procedure for isolation of human hepatocytes is described. This method can also be applied to other mammalian livers. The isolated hepatocytes are available in high yield and viability, making them a suitable model for scientific research in areas such as liver regeneration, pharmacokinetics and toxicology.

The liver, an organ with an exceptional regeneration capacity, carries out a wide range of functions, such as detoxification, metabolism and homeostasis. ...

Protocol for Isolation of Primary Human Hepatocytes and Corresponding Major Populations of Non-parenchymal Liver Cells

Beside parenchymal hepatocytes, the liver consists of non-parenchymal cells (NPC) namely Kupffer cells (KC), liver endothelial cells (LEC) and hepatic Stellate cells (HSC). Two-dimensional (2D) culture of primary human hepatocyte (PHH) is still considered as the &#34;gold standard&#34; for in vitro testing of drug metabolism and hepatotoxicity. It is well-known that the 2D monoculture of PHH suffers from dedifferentiation and loss of function. Recently it was shown that hepatic NPC play a central role in liver (patho-) physiology and the maintenance of PHH functions. Current research focuses on the reconstruction of in vivo tissue architecture by 3D- and co-culture models to overcome the limitations of 2D monocultures. Previously we published a method to isolate human liver cells and investigated the suitability of these cells for their use in cell cultures in Experimental Biology and Medicine1. Based on the broad interest in this technique the aim of this article was to provide a more detailed protocol for the liver cell isolation process including a video, which will allow an easy reproduction of this technique.
Human liver cells were isolated from human liver tissue samples of surgical interventions by a two-step EGTA/collagenase P perfusion technique. PHH were separated from the NPC by an initial centrifugation at 50 x g. Density gradient centrifugation steps were used for removal of dead cells. Individual liver cell populations were isolated from the enriched NPC fraction using specific cell properties and cell sorting procedures. Beside the PHH isolation we were able to separate KC, LEC and HSC for further cultivation.
Taken together, the presented protocol allows the isolation of PHH and NPC in high quality and quantity from one donor tissue sample. The access to purified liver cell populations could allow the creation of in vivo like human liver models.

A technique to isolate human hepatocytes and non-parenchymal liver cells from the same donor is described. The different liver cell types build the basis for functional liver models and tissue engineering. This new method aims to isolate liver cells in a high yield and viability.

Beside parenchymal hepatocytes, the liver consists of non-parenchymal cells (NPC) namely Kupffer cells (KC), liver endothelial cells (LEC) and hepatic ...

Isolation and 3D Collagen Sandwich Culture of Primary Mouse Hepatocytes to Study the Role of Cytoskeleton in Bile Canalicular Formation In Vitro

Hepatocytes are the central cells of the liver responsible for its metabolic function. As such, they form a uniquely polarized epithelium, in which two or more hepatocytes contribute apical membranes to form a bile canalicular network through which bile is secreted. Hepatocyte polarization is essential for correct canalicular formation and depends on interactions between the hepatocyte cytoskeleton, cell-cell contacts, and the extracellular matrix. In vitro studies of hepatocyte cytoskeleton involvement in canaliculi formation and its response to pathological situations are handicapped by the lack of cell culture, which would closely resemble the canaliculi network structure in vivo. Described here is a protocol for the isolation of mouse hepatocytes from the adult mouse liver using a modified collagenase perfusion technique. Also described is the production of culture in a 3D collagen sandwich setting, which is used for immunolabeling of cytoskeletal components to study bile canalicular formation and its response to treatments in vitro. It is shown that hepatocyte 3D collagen sandwich cultures respond to treatments with toxins (ethanol) or actin cytoskeleton altering drugs (e.g., blebbistatin) and serve as a valuable tool for in vitro studies of bile canaliculi formation and function.

Presented here is a protocol for the isolation of mouse hepatocytes from adult mouse livers using a modified collagenase perfusion technique. Also described is the long-term culture of hepatocytes in a 3D collagen sandwich setting as well as immunolabeling of the cytoskeletal components to study bile canalicular formation and its response to treatment.

Hepatocytes are the central cells of the liver responsible for its metabolic function. As such, they form a uniquely polarized epithelium, in which two or ...

An Improved Time- and Labor- Efficient Protocol for Mouse Primary Hepatocyte Isolation

Primary hepatocytes are used extensively in liver in vitro research, especially in glucose metabolism studies. A base technique has been adapted based on different needs, like time, labor, cost, and primary hepatocyte usage, resulting in various primary hepatocyte isolation protocols. However, the numerous steps and time-consuming reagent preparations in primary hepatocyte isolation are major drawbacks for efficiency. After comparing different protocols for their pros and cons, the advantages of each were combined, and a rapid and efficient primary hepatocyte isolation protocol was formulated. Within only ~35 min, this protocol could yield as much, if not more, healthy primary hepatocytes as other protocols. Further, glucose metabolism experiments performed using the isolated primary hepatocytes validated the usefulness of this protocol in in vitro liver metabolism studies. We also extensively reviewed and analyzed the significance and purpose of each step in this study so that future researchers can further optimize this protocol based on needs.

Primary hepatocytes are a valuable tool to study liver response and metabolism in vitro. Utilizing commercially available reagents, an improved time- and labor-efficient protocol for mouse primary hepatocyte isolation was developed.

Primary hepatocytes are used extensively in liver in vitro research, especially in glucose metabolism studies. A base technique has been adapted based on ...

Isolation of Primary Rat Hepatocytes with Multiparameter Perfusion Control

Primary hepatocytes are widely used in basic research on liver diseases and for toxicity testing in vitro. The two-step collagenase perfusion procedure for primary hepatocyte isolation is technically challenging, especially in portal vein cannulation. The procedure is also prone to occasional contamination and variations in perfusion conditions due to difficulties in the assembly, optimization, or maintenance of the perfusion setup. Here, a detailed protocol for an improved two-step collagenase perfusion procedure with multiparameter perfusion control is presented. Primary rat hepatocytes were successfully and reliably isolated by taking the necessary technical precautions at critical steps of the procedure, and by reducing the operational difficulty and mitigating the variability of perfusion parameters through the adoption of a special intravenous catheter, standardized sterile disposable tubing, temperature control, and real-time monitoring and alarm system. The isolated primary rat hepatocytes consistently exhibit high cell viability (85%-95%), yield (2-5 x 108 cells per 200-300 g rat) and functionality (albumin, urea and CYP activity). The procedure was complemented by an integrated perfusion system, which is compact enough to be set up in the laminar flow hood to ensure aseptic operation.

This protocol details the use of a special intravenous catheter, standardized sterile disposable tubing, temperature control complemented by real-time monitoring, and an alarm system for two-step collagenase perfusion procedure to improve the consistency in the viability, yield, and functionality of isolated primary rat hepatocytes.

Primary hepatocytes are widely used in basic research on liver diseases and for toxicity testing in vitro. The two-step collagenase perfusion procedure ...

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

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