This work was supported by the Grant Agency of the Czech Republic (18-02699S); the Grant Agency of the Ministry of Health of the Czech Republic (17-31538A); the Institutional Research Project of the Czech Academy of Sciences (RVO 68378050) and MEYS CR projects (LQ1604 NPU II, LTC17063, LM2015040, OP RDI CZ.1.05/2.1.00/19.0395, and OP RDE CZ.02.1.01/0.0/0.0/16_013/0001775); Charles University (personal stipend to K.K.), and an Operational Program Prague&#8211;Competitiveness project (CZ.2.16/3.1.00/21547). We acknowledge the Light Microscopy Core Facility, IMG CAS, Prague, Czech Republic (supported MEYS CR projects LM2015062 and LO1419) for support with the microscopy imaging presented.

All animal experiments were performed in accordance with European Directive 2010/63/EU and they were approved by the Czech Central Commission for Animal Welfare.
1. Materials
<ol>
	<li>House animals under specific pathogen-free conditions according to the guidelines of the Federation for Laboratory Animal Science Associations with free access to regular chow and drinking water. House animals under a 12 h/12 h dark/light cycle. For hepatocyte isolation and culture, use 8&#8211;12 week-old animals.</li>
	<li>Stock solutions A and B
	<ol>
		<li>Prepare stock solution A and stock solution B according to Table 1 and Table 2, respectively, in advance. Dissolve all components in 1 L of distilled H2O (dH2O).</li>
		<li>Adjust the pH of solutions to 7.2 and filter the solutions through a 0.2 &#956;m filter. Both solutions can be stored at 4 &#176;C for up to 6 months.</li>
	</ol>
	</li>
	<li>Stock solution C
	<ol>
		<li>Prepare solution C on the day of primary hepatocyte isolation.</li>
		<li>Add all components according to Table 3, fill to a 50 mL total volume with dH2O, and dissolve. Adjust the pH to 7.3.</li>
		<li>Aliquot 50 mL of the solution in 50 mL tubes. Use one tube per animal. Place all required aliquots in a pre-warmed water bath (37 &#176;C).</li>
	</ol>
	</li>
	<li>Stock solution D
	<ol>
		<li>Prepare solution D on the day of primary hepatocyte isolation.</li>
		<li>Add all components according to Table 4, fill to a 30 mL total volume with dH2O, and dissolve. Adjust the pH to 7.3.</li>
		<li>Aliquot 30 mL of the solution in 50 mL tubes. Add collagenase I (5 mg/30 mL) into solution D. Use one aliquot per animal. Place all aliquots in a pre-warmed water bath (37 &#176;C).</li>
	</ol>
	</li>
	<li>Stock solution E
	<ol>
		<li>Prepare solution E on the day of primary hepatocyte isolation.</li>
		<li>Add all components according to Table 5, fill to a 50 mL total volume with dH2O, and dissolve. Adjust the pH to 7.3.</li>
		<li>Aliquot 50 mL of the solution in 50 mL tubes. Add albumin V (0.65 g/50 mL) into solution E. Use one aliquot per animal. Place all aliquots in a pre-warmed water bath (37 &#176;C).</li>
	</ol>
	</li>
</ol>
2. Preparation of collagen sandwiches
<ol>
	<li>One day before primary hepatocyte isolation, prepare the first layer of the collagen I sandwich. 
	NOTE: Work on ice and use pre-chilled solutions, tips, plates, and tubes to minimize unwanted gelation of collagen I.</li>
	<li>Neutralize the required amount of collagen I (from rat tail) according to the manufacturer&#39;s protocol. A volume of 100 &#181;L of neutralized collagen (1.5 mg/mL) per experimental sample (3.5 cm dish) is required. To prepare 1 mL of neutralized collagen (1.5 mg/mL), add 100 &#956;L of 10x DMEM, 1.5 &#956;L of 1M NaOH, and 48.5 &#956;L of dH2O into 500 &#956;L of collagen (stock concentration = 3 mg/mL). Check the pH of neutralized collagen with litmus paper (the pH should be ~7.5).</li>
	<li>Disperse 100 &#956;L of neutralized collagen solution evenly using a pre-chilled 200 &#956;L tip over the surface of a 3.5 cm dish set on ice.</li>
	<li>Incubate overnight under standard culture conditions (incubator with 5% CO2 at 37 &#176;C). On the day of primary hepatocyte isolation, add 1 mL of pre-warmed (37 &#176;C) PBS to the first collagen layer. Allow the collagen to rehydrate for 2-3 h at 37 &#176;C.</li>
</ol>
3. Equipment set up
<ol>
	<li>Prepare all equipment as listed in Table of Materials and set up as shown in Figure 1.</li>
</ol>
4. Surgical procedure
<ol>
	<li>Anesthetize the mouse by intramuscular injection of tiletamine (60 mg/kg of body weight), zolazepam (60 mg/kg), and xylazine (4.5 mg/kg). After several minutes, confirm proper anesthetization by the toe pinch. If animal does not respond to the pinch, proceed to 4.2.</li>
	<li>Place the anesthetized mouse on a dissection mat and tape the lower and upper extremities to fix the mouse in a supine position. Swab the abdomen with 70% ethanol and open the abdomen with a V-shape incision from the pubic area to front legs. Fold the skin over the chest to uncover the abdominal cavity. Place the dissecting mat under a dissecting microscope.</li>
	<li>Bend an insulin syringe needle (30 G) to a 45&#176; angle. Expose the inferior vena cava (IVC) by moving the intestines and colon in the caudal direction.</li>
	<li>Fill 2.5 mL of pre-warmed (37 &#176;C) solution C into a 2 mL syringe with a cannula. Ensure that there are no air bubbles in the cannula or syringe.</li>
	<li>Before cannulation of the IVC, reposition the liver lobes by pressing them up to the diaphragm with a wet (PBS) cotton swab. Place a silk suture around the IVC just below the liver (Figure 2A,B).</li>
	<li>Inject 10 &#181;L of heparin (5000 U/mL) into the portal vein using an insulin syringe with a 30 G needle bent at a 45&#176; angle (Figure 2C).</li>
	<li>To cannulate the liver, make a small incision with microsurgical scissors to the IVC directly next to the liver (below the suture; Figure 2D) that is large enough to insert the cannula. Secure the cannula in the position using sutures and two surgical knots (Figure 2E).</li>
	<li>Cut the portal vein (Figure 2F) to allow the perfusion buffers to flow out from the liver to prevent expansion of the liver.</li>
	<li>Manually perfuse the liver with 1.5 mL of pre-warmed solution C by slowly pressing the syringe connected to the cannula (this should take ~15 s). The removal of blood from the liver by discoloration of the liver can now be observed.</li>
	<li>Pre-fill a peristaltic pump with pre-warmed (37 &#176;C) solution C. Check the perfusion apparatus and ensure that there are no air bubbles in the system.</li>
	<li>Cautiously disconnect the cannula from the syringe and connect it to the tubing of the peristaltic pump running at flow rate of 2.5 mL/min. Work quickly but carefully and ensure that the cannula remains in position and that no bubbles enter the tubing or cannula.</li>
	<li>Perfuse the liver with solution C for 2 min (5 mL of solution C). Change to solution D and continue the perfusion for an additional 10 min (25 mL of solution D).</li>
	<li>Once the liver has been perfused, remove it from the abdominal cavity. The liver will now be very fragile and pale in color (Figure 3).</li>
</ol>
5. Isolation of primary hepatocytes
<ol>
	<li>Remove the liver from the mouse. The cannula will still be tied to the liver, so use the forceps to lift the cannula with the liver, and carefully cut off all fascia connections. Transfer the liver to a 50 mL tube containing 20 mL of pre-warmed solution E.</li>
	<li>Hold the cannula with the liver using forceps and disassociate the tissue by rubbing the liver around the wall of the tube to transfer the isolated hepatocytes into buffer. Keep the isolated cells on ice. 
	NOTE: Isolated primary hepatocytes are viable for several hours while kept on ice. Users can repeat steps 4.1 through 5.2 isolating primary hepatocytes from another donor mouse, if necessary, or proceed to the next step.</li>
	<li>Place a 70 &#181;m nylon cell strainer on top of a 50 mL tube and filter the isolated cells.</li>
	<li>Centrifuge the tube at 50 x g and 4 &#176;C for 5 min. Aspirate the supernatant. To remove dead cells and increase the percentage of viable cells, resuspend the pellet in 20 mL of 40% percol in DMEM.</li>
	<li>Centrifuge the tube at 50 x g and 4 &#176;C for 5 min. Aspirate the supernatant containing dead cells and resuspend the pellet in 20 mL of solution E.</li>
	<li>Centrifuge tubes at 50 x g and 4 &#176;C for 5 min. Aspirate the supernatant and resuspend the pellet in 10 mL of solution E.</li>
	<li>Check the primary hepatocyte yield and viability using trypan blue staining. Count the cell number using a Neubauer cell counting chamber and adjust the cell concentration to 3.75 x 105 viable cells/mL. Keep the cells on ice.</li>
</ol>
6. Cultivation of primary hepatocytes in 3D collagen sandwiches
<ol>
	<li>Prepare hepatocyte culture medium (HCM). Add 15 &#181;L of glucagon (1 mg/mL), 15 &#181;L of hydrocortisone (50 mg/mL), and 40 &#181;L of insulin (10 mg/mL) to 50 mL of complete medium (DMEM, high glucose, 10% FBS, 1% penicillin-streptomycin).</li>
	<li>Evenly disperse 2 mL (5 x 105 cells/mL) of viable primary hepatocytes in a pre-coated 3.5 cm dish. Incubate the cells with 5% CO2 at 37 &#176;C for 3 h. Important: Evenly distribute the cells by tilting the dish in all directions just before putting the dish into incubator.</li>
	<li>Prepare neutralized collagen I (100 &#181;L/3.5 cm dish; i.e., a sufficient volume for all dishes as described above [step 2.1]). Keep on ice till use.</li>
	<li>After 3 h, carefully remove the medium and unattached cells and add 100 &#181;L of neutralized collagen I to each 3.5 cm dish to form the top layer of collagen sandwich on cells. Incubate the collagen sandwich under standard cell-culture conditions (5% CO2 at 37 &#176;C) for 1 h. After a 1 h incubation, carefully add 2 mL of HCM.</li>
	<li>After 24 h of culture, change the HCM medium for a fresh one.</li>
	<li>Culture for 3&#8211;8 days, depending on the formation of bile canaliculi. Check the culture every day under a microscope (Figure 4). Change the HCM every second day.</li>
</ol>
7. Immunolabeling of primary hepatocytes in 3D collagen sandwiches
<ol>
	<li>Remove the media from the hepatocyte sandwiches, then wash carefully with pre-warmed PBS. Fix the sandwich cultures with 1 mL of 4% paraformaldehyde in PBS for 30 min at room temperature (RT).</li>
	<li>After fixation, wash the sandwiches 3x for 10 min in 2 mL of PBS + 0.1% Tween 20 (PBS-T).</li>
	<li>Permeabilize cells with 1 mL of 0.1 M glycine, 0.2% Triton X-100 in PBS at RT for 1 h. Wash 3x for 10 min in PBS-T.</li>
	<li>Gently disturb the top layer of collagen using a 10 &#956;L loading tip connected to a vacuum aspiration to ensure sufficient antibody penetration.</li>
	<li>Block with 1 mL of 5% BSA in PBS-T (i.e., blocking solution) for 2 h. Incubate with primary antibodies diluted in blocking solution overnight at 37 &#176;C. Wash 3x for 15 min in PBS-T.</li>
	<li>After washing, incubate with secondary antibody at 37 &#176;C for 5 h. Wash 3x for 15 min in PBS-T followed by 1x wash with distilled H2O.</li>
	<li>Mount with anti-fade mounting media (see Table of Materials) for microscopy.</li>
</ol>

<ol>
	<li>Gissen, P., Arias, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+and+functional+hepatocyte+polarity+and+liver+disease.">Structural and functional hepatocyte polarity and liver disease.</a> Journal of Hepatology. 63 (4), 1023-1037 (2015).</li><li>LeCluyse, E. L., Fix, J. A., Audus, K. L., Hochman, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+and+maintenance+of+bile+canalicular+networks+in+collagen-sandwiched+hepatocytes.">Regeneration and maintenance of bile canalicular networks in collagen-sandwiched hepatocytes.</a> Toxicology In Vitro. 14 (2), 117-132 (2000).</li><li>Tsukada, N., Ackerley, C. A., Phillips, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+structure+and+organization+of+the+bile+canalicular+cytoskeleton+with+special+reference+to+actin+and+actin-binding+proteins.">The structure and organization of the bile canalicular cytoskeleton with special reference to actin and actin-binding proteins.</a> Hepatology. 21 (4), 1106-1113 (1995).</li><li>Herr, K. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+alpha-catenin+elicits+a+cholestatic+response+and+impairs+liver+regeneration.">Loss of alpha-catenin elicits a cholestatic response and impairs liver regeneration.</a> Scientific Reports. 4, 6835 (2014).</li><li>Yeh, T. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver-specific+beta-catenin+knockout+mice+have+bile+canalicular+abnormalities,+bile+secretory+defect,+and+intrahepatic+cholestasis.">Liver-specific beta-catenin knockout mice have bile canalicular abnormalities, bile secretory defect, and intrahepatic cholestasis.</a> Hepatology. 52 (4), 1410-1419 (2010).</li><li>Pradhan-Sundd, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual+catenin+loss+in+murine+liver+causes+tight+junctional+deregulation+and+progressive+intrahepatic+cholestasis.">Dual catenin loss in murine liver causes tight junctional deregulation and progressive intrahepatic cholestasis.</a> Hepatology. 67 (6), 2320-2337 (2018).</li><li>Theard, D., Steiner, M., Kalicharan, D., Hoekstra, D., van Ijzendoorn, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+polarity+development+and+protein+trafficking+in+hepatocytes+lacking+E-cadherin/beta-catenin-based+adherens+junctions.">Cell polarity development and protein trafficking in hepatocytes lacking E-cadherin/beta-catenin-based adherens junctions.</a> Molecular Biology of the Cell. 18 (6), 2313-2321 (2007).</li><li>Jirouskova, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plectin+controls+biliary+tree+architecture+and+stability+in+cholestasis.">Plectin controls biliary tree architecture and stability in cholestasis.</a> Journal of Hepatology. 68 (5), 1006-1017 (2018).</li><li>Fu, D., Wakabayashi, Y., Ido, Y., Lippincott-Schwartz, J., Arias, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+bile+canalicular+network+formation+and+maintenance+by+AMP-activated+protein+kinase+and+LKB1.">Regulation of bile canalicular network formation and maintenance by AMP-activated protein kinase and LKB1.</a> Journal of Cell Science. 123, 3294-3302 (2010).</li><li>Woods, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LKB1+is+required+for+hepatic+bile+acid+transport+and+canalicular+membrane+integrity+in+mice.">LKB1 is required for hepatic bile acid transport and canalicular membrane integrity in mice.</a> Biochemical Journal. 434 (1), 49-60 (2011).</li><li>Porat-Shliom, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver+kinase+B1+regulates+hepatocellular+tight+junction+distribution+and+function+in+vivo.">Liver kinase B1 regulates hepatocellular tight junction distribution and function in vivo.</a> Hepatology. 64 (4), 1317-1329 (2016).</li><li>Sarnova, L., Gregor, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biliary+system+architecture:+experimental+models+and+visualization+techniques.">Biliary system architecture: experimental models and visualization techniques.</a> Physiological Research. 66 (3), 383-390 (2017).</li><li>Talamini, M. A., Kappus, B., Hubbard, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repolarization+of+hepatocytes+in+culture.">Repolarization of hepatocytes in culture.</a> Hepatology. 25 (1), 167-172 (1997).</li><li>Bhandari, R. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver+tissue+engineering:+a+role+for+co-culture+systems+in+modifying+hepatocyte+function+and+viability.">Liver tissue engineering: a role for co-culture systems in modifying hepatocyte function and viability.</a> Tissue Engineering. 7 (3), 345-357 (2001).</li><li>Godoy, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+2D+and+3D+in+vitro+systems+using+primary+hepatocytes,+alternative+hepatocyte+sources+and+non-parenchymal+liver+cells+and+their+use+in+investigating+mechanisms+of+hepatotoxicity,+cell+signaling+and+ADME.">Recent advances in 2D and 3D in vitro systems using primary hepatocytes, alternative hepatocyte sources and non-parenchymal liver cells and their use in investigating mechanisms of hepatotoxicity, cell signaling and ADME.</a> Archives of Toxicology. 87 (8), 1315 (2013).</li><li>Wilkening, S., Stahl, F., Bader, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+primary+human+hepatocytes+and+hepatoma+cell+line+Hepg2+with+regard+to+their+biotransformation+properties.">Comparison of primary human hepatocytes and hepatoma cell line Hepg2 with regard to their biotransformation properties.</a> Drug Metabolism and Disposition. 31 (8), 1035-1042 (2003).</li><li>Strnad, P., Windoffer, R., Leube, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+detection+of+cytokeratin+filament+network+breakdown+in+cells+treated+with+the+phosphatase+inhibitor+okadaic+acid.">In vivo detection of cytokeratin filament network breakdown in cells treated with the phosphatase inhibitor okadaic acid.</a> Cell and Tissue Research. 306 (2), 277-293 (2001).</li><li>Chalupsky, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ADAM10/17-Dependent+Release+of+Soluble+c-Met+Correlates+with+Hepatocellular+Damage.">ADAM10/17-Dependent Release of Soluble c-Met Correlates with Hepatocellular Damage.</a> Folia Biologica. 59 (2), 76-86 (2013).</li><li>Li, W. C., Ralphs, K. L., Tosh, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+adult+mouse+hepatocytes.">Isolation and culture of adult mouse hepatocytes.</a> Methods in Molecular Biology. 633, 185-196 (2010).</li><li>Horner, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+Percoll+purification+on+isolation+of+primary+human+hepatocytes.">Impact of Percoll purification on isolation of primary human hepatocytes.</a> Scientific Reports. 9 (1), 6542 (2019).</li><li>Severgnini, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+two-step+method+for+isolation+of+functional+primary+mouse+hepatocytes:+cell+characterization+and+asialoglycoprotein+receptor+based+assay+development.">A rapid two-step method for isolation of functional primary mouse hepatocytes: cell characterization and asialoglycoprotein receptor based assay development.</a> Cytotechnology. 64 (2), 187-195 (2012).</li></ol>

The authors have nothing to disclose.

The use of mouse primary hepatocyte cultures is important for in vitro studies to better understand the signaling processes involved in the establishment of hepatocyte polarization, proper canalicular structure formation, and bile secretion. The challenges in isolation and long-term culture of mouse primary hepatocytes in 2D culture have driven the invention of several technical approaches with increased isolation effectivity and longevity of isolated cells, each with several advantages and disadvantages. It is now widely accepted that 2D cultures of primary hepatocytes mimic only limited number of attributes of liver biology for a short period of time. Thus, 3D cultivation in a collagen sandwich arrangement is widely replacing the 2D conditions, particularly when focused on function of the cytoskeleton in liver biology (e.g., toxic drug effects or spatial organization of bile transport).
Since the 1980s, several protocols for isolation of mouse hepatocytes with various modifications have been described. The two-step collagenase perfusion approach has become widely used in many laboratories. The addition of gradient centrifugation into the isolation protocol allows the removal of dead cells19,20 and significantly increases the number of viable cells (here, routinely to ~93%). Even though this step extends the handling time of the cells and results in reduced cell numbers21, this step is viewed as necessary in the 3D collagen sandwich culture for proper bile canalicular network formation. Additionally, it is more important to proceed quickly and accurately during perfusion steps, which shortens the time the cells are handled.
Other important factors that increase the viability of the cells and their ability to form canalicular networks in 3D is the usage of freshly prepared solutions and avoidance of bubbles during perfusion. Therefore, solutions should be prepared on the day of mouse hepatocyte isolation, and the peristaltic pump and tubing should be checked when changing solutions. If the protocol is closely followed, the isolation of primary hepatocytes should be successful with a high yield of viable cells.
Another critical factor in long-term 3D hepatocyte culture is the initial source of primary hepatocytes that are used. It is important to use animals that are 8&#8211;12 weeks old, which serve as optimal donors of hepatocytes. The use of hepatocytes from older animals was not as successful in long-term culture, as these hepatocytes changed their morphology more often, depolarized, and ceased to form canalicular networks. Also, plating hepatocytes on properly neutralized collagen gel formed from relatively highly concentrated solution is a vital step. In most protocols, concentrations of about 1 mg/mL are used. After many optimizations, a concentration of 1.5 mg/mL is optimal for long-term hepatocyte cultivation and provides highly organized hepatocytes with formed bile canaliculi.
This easy-to-follow protocol allows long-term cultivation of primary mouse hepatocytes. Representative results demonstrate a broad spectrum of use for 3D cultured primary mouse hepatocytes when studying the role of cytoskeletal components in bile canaliculi formation.

Hepatocytes, the central cellular structures of the liver that are responsible for its metabolic functions, are uniquely polarized epithelial cells. Their polarization, appearing in mammals shortly after birth, results in formation of the biliary canalicular network and is essential for proper bile secretion. Apical membranes of hepatocytes collectively form bile canaliculi, whereas basal membranes remain in contact with the endothelium of sinusoids. The loss of hepatocyte polarization leads to redistribution of bile transporters and results in pathological processes connected with bile retention in the liver (i.e., cholestasis).
The establishment and maintenance of hepatocyte polarization and the development of bile canaliculi entail complex mechanisms. The underlying processes depend on collective interplay among the hepatocyte cytoskeleton, cell-cell contacts, and interactions with the extracellular matrix1. The hepatocyte cytoskeleton consists of all three filament networks, the actin cytoskeleton, microtubules, and intermediate filaments, which provide structural support for canalicular formation. The differential role of cytoskeletal components in the regeneration and maintenance of bile canalicular networks has been previously illustrated in vitro in 3D collagen-sandwich hepatocyte cultures2.
Actin microfilaments and microtubules are important during the initial stages of hepatocyte membrane polarization at the sites of canaliculus generation2. The actin cytoskeleton establishes the structure and function of bile canaliculi, forming membrane-associated microfilaments and the circumferential ring, thus supporting the canalicular architecture and inserting the actin cytoskeleton into tight and adherens junctions3. The ring of keratin intermediate filaments outside the actin cytoskeleton further stabilizes the canalicular structure3.
The importance of proteins in hepatocyte junctional complexes in the organization of bile canaliculi architecture has been well-documented in several knock-out mouse models, which show distorted canaliculi in mice lacking both tight and/or adherens junctional proteins4,5,6. The deletion of the adherens junction protein &#945;-catenin has been shown to lead to disorganization of the hepatocyte actin cytoskeleton, dilatation of bile canalicular lumens, leaky tight junctions, and effectively to a cholestatic phenotype4. Moreover, in vitro studies have shown the importance of adherens junction components E-cadherin and &#946;-catenin in remodeling of the hepatocellular apical lumen and protein trafficking7.
Strikingly, ablation of the cytoskeleton crosslinking protein plectin, which is the major keratin organizer, has revealed phenotypes comparable to those linked to the actin cytoskeleton8. This suggests a critical role of keratin intermediate filaments in supporting of the canalicular structure. In vitro studies utilizing 3D hepatocyte collagen sandwiches have also shown the importance of the AMP-activated protein kinase and its upstream activator LKB1 in bile canalicular network formation9. These findings were then further confirmed by subsequent in vivo studies10,11. Thus, it has become clear that in vitro studies are necessary to further the understanding of signaling processes involved in the establishment of hepatocyte polarization, proper canalicular network formation, and bile secretion.
A major challenge in studying processes connected with bile canalicular formation and its response to pathological situations in vitro is using a cell culture conditions that closely resemble the situation in vivo12. Freshly isolated primary hepatocytes are not polarized; thus, they lose their function, morphology, and functional bile canaliculi in 2D culture conditions (e.g., changes in gene regulation, polarization, and de-differentiation13,14,15). Despite this fact, freshly isolated hepatocytes most closely reflect the nature of the liver in vivo, unlike liver-derived cell lines16. Even though they have been used in the past, immortalized cell lines do not exert the epithelial-like characteristic morphology of hepatocytes, and the bile canalicular lumens formed by these cells resemble liver canaliculi poorly7. Recently, 3D cultures of primary hepatocytes, from both mice and rats, have become a useful tool to investigate processes involved in bile canalicular network formation in vitro9. Primary hepatocytes cultured between two layers of collagen (referred to as a 3D collagen sandwich culture) can repolarize in several days. Because of high technical demands required when culturing mouse hepatocytes in 3D collagen sandwiches, here we present a complex protocol to isolate, to cultivate, and to immunolabel mouse hepatocytes embedded in 3D collagen sandwiches in order to characterize the involvement of cytoskeletal components during bile canalicular formation.

<table><tbody><tr><td>35 mm TC-treated culture dish</td><td>Corning</td><td>430165</td><td></td></tr><tr><td>50 mL Centrifuge tubes, SuperClear, Ultra High Performance</td><td>VWR</td><td>525-0156</td><td></td></tr><tr><td>70 &amp;micro;m nylon cell strainer</td><td>Biologix</td><td>15-1070</td><td></td></tr><tr><td>Albumin Fraction V</td><td>ROTH</td><td>8076.4</td><td></td></tr><tr><td>Arteriotomy Cannula (1mm)</td><td>Medtronic</td><td>31001</td><td></td></tr><tr><td>Calcium Chloride</td><td>Sigma-Aldrich</td><td>P5655</td><td></td></tr><tr><td>Collagen I. from Rat tail (3mg/ml)</td><td>Corning</td><td>354249</td><td></td></tr><tr><td>Collagenase (from Clostridium Hystolyticum)</td><td>Sigma-Aldrich</td><td>C5138</td><td></td></tr><tr><td>D(+) glucose monohydrate</td><td>ROTH</td><td>6780.1</td><td></td></tr><tr><td>Dissecting microscope</td><td>Zeiss</td><td>Stemi 508</td><td></td></tr><tr><td>DMEM, high glucose</td><td>Sigma-Aldrich</td><td>D6429</td><td></td></tr><tr><td>EGTA</td><td>ROTH</td><td>3054.2</td><td></td></tr><tr><td>FBS</td><td>Gibco</td><td>10270-106</td><td></td></tr><tr><td>Glucagon</td><td>Sigma-Aldrich</td><td>G-2044</td><td></td></tr><tr><td>Glycine</td><td>Pufferan</td><td>G 05104</td><td></td></tr><tr><td>Heparin (5000 U/mL)</td><td>Zentiva</td><td>8594739026131</td><td></td></tr><tr><td>Hydrocortisone</td><td>Sigma-Aldrich</td><td>H0888</td><td></td></tr><tr><td>Insulin</td><td>Sigma-Aldrich</td><td>I9278</td><td></td></tr><tr><td>Insulin syringe (30G)</td><td>BD Medical</td><td>320829</td><td></td></tr><tr><td>Magnesium Sulphate Heptahydrate</td><td>ROTH</td><td>T888.1</td><td></td></tr><tr><td>microsurgical forceps</td><td>FST</td><td>11252-20</td><td></td></tr><tr><td>microsurgical forceps</td><td>FST</td><td>11251-35</td><td></td></tr><tr><td>microsurgical scissor</td><td>FST</td><td>15025-10</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma-Aldrich</td><td>P6148</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Sigma-Aldrich</td><td>P4333</td><td></td></tr><tr><td>Percoll</td><td>Sigma-Aldrich</td><td>P1644</td><td></td></tr><tr><td>Peristaltic Pump Minipuls Evolution</td><td>Gilson</td><td>F110701, F110705</td><td></td></tr><tr><td>Potassium Chloride</td><td>ROTH</td><td>6781.1</td><td></td></tr><tr><td>Potassium Phosphate monobasic</td><td>Sigma-Aldrich</td><td>P5655</td><td></td></tr><tr><td>ProLong Gold Antifade Mountant</td><td>Thermo Fischer Scientific</td><td>P10144</td><td></td></tr><tr><td>Refrigerated centrifuge</td><td>Eppendorf</td><td>5810 R</td><td></td></tr><tr><td>Round cover glasses, 30 mm, thickness 1.5</td><td>VWR</td><td>630-2124</td><td></td></tr><tr><td>Silk braided black</td><td>Chirmax</td><td>EP 0.5 &amp;ndash; USP 7/0</td><td></td></tr><tr><td>Sodium Chloride</td><td>ROTH</td><td>3957.1</td><td></td></tr><tr><td>Sodium Hydrogen Carbonate</td><td>Sigma-Aldrich</td><td>30435</td><td></td></tr><tr><td>Sodium hydroxide</td><td>Sigma-Aldrich</td><td>221465</td><td></td></tr><tr><td>Sodium Hydroxide</td><td>Sigma-Aldrich</td><td>S8045</td><td></td></tr><tr><td>Sodium Phosphate Dibasic Dihydrate</td><td>Sigma-Aldrich</td><td>30435</td><td></td></tr><tr><td>Syringe 2 ml</td><td>Chirana</td><td>CH002L</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>X100</td><td></td></tr><tr><td>Tween 20</td><td>Sigma-Aldrich</td><td>P1379</td><td></td></tr><tr><td>Water bath</td><td>Nuve</td><td>NB 9</td><td></td></tr><tr><td>Whatman membrane filters nylon, pore size 0.2 &amp;mu;m, diam. 47 mm</td><td>Sigma-Aldrich</td><td>WHA7402004</td><td></td></tr><tr><td>Whatman pH indicator papers, pH 6.0-8.1</td><td>GE Healthcare Life Sciences</td><td>2629-990</td><td></td></tr><tr><td>Zoletil</td><td>Vibrac</td><td>VET00083</td><td></td></tr></tbody></table>

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.

Mouse primary hepatocytes were isolated and seeded in 3D collagen sandwiches. Bile canaliculi between two adjacent cells started to form within several hours after seeding. Cells formed clusters and self-organized in an approximately regular network of bile canaliculi within 1 day (Figure 4). Within 3&#8211;6 days, clusters of 5&#8211;10 cells were usually observed, with fully polarized hepatocytes forming a canalicular network (Figure 4).
Treatment of primary mouse hepatocytes in 3D collagen sandwiches with either toxin (ethanol) or cytoskeleton-altering drugs (e.g., blebbistatin, okadaic acid) resulted in changes in the hepatocyte cytoskeleton, canaliculi width, shape, and number of bile canaliculi illustrated by immunolabeling with an antibody to keratin 8 (the most abundant keratin in hepatocytes), phalloidin (visualizing F-actin), and antibody to tight junction protein zonula occludens-1 (ZO-1; Figure 5).
Ethanol treatment had only a mild effect on organization of keratin 8; however, it increased the tortuosity (as seen from F-actin staining) and distribution of bile canalicular widths (Figure 5). The signal intensity of ZO-1 staining was decreased in ethanol-treated bile canaliculi compared to untreated controls, suggesting a loss of tight junctions after ethanol treatment. The inhibition of actomyosin contractility with blebbistatin significantly affected the shape and number of bile canaliculi. The regular canalicular network was reorganized, compared to untreated hepatocytes, into disorderly shaped bile canaliculi with an increased incidence of thick rounded bile canaliculi instead of thin long ones (as seen in the histogram of canalicular widths).
Additionally, treatment with okadaic acid (OA) inhibiting phosphatases strongly affected the physical properties of keratins, as previously shown8,17. OA changes the solubility of keratin filaments; thus, the treatment resulted in profound reorganization of the keratin meshwork, which collapsed into large perinuclear aggregates. Both F-actin and tight junction protein ZO-1 were not localized into any particular structures, suggesting almost complete disappearance of organized bile canaliculi and a complete loss of hepatocyte polarity. The remaining bile canaliculi were significantly narrowed compared to untreated controls, as seen in the canalicular width histogram (Figure 5).
To correlate microscopic observations of changes in the hepatocyte cytoskeleton with the hepatocellular biochemical response to treatment, the protocol also measured levels of alanine aminotransferase (ALT) and aspartate transaminase (AST) (two liver enzymes that are commonly used as hepatocellular injury markers) in supernatant from the 3D collagen sandwiches (Figure 6)18. Ethanol treatment significantly elevated the levels of both ALT and AST, suggesting severe hepatocellular injury. Blebbistatin treatment did not lead to any considerable changes in both ALT and AST levels compared to okadaic acid treatment, which triggered mild biochemical changes with increased levels of ALT, but no change in levels of AST. Thus, biochemical markers of hepatocellular injury measured in vitro from hepatocyte supernatant correlate with the cytoskeletal changes observed by immunostaining.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60507/60507fig1v2.jpg" /> 
Figure 1: Experimental set up. (A) Mouse fixed in a supine position, is placed under a dissecting microscope before the surgery. All surgical instruments required are placed on a tray. (B) The perfusion suite during mouse liver perfusion showing silicone tubing connecting the reservoir with warm buffer and the perfused mouse. (C) Schematic representation of B. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60507/60507fig1v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60507/60507fig2v2.jpg" /> 
Figure 2: Opening of the abdominal cavity and cannulation of the IVC. The abdomen is opened with a V-shape incision from the pubic area to front legs. The skin is folded over the chest to expose and enlarge the abdominal cavity. To expose the IVC, the intestines and colon are carefully moved caudally. (A, B) Prior to cannulation of the IVC, liver lobes should be repositioned by pressing them upwards to the diaphragm with a PBS-wetted cotton swab. The IVC is then carefully separated from surrounding tissues, and a silk suture is placed around the IVC in close proximity of the liver. Panel B represents schematics of the abdominal cavity shown in panel A. The liver lobes, gut, inferior vena cava (IVC, red), and sutures are indicated. (C) Heparin is injected into the portal vein (PV, arrow) with an insulin syringe (30 G needle bent at 45&#176; angle). (D) To cannulate the liver, the IVC is incised directly next to the liver (below the suture). (E) The cannula is inserted and secured with sutures by tying two surgical knots. (F) The portal vein is fully cut to allow free buffer outflow, preventing liver expansion. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60507/60507fig2alarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60507/60507fig3v2.jpg" /> 
Figure 3: Representative liver images before and after perfusion. (A, B) The cannulated liver was resected and perfused for 12 min at a flow rate of 2.5 mL/min. Note the significantly discolored liver after perfusion (B) compared to the freshly resected liver (A). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60507/60507fig3alarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/60507/60507fig4v2.jpg" /> 
Figure 4: 3D collagen sandwich culture of primary mouse hepatocytes. Representative bright-field images of mouse primary hepatocytes cultured for 1, 2, and 3 days in 3D conditions. It should be noted that larger clusters of highly organized cells are formed after 3 days in culture. Boxed areas show ~3x magnified images. Arrowheads indicate the bile canaliculi. Scale bar = 100 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60507/60507fig4alarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/60507/60507fig5v2.jpg" /> 
Figure 5: Evaluation of the morphological response to toxic stress by immunofluorescent microscopy. Primary mouse hepatocytes cultured in 3D collagen sandwiches were treated with toxins (ethanol, blebbistatin, or okadaic acid) on day 3 of culture. Fixed cells were stained to visualize cytoskeletal components: keratin 8 (green), F-actin (red), and zonula occludens-1 (ZO-1, magenta) by immunofluorescence. The toxic treatment led to disorganization of visualized cytoskeletal components, and it reduced the number and increased the tortuosity of bile canaliculi. Canalicular widths were measured in both untreated and treated hepatocytes and are depicted as histograms of widths distribution. Arrowheads indicate the bile canaliculi. Scale bar = 100 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60507/60507fig5alarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/60507/60507fig6v2.jpg" /> 
Figure 6: Biochemical analysis of the response of 3D hepatocyte collagen sandwiches to toxic injury in vitro. ALT and AST, well-established markers of hepatocellular injury, were measured in supernatant from 3D hepatocyte collagen sandwiches treated with toxins (ethanol, blebbistatin, and okadaic acid). ALT and AST were elevated in treated cells compared to untreated ones. Data are reported as arithmetic means &#177; SEM. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60507/60507fig6alarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="2">Stock Solution A (10x)</td>
		</tr>
		<tr>
			<td>Reagent</td>
			<td>Final concentration (g/liter)</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>80</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>4</td>
		</tr>
		<tr>
			<td>MgSO4&#183;7H2O</td>
			<td>1.97</td>
		</tr>
		<tr>
			<td>Na2HPO4&#183;2H2O</td>
			<td>0.598</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>0.6</td>
		</tr>
	</tbody>
</table>
Table 1: Stock solution A recipe.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="2">Stock Solution B (10x)</td>
		</tr>
		<tr>
			<td>Reagent</td>
			<td>Final concentration (g/liter)</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>69</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>3.6</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>1.30</td>
		</tr>
		<tr>
			<td>MgSO4&#183;7H2O</td>
			<td>2.94</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>2.772</td>
		</tr>
	</tbody>
</table>
Table 2: Stock solution B recipe.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="2">Solution C</td>
		</tr>
		<tr>
			<td>Reagent</td>
			<td></td>
		</tr>
		<tr>
			<td>Stock Solution A (10x)</td>
			<td>5 mL</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>0.1094 g</td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>0.0095 g</td>
		</tr>
		<tr>
			<td>dH2O</td>
			<td>to 50 mL</td>
		</tr>
	</tbody>
</table>
Table 3: Stock solution C recipe.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="2">Solution D</td>
		</tr>
		<tr>
			<td>Reagent</td>
			<td></td>
		</tr>
		<tr>
			<td>Stock solution A (10x)</td>
			<td>3 mL</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>0.065 g</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>0.0125 g</td>
		</tr>
		<tr>
			<td>dH2O</td>
			<td>to 30 mL</td>
		</tr>
	</tbody>
</table>
Table 4: Stock solution D recipe.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="2">Solution E</td>
		</tr>
		<tr>
			<td>Reagent</td>
			<td></td>
		</tr>
		<tr>
			<td>Stock solution B (10x)</td>
			<td>5 mL</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>0.1 g</td>
		</tr>
		<tr>
			<td>glucose</td>
			<td>0.045 g</td>
		</tr>
		<tr>
			<td>dH2O</td>
			<td>to 50 mL</td>
		</tr>
	</tbody>
</table>
Table 5: Stock solution E recipe.

Watch this Scientific Journal Video about Isolation and 3D Collagen Sandwich Culture of Primary Mouse Hepatocytes to Study the Role of Cytoskeleton in Bile Canalicular Formation In Vitro at JoVE.com

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

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 ...

isolation-3d-collagen-sandwich-culture-primary-mouse-hepatocytes-to

Nanyang Technological University

Research

JoVE Journal

Biology

11.4K Views.  Institute of Molecular Genetics of the Czech Academy of Sciences. 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.

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

Generation of Organoids from Mouse Extrahepatic Bile Ducts

Cholangiopathies, which affect extrahepatic bile ducts (EHBDs), include biliary atresia, primary sclerosing cholangitis, and cholangiocarcinoma. They have no effective therapeutic options. Tools to study EHBD are very limited. Our purpose was to develop an organ-specific, versatile, adult stem cell-derived, preclinical cholangiocyte model that can be easily generated from wild type and genetically engineered mice. Thus, we report on the novel technique of developing an EHBD organoid (EHBDO) culture system from adult mouse EHBDs. The model is cost-efficient, able to be readily analyzed, and has multiple downstream applications. Specifically, we describe the methodology of mouse EHBD isolation and single cell dissociation, organoid culture initiation, propagation, and long-term maintenance and storage. This manuscript also describes EHBDO processing for immunohistochemistry, fluorescent microscopy, and mRNA abundance quantitation by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). This protocol has significant advantages in addition to producing EHBD-specific organoids. The use of a conditioned medium from L-WRN cells significantly reduces the cost of this model. The use of mouse EHBDs provides almost unlimited tissue for culture generation, unlike human tissue. Generated mouse EHBDOs contain a pure population of epithelial cells with markers of endodermal progenitor and differentiated biliary cells. Cultured organoids maintain homogenous morphology through multiple passages and can be recovered after a long-term storage period in liquid nitrogen. The model allows for the study of biliary progenitor cell proliferation, can be manipulated pharmacologically, and may be generated from genetically engineered mice. Future studies are needed to optimize culture conditions in order to increase plating efficiency, evaluate functional cell maturity, and direct cell differentiation. Development of co-culture models and a more biologically neutral extracellular matrix are also desirable.

This protocol describes the production of a mouse extrahepatic bile duct 3-dimensional organoid system. These biliary organoids can be maintained in culture to study cholangiocyte biology. Biliary organoids express markers of both progenitor and biliary cells and are composed of polarized epithelial cells.

Cholangiopathies, which affect extrahepatic bile ducts (EHBDs), include biliary atresia, primary sclerosing cholangitis, and cholangiocarcinoma. They have ...

Developmental Biology

Measurement of Fatty Acid &#946;-Oxidation in a Suspension of Freshly Isolated Mouse Hepatocytes

Fatty acid &#946;-oxidation is a key metabolic pathway to meet the energy demands of the liver and provide substrates and cofactors for additional processes, such as ketogenesis and gluconeogenesis, which are essential to maintain whole-body glucose homeostasis and support extra-hepatic organ function in the fasted state. Fatty acid &#946;-oxidation occurs within the mitochondria and peroxisomes and is regulated through multiple mechanisms, including the uptake and activation of fatty acids, enzyme expression levels, and availability of cofactors such as coenzyme A and NAD+. In assays that measure fatty acid &#946;-oxidation in liver homogenates, cell lysis and the common addition of supraphysiological levels of cofactors mask the effects of these regulatory mechanisms. Furthermore, the integrity of the organelles in the homogenates is hard to control and can vary significantly between preparations. The measurement of fatty acid &#946;-oxidation in intact primary hepatocytes overcomes the above pitfalls. This protocol describes a method for the measurement of fatty acid &#946;-oxidation in a suspension of freshly isolated primary mouse hepatocytes incubated with 14C-labeled palmitic acid. By avoiding hours to days of culture, this method has the advantage of better preserving the protein expression levels and metabolic pathway activity of the original liver, including the activation of fatty acid &#946;-oxidation observed in hepatocytes isolated from fasted mice compared to fed mice.

Fatty acid &#946;-oxidation is an essential metabolic pathway responsible for generating energy in many different cell types, including hepatocytes. Here, we describe a method to measure fatty acid &#946;-oxidation in freshly isolated primary hepatocytes using 14C-labeled palmitic acid.

Fatty acid &#946;-oxidation is a key metabolic pathway to meet the energy demands of the liver and provide substrates and cofactors for additional ...

Biochemistry

Induction of an Inflammatory Response in Primary Hepatocyte Cultures from Mice

The liver plays a decisive role in the regulation of systemic inflammation. In chronic kidney disease in particular, the liver reacts in response to the uremic milieu, oxidative stress, endotoxemia and the decreased clearance of circulating proinflammatory cytokines by producing a large number of acute-phase reactants. Experimental tools to study inflammation and the underlying role of hepatocytes are crucial to understand the regulation and contribution of hepatic cytokines to a systemic acute phase response and a prolonged pro-inflammatory scenario, especially in an intricate setting such as chronic kidney disease. Since studying complex mechanisms of inflammation in vivo remains challenging, resource-intensive and usually requires the usage of transgenic animals, primary isolated hepatocytes provide a robust tool to gain mechanistic insights into the hepatic acute-phase response. Since this in vitro technique features moderate costs, high reproducibility and common technical knowledge, primary isolated hepatocytes can also be easily used as a screening approach. Here, we describe an enzymatic-based method to isolate primary murine hepatocytes, and we describe the assessment of an inflammatory response in these cells using ELISA and quantitative real-time PCR.

Here, we show an enzymatic approach to isolate primary hepatocytes from adult mice, and we describe the quantification of an inflammatory response using ELISA and real-time PCR.

The liver plays a decisive role in the regulation of systemic inflammation. In chronic kidney disease in particular, the liver reacts in response to the ...

Immunology and Infection

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 ...

Isolation of Primary Mouse Hepatocytes for Nascent Protein Synthesis Analysis by Non-radioactive L-azidohomoalanine Labeling Method

Hepatocytes are parenchymal cells of the liver and engage multiple metabolic functions, including synthesis and secretion of proteins essential for systemic energy homeostasis. Primary hepatocytes isolated from the murine liver constitute a valuable biological tool to understand the functional properties or alterations occurring in the liver. Herein we describe a method for the isolation and culture of primary mouse hepatocytes by performing a two-step collagenase perfusion technique and discuss their utilization for investigating protein metabolism. The liver of an adult mouse is sequentially perfused with ethylene glycol-bis tetraacetic acid (EGTA) and collagenase, followed by the isolation of hepatocytes with the density gradient buffer. These isolated hepatocytes are viable on culture plates and maintain the majority of endowed characteristics of hepatocytes. These hepatocytes can be used for assessments of protein metabolism including nascent protein synthesis with non-radioactive reagents. We show that the isolated hepatocytes are readily controlled and comprise a higher quality and volume stability of protein synthesis linked to energy metabolism by utilizing the chemo-selective ligation reaction with a Tetramethylrhodamine (TAMRA) protein detection method and western blotting analyses. Therefore, this method is valuable for investigating hepatic nascent protein synthesis linked to energy homeostasis. The following protocol outlines the materials and methods for the isolation of high-quality primary mouse hepatocytes and detection of nascent protein synthesis.

Here, we present a protocol for the isolation of healthy and functional primary mouse hepatocytes. Instructions for detecting hepatic nascent protein synthesis by non-radioactive labeling substrate were provided to help understand the mechanisms underlying protein synthesis in the context of energy-metabolism homeostasis in the liver.

Hepatocytes are parenchymal cells of the liver and engage multiple metabolic functions, including synthesis and secretion of proteins essential for ...

Medicine

Isolation of Neonatal Extrahepatic Cholangiocytes

The intra and extrahepatic bile ducts of the liver are developmentally distinct, and may be differentially affected by certain diseases. However, differences between intra and extrahepatic cholangiocytes, and between neonatal and adult cells, are not well understood.
Methods for the isolation of cholangiocytes from intrahepatic bile ducts are well established1-4. Isolation of extrahepatic ductal cells, especially from the neonate, has not yet been described, although this would be of great benefit in understanding the differences between distinct cholangiocyte populations and in studying diseases such as biliary atresia that appear to target the extrahepatic ducts. Described here is an optimized technique to isolate both neonatal and adult mouse extrahepatic bile duct cells. This technique yields a pure cell population with minimal contamination from mesenchymal cells like fibroblasts.
This method is based on the removal of the extrahepatic ducts and gallbladder, followed by meticulous dissection and scraping to remove fat and fibroblast layers. Structures are embedded in thick layers of collagen and cultured for approximately 3 weeks to allow outgrowth of cholangiocytes in monolayers, which can then be trypsinized and re plated for experimental use.

A technique to isolate cholangiocytes from the extrahepatic bile ducts of neonatal mice is described. The ducts are meticulously dissected, and then cells are isolated by outgrowth in thick collagen gels. This method provides a useful tool for studying extrahepatic bile duct development and pathology.

The intra and extrahepatic bile ducts of the liver are developmentally distinct, and may be differentially affected by certain diseases. However, ...

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 ...

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 ...

Isolation and Characterization of Mouse Primary Liver Sinusoidal Endothelial Cells

Liver sinusoidal endothelial cells (LSECs) are specialized endothelial cells located at the interface between the circulation and the liver parenchyma. LSECs have a distinct morphology characterized by the presence of fenestrae and the absence of basement membrane. LSECs play essential roles in many pathological disorders in the liver, including metabolic dysregulation, inflammation, fibrosis, angiogenesis, and carcinogenesis. However, little has been published about the isolation and characterization of the LSECs. Here, this protocol discusses the isolation of LSEC from both healthy and nonalcoholic fatty liver disease (NAFLD) mice. The protocol is based on collagenase perfusion of the mouse liver and magnetic beads positive selection of nonparenchymal cells to purify LSECs. This study characterizes LSECs using specific markers by flow cytometry and identifies the characteristic phenotypic features by scanning electron microscopy. LSECs isolated following this protocol can be used for functional studies, including adhesion and permeability assays, as well as downstream studies for a particular pathway of interest. In addition, these LSECs can be pooled or used individually, allowing multi-omics data generation including RNA-seq bulk or single cell, proteomic or phospho-proteomics, and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq), among others. This protocol will be useful for investigators studying LSECs&#39; communication with other liver cells in health and disease and allow an in-depth understanding of the role of LSECs in the pathogenic mechanisms of acute and chronic liver injury.

Here we outline and demonstrate a protocol for primary mouse liver sinusoidal endothelial cell (LSEC) isolation. The protocol is based on liver collagenase perfusion, nonparenchymal cell purification by low-speed centrifugation, and CD146 magnetic bead selection. We also phenotype and characterize these isolated LSECs using flow cytometry and scanning electron microscopy.

Liver sinusoidal endothelial cells (LSECs) are specialized endothelial cells located at the interface between the circulation and the liver parenchyma. ...