This work was supported by the NIH (R01HL128714 to C.F.) and (F31DK10236101 to K.S) and the American Heart Association (C.F. and A.G.).

All animal protocols and experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Miami Miller School of Medicine.
1. Preparation
<ol>
	<li>Preheat liver perfusion media and liver digest media in a water bath at 37 &#176;C.</li>
	<li>Set up a perfusion pump (30 mL/min). Carefully prefill the tubing system of the pump with liver perfusion media. Avoid any air bubbles in the system and prepare the stereotactic microscope.</li>
	<li>Coat cell culture dishes using collagen type I according to the manufacturer&#39;s protocol.</li>
</ol>
2. Liver Recovery
<ol>
	<li>Anesthetize the donor mouse using oxygen/isoflurane inhalation (isoflurane 4%/2 L/min oxygen).
	<ol>
		<li>Maintain anesthesia by lowering isoflurane to 2-2.5% or by injecting ketamine (100 mg/kg of body weight intraperitoneally) and xylazine (10 mg/kg of body weight intraperitoneally). Continuous isoflurane anesthesia is preferred since ketamine lowers blood pressure and can cause hepatotoxicity.</li>
	</ol>
	</li>
	<li>Place the anesthetized mouse on an absorbent pad. Fix the mouse&#39;s extremities with adhesive tape.</li>
	<li>Shave and disinfect the abdomen of the mouse and perform a ventral laparotomy from the pubis to the cranial border of the liver using surgical scissors with sharp/blunt tips. Incise the abdominal wall to both sides, caudal of the diaphragm.</li>
	<li>Expose the inferior vena cava (IVC) by carefully moving the intestine to the right side. Carefully dissect fat tissue around the IVC using sterile cotton tip swabs and angled tip forceps.</li>
	<li>Incise the suprahepatic diaphragm and open the thoracic cavity by two lateral incisions using fine surgical scissors with sharp/sharp tips. Ligate the thoracic IVC using surgical silk (5/0).</li>
	<li>Cannulate the infrarenal IVC using a shielded i.v. catheter, carefully remove the needle, connect the perfusion pump and start perfusing the liver. If performed properly, the liver should immediately begin to blanch and swell.</li>
	<li>Transect the portal vein allowing suitable drainage of blood and perfusion media using fine surgical scissors.</li>
	<li>Perfuse the liver with approximately 30 mL of liver perfusion media, and then switch to liver digest media (30 mL). Turn off the perfusion pump and remove the cannula once perfusion is complete.</li>
	<li>Gently expose the central region of the liver and locate the connecting tissue linking the liver lobes. Grab the central connecting fibers and carefully dissect all tissues holding the liver in place and gently remove the liver.</li>
	<li>Immediately transfer the liver into a 50 mL polypropylene conical tube containing 15 mL of liver digest media. 
	NOTE: &#160;There is no specific time-line for this step. After perfusion, the liver remains in digest media for the transport to a sterile cell culture hood.&#160;All subsequent steps should be performed in a sterile environment.</li>
</ol>
3. Isolation and Treatment of Cells
<ol>
	<li>Within a cell culture hood, transfer the liver from the 50 mL polypropylene conical tube containing digestion media into a sterile tissue culture dish.</li>
	<li>Using sterile forceps, gently tear the four lobes of the liver. If performed properly, digestion medium should become turbid due to isolating hepatocytes from the liver.</li>
	<li>Using a 25 mL sterile pipette tip, filter the turbid digestion medium through a 70 &#181;m nylon cell strainer into a new 50 mL polypropylene conical tube to remove undigested tissue particles and cell debris.</li>
	<li>Repeat steps 3.2 and 3.3 with sterile phosphate-buffered saline (PBS). This assists with removing any residual hepatocytes from the liver.</li>
	<li>Centrifuge at 50 x g for 2 min at 4 &#176;C. Aspirate the supernatant containing excess cell debris and gently re-suspend cells in 25 mL cold 1x Williams&#39; Medium E.</li>
	<li>Using a 25 mL sterile pipette tip, filter the re-suspension through a new 70 &#181;m nylon cell strainer into a 50 mL polypropylene conical tube to achieve a cleaner suspension.</li>
	<li>Repeat steps 3.5 and 3.6 for three additional washes using cold Williams&#39; Medium E 1x to obtain a clearer cell suspension.</li>
	<li>After the final washing step, aspirate supernatant to remove excess cell debris. Re-suspend and filter cells in 25 mL warm Williams&#39; Media E 1x that contains primary hepatocyte thawing and plating supplements through a new 70 &#181;m nylon cell strainer into a 50 mL polypropylene conical tube.</li>
	<li>Count cells within the 25 mL cell suspension using a hemocytometer. On average expect 15 - 20 million cells per adult liver. Determine cell viability using trypan blue staining.</li>
	<li>Plate cells in 2 mL of Williams&#39; Media E 1x that contains primary hepatocyte thawing and plating supplements on a collagen coated 6-well cell culture cluster (9 x 105 cells per well).</li>
	<li>After 4 h, change media to Williams&#39; Media E 1x that contains primary hepatocyte maintenance supplements.</li>
	<li>After 24 h, serum starve cells with Dulbecco&#39;s modified Eagle medium (DMEM) media 1x for 6 h.</li>
	<li>Treat cells with PBS as vehicle, FGF23 (25 ng/mL), LPS (100 &#181;g/mL) or IL-6 (50 ng/mL) in serum-free medium and incubate for 24 h.</li>
</ol>
4. Isolation of RNA
<ol>
	<li>Prepare RNA using a commercial spin column kit according to the manufacturer&#39;s instructions.</li>
</ol>
5. Generating cDNA from isolated RNA by Reverse Transcription
<ol>
	<li>Add 200 ng of isolated RNA sample to a PCR tube.</li>
	<li>Add 14 &#181;L of endonuclease-free H2O to PCR tube from step 5.1.</li>
	<li>Add 4 &#181;L of reverse transcriptase to PCR tube from step 5.1.</li>
	<li>Re-suspend contents gently to make mixture homogenous; centrifuge gently.</li>
	<li>Place PCR tube containing the mixture into a thermocycler. Run thermocycler for reverse transcription: 1 cycle of 25 &#176;C (5 min), 42 &#176;C (30 min), 85 &#176;C (5 min) and then hold at 4 &#176;C. The final product will be the desired cDNA.</li>
</ol>
6. Analysis of Cells by Quantitative Real-time PCR (qPCR)
<ol>
	<li>Prepare the 96-well plate.
	<ol>
		<li>Place all components of qPCR reaction on ice: 1 &#181;L cDNA, .25 &#181;M forward primer, .25 &#181;M reverse primer, 5 &#181;L SYBR Green, 3.5 &#181;L PCR grade water to a total volume of 10 &#181;L. Using SYBR Green, prepare master mix to run all samples in triplicate.</li>
		<li>Once the master mix is complete, vortex to make homogenous. Aliquot 9 &#181;L of qPCR master mix into each well of a 96-wll plate. After, carefully add 1 &#181;L of cDNA into each well. Total reaction volume will become 10 &#181;L.</li>
		<li>After loading is complete, seal 96-well plate with optical adhesive film and briefly centrifuge 96-well plate (50 x g for 1 min).</li>
	</ol>
	</li>
	<li>Run samples in a Real Time-PCR instrument. Place sealed 96-well plate into Real Time-PCR instrument and run samples as per instrument manufacturer recommendations. Examples of standard and fast cycles are included below.
	<ol>
		<li>For standard cycling parameters, use the following: initial denaturation at 94 &#176;C for 120 s, then 40 cycles of 94 &#176;C for 15 s, 60 &#176;C for 60 s, and then optionally hold at 4 &#176;C.</li>
		<li>For fast cycling parameters: use the following: initial denaturation at 94 &#176;C for 30 s, then 40 cycles of 95 &#176;C for 5 s, 58 &#176;C for 15 s, and then 72 &#176;C for 10 s.</li>
	</ol>
	</li>
	<li>Refer to instrument manual for guidance on how to analyze data.</li>
</ol>
7. Analysis of Cell Supernatants by ELISA
NOTE: All steps are performed according to manufacturer&#39;s protocol.
<ol>
	<li>Sample Preparation
	<ol>
		<li>Dispense 398 &#181;L of diluent into separate tubes. Pipette 2 &#181;L of the cell supernatant sample into the 398 &#181;L diluent tube. This will provide a dilution fold of 200.</li>
		<li>Repeat step 7.1.1 for each sample to be tested.</li>
	</ol>
	</li>
	<li>Procedure
	<ol>
		<li>Pipette 100 &#181;L of 200-fold diluted sample and a standard into the wells of a 96-well plate.</li>
		<li>Incubate the 96-well plate at 25 &#176;C for 45 min on a microplate shaker at 150 rpm.</li>
		<li>Wash the wells 5 times with 1x wash buffer solution. Once the last&#160;wash is completed, remove all residual wash solution with absorbent paper.</li>
		<li>Add 500 &#181;L of HRP-conjugate reagent into each well.</li>
		<li>Repeat steps 7.2.2 - 7.2.3.</li>
		<li>Pipette 100 &#181;L of tetramethylbenzidine (TMB) reagent into each well of 96-well plate.</li>
		<li>Gently mix 96-well plate at 25 &#176;C for 20 min on a microplate shaker at 150 rpm. Stop the reaction by directly adding 100 &#181;L of stop solution to each well and mix gently. Once the stop solution is added the color should turn yellow. This informs that the stop solution has been correctly mixed.</li>
		<li>Using a microtiter plate reader, determine the optical density at 450 nm within the first 5 min.</li>
	</ol>
	</li>
	<li>Calculation of Results
	<ol>
		<li>Calculate a table of average absorbance values (A450) for each sample and each standard.</li>
		<li>Assemble a standard curve by plotting the A450 mean of each standard against its standard concentration in ng/mL on a linear graph. Allow the x-axis to represent the concentration and the y-axis to represent A450 values.</li>
		<li>Utilizing the mean A450 from each diluted sample, determine the concentration of the desired protein in ng/mL from the standard curve.</li>
		<li>Multiply the sample concentration by the dilution factor. This will allow for determining the actual concentration of the desired protein in the cell supernatant sample.</li>
		<li>Plot the constructed table from samples into a line graph. If OD450 values fall outside of the standard curve, samples should be diluted appropriately and re-tested.</li>
		<li>Normalize results to 500,000 cells.</li>
	</ol>
	</li>
</ol>

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	<li>Silverstein, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammation+in+chronic+kidney+disease:+role+in+the+progression+of+renal+and+cardiovascular+disease.">Inflammation in chronic kidney disease: role in the progression of renal and cardiovascular disease.</a> Pediatr Nephro. 24 (8), 1445-1452 (2008).</li><li>Isakova, 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=Fibroblast+growth+factor+23+is+elevated+before+parathyroid+hormone+and+phosphate+in+chronic+kidney+disease.">Fibroblast growth factor 23 is elevated before parathyroid hormone and phosphate in chronic kidney disease.</a> Kidney Int. 79 (12), 1370-1378 (2011).</li><li>Faul, C., 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=FGF23+induces+left+ventricular+hypertrophy.">FGF23 induces left ventricular hypertrophy.</a> J Clin Invest. 121 (11), 4393-4408 (2011).</li><li>Guti&#233;rrez, O. 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=Fibroblast+growth+factor+23+and+mortality+among+patients+undergoing+hemodialysis.">Fibroblast growth factor 23 and mortality among patients undergoing hemodialysis.</a> N England J Med. 359 (6), 584-592 (2008).</li><li>Munoz Mendoza, 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=Fibroblast+growth+factor+23+and+Inflammation+in+CKD.">Fibroblast growth factor 23 and Inflammation in CKD.</a> Clin J Am Soc Nephrol. 7 (7), 1155-1162 (2012).</li><li>Hanks, L. J., Casazza, K., Judd, S. E., Jenny, N. S., Guti&#233;rrez, O. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Associations+of+Fibroblast+Growth+Factor-23+with+Markers+of+Inflammation,+Insulin+Resistance+and+Obesity+in+Adults.">Associations of Fibroblast Growth Factor-23 with Markers of Inflammation, Insulin Resistance and Obesity in Adults.</a> PLoS ONE. 10 (3), e0122885 (2015).</li><li>Singh, S., 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=Fibroblast+growth+factor+23+directly+targets+hepatocytes+to+promote+inflammation+in+chronic+kidney+disease.">Fibroblast growth factor 23 directly targets hepatocytes to promote inflammation in chronic kidney disease.</a> Kidney Int. , 1-12 (2016).</li><li>Howard, R. B., Christensen, A. K., Gibbs, F. A., Pesch, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+enzymatic+preparation+of+isolated+intact+parenchymal+cells+from+rat+liver.">The enzymatic preparation of isolated intact parenchymal cells from rat liver.</a> J Cell Biol. 35 (3), 675-684 (1967).</li><li>Moshage, H. 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=The+effect+of+interleukin-1,+interleukin-6+and+its+interrelationship+on+the+synthesis+of+serum+amyloid+A+and+C-reactive+protein+in+primary+cultures+of+adult+human+hepatocytes.">The effect of interleukin-1, interleukin-6 and its interrelationship on the synthesis of serum amyloid A and C-reactive protein in primary cultures of adult human hepatocytes.</a> Bioch Biophysi Res Commun. 155 (1), 112-117 (1988).</li><li>Soldatow, V. Y., LeCluyse, E. L., Griffith, L. G., Rusyn, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+models+for+liver+toxicity+testing.">In vitro models for liver toxicity testing.</a> Toxicol. Res. 2 (1), 23-39 (2013).</li><li>Lee, S. M. L., Schelcher, C., Demmel, M., Hauner, M., Thasler, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+Human+Hepatocytes+by+a+Two-step+Collagenase+Perfusion+Procedure.">Isolation of Human Hepatocytes by a Two-step Collagenase Perfusion Procedure.</a> J Vis Exp. (79), (2013).</li><li>Kegel, V., 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=Protocol+for+Isolation+of+Primary+Human+Hepatocytes+and+Corresponding+Major+Populations+of+Non-parenchymal+Liver+Cells.">Protocol for Isolation of Primary Human Hepatocytes and Corresponding Major Populations of Non-parenchymal Liver Cells.</a> J Vis Exp. (109), (2016).</li><li>Moshage, 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=Cytokines+and+the+hepatic+acute+phase+response.">Cytokines and the hepatic acute phase response.</a> J Pathol. 181 (3), 257-266 (1997).</li><li>Klaunig, J. E., 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=Mouse+liver+cell+culture.+I.+Hepatocyte+isolation.">Mouse liver cell culture. I. Hepatocyte isolation.</a> In vitro. 17 (10), 913-925 (1981).</li><li>Lu, Y. C., Yeh, W. C., Ohashi, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LPS/TLR4+signal+transduction+pathway.">LPS/TLR4 signal transduction pathway.</a> Cytokine. 42 (2), 145-151 (2008).</li><li>Schmidt-Arras, D., Rose-John, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL-6+pathway+in+the+liver:+From+physiopathology+to+therapy.">IL-6 pathway in the liver: From physiopathology to therapy.</a> J Hepatol. 64 (6), 1403-1415 (2016).</li><li>Grabner, 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=Activation+of+Cardiac+Fibroblast+Growth+Factor+Receptor+4+Causes+Left+Ventricular+Hypertrophy.">Activation of Cardiac Fibroblast Growth Factor Receptor 4 Causes Left Ventricular Hypertrophy.</a> Cell Metab. 22 (6), 1020-1032 (2015).</li><li>Shulman, M., Nahmias, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+17,+Long-Term+Culture+and+Coculture+of+Primary+Rat+and+Human+Hepatocytes.">Chapter 17, Long-Term Culture and Coculture of Primary Rat and Human Hepatocytes.</a> Methods Mol Biol. 945, 287-302 (2012).</li></ol>

The authors have no conflict of interests to declare.

Isolating primary hepatocytes from mice is a fast, inexpensive and reliable tool to study inflammatory responses ex vivo. If performed correctly, results can be easily generated and reproduced in a timely and cost-efficient manner. The following points should be carefully assessed in order to ensure a successful isolation.
The surgical incision and the cannulation of the IVC should be performed under general anesthesia and not after euthanasia. A young, inexperienced investigator will...

Chronic kidney disease (CKD) can be defined as a state of acute and chronic inflammation1. In patients with CKD, serum levels of the phosphaturic hormone fibroblast growth factor 23 (FGF23) progressively rise in order to maintain serum phosphate homeostasis2. Increased serum FGF23 levels are independently associated with cardiovascular morbidity and mortality among patients who are beginning hemodialysis treatment3,4. Furthermore, several clinical studies have shown a strong correlation between elevated FGF23 levels and serum levels of C-reactive protein (CRP), Interleukin-6 (IL-6) and Tumor Necrosis Factor &#945; (TNF&#945;)5,6. Moreover, in an experimental study, we have recently demonstrated that FGF23 can directly target hepatocytes and cause an inflammatory response by increasing CRP and IL-6 production in the liver7. Hence, FGF23 might act as a circulating factor that contributes to systemic inflammation in CKD.
In the early 70&#39;s, primary hepatocytes were isolated and studied for the first time8. Since then primary cultured hepatic cells have been extensively used to examine metabolic processing, hormonal function, drug metabolism and toxicity as well as immunity and inflammatory responses9,10. Previous protocols have mainly described the enzymatic isolation of primary hepatocytes from human liver tissue11,12. While an excellent model, this leaves out the ability to study how genetic manipulation affects complex hepatic signaling mechanisms as well as functional consequences upon different types of stimuli. In the following, we describe the isolation of murine primary hepatocytes. Notably, the effect of several mediators of the hepatic acute-phase response, such as lipopolysaccharide (LPS), IL-6 and FGF23 can be analyzed in an easy, fast and reproducible manner13.
Herein, we present a protocol for the enzymatic isolation of hepatocytes from adult mice, and we demonstrate that established inducers of inflammation, such as LPS and IL-6, as well as novel inflammatory mediators such as FGF23, can directly stimulate expression and secretion of inflammatory cytokines, such as CRP and IL-6 in cultured hepatocytes.

<table><tbody><tr><td>&lt;strong&gt;Consumables&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Cell Strainer 70 &amp;mu;m Nylon cell strainer</td><td>Falcon</td><td>352350</td><td></td></tr><tr><td>BD Insyte Autoguard</td><td>BD</td><td>381412</td><td></td></tr><tr><td>50 mL Polypropylene Conical Tube</td><td>Falcon</td><td>352098</td><td></td></tr><tr><td>100 6-inch Cotton Tipped Applicators&amp;nbsp;</td><td>Puritan</td><td>806-WC</td><td></td></tr><tr><td>1cc U-100 Insulin Syringe 28 G 1/2</td><td>Becton Dickinson</td><td>329420</td><td></td></tr><tr><td>Tissue Culture Dish 100 x 20 mm Style</td><td>Corning</td><td>353003</td><td></td></tr><tr><td>6 well Cell Culture Cluster</td><td>Costar</td><td>3516</td><td></td></tr><tr><td>5/0 Black Braided Surgical Silk (100 yards)</td><td>LOOK</td><td>SP115</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Minipuls 3 Perfusion Pump</td><td>Gilson</td><td>F155007</td><td></td></tr><tr><td>Hemacytometer Kits, Propper</td><td>VWR</td><td>48300-474</td><td></td></tr><tr><td>Hemacytometer Cover Glasses, Propper</td><td>VWR</td><td>48300-470</td><td></td></tr><tr><td>Surgical Scissers - Sharp/Blunt</td><td>F.S.T.</td><td>14001-12</td><td></td></tr><tr><td>Iris Scissors-ToughCut Straight</td><td>F.S.T.</td><td>14058-11</td><td></td></tr><tr><td>Dumont SS-45 Forceps</td><td>F.S.T.</td><td>11203-25</td><td></td></tr><tr><td>Student Tissue Forceps</td><td>F.S.T.</td><td>991121-12</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Acetic acid solution, 2.0 N</td><td>Sigma</td><td>&amp;nbsp;&amp;nbsp;&amp;nbsp; A8976-100ML</td><td></td></tr><tr><td>Isoflurane, USP 250 mL</td><td>Piramal Healthcare</td><td>&amp;nbsp;&amp;nbsp; 66794-013-25</td><td></td></tr><tr><td>KetaVed 1,000 mg/10 mL (100 mg/mL)</td><td>VEDCO</td><td>&amp;nbsp;&amp;nbsp;&amp;nbsp; 50989-161-06</td><td></td></tr><tr><td>Xylazine 100 mg/mL</td><td>AnaSed Injection</td><td>139-236</td><td></td></tr><tr><td>Willams&#039; Medium E (1x)</td><td>gibco</td><td>12551-032</td><td></td></tr><tr><td>Liver Perfusion Medium (1x)</td><td>gibco</td><td>17701-038</td><td></td></tr><tr><td>Liver Digest Medium (1x)</td><td>Life Technologies</td><td>17703034</td><td></td></tr><tr><td>Primary Hepatocyte Thawing and Plating Supplements</td><td>Life Technologies</td><td>CM3000</td><td></td></tr><tr><td>Primary Hepatocyte Maintenance Supplements</td><td>Life Technologies</td><td>CM4000</td><td></td></tr><tr><td>Phosphate Buffer Saline (PBS) pH 7.4</td><td>ThermoFisher scientific</td><td>10010031</td><td></td></tr><tr><td>Collagen Type 1</td><td>Corning</td><td>354236</td><td></td></tr><tr><td>Trypan Blue Solution</td><td>VWR</td><td>45000-717</td><td></td></tr></tbody></table>

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.

Histology
Representative light microscopy images of primary isolated and cultured cells are depicted in Figure 1A. Immunocytochemical analysis demonstrates that isolated hepatocytes highly express albumin (red) as well as fibroblast growth factor receptor 4 (FGFR4) (green). Nuclei are stained with 4&#39;,6-diamidino-2-phenylindole (DAPI) (blue). (Figure 1B).
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Induction of an Inflammatory Response in Primary Hepatocyte Cultures from Mice

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 ...

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Immunology and Infection

9.9K Views.  University of Miami Leonard M. Miller School of Medicine. 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.

Video: Induction of an Inflammatory Response in Primary Hepatocyte Cultures from Mice

The CYP2D6 Animal Model: How to Induce Autoimmune Hepatitis in Mice

Autoimmune hepatitis is a rare but life threatening autoimmune disease of the liver of unknown etiology1,2. In the past many attempts have been made to generate an animal model that reflects the characteristics of the human disease 3-5. However, in various models the induction of disease was rather complex and often hepatitis was only transient3-5. Therefore, we have developed a straightforward mouse model that uses the major human autoantigen in type 2 autoimmune hepatitis (AIH-2), namely hCYP2D6, as a trigger6. Type 1 liver-kidney microsomal antibodies (LKM-1) antibodies recognizing hCYP2D6 are the hallmark of AIH-27,8. Delivery of hCYP2D6 into wildtype FVB or C57BL/6 mice was by an Adenovirus construct (Ad-2D6) that ensures a direct delivery of the triggering antigen to the liver. Thus, the ensuing local inflammation generates a fertile field9 for the subsequent development of autoimmunity. A combination of intravenous and intraperitoneal injection of Ad-2D6 is the most effective route to induce a long-lasting autoimmune damage to the liver (section 1). Here we provide a detailed protocol on how autoimmune liver disease is induced in the CYP2D6 model and how the different aspects of liver damage can be assessed. First, the serum levels of markers indicating hepatocyte destruction, such as aminotransferases, as well as the titers of hCYP2D6 antibodies are determined by sampling blood retroorbitaly (section 2). Second, the hCYP2D6-specific T cell response is characterized by collecting lymphocytes from the spleen and the liver. In order to obtain pure liver lymphocytes, the livers are perfused by PBS via the portal vein (section 3), digested in collagen and purified over a Percoll gradient (section 4). The frequency of hCYP2D6-specific T cells is analyzed by stimulation with hCYP2D6 peptides and identification of IFN&gamma;-producing cells by flow cytometry (section 5). Third, cellular infiltration and fibrosis is determined by immunohistochemistry of liver sections (section 6). Such analysis regimen has to be conducted at several times after initiation of the disease in order to prove the chronic nature of the model. The magnitude of the immune response characterized by the frequency and activity of hCYP2D6-specific T and/or B cells and the degree of the liver damage and fibrosis have to be assessed for a subsequent evaluation of possible treatments to prevent, delay or abrogate the autodestructive process of the liver.

Infection of mice with an Adenovirus expressing the major human autoantigen cytochrome P450 2D6 (hCYP2D6) recognized by sera of patients suffering from type 2 autoimmune hepatitis results in a persistent form of autoimmune-mediated liver disease characterized by extensive hepatitis, fibrosis and generation of a CYP2D6-specific immune response.

Autoimmune hepatitis is a rare but life threatening autoimmune disease of the liver of unknown etiology1,2. In the past many attempts have been made to ...

Medicine

An Oncogenic Hepatocyte-Induced Orthotopic Mouse Model of Hepatocellular Cancer Arising in the Setting of Hepatic Inflammation and Fibrosis

The absence of a clinically relevant animal model addressing the typical immune characteristics of hepatocellular cancer (HCC) has significantly impeded elucidation of the underlying mechanisms and development of innovative immunotherapeutic strategies. To develop an ideal animal model recapitulating human HCC, immunocompetent male C57BL/6J mice first receive a carbon tetrachloride (CCl4) injection to induce liver fibrosis, then receive histologically-normal oncogenic hepatocytes from young male SV40 T antigen (TAg)-transgenic mice (MTD2) by intra-splenic (ISPL) inoculation. Androgen generated in recipient male mice at puberty initiates TAg expression under control of a liver-specific promoter. As a result, the transferred hepatocytes become cancer cells and form tumor masses in the setting of liver fibrosis/cirrhosis. This novel model mimics human HCC initiation and progression in the context of liver fibrosis/cirrhosis and reflects the most typical features of human HCC including immune dysfunction.

Here, we describe the development of a clinically relevant murine model of liver cancer recapitulating the typical immune features of hepatocellular cancer (HCC).

The absence of a clinically relevant animal model addressing the typical immune characteristics of hepatocellular cancer (HCC) has significantly impeded ...

Cancer Research

Bile Duct Ligation in Mice: Induction of Inflammatory Liver Injury and Fibrosis by Obstructive Cholestasis

In most vertebrates, the liver produces bile that is necessary to emulsify absorbed fats and enable the digestion of lipids in the small intestine as well as to excrete bilirubin and other metabolic products. In the liver, the experimental obstruction of the extrahepatic biliary system initiates a complex cascade of pathological events that leads to cholestasis and inflammation resulting in a strong fibrotic reaction originating from the periportal fields. Therefore, surgical ligation of the common bile duct has become the most commonly used model to induce obstructive cholestatic injury in rodents and to study the molecular and cellular events that underlie these pathophysiological mechanisms induced by inappropriate bile flow. In recent years, different surgical techniques have been described that either allow reconnection or reanastomosis after bile duct ligation (BDL), e.g., partial BDL, or other microsurgical methods for specific research questions. However, the most frequently used model is the complete obstruction of the common bile duct that induces a strong fibrotic response after 21 to 28 days. The mortality rate can be high due to infectious complications or technical inaccuracies. Here we provide a detailed surgical procedure for the BDL model in mice that induce a highly reproducible fibrotic response in accordance to the 3R rule for animal welfare postulated by Russel and Burch in 1959.

Disruption of bile flow results in severe inflammatory cholestatic liver injury with a characteristic time-dependent sequence of morphological alterations. Here we present a protocol for the surgical ligation of the common bile duct in mice that allows to induce a strong fibrotic response after 21 to 28 days.

In most vertebrates, the liver produces bile that is necessary to emulsify absorbed fats and enable the digestion of lipids in the small intestine as well ...

Induction of Drug-Induced, Autoimmune Hepatitis in BALB/c Mice for the Study of Its Pathogenic Mechanisms

Drug-induced autoimmune hepatitis (DIH) is the most common hepatic drug-induced hypersensitization process observed in approximately 9 to 12% of patients with autoimmune hepatitis. The overwhelming majority of patients with DIH are women. The underlying mechanisms of these sex differences in prevalence are unclear because of the paucity of animal models that mimic human disease. Even so, underlying mechanisms are widely believed to be associated with human leukocyte antigen haplotypes and sex hormones. In contrast, using a DIH mouse model, we have uncovered that IL-4 initiated&#160;CD4+ T cells directed against an epitope of cytochrome P450 2E1 induces influx of neutrophils, macrophages and mast cells into the livers of female BALB/c mice. Using this model, we have also shown that IL-33-induced FoxP3+regulatory T cells confer protection against DIH in female and male mice. This DIH model is induced by immunizing mice with an epitope of CYP2E1 that has been covalently altered with a drug metabolite that has been associated with DIH. This epitope is recognized by patients with DIH. Our method induces robust and reproducible hepatitis and autoantibodies that can be utilized to study the pathogenesis of DIH. While in vivo studies can cause undue pain and distress in mice when done improperly, the advantage of an in vivo model is the ability to evaluate the pathogenesis of disease in a large number of mice. Additionally, biological effects of the altered liver proteins can be studied using invasive procedures. The addition of in vitro studies to the experimental design allows rapid repetition and mechanistic analysis at a cellular level. Thus, we will demonstrate our model protocol and how it can be utilized to study in vivo and in vitro mechanisms of DIH.

We describe an in vivo immunization, translational hepatitis model in BALB/c mice that can be utilized to study the pathogenesis of drug-induced autoimmune hepatitis including sex differences seen in this disease. We will describe how this model demonstrates reproducible analyses using in vivo and in vitro experimental techniques.

Drug-induced autoimmune hepatitis (DIH) is the most common hepatic drug-induced hypersensitization process observed in approximately 9 to 12% of patients ...

An Advanced Murine Model for Nonalcoholic Steatohepatitis in Association with Type 2 Diabetes

Obesity is associated with chronic low-grade inflammation and insulin resistance, contributing to an increasing prevalence of chronic metabolic diseases, such as type 2 diabetes and nonalcoholic steatohepatitis (NASH). Recent research has established that pro-inflammatory immune cells infiltrate obese hypertrophic adipose tissue and liver. Given the emerging importance of immune cells in the context of metabolic homeostasis, there is a critical need to quantify and characterize their modification during the development of type 2 diabetes and NASH. However, animal models that induce pathophysiological features typical of human NASH are sparse.
In this article, we provide a detailed protocol to identify immune cell subsets isolated from liver and adipose tissue in a reliable mouse model of NASH, established by housing high-fat diet (HFD) mice under non-specific pathogen-free (SPF) conditions without a barrier for at least seven weeks. We demonstrate the handling of mice in non-SPF conditions, digestion of the tissues and identification of macrophages, natural killer (NK) cells, dendritic cells, B and T cell subsets by flow cytometry. Representative flow cytometry plots from SPF HFD mice and non-SPF mice are provided. To obtain reliable and interpretable data, the use of antibodies, accurate and precise methods for tissue digestion and proper gating in flow cytometry experiments are critical elements.
The intervention to restore physiological antigen exposure in mice by housing them in non-SPF conditions and unspecific exposure to microbial antigens could provide a relevant tool for investigating the link between immunological alterations, diet-induced obesity and related long term complications.

A simple and reliable diet-induced rodent animal model for nonalcoholic steatohepatitis (NASH) is described, achieved through non-SPF housing of the animals and administration of a specific high-fat diet. We describe identification of hepatic and adipose immune cell subsets to recapitulate human immunological conditions by exposing mice to environmental germs.

Obesity is associated with chronic low-grade inflammation and insulin resistance, contributing to an increasing prevalence of chronic metabolic diseases, ...

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 ...

Inducing and Characterizing Vesicular Steatosis in Differentiated HepaRG Cells

Hepatic steatosis represents a metabolic dysfunction that results from an accumulation of triglyceride-containing lipid droplets in hepatocytes. Excessive fat accumulation leads to non-alcoholic fatty liver disease (NAFLD), &#160;which is potentially reversible and may evolve into non-alcoholic steatohepatitis (NASH) and eventually cirrhosis and hepatocellular carcinoma (HCC). The molecular mechanisms linking lipid accumulation in hepatocytes with the progression to NASH, irreversible liver damage, fibrosis, cirrhosis, and even HCC still remains unclear. To this end, several in vitro and in vivo models have been developed to elucidate the pathological processes that cause NAFLD. In the present study, we describe a cellular model for the induction of liver vesicular steatosis that consists of DMSO-differentiated human hepatic HepaRG cells treated with the fatty acid salt sodium oleate. Indeed, sodium oleate-treated HepaRG cells accumulate lipid droplets in the cytoplasm and show typical features of steatosis. This in vitro human model represents a valuable alternative to in vivo mice models as well as to the primary human hepatocytes. We also present a comparison of several methods for the quantification and evaluation of fat accumulation in HepaRG cells, including Oil Red O staining, cytofluorimetric Bodipy measurement, metabolic gene expression analysis by qPCR, and coherent anti-Stokes Raman scattering (CARS) microscopy. CARS imaging combines the chemical specificity of Raman spectroscopy, a chemical analysis technique well-known in materials science applications, with the benefits of high-speed, high-resolution non-linear optical microscopies to allow precise quantification of lipid accumulation and lipid droplet dynamics. The establishment of an efficient in vitro model for the induction of vesicular steatosis, alongside an accurate method for the quantification and characterization of lipid accumulation, could lead to the development of early stage diagnosis of NAFLD via the identification of molecular markers, and to the generation of new treatment strategies.

In this study, we describe a detailed protocol for inducing liver vesicular steatosis in differentiated HepaRG cells with the fatty acid salt sodium oleate and employ methods for detection and quantification of lipid accumulation, including coherent anti-Stokes Raman scattering (CARS) microscopy, cytofluorimetric analysis, Oil red O staining, and qPCR.&#160;

Hepatic steatosis represents a metabolic dysfunction that results from an accumulation of triglyceride-containing lipid droplets in hepatocytes. Excessive ...

Murine Precision-Cut Liver Slices as an Ex Vivo Model of Liver Biology

Understanding the mechanisms of liver injury, hepatic fibrosis, and cirrhosis that underlie chronic liver diseases (i.e., viral hepatitis, non-alcoholic fatty liver disease, metabolic liver disease, and liver cancer) requires experimental manipulation of animal models and in vitro cell cultures. Both techniques have limitations, such as the requirement of large numbers of animals for in vivo manipulation. However, in vitro cell cultures do not reproduce the structure and function of the multicellular hepatic environment. The use of precision-cut liver slices is a technique in which uniform slices of viable mouse liver are maintained in laboratory tissue culture for experimental manipulation. This technique occupies an experimental niche that exists between animal studies and in vitro cell culture methods. The presented protocol describes a straightforward and reliable method to isolate and culture precision-cut liver slices from mice. As an application of this technique, ex vivo liver slices are treated with bile acids to simulate cholestatic liver injury and ultimately assess the mechanisms of hepatic fibrogenesis.

This protocol provides a simple and reliable method for the production of viable precision-cut liver slices from mice. The ex vivo tissue samples can be maintained under laboratory tissue culture conditions for multiple days, providing a flexible model to examine liver pathobiology.

Understanding the mechanisms of liver injury, hepatic fibrosis, and cirrhosis that underlie chronic liver diseases (i.e., viral hepatitis, non-alcoholic ...

The Murine Choline-Deficient, Ethionine-Supplemented (CDE) Diet Model of Chronic Liver Injury

Chronic liver diseases, such as viral hepatitis, alcoholic liver disease, or non-alcoholic fatty liver disease, are characterized by continual inflammation, progressive destruction and regeneration of the hepatic parenchyma, liver progenitor cell proliferation, and fibrosis. The end-stage of every chronic liver disease is cirrhosis, a major risk factor for the development of hepatocellular carcinoma. To study processes regulating disease initiation, establishment, and progression, several animal models are used in laboratories. Here we describe a six-week time course of the choline-deficient and ethionine-supplemented (CDE) mouse model, which involves feeding six-week old male C57BL/6J mice with choline-deficient chow and 0.15% DL-ethionine-supplemented drinking water. Monitoring of animal health and a typical body weight loss curve are explained. The protocol demonstrates the gross examination of a CDE-treated liver and blood collection by cardiac puncture for subsequent serum analyses. Next, the liver perfusion technique and collection of different hepatic lobes for standard evaluations are shown, including liver histology assessments by hematoxylin and eosin or Sirius Red stainings, immunofluorescent detection of hepatic cell populations as well as transcriptome profiling of the liver microenvironment. This mouse model is suitable for studying inflammatory, fibrogenic, and liver progenitor cell dynamics induced through chronic liver disease and can be used to test potential therapeutic agents that may modulate these processes.

The Murine Choline-Deficient, Ethionine-Supplemented CDE Diet Model of Chronic Liver Injury

Here we describe a common method to induce chronic liver injury in mice by feeding of a choline-deficient and ethionine-supplemented (CDE) diet. We demonstrate health monitoring, liver perfusion, isolation, and preservation. A time course of six weeks can inform about liver injury, pathohistology, fibrosis, inflammatory, and liver progenitor cell responses.

Chronic liver diseases, such as viral hepatitis, alcoholic liver disease, or non-alcoholic fatty liver disease, are characterized by continual ...

Biology

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 ...