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>C...

<ol>
	<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 border="0" cellpadding="0" cellspacing="0" width="924">
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		<tr height="21">
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			<td height="21" style="height:21px;">Tissue Culture Dish 100 x 20 mm Style</td>
			<td>Corning</td>
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			<td height="21" style="height:21px;">6 well Cell Culture Cluster</td>
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			<td>F.S.T.</td>
			<td>14001-12</td>
			<td></td>
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		<tr height="22">
			<td height="22" style="height:23px;">Iris Scissors-ToughCut Straight</td>
			<td>F.S.T.</td>
			<td>14058-11</td>
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			<td height="22" style="height:23px;">Dumont SS-45 Forceps</td>
			<td>F.S.T.</td>
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			<td height="22" style="height:23px;">Student Tissue Forceps</td>
			<td>F.S.T.</td>
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			<td></td>
			<td></td>
			<td></td>
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			<td height="21" style="height:21px;">Acetic acid solution, 2.0 N</td>
			<td>Sigma</td>
			<td>&#160;&#160;&#160; A8976-100ML</td>
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			<td height="21" style="height:21px;">Isoflurane, USP 250 mL</td>
			<td>Piramal Healthcare</td>
			<td>&#160;&#160; 66794-013-25</td>
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			<td>VEDCO</td>
			<td>&#160;&#160;&#160; 50989-161-06</td>
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			<td height="21" style="height:21px;">Xylazine 100 mg/mL</td>
			<td>AnaSed Injection</td>
			<td>139-236</td>
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			<td height="21" style="height:21px;">Willams&#39; Medium E (1x)</td>
			<td>gibco</td>
			<td>12551-032</td>
			<td></td>
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			<td height="21" style="height:21px;">Liver Perfusion Medium (1x)</td>
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			<td>17701-038</td>
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			<td>Life Technologies</td>
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			<td>ThermoFisher scientific</td>
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			<td>354236</td>
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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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Watch this Scientific Journal Video about Induction of an Inflammatory Response in Primary Hepatocyte Cultures from Mice at JoVE.com

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 ...

The overall goal of this procedure is to successfully isolate functional murine hepatocytes for studying the expression and secretion of inflammatory cytokines such as CRP and IL6 in response to mediators of the hepatic acute phase response. This method could help answer key questions of hepatocellular function and the contribution of hepatic cytokines to a prolonged inflammatory environment as found in chronic kidney disease. The main advantage of this technique is that it provides a robust tool for a simple, fast, and reproducible analysis of hepatic metabolomics and inflammation as well as a response to drugs.To begin, preheat the liver perfusion medium and liver digest medium in a water bath at 37 degrees Celsius. Set up a perfusion pump as shown here. Then use liver perfusion medium to carefully pre-fill the tubing system of the pump.Try avoiding any air bubbles in the system. Prepare the stereotactic microscope for the perfusion procedure. Then after anesthetizing a donor mouse according to the text protocol, place the anesthetized animal on an absorbent pad.Use adhesive tape to fix the mouse's extremities. Next, shave and disinfect the abdomen of the mouse. Then use surgical scissors with sharp blunt tips to perform a ventral laparotomy from the pubis to the cranial border of the liver.Incise the abdominal wall on both sides at a position caudal to the diaphragm. Expose the inferior vena cava or IVC by carefully moving the intestine to the right side using sterile cotton swabs. With fine surgical scissors and sharp sharp tips, make lateral incisions across the suprahepatic diaphragm and open the thoracic cavity.Then use 5-0 surgical silk to ligate the thoracic IVC. With a shielded IV catheter, cannulate the infrarenal IVC. Connect the perfusion pump and start perfusing the liver.If performed properly, the liver should immediately begin to blanch and swell. Next, with fine surgical scissors, transect the portal vein, allowing suitable drainage of blood and perfusion medium. After perfusing the liver with approximately 30 milliliters of liver perfusion medium, stop the perfusion pump.Switch to liver digest medium and resume the pump. Once perfusion is complete, remove the cannula. 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 the tissues holding the liver in place. Then gently remove the liver. Immediately transfer the organ into a 50 milliliter polypropylene conical tube containing 15 milliliters of liver digest medium.While working under a cell culture hood, transfer the liver from the 50 milliliter polypropylene conical tube into a sterile tissue culture dish. With sterile forceps, gently tear the four lobes of the liver. If performed properly, the digestion medium should become turbid duet to the isolation of hepatocytes from the liver.Using a 25 milliliter sterile pipette tip, filter the turbid digestion medium through a 70 micrometer nylon cell strainer into a new 50 milliliter polypropylene conical tube to remove undigested tissue particles and cell debris. Centrifuge the combined filtrate at 50 times g in four degrees Celsius for two minutes, then aspirate the supernatant containing excess cell debris and use 25 milliliters of cold 1X William's Medium E to gently resuspend cells. After repeating the filtering and washing of the hepatocytes three times, aspirate the supernatant to remove excess cell debris.Resuspend the cells in 25 milliliters of warm 1X William's Medium E that contains primary hepatocyte thawing and plating supplements and filter the suspension through a new 70 micrometer nylon cell strainer into a 50 milliliter conical tube. Once the cells have been counted, plate them in two milliliters of William's Medium E that contains primary hepatocyte thawing and plating supplements on a collagen-coated six well cell culture cluster. Following two changes of medium to eventually starve the cells, treat the cells with PBS as vehicle, 25 nanograms per milliliter of FGF23, 100 micrograms per milliliter of LPS, or 50 nanograms per milliliter of IL6 in serum-free medium.Incubate the cultures for 24 hours. After preparing RNA using a spin column kit according to the manufacturer's instructions, add 200 nanograms of the isolated RNA to a PCR tube. Then add 14 microliters of endonuclease-free water and four microliters of reverse transcriptase to the tube.Place the PCR tube into the thermocycler and run the following reverse transcription program to generate cDNA. To carry out quantitative PCR, combine the following QPCR master mix components in a tube on ice. Vortex the master mix.Then aliquot nine microliters of the solution into each well of a 96 well plate. Carefully add one microliter of cDNA into each well, preparing three wells for each sample. Finally, run the samples in a real-time PCR instrument and analyze the data according to the text protocol.A representative light microscopy image of primary isolated and cultured cells are depicted here. Immunocytochemical analysis demonstrates that isolated hepatocytes highly express albumin as well as fibroblast growth factor receptor 4. In this experiment, primary isolated hepatocytes were treated with FGF23, LPS, or IL6 for 24 hours and mRNA levels of CRP and IL6 were analyzed by quantitative real-time PCR.LPS, IL6, and FGF23 significantly increased expression of CRP and IL6. As shown here, cell culture supernatants from isolated primary hepatocytes were analyzed by ELISA for CRP levels. As demonstrated with QPCR analysis, LPS, IL6, and FGF23 treatments significantly increased CRP levels in cell supernatants when compared to PBS-treated cells.Once mastered, this technique can be done in just a few hours if it is performed properly. While attempting this procedure, it's important to remember to maintain a sterile environment. Due to working with primary culture, minimizing the risk of possible contamination is most important for achieving optimal results.After watching this video, you should have a good understanding of how to successfully isolate murine hepatocytes for studying various hepatocellular functions such as expression and secretion of hepatic cytokines.

induction-an-inflammatory-response-primary-hepatocyte-cultures-from

Research

JoVE Journal

Immunology and Infection

9.8K visualizações.  University of Miami Leonard M. Miller School of Medicine. The overall goal of this procedure is to successfully isolate functional murine hepatocytes for studying the expression and secretion of inflammatory cytokines such as CRP and IL6 in response to mediators of the hepatic acute phase response. This method could help answer key questions of hepatocellular function and the contribution of hepatic cytokines to a prolonged inflammatory environment as found in chronic kidney disease. The main advantage of this technique is that it provides a robust ...