Name: Induction of an Inflammatory Response in Primary Hepatocyte Cultures from Mice (Video) | JoVE Journal
Uploaded: 2017-03-10T15:00:00.000+00:00
Duration: 8 min 32 s
Description: Learn to isolate functional murine hepatocytes using enzymatic perfusion for studying inflammatory cytokine expression and secretion. This method enables analysis of hepatic acute phase responses in liver disease research.

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5 minutes

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>

<ol><li>Silverstein, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Inflammation+in+chronic+kidney+disease%3A+role+in+the+progression+of+renal+and+cardiovascular+disease%5BTitle%5D)+AND+%22Pediatr+Nephro%22%5BJournal%5D)">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/pubmed?term=((Fibroblast+growth+factor+23+is+elevated+before+parathyroid+hormone+and+phosphate+in+chronic+kidney+disease%5BTitle%5D)+AND+%22Kidney+Int%22%5BJournal%5D)">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/pubmed?term=((FGF23+induces+left+ventricular+hypertrophy%5BTitle%5D)+AND+%22J+Clin+Invest%22%5BJournal%5D)">FGF23 induces left ventricular hypertrophy.</a> J Clin Invest. 121 (11), 4393-4408 (2011).</li>
<li>Gutiérrez, O. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fibroblast+growth+factor+23+and+mortality+among+patients+undergoing+hemodialysis%5BTitle%5D)+AND+%22N+England+J+Med%22%5BJournal%5D)">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/pubmed?term=((Fibroblast+growth+factor+23+and+Inflammation+in+CKD%5BTitle%5D)+AND+%22Clin+J+Am+Soc+Nephrol%22%5BJournal%5D)">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., Gutiérrez, O. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Associations+of+Fibroblast+Growth+Factor-23+with+Markers+of+Inflammation%2C+Insulin+Resistance+and+Obesity+in+Adults%5BTitle%5D)+AND+%22PLoS+ONE%22%5BJournal%5D)">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/pubmed?term=((Fibroblast+growth+factor+23+directly+targets+hepatocytes+to+promote+inflammation+in+chronic+kidney+disease%5BTitle%5D)+AND+%22Kidney+Int%22%5BJournal%5D)">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/pubmed?term=((The+enzymatic+preparation+of+isolated+intact+parenchymal+cells+from+rat+liver%5BTitle%5D)+AND+%22J+Cell+Biol%22%5BJournal%5D)">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/pubmed?term=((The+effect+of+interleukin-1%2C+interleukin-6+and+its+interrelationship+on+the+synthesis+of+serum+amyloid+A+and+C-reactive+protein+in+primary+cultures+of+adult+human+hepatocytes%5BTitle%5D)+AND+%22Bioch+Biophysi+Res+Commun%22%5BJournal%5D)">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/pubmed?term=((In+vitro+models+for+liver+toxicity+testing%5BTitle%5D)+AND+%22Toxicol.+Res%22%5BJournal%5D)">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/pubmed?term=((Isolation+of+Human+Hepatocytes+by+a+Two-step+Collagenase+Perfusion+Procedure%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">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/pubmed?term=((Protocol+for+Isolation+of+Primary+Human+Hepatocytes+and+Corresponding+Major+Populations+of+Non-parenchymal+Liver+Cells%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">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/pubmed?term=((Cytokines+and+the+hepatic+acute+phase+response%5BTitle%5D)+AND+%22J+Pathol%22%5BJournal%5D)">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/pubmed?term=((Mouse+liver+cell+culture.+I.+Hepatocyte+isolation%5BTitle%5D)+AND+%22In+vitro%22%5BJournal%5D)">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="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1782563/">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/pubmed?term=((IL-6+pathway+in+the+liver%3A+From+physiopathology+to+therapy%5BTitle%5D)+AND+%22J+Hepatol%22%5BJournal%5D)">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/pubmed?term=((Activation+of+Cardiac+Fibroblast+Growth+Factor+Receptor+4+Causes+Left+Ventricular+Hypertrophy%5BTitle%5D)+AND+%22Cell+Metab%22%5BJournal%5D)">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. Chapter 17, Long-Term Culture and Coculture of Primary Rat and Human Hepatocytes. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aShulman%2C+M+%22Methods+Mol+Biol%22">Methods Mol Biol</a>. 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

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',6-diamidino-2-phenylindole (DAPI) (blue). (Figure 1B).
<p class="jove_step" fo:keep-together.wi...

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.

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