Name: induction of an inflammatory response in primary hepatocyte cultures from mice
Uploaded: 2017-03-10T15:00:00
Description: Watch this Scientific Journal Video about Induction of an Inflammatory Response in Primary Hepatocyte Cultures from Mice at JoVE.com

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>

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">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Consumables</td>
			<td style="width:283px;"></td>
			<td style="width:175px;"></td>
			<td style="width:103px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell Strainer 70 &#956;m Nylon cell strainer</td>
			<td>Falcon</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BD Insyte Autoguard</td>
			<td>BD</td>
			<td>381412</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">50 mL Polypropylene Conical Tube</td>
			<td>Falcon</td>
			<td>352098</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100 6-inch Cotton Tipped Applicators&#160;</td>
			<td>Puritan</td>
			<td>806-WC</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1cc U-100 Insulin Syringe 28 G 1/2</td>
			<td>Becton Dickinson</td>
			<td>329420</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tissue Culture Dish 100 x 20 mm Style</td>
			<td>Corning</td>
			<td>353003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">6 well Cell Culture Cluster</td>
			<td>Costar</td>
			<td>3516</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5/0 Black Braided Surgical Silk (100 yards)</td>
			<td>LOOK</td>
			<td>SP115</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Minipuls 3 Perfusion Pump</td>
			<td>Gilson</td>
			<td>F155007</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hemacytometer Kits, Propper</td>
			<td>VWR</td>
			<td>48300-474</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hemacytometer Cover Glasses, Propper</td>
			<td>VWR</td>
			<td>48300-470</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Surgical Scissers - Sharp/Blunt</td>
			<td>F.S.T.</td>
			<td>14001-12</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;">Iris Scissors-ToughCut Straight</td>
			<td>F.S.T.</td>
			<td>14058-11</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;">Dumont SS-45 Forceps</td>
			<td>F.S.T.</td>
			<td>11203-25</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;">Student Tissue Forceps</td>
			<td>F.S.T.</td>
			<td>991121-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Acetic acid solution, 2.0 N</td>
			<td>Sigma</td>
			<td>&#160;&#160;&#160; A8976-100ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isoflurane, USP 250 mL</td>
			<td>Piramal Healthcare</td>
			<td>&#160;&#160; 66794-013-25</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KetaVed 1000mg/10mL (100mg/mL)</td>
			<td>VEDCO</td>
			<td>&#160;&#160;&#160; 50989-161-06</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Xylazine 100 mg/mL</td>
			<td>AnaSed Injection</td>
			<td>139-236</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Willams&#39; Medium E (1x)</td>
			<td>gibco</td>
			<td>12551-032</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Liver Perfusion Medium (1x)</td>
			<td>gibco</td>
			<td>17701-038</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Liver Digest Medium (1x)</td>
			<td>Life Technologies</td>
			<td>17703034</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Primary Hepatocyte Thawing and Plating Supplements</td>
			<td>Life Technologies</td>
			<td>CM3000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Primary Hepatocyte Maintenance Supplements</td>
			<td>Life Technologies</td>
			<td>CM4000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate Buffer Saline (PBS) pH 7.4</td>
			<td>ThermoFisher scientific</td>
			<td>10010031</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Collagen Type 1</td>
			<td>Corning</td>
			<td>354236</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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).
<p class="jove_step" fo:keep-togethe...

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 ...

Research

JoVE Journal

Immunology and Infection

Induction of Experimental Autoimmune Hypophysitis in SJL Mice

Autoimmune hypophysitis can be reproduced experimentally by the injection of pituitary proteins mixed with an adjuvant into susceptible mice1. Mouse ...

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

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

Generation of Organotypic Raft Cultures from Primary Human Keratinocytes

The development of organotypic epithelial raft cultures has provided researchers with an efficient in vitro system that faithfully recapitulates ...

Primary Microglia Isolation from Mixed Glial Cell Cultures of Neonatal Rat Brain Tissue

Microglia account for approximately 12% of the  total cellular population in the mammalian brain. While neurons and astrocytes are considered  the major ...

Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of ...

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages ...

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

Aspergillus fumigatus is an opportunistic fungal pathogen causing invasive infections in immunocompromised hosts with a high case-fatality rate. Research ...

Screening Assays to Characterize Novel Endothelial Regulators Involved in the Inflammatory Response

The endothelial layer is essential for maintaining homeostasis in the body by controlling many different functions. Regulation of the inflammatory ...

Induction of Paralysis and Visual System Injury in Mice by T Cells Specific for Neuromyelitis Optica Autoantigen Aquaporin-4

While it is recognized that aquaporin-4 (AQP4)-specific T cells and antibodies participate in the pathogenesis of neuromyelitis optica (NMO), a human ...

Adoptive Immunotherapy of iNKT Cells in Glucose-6-Phosphate Isomerase (G6PI)-Induced RA Mice

Adoptive Immunotherapy of iNKT Cells in Glucose-6-Phosphate Isomerase G6PI-Induced RA Mice

Rheumatoid arthritis (RA) is a complex chronic inflammatory autoimmune disease. The pathogenesis of the disease is related to invariant natural killer T ...