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

Questo lavoro è stato supportato dal NIH (R01HL128714 a CF) e (F31DK10236101 a KS) e l&#39;American Heart Association (CF e AG).

Tutti i protocolli di animali e procedure sperimentali sono state approvate dal Comitato Istituzionale Animal Care e Usa (IACUC) dell &#39;Università di Miami Miller School of Medicine. 1. Preparazione <ol><li> mezzi di perfusione epatica di preriscaldamento e fegato digerire media in un bagno d&#39;acqua a 37 ° C. </li><li> Impostare una pompa di perfusione (30 mL / min). precompilare con attenzione il sistema di tubi della pompa con mezzi di perfusione epatica. Evitare eventuali bolle ...

<ol>
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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. 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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>

Isolando epatociti primari da topi è uno strumento veloce, economico e affidabile per studiare le risposte infiammatorie ex vivo. Se eseguita correttamente, i risultati possono essere facilmente generati e riprodotti in modo tempestivo ed economicamente efficiente. I seguenti punti devono essere attentamente valutati per assicurare un isolamento efficace. L&#39;incisione chirurgica e incannulamento della IVC devono essere eseguite in anestesia generale e non dopo l&#39;eutanasia. U...

Malattia renale cronica (CKD) può essere definita come uno stato di infiammazione acuta e cronica 1. Nei pazienti con insufficienza renale cronica, i livelli sierici del fattore di crescita dei fibroblasti ormone phosphaturic 23 (FGF23) progressivo aumento al fine di mantenere l&#39;omeostasi fosfato sierico 2. L&#39;aumento dei livelli sierici di FGF23 sono indipendentemente associati con la morbilità e mortalità cardiovascolare tra i pazienti che iniziano il trattamento di emodialisi 3, 4. Inoltre, diversi studi clinici hanno dimostrato una forte correlazione tra i livelli di FGF23 elevate e livelli sierici di proteina C-reattiva (CRP), interleuchina-6 (IL-6) e Tumor Necrosis Factor α (TNF-alfa) 5, 6. Inoltre, in uno studio sperimentale, abbiamo recentemente dimostrato che FGF23 può indirizzare direttamente epatociti e provocare una risposta infiammatoria aumentando CRP e IL-6 produzione nel fegato 7. Quindi, FGF23 potrebbe agire come un fattore che contribuisce alla circolazione infiammazione sistemica in CKD. Nei primi anni &#39;70, epatociti primari sono stati isolati e studiati per la prima volta 8. Da allora le cellule epatiche in coltura primaria sono stati ampiamente utilizzati per esaminare il trattamento metabolico, la funzione ormonale, il metabolismo dei farmaci e la tossicità, nonché immunità e le risposte infiammatorie 9, 10. Protocolli precedenti hanno descritto principalmente l&#39;isolamento enzimatica di epatociti primari da tessuto epatico umano 11, 12. Mentre un modello eccellente, questo lascia la possibilità di studiare come genetica manipolazione riguarda i meccanismi di segnalazione epatica complessi così come conseguenze funzionali su diversi tipi di stimoli. Nel seguito si descrive l&#39;isolamento di epatociti primari murini. In particolare, l&#39;effetto di diversi mediatori della risposta di fase acuta epatica, come lipopolisaccaride (LPS), IL-6 e FGF23 può essere analizzato in modo facile, veloce e riproducibile 13. Qui, vi presentiamo un protocollo per l&#39;isolamento enzimatica di epatociti di topi adulti, e dimostriamo che induttori stabiliti di infiammazione, come LPS e IL-6, così come mediatori infiammatori innovative come le FGF23, in grado di stimolare direttamente l&#39;espressione e la secrezione di citochine infiammatorie, come la PCR e iL-6 in epatociti coltivati.

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			<td height="21" style="height:21px;width:363px;">Consumables</td>
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			<td height="21" style="height:21px;">Cell Strainer 70 &#956;m Nylon cell strainer</td>
			<td>Falcon</td>
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			<td>Falcon</td>
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			<td>Becton Dickinson</td>
			<td>329420</td>
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			<td height="21" style="height:21px;">Tissue Culture Dish 100 x 20 mm Style</td>
			<td>Corning</td>
			<td>353003</td>
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			<td height="21" style="height:21px;">6 well Cell Culture Cluster</td>
			<td>Costar</td>
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			<td height="21" style="height:21px;">5/0 Black Braided Surgical Silk (100 yards)</td>
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			<td>Gilson</td>
			<td>F155007</td>
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			<td>F.S.T.</td>
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			<td>F.S.T.</td>
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			<td height="22" style="height:23px;">Dumont SS-45 Forceps</td>
			<td>F.S.T.</td>
			<td>11203-25</td>
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			<td height="22" style="height:23px;">Student Tissue Forceps</td>
			<td>F.S.T.</td>
			<td>991121-12</td>
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			<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>
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		<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>
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		<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>
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		<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>
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		<tr height="21">
			<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>
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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>17703034</td>
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			<td>CM3000</td>
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			<td>Life Technologies</td>
			<td>CM4000</td>
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			<td>10010031</td>
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			<td>Corning</td>
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			<td>VWR</td>
			<td>45000-717</td>
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induction of an inflammatory response in primary hepatocyte cultures from mice

Il fegato svolge un ruolo determinante nella regolazione dell&#39;infiammazione sistemica. Nella malattia renale cronica in particolare, il fegato reagisce in risposta al ambiente uremico, stress ossidativo, endotossiemia e la clearance dell&#39;amlodipina circolante citochine proinfiammatorie producendo un gran numero di proteine ​​della fase acuta. strumenti sperimentali per studiare l&#39;infiammazione e il ruolo di fondo di epatociti sono cruciali per comprendere la regolamentazione e il contributo di citochine epatiche ad una risposta di fase acuta sistemica e uno scenario pro-infiammatoria prolungata, particolarmente in un contesto complicato come la malattia renale cronica. Dal momento che lo studio complessi meccanismi di infiammazione in vivo rimane difficile, e di solito ad alta intensità di risorse richiede l&#39;utilizzo di animali transgenici, epatociti isolati primari forniscono uno strumento robusto per acquisire conoscenze meccanicistici nella risposta della fase acuta epatica. Poiché questa tecnica in vitro offre costi contenuti, alta reproducibilità e la conoscenza tecnica comune, epatociti isolati primari possono anche essere facilmente utilizzato come un approccio di screening. Qui, descriviamo un metodo enzimatico-based per isolare epatociti murini primari, e descriviamo la valutazione di una risposta infiammatoria in queste cellule utilizzando ELISA e quantitativa real-time PCR.

Istologia Immagini luce microscopia rappresentativi di cellule isolate e coltivate primarie sono descritte nella Figura 1A. Immunocytochemical analisi dimostra che epatociti isolati altamente esprimono albumina (rosso) e fattore di crescita dei fibroblasti receptor 4 (FGFR4) (verde). I nuclei sono colorati con 4 &#39;, 6-diamidino-2-phenylindole (DAPI) (blu). (Figura 1B). <p class="jove_step"...

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

Qui vi mostriamo un approccio enzimatico per isolare epatociti primari da topi adulti, e descriviamo la quantificazione di una risposta infiammatoria con ELISA e PCR in tempo reale.

L&#39;induzione di una risposta infiammatoria in colture primarie epatociti di topi

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.

Research

JoVE Journal

Immunology and Infection

Induction of Experimental Autoimmune Hypophysitis in SJL Mice

Induzione di ipofisite autoimmune sperimentale nei topi SJL

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

Ricerca

Immunologia e infezioni

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

Un metodo semplice ed efficace per isolare i macrofagi da Mixed colture primarie di cellule del fegato degli adulti

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

Generazione di culture Raft organotipiche da cheratinociti umani primari

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

Isolamento primario Microglia misti da colture cellulari gliali del tessuto neonatale cervello di ratto

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

Differentiating Functional Roles of Gene Expression from Immune and Non-immune Cells in Mouse Colitis by Bone Marrow Transplantation

Differenziare i ruoli funzionali di espressione genica di cellule immunitarie e non immunitarie in Colite mouse da trapianto di midollo osseo

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice ...

Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

Isolamento e iniezione endovenosa di Murine derivate dal midollo osseo monociti

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

Induzione di Murine infiammazione intestinale per trasferimento adottivo di Effector CD4<sup&gt; +</sup&gt; CD45RB<sup&gt; Alto</sup&gt; T Cells in topi immunodeficienti

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

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

Saggi per caratterizzare nuovi endoteliale regolatori coinvolti nella risposta infiammatoria di screening

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

Induzione di paralisi e di lesioni del sistema visivo in topi da specifiche cellule T per neuromielite ottica Autoantigen acquaporina-4

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

Effects of Taste Signaling Protein Abolishment on Gut Inflammation in an Inflammatory Bowel Disease Mouse Model

Effetti del gusto segnalazione proteina Abolishment su infiammazione dell'intestino in un modello murino di malattia infiammatoria intestinale

Inflammatory bowel disease (IBD) is one of the immune-related gastrointestinal disorders, including ulcerative colitis and Crohn's disease, that affects ...