Name: steps for the autologous ex vivo perfused porcine liver-kidney experiment
Uploaded: 2013-12-18T13:00:00
Description: Watch this Scientific Journal Video about Steps for the Autologous Ex vivo Perfused Porcine Liver-kidney Experiment at JoVE.com

We would like to thank Sarah Hosgood from the Department of Transplantation, University Hospitals of Leicester, for technical support with the porcine kidney in this model.

1. Preparation of the Procedure Equipment, Solutions, and Experiment Setup
<ol>
	<li>Equipment: 
	The following equipment is required:
	<ol>
		<li>Dissecting instruments: scissors, scalpels, artery forceps, 6x artery clips.</li>
		<li>4x 12-14F catheters (for portal vein, hepatic artery, bile duct and renal artery), 1x 12F catheter (ureter), suture material (20x Vicryl stitches and Vicryl ties), stitch scissors, 3x infusion sets, ice in large insulated transport container, gloves, a large sterile ...

<ol>
	<li>Gravante, G., Ong, S. L., Metcalfe, M. S., Lloyd, D. M., Dennison, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+porcine+hepatic+arterial+supply,+its+variations+and+their+influence+on+the+extracorporeal+perfusion+of+the+liver.">The porcine hepatic arterial supply, its variations and their influence on the extracorporeal perfusion of the liver.</a> J. Surg. Res. 1, 56-61 (2011).</li><li>Gravante, G., 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=Cytokine+response+of+electrolytic+ablation+in+an+ex+vivo+perfused+liver+model.">Cytokine response of electrolytic ablation in an ex vivo perfused liver model.</a> ANZ J. Surg. , 7-8 (2010).</li><li>Gravante, G., 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=Cytokine+response+to+ischemia/reperfusion+injury+in+an+ex+vivo+perfused+porcine+liver+model.">Cytokine response to ischemia/reperfusion injury in an ex vivo perfused porcine liver model.</a> Transplant Proc. 4, 1107-1112 (2009).</li><li>Gravante, G., 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=Patterns+of+histological+changes+following+hepatic+electrolytic+ablation+in+an+ex-vivo+perfused+model.">Patterns of histological changes following hepatic electrolytic ablation in an ex-vivo perfused model.</a> Pathol. Oncol. Res. 4, 1085-1089 (2012).</li><li>Gravante, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+acid-base+balance+during+electrolytic+ablation+in+an+ex+vivo+perfused+liver+model.">Changes in acid-base balance during electrolytic ablation in an ex vivo perfused liver model.</a> Am. J. Surg. 5, 666-670 (2012).</li><li>Chung, W. Y., 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=Addition+of+a+kidney+to+the+normothermic+ex+vivo+perfused+porcine+liver+model+does+not+increase+cytokine+response.">Addition of a kidney to the normothermic ex vivo perfused porcine liver model does not increase cytokine response.</a> J. Artif. Organs. 3, 290-294 (2012).</li><li>Chung, W. Y., 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+autologous+normothermic+ex+vivo+perfused+porcine+liver-kidney+model:+improving+the+circuit's+biochemical+and+acid-base+environment.">The autologous normothermic ex vivo perfused porcine liver-kidney model: improving the circuit's biochemical and acid-base environment.</a> Am. J. Surg. 4, 518-526 (2012).</li><li>Yanaga, K., Makowka, L., Lebeau, G., Hwang, R. R., Shimada, M., Kakizoe, 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=A+new+liver+perfusion+and+preservation+system+for+transplantation+research+in+large+animals.">A new liver perfusion and preservation system for transplantation research in large animals.</a> J. Invest. Surg. 3 (1), 65-75 (1990).</li><li>Chung, W. Y., 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+autologous+normothermic+ex+vivo+perfused+porcine+liver-kidney+model:+improving+the+circuit's+biochemical+and+acid-base+environment.">The autologous normothermic ex vivo perfused porcine liver-kidney model: improving the circuit's biochemical and acid-base environment.</a> Am. J. Surg. 4, 518-526 (2012).</li><li>Chung, W. Y., 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=Addition+of+a+kidney+to+the+normothermic+ex+vivo+perfused+porcine+liver+model+does+not+increase+cytokine+response.">Addition of a kidney to the normothermic ex vivo perfused porcine liver model does not increase cytokine response.</a> J. Artif. Organs. 3, 290-294 (2012).</li><li>Gravante, G., 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=Effects+of+hypoxia+due+to+isovolemic+hemodilution+on+an+ex+vivo+normothermic+perfused+liver+model.">Effects of hypoxia due to isovolemic hemodilution on an ex vivo normothermic perfused liver model.</a> J. Surg. Res. 1, 73-80 (2010).</li><li>Butler, A. 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=Successful+extracorporeal+porcine+liver+perfusion+for+72+hr.">Successful extracorporeal porcine liver perfusion for 72 hr.</a> Transplantation. 8, 1212-1218 (2002).</li><li>St Peter, S. D., Imber, C. J., Lopez, I., Hughes, D., Friend, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extended+preservation+of+non-heart-beating+donor+livers+with+normothermic+machine+perfusion.">Extended preservation of non-heart-beating donor livers with normothermic machine perfusion.</a> Br. J. Surg. 5, 609-616 (2002).</li><li>Schon, M. R., 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=Liver+transplantation+after+organ+preservation+with+normothermic+extracorporeal+perfusion.">Liver transplantation after organ preservation with normothermic extracorporeal perfusion.</a> Ann. Surg. 1, 114-123 (2001).</li><li>Siedlecki, A., Irish, W., Brennan, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delayed+graft+function+in+the+kidney+transplant.">Delayed graft function in the kidney transplant.</a> Am. J. Transplant. 11, 2279-2296 (2011).</li><li>Monbaliu, D., 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=Primary+graft+nonfunction+and+Kupffer+cell+activation+after+liver+transplantation+from+non-heart-beating+donors+in+pigs.">Primary graft nonfunction and Kupffer cell activation after liver transplantation from non-heart-beating donors in pigs.</a> Liver Transpl. 2, 239-247 (2007).</li><li>Skaro, A. I., Jay, C. L., Ladner, D., Abecassis, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trends+in+donation+after+cardiac+death+and+donation+after+brain+death--reading+between+the+lines.">Trends in donation after cardiac death and donation after brain death--reading between the lines.</a> Am. J. Transplant. 11, 2390-2391 (2010).</li><li>Brandhorst, D., Iken, M., Bretzel, R. G., Brandhorst, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pancreas+storage+in+oxygenated+perfluorodecalin+does+not+restore+post-transplant+function+of+isolated+pig+islets+pre-damaged+by+warm+ischemia.">Pancreas storage in oxygenated perfluorodecalin does not restore post-transplant function of isolated pig islets pre-damaged by warm ischemia.</a> Xenotransplantation. 5, 465-470 (2006).</li><li>Lin, Q. Y., Chui, K. K., Rao, A. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+donor+liver+procurement+with+only+aortic+perfusion.">Rapid donor liver procurement with only aortic perfusion.</a> World. J. Gastroenterol. 6, 884-886 (2001).</li><li>Chung, W. Y., 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+development+of+a+multiorgan+ex+vivo+perfused+model:+results+with+the+porcine+liver-kidney+circuit+over+24+hours.">The development of a multiorgan ex vivo perfused model: results with the porcine liver-kidney circuit over 24 hours.</a> Artif. Organs. 5, 457-466 (2013).</li></ol>

In our group the experience and the results achieved with the ex&#160;vivo perfusion of the liver have been paralleled over the years by concomitant modifications in the retrieval technique adopted. In the last few years our group has achieved considerable experience using an ex vivo porcine model to study the hepatic physiology5,11, new liver ablation techniques2,5, and liver immunology3. Despite the technical challenges derived from an additional organ, corrections of ...

Studying a large animal organ physiology in an artificial environment is challenging. Furthermore, maintaining normal physiological and biochemical parameters for a prolonged period requires a closely monitored perfusion method and model. We have previously described our ex vivo autologous perfused porcine liver model for the study of hepatic physiology and significant progress has been achieved, since we began our liver reperfusion ex vivo model in 2005, in areas such as harvesting technique, understanding hepatic artery anatomical variations1, inflammatory response2,3, histological changes following liver electrolytic ablation (EA)4, and changes in acid base balance during EA5. Unfortunately hyperglycemia and metabolic acidosis require external supplements of insulin and bicarbonate to maintain an accurate physiological environment. Stable blood sugar levels and acid base balance are crucial when studying islet auto-transplantation, especially in the early implantation phase. We have developed a new model incorporating the kidney to clear excess metabolites and improve the environment for the islet transplantation6,7.
Extracorporeal perfusion experiments lasting longer than 24 hr can also be performed, but these require a larger team to perform the experiments8.
Animals used in this study received human care and study protocols were in accordance with the United Kingdom laws. They were euthanized with final exsanguinations during the blood-harvesting procedure according to United Kingdom regulations. Porcine liver and a left kidney were retrieved from animals in the food chain and that would otherwise be discarded. In this way important ethical implications can be overcome and the economic burden is lower than more common in vivo studies. The short duration of liver and kidney perfusion time in this model, lasting from 6-24 hr, has been a major limitation of our studies; this can be improved by minimizing the exposure of the organs to the ischemic injury (cold and warm ischemia).

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Epoprostenol sodium (Flolan)</td>
			<td style="width:259px;">Glaxo Smith Kline</td>
			<td style="width:137px;">PL10949/0310</td>
			<td style="width:211px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Bicarbonate 8.4%</td>
			<td>Polyfusor, Fresenius Kabi Ltd</td>
			<td style="width:137px;">PL8828/0043</td>
			<td style="width:211px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cefuroxime sodium (Zynacef)</td>
			<td>Flynn Pharma Ltd</td>
			<td style="width:137px;">PL13621/0018</td>
			<td style="width:211px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Calcium Chloride 14.7%</td>
			<td>Martindale Pharmaceuticals</td>
			<td style="width:137px;">PL1883/6174R</td>
			<td style="width:211px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heparin&#160;</td>
			<td style="width:259px;">LEO Laboratories Limited</td>
			<td style="width:137px;">PL0043/0038R</td>
			<td style="width:211px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium taurocholate</td>
			<td>Sigma-Aldrich</td>
			<td style="width:137px;">T4009-25G</td>
			<td style="width:211px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Insulin (Actrapid)</td>
			<td>Novo Nordisk</td>
			<td style="width:137px;">EU/1/02/230/003</td>
			<td style="width:211px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:195px;">Soltran (preservation solution)</td>
			<td style="width:259px;">Baxter Health Care</td>
			<td style="width:137px;">FKB4708G</td>
			<td style="width:211px;">preservation solution</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:195px;">Atraumatic centrifugal pump (Bio-console 560)</td>
			<td style="width:259px;">Medtronic Inc., Minneapolis, Minnesota- United States; custom pack, cardiac surgery division Europe</td>
			<td style="width:137px;"></td>
			<td style="width:211px;"></td>
		</tr>
	</tbody>
</table>

steps for the autologous ex vivo perfused porcine liver-kidney experiment

The use of ex vivo perfused models can mimic the physiological conditions of the liver for short periods, but to maintain normal homeostasis for an extended perfusion period is challenging. We have added the kidney to our previous ex vivo perfused liver experiment model to reproduce a more accurate physiological state for prolonged experiments without using live animals. Five intact livers and kidneys were retrieved post-mortem from sacrificed pigs on different days and perfused for a minimum of 6 hr. Hourly arterial blood gases were obtained to analyze pH, lactate, glucose and renal parameters. The primary endpoint was to investigate the effect of adding one kidney to the model on the acid base balance, glucose, and electrolyte levels. The result of this liver-kidney experiment was compared to the results of five previous liver only perfusion models. In summary, with the addition of one kidney to the ex vivo liver circuit, hyperglycemia and metabolic acidosis were improved. In addition this model reproduces the physiological and metabolic responses of the liver sufficiently accurately to obviate the need for the use of live animals. The ex vivo liver-kidney perfusion model can be used as an alternative method in organ specific studies. It provides a disconnection from numerous systemic influences and allows specific and accurate adjustments of arterial and venous pressures and flow.

Liver Function
The liver function tests remained stable for the first 6 hr. However, there were significant changes present only from the 19th hour of the 24 hr experiment (P&#60;0.001). Factor V and X were progressively diminished during the whole experiment and significant changes were evident from the 5th hour of perfusion for factor V and from the 3rd hour for the factor X onward, (P&#60;0.05; Figure&#160;2)20.
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Watch this Scientific Journal Video about Steps for the Autologous Ex vivo Perfused Porcine Liver-kidney Experiment at JoVE.com

Steps for the Autologous Ex vivo Perfused Porcine Liver-kidney Experiment

Study of the animal organ physiology can be achieved by performing an experimental ex vivo perfusion system. The addition of a porcine kidney, as a homeostatic organ to our previously developed ex vivo liver perfusion model can be a principal step to achieve a better physiological environment.

Steps for the Autologous Ex vivo Perfused Porcine Liver-kidney Experiment

The use of ex vivo perfused models can mimic the physiological conditions of the liver for short periods, but to maintain normal homeostasis for an ...

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