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 container for the blood collection. 
		At least three researchers are required to perform the organ retrieval.</li>
	</ol>
	</li>
	<li>Solutions and/ or drugs 
	Table 1 and the table of specific reagents and materials summarize all the required solutions and drugs required for the experiment.</li>
	<li>Extracorporeal circuit preparation 
	Setup the circuit before the organ is harvested. The circuit is similar to those used for cardiopulmonary extracorporeal bypass surgery. It consists of nontraumatic centrifugal pump that provides the hepatic/renal arterial blood flow and pressure readings, an oxygenator, a heat exchanger unit and a venous reservoir to stimulate the venous flow and pressures. After passage through the liver and kidney, the venous blood is collected from both the Inferior Vena Cava (IVC) and&#160;renal vein into a container simulating the systemic venous reservoir and returned to the centrifugal pump (Figure&#160;1).
	<ol>
		<li>Remove the circuit from the packaging ensuring its sterility. The circuit consists of two reservoirs; one supplying the pseudo portal circulation and one supplying the pseudo systemic circulation as per Figure 1.</li>
		<li>Connect the drainage lines from the IVC and renal vein to the systemic reservoir.</li>
		<li>Connect the systemic reservoir to the pump and then the oxygenator.</li>
		<li>There are 3 lines exiting the oxygenator. Connect one to each of the following: the hepatic artery, renal artery, and the reservoir supplying the hepatic portal vein. Connect this reservoir to the hepatic portal vein.</li>
	</ol>
	</li>
</ol>
2. Harvesting the Porcine Kidney and&#160;Liver and Washing the Organs with Preservation Solution
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.
<ol>
	<li>Collect the autologous blood into a preheparinized nonpyogenic container with 25,000 units of Heparin.</li>
	<li>Immediately following the laparotomy and sternotomy incision, shift all bowel loops to the right side, open the left retroperitoneal space and free up the left kidney from its attachments</li>
	<li>Identify the renal artery and vein then resect them at their origin from the aorta and IVC.</li>
	<li>Identify the ureter and cut approximately 10-15 cm distal to origin. 
	One team member should take the kidney to a backbench table and place it in a kidney dish prefilled with ice and preservation solution (50:50).</li>
	<li>Cannulate the renal artery as soon as possible and start an infusion of 500 ml (4 &#176;C) preservation solution. Take all precautions to avoid any kink in the ureter as this will lead to hydronephrosis. On completion of the preservation infusion solution, place the kidney in a bag filled with preservation solution and store on ice, in an insulated container for transport to the laboratory.</li>
	<li>Identify both the gastrocolic and treitz ligament and divide them; the Superior Mesenteric Vein (SMV) is found close to treitz ligament.</li>
	<li>Cannulate the SMV to reach the portal vein and fix with a 0 Vicryl tie. Ligate the splenic vein (SV), and infuse cold 0.9% Sodium Chloride solution through the portal vein to remove any residual blood clots.</li>
	<li>Identify the third part of the duodenum, ligate and then divided it. Mobilize the stomach, identify and resect the infra-hepatic IVC at any level. Identify the suprahepatic IVC and divide it along with the aorta and esophagus. Detach the aorta from the vertebral column attachments up to the level of the renal vessels and divide it at this level. Dissect any nonvascular attachments allowing the liver, stomach, second part of duodenum, pancreas, and spleen to be removed en-bloc, and then move the specimen on to a tray filled with ice on a separate bench table.</li>
	<li>Start an infusion of approximately 1.5 L of the cold preservation solution (4 &#176;C) through the portal vein.</li>
	<li>Identify and cannulate the hepatic artery. Secure the cannula with a Vicryl tie and commence an infusion of 1 L of cold (4 &#176;C) preservation solution. 
	All of the above steps should be completed as fast as possible to minimize the warm ischemic injury time to the organs.</li>
	<li>Mobilize the spleen and divide the gastrosplenic ligament, ligate the right gastric artery and then discard the spleen and&#160;stomach.</li>
	<li>Finally, carefully dissect the pancreas from the splenic artery and free the pancreatic branches of the hepatic artery and ligate them, then identify the common bile duct and cut it long.</li>
	<li>Thoroughly flush the retrieved liver to remove all blood clots, transfer the liver into a bag containing cold preservation solution and then place it inside an ice box for transport to the laboratory.</li>
</ol>
3. Priming the Circuit
<ol>
	<li>Add approximately 1.5-2.5 L of the heparinized autologous porcine blood to the systemic venous reservoir of the circuit during the backbench preparation. This blood is the perfusate for the ex vivo liver and kidney experiment. Start the centrifugal pump and allow the perfusate to run through the intra-luminal and extra-luminal compartments and fill in all circuit lines as well as the hollow fiber filter</li>
	<li>Add the following to the above perfusate: 40 ml of 8.4% sodium bicarbonate, 20 ml of epoprostenol sodium (500 &#956;g in 50 ml of diluent, diluted with 200 ml of 0.9% sodium chloride), 750 mg of cefuroxime sodium in 10 ml water, 10 mmol in 10 ml calcium chloride 14.7%, and 50 units of insulin actrapid.</li>
	<li>Prime the centrifugal pump with perfusate under the effect of gravity. Manually tap the circuit to remove any air bubbles toward the venous reservoir.</li>
	<li>Start the centrifugal pump at a pump speed of 1,200 rpm to prime the remaining part of the circuit with perfusate.</li>
	<li>Supply the oxygen (mixture of 95% oxygen and&#160;5% carbon dioxide) to the oxygenator at a rate of 2 L/min and connect the oxygenator to the water bath/ heat exchanger preset at temperature of 38 &#176;C (the physiologic temperature of pig).</li>
	<li>Zero the pressure measuring device on the centrifugal pump to air (to zero the flow, both the inlet and outlet into the flow transducer should be fully clamped).</li>
	<li>Once the perfusion has started maintain a continuous infusion of epoprostenol sodium 20 ml/hr and sodium taurocholate at 10 ml/hr (10 units/ml).</li>
	<li>Administer boluses of 40 ml 8.4% sodium bicarbonate and 50 units of insulin actrapid to maintain physiological parameters of glucose and acid base balance.</li>
	<li>If the metabolic acidosis and hyperglycemia are not corrected with boluses after the first hour, administer sodium bicarbonate 8.4% as an infusion at a rate of 40 ml/hr and titrate it according to the ABG and 100 units/hr Insulin. 
	These continuous infusions need to be started to mimic the normal physiologic conditions and to maintain the pH within the physiologic range after reperfusion, ameliorating the acidotic changes derived from the ischemic reperfusion injury.</li>
</ol>
4. Backbench Preparation of Liver and Kidney for the Perfusion
<ol>
	<li>Identify both the ureter and the common bile duct, cannulate and secure them.</li>
	<li>Infuse 1 L of 0.9% sodium chloride solution through the portal vein, 500 ml through the hepatic artery and 500 ml through the renal artery to remove any blood clots from the organs and the cannulas and to flush out the preservation solution.</li>
	<li>Identify any leaks from the vascular branches (as these will bleed during the perfusion) and ligate them with 2/0 Vicryl tie or stitches.</li>
</ol>
5. Ex vivo Liver-kidney Perfusion
<ol>
	<li>Once all the lines are primed and the perfusate is optimized, collect a baseline blood sample for full blood count, biochemical and blood gas analysis before connecting the first organ. Take a tissue biopsy if performing histological examination.</li>
	<li>Transfer the retrieved kidney into the allocated nonpyogenic perfusion chamber; connect the renal artery catheter to the respective line of the extracorporeal circuit and the catheterized ureter into an hourly urometer for urine output monitoring.</li>
	<li>Adjust the perfusion pressure of the kidney and maintain the renal arterial pressure between 80-90 mmHg.</li>
	<li>Transfer the liver into the allocated perfusion chamber, and immediately connect the hepatic artery and portal vein to their respective lines of the extracorporeal circuit. Allow the cannulated bile duct to drain freely into an allocated bile bag for monitoring.</li>
	<li>Adjust the perfusion pressure and maintain the hepatic artery pressure between 80-100 mmHg and the portal vein pressure at &#60;10 mmHg. 
	The resistance in the portal venous system is low compared to the arterial channels, therefore, apply a clamp on the inlet tubing of the portal reservoir to control the inflow into the portal reservoir and maintain physiological pressures in the hepatic artery and portal vein. Tightening the clamp will decrease the inflow into the portal reservoir and increase the flow and pressure into the arterial lines, and vice versa.</li>
	<li>To maintain the perfusion within physiological values of the arterial and venous flows and pressure, alter both the pump speed and change the inflow resistance of the reservoir aiming for a hepatic and renal artery flow of between 0.2-0.3 L/min&#160;and the portal vein flow above 1 L/min.</li>
	<li>Collect blood samples hourly for hematological, biochemical and blood gas analysis and correct any electrolyte, glycemic and metabolic changes using the relevant solution (Table 1). Collect the last blood sample after 6 hr of perfusion and discard the organs according to your local policies.</li>
</ol>

<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>

Authors have nothing to disclose.

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).

<table border="0" cellpadding="0" cellspacing="0" width="800">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<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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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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7.3K vistas.  University Hospitals of Leicester. 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.

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

Ex Vivo Infection of Live Tissue with Oncolytic Viruses

Oncolytic Viruses (OVs) are novel therapeutics that selectively replicate in and kill tumor cells1. Several clinical trials evaluating the effectiveness of a variety of oncolytic platforms including HSV, Reovirus, and Vaccinia OVs as treatment for cancer are currently underway2-5. One key characteristic of oncolytic viruses is that they can be genetically modified to express reporter transgenes which makes it possible to visualize the infection of tissues by microscopy or bio-luminescence imaging6,7. This offers a unique advantage since it is possible to infect tissues from patients ex vivo prior to therapy in order to ascertain the likelihood of successful oncolytic virotherapy8. To this end, it is critical to appropriately sample tissue to compensate for tissue heterogeneity and assess tissue viability, particularly prior to infection9. It is also important to follow viral replication using reporter transgenes if expressed by the oncolytic platform as well as by direct titration of tissues following homogenization in order to discriminate between abortive and productive infection. The object of this protocol is to address these issues and herein describes 1. The sampling and preparation of tumor tissue for cell culture 2. The assessment of tissue viability using the metabolic dye alamar blue 3. Ex vivo infection of cultured tissues with vaccinia virus expressing either GFP or firefly luciferase 4. Detection of transgene expression by fluorescence microscopy or using an In Vivo Imaging System (IVIS) 5. Quantification of virus by plaque assay. This comprehensive method presents several advantages including ease of tissue processing, compensation for tissue heterogeneity, control of tissue viability, and discrimination between abortive infection and bone fide viral replication.

Ex Vivo Infection of Live Tissue with Oncolytic Viruses

Oncolytic viruses are promising for cancer therapeutics. The ability to ascertain the infectability of live tissue specimens obtained from patients prior to treatment is a unique advantage of this therapeutic approach. This protocol describes how to process tissues for ex vivo infection with oncolytic virus and subsequent viral quantification.

Oncolytic Viruses (OVs) are novel therapeutics that selectively replicate in and kill tumor cells1. Several clinical trials evaluating the effectiveness ...

An Ex vivo Model to Study Hormone Action in the Human Breast

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial cells tend to lose hormone receptor expression. Widely used hormone receptor positive breast cancer cell lines are of limited relevance to the in vivo situation. Here, we describe an ex vivo model to study hormone action in the human breast. Fresh human breast tissue specimens from surgical discard material such as reduction mammoplasties or mammectomies are mechanically and enzymatically digested to obtain tissue fragments containing ducts and lobules and multiple stromal cell types. These tissue microstructures kept in basal medium without growth factors preserve their intercellular contacts, the tissue architecture, and remain hormone responsive for several days. They are readily processed for RNA and protein extraction, histological analysis or stored in freezing medium. Fluorescence activated cell sorting (FACS) can be used to enrich for specific cell populations. This protocol provides a straightforward, standard approach for translational studies with highly complex, varied human specimens.

An Ex vivo Model to Study Hormone Action in the Human Breast

We have developed a novel ex vivo model to study hormone action in the human breast. It is based on tissue microstructures isolated from surgical breast tissue specimens which preserve tissue architecture, intercellular interactions, and paracrine signaling.

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial ...

Human Dupuytren's Ex Vivo Culture for the Study of Myofibroblasts and Extracellular Matrix Interactions

Organ fibrosis or &#8220;scarring&#8221; is known to account for a high death toll due to the extensive amount of disorders and organs affected (from cirrhosis to cardiovascular diseases). There is no effective treatment and the in vitro tools available do not mimic the in vivo situation rendering the progress of the out of control wound healing process still enigmatic.
To date, 2D and 3D cultures of fibroblasts derived from DD patients are the main experimental models available. Primary cell cultures have many limitations; the fibroblasts derived from DD are altered by the culture conditions, lack cellular context and interactions, which are crucial for the development of fibrosis and weakly represent the derived tissue. Real-time PCR analysis of fibroblasts derived from control and DD samples show that little difference is detectable. 3D cultures of fibroblasts include addition of extracellular matrix that alters the native conditions of these cells. 
As a way to characterize the fibrotic, proliferative properties of these resection specimens we have developed a 3D culture system, using intact human resections of the nodule part of the cord. The system is based on transwell plates with an attached nitrocellulose membrane that allows contact of the tissue with the medium but not with the plastic, thus, preventing the alteration of the tissue. No collagen gel or other extracellular matrix protein substrate is required. The tissue resection specimens maintain their viability and proliferative properties for 7 days. This is the first &#8220;organ&#8221; culture system that allows human resection specimens from DD patients to be grown ex vivo and functionally tested, recapitulating the in vivo situation.

Human Dupuytren&#039;s Ex Vivo Culture for the Study of Myofibroblasts and Extracellular Matrix Interactions

Dupuytren&#8217;s disease (DD) is a fibroproliferative disease of the palm of the hand. Here, we present a protocol to culture resection specimens from DD in a three-dimensional (3D) culture system. Such short-term culture system allows preservation of the 3D structure and molecular properties of the fibrotic tissue.

Organ fibrosis or &#8220;scarring&#8221; is known to account for a high death toll due to the extensive amount of disorders and organs affected (from ...

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of women are alive 5 years after diagnosis. The omentum is a preferred site of HGSC metastasis formation. Despite the clinical importance of this microenvironment, the contribution of omental adipose tissue to ovarian cancer progression remains understudied. Omental adipose is unusual in that it contains structures known as milky spots, which are comprised of B, T, and NK cells, macrophages, and progenitor cells surrounding dense nests of vasculature. Milky spots play a key role in the physiologic functions of the omentum, which are required for peritoneal homeostasis. We have shown that milky spots also promote ovarian cancer metastatic colonization of peritoneal adipose, a key step in the development of peritoneal metastases. Here we describe the approaches we developed to evaluate and quantify milky spots in peritoneal adipose and study their functional contribution to ovarian cancer cell metastatic colonization of omental tissues both in vivo and ex vivo. These approaches are generalizable to additional mouse models and cell lines, thus enabling the study of ovarian cancer metastasis formation from initial localization of cells to milky spot structures to the development of widespread peritoneal metastases.

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

We outline a protocol that implements both in vivo and ex vivo approaches to study ovarian cancer colonization of peritoneal adipose tissues, particularly the omentum. Furthermore, we present a protocol to quantitate and analyze immune cell-structures in the omentum known as milky spots, which promote metastases of peritoneal adipose.

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of ...

Normothermic Ex Vivo Kidney Perfusion for the Preservation of Kidney Grafts prior to Transplantation

Kidney transplantation has become a well-established treatment option for patients with end-stage renal failure. The persisting organ shortage remains a serious problem. Therefore, the acceptance criteria for organ donors have been extended leading to the usage of marginal kidney grafts. These marginal organs tolerate cold storage poorly resulting in increased preservation injury and higher rates of delayed graft function. To overcome the limitations of cold storage, extensive research is focused on alternative normothermic preservation methods.
Ex vivo normothermic organ perfusion is an innovative preservation technique. The first experimental and clinical trials for ex vivo lung, liver, and kidney perfusions demonstrated favorable outcomes.
In addition to the reduction of cold ischemic injury, the method of normothermic kidney storage offers the opportunity for organ assessment and repair. This manuscript provides information about kidney retrieval, organ preservation techniques, and isolated ex vivo normothermic kidney perfusion (NEVKP) in a porcine model. Surgical techniques, set up for the perfusion solution and the circuit, potential assessment options, and representative results are demonstrated.

Normothermic Ex Vivo Kidney Perfusion for the Preservation of Kidney Grafts prior to Transplantation

The severe organ shortage has resulted in increased use of marginal kidney grafts for transplantation. This has triggered interest in alternative storage methods, since marginal grafts especially tolerate cold storage poorly. The technique of normothermic ex vivo kidney perfusion (NEVKP) represents a novel preservation method for kidney grafts prior to &#160;transplantation.

Kidney transplantation has become a well-established treatment option for patients with end-stage renal failure. The persisting organ shortage remains a ...

Preparation Steps for Measurement of Reactivity in Mouse Retinal Arterioles Ex Vivo

Vascular insufficiency and alterations in normal retinal perfusion are among the major factors for the pathogenesis of various sight-threatening ocular diseases, such as diabetic retinopathy, hypertensive retinopathy, and possibly glaucoma. Therefore, retinal microvascular preparations are pivotal tools for physiological and pharmacological studies to delineate the underlying pathophysiological mechanisms and to design therapies for the diseases. Despite the wide use of mouse models in ophthalmic research, studies on retinal vascular reactivity are scarce in this species. A major reason for this discrepancy is the challenging isolation procedures owing to the small size of these retinal blood vessels, which is ~ &#8804; 30 &#181;m in luminal diameter. To circumvent the problem of direct isolation of these retinal microvessels for functional studies, we established an isolation and preparation technique that enables ex vivo studies of mouse retinal vasoactivity under near-physiological conditions. Although the present experimental preparations will specifically refer to the mouse retinal arterioles, this methodology can readily be employed to microvessels from rats.

Preparation Steps for Measurement of Reactivity in Mouse Retinal Arterioles Ex Vivo

Many vision-threatening ocular diseases are associated with dysfunctional retinal microvessels. Therefore, the measurement of retinal arteriole responses is important to investigate the underlying pathophysiological mechanisms. This article describes a detailed protocol for mouse retinal arteriole isolation and preparation to assess the effects of vasoactive substances on vascular diameter.

Vascular insufficiency and alterations in normal retinal perfusion are among the major factors for the pathogenesis of various sight-threatening ocular ...

Lentiviral Vector-mediated Gene Therapy of Hepatocytes Ex Vivo for Autologous Transplantation in Swine

Gene therapy is an ideal choice to cure many inborn errors of metabolism of the liver. Ex-vivo, lentiviral vectors have been used successfully in the treatment of many hematopoietic diseases in humans, as their use offers stable transgene expression due to the vector&#39;s ability to integrate into the host genome. This method demonstrates the application of ex vivo gene therapy of hepatocytes to a large animal model of hereditary tyrosinemia type I. This process consists of 1) isolation of primary hepatocytes from the autologous donor/recipient animal, 2) ex vivo gene delivery via hepatocyte transduction with a lentiviral vector, and 3) autologous transplant of corrected hepatocytes via portal vein injection. Success of the method generally relies upon efficient and sterile removal of the liver resection, careful handling of the excised specimen for isolation of viable hepatocytes sufficient for re-engrafting, high-percentage transduction of the isolated cells, and aseptic surgical procedures throughout to prevent infection. Technical failure at any of these steps will result in low yield of viable transduced hepatocytes for autologous transplant or infection of the donor/recipient animal. The pig model of human type 1 hereditary tyrosinemia (HT-1) chosen for this approach is uniquely amenable to such a method, as even a small percentage of engraftment of corrected cells will lead to repopulation of the liver with healthy cells based on a powerful selective advantage over native-diseased hepatocytes. Although this growth selection will not be true for all indications, this approach is a foundation for expansion into other indications and allows for manipulation of this environment to address additional diseases, both within the liver and beyond, while controlling for exposure to viral vector and opportunity for off-target toxicity and tumorigenicity.

Lentiviral Vector-mediated Gene Therapy of Hepatocytes Ex Vivo for Autologous Transplantation in Swine

This protocol is intended to describe porcine hepatocyte isolation and ex vivo gene delivery to cure models of metabolic diseases via autologous cell transplantation. Although this particular model enjoys unique advantages that favor successful therapy, the application is a relevant foundation to address additional diseases and indications.

Gene therapy is an ideal choice to cure many inborn errors of metabolism of the liver. Ex-vivo, lentiviral vectors have been used successfully in the ...

Mechanisms Underlying Gut Hormone Secretion Using the Isolated Perfused Rat Small Intestine

The gut is the largest endocrine organ of the body, producing more than 15 different peptide hormones that regulate appetite and food intake, digestion, nutrient absorption and distribution, and post-prandial glucose excursions. Understanding the molecular mechanisms that regulate gut hormone secretion is fundamental for understanding and translating gut hormone physiology. Traditionally, the mechanisms underlying gut hormone secretion are either studied in vivo (in experimental animals or humans) or using gut hormone-secreting primary mucosal cell cultures or cell lines. Here, we introduce an isolated perfused rat small intestine as an alternative method for studying gut hormone secretion. The virtues of this model are that it relies on the intact gut, meaning that it recapitulates most of the physiologically important parameters responsible for the secretion in in vivo studies, including mucosal polarization, paracrine relationships and routes of perfusion/stimulus exposure. In addition, and unlike in vivo studies, the isolated perfused rat small intestine allows for almost complete experimental control and direct assessment of secretion. In contrast to in vitro studies, it is possible to study both the magnitude and the dynamics of secretion and to address important questions, such as what stimuli cause secretion of different gut hormones, from which side of the gut (luminal or vascular) is secretion stimulated, and to analyze in detail molecular sensors underlying the secretory response. In addition, the preparation is a powerful model for the study of intestinal absorption and details regarding the dynamics of intestinal absorption including the responsible transporters.

Here, we present a powerful and physiological model to study the molecular mechanisms underlying gut hormone secretion and intestinal absorption &#8212; the isolated perfused rat small intestine.

The gut is the largest endocrine organ of the body, producing more than 15 different peptide hormones that regulate appetite and food intake, digestion, ...

Ex Vivo Corneal Organ Culture Model for Wound Healing Studies

The cornea has been used extensively as a model system to study wound healing. The ability to generate and utilize primary mammalian cells in two dimensional (2D) and three dimensional (3D) culture has generated a wealth of information not only about corneal biology but also about wound healing, myofibroblast biology, and scarring in general. The goal of the protocol is an assay system for quantifying myofibroblast development, which characterizes scarring. We demonstrate a corneal organ culture ex vivo model using pig eyes. In this anterior keratectomy wound, corneas still in the globe are wounded with a circular blade called a trephine. A plug of approximately 1/3 of the anterior cornea is removed including the epithelium, the basement membrane, and the anterior part of the stroma. After wounding, corneas are cut from the globe, mounted on a collagen/agar base, and cultured for two weeks in supplemented-serum free medium with stabilized vitamin C to augment cell proliferation and extracellular matrix secretion by resident fibroblasts. Activation of myofibroblasts in the anterior stroma is evident in the healed cornea. This model can be used to assay wound closure, the development of myofibroblasts and fibrotic markers, and for toxicology studies. In addition, the effects of small molecule inhibitors as well as lipid-mediated siRNA transfection for gene knockdown can be tested in this system.

A protocol for an ex vivo corneal organ culture model useful for wound healing studies is described. This model system can be used to assess the effects of agents to promote regenerative healing or drug toxicity in an organized 3D multicellular environment.

The cornea has been used extensively as a model system to study wound healing. The ability to generate and utilize primary mammalian cells in two ...

Optocardiography and Electrophysiology Studies of Ex Vivo Langendorff-perfused Hearts

Small animal models are most commonly used in cardiovascular research due to the availability of genetically modified species and lower cost compared to larger animals. Yet, larger mammals are better suited for translational research questions related to normal cardiac physiology, pathophysiology, and preclinical testing of therapeutic agents. To overcome the technical barriers associated with employing a larger animal model in cardiac research, we describe an approach to measure physiological parameters in an isolated, Langendorff-perfused piglet heart. This approach combines two powerful experimental tools to evaluate the state of the heart: electrophysiology (EP) study and simultaneous optical mapping of transmembrane voltage and intracellular calcium using parameter sensitive dyes (RH237, Rhod2-AM). The described methodologies are well suited for translational studies investigating the cardiac conduction system, alterations in action potential morphology, calcium handling, excitation-contraction coupling and the incidence of cardiac alternans or arrhythmias.

The objective of this study was to establish a method for investigating cardiac dynamics using a translational animal model. The described experimental approach incorporates dual-emission optocardiography in conjunction with an electrophysiological study to assess electrical activity in an isolated, intact porcine heart model.

Small animal models are most commonly used in cardiovascular research due to the availability of genetically modified species and lower cost compared to ...