Name: porcine liver transplantation without veno-venous bypass as an extended criteria donor model
Uploaded: 2022-08-17T14:00:00
Description: Watch this Scientific Journal Video about Porcine Liver Transplantation Without Veno-Venous Bypass As an Extended Criteria Donor Model at JoVE.com

The authors thank Britta Trautewig, Corinna L&#246;bbert, Astrid Dinkel, and Ingrid Meder for their diligence and commitment. Furthermore, the authors thank Tom Figiel for producing the picture material.

This study was performed at the Laboratory for Animal Science of Hannover Medical School after approval by the Lower Saxony regional authority for consumer protection and food safety (Nieders&#228;chsisches Landesamt f&#252;r Verbraucherschutz und Lebensmittelsicherheit [LAVES]; 19/3146)
1. Donor liver procurement
NOTE: The liver donors were female domestic pigs (Sus scrofa domesticus), aged 4-5 months old and with an average body weight of a...

<ol>
	<li>Zarrinpar, A., Busuttil, R. W. <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:+Past,+present+and+future.">Liver transplantation: Past, present and future.</a> Nature Reviews Gastroenterology &amp; Hepatology. 10 (7), 434-440 (2013).</li><li>Song, A. 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=Liver+transplantation:+Fifty+years+of+experience.">Liver transplantation: Fifty years of experience.</a> World Journal of Gastroenterology. 20 (18), 5363-5374 (2014).</li><li>Jakubauskas, 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=Machine+perfusion+in+liver+transplantation:+A+systematic+review+and+meta-analysis.">Machine perfusion in liver transplantation: A systematic review and meta-analysis.</a> Visceral Medicine. , (2021).</li><li>Serifis, N., 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=Machine+perfusion+of+the+liver:+A+review+of+clinical+trials.">Machine perfusion of the liver: A review of clinical trials.</a> Frontiers in Surgery. 8, 625394 (2021).</li><li>Ceresa, C. D. L., Nasralla, D., Pollok, J. -. M., 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=Machine+perfusion+of+the+liver:+Applications+in+transplantation+and+beyond.">Machine perfusion of the liver: Applications in transplantation and beyond.</a> Nature Reviews Gastroenterology &amp; Hepatology. 19 (3), 199-209 (2022).</li><li>Schlegel, A., Muller, X., Dutkowski, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Machine+perfusion+strategies+in+liver+transplantation.">Machine perfusion strategies in liver transplantation.</a> Hepatobiliary Surgery and Nutrition. 8 (5), 490-501 (2019).</li><li>Wenzel, N., Blasczyk, R., Figueiredo, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+in+allogenic+solid+organ+transplantation.">Animal models in allogenic solid organ transplantation.</a> Transplantology. 2 (4), 412-424 (2021).</li><li>Kamada, N., Calne, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+surgical+experience+with+five+hundred+thirty+liver+transplants+in+the+rat.">A surgical experience with five hundred thirty liver transplants in the rat.</a> Surgery. 93 (1), 64-69 (1983).</li><li>Oldani, G., Lacotte, S., Morel, P., Mentha, G., Toso, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthotopic+liver+transplantation+in+rats.">Orthotopic liver transplantation in rats.</a> Journal of Visualized Experiments. (65), e4143 (2012).</li><li>Yang, L., 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+rat+model+of+orthotopic+liver+transplantation+using+a+novel+magnetic+anastomosis+technique+for+suprahepatic+vena+cava+reconstruction.">A rat model of orthotopic liver transplantation using a novel magnetic anastomosis technique for suprahepatic vena cava reconstruction.</a> Journal of Visualized Experiments. (133), e56933 (2018).</li><li>Chen, X. -. 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=Reduced+complications+after+arterial+reconnection+in+a+rat+model+of+orthotopic+liver+transplantation.">Reduced complications after arterial reconnection in a rat model of orthotopic liver transplantation.</a> Journal of Visualized Experiments. (165), e60628 (2020).</li><li>Qian, S. G., Fung, J. J., Demetris, A. V., Ildstad, S. T., Starzl, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthotopic+liver+transplantation+in+the+mouse.">Orthotopic liver transplantation in the mouse.</a> Transplantation. 52 (3), 562-564 (1991).</li><li>Li, X., Wang, Y., Yang, H., Dai, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver+and+hepatocyte+transplantation:+What+can+pigs+contribute.">Liver and hepatocyte transplantation: What can pigs contribute.</a> Frontiers in Immunology. 12, 802692 (2022).</li><li>Reardon, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=First+pig-to-human+heart+transplant:+what+can+scientists+learn.">First pig-to-human heart transplant: what can scientists learn.</a> Nature. 601 (7893), 305-306 (2022).</li><li>Garnier, 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=Liver+transplantation+in+the+pig:+Surgical+approach.">Liver transplantation in the pig: Surgical approach.</a> Comptes Rendus Hebdomadaires des Seances de l'Academie des Sciences. Serie d: Sciences Naturelles. 260 (21), 5621-5623 (1965).</li><li>Calne, R. 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=Observations+of+orthotopic+liver+transplantation+in+the+pig.">Observations of orthotopic liver transplantation in the pig.</a> British Medical Journal. 2 (5550), 478-480 (1967).</li><li>Chalstrey, L. 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=Technique+of+orthotopic+liver+transplantation+in+the+pig.">Technique of orthotopic liver transplantation in the pig.</a> The British Journal of Surgery. 58 (8), 585-588 (1971).</li><li>Filipponi, F., Falcini, F., Benassai, C., Martini, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthotopic+liver+transplant+in+pigs:+Several+variations+of+the+surgical+technic.">Orthotopic liver transplant in pigs: Several variations of the surgical technic.</a> Il Giornale di Chirurgia. 10 (7-8), 374-378 (1989).</li><li>Spetzler, V. N., 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=Technique+of+porcine+liver+procurement+and+orthotopic+transplantation+using+an+active+porto-caval+shunt.">Technique of porcine liver procurement and orthotopic transplantation using an active porto-caval shunt.</a> Journal of Visualized Experiments. (99), e52055 (2015).</li><li>Oldhafer, K. J., Hauss, J., Gubernatis, G., Pichlmayr, R., Spiegel, H. U. <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+in+pigs:+A+model+for+studying+reperfusion+injury.">Liver transplantation in pigs: A model for studying reperfusion injury.</a> Journal of Investigative Surgery. 6 (5), 439-450 (1993).</li><li>Oldhafer, K. 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=Analysis+of+liver+hemodynamics+in+severe+ischemia+and+reperfusion+injury+after+liver+transplantation.">Analysis of liver hemodynamics in severe ischemia and reperfusion injury after liver transplantation.</a> Zentralblatt fur Chirurgie. 119 (5), 317-321 (1994).</li><li>Vogel, 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=Successful+transplantation+of+porcine+liver+grafts+following+48-hour+normothermic+preservation.">Successful transplantation of porcine liver grafts following 48-hour normothermic preservation.</a> PLoS One. 12 (11), 0188494 (2017).</li><li>Leal, 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=A+simplified+experimental+model+of+large-for-size+liver+transplantation+in+pigs.">A simplified experimental model of large-for-size liver transplantation in pigs.</a> Clinics. 68 (8), 1152-1156 (2013).</li><li>Schiefer, 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=Regulation+of+histamine+and+diamine+oxidase+in+patients+undergoing+orthotopic+liver+transplantation.">Regulation of histamine and diamine oxidase in patients undergoing orthotopic liver transplantation.</a> Scientific Reports. 10 (1), 822 (2020).</li><li>Esmaeilzadeh, 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=Technical+guidelines+for+porcine+liver+allo-transplantation:+A+review+of+literature.">Technical guidelines for porcine liver allo-transplantation: A review of literature.</a> Annals of Transplantation. 17 (2), 101-110 (2012).</li><li>Oldhafer, F., 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=Supportive+hepatocyte+transplantation+after+partial+hepatectomy+enhances+liver+regeneration+in+a+preclinical+pig+model.">Supportive hepatocyte transplantation after partial hepatectomy enhances liver regeneration in a preclinical pig model.</a> European Surgical Research. 62 (4), 238-247 (2021).</li><li>Stockmann, 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=The+LiMAx+test:+A+new+liver+function+test+for+predicting+postoperative+outcome+in+liver+surgery.">The LiMAx test: A new liver function test for predicting postoperative outcome in liver surgery.</a> HPB. 12 (2), 139-146 (2010).</li><li>Lapisatepun, W., Lapisatepun, W., Agopian, V., Xia, V. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Venovenous+bypass+during+liver+transplantation:+A+new+look+at+an+old+technique.">Venovenous bypass during liver transplantation: A new look at an old technique.</a> Transplantation Proceedings. 52 (3), 905-909 (2020).</li><li>Falcini, F., 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=Veno-venous+bypass+in+experimental+liver+transplantation:+portal-jugular+versus+caval-portal-jugular.">Veno-venous bypass in experimental liver transplantation: portal-jugular versus caval-portal-jugular.</a> Il Giornale di Chirurgia. 11 (4), 206-210 (1990).</li><li>Copca, N., 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=Experimental+liver+transplantation+on+pigs+--+Technical+considerations.">Experimental liver transplantation on pigs -- Technical considerations.</a> Chirurgia. 108 (4), 542-546 (2013).</li><li>Torres, O. 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=Hemodynamic+alterations+during+orthotopic+liver+experimental+transplantation+in+pigs.">Hemodynamic alterations during orthotopic liver experimental transplantation in pigs.</a> Acta Cirurgica Brasileria. 23 (2), 135-139 (2008).</li><li>Canedo, B. F., 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+autotransplantation+in+pigs+without+venovenous+bypass:+A+simplified+model+using+a+supraceliac+aorta+cross-clamping+maneuver.">Liver autotransplantation in pigs without venovenous bypass: A simplified model using a supraceliac aorta cross-clamping maneuver.</a> Annals of Transplantation. 20, 320-326 (2015).</li><li>Battersby, C., Hickman, R., Saunders, S. J., Terblanche, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver+function+in+the+pig.+1.+The+effects+of+30+minutes'+normothermic+ischaemia.">Liver function in the pig. 1. The effects of 30 minutes' normothermic ischaemia.</a> The British Journal of Surgery. 61 (1), 27-32 (1974).</li><li>Kaiser, G. M., Heuer, M. M., Fr&#252;hauf, N. R., K&#252;hne, C. A., Broelsch, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=General+handling+and+anesthesia+for+experimental+surgery+in+pigs.">General handling and anesthesia for experimental surgery in pigs.</a> Journal of Surgical Research. 130 (1), 73-79 (2006).</li><li>Oike, F., 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=Simplified+technique+of+orthotopic+liver+transplantation+in+pigs.">Simplified technique of orthotopic liver transplantation in pigs.</a> Transplantation. 71 (2), 328-331 (2001).</li><li>Heuer, 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=Liver+transplantation+in+swine+without+venovenous+bypass.">Liver transplantation in swine without venovenous bypass.</a> European Surgical Research. 45 (1), 20-25 (2010).</li><li>Fondevila, 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=Step-by-step+guide+for+a+simplified+model+of+porcine+orthotopic+liver+transplant.">Step-by-step guide for a simplified model of porcine orthotopic liver transplant.</a> The Journal of Surgical Research. 167 (1), 39-45 (2011).</li></ol>

Recent technical developments such as the introduction of machine perfusion have the potential to revolutionize the field of liver transplantation. In order to translate graft reconditioning or modification concepts into clinical settings, reproducible transplant models in large animals are inevitable.
After the initial introduction of porcine orthotopic liver transplantation, several authors have worked on the improvement of these techniques over the past five&#160;decades. Differences within...

Liver transplantation remains to be the only chance for survival in a variety of different diseases leading to acute or chronic hepatic failure. Since its first successful application in mankind in 1963 by Thomas E. Starzl, the concept of liver transplantation has evolved into a reliable treatment option applied worldwide, mainly as a result of advancements in the understanding of the immune system, the development of modern immunosuppression, and the optimization of perioperative care and surgical techniques1,2. However, aging populations and a higher demand for organs have resulted in donor shortages, with increased use of marginal grafts from extended criteria donors and the emergence of new challenges in the past decades. The introduction and widespread implementation of organ machine perfusion is believed to open up an array of possibilities with regard to graft reconditioning and modulation and to help mitigate organ shortages and reduce waiting list mortality3,4,5,6.
In order to evaluate these concepts and their effects in vivo, translational transplant models are necessary7. In 1983, Kamada et al. introduced an efficient orthotopic liver transplant model in rats that has since been extensively modified and applied by workgroups around the globe8,9,10,11. The orthotopic liver transplant model in mice is technically more demanding, but also more valuable in terms of immunological transferability, and was first reported in 1991 by Qian et al.12. Despite advantages regarding availability, animal welfare, and costs, rodent models are limited in their applicability in clinical settings7. Hence, large animal models are required.
In recent years, pigs have become the main animal species used for translational research due to their anatomical and physiological similarities with humans. Furthermore, current progress in the field of xenotransplantation might further increase the importance of pigs as research objects13,14.
Garnier et al. described a liver transplant model in pigs as early as 196515. Several authors, including Calne et al. in 1967 and Chalstrey et al. in 1971, subsequently reported modifications, ultimately leading to a safe and feasible concept of experimental porcine liver transplantation in the decades to follow16,17,18,19,20,21.
More recently, different work groups have provided data with regard to current issues in liver transplantation using a technique of porcine orthotopic liver transplantation, almost invariably including an active or passive veno-venous, i.e., porto-caval, bypass19,22. The reason for this is a species-specific intolerance to the clamping of the vena cava inferior and the portal vein during the anhepatic phase due to a comparatively larger intestine and fewer porto-caval or cavo-caval shunts (e.g., lack of a vena azygos), resulting in increased perioperative morbidity and mortality23. Vena cava inferior-sparing transplant techniques applied in human recipients as an alternative are not feasible as the porcine vena cava inferior is encased by hepatic tissue23.
However, the usage of a veno-venous bypass further increases technical and logistical complexity in an already demanding surgical procedure, therefore possibly preventing workgroups from attempting implementation of the model altogether. Apart from the direct physiological and immunological effects of a bypass, some authors have pointed out the significant morbidity such as blood loss or air embolism during shunt placement and the need for a simultaneous splenectomy, potentially affecting short- and long-term results after engraftment24,25.
The following protocol describes a simple technique of porcine orthotopic liver transplantation after static cold storage of donor organs for 20 h, representing extended criteria donor conditions without the usage of a veno-venous bypass during engraftment, including donor liver procurement, back-table preparation, recipient hepatectomy, and anesthesiological pre- and intraoperative management.
This model should be of special interest for surgical workgroups focusing on the immediate postoperative course, ischemia-reperfusion injury, the reconditioning of extended criteria donor organs, and associated immunological mechanisms.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Abdominal retractor</td>
			<td>No Company Name available</td>
			<td>No Catalog Number available</td>
			<td></td>
		</tr>
		<tr>
			<td>Aortic clamp, straight</td>
			<td>Firma Martin</td>
			<td>No Catalog Number available</td>
			<td></td>
		</tr>
		<tr>
			<td>Arterial Blood Sampler Aspirator (safePICOAspirator) 1.5 mL</td>
			<td>Radiometer Medical ApS</td>
			<td>956-622</td>
			<td></td>
		</tr>
		<tr>
			<td>Atropine (Atropinsulfat 0.5 mg/1 mL)</td>
			<td>B.Braun</td>
			<td>648037</td>
			<td></td>
		</tr>
		<tr>
			<td>Backhaus clamp</td>
			<td>Bernshausen</td>
			<td>BF432</td>
			<td></td>
		</tr>
		<tr>
			<td>Bipolar forceps, 23 cm&#160;</td>
			<td>SUTTER</td>
			<td>780222 SG</td>
			<td></td>
		</tr>
		<tr>
			<td>Bowl 5 L, 6 L, 9 L</td>
			<td>Chiru-Instrumente</td>
			<td>35-114327</td>
			<td></td>
		</tr>
		<tr>
			<td>Braunol Braunoderm</td>
			<td>B.Braun</td>
			<td>3881059</td>
			<td></td>
		</tr>
		<tr>
			<td>Bulldog clamp</td>
			<td>Aesculap</td>
			<td>No Catalog Number available</td>
			<td></td>
		</tr>
		<tr>
			<td>Button canula</td>
			<td>Krauth + Timmermann GmbH</td>
			<td>1464LL1B</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium gluconate (2.25 mmol/10 mL (10%))</td>
			<td>B.Braun</td>
			<td>2353745</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Saver (Autotransfusion Reservoir)</td>
			<td>Fresenius Kabi AG</td>
			<td>9108471</td>
			<td></td>
		</tr>
		<tr>
			<td>Central venous catheter 7Fr., 3 Lumina, 30 cm 0.81 mm</td>
			<td>Arrow</td>
			<td>AD-24703</td>
			<td></td>
		</tr>
		<tr>
			<td>Clamp</td>
			<td>INOX</td>
			<td>B-17845&#160; /&#160; BH110&#160; / B-481</td>
			<td></td>
		</tr>
		<tr>
			<td>Clamp</td>
			<td>Aesculap</td>
			<td>AN909R</td>
			<td></td>
		</tr>
		<tr>
			<td>Clamp, 260 mm</td>
			<td>Fehling Instruments GMbH &#38;Co.KG</td>
			<td>ZAU-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Clip Forceps, medium</td>
			<td>Ethicon</td>
			<td>LC207</td>
			<td></td>
		</tr>
		<tr>
			<td>Clip forceps, small</td>
			<td>Ethicon&#160;</td>
			<td>LC107</td>
			<td></td>
		</tr>
		<tr>
			<td>CPDA-1 solution</td>
			<td>Fresenius Kabi AG</td>
			<td>41SD09AA00</td>
			<td></td>
		</tr>
		<tr>
			<td>Custodiol (Histidin-Tryptophan-Ketogluterat-Solution)</td>
			<td>Dr.Franz K&#246;hler Chemie GmbH</td>
			<td>2125921</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting scissors</td>
			<td>LAWTON&#160; 05-0641&#160;</td>
			<td>No Catalog Number available</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting scissors, 180 mm</td>
			<td>Metzenbaum&#160;</td>
			<td>BC606R</td>
			<td></td>
		</tr>
		<tr>
			<td>Endotracheal tube 8.0 mm</td>
			<td>Covetrus</td>
			<td>800764</td>
			<td></td>
		</tr>
		<tr>
			<td>Epinephrine (Adrenalin 1:1000)</td>
			<td>InfectoPharm</td>
			<td>9508734</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon Tubes 50ml</td>
			<td>Greiner&#160;</td>
			<td>227 261 L</td>
			<td></td>
		</tr>
		<tr>
			<td>Femoralis clamp</td>
			<td>Ulrich&#160;</td>
			<td>No Catalog Number available</td>
			<td></td>
		</tr>
		<tr>
			<td>Fentanyl 0.1mg</td>
			<td>PanPharma</td>
			<td>00483</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps, anatomical</td>
			<td>Martin</td>
			<td>12-100-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps, anatomical, 250 mm</td>
			<td>Aesculap</td>
			<td>BD052R</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps, anatomical, 250 mm</td>
			<td>Aesculap</td>
			<td>BD032R</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps, anatomical, 250 mm&#160;</td>
			<td>Aesculap</td>
			<td>BD240R</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps, surgical</td>
			<td>Bernshausen</td>
			<td>BD 671</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps, surgical</td>
			<td>INOX</td>
			<td>B-1357</td>
			<td></td>
		</tr>
		<tr>
			<td>G40 solution</td>
			<td>Serag Wiessner</td>
			<td>10755AAF</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelafundin ISO solution 40 mg/mL</td>
			<td>B. Braun</td>
			<td>210257641</td>
			<td></td>
		</tr>
		<tr>
			<td>Guidewire with marker</td>
			<td>Arrow</td>
			<td>14F21E0236</td>
			<td></td>
		</tr>
		<tr>
			<td>Haemostatic gauze (&#34;Tabotamp&#34;&#160; 5 x 7.5 cm)</td>
			<td>Ethicon</td>
			<td>474273</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin sodium 25,000IE</td>
			<td>Ratiopharm</td>
			<td>W08208A</td>
			<td></td>
		</tr>
		<tr>
			<td>Hico-Aquatherm 60</td>
			<td>Hospitalwerk</td>
			<td>No Catalog Number available</td>
			<td></td>
		</tr>
		<tr>
			<td>Infusion Set Intrafix</td>
			<td>B.Braun</td>
			<td>4062981 L</td>
			<td></td>
		</tr>
		<tr>
			<td>Intrafix SafeSet 180 cm</td>
			<td>B.Braun</td>
			<td>4063000</td>
			<td></td>
		</tr>
		<tr>
			<td>Introcan Safety, 18 G&#160;</td>
			<td>B.Braun</td>
			<td>4251679-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Isofluran CP</td>
			<td>CP-Pharma</td>
			<td>No Catalog Number available</td>
			<td></td>
		</tr>
		<tr>
			<td>Large-bore venous catheter, 7Fr.</td>
			<td>Edwards Lifesciences</td>
			<td>I301F7</td>
			<td></td>
		</tr>
		<tr>
			<td>Ligaclip, medium</td>
			<td>Ethicon</td>
			<td>LT200</td>
			<td></td>
		</tr>
		<tr>
			<td>Ligaclip, small</td>
			<td>Ethicon&#160;</td>
			<td>LT100</td>
			<td></td>
		</tr>
		<tr>
			<td>Material scissors</td>
			<td>Martin&#160;</td>
			<td>11-285-23</td>
			<td></td>
		</tr>
		<tr>
			<td>Methylprednisolone (Urbason solubile forte 250 mg)</td>
			<td>Sanofi</td>
			<td>7823704</td>
			<td></td>
		</tr>
		<tr>
			<td>Monopolar ERBE ICC 300</td>
			<td>Fa. Erbe</td>
			<td>No Catalog Number available</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl solution (0.9%)</td>
			<td>Baxter</td>
			<td>1533</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle holder</td>
			<td>Aesculap</td>
			<td>BM36</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle holder</td>
			<td>Aesculap</td>
			<td>BM035R</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle holder</td>
			<td>Aesculap</td>
			<td>BM 67</td>
			<td></td>
		</tr>
		<tr>
			<td>Neutral electrode</td>
			<td>Erbe Elektromedizin GmbH T&#252;bingen</td>
			<td>21191 - 060</td>
			<td></td>
		</tr>
		<tr>
			<td>Norepinephrine (Sinora)</td>
			<td>Sintetica GmbH</td>
			<td>04150124745717</td>
			<td></td>
		</tr>
		<tr>
			<td>Omniflush Sterile Filed 10 mL</td>
			<td>B.Braun</td>
			<td>3133335</td>
			<td></td>
		</tr>
		<tr>
			<td>Original Perfusorline 300 cm</td>
			<td>B.Braun</td>
			<td>21E26E8SM3</td>
			<td></td>
		</tr>
		<tr>
			<td>Overhold clamp</td>
			<td>INOX</td>
			<td>BH 959</td>
			<td></td>
		</tr>
		<tr>
			<td>Overhold clamp</td>
			<td>Ulrich</td>
			<td>CL 2911</td>
			<td></td>
		</tr>
		<tr>
			<td>Pentobarbital sodium(Release 500 mg/mL)</td>
			<td>WDT, Garbsen</td>
			<td>21217</td>
			<td></td>
		</tr>
		<tr>
			<td>Perfusers</td>
			<td>B.Braun</td>
			<td>49-020-031</td>
			<td></td>
		</tr>
		<tr>
			<td>Perfusor Syringe 50 mL</td>
			<td>B.Braun</td>
			<td>8728810F</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dishes&#160; 92 x 17 mm</td>
			<td>Nunc</td>
			<td>150350</td>
			<td></td>
		</tr>
		<tr>
			<td>Poole Suction Instrument Argyle flexibel</td>
			<td>Covidien, Mansfield USA</td>
			<td>20C150FHX</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride (7.45%)</td>
			<td>B.Braun</td>
			<td>4030539078276</td>
			<td></td>
		</tr>
		<tr>
			<td>Pressure measurement set</td>
			<td>Codan pvb Medical GmbH</td>
			<td>957179</td>
			<td></td>
		</tr>
		<tr>
			<td>Propofol (1%)</td>
			<td>CP-Pharma</td>
			<td>No Catalog Number available</td>
			<td></td>
		</tr>
		<tr>
			<td>S-Monovette 2.6 mL K3E</td>
			<td>Sarstedt</td>
			<td>04.1901</td>
			<td></td>
		</tr>
		<tr>
			<td>S-Monovette 2.9 mL 9NC</td>
			<td>Sarstedt</td>
			<td>04.1902</td>
			<td></td>
		</tr>
		<tr>
			<td>S-Monovette 7.5 mL Z-Gel</td>
			<td>Sarstedt</td>
			<td>11602</td>
			<td></td>
		</tr>
		<tr>
			<td>Sartinski clamp</td>
			<td>Aesculap</td>
			<td>No Catalog Number available</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel&#160; No.11</td>
			<td>Feather Safety Razor Co.LTD</td>
			<td>02.001.40.011</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>INOX&#160;</td>
			<td>BC 746</td>
			<td></td>
		</tr>
		<tr>
			<td>Seldinger Arterial catheter</td>
			<td>Arrow</td>
			<td>SAC-00520</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium bicarbonate (8.4%)</td>
			<td>B.Braun</td>
			<td>212768082</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterilization Set (&#34;ProSet Preparation Kit CVC&#34;)</td>
			<td>B.Braun</td>
			<td>4899719</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterofundin ISO solution</td>
			<td>B.Braun</td>
			<td>No Catalog Number available</td>
			<td></td>
		</tr>
		<tr>
			<td>Suction</td>
			<td>Dahlhausen</td>
			<td>07.068.25.301</td>
			<td></td>
		</tr>
		<tr>
			<td>Suction Aesculap Securat 80</td>
			<td>Aesculap</td>
			<td>No Catalog Number available</td>
			<td></td>
		</tr>
		<tr>
			<td>Suction catheter</td>
			<td>ConvaTec</td>
			<td>5365049</td>
			<td></td>
		</tr>
		<tr>
			<td>Sultamicillin (Unacid: 2000 mg Ampicillin/1000 mg Sulbactam)</td>
			<td>Pfizer</td>
			<td>DL253102</td>
			<td></td>
		</tr>
		<tr>
			<td>Suprapubic urinary catheter, &#34;bronchialis&#34;, 50 cm</td>
			<td>ConvaTec</td>
			<td>UK&#160; 1F02772</td>
			<td></td>
		</tr>
		<tr>
			<td>Suprasorb (&#34;Toptex lite RK&#34;)</td>
			<td>Lohmann &#38; Rauscher</td>
			<td>31654</td>
			<td></td>
		</tr>
		<tr>
			<td>Suture Vicryl 3-0</td>
			<td>Ethicon</td>
			<td>VCP 1218 H</td>
			<td></td>
		</tr>
		<tr>
			<td>Suture Vicryl 4-0</td>
			<td>Ethicon</td>
			<td>V392H</td>
			<td></td>
		</tr>
		<tr>
			<td>Suture, Prolene 4-0</td>
			<td>Ethicon</td>
			<td>7588 H</td>
			<td></td>
		</tr>
		<tr>
			<td>Suture, Prolene 5-0, double armed</td>
			<td>Ethicon&#160;</td>
			<td>8890 H</td>
			<td></td>
		</tr>
		<tr>
			<td>Suture, Prolene 5-0, single armed</td>
			<td>Ethicon&#160;</td>
			<td>8720 H</td>
			<td></td>
		</tr>
		<tr>
			<td>Suture, Prolene 6-0, double armed</td>
			<td>Ethicon&#160;</td>
			<td>7230 H</td>
			<td></td>
		</tr>
		<tr>
			<td>Suture, Prolene 6-0, single armed</td>
			<td>Ethicon</td>
			<td>EH 7406 H</td>
			<td></td>
		</tr>
		<tr>
			<td>Suture, Prolene: blau 3-0&#160;</td>
			<td>Ethicon</td>
			<td>EH 7499H</td>
			<td></td>
		</tr>
		<tr>
			<td>Suture, Safil 2/0</td>
			<td>Aesculap</td>
			<td>C 1038446</td>
			<td></td>
		</tr>
		<tr>
			<td>Suture, Terylene 0</td>
			<td>Serag Wiessner</td>
			<td>353784</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe 2 mL, 5 mL, 10 mL, 20 mL</td>
			<td>B.Braun</td>
			<td>4606027V</td>
			<td></td>
		</tr>
		<tr>
			<td>TransferSet &#34;1D/X-double&#34; steril 330 cm</td>
			<td>Fresenius Kabi AG</td>
			<td>2877101</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasound Butterfly IQ+</td>
			<td>Butterfly Network Inc.</td>
			<td>850-20014</td>
			<td></td>
		</tr>
		<tr>
			<td>Ventilator &#34;Oxylog Dr&#228;ger Fl&#34;</td>
			<td>Dr&#228;ger Medical AG</td>
			<td>No Catalog Number available</td>
			<td></td>
		</tr>
		<tr>
			<td>Yankauer Suction</td>
			<td>Medline</td>
			<td>RA19GMD</td>
			<td></td>
		</tr>
		<tr>
			<td>Zoletil 100 mg/mL&#160; (50 mg Zolazepam, 50 mg tiletamin)</td>
			<td>Virbac</td>
			<td>794-861794861</td>
			<td></td>
		</tr>
	</tbody>
</table>

porcine liver transplantation without veno-venous bypass as an extended criteria donor model

Liver transplantation is regarded as the gold standard for the treatment of a variety of fatal hepatic diseases. However, unsolved issues of chronic graft failure, ongoing organ donor shortages, and the increased use of marginal grafts call for the improvement of current concepts, such as the implementation of organ machine perfusion. In order to evaluate new methods of graft reconditioning and modulation, translational models are required. With respect to anatomical and physiological similarities to humans and recent progress in the field of xenotransplantation, pigs have become the main large animal species used in transplantation models. After the initial introduction of a porcine orthotopic liver transplant model by Garnier et al. in 1965, several modifications have been published over the past 60 years.
Due to specifies-specific anatomical traits, a veno-venous bypass during the anhepatic phase is regarded as a necessity to reduce intestinal congestion and ischemia resulting in hemodynamic instability and perioperative mortality. However, the implementation of a bypass increases the technical and logistical complexity of the procedure. Furthermore, associated complications such as air embolism, hemorrhage, and the need for a simultaneous splenectomy have been reported previously.
In this protocol, we describe a model of porcine orthotopic liver transplantation without the use of a veno-venous bypass. The engraftment of donor livers after static cold storage of 20 h - simulating extended criteria donor conditions - demonstrates that this simplified approach can be performed without significant hemodynamic alterations or intraoperative mortality and with regular uptake of liver function (as defined by bile production and liver-specific CYP1A2 metabolism). The success of this approach is ensured by an optimized surgical technique and a sophisticated anesthesiologic volume and vasopressor management.
This model should be of special interest for workgroups focusing on the immediate postoperative course, ischemia-reperfusion injury, associated immunological mechanisms, and the reconditioning of extended criteria donor organs.

The technique presented in this protocol has provided reliable and reproducible results in terms of hemodynamic stability and animal survival throughout the procedure, as well as graft function in the postoperative course.
Most recently, we applied the model for the study of ischemia-reperfusion injury and therapeutic interventions mitigating detrimental effects in the immediate postoperative course. Upon retrieval and 20 h of static cold storage, liver grafts (with a mean weight of 983.38 g) ...

Watch this Scientific Journal Video about Porcine Liver Transplantation Without Veno-Venous Bypass As an Extended Criteria Donor Model at JoVE.com

Porcine Liver Transplantation Without Veno-Venous Bypass As an Extended Criteria Donor Model

In this protocol, a model of porcine orthotopic liver transplantation after static cold storage of donor organs for 20 h without the use of a veno-venous bypass during engraftment is described. The approach uses a simplified surgical technique with minimization of the anhepatic phase and sophisticated volume and vasopressor management.

Liver transplantation is regarded as the gold standard for the treatment of a variety of fatal hepatic diseases. However, unsolved issues of chronic graft ...

Current issues in liver transplantation require the implementation of large animal models. This protocol describes the technique of porcine orthotopic liver transplantation without the use of a veno-venous bypass. This technique significantly reduces the logistical and technical complexity of the procedure.Furthermore, by engrafting livers after 20 hours of static cold storage, our model simulates extended criteria donor conditions. Begin by placing the anesthetized donor pig in a supine position and fix the limbs at the base of the operation table with elastic bands. Scrub the skin with an antiseptic agent and cover the animal with sterile drapes.After confirming the loss of petal reflex, perform a midline laparotomy beginning at the xiphoid process by using monopolar cautery and sever the falciform and the triangular ligaments using scissors and bipolar cautery. After sufficient liver dissection, incise the left portion of the diaphragm over a distance of 5 to 10 centimeters using scissors to locate the thoracic segment of the descending aorta. Encircle and place a 3-0 polyfilament ligature without tightening.Incise the right portion of the diaphragm over a distance of 5 to 10 centimeters using scissors and identify the suprahepatic vena cava inferior. Relocate the intestine to the upper left of the donor and enter the retroperitoneal space by transverse incision of the peritoneum over a distance of 5 to 10 centimeters using scissors. Place two 3-0 polyfilament ligatures around the abdominal aorta.One cranial of the iliac bifurcation and one approximately 3 centimeters cranially without tightening. Place another ligature around the infrahepatic vena cava inferior without tightening. Tighten the caudally-located first ligature around the abdominal aorta.After occluding the abdominal aorta cranially of the second ligature, make a transverse incision between both the ligatures using scissors. Insert the cannula into the incision and secure it with the remaining ligature. Using scissors, sever the suprahepatic inferior vena cava far cranially close to the right atrium.After blood loss of approximately 1, 500 to 2, 000 milliliters, cross-clamp the thoracic segment of the descending aorta by tying the ligature and start to antegrade perfusion. Tighten the ligature placed around the infrahepatic vena cava inferior. Incise the vessel cranially of the ligature and insert the surgical aspirator.After perfusion with 3, 500 milliliters of preservation solution over the course of approximately 10 to 15 minutes, sever the inside supra hepatic vena cava inferior of the donor pig, then sever the infrahepatic vena cava inferior at the level of the left renal vein. Sever the bile duct cranial of the pancreatic tissue between two 3-0 polyfilament ligatures to avoid bile spillage, then sever the portal vein cranial of the pancreas. Locate the celiac artery after blunt preparation and follow dorsally to the abdominal aorta.Excise the respective aortic segment to create a patch for the engraftment. Excise the diaphragm around the super hepatic vena cava inferior and sever the remaining adhesions using scissors. Extract the liver and place on ice.Perform a cholecystectomy or tighten a ligature around the cystic duct, then flush the common bile duct with at least 20 milliliters of preservation solution. Place the perfusion cannula into the portal vein and flush the graft with 500 milliliters of preservation solution. Place the graft in a sterile bowl placed on ice.Remove the lymphatic tissue of the aortic segment and the hepatic artery, thereby identifying and occluding the arterial side branches and lymphatic vessels with either clips, ligatures, or sutures. Proceed similarly with the portal vein. Perform shortening of the vessels and preparation of the aortic patch only upon and engraftment to take into account the individual anatomic circumstances.Identify the super hepatic vena cava inferior and place sutures around both diaphragmatic veins after removing surrounding diaphragmatic tissue. Place the anesthetized recipient pig in a supine position. Enter the abdominal cavity and sever the falciform and triangular ligaments of the anesthetized animal using scissors and bipolar cautery as demonstrated earlier.After sufficient liver dissection, encircle both the supra and infrahepatic vena cavae inferior close to the liver parenchyma. Incise the superficial peritoneal layer covering the hepatoduodenal ligament and identify the hepatic arteries before entering the liver parenchyma. Dissect using bipolar cautery or placing the clips, ligatures, or sutures.Dissect and sever the common bile duct below the cystic duct junction between two ligatures. Identify the portal vein and encircle close to the liver parenchyma. Identify the abdominal aorta after incising the avascular layer between the right and left diaphragmatic muscles.Prepare the aorta for aortic anastomosis by removal of the surrounding tissue. Perform recipient hepatectomy by placing in a traumatic vascular clamp on the portal vein and sever the portal vein close to the liver parenchyma followed by the infrahepatic vena cava. Place an atraumatic vascular clamp on the suprahepatic vena cava including the surrounding diaphragm while caudally retracting the liver and sever the vessel close to the liver parenchyma.Remove the recipient liver from the abdominal cavity. Place the donor liver into the abdominal cavity and shorten the donor or recipient suprahepatic vena cava inferior to an adequate length while avoiding kinking or too much tension on the anastomosis. Place a single 5-0 monofilament suture as a supporting thread adapting the right corner of the donor and recipient supra hepatic vena cavae inferior.Begin the dorsal side of the anastomosis from the left corner of the vessels with a 5-0 monofilament double-armed running suture. Remove the supporting thread when reaching the right corner and secure the running suture with a clamp, then continue with the ventral side of the anastomosis, again, beginning from the left corner of the vessels. After completing the ventral side of the anastomosis, tighten the suture with multiple knots without constricting the vessel diameter to avoid stenosis.Perform a vascular anastomosis of the donor and recipient portal vein as demonstrated earlier using a 6-0 monofilament double-armed suture. Begin the portal venous reperfusion by removing the vascular clamp, occluding the recipient portal vein and occlude the donor infrahepatic vena cava inferior with a vascular clamp after draining approximately 200 to 400 milliliters of blood. Slowly remove the vascular clamp occluding the recipient supra hepatic vena cava inferior and search for active bleeding.Shorten the donor and/or recipient infrahepatic vena cava inferior. then perform a vascular anastomosis of the donor and recipient infrahepatic vena cavae inferior. After clamping the abdominal aorta and making an incision to fit the patch, begin the aortic anastomosis with a 6-0 monofilament double-armed running suture at the cranial corner of the incision or patch.When reaching the caudal corner, secure the running suture with a clamp and complete the anastomosis again beginning at the cranial corner. Tighten the suture with multiple knots and slowly remove the vascular clamp after checking all vascular anastomoses. Place a catheter in the common bile duct and secure it with a single ligature.Close the abdomen temporarily by adapting the muscular fascia and the skin with a running suture and cover the abdomen with clinging film or drapes to avoid thermal loss. The mean operation time was 103.50 minutes including a mean and hepatic phase of 27.13 minutes. Only two recipients underwent an anhepatic phase of more than 30 minutes while all recipients showed declining lactate serum concentrations 4 hours after portal venous reperfusion.Recipients required increased concentrations of norepinephrine immediately before and throughout the anhepatic phase to stabilize the mean arterial pressure at 60 millimeters of mercury or higher. Low epinephrine concentrations were simultaneously used to increase cardiac output in this vulnerable time period. After engraftment, the mean arterial pressure and required doses of vasopressors remained stable.The LiMAx values obtained 6 hours after portal venous reperfusion were comparable to the values measured in the liver donors before organ procurement in all but one recipient with an anhepatic phase of 34 minutes. This model can be of special interest as a readout for workups focusing on mechanisms of ischemia and reperfusion injury and the reconditioning of extended criteria donor organs.

porcine-liver-transplantation-without-veno-venous-bypass-as-an

Research

JoVE Journal

Medicine

Orthotopic Liver Transplantation in Rats

Clinical progress in the field of liver transplantation has been largely supported by animal models1,2. Since the publication of the first orthotopic rat liver transplantation in 1979 by Kamada et al.3, this model has remained the gold standard despite various proposed alternative techniques4. Nevertheless, its broader use is limited by its steep learning curve5.
In this video paper, we show a simple and easy-to-establish revision of Kamada's two-cuff technique. The suprahepatic vena cava anastomosis is performed manually with a running suture, and the vena porta and infrahepatic vena cava anastomoses are performed utilizing a quick-linker cuff system6. Manufacturing the quick-linker kit is shown in a separate video paper.

We present an easy-to-establish revision of the classical two-cuff technique for orthotopic liver transplantation in rat.

Clinical progress in the field of liver transplantation has been largely supported by animal models1,2. Since the publication of the first orthotopic rat ...

Using Quantitative Real-time PCR to Determine Donor Cell Engraftment in a Competitive Murine Bone Marrow Transplantation Model

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules that regulate HSCs. In these transplant model systems, the function of HSCs is determined by the ability of these cells to engraft and reconstitute lethally irradiated recipient mice. Commonly, the donor cell contribution/engraftment is measured by antibodies to donor- specific cell surface proteins using flow cytometry. However, this method heavily depends on the specificity and the ability of the cell surface marker to differentiate donor-derived cells from recipient-originated cells, which may not be available for all mouse strains. Considering the various backgrounds of genetically modified mouse strains in the market, this cell surface/ flow cytometry-based method has significant limitations especially in mouse strains that lack well-defined surface markers to separate donor cells from congenic recipient cells. Here, we reported a PCR-based technique to determine donor cell engraftment/contribution in transplant recipient mice. We transplanted male donor bone marrow HSCs to lethally irradiated congenic female mice. Peripheral blood samples were collected at different time points post transplantation. Bone marrow samples were obtained at the end of the experiments. Genomic DNA was isolated and the Y chromosome specific gene, Zfy1, was amplified using quantitative Real time PCR. The engraftment of male donor-derived cells in the female recipient mice was calculated against standard curve with known percentage of male vs. female DNAs. Bcl2 was used as a reference gene to normalize the total DNA amount. Our data suggested that this approach reliably determines donor cell engraftment and provides a useful, yet simple method in measuring hematopoietic cell reconstitution in murine bone marrow transplantation models. Our method can be routinely performed in most laboratories because no costly equipment such as flow cytometry is required.

Determining donor cell engraftment presents a challenge in mouse bone marrow transplant models that lack well-defined phenotypical markers. We described a methodology to quantify male donor cell engraftment in female transplant recipient mice. This method can be used in all mouse strains for the study of HSC functions.

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules ...

Technique of Porcine Liver Procurement and Orthotopic Transplantation using an Active Porto-Caval Shunt

The success of liver transplantation has resulted in a dramatic organ shortage. Each year, a considerable number of patients on the liver transplantation waiting list die without receiving an organ transplant or are delisted due to disease progression. Even after a successful transplantation, rejection and side effects of immunosuppression remain major concerns for graft survival and patient morbidity. 
Experimental animal research has been essential to the success of liver transplantation and still plays a pivotal role in the development of clinical transplantation practice. In particular, the porcine orthotopic liver transplantation model (OLTx) is optimal for clinically oriented research for its close resemblance to human size, anatomy, and physiology. 
Decompression of intestinal congestion during the anhepatic phase of porcine OLTx is important to guarantee reliable animal survival. The use of an active porto-caval-jugular shunt achieves excellent intestinal decompression. The system can be used for short-term as well as long-term survival experiments. The following protocol contains all technical information for a stable and reproducible liver transplantation model in pigs including post-operative animal care.

Experimental animal research plays a pivotal role in the development of clinical transplantation practice. The porcine orthotopic liver transplantation model (OLTx) closely resembles human conditions and is frequently used in clinically oriented research. The following protocol contains all information for a reliable porcine OLTx model using an active porto-caval-jugular shunt.

The success of liver transplantation has resulted in a dramatic organ shortage. Each year, a considerable number of patients on the liver transplantation ...

Ex Situ Normothermic Machine Perfusion of Donor Livers

In contrast to conventional static cold preservation (0-4 &#176;C), ex situ machine perfusion may provide better preservation of donor livers. Continuous perfusion of organs provides the opportunity to improve organ quality and allows ex situ viability assessment of donor livers prior to transplantation. This video article provides a step by step protocol for ex situ normothermic machine perfusion (37 &#176;C) of human donor livers using a device that provides a pressure and temperature controlled pulsatile perfusion of the hepatic artery and continuous perfusion of the portal vein. The perfusion fluid is oxygenated by two hollow fiber membrane oxygenators and the temperature can be regulated between 10 &#176;C and 37 &#176;C. During perfusion, the metabolic activity of the liver as well as the degree of injury can be assessed by biochemical analysis of samples taken from the perfusion fluid. Machine perfusion is a very promising tool to increase the number of livers that are suitable for transplantation.

Ex Situ Normothermic Machine Perfusion of Donor Livers

Here we present a protocol describing oxygenated ex situ machine perfusion of donor liver grafts. This article contains a step by step protocol to procure and prepare the liver graft for machine perfusion, prepare the perfusion fluid, prime the perfusion machine and perform oxygenated normothermic machine perfusion of the liver graft.

In contrast to conventional static cold preservation (0-4 &#176;C), ex situ machine perfusion may provide better preservation of donor livers. Continuous ...

Cardiopulmonary Bypass in a Mouse Model: A Novel Approach

As prolonged cardiopulmonary bypass becomes more essential during cardiac interventions, an increasing clinical demand arises for procedure optimization and for minimizing organ damage resulting from prolonged extracorporal circulation. The goal of this paper was to demonstrate a fully functional and clinically relevant model of cardiopulmonary bypass in a mouse. We report on the device design, perfusion circuit optimization, and microsurgical techniques. This model is an acute model, which is not compatible with survival due to the need for multiple blood drawings. Because of the range of tools available for mice (e.g., markers, knockouts, etc.), this model will facilitate investigation into the molecular mechanisms of organ damage and the effect of cardiopulmonary bypass in relation to other comorbidities.

This paper describes how to perform cardiopulmonary bypass in mice. This novel model will facilitate the investigation of the molecular mechanisms involved in organ damage.

As prolonged cardiopulmonary bypass becomes more essential during cardiac interventions, an increasing clinical demand arises for procedure optimization ...

Veno-Venous Extracorporeal Membrane Oxygenation in a Mouse

The use of extracorporeal membrane oxygenation (ECMO) has increased substantially in recent years. ECMO has become a reliable and effective therapy for acute as well as end-stage lung diseases. With the increase in clinical demand and prolonged use of ECMO, procedural optimization and prevention of multi-organ damage are of critical importance. The aim of this protocol is to present a detailed technique of veno-venous ECMO in a non-intubated, spontaneously breathing mouse. This protocol demonstrates the technical design of the ECMO and surgical steps. This murine ECMO model will facilitate the study of pathophysiology related to ECMO (e.g., inflammation,bleeding and thromboembolic events). Due to the abundance of genetically modified mice, the molecular mechanisms involved in ECMO-related complications can also be dissected.

Here we present a protocol describing the technique of veno-venous extracorporeal membrane oxygenation (ECMO) in a non-intubated, spontaneously breathing mouse. This murine model of ECMO can be effectively implemented in experimental studies of acute and end-stage lung diseases.

The use of extracorporeal membrane oxygenation (ECMO) has increased substantially in recent years. ECMO has become a reliable and effective therapy for ...

A Pre-Clinical Porcine Model of Orthotopic Heart Transplantation

Fifty-years following the first successful report, cardiac transplantation remains the gold-standard treatment for eligible patients with advanced heart failure. Multiple small-animal models of heart transplantation have been used to study the acute and long-term effects of novel therapies. However, few are tested and demonstrated success in clinical trials. It is of critical importance to evaluate new therapies in a clinically relevant large-animal model for efficient and reliable translation of basic studies&#39;&#160;findings. Here, we describe a pre-clinical large-animal (porcine) model of orthotopic heart transplantation that has been firmly established and previously used to investigate novel cardioprotective strategies. This procedure focuses on acute ischemia-reperfusion injury and is a reliable method to investigate novel interventions which have been tested and validated in smaller experimental models, such as the murine model. We demonstrate its usefulness in assessing cardiac performance during the early post-transplantation period and other potential possibilities enabled by the model.

Here, we describe a pre-clinical large-animal (porcine) model of orthotopic heart transplantation that has been firmly established and utilized to investigate novel cardioprotective strategies.

Fifty-years following the first successful report, cardiac transplantation remains the gold-standard treatment for eligible patients with advanced heart ...

Murine Precision-Cut Liver Slices as an Ex Vivo Model of Liver Biology

Understanding the mechanisms of liver injury, hepatic fibrosis, and cirrhosis that underlie chronic liver diseases (i.e., viral hepatitis, non-alcoholic fatty liver disease, metabolic liver disease, and liver cancer) requires experimental manipulation of animal models and in vitro cell cultures. Both techniques have limitations, such as the requirement of large numbers of animals for in vivo manipulation. However, in vitro cell cultures do not reproduce the structure and function of the multicellular hepatic environment. The use of precision-cut liver slices is a technique in which uniform slices of viable mouse liver are maintained in laboratory tissue culture for experimental manipulation. This technique occupies an experimental niche that exists between animal studies and in vitro cell culture methods. The presented protocol describes a straightforward and reliable method to isolate and culture precision-cut liver slices from mice. As an application of this technique, ex vivo liver slices are treated with bile acids to simulate cholestatic liver injury and ultimately assess the mechanisms of hepatic fibrogenesis.

This protocol provides a simple and reliable method for the production of viable precision-cut liver slices from mice. The ex vivo tissue samples can be maintained under laboratory tissue culture conditions for multiple days, providing a flexible model to examine liver pathobiology.

Understanding the mechanisms of liver injury, hepatic fibrosis, and cirrhosis that underlie chronic liver diseases (i.e., viral hepatitis, non-alcoholic ...

Left Lung Orthotopic Transplantation in a Juvenile Porcine Model for ESLP

Lung transplantation is the gold-standard treatment for end-stage lung disease, with over 4,600 lung transplantations performed worldwide annually. However, lung transplantation is limited by a shortage of available donor organs. As such, there is high waitlist mortality. Ex situ lung perfusion (ESLP) has increased donor lung utilization rates in some centers by 15%-20%. ESLP has been applied as a method to assess and recondition marginal donor lungs and has demonstrated acceptable short- and long-term outcomes following transplantation of extended criteria donor (ECD) lungs. Large animal (in vivo) transplantation models are required to validate ongoing in vitro research findings. Anatomic and physiologic differences between humans and pigs pose significant technical and anesthetic challenges. An easily reproducible transplant model would permit the&#160;in vivo&#160;validation of current ESLP strategies and the preclinical evaluation of various interventions designed to improve donor lung function. This protocol describes a porcine model of orthotopic left lung allotransplantation. This includes anesthetic and surgical techniques, a customized surgical checklist, troubleshooting, modifications, and the benefits and limitations of the approach.

This protocol describes a juvenile porcine model of orthotopic left lung allotransplantation designed for use with ESLP research. Focus is made on anesthetic and surgical techniques, as well as critical steps and troubleshooting.

Lung transplantation is the gold-standard treatment for end-stage lung disease, with over 4,600 lung transplantations performed worldwide annually. ...

A Porcine Heterotopic Heart Transplantation Protocol for Delivery of Therapeutics to a Cardiac Allograft

Cardiac transplantation is the gold standard treatment for end-stage heart failure. However, it remains limited by the number of available donor hearts and complications such as primary graft dysfunction and graft rejection. The recent clinical use of an ex vivo perfusion device in cardiac transplantation introduces a unique opportunity for treating cardiac allografts with therapeutic interventions to improve function and avoid deleterious recipient responses. Establishing a translational, large-animal model for therapeutic delivery to the entire allograft is essential for testing novel therapeutic approaches in cardiac transplantation. The porcine, heterotopic heart transplantation model in the intraabdominal position serves as an excellent model for assessing the effects of novel interventions and the immunopathology of graft rejection. This model additionally offers long-term survival for the pig, given that the graft is not required to maintain the recipient's circulation. The aim of this protocol is to provide a reproducible and robust approach for achieving ex vivo delivery of a therapeutic to the entire cardiac allograft prior to transplantation and provide technical details to perform a survival heterotopic transplant of the ex vivo perfused heart.

We present a protocol for utilizing a normothermic ex vivo sanguinous perfusion system for the delivery of therapeutics to an entire cardiac allograft in a porcine heterotopic heart transplant model.

Cardiac transplantation is the gold standard treatment for end-stage heart failure. However, it remains limited by the number of available donor hearts ...