Name: a novel surgical technique as a foundation for in vivo partial liver engineering in rat
Uploaded: 2018-10-06T14:00:00
Description: Watch this Scientific Journal Video about A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat at JoVE.com

The authors would like to thank Jens Geiling from the Institute of Anatomy I, Jena University Hospital, for producing the schematic drawings of rat liver anatomy.

The housing and all procedures carried out were in accordance with German animal welfare legislation. All gauze, covering clothes, and surgical instruments are autoclaved and prepared before the operation. All procedures are carried out under sterile conditions.
1. Preparation of the Rat for the Surgical Procedure
<ol>
	<li>Place the rat in an induction chamber and anesthetize the rat with 4% vaporized isoflurane and 100% oxygen at 0.5 L/min for about 3 min, until the rat is completely anest...

<ol>
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K., 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=Bioengineered+transplantable+porcine+livers+with+re-endothelialized+vasculature.">Bioengineered transplantable porcine livers with re-endothelialized vasculature.</a> Biomaterials. 40, 72-79 (2015).</li><li>Bao, 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=Construction+of+a+portal+implantable+functional+tissue-engineered+liver+using+perfusion-decellularized+matrix+and+hepatocytes+in+rats.">Construction of a portal implantable functional tissue-engineered liver using perfusion-decellularized matrix and hepatocytes in rats.</a> Cell Transplantation. 20, 753-766 (2011).</li><li>Pan, 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=In-vivo+organ+engineering:+Perfusion+of+hepatocytes+in+a+single+liver+lobe+scaffold+of+living+rats.">In-vivo organ engineering: Perfusion of hepatocytes in a single liver lobe scaffold of living rats.</a> The International Journal of Biochemistry &amp; Cell Biology. 80, 124-131 (2016).</li><li>Madrahimov, 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=Marginal+hepatectomy+in+the+rat:+from+anatomy+to+surgery.">Marginal hepatectomy in the rat: from anatomy to surgery.</a> Annals of Surgery. 244, 89-98 (2006).</li><li>Mussbach, F., Settmacher, U., Dirsch, O., Dahmen, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioengineered+Livers:+A+New+Tool+for+Drug+Testing+and+a+Promising+Solution+to+Meet+the+Growing+Demand+for+Donor+Organs.">Bioengineered Livers: A New Tool for Drug Testing and a Promising Solution to Meet the Growing Demand for Donor Organs.</a> European Surgical Research. 57, 224-239 (2016).</li><li>Mussbach, F., Settmacher, U., Dirsch, O., Dahmen, 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+engineering+as+a+new+source+of+donor+organs:+A+systematic+review.">Liver engineering as a new source of donor organs: A systematic review.</a> Der Chirurg. 87, 504-513 (2016).</li><li>Xie, 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=Monitoring+of+systemic+and+hepatic+hemodynamic+parameters+in+mice.">Monitoring of systemic and hepatic hemodynamic parameters in mice.</a> Journal of Visualized Experiments. 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By blocking and cannulating the left portal vein with a catheter as a fluid inlet and the left lateral hepatic vein with another catheter as a fluid outlet, we successfully generated an in vivo fluid bypass within the left lateral lobe, indicating that although the technique is highly challenging due to the small size of the vessels for cannulation and a high risk of causing bleeding, it is feasible. Even the rats undergoing a long perfusion period of 4 hours survived at least 1 week, showing that the rats could...

The indications for organ transplantation are constantly expanding. In contrast, organ donation rates and overall quality of organs are declining, leading to an increasing demand for grafts. The number of candidates added to the liver transplant waiting list continued to increase (e.g., in the United States, 11,340 patients were added in 2016, compared with 10,636 in 2015)1. Despite substantial efforts, the number of available organs does not meet clinical needs. Due to the increased incidence of liver disease, many patients with end-stage liver diseases die on the transplant waiting list before a donor organ becomes available. To meet the huge demand for donor liver grafts, alternative approaches using liver tissue engineering principles are being actively pursued2. Nowadays, a newly developed biological technique of liver engineering could potentially overcome this shortage.
Liver engineering consists of two steps: the generation of an acellular scaffold, followed by a repopulation of the scaffold. To obtain a biological acellular liver scaffold, the explanted liver is perfused via the vascular system with ionic or nonionic detergents, which can remove the cellular material from the liver. In most previous studies, a biological acellular liver scaffold was achieved by perfusion of the liver with a combination of sodium dodecyl sulfate and TritonX100. As a result, all cells were removed, whereas the structure of the extracellular matrix was maintained. The organ scaffolds were reseeded with mature cells, hepatocellular, as well as endothelial cell lines, and primary hepatocytes with or without the simultaneous application of endothelial cells or mesenchymal stem cells (MSC). Most researchers focus on ex vivo liver engineering3,4,5,6,7,8,9,10,11,12,13,14. However, in most previous studies, only small pieces of repopulated scaffold cubes were transplanted into different heterotopic implantation sites. In a few studies, partial repopulated scaffolds were transplanted as an auxiliary graft. However, the maximal reported survival time was only 72 h8,14. As far as we know, orthotopic transplantation of a repopulated full liver graft has not yet been performed or published about. The long-term function and transplantation of engineered organs are still in their infancy. Therefore, an alternative approach to ex vivo liver engineering is needed.
In vivo liver engineering may represent an alternative to study hepatic repopulation under physiological conditions. The advantages of in vivo liver engineering compared to ex vivo liver engineering are manifold. The in vivo repopulated partial liver scaffold is subjected to physiological blood perfusion with proper temperature, sufficient oxygen, nutrients, and growth factors in contrast to ex vivo perfusion with artificial culture medium. Furthermore, the remaining partial normal liver maintains the hepatic function, principally allowing long-term survival. Since an implanted ex vivo engineered liver graft is still incapable of sustaining the long-term survival of experimental animals by its liver function8,&#160;we envision that in vivo partial liver engineeringwould ultimately become a promising model to further study the evolution of engineered livers with longer survival observations than ex vivo.
Recently, one research group (Pan and colleagues) presented, for the first time, a technique of in vivo liver engineering15. They achieved the isolated perfusion of the right inferior liver lobe in living rats despite anatomic and technical challenges. They reported the first intraoperative results of in vivo repopulation using a rat primary hepatocyte cell line. However, the in vivo surgical perfusion model of Pan et al. has disadvantages. They achieved single liver lobe perfusion in rats at the expense of completely blocking the portal vein and inferior vena cava, which may cause severe harm to the animal. The experimental rats were sacrificed after only 6 hours of intraoperative observation time. Therefore, the in vivo liver lobe perfusion technique needs further improvement to achieve postoperative survival.
We developed a novel survival model for in vivo liver lobe perfusion, based on previous studies of the hepatic anatomy of rat16, the portal vein cannulation technique for hemodynamic monitoring in mice17, and liver bioengineering18,19. The key steps for the procedure are illustrated in Figure&#160;1A - 1E.
This technique is suitable for those who want to use this experimental in vivo perfusion model for basic research on partial organ treatment by infusion with drugs, in vivo decellularization as a chemical resection for organ diseases (e.g., liver cancer), in vivo cell culture in a decellularized matrix comparing ex vivo two-dimensional and three-dimensional cell culture systems20,21,22,23,24,25,26, and in vivo liver engineering by decellularization and repopulation.

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			<td height="21" style="height:21px;width:216px;">Perfusion Pump</td>
			<td style="width:104px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Perfusor VI</td>
			<td colspan="2">B. Braun, Melsungen</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Catheter</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Versatus-W&#160; Catheter</td>
			<td>Terumo</td>
			<td>SR+DU2419PX</td>
			<td>24G, 0.74&#215;19mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Versatus-W&#160; Catheter</td>
			<td>Terumo</td>
			<td>SR+DU2225PX</td>
			<td>22G, 0.9&#215;25mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">micro surgical instrument</td>
			<td></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">micro scissors</td>
			<td>F&#183;S&#183;L</td>
			<td>No. 14058-09</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">micro serrefine</td>
			<td>F&#183;S&#183;L</td>
			<td>No.18055-05</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micro clamps applicator</td>
			<td>F&#183;S&#183;L</td>
			<td>No. 18057-14</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Straight micro forceps</td>
			<td>F&#183;S&#183;L</td>
			<td>No. 00632-11</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Curved micro forceps</td>
			<td>F&#183;S&#183;L</td>
			<td>No. 00649-11</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">micro needle-holder</td>
			<td>F&#183;S&#183;L</td>
			<td style="width:119px;">No. 12061-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">general surgical instruments</td>
			<td></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">standard sissors</td>
			<td>F&#183;S&#183;L</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">mosquito clamp</td>
			<td>F&#183;S&#183;L</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">serrated forcep</td>
			<td>F&#183;S&#183;L</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">teethed forcep</td>
			<td>F&#183;S&#183;L</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">needle-holder</td>
			<td>F&#183;S&#183;L</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">suture</td>
			<td style="width:104px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">4-0 prolene</td>
			<td style="width:104px;">ethicon</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">4-0 ETHICON*II</td>
			<td style="width:104px;">ethicon</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">6-0 silk</td>
			<td style="width:104px;">ethicon</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">11-0 polyamide</td>
			<td style="width:104px;">ethicon</td>
			<td style="width:119px;"></td>
			<td></td>
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a novel surgical technique as a foundation for in vivo partial liver engineering in rat

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver engineering, including in vivo organ decellularization followed by repopulation, has emerged as a promising approach over ex vivo liver engineering. However, postoperative survival was not achieved. The aim of this study is to develop a novel surgical technique of in vivo selective liver lobe perfusion in rats as a prerequisite for in vivo liver engineering. We generate a circuit bypass only through the left lateral lobe. Then, the left lateral lobe is perfused with heparinized saline. The experiment is performed with 4 groups (n = 3 rats per group) based on different perfusion times of 20 min, 2 h, 3 h, and 4 h. Survival, as well as the macroscopically visible change of color and the histologically determined absence of blood cells in the portal triad and the sinusoids, is taken as an indicator for a successful model establishment. After selective perfusion of the left lateral lobe, we observe that the left lateral lobe, indeed, turned from red to faint yellow. In a histological assessment, no blood cells are visible in the branch of the portal vein, the central vein, and the sinusoids. The left lateral lobe turns red after reopening the blocked vessels. 12/12 rats survived the procedure for more than one week. We are the first to report a surgical model for in vivo single liver lobe perfusion with a long survival period of more than one week. In contrast to the previously published report, the most important advantage of the technique presented here is that perfusion of 70% of the liver is maintained throughout the whole procedure. The establishment of this technique provides a foundation for in vivo partial liver engineering in rats, including decellularization and recellularization.

Twelve male (aged 12 - 13 weeks) Lewis rats were used to assess the effect of selective liver lobe perfusion. The experiment was performed in four groups (n = 3 rats per group). Using different perfusion periods of 20 minutes, 2 hours, 3 hours, and 4 hours, following the steps described above, we successfully achieved in vivo single lobe perfusion.
In Vivo Perfusion of the Left...

Watch this Scientific Journal Video about A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat at JoVE.com

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

We establish a novel surgical technique for an in vivo single liver lobe perfusion model in rat as a prerequisite for further studying in vivo partial liver engineering in the future.

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver ...

Over the last few years, liver engineering has become a hot topic around the world due to it's potential of generating transplant able liver grafts. However viable long term function and the transplantation of engineered organs are still a vision of the future. Recently in-vivo liver lobe profusion has become a promising strategy to start liver engineering.A major advantage of this technique is that the in-vivo repopulated partial liver scaffold subjected scatological blood profusion. This provides the scaffold with the proper temperature, sufficient oxygen, nutrients and growth factors in contrast to ex-vivo profusion with artificial culture medium. Another advantage is that remaining liver maintains hepatic function and thereby principally allows long term survival.However the in-vivo single lobe profusion model is technically challenging and post operative survival has yet to be achieved. Here we are going to present another surgical model with long term survival as a foundation for further in-vivo single liver lobe engineering. Before we demonstrate the surgical model, we'd like to give a brief introduction to rat liver anatomy.To establish our single liver lobe profusion model we selected the left lateral lobe. This is the only liver lobe with a distinct vascular supply and drainage allowing to create a bypass. This is a 3-D reconstruction of the portal venous system in rat.Our focus here is on the left portal vein which will later serve as the fluid inlet. This is a 3-D reconstruction of the hepatic venous system in rat. Here our focus is on the left lateral hepatic vein which will later serve as a fluid outlet.This illustration of liver vascular anatomy highlights the vascular access points used for out partial perfusion model. This animation illustrates the scheme of generating the in-vivo left lateral liver lobe perfusion model. In order to establish the surgical model we are going to generate a circuit bypass within the left lateral lobe.For this purpose we need to block the following vessels the left portal vein, the left hepatic artery, the left bile duct, the left median protal vein, hepatic artery and bile duct, and the left lateral hepatic vein. Then we need to cannulate the left portal vein with a 24 gage catheter. Afterwards the left lateral hepatic vein is cannulated using a 22 gage catheter.Then the catheter in the left portal vein is connected to a perfusion pump. Dry gauze is placed at the outlet of the catheter in the left lateral hepatic vein to absorb waste fluid. This way a bypass is created.Then the left lateral lobe perfused with heparinized saline At a flow rate of 0.5 milliliters per minute. Physiological perfusion to the left lateral lobe is restored after reopening the blocked vessels. Now the surgical perfusion model is established.All procedures demonstrated in this video were assessed and approved by the local authorities. The operation field of the abdomen is disinfected with three rounds of iodine tincture followed by two rounds of 70%alcohol. Sterile gauze is placed on the abdomen leaving only the operation field exposed.A transverse incision is made on the abdominal wall of the rat. A 4-0 prolene suture is used to fix the xiphoid process and pull it toward the head. Both sides of the upper abdominal walls are retracted using two subcoastal hooks to reach full exposure of the organ.Then we can see the liver lobes. Here's the left lateral lobe which is used for selective perfusion. We cover the duodenum and the small intestine in the abdominal cavity with moistened gauze to ovoid dryness.We expose the hilum of the liver by lifting up the median lobes with moistened gauze. The rat is placed under a sterile microscope. We dissect the left portal vein.We then ligate the left protal vein with a 6-0 silk suture. The left hepatic artery, the left bile duct, as well as the left median portal vein, the left median hepatic artery, and the left median bile duct are blocked using micro-clamps. We block the left lateral hepatic vein with micro-clamps at the base of the left lateral lobe.To create the fluid inlet, the stem of the portal vein is punctured with a 24 gage needle dwelling catheter. But the catheter is not inserted. We then remove the needle from the catheter and connect the catheter to a perfusion pump.To expel air from the tube and the catheter, we perfuse with heparinized saline for a minute. We then turn on off the pump. Next the needle free catheter, which is now connected to the perfusion pump, is inserted into the left portal vein.In this way we minimize manipulations of the cannulated vessel. Here's an exposed region of the left lateral hepatic vein we are going to cannulate in a similar way. First we create a drainage hole in the left lateral hepatic vein by puncturing it with a 22 gage or 24 gage needle dwelling catheter.We recommend that catheter is slightly smaller than the vessel. We then start to perfuse the left lateral lobe with heparinized saline at a flow rate of 0.5 milliliters per minute. The out flowing waste fluid is absorbed with gauze.To minimize intraabdominal contamination with waste fluid we reinsert the needle free catheter into the left lateral hepatic vein. Thus the cannulated left lateral hepatic vein serves as a fluid outlet. We continue perfusing the left lateral lobe with heparinized saline.Bloody fluid can be seen dripping from the outlet. The arrow indicates that the left lateral lobe turned yellow during perfusion with heparinized saline. At the end of perfusion the catheters are taken out of the vessels.The drainage opening on the left lateral hepatic vein is closed with a single 11-0 pollumite suture. The inlet opening on the left portal vein is closed with an 11-0 pollumite suture as well. Re perfusion starts with backflow from the suprahepatic vena cava after removing the clamp on the left lateral hepatic vein.Physiological perfusion is further restored after removing the clamp from the left hepatic artery and bile duct, as well as the left median vascular structures. Complete re perfusion is achieved after reopening the left portal vein as indicated by a color change to dark red. Perfusion of left later lobe.The white arrow in this image indicates that the target liver lobe was indeed selectively perfused. Here the white arrows show that the remaining lobes, which represent about 70%of the liver retained physiological perfusion throughout the whole procedure. Re perfusion of the left lateral lobe.Here the red arrow indicates that the left lateral lobe is physiologically re perfused after reopening the blocked vessels. The blue arrow shows that the left median lobe sustained ischemia. The selectively perfused left lateral lobe was histologically examined using H E staining.Figure A shows that in the perfused left lateral lobe no blood cells are detectable in the branch of the portal vein and the sinusoids. As expected red cells are visible in the branch of the hepatic artery. In figure B the inferior caudate lobe serves as control.Blood cells are clearly visible and the branches of the protal vein and hepatic artery as well as the sinusoids. Figure C demonstrates that in the perfused left lateral lobe there are no blood cells visible in the central vein. In contrast, figure D demonstrates that in the control inferior caudate lobe blood cells are visible in the central vein.We achieved a 100%one week survival rate in 12 consecutive procedures. We compared our in-vivo liver lobe perfusion model with the model of Pan and colleagues. The main differences in methodology and results between Pan and our group are:they selected the right inferior lobe while we chose the left lateral lobe as target liver lobe;they blocked vena cava and main portal vein leading to portal hypertension.In contrast we maintained portal perfusion of 70%of the liver;they sacrificed the rats inter operatively while our one week survival rate is 100%The advantages of our novel surgical model are:that it's technically challenging but feasible;and that it is well tolerated as demonstrated by the 100%survival rate. However one limitation is that the left median lobe sustained ischemia due to the blockage of the portal vein. Potential applications of our technique include:that is can be used for in-vivo partial organ treatment by perfusion with drugs;in-vivo partial organ decellularization as chemical resection;in-vivo cell culture system in comparison with ex-vivo;and in-vivo liver engineering.

Research

JoVE Journal

Bioengineering

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

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