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

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.