Name: A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat (Video) | JoVE Journal
Uploaded: 2018-10-06T14:00:00.000+00:00
Duration: 13 min 27 s
Description: Learn a novel surgical technique for in vivo single liver lobe perfusion in rats. This method establishes a foundation for partial liver engineering research and supports long-term survival.

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5 minutes

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 anesthetized.</li>
	<li>Take the rat out of the induction chamber and measure its body weight.</li>
	<li>Shave the fur of the surgical region on the abdomen.</li>
	<li>Place the animal back into the isoflurane chamber for an additional 2 min to deepen anesthesia.</li>
	<li>Place the rat on the operation table in supine position.</li>
	<li>Fix the anesthesia mask to the mouth region of the rat and keep the animal anesthetized with a continuous gas flow of 2% vaporized isoflurane and 100% oxygen at a flow rate of 0.5 L/min.</li>
	<li>Fix the limbs with pieces of tape.</li>
	<li>Apply vet ointment on both eyes to prevent dryness.</li>
	<li>Administer buprenorphine 0.05 mg/kg subcutaneously, to relieve pain during the operation period.</li>
	<li>Disinfect the surgical field of the abdomen with 3 rounds of iodine tincture followed by 2 rounds of 70% alcohol.</li>
	<li>Place sterilized gauze around the area where the incision will be made to only leave the operation field of the abdomen exposed.</li>
	<li>Proceed to perform the operation when the toe-pinch withdrawal reflex of the rat is absent.</li>
</ol>
2. Laparotomy of the Rat
<ol>
	<li>Make a transverse abdominal skin and muscle incision using scissors and an electrical coagulator.</li>
	<li>Fix and pull the xiphoid process toward the head using a 4-0 polypropylene suture. 
	NOTE: Pay attention to lift up the xiphoid process vertically to better expose the liver, but proceed with caution to avoid severe respiratory restriction and suffocation.</li>
	<li>Open the peritoneal cavity by pulling both sides of the abdominal walls towards the head with two subcostal hooks to expose the liver.</li>
	<li>Cover the duodenum and small intestine in the abdominal cavity with a moistened gauze to avoid drying.</li>
	<li>Lift left and right median lobes up by using a moistened gauze and hold them against the thorax to better expose the hilum of the liver.</li>
	<li>Place the rat under a stereomicroscope (8X magnification).</li>
	<li>Drop some warm saline into the abdomen and onto the surface of the liver and intestines every several minutes, to prevent drying during the whole procedure.</li>
</ol>
3. Establishment of a Bypass Passage Within the Left Lateral Lobe
<ol>
	<li>Dissect the left portal vein and ligate it with a 6-0 silk suture at the base (Figure 2A).</li>
	<li>Block the left hepatic artery, the left bile duct along with the left median portal vein, the left median hepatic artery, and the left median bile duct with micro clamps to prevent a flow of the perfusate to the left median lobe (Figure 2B).</li>
	<li>Separate the left lateral lobe by cutting off the surrounding ligaments of the lobe with micro scissors.</li>
	<li>Block the left lateral hepatic vein by clamping at the base of the left lateral lobe with micro clamps (Figure 2C). 
	NOTE: Make sure not to clamp the left portal vein as well by mistake.</li>
	<li>Use mosquito clamps to hold the ligature of the left portal vein and keep the vein with proper tension for later cannulation.</li>
	<li>Carefully make an incision in the front wall of the left portal vein by puncturing it with a 24-G needle-dwelling catheter ( Figure 3A). 
	NOTE: To create a bypass, vascular access points on the left portal vein and the left hepatic vein are needed. For this step, it is preferred to create the vascular access by puncturing the vessels with a needle rather than making a larger incision using scissors. This reduces the risk of bleeding and later stenosis.</li>
	<li>Withdraw the catheter and take the needle out of the catheter to obtain a needle-free 24-G catheter.</li>
	<li>Connect the catheter to a perfusion tube, of which the other endpoint connects to a 20-mL syringe with 15 mL of 40 U/mL heparinized saline on a perfusion pump.</li>
	<li>Turn on the pump for perfusing the tube to expel air out from the tube and the needle-free catheter.</li>
	<li>Turn off the perfusion pump.</li>
	<li>Again, insert the needle-free catheter into the left portal vein via the punctured incision on the vein (Figure 3B). 
	NOTE: Owing to the fact there is very limited space for fixing the catheter, it is not fixed at this point. Therefore, the surgeon should use care to avoid the displacement of the cannulated catheter.</li>
	<li>Carefully make another incision at the margin of the exposed region of the left lateral hepatic vein by puncturing it with a 22- or 24-G needle-dwelling catheter (Figure 3C). 
	NOTE: It is recommended that the catheter be slightly smaller than the vessel.</li>
	<li>Withdraw the catheter and take the needle out of the catheter to obtain a needle-free 22-G catheter.</li>
	<li>Turn on the perfusion pump to perfuse heparinized saline into the left lateral lobe via the 24-G cannulated needle-free catheter of the left portal vein at a flow rate of 0.5 mL/min.</li>
	<li>Use dry gauze to absorb out-flowing waste fluid around the incision area of the left lateral hepatic vein.</li>
	<li>Cannulate the left lateral hepatic vein via the punctured incision of the vein with the 22-G needle-free catheter, to generate a fluid outlet to minimize intra-abdominal contamination (Figure 3D). 
	NOTE: It is technically difficult to fix the cannulated catheter to the liver lobe. Therefore, the surgeon should pay attention to avoid displacement of the catheter. Alternatively, without cannulation of the left lateral hepatic vein with a catheter, waste fluid can also be absorbed at the incision region of the vein only with a dry gauze.</li>
	<li>Keep perfusing the left lateral lobe with heparinized saline for around 20 min (group 1) and then only with saline for 2 h, 3 h, or 4 h (group 2, group 3, and group 4, respectively).</li>
	<li>Turn off the pump to stop the perfusion.</li>
</ol>
4. Physiological Reperfusion of the Left Lateral Lobe
<ol>
	<li>Take off both catheters from the left portal vein and the left lateral hepatic vein.</li>
	<li>Close the incision of the left portal vein with an 11-0 polyamide suture.</li>
	<li>Close the incision of the left lateral hepatic vein with an 11-0 polyamide suture as well.</li>
	<li>Unclamp the left lateral hepatic vein.</li>
	<li>Unclamp the left median portal vein, left bile duct, and left hepatic artery.</li>
	<li>Cut off the ligature on the left portal vein to reopen the vein.</li>
</ol>
5. Closure of the Abdominal Wall
<ol>
	<li>Close the muscle layer of the abdominal wall by interrupted suturing with a 4-0 absorbable polyglactin 910 suture.</li>
	<li>Close the skin layer of the abdominal wall by interrupted suturing with a 4-0 absorbable polydioxanone suture.</li>
	<li>After closing the abdomen, disinfect the skin incision with 70% alcohol.</li>
</ol>
6. Postoperative Treatment of the Rat
<ol>
	<li>Place the animal on a warming pad for resuscitation for about 10 min and then put it into a new cage.</li>
	<li>Administer buprenorphine 0.05 mg/kg subcutaneously 2x a day for a consecutive 3 d postoperatively to release pain.</li>
</ol>

<ol><li>Kim, W. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((OPTN+%2F+SRTR+2016+Annula+Data+Report%3A+Liver%5BTitle%5D)+AND+%22American+Journal+of+Transplantation.+Suppl%22%5BJournal%5D)">OPTN / SRTR 2016 Annula Data Report: Liver.</a> American Journal of Transplantation. Suppl. 1, 172-253 (2018).</li>
<li>Palakkan, A. A., Hay, D. C., Anil Kumar, P. R., Kumary, T. V., Ross, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Liver+tissue+engineering+and+cell+sources%3A+issues+and+challenges%5BTitle%5D)+AND+%22Liver+International%22%5BJournal%5D)">Liver tissue engineering and cell sources: issues and challenges.</a> Liver International. 33, 666-676 (2013).</li>
<li>Hynes, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+extracellular+matrix%3A+not+just+pretty+fibrils%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">The extracellular matrix: not just pretty fibrils.</a> Science. 326, 1216-1219 (2009).</li>
<li>Flaim, C. J., Chien, S., Bhatia, S. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+extracellular+matrix+microarray+for+probing+cellular+differentiation%5BTitle%5D)+AND+%22Nature+Methods%22%5BJournal%5D)">An extracellular matrix microarray for probing cellular differentiation.</a> Nature Methods. 2, 119-125 (2005).</li>
<li>Wells, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+role+of+matrix+stiffness+in+regulating+cell+behavior%5BTitle%5D)+AND+%22Hepatology%22%5BJournal%5D)">The role of matrix stiffness in regulating cell behavior.</a> Hepatology. 47, 1394-1400 (2008).</li>
<li>Ren, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evaluation+of+two+decellularization+methods+in+the+development+of+a+whole-organ+decellularized+rat+liver+scaffold%5BTitle%5D)+AND+%22Liver+International%22%5BJournal%5D)">Evaluation of two decellularization methods in the development of a whole-organ decellularized rat liver scaffold.</a> Liver International. 33, 448-458 (2013).</li>
<li>Yagi, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Human-scale+whole-organ+bioengineering+for+liver+transplantation%3A+a+regenerative+medicine+approach%5BTitle%5D)+AND+%22Cell+Transplantation%22%5BJournal%5D)">Human-scale whole-organ bioengineering for liver transplantation: a regenerative medicine approach.</a> Cell Transplantation. 22, 231-242 (2013).</li>
<li>Jiang, W. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cryo-chemical+decellularization+of+the+whole+liver+for+mesenchymal+stem+cells-based+functional+hepatic+tissue+engineering%5BTitle%5D)+AND+%22Biomaterials%22%5BJournal%5D)">Cryo-chemical decellularization of the whole liver for mesenchymal stem cells-based functional hepatic tissue engineering.</a> Biomaterials. 35, 3607-3617 (2014).</li>
<li>Uygun, B. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Organ+reengineering+through+development+of+a+transplantable+recellularized+liver+graft+using+decellularized+liver+matrix%5BTitle%5D)+AND+%22Nature+Medicine%22%5BJournal%5D)">Organ reengineering through development of a transplantable recellularized liver graft using decellularized liver matrix.</a> Nature Medicine. 16, 814-820 (2010).</li>
<li>Baptista, P. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+use+of+whole+organ+decellularization+for+the+generation+of+a+vascularized+liver+organoid%5BTitle%5D)+AND+%22Hepatology%22%5BJournal%5D)">The use of whole organ decellularization for the generation of a vascularized liver organoid.</a> Hepatology. 53, 604-617 (2011).</li>
<li>Bruinsma, B. G., Kim, Y., Berendsen, T. A., Yarmush, M. L., Uygun, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Layer-by-layer+heparinization+of+decellularized+liver+matrices+to+reduce+thrombogenicity+of+tissue+engineered+grafts%5BTitle%5D)+AND+%22Journal+of+Clinical+and+Translational+Research%22%5BJournal%5D)">Layer-by-layer heparinization of decellularized liver matrices to reduce thrombogenicity of tissue engineered grafts.</a> Journal of Clinical and Translational Research. 1 (1), (2015).</li>
<li>Park, K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Decellularized+Liver+Extracellular+Matrix+as+Promising+Tools+for+Transplantable+Bioengineered+Liver+Promotes+Hepatic+Lineage+Commitments+of+Induced+Pluripotent+Stem+Cells%5BTitle%5D)+AND+%22Tissue+Engineering+Part+A%22%5BJournal%5D)">Decellularized Liver Extracellular Matrix as Promising Tools for Transplantable Bioengineered Liver Promotes Hepatic Lineage Commitments of Induced Pluripotent Stem Cells.</a> Tissue Engineering Part A. 22, 449-460 (2014).</li>
<li>Ko, I. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bioengineered+transplantable+porcine+livers+with+re-endothelialized+vasculature%5BTitle%5D)+AND+%22Biomaterials%22%5BJournal%5D)">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/pubmed?term=((Construction+of+a+portal+implantable+functional+tissue-engineered+liver+using+perfusion-decellularized+matrix+and+hepatocytes+in+rats%5BTitle%5D)+AND+%22Cell+Transplantation%22%5BJournal%5D)">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/pubmed?term=((In-vivo+organ+engineering%3A+Perfusion+of+hepatocytes+in+a+single+liver+lobe+scaffold+of+living+rats%5BTitle%5D)+AND+%22The+International+Journal+of+Biochemistry+%26+Cell+Biology%22%5BJournal%5D)">In-vivo organ engineering: Perfusion of hepatocytes in a single liver lobe scaffold of living rats.</a> The International Journal of Biochemistry & Cell Biology. 80, 124-131 (2016).</li>
<li>Madrahimov, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Marginal+hepatectomy+in+the+rat%3A+from+anatomy+to+surgery%5BTitle%5D)+AND+%22Annals+of+Surgery%22%5BJournal%5D)">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/pubmed?term=((Bioengineered+Livers%3A+A+New+Tool+for+Drug+Testing+and+a+Promising+Solution+to+Meet+the+Growing+Demand+for+Donor+Organs%5BTitle%5D)+AND+%22European+Surgical+Research%22%5BJournal%5D)">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/pubmed?term=((Liver+engineering+as+a+new+source+of+donor+organs%3A+A+systematic+review%5BTitle%5D)+AND+%22Der+Chirurg%22%5BJournal%5D)">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/pubmed?term=((Monitoring+of+systemic+and+hepatic+hemodynamic+parameters+in+mice%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Monitoring of systemic and hepatic hemodynamic parameters in mice.</a> Journal of Visualized Experiments. (92), e51955(2014).</li>
<li>Zhou, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Decellularization+and+Recellularization+of+Rat+Livers+With+Hepatocytes+and+Endothelial+Progenitor+Cells%5BTitle%5D)+AND+%22Artificial+Organs%22%5BJournal%5D)">Decellularization and Recellularization of Rat Livers With Hepatocytes and Endothelial Progenitor Cells.</a> Artificial Organs. 40, E25-E38 (2016).</li>
<li>Yagi, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Human-scale+whole-organ+bioengineering+for+liver+transplantation%3A+a+regenerative+medicine+approach%5BTitle%5D)+AND+%22Cell+Transplantation%22%5BJournal%5D)">Human-scale whole-organ bioengineering for liver transplantation: a regenerative medicine approach.</a> Cell Transplantation. 22, 231-242 (2013).</li>
<li>Otsuka, H., Sasaki, K., Okimura, S., Nagamura, M., Nakasone, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Micropatterned+co-culture+of+hepatocyte+spheroids+layered+on+non-parenchymal+cells+to+understand+heterotypic+cellular+interactions%5BTitle%5D)+AND+%22Science+and+Technology+of+Advanced+Materials%22%5BJournal%5D)">Micropatterned co-culture of hepatocyte spheroids layered on non-parenchymal cells to understand heterotypic cellular interactions.</a> Science and Technology of Advanced Materials. 14, 065003(2013).</li>
<li>Bale, S. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Long-term+coculture+strategies+for+primary+hepatocytes+and+liver+sinusoidal+endothelial+cells%5BTitle%5D)+AND+%22Tissue+Engineering+Part+C%3A+Methods%22%5BJournal%5D)">Long-term coculture strategies for primary hepatocytes and liver sinusoidal endothelial cells.</a> Tissue Engineering Part C: Methods. 21, 413-422 (2015).</li>
<li>Wu, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optimizing+perfusion-decellularization+methods+of+porcine+livers+for+clinical-scale+whole-organ+bioengineering%5BTitle%5D)+AND+%22BioMed+Research+International%22%5BJournal%5D)">Optimizing perfusion-decellularization methods of porcine livers for clinical-scale whole-organ bioengineering.</a> BioMed Research International. , 785474(2015).</li>
<li>Barakat, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Use+of+decellularized+porcine+liver+for+engineering+humanized+liver+organ%5BTitle%5D)+AND+%22Journal+of+Surgical+Research%22%5BJournal%5D)">Use of decellularized porcine liver for engineering humanized liver organ.</a> Journal of Surgical Research. 173 (1), e11-e25 (2012).</li>
<li>Navarro-Tableros, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Recellularization+of+rat+liver+scaffolds+by+human+liver+stem+cells%5BTitle%5D)+AND+%22Tissue+Engineering+Part+A%22%5BJournal%5D)">Recellularization of rat liver scaffolds by human liver stem cells.</a> Tissue Engineering Part A. 21 (11-12), 1929-1939 (2015).</li>
</ol>

The authors have nothing to disclose.

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.

<table><tbody><tr><td>Perfusion Pump</td><td></td><td></td><td></td></tr><tr><td>Perfusor VI</td><td>B. Braun, Melsungen</td><td></td><td></td></tr><tr><td>Catheter</td><td></td><td></td><td></td></tr><tr><td>Versatus-W&amp;nbsp; Catheter</td><td>Terumo</td><td>SR+DU2419PX</td><td>24G, 0.74&amp;times;19mm</td></tr><tr><td>Versatus-W&amp;nbsp; Catheter</td><td>Terumo</td><td>SR+DU2225PX</td><td>22G, 0.9&amp;times;25mm</td></tr><tr><td>micro surgical instrument</td><td></td><td></td><td></td></tr><tr><td>micro scissors</td><td>F&amp;middot;S&amp;middot;L</td><td>No. 14058-09</td><td></td></tr><tr><td>micro serrefine</td><td>F&amp;middot;S&amp;middot;L</td><td>No.18055-05</td><td></td></tr><tr><td>Micro clamps applicator</td><td>F&amp;middot;S&amp;middot;L</td><td>No. 18057-14</td><td></td></tr><tr><td>Straight micro forceps</td><td>F&amp;middot;S&amp;middot;L</td><td>No. 00632-11</td><td></td></tr><tr><td>Curved micro forceps</td><td>F&amp;middot;S&amp;middot;L</td><td>No. 00649-11</td><td></td></tr><tr><td>micro needle-holder</td><td>F&amp;middot;S&amp;middot;L</td><td>No. 12061-01</td><td></td></tr><tr><td>general surgical instruments</td><td></td><td></td><td></td></tr><tr><td>standard sissors</td><td>F&amp;middot;S&amp;middot;L</td><td></td><td></td></tr><tr><td>mosquito clamp</td><td>F&amp;middot;S&amp;middot;L</td><td></td><td></td></tr><tr><td>serrated forcep</td><td>F&amp;middot;S&amp;middot;L</td><td></td><td></td></tr><tr><td>teethed forcep</td><td>F&amp;middot;S&amp;middot;L</td><td></td><td></td></tr><tr><td>needle-holder</td><td>F&amp;middot;S&amp;middot;L</td><td></td><td></td></tr><tr><td>suture</td><td></td><td></td><td></td></tr><tr><td>4-0 prolene</td><td>ethicon</td><td></td><td></td></tr><tr><td>4-0 ETHICON*II</td><td>ethicon</td><td></td><td></td></tr><tr><td>6-0 silk</td><td>ethicon</td><td></td><td></td></tr><tr><td>11-0 polyamide</td><td>ethicon</td><td></td><td></td></tr></tbody></table>

a novel surgical technique as a foundation for in vivo partial liver engineering in rat

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...

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.

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

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Research

JoVE Journal

Bioengineering

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Learn a novel surgical technique for in vivo single liver lobe perfusion in rats. This method establishes a foundation for partial liver engineering research and supports long-term survival.

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

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