This work was supported by the core grant received from the Department of Biotechnology, Government of India to the National Institute of Immunology, New Delhi. Dr. Bhattacharjee&#39;s current address is Division of Gastroenterology, Hepatology and Nutrition, Children&#39;s Hospital Los Angeles.

Procedures presented in this protocol have been approved by the Institutional Animal Ethics Committee of the National Institute of Immunology, New Delhi. The serial reference number of the approval is IAEC#319/13. 
Note: There are excellent resources on general surgery procedures20 and specific protocols for rodent surgery21. For those doing animal surgery for the first time, it is recommended to extensively practice surgical procedures on dummies before operating on animals.
1. Preparation
<ol>
	<li>Prior to experiment, keep sterile phosphate buffered saline (PBS) or saline ready.</li>
	<li>Assemble a surgery kit containing scissors, serrated forceps, tissue forceps, cotton, cotton buds, nylon threads, and different micro needle holders. Autoclave the surgery kit. Special care should be taken if an immune-compromised NOD.SCID mouse is included in the protocol.</li>
	<li>Perform the complete experimental procedure, from the preparation to the end of surgery, in the bio-safety class I cabinet.</li>
	<li>Weigh a NOD.SCID mouse of 6-8 weeks old prior to the surgery. Mice weighing between 14-18 g are used in this study.</li>
	<li>Shave the upper central and hypochondriac abdominal region of the mouse with the hair trimmer. Apply hair removing cream evenly throughout the region with a spatula to completely remove the trimmed hairs. Remove the hairs gently with the help of a piece of wet sterile cotton after 2-5 min.</li>
	<li>Place the mouse in the isoflurane chamber and unlock the valve of the oxygen cylinder. Maintain the oxygen flow at the rate of 4 L/min and the isoflurane vaporization at 4% to induce anesthesia.
	<ol>
		<li>Ensure that the mouse has been anesthetized properly by gentle toe pinching.</li>
	</ol>
	</li>
	<li>Place a surgery board inside a biosafety cabinet. Place the animal on the surgery board, such that the ventral portion of the mouse is facing up and the anterior portion of the mouse is placed inside the nose cone connected to the isoflurane and oxygen supply.</li>
	<li>Reduce the isoflurane vaporization to 2%, and maintain it throughout the surgical procedure.</li>
	<li>Disinfect the skin of the mouse and sterilize it by wiping with 70% ethanol soaked sterile cotton.</li>
</ol>
2. Surgical Procedure
<ol>
	<li>Partial Hepatectomy
	<ol>
		<li>Make a transverse incision of around 1 cm in the skin just beneath the sternum, perpendicular to the xiphoid process and parallel to the ribcage, with the help of straight operating scissors.</li>
		<li>Gently separate the skin attached with the abdominal muscle layer in the vicinity of the incised area with forceps or sterile moistened cotton tips to distinguish between skin and abdominal muscle layer. Soak the intradermal region with PBS using sterile cotton tips to evade desiccation.</li>
		<li>Expose the area of the left lobe smoothly by making a transverse incision through the peritoneal layer just beneath the xiphoid. Use two moistened cotton tips to expose and lift the left lobe of the liver.</li>
		<li>Place one of the cotton tips on the abdominal side of the cut, and place another cotton tip on the diaphragm side. Gently press the tip placed towards the diaphragm, and give a sliding push by the other tip to lift the left lobe of the liver.</li>
		<li>Slip a nylon thread with a loop through the lifted left lobe, and slide the loop towards the base of the left lobe close to the hilum with the help of the micro-forceps or cotton tips. Gently push down the nylon thread loop to the base of the left lobe.</li>
		<li>Tie two ends of the nylon thread over the top of the left lobe using a microsurgery needle holder and the micro forceps. Make two additional knots on the other side.</li>
		<li>Dissect out the tied lobe with the help of scissors. Do not attempt to cut very close to the thread. In case the procedure lasts more than 5 min, keep the peritoneal cavity and organs moist with sterile PBS to avoid desiccation due to fluid loss.</li>
		<li>Sew the peritoneum by continuous suturing using a 4-0 Catgut suture. Subsequently, close the skin by discontinuous suturing as quickly as possible.</li>
	</ol>
	</li>
	<li>Cell Transplantation 
	Note: The GFP expressing hepatocytes were isolated from transgenic GFP mice (C57BL/6-Tg (UBC-GFP) 30 Scha/J), according to the procedure described by Lee et al.22 and Shen et al.23 Hepatocyte-like cells (NeoHep) of human origin differentiated from monocytes24 were also used for transplantation. However, cells derived from any other sources may also be used in the protocol.
	<ol>
		<li>Suspend 0.2 million viable cells in around 50 &#181;L of Iscove&#39;s Modified Dulbecco&#39;s Medium (IMDM), and aspirate it in a 1 mL insulin syringe capped with 30G needle. Keep the syringe cold by placing it on ice.</li>
		<li>Place the mouse in a way that the left lateral portion faces up toward the person performing surgery. Identify the splenic area and transversely dissect the skin near the hypochondriac region, followed by a short incision through the peritoneal layer just to expose the spleen.</li>
		<li>Gently lift the spleen and hold it outside the cavity with the help of two PBS moistened cotton tips.</li>
		<li>Hold the spleen carefully with two cotton tips in one hand, and place the needle of the syringe exactly vertical to the spleen. Gently pierce the spleen and push the needle very slowly inside; the needle should not get deeper than 2 mm.</li>
		<li>Push down the piston of the syringe slowly to inject the cells into the spleen. After transplantation, keep the needle of the syringe stable and remove it slowly from the spleen to avoid bleeding or loss of cells.</li>
		<li>After placing the spleen back into the peritoneal cavity with the cotton tips, close the peritoneal layer by continuous suturing with a 4-0 Catgut suture. Sew the skin discontinuously with the same suture. Avoid using wound clips for closing skin; instead, close it by a 4-0 suture. The wound clips restrict the natural movement of the mouse, and often clips become loose and come out quickly.</li>
	</ol>
	</li>
</ol>
3. Post-Operative Care
<ol>
	<li>After closing the skin, wipe the surroundings of both the suture sites with iodine solution (betadine) using a sterile cotton tip.</li>
	<li>Inject a dose of antibiotic cefotaxime as 600 mg/kg body weight (typically 12 mg cefotaxime in 100 &#181;L of saline/mouse)intraperitoneally using 1 mL syringe.</li>
	<li>Give daily doses of analgesic Meloxicam as 1 mg/kg body weight (typically 12 &#181;g Meloxicam in 100 &#181;L of saline/mouse) to the animal intraperitoneally, for up to three days after the surgery.</li>
	<li>After completion of the surgery, stop the flow of isoflurane gas and place the mouse back in the individually ventilated cage.</li>
</ol>
4. Euthanization and Characterizations
<ol>
	<li>After the experimental end-points (1 day and 10 days post-surgery), euthanize the mice according to the institutional animal ethics guidelines.</li>
	<li>Collect blood by terminally bleeding the animals, puncturing the ocular terminal plexus.</li>
	<li>Isolate serum from the blood.</li>
</ol>

<ol>
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U.S.A. 88 (4), 1217-1221 (1991).</li><li>Kokudo, N., Horimoto, H., Ishida, K., Takahashi, S., Nozawa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allogeneic+hepatocyte+and+fetal+liver+transplantation+and+xenogeneic+hepatocyte+transplantation+for+Nagase's+analbuminemic+rats.">Allogeneic hepatocyte and fetal liver transplantation and xenogeneic hepatocyte transplantation for Nagase's analbuminemic rats.</a> Cell Transplant. 5 (5 Suppl 1), S21-S22 (1996).</li><li>Christ, B., Bruckner, S., Stock, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatic+transplantation+of+mesenchymal+stem+cells+in+rodent+animal+models.">Hepatic transplantation of mesenchymal stem cells in rodent animal models.</a> Methods Mol. Biol. 698, 315-330 (2011).</li><li>Fox, I. J., Roy-Chowdhury, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatocyte+transplantation.">Hepatocyte transplantation.</a> J. Hepatol. 40 (6), 878-886 (2004).</li><li>Glanemann, 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=Transplantation+of+monocyte-derived+hepatocyte-like+cells+(NeoHeps)+improves+survival+in+a+model+of+acute+liver+failure.">Transplantation of monocyte-derived hepatocyte-like cells (NeoHeps) improves survival in a model of acute liver failure.</a> Ann. Surg. 249 (1), 149-154 (2009).</li><li>Rahman, T. M., Hodgson, H. J. <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+of+acute+hepatic+failure.">Animal models of acute hepatic failure.</a> Int. J. Exp. 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The authors have nothing to disclose.

Partial hepatectomy is an established technique for investigating liver regeneration, and excessive hepatectomy is reported to mimic the AHF model. Among animal models of AHF, rodents, particularly mice, are the most researched model. To obtain a liver injury model in mice, up to a 70% hepatectomy has been reported with a good survival rate25,26. However in nude and other immunodeficient mouse, a 70% hepatectomy was reported as fatal and animals died within 24 ho...

Currently, hepatocyte transplantation is proposed as an alternative to whole organ transplantation for treating patients having severe liver disorders. It is believed that it can bridge patients to whole organ transplantation1. In addition to the allogenic hepatocytes2, xenogenic hepatocytes3 and hepatocytes derived from stem cells4 are also being investigated in animal models. In this context, the homing and engraftment potential of the transplanted cells in the recipient is an important criterion for cell based therapy in acute hepatic failure (AHF).
For investigating the transplantation of hepatocytes or hepatocyte-like cells5, AHF is created in an animal model either by surgical6 or pharmacological7 procedures, followed by transplanting cells. To make an AHF animal model by pharmacological reagents, many hepatotoxins such as d-galactosamine8, acetaminophen9, carbon tetrachloride10, thioacetamide11, Concanavalin A12, lipopolysaccharide13, etc., have been used. From this list, every reagent generates a unique set of features for AHF, but unfortunately no single reagent mimics the human AHF. Moreover, the AHF induced by hepatotoxins takes a long time, which puts animals under chronic stress, and reproducible results are difficult to obtain.
On the other hand, the surgical procedure of partial hepatectomy (PHx) is skill dependent, and reproducible results are easy to obtain after developing required skills. To induce AHF by surgical intervention alone, resection of more than 70% of the liver is required; however, less than a 70% hepatectomy can still be utilized to study engraftment and proliferation of transplanted cells in the liver for analyzing their therapeutic capacity during liver damage14. The transplantation of hepatocytes have been performed post hepatectomy through the peritoneum15, tail vein16, hepatic vein17, or the spleen18. Currently, hepatic vein infusion and intrasplenic transplantation of hepatocytes are the preferred procedures, as they are easier to reproduce.
In this paper, we have described a procedure for a 30% partial hepatectomy in NOD.SCID (NOD.CB17-Prkdcscid/J) mice in which the left lobe of the liver is excised. It is followed by transplantation of 0.2 million GFP expressing mouse (C57BL/6-Tg (UBC-GFP) 30Scha/J) hepatocytes as well as human origin NeoHep19 in the spleen. This procedure leads to engraftment of the transplanted cells in the liver. This procedure is the least invasive and a minimally painful technique.

<table border="0" cellpadding="0" cellspacing="0" width="1002">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Gas Anesthesia System</td>
			<td style="width:255px;">Ugo Basile; Italy</td>
			<td style="width:119px;">211000</td>
			<td style="width:217px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Weighing machine</td>
			<td style="width:255px;">Goldtech ; India</td>
			<td style="width:119px;"></td>
			<td>Local Procurement</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Biological safety cabinet ( Class I)</td>
			<td style="width:255px;">Kartos international;&#160; India</td>
			<td style="width:119px;"></td>
			<td>Local Procurement</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hair Trimmer</td>
			<td>Panasonic ;&#160; Japan&#160;</td>
			<td style="width:119px;">ER-GY10&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Straight operating scissor with sharp /sharp blades</td>
			<td style="width:255px;">Major Surgicals; India</td>
			<td style="width:119px;"></td>
			<td>Local Procurement</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Forceps with Serrations</td>
			<td style="width:255px;">Major Surgicals; India</td>
			<td style="width:119px;"></td>
			<td>Local Procurement</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micro needle holders&#160; straight &#38; curved&#160;</td>
			<td>Mercian ;&#160; England&#160;</td>
			<td style="width:119px;">BS-13-8</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1 ml insulin syringe with 30G *5/16 needles&#160;</td>
			<td>Dispo Van; India</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1 ml syringe with 26 G * 1/2 needle</td>
			<td>BD ; US&#160;</td>
			<td>REF 303060</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nylon Threads&#160;&#160;</td>
			<td style="width:255px;">Mighty ; India</td>
			<td style="width:119px;"></td>
			<td>(1-0) Local Procurement</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MERSUTURES 4-0 Sterilised Surgical Needled Suture</td>
			<td style="width:255px;">Ethicon, Johnson &#38; Johnson, India</td>
			<td style="width:119px;">NW 5047</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TRUGUT 76 cm 4-0 absorbable surgical suture</td>
			<td>Sutures India Pvt. Ltd; India</td>
			<td>SN 5048</td>
			<td>Sterilised Surgical Needled Suture Catgut Chromic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Cotton Buds</td>
			<td style="width:255px;">Pure Swabs Pvt Ltd ;&#160; India</td>
			<td style="width:119px;"></td>
			<td>Local Procurement</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Surgical Tape</td>
			<td style="width:255px;">3M India ; India</td>
			<td style="width:119px;">1530-1</td>
			<td>Micropore Surgical Tape</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Microtome</td>
			<td style="width:255px;">Histo-Line Laboratories, Italy</td>
			<td style="width:119px;">MRS3500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Shandon Cryotome E Cryostat</td>
			<td style="width:255px;">Thermo Electron Corporation ; US</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Confocal laser scanning microscope</td>
			<td style="width:255px;">Carl Zeiss ; Germany&#160;</td>
			<td style="width:119px;">LSM 510 META</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Bright Field Microscope</td>
			<td style="width:255px;">Olympus, Japan</td>
			<td style="width:119px;">LX51</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:411px;">Automated analyser</td>
			<td>Tulip, Alto Santracruz, India</td>
			<td style="width:119px;">Screen Maaster 3000</td>
			<td>Biochemical analyser for liver functional test</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Flow Cytometer</td>
			<td style="width:255px;">BD ; US&#160;</td>
			<td style="width:119px;">BD FACSverse</td>
			<td>Assesment of presence of cells post transplantation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Veet hair removal cream&#160;</td>
			<td>Reckitt Benckiser , India</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FORANE</td>
			<td style="width:255px;">Abbott ; US</td>
			<td style="width:119px;"></td>
			<td>isoflurane USP 99.9%&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Taxim</td>
			<td style="width:255px;">AlKem ; India</td>
			<td style="width:119px;"></td>
			<td>cefotaxime sodium injection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Povidone-Iodine solution&#160;</td>
			<td style="width:255px;">Win-Medicare;&#160; India</td>
			<td style="width:119px;"></td>
			<td>Betadine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Paraformaldehyde</td>
			<td style="width:255px;">Himedia; India</td>
			<td style="width:119px;">GRM 3660</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:411px;">Iscove&#39;s Modified Dulbecco&#39;s Medium (IMDM)</td>
			<td style="width:255px;">Life technologies, Thermo Fisher scientific ; US</td>
			<td style="width:119px;">12200-036</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Sucrose</td>
			<td style="width:255px;">Sigma ; US</td>
			<td style="width:119px;">S0389</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Tissue-Tek</td>
			<td style="width:255px;">Sakura; US</td>
			<td>25608-930</td>
			<td>O.C.T compound</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">DAPI</td>
			<td style="width:255px;">Himedia; India</td>
			<td style="width:119px;">MB 097</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">anti-Albumin goat Polyclonal</td>
			<td style="width:255px;">Thermo Scientific,Pierce, US</td>
			<td style="width:119px;">PA126081</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">anti-connexin 32/GJB1 Polyclonal</td>
			<td style="width:255px;">abcam, UK</td>
			<td style="width:119px;">ab64609-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">antiGFP rabbit polyclonal&#160;</td>
			<td style="width:255px;">Santa Cruz biotechnology; US</td>
			<td style="width:119px;">SC 8334</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:411px;">Alexa Fluor 594 donkey anti-goat</td>
			<td style="width:255px;">&#160;Molecular Probes , Thermo Fisher Scientific ;&#160; US</td>
			<td style="width:119px;">A11058</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:411px;">Alexa Fluor 488 donkey anti-sheep</td>
			<td style="width:255px;">&#160;Molecular Probes , Thermo Fisher Scientific ;&#160; US</td>
			<td style="width:119px;">A11015</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:411px;">Alexa Fluor 594 chicken anti rabbit</td>
			<td style="width:255px;">&#160;Molecular Probes , Thermo Fisher Scientific ;&#160; US</td>
			<td style="width:119px;">A21442</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:411px;">Goat anti rabbit IgG HRP</td>
			<td style="width:255px;">Invitrogen, Thermo Fisher Scientific; US&#160;</td>
			<td>65-6120</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">anti-Ki67 antibody</td>
			<td style="width:255px;">abcam, UK</td>
			<td style="width:119px;">ab15580</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Antigen Unmasking Solution, Citric acid base</td>
			<td style="width:255px;">Vector laboratories, US</td>
			<td style="width:119px;">H-3300</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">ProLong Diamond antifade mountant</td>
			<td style="width:255px;">Life technologies, Thermo Fisher scientific ; US</td>
			<td style="width:119px;">P36966</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">SGOT (ASAT) KIT</td>
			<td style="width:255px;">Coral Clinical System, India</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">SGPT (ALAT) KIT</td>
			<td style="width:255px;">Coral Clinical System, India</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Alkaline Phosphatase Kit (DEA)</td>
			<td style="width:255px;">Coral Clinical System, India</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Hematoxylin Solution, Mayer&#39;s</td>
			<td style="width:255px;">Sigma ; US</td>
			<td style="width:119px;">MHS16</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Eosin Y solution, alcoholic</td>
			<td style="width:255px;">Sigma ; US</td>
			<td style="width:119px;">HT110132</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">DPX Mountant&#160;</td>
			<td style="width:255px;">Sigma ; US</td>
			<td align="right" style="width:119px;">6522</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Melonex (Pain Killer)</td>
			<td style="width:255px;">Intas Pharmaceuticals Ltd; India</td>
			<td style="width:119px;"></td>
			<td>Meloxicam injection&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">DAB enhanced liquid substrate system tetrahydrochloride</td>
			<td style="width:255px;">Sigma ; US</td>
			<td style="width:119px;">D3939</td>
			<td></td>
		</tr>
	</tbody>
</table>

intrasplenic transplantation of hepatocytes after partial hepatectomy in nod.scid mice

Partial hepatectomy is a versatile and reproducible method to study liver regeneration and the effect of cell based therapeutics in various pathological conditions. Partial hepatectomy also facilitates the increased engraftment and proliferation of transplanted cells by accelerating neovascularization and cell migration towards the liver. Here, we describe a simple protocol for performing 30% hepatectomy and transplantation of cells in the spleen of a non-obese diabetic/severe combined immunodeficient NOD.SCID (NOD.CB17-Prkdcscid/J) mouse. 
In this procedure, two small incisions are made. The first incision is to expose and resect the left lobe of the liver, and another small incision is made to expose the spleen for the intrasplenic transplantation of cells. This procedure does not require any specialized surgical skills, and it can be completed in 5-7 minutes with less stress and pain, faster recovery, and better survival. We have demonstrated the transplantation of hepatocytes isolated from a green fluorescent protein (GFP) expressing mouse (Transgenic C57BL/6-Tg (UBC-GFP) 30Scha/J), as well as hepatocyte like cells of human origin (NeoHep) in partially hepatectomized NOD.SCID mice.

Hepatocyte proliferation after 30% partial hepatectomy: The proliferation of hepatocytes in the remaining liver after 30% hepatectomy was examined by immunohistochemical (IHC) staining for a cell proliferation marker, Ki-67. One-day post hepatectomy, the mice were euthanized, the remaining liver lobes were excised, and paraffin sections were obtained. The sections were stained with Ki-67 antibody, followed by labelling with horseradish peroxidase (HRP) conjugated secondary antibody. Di-Amino Benzidine (D...

Watch this Scientific Journal Video about Intrasplenic Transplantation of Hepatocytes After Partial Hepatectomy in NOD.SCID Mice at JoVE.com

Intrasplenic Transplantation of Hepatocytes After Partial Hepatectomy in NOD.SCID Mice

We have described a protocol for performing partial hepatectomy (PHx) and cell transplantation via spleen in NOD.SCID (NOD.CB17-Prkdcscid/J) mice. In this protocol, an incision is made to expose and resect the left lobe of the liver followed by another incision for the intrasplenic transplantation of cells.

Partial hepatectomy is a versatile and reproducible method to study liver regeneration and the effect of cell based therapeutics in various pathological ...

intrasplenic-transplantation-hepatocytes-after-partial-hepatectomy

Research

JoVE Journal

Medicine

13.9K Views.  National Institute of Immunology. We have described a protocol for performing partial hepatectomy (PHx) and cell transplantation via spleen in NOD.SCID (NOD.CB17-Prkdcscid/J) mice. In this protocol, an incision is made to expose and resect the left lobe of the liver followed by another incision for the intrasplenic transplantation of cells.

Video: Intrasplenic Transplantation of Hepatocytes After Partial Hepatectomy in NOD.SCID Mice

Mammary Transplantation of Stromal Cells and Carcinoma Cells in C57BL/6J Mice

The influence of stromal cells, including fibroblasts on mammary tumor progression has been well documented through the use of mouse models, in particular through transplantation of stromal cells and epithelial cells in the mammary gland of mice. Current transplantation models often involve the use of immunocompromised mice due to the different genetic backgrounds of stromal cells and epithelial cells. Extracellular matrices are often used to embed the two different cell types for consistent cell-cell interactions, but involve the use of Matrigel or rat tail collagen, which are immunogenic substrates. The lack of functional T cells from immunocompromised mice prevents accurate assessment of stromal cells on mammary tumor progression in vivo, with important implications on drug development and efficacy. Moreover, immunocompromised mice are costly, hard to breed and require special care conditions. To overcome these obstacles, we have developed an approach to orthotopically transplant stromal cell and epithelial cells into mice from the same genetic background to induce consistent tumor formation. This system involves harvesting normal, carcinoma associated fibroblasts, PyVmT mammary carcinoma cells and collagen from donor C57BL/6J mice. The cells are then embedded in collagen and transplanted in the inguinal mammary glands of female C57BL/6J mice. Transplantation of PyVmT cells alone form palpable tumors 30-40 days post transplantation. Endpoint analysis at 60 days indicates that co-transplantation with fibroblasts enhances mammary tumor growth compared to PyVmT cells transplanted alone. While cells and matrix from C57BL/6J mice were used in these studies, the isolation of cells and matrix and transplantation approach may be applied towards mice from different genetic backgrounds demonstrating versatility. In summary, this system may be used to investigate molecular interactions between stromal cells and epithelial cells, and overcomes critical limitations in immunocompromised mouse models.

In this report, we demonstrate a system to isolate and culture donor cells from the mouse mammary gland, and orthotopically transplant these cells in recipient mice to analyze stromal: epithelial interactions during mammary tumor development.

The influence of stromal cells, including fibroblasts on mammary tumor progression has been well documented through the use of mouse models, in particular ...

Orthotopic Aortic Transplantation in Mice for the Study of Vascular Disease

Vascular procedures involving anastomoses in the mouse are generally thought to be difficult and highly dependent on the skill of the individual surgeon. This is largely true, but there are a number of important principles that can reduce the difficulty of these procedures and enhance reproducibility. Orthotopic aortic transplantation is an excellent procedure in which to learn these principles because it involves only two end-to-end anastomoses, but requires good suturing technique and handling of the vessels for consistent success. This procedure begins with the procurement of a length of abdominal aorta from a donor animal, followed by division of the native aorta in the recipient. The procured aorta is then placed between the divided ends of the recipient aorta and sutured into place using end-to-end anastomoses. To accomplish this objective successfully requires a high degree of concentration, good tools, a steady hand, and an appreciation of how easily the vasculature of a mouse can be damaged, resulting in thrombosis. Learning these important principles is what occupies most of the beginner's time when learning microsurgery in small rodents. Throughout this protocol, we refer to these important points. This model can be used to study vascular disease in a variety of different experimental systems1-8. In the context shown here, it is most often used for the study of post-transplant vascular disease, a common long-term complication of solid organ transplantation in which intimal hyperplasia occurs within the allograft. The primary advantage of the model is that it facilitates quantitative morphometric analyses and the transplanted vessel lies contiguous to the endogenous vessel, which can serve as an additional control9. The technique shown here is most often used for mice weighing 18-25 grams. We have accumulated most of our experience using the C57BL/6J, BALB/cJ, and C3H/HeJ strains.

We describe a technique in which a section of the abdominal aorta from a mouse is transplanted orthotopically to just below the renal arteries in an allogeneic or syngeneic recipient. This technique can be useful in studies in which transplantation of large arteries of uniform size is deemed advantageous.

Vascular procedures involving anastomoses in the mouse are generally thought to be difficult and highly dependent on the skill of the individual surgeon. ...

Surgical Procedures for a Rat Model of Partial Orthotopic Liver Transplantation with Hepatic Arterial Reconstruction

Orthotopic liver transplantation (OLT) in rats using a whole or partial graft is an indispensable experimental model for transplantation research, such as studies on graft preservation and ischemia-reperfusion injury 1,2, immunological responses 3,4, hemodynamics 5,6, and small-for-size syndrome 7. The rat OLT is among the most difficult animal models in experimental surgery and demands advanced microsurgical skills that take a long time to learn. Consequently, the use of this model has been limited. Since the reliability and reproducibility of results are key components of the experiments in which such complex animal models are used, it is essential for surgeons who are involved in rat OLT to be trained in well-standardized and sophisticated procedures for this model.
While various techniques and modifications of OLT in rats have been reported 8 since the first model was described by Lee et al. 9 in 1973, the elimination of the hepatic arterial reconstruction 10 and the introduction of the cuff anastomosis technique by Kamada et al. 11 were a major advancement in this model, because they simplified the reconstruction procedures to a great degree. In the model by Kamada et al., the hepatic rearterialization was also eliminated. Since rats could survive without hepatic arterial flow after liver transplantation, there was considerable controversy over the value of hepatic arterialization. However, the physiological superiority of the arterialized model has been increasingly acknowledged, especially in terms of preserving the bile duct system 8,12 and the liver integrity 8,13,14.
In this article, we present detailed surgical procedures for a rat model of OLT with hepatic arterial reconstruction using a 50% partial graft after ex vivo liver resection. The reconstruction procedures for each vessel and the bile duct are performed by the following methods: a 7-0 polypropylene continuous suture for the supra- and infrahepatic vena cava; a cuff technique for the portal vein; and a stent technique for the hepatic artery and the bile duct.

Orthotopic liver transplantation in rats is an indispensable experimental model for biomedical research. Here we present our surgical procedures for orthotopic rat liver transplantation with hepatic arterial reconstruction using a 50% partial graft.

Orthotopic liver transplantation (OLT) in rats using a whole or partial graft is an indispensable experimental model for transplantation research, such as ...

Heterotopic Cervical Heart Transplantation in Mice

The heterotopic cervical heart transplantation in mice is a valuable tool in transplant and cardiovascular research. The cuff technique greatly simplifies this model by avoiding challenging suture anastomoses of small vessels thereby reducing warm ischemia time. In comparison to abdominal graft implantation the cervical model is less invasive and the implanted graft is easily accessible for further follow-up examinations. Anastomoses are performed by pulling the ascending aorta of the graft over the cuff with the recipient&#8217;s common carotid artery and by pulling the main pulmonary artery over the cuff with the external jugular vein. Selection of appropriate cuff size and complete mobilization of the vessels are important for successful revascularization. Ischemia-reperfusion (I/R) injury can be minimized by perfusing the graft with a cardioplegic solution and by hypothermia. In this article, we provide technical details for a simplified and improved cuff technique, which should allow surgeons with basic microsurgical skills to perform the procedure with a high success rate.

The cuff technique shortens and facilitates the model of heterotopic cervical heart transplantation in mice by avoiding technically challenging suture anastomoses of small vessels. The technical details shown in this video paper should allow researches to establish this model in their laboratories.

The heterotopic cervical heart transplantation in mice is a valuable tool in transplant and cardiovascular research. The cuff technique greatly simplifies ...

Visualization of Vascular and Parenchymal Regeneration after 70% Partial Hepatectomy in Normal Mice

A modified silicone injection procedure was used for visualization of the hepatic vascular tree. This procedure consisted of in-vivo injection of the silicone compound, via a 26 G catheter, into the portal or hepatic vein. After silicone injection, organs were explanted and prepared for ex-vivo micro-CT (&#181;CT) scanning. The silicone injection procedure is technically challenging. Achieving a successful outcome requires extensive microsurgical experience from the surgeon. One of the challenges of this procedure involves determining the adequate perfusion rate for the silicone compound. The perfusion rate for the silicone compound needs to be defined based on the hemodynamic of the vascular system of interest. Inappropriate perfusion rate can lead to an incomplete perfusion, artificial dilation and rupturing of vascular trees.
The 3D reconstruction of the vascular system was based on CT scans and was achieved using preclinical software such as HepaVision. The quality of the reconstructed vascular tree was directly related to the quality of silicone perfusion. Subsequently computed vascular parameters indicative of vascular growth, such as total vascular volume, were calculated based on the vascular reconstructions. Contrasting the vascular tree with silicone allowed for subsequent histological work-up of the specimen after &#181;CT scanning. The specimen can be subjected to serial sectioning, histological analysis and whole slide scanning, and thereafter to 3D reconstruction of the vascular trees based on histological images. This is the prerequisite for the detection of molecular events and their distribution with respect to the vascular tree. This modified silicone injection procedure can also be used to visualize and reconstruct the vascular systems of other organs. This technique has the potential to be extensively applied to studies concerning vascular anatomy and growth in various animal and disease models.

Tools used for visualizing vascular regeneration require methods for contrasting the vascular trees. This film demonstrated a delicate injection technique used to achieve optimal contrasting of the vascular trees and illustrate the potential benefits resulting from a detailed analysis of the resulting specimen using &#181;CT and histological serial sections.

A modified silicone injection procedure was used for visualization of the hepatic vascular tree. This procedure consisted of in-vivo injection of the ...

Re-Arterialized Rat Partial Liver Transplantation with an in vivo Vessel-Oriented 70% Hepatectomy

Split liver transplantation and living liver donor liver transplantation were developed in the clinic to utilize liver organs in a more efficient manner. To better understand the mechanism behind these surgical procedures, a rat partial liver transplantation (PLTx) model was established for relevant surgical studies. Because of the complexity of the rat PLTx model, a protocol with detailed descriptions is required. An article published previously reported a protocol in which ex vivo hepatectomy was used to achieve 50% rat PLTx. In contrast to this protocol, we introduced a re-arterialized PLTx with an in vivo 70% hepatectomy. An updated vessel-oriented hepatectomy was incorporated into the rat PLTx to refine the microsurgical procedure. The portal veins and hepatic arteries of the left lateral lobe and the median lobe were individually dissected and ligated before removal of the liver parenchyma, thereby decreasing the probability of bleeding in the remnant liver stump. Furthermore, an end-to-side vessel anastomosis between the common hepatic artery and the enlarged proper hepatic artery was introduced to re-arterialize the hepatic artery. By using this end-to-side vessel anastomosis technique, the diameter of the anastomosis was enlarged, thereby decreasing the difficulty of hand suture and maintaining a high rate of anastomotic patency. Moreover, the cuff anastomosis of the infrahepatic vena cava was slightly modified. A section of circumferential liver parenchyma around the vena cava of a recipient was preserved during cuff anastomosis to maintain the three-dimensional shape of the vascular lumen. This section of liver parenchyma was removed after completing the anastomosis. With this modification, the step involving placement of stay sutures was omitted, thereby further shortening the cuff anastomosis time. By using this protocol of rat PLTx, a low liver enzyme level, an intact liver lobular architecture and a high survival rate were achieved after microsurgery.

Re-Arterialized Rat Partial Liver Transplantation with an in vivo Vessel-Oriented 70% Hepatectomy

Here, a protocol involving re-arterialized rat partial liver transplantation is presented. Specifically, 70% liver was resected in vivo by using an updated technique of vessel-oriented hepatectomy. The hepatic artery was reconstructed in an end-to-side manner. The cuff technique was modified to shorten the anastomosis time of the infrahepatic vena cava.

Split liver transplantation and living liver donor liver transplantation were developed in the clinic to utilize liver organs in a more efficient manner. ...

Transplantation of Adipose Tissue-Derived Stem Cell Sheet to Reduce Leakage After Partial Colectomy in A Rat Model

Anastomotic leakage is a disastrous complication after colorectal surgery. Although current methods for leakage prevention have different levels of clinical efficacy, they are until now imperfect solutions. Stem cell therapy using ASC sheets could provide a solution to this problem. ASCs are considered as promising candidates for promoting tissue healing because of their trophic and immunomodulatory properties. Here, we provide methods to produce high-density ASC sheets, that are transplanted onto a colorectal anastomosis in a rat model to reduce the leakage. ASCs formed cell sheets in thermo-responsive culture dishes that could be easily detached. On the day of the transplantation, a partial colectomy with a 5-suture colorectal anastomosis was performed. Animals were immediately transplanted with 1 ASC sheet per rat. ASC sheets adhered spontaneously to the anastomosis without any glue, suture, or any biomaterial. Animal groups were sacrificed 3 and 7 days postoperatively. Compared to transplanted animals, the incidence of anastomotic abscesses and leakage was higher in control animals. In our model, the transplantation of ASC sheets after colorectal anastomosis was successful and associated with a lower leakage rate.

The preparation and transplantation of adipose tissue derived stem cell (ASC) sheets onto insufficiently sutured colorectal anastomoses in a rat model is presented. This novel application shows that ASC sheets can reduce colorectal anastomosis leakage.

Anastomotic leakage is a disastrous complication after colorectal surgery. Although current methods for leakage prevention have different levels of ...

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

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

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

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

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

Generation of a Rat Model of Acute Liver Failure by Combining 70% Partial Hepatectomy and Acetaminophen

Acute liver failure (ALF) is a clinical condition caused by various etiologies resulting in the loss of metabolic, biochemical, synthesizing, and detoxifying functions of the liver. In most irreversible liver damage cases, orthotropic liver transplant (OLT) remains the only available treatment. To study the therapeutic potential of a treatment for ALF, its prior testing in an animal model of ALF is essential. In the current study, an ALF model in rats was developed by combining 70% partial hepatectomy (PHx) and injections of acetaminophen (APAP) that provides a therapeutic window of 48 h. The median and left lateral lobes of the liver were removed to excise 70% of the liver mass and APAP was given 24 h postsurgically for 2 days. Survival in ALF-induced animals was found to be severely decreased. The development of ALF was confirmed by altered serum levels of the enzymes alanine amino transferase (ALT), aspartate amino transferase (AST), alkaline phosphatase (ALP); changes in prothrombin time (PT); and assessment of the international normalized ratio (INR). Study of the gene expression profile by qPCR revealed an increase in expression levels of genes involved in apoptosis, inflammation, and in the progression of liver injury. Diffused degeneration of hepatocytes and infiltration of immune cells was observed by histological evaluation. The reversibility of ALF was confirmed by the restoration of survival and serum levels of ALT, AST, and ALP after intrasplenic transplantation of syngeneic healthy rat hepatocytes. This model presents a reliable alternative to the available ALF animal models to study the pathophysiology of ALF as well as to evaluate the potential of a novel therapy for ALF. The use of two different approaches also makes it possible to study the combined effect of physical and drug-induced liver injury. The reproducibility and feasibility of current procedure is an added benefit of the model.

The acute liver failure animal model developed in the current study presents a feasible alternative for the study of potential therapies. The current model employs the combined effect of physical and drug-induced hepatic injury and provides a suitable time window to study the potential of novel therapies.

Acute liver failure (ALF) is a clinical condition caused by various etiologies resulting in the loss of metabolic, biochemical, synthesizing, and ...

Reduced Complications after Arterial Reconnection in a Rat Model of Orthotopic Liver Transplantation

The rat orthotopic liver transplantation (OLT) model is a powerful tool to study acute and chronic rejection. However, it is not a complete representation of human liver transplantation due to the absence of arterial reconnection. Described here is a modified transplantation procedure that includes the incorporation of hepatic artery (HA) reconnection, leading to a marked improvement in transplant outcomes. With a mean anhepatic time of 12 min and 14 s, HA reconnection results in improved perfusion of the transplanted liver and an increase in long-term recipient survival from 37.5% to 88.2%. This protocol includes the use of 3D-printed cuffs and holders to connect the portal vein and infrahepatic inferior vena cava. It can be implemented for studying multiple aspects of liver transplantation, from immune response and infection to technical aspects of the procedure. By incorporating a simple and practical method for arterial reconnection using a microvascular technique, this modified rat OLT protocol closely mimics aspects of human liver transplantation and will serve as a valuable and clinically relevant research model.

The goal of this study is to modify the rat orthotopic liver transplant model to better represent human liver transplantation and improve recipient survival. The presented method reestablishes hepatic arterial inflow by connecting the donor liver&#39;s common hepatic artery to the recipient liver&#39;s proper hepatic artery.

The rat orthotopic liver transplantation (OLT) model is a powerful tool to study acute and chronic rejection. However, it is not a complete representation ...