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>

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</ol>

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><tbody><tr><td>Gas Anesthesia System</td><td>Ugo Basile; Italy</td><td>211000</td><td></td></tr><tr><td>Weighing machine</td><td>Goldtech ; India</td><td></td><td>Local Procurement</td></tr><tr><td>Biological safety cabinet ( Class I)</td><td>Kartos international;&amp;nbsp; India</td><td></td><td>Local Procurement</td></tr><tr><td>Hair Trimmer</td><td>Panasonic ;&amp;nbsp; Japan&amp;nbsp;</td><td>ER-GY10&amp;nbsp;</td><td></td></tr><tr><td>Straight operating scissor with sharp /sharp blades</td><td>Major Surgicals; India</td><td></td><td>Local Procurement</td></tr><tr><td>Forceps with Serrations</td><td>Major Surgicals; India</td><td></td><td>Local Procurement</td></tr><tr><td>Micro needle holders&amp;nbsp; straight &amp;amp; curved&amp;nbsp;</td><td>Mercian ;&amp;nbsp; England&amp;nbsp;</td><td>BS-13-8</td><td></td></tr><tr><td>1 ml insulin syringe with 30G *5/16 needles&amp;nbsp;</td><td>Dispo Van; India</td><td></td><td></td></tr><tr><td>1 ml syringe with 26 G * 1/2 needle</td><td>BD ; US&amp;nbsp;</td><td>REF 303060</td><td></td></tr><tr><td>Nylon Threads&amp;nbsp;&amp;nbsp;</td><td>Mighty ; India</td><td></td><td>(1-0) Local Procurement</td></tr><tr><td>MERSUTURES 4-0 Sterilised Surgical Needled Suture</td><td>Ethicon, Johnson &amp;amp; Johnson, India</td><td>NW 5047</td><td></td></tr><tr><td>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><td>Cotton Buds</td><td>Pure Swabs Pvt Ltd ;&amp;nbsp; India</td><td></td><td>Local Procurement</td></tr><tr><td>Surgical Tape</td><td>3M India ; India</td><td>1530-1</td><td>Micropore Surgical Tape</td></tr><tr><td>Microtome</td><td>Histo-Line Laboratories, Italy</td><td>MRS3500</td><td></td></tr><tr><td>Shandon Cryotome E Cryostat</td><td>Thermo Electron Corporation ; US</td><td></td><td></td></tr><tr><td>Confocal laser scanning microscope</td><td>Carl Zeiss ; Germany&amp;nbsp;</td><td>LSM 510 META</td><td></td></tr><tr><td>Bright Field Microscope</td><td>Olympus, Japan</td><td>LX51</td><td></td></tr><tr><td>Automated analyser</td><td>Tulip, Alto Santracruz, India</td><td>Screen Maaster 3000</td><td>Biochemical analyser for liver functional test</td></tr><tr><td>Flow Cytometer</td><td>BD ; US&amp;nbsp;</td><td>BD FACSverse</td><td>Assesment of presence of cells post transplantation</td></tr><tr><td>Veet hair removal cream&amp;nbsp;</td><td>Reckitt Benckiser , India</td><td></td><td></td></tr><tr><td>FORANE</td><td>Abbott ; US</td><td></td><td>isoflurane USP 99.9%&amp;nbsp;</td></tr><tr><td>Taxim</td><td>AlKem ; India</td><td></td><td>cefotaxime sodium injection</td></tr><tr><td>Povidone-Iodine solution&amp;nbsp;</td><td>Win-Medicare;&amp;nbsp; India</td><td></td><td>Betadine</td></tr><tr><td>Paraformaldehyde</td><td>Himedia; India</td><td>GRM 3660</td><td></td></tr><tr><td>Iscove&#039;s Modified Dulbecco&#039;s Medium (IMDM)</td><td>Life technologies, Thermo Fisher scientific ; US</td><td>12200-036</td><td></td></tr><tr><td>Sucrose</td><td>Sigma ; US</td><td>S0389</td><td></td></tr><tr><td>Tissue-Tek</td><td>Sakura; US</td><td>25608-930</td><td>O.C.T compound</td></tr><tr><td>DAPI</td><td>Himedia; India</td><td>MB 097</td><td></td></tr><tr><td>anti-Albumin goat Polyclonal</td><td>Thermo Scientific,Pierce, US</td><td>PA126081</td><td></td></tr><tr><td>anti-connexin 32/GJB1 Polyclonal</td><td>abcam, UK</td><td>ab64609-500</td><td></td></tr><tr><td>antiGFP rabbit polyclonal&amp;nbsp;</td><td>Santa Cruz biotechnology; US</td><td>SC 8334</td><td></td></tr><tr><td>Alexa Fluor 594 donkey anti-goat</td><td>&amp;nbsp;Molecular Probes , Thermo Fisher Scientific ;&amp;nbsp; US</td><td>A11058</td><td></td></tr><tr><td>Alexa Fluor 488 donkey anti-sheep</td><td>&amp;nbsp;Molecular Probes , Thermo Fisher Scientific ;&amp;nbsp; US</td><td>A11015</td><td></td></tr><tr><td>Alexa Fluor 594 chicken anti rabbit</td><td>&amp;nbsp;Molecular Probes , Thermo Fisher Scientific ;&amp;nbsp; US</td><td>A21442</td><td></td></tr><tr><td>Goat anti rabbit IgG HRP</td><td>Invitrogen, Thermo Fisher Scientific; US&amp;nbsp;</td><td>65-6120</td><td></td></tr><tr><td>anti-Ki67 antibody</td><td>abcam, UK</td><td>ab15580</td><td></td></tr><tr><td>Antigen Unmasking Solution, Citric acid base</td><td>Vector laboratories, US</td><td>H-3300</td><td></td></tr><tr><td>ProLong Diamond antifade mountant</td><td>Life technologies, Thermo Fisher scientific ; US</td><td>P36966</td><td></td></tr><tr><td>SGOT (ASAT) KIT</td><td>Coral Clinical System, India</td><td></td><td></td></tr><tr><td>SGPT (ALAT) KIT</td><td>Coral Clinical System, India</td><td></td><td></td></tr><tr><td>Alkaline Phosphatase Kit (DEA)</td><td>Coral Clinical System, India</td><td></td><td></td></tr><tr><td>Hematoxylin Solution, Mayer&#039;s</td><td>Sigma ; US</td><td>MHS16</td><td></td></tr><tr><td>Eosin Y solution, alcoholic</td><td>Sigma ; US</td><td>HT110132</td><td></td></tr><tr><td>DPX Mountant&amp;nbsp;</td><td>Sigma ; US</td><td>6522</td><td></td></tr><tr><td>Melonex (Pain Killer)</td><td>Intas Pharmaceuticals Ltd; India</td><td></td><td>Meloxicam injection&amp;nbsp;</td></tr><tr><td>DAB enhanced liquid substrate system tetrahydrochloride</td><td>Sigma ; US</td><td>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 hebben een protocol voor het uitvoeren van gedeeltelijke hepatectomy (PHx) en cel transplantatie via milt in KNIPOOG beschreven. SCID (NOD. CB17-Prkdcscid/j) muizen. In dit protocol wordt een incisie gemaakt bloot en resect de linker kwab van de lever, gevolgd door een andere snede voor intrasplenic transplantatie van cellen.

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

An Oncogenic Hepatocyte-Induced Orthotopic Mouse Model of Hepatocellular Cancer Arising in the Setting of Hepatic Inflammation and Fibrosis

The absence of a clinically relevant animal model addressing the typical immune characteristics of hepatocellular cancer (HCC) has significantly impeded elucidation of the underlying mechanisms and development of innovative immunotherapeutic strategies. To develop an ideal animal model recapitulating human HCC, immunocompetent male C57BL/6J mice first receive a carbon tetrachloride (CCl4) injection to induce liver fibrosis, then receive histologically-normal oncogenic hepatocytes from young male SV40 T antigen (TAg)-transgenic mice (MTD2) by intra-splenic (ISPL) inoculation. Androgen generated in recipient male mice at puberty initiates TAg expression under control of a liver-specific promoter. As a result, the transferred hepatocytes become cancer cells and form tumor masses in the setting of liver fibrosis/cirrhosis. This novel model mimics human HCC initiation and progression in the context of liver fibrosis/cirrhosis and reflects the most typical features of human HCC including immune dysfunction.

Here, we describe the development of a clinically relevant murine model of liver cancer recapitulating the typical immune features of hepatocellular cancer (HCC).

The absence of a clinically relevant animal model addressing the typical immune characteristics of hepatocellular cancer (HCC) has significantly impeded ...

Cancer Research

Generation of Subcutaneous and Intrahepatic Human Hepatocellular Carcinoma Xenografts in Immunodeficient Mice

In vivo experimental models of hepatocellular carcinoma (HCC) that recapitulate the human disease provide a valuable platform for research into disease pathophysiology and for the preclinical evaluation of novel therapies. We present a variety of methods to generate subcutaneous or orthotopic human HCC xenografts in immunodeficient mice that could be utilized in a variety of research applications. With a focus on the use of primary tumor tissue from patients undergoing surgical resection as a starting point, we describe the preparation of cell suspensions or tumor fragments for xenografting. We describe specific techniques to xenograft these tissues i) subcutaneously; or ii) intrahepatically, either by direct implantation of tumor cells or fragments into the liver, or indirectly by injection of cells into the mouse spleen. We also describe the use of partial resection of the native mouse liver at the time of xenografting as a strategy to induce a state of active liver regeneration in the recipient mouse that may facilitate the intrahepatic engraftment of primary human tumor cells. The expected results of these techniques are illustrated. The protocols described have been validated using primary human HCC samples and xenografts, which typically perform less robustly than the well-established human HCC cell lines that are widely used and frequently cited in the literature. In comparison with cell lines, we discuss factors which may contribute to the relatively low chance of primary HCC engraftment in xenotransplantation models and comment on technical issues that may influence the kinetics of xenograft growth. We also suggest methods that should be applied to ensure that xenografts obtained accurately resemble parent HCC tissues.

Human tumor xenografts in immunodeficient mice are valuable tools to study cancer biology. Specific protocols to generate subcutaneous and intrahepatic xenografts from human hepatocellular carcinoma cells or tumor fragments are described. Liver regeneration induced by partial hepatectomy in recipient mice is presented as a strategy to facilitate intrahepatic engraftment.

In  vivo experimental models of hepatocellular carcinoma (HCC) that recapitulate  the human disease provide a valuable platform for research into disease  ...

Partial Lobular Hepatectomy: A Surgical Model for Morphologic Liver Regeneration

Morphological organ regeneration following acute tissue loss is common among lower vertebrates, but is rarely observed in mammalian postnatal life. Adult liver regeneration after 70% partial hepatectomy results in hepatocyte hypertrophy with some replication in remaining lobes with restoration of metabolic activity, but with permanent loss of the injured lobe&#39;s morphology and architecture. Here, we detail a new surgical method in the neonate that leaves a physiologic environment conducive to regeneration. This model involves amputation of the left lobe apex and a subsequent conservative management regimen, and lacks the necessity for ligation of major liver vessels or chemical injury, leaving a physiologic environment where regeneration may occur. We extend this protocol to amputations on juvenile (P7-14) mice, during which the injured liver transitions from organ regeneration to compensatory growth by hypertrophy. The presented, brief 30 min protocol provides a framework to study the mechanisms of regeneration, its age-associated decline in mammals, and the characterization of putative hepatic stem or progenitors.

Here, we present a new method for partial resection of the left hepatic lobe in neonatal (day 0) mice. This new protocol is suitable for studying acute liver injury and injury response in the neonatal setting.

Morphological organ regeneration following acute tissue loss is common among lower vertebrates, but is rarely observed in mammalian postnatal life. Adult ...

Developmental Biology

A Familial Hypercholesterolemia Human Liver Chimeric Mouse Model Using Induced Pluripotent Stem Cell-derived Hepatocytes

Familial hypercholesterolemia (FH) is mostly caused by low-density lipoprotein receptor (LDLR) mutations and results in an increased risk of early-onset cardiovascular disease due to marked elevation of LDL cholesterol (LDL-C) in blood. Statins are the first line of lipid-lowering drugs for treating FH and other types of hypercholesterolemia, but new approaches are emerging, in particular PCSK9 antibodies, which are now being tested in clinical trials. To explore novel therapeutic approaches for FH, either new drugs or new formulations, we need appropriate in vivo models. However, differences in the lipid metabolic profiles compared to humans are a key problem of the available animal models of FH. To address this issue, we have generated a human liver chimeric mouse model using FH induced pluripotent stem cell (iPSC)-derived hepatocytes (iHeps). We used Ldlr-/-/Rag2-/-/Il2rg-/- (LRG) mice to avoid immune rejection of transplanted human cells and to assess the effect of LDLR-deficient iHeps in an LDLR null background. Transplanted FH iHeps could repopulate 5-10% of the LRG mouse liver based on human albumin staining. Moreover, the engrafted iHeps responded to lipid-lowering drugs and recapitulated clinical observations of increased efficacy of PCSK9 antibodies compared to statins. Our human liver chimeric model could thus be useful for preclinical testing of new therapies to FH. Using the same protocol, similar human liver chimeric mice for other FH genetic variants, or mutations corresponding to other inherited liver diseases, may also be generated.

Here, we present a protocol to generate a human liver chimeric mouse model of familial hypercholesterolemia using human induced pluripotent stem cell-derived hepatocytes. This is a valuable model for testing new therapies for hypercholesterolemia.

Familial hypercholesterolemia (FH) is mostly caused by low-density lipoprotein receptor (LDLR) mutations and results in an increased risk of early-onset ...

Generation of a Humanized Mouse Liver Using Human Hepatic Stem Cells

A novel animal model involving chimeric mice with humanized livers established via human hepatocyte transplantation has been developed. These mice, in which the liver has been repopulated with functional human hepatocytes, could serve as a useful tool for investigating human hepatic cell biology, drug metabolism, and other preclinical applications. One of the key factors required for successful transplantation of human hepatocytes into mice is the elimination of the endogenous hepatocytes to prevent competition with the human cells and provide a suitable space and microenvironment for promoting human donor cell expansion and differentiation. To date, two major liver injury mouse models utilizing fumarylacetoacetate hydrolase (Fah) and uroplasminogen activator (uPA) mice have been established. However, Fah mice are used mainly with mature hepatocytes and the application of the uPA model is limited by decreased breeding. To overcome these limitations, Alb-toxin receptor mediated cell knockout (TRECK)/SCID mice were used for in vivo differentiation of immature human hepatocytes and humanized liver generation. Human hepatic stem cells (HpSCs) successfully repopulated the livers of Alb-TRECK/SCID mice that had developed lethal fulminant hepatic failure following diphtheria toxin (DT) treatment. This model of a humanized liver in Alb-TRECK/SCID mice will have functional applications in studies involving drug metabolism and drug-drug interactions and will promote other in vivo and in vitro studies.

Here, we present a novel humanized mouse liver model generated in Alb-toxin receptor mediated cell knockout (TRECK)/SCID mice following the transplantation of immature and expandable human hepatic stem cells.

A novel animal model involving chimeric mice with humanized livers established via human hepatocyte transplantation has been developed. These mice, in ...

Establishment of the Dual Humanized TK-NOG Mouse Model for HIV-associated Liver Pathogenesis

Despite the increased life expectancy of patients infected with human immunodeficiency virus-1 (HIV-1), liver disease has emerged as a common cause of their morbidity. The liver immunopathology caused by HIV-1 remains elusive. Small xenograft animal models with human hepatocytes and human immune system can recapitulate the human biology of the disease&#39;s pathogenesis. Herein, a protocol is described to establish a dual humanized mouse model through human hepatocytes and CD34+ hematopoietic stem/progenitor cells (HSPCs) transplantation, to study liver immunopathology as observed in HIV-infected patients. To achieve dual reconstitution, male TK-NOG (NOD.Cg-Prkdcscid Il2rgtm1Sug Tg(Alb-TK)7-2/ShiJic) mice are intraperitoneally injected with ganciclovir (GCV) doses to eliminate mouse transgenic liver cells, and with treosulfan for nonmyeloablative conditioning, both of which facilitate human hepatocyte (HEP) engraftment and human immune system (HIS) development. Human albumin (ALB) levels are evaluated for liver engraftment, and the presence of human immune cells in blood detected by flow cytometry confirms the establishment of human immune system. The model developed using the protocol described here resembles multiple components of liver damage from HIV-1 infection. Its establishment could prove to be essential for studies of hepatitis virus co-infection and for the evaluation of antiviral and antiretroviral drugs.

This protocol provides a reliable method to establish humanized mice with both human immune system and liver cells. Dual reconstituted immunodeficient mice achieved via intrasplenic injection of human hepatocytes and CD34+ hematopoietic stem cells are susceptible to human immunodeficiency virus-1 infection and recapitulate liver damage as observed in HIV-infected patients.

Despite the increased life expectancy of patients infected with human immunodeficiency virus-1 (HIV-1), liver disease has emerged as a common cause of ...

Immunology and Infection

A Mouse Model of Vascularized Heterotopic Spleen Transplantation for Studying Spleen Cell Biology and Transplant Immunity

The spleen is a unique lymphoid organ that plays a critical role in the homeostasis of the immune and hematopoietic systems. Patients that have undergone splenectomy regardless of precipitating causes are prone to develop an overwhelming post-splenectomy infection and experience increased risks of deep venous thrombosis and malignancies. Recently, epidemiological studies indicated that splenectomy might be associated with the occurrence of cardiovascular diseases, suggesting that physiological functions of the spleen have not yet been fully recognized. Here, we introduce a mouse model of vascularized heterotopic spleen transplantation, which not only can be utilized to study the function and behavioral activity of splenic immune cell subsets in different biologic processes, but also can be a powerful tool to test the therapeutic potential of spleen transplantation in certain diseases. The main surgical steps of this model include donor spleen harvest, the removal of recipient native spleen, and spleen graft revascularization. Using congenic mouse strains (e.g., mice with CD45.1/CD45.2 backgrounds), we observed that after syngeneic transplantation, both donor-derived splenic lymphocytes and myeloid cells migrated out of the graft as early as post-operative day 1, concomitant with the influx of multiple types of recipient cells, thus generating a unique chimera. &#160;Despite relatively challenging techniques, this procedure can be performed with &#62;90% success rate. This model allows tracking the fate, longevity, and function of splenocytes during steady state and in a disease setting following a spleen transplantation, thereby offering a great opportunity to discover the distinct role for spleen-derived immune cells in different disease processes.

This protocol details the surgical steps of a mouse model of vascularized heterotopic spleen transplantation, a technically challenging model that can serve as a powerful tool in studying the fate and longevity of spleen cells, the mechanisms of distinct spleen cell populations in disease progression, and transplant immunity.

The spleen is a unique lymphoid organ that plays a critical role in the homeostasis of the immune and hematopoietic systems. Patients that have undergone ...

Minimizing Post-Infusion Portal Vein Bleeding during Intrahepatic Islet Transplantation in Mice

Although the liver is currently accepted as the primary transplantation site for human islets in clinical settings, islets are transplanted under the kidney capsule in most rodent preclinical islet transplantation studies. This model is commonly used because murine intrahepatic islet transplantation is technically challenging, and a high percentage of mice could die from surgical complications, especially bleeding from the injection site post-transplantation. In this study, two procedures that can minimize the incidence of post-infusion portal vein bleeding are demonstrated. The first method applies an absorbable hemostatic gelatin sponge to the injection site, and the second method involves penetrating the islet injection needle through the fat tissue first and then into the portal vein by using the fat tissue as a physical barrier to stop bleeding. Both methods could effectively prevent bleeding-induced mouse death. The whole liver section showing islet distribution and evidence of islet thrombosis post-transplantation, a typical feature for intrahepatic islet transplantation, were presented. These improved protocols refine the intrahepatic islet transplantation procedures and may help laboratories set up the procedure to study islet survival and function in pre-clinical settings.

Here we present refined surgical procedures on successfully performing intraportal islet transplantation, a clinically relevant but technically challenging surgical procedure, in mice.

Although the liver is currently accepted as the primary transplantation site for human islets in clinical settings, islets are transplanted under the ...

Isolation of Regenerating Hepatocytes after Partial Hepatectomy in Mice

Partial hepatectomy has been widely used to investigate liver regeneration in mice, but the isolation of high yields of viable hepatocytes for downstream single-cell applications is challenging. A marked accumulation of lipids within regenerating hepatocytes is observed during the first 2 days of normal liver regeneration in mice. This so-called transient regeneration-associated steatosis (TRAS) is temporary but partially overlaps the major proliferative phase. Density-gradient purification is the backbone of most existing protocols for the isolation of primary hepatocytes. As gradient purification relies on the density and size of cells, it separates non-steatotic from steatotic hepatocyte populations. Therefore, fatty hepatocytes often are lost, yielding non-representative hepatocyte fractions. 
The presented protocol describes an easy and reliable method for the in vivo isolation of regenerating hepatocytes regardless of their lipid content. Hepatocytes from male C57BL/6 mice are isolated 24-48 h after hepatectomy by a classic two-step collagenase perfusion approach. A standard peristaltic pump drives the warmed solutions via the catheterized inferior vena cava into the remnant, using a retrograde perfusion technique with outflow through the portal vein. Hepatocytes are dissociated by collagenase for their release from the Glisson's capsule. After washing and careful centrifugation, the hepatocytes can be used for any downstream analyses. In conclusion, this paper describes a straightforward and reproducible technique for the isolation of a representative population of regenerating hepatocytes after partial hepatectomy in mice. The method may also aid the study of fatty liver disease.

Lipid-laden hepatocytes are inherent to liver regeneration but are usually lost upon density-gradient centrifugation. Here, we present an optimized cell isolation protocol that retains steatotic hepatocytes, yielding representative populations of regenerating hepatocytes after partial hepatectomy in mice.

Partial hepatectomy has been widely used to investigate liver regeneration in mice, but the isolation of high yields of viable hepatocytes for downstream ...

Liver Transplantation in Mouse and Analyzing Allograft Patency

Successful Orthotopic Liver Transplantation in Mice Utilizing Microcomputed Tomography Angiography

Microcomputed tomography (microCT) angiography is an invaluable resource to researchers. New advances in this technology have allowed for high-quality images to be obtained of micro-vasculature and are high-fidelity tools in the field of organ transplantation. In this model of orthotopic liver transplantation (OLT) in mice, microCT affords the opportunity to evaluate allograft anastomosis in real time and has the added benefit of not having to sacrifice study animals. The choice of contrast, as well as image acquisition settings, create a high-definition image, which gives researchers invaluable information. This allows for evaluation of the technical aspects of the procedure as well as potentially evaluating different therapeutics over an extended duration of time. In this protocol, we detail an OLT model in mice in a stepwise fashion and finally describe a microCT protocol that can give high-quality images, which aid researchers in in-depth analysis of solid organ transplantation. We provide a step-by-step guide for liver transplantation in a mouse, as well as briefly discuss a protocol for evaluating the patency of the graft through microCT angiography.

Author Spotlight: Enhancing Transplantation Research Through MicroCT Angiography in Murine Models 

In this protocol, we discuss the implementation of a model of successful orthotopic liver transplantation (OLT) in mice. Additionally, adjuvants to further analyze allograft patency after successful OLT in a mouse are discussed as well, specifically utilizing microcomputed tomography (microCT) scans.

Microcomputed tomography (microCT) angiography is an invaluable resource to researchers. New advances in this technology have allowed for high-quality ...

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

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