Este trabajo fue apoyado por la concesión de la base recibida del Departamento de biotecnología, gobierno de la India en el Instituto Nacional de Inmunología, en Nueva Delhi. Dirección actual del Dr. Bhattacharjee es la división de Gastroenterología, Hepatología y nutrición, Hospital Los Ángeles de los niños.

En este protocolo los procedimientos han sido aprobados por el Comité institucional de ética Animal del Instituto Nacional de Inmunología, en Nueva Delhi. El número de serie de referencia de la aprobación es AICE #319/13.
Nota: Hay excelentes recursos en procedimientos de cirugía general20 y protocolos específicos para roedores cirugía21. Para aquellos haciendo cirugía animal por primera vez, se recomienda ampliamente practicar procedimientos quirúrgicos en maniquíes antes de operar a los animales.
1. preparación
<ol><li>Antes del experimento, se mantenga estéril tamponada de fosfato salino (PBS) o solución salina preparada.</li>
	<li>Montar un kit de cirugía contiene tijeras, pinzas dentadas, pinzas de tejido, algodón, bastoncillos de algodón, hilos de nylon y portaagujas diferentes micro. Autoclave el kit de cirugía. Debe tener especial cuidado si un GUIÑO inmuno-comprometidos. Ratón SCID está incluido en el protocolo.</li>
	<li>Realizar el procedimiento experimental completo, desde la preparación hasta el final de la cirugía, en la bio-seguridad clase I gabinete.</li>
	<li>Pesar un GUIÑO. Ratón SCID de 6-8 semanas antes de la cirugía. Ratones que pesen entre 14-18 g son utilizados en este estudio.</li>
	<li>Afeitado de la región abdominal central e hipocondríaco superior del ratón con el recortador de vellos. Aplicar el cabello eliminación crema uniformemente en toda la región con una espátula para eliminar completamente el vello recortado. Quite los pelos con la ayuda de un trozo de algodón estéril húmedo después de 2-5 minutos.</li>
	<li>Colocar el ratón en la cámara de isoflurano y abrir la válvula de la botella de oxígeno. Mantener el flujo de oxígeno a razón de 4 L/min y la vaporización de isoflurano en el 4% para inducir anestesia.<ol>
<li>Asegúrese de que el ratón ha sido anestesiado correctamente pellizcando suave del dedo del pie.</li>
	</ol>
</li>
	<li>Coloque un tablero de cirugía dentro de una bioseguridad gabinete. Coloque el animal en el tablero de la cirugía, tal que la porción ventral del ratón es hacia arriba y la porción anterior del ratón se coloca dentro del cono de nariz conectado a la fuente de isoflurano y el oxígeno.</li>
	<li>Reducir la vaporización de isoflurano al 2% y mantener durante todo el procedimiento quirúrgico.</li>
	<li>Desinfectar la piel del ratón y esterilizar limpiándolo con algodón estéril empapado etanol al 70%.</li>
</ol>2. quirúrgico
<ol><li>
Hepatectomía parcial
	<ol>
<li>Realizar una incisión transversal de aproximadamente 1 cm en la piel justo debajo del esternón, perpendicular en el proceso de xiphoid y paralelo a la caja torácica, con la ayuda de tijeras de operación recta.</li>
		<li>Separe suavemente la piel atada con la capa del músculo abdominal en las cercanías de la zona Incisa con puntas de algodón estéril humedecido para distinguir entre la piel y la capa de músculo abdominal o fórceps. Empape la región intradérmica con PBS utilizando puntas de algodón estéril para evitar la desecación.</li>
		<li>Exponga el área del lóbulo izquierdo suavemente haciendo una incisión transversal a través de la capa peritoneal justo por debajo del xifoides. Utilizar dos puntas de algodón humedecido para exponer y levantar el lóbulo izquierdo del hígado.</li>
		<li>Una de las puntas de algodón en el lado abdominal del corte y otra punta de algodón en el lado del diafragma. Suavemente presione la punta a hacia el diafragma y dar un impulso corredero por el otro extremo para levantar el lóbulo izquierdo del hígado.</li>
		<li>Deslice un hilo de nylon con un bucle el lóbulo izquierdo levantado y deslice el lazo hacia la base del lóbulo izquierdo cerca del hilio con la ayuda de los consejos micro pinzas o algodón. Presione suavemente el bucle de hilo de nylon a la base del lóbulo izquierdo.</li>
		<li>Atar dos extremos del hilo de nylon en la parte superior del lóbulo izquierdo con un Portaagujas de microcirugía y las micro pinzas. Hacer dos nudos adicionales en el otro lado.</li>
		<li>Disecan hacia fuera del lóbulo atado con la ayuda de tijeras. No intente cortar muy cerca del hilo. En caso de que el procedimiento dura más de 5 min, mantener la cavidad peritoneal y órganos húmedos con PBS estéril para evitar la desecación debido a la pérdida de líquidos.</li>
		<li>Coser el peritoneo utilizando sutura continua sutura Catgut 4-0. Posteriormente, cerca de la piel por la sutura discontinua lo antes posible.</li>
	</ol>
</li>
	<li>
Trasplante de la célula Nota: Los hepatocitos expresan GFP fueron aislados de ratones transgénicos GFP (C57BL/6-Tg (UBC-GFP) 30 Scha/J), según el procedimiento descrito por Lee et al. 22 y Shen et al. 23 hepatocito-como las células (NeoHep) de origen humano diferenciado de monocitos24 también fueron utilizadas para el trasplante. Sin embargo, las células derivadas de otras fuentes pueden utilizarse también en el protocolo.<ol>
<li>Suspender las células viables 0,2 millones en alrededor de 50 μl de medio de modificado Dulbecco de Iscove (IMDM) y aspirar en una jeringa de insulina 1 mL con aguja de 30 G. Mantenga la jeringa fría puesta en hielo.</li>
		<li>Colocar el ratón de manera que la parte lateral izquierda se enfrenta hacia la persona que realiza la cirugía. Identificar el área esplénico y transversalmente disecar la piel cerca de la región hipocondríaco, seguida de una corta incisión a través de la capa peritoneal para exponer el bazo.</li>
		<li>Levante el bazo suavemente y mantener que fuera de la cavidad con la ayuda de dos PBS humedecido puntas de algodón.</li>
		<li>Sostenga el bazo cuidadosamente con las dos puntas de algodón en una mano y coloque la aguja de la jeringa vertical exactamente en el bazo. Perforar el bazo y empuje la aguja muy lentamente en el interior; la aguja no debe recibir más de 2 mm.</li>
		<li>Empuje el émbolo de la jeringa lentamente para inyectar las células en el bazo. Después del trasplante, mantener estable la aguja de la jeringa y retire lentamente desde el bazo para evitar sangrado o pérdida de las células.</li>
		<li>Después de colocar el bazo hacia la cavidad peritoneal con las puntas de algodón, cerca de la capa peritoneal continua de sutura con una sutura de Catgut 4-0. Coser la piel discontinuo con la misma sutura. Evitar el uso de clips de herida para el cierre de la piel; en cambio, cerrarla por una sutura de 4-0. Los clips de herida restringen el movimiento natural del ratón, y a menudo clips soltarse y salen rápidamente.</li>
	</ol>
</li>
</ol>3. postoperatorio cuidado
<ol><li>Tras el cierre de la piel, limpie los alrededores de los sitios de sutura con solución de yodo (betadine) usando una punta de algodón estéril.</li>
	<li>Inyectar una dosis de antibiótico cefotaxima como 600 mg/kg de peso (típicamente 12 mg cefotaxima en 100 μl de solución salina y el ratón) corporal por vía intraperitoneal utilizando jeringa de 1 mL.</li>
	<li>Dar dosis diarias de analgésico Meloxicam 1 mg/kg peso corporal (típicamente 12 μg Meloxicam en 100 μl de solución salina y el ratón) a los animales por vía intraperitoneal, hasta tres días después de la cirugía.</li>
	<li>Después de la terminación de la cirugía, detener el flujo de gas isoflurano y volver a colocar el ratón en la jaula individualmente ventilada.</li>
</ol>4. euthanization y caracterizaciones
<ol><li>Después de las variables experimentales (1 día y 10 días post cirugía), eutanasia a los ratones según las pautas del ética animal institucional.</li>
	<li>Recoger la sangre por hemorragia terminal los animales, pinchar el plexo terminal ocular.</li>
	<li>Aislar el suero de la sangre.</li>
</ol>

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Los autores no tienen nada que revelar.

Hepatectomía parcial es una técnica establecida para investigar la regeneración hepática y hepatectomía excesiva se divulga para imitar el modelo de AHF. Entre los modelos animales de AHF, roedores, especialmente ratones, son el modelo más investigado. Para obtener un modelo de daño hepático en ratones, hasta una hepatectomía del 70% se ha divulgado con una buena supervivencia tasa25,26. Sin embargo en nude y otro ratón inmunodeficiente, una hepatectom?...

Actualmente, trasplante de hepatocitos es propuesto como una alternativa al trasplante de órgano entero para tratar a los pacientes tener severos trastornos hepáticos. Se cree que puede superar a los pacientes de trasplante de órgano entero1. Además de los hepatocitos alogénicos2, hepatocitos xenogénica3 y hepatocitos derivados de las células4 también están siendo investigados en modelos animales. En este contexto, el engraftment y autoguiado hacia el blanco potencial de las células trasplantadas en el receptor es un criterio importante para la terapia celular basada en la insuficiencia hepática aguda (AHF).
Para investigando el trasplante de hepatocitos o de hepatocitos como las células5, AHF se crea en un modelo animal por quirúrgico6 o procedimientos farmacológicos7 , seguidos de transplante de células. Para hacer un modelo animal de la AHF por reactivos farmacológicos, muchas hepatotoxins como la d-galactosamina8, acetaminofén9, tetracloruro de carbono10, tioacetamida11, concanavalina A12, lipopolysaccharide13 , etcetera., se han utilizado. De esta lista, cada reactivo genera un conjunto de características únicas para AHF, pero lamentablemente no solo reactivo imita el humano AHF. Por otra parte, el AHF inducido por hepatotoxins lleva mucho tiempo, que pone a los animales bajo estrés crónico, y son difíciles de obtener resultados reproducibles.
Por otra parte, la intervención quirúrgica de hepatectomía parcial (PHx) es dependiente de la habilidad y la reproducibilidad es fácil de obtener después de desarrollar las habilidades necesarias. Para inducir AHF por solo la intervención quirúrgica, la resección de más del 70% del hígado se requiere; sin embargo, menos de una hepatectomía del 70% todavía puede ser utilizada para estudiar el engraftment y proliferación de trasplantar las células en el hígado para analizar su capacidad terapéutica en daño hepático14. El trasplante de hepatocitos han sido realizadas post hepatectomía por el peritoneo15, cola vena16, vena hepática17o el18del bazo. Actualmente, la infusión de la vena hepática y intrasplenic trasplante de hepatocitos son los procedimientos preferidos, ya que son fáciles de reproducir.
En este trabajo hemos descrito un procedimiento de una hepatectomía parcial del 30% en cabeceo. SCID (CABECEO. CB17-Prkdcscid/J) ratones en el que el lóbulo izquierdo del hígado es suprimido. Es seguida por el trasplante de hepatocitos de ratón (C57BL/6-Tg (UBC-GFP) 30Scha/J) expresando GFP 0,2 millones así como origen humano NeoHep19 en el bazo. Este procedimiento conduce al engraftment de células trasplantadas en el hígado. Este procedimiento es menos invasivo y una técnica mínimamente dolorosa.

<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

Hepatectomía parcial es un método versátil y reproducible para el estudio de la regeneración hepática y el efecto de la terapéutica celular basado en diversas condiciones patológicas. Hepatectomía parcial también facilita el engraftment aumento y proliferación de las células trasplantadas por neovascularización aceleración y migración de células hacia el hígado. Aquí, describimos un protocolo simple para realizar 30% hepatectomía y trasplante de células en el bazo de un no obeso diabético grave había combinado NOD inmunodeficiente. SCID (CABECEO. CB17-Prkdcscid/j) ratón.
En este procedimiento, se realizan dos pequeñas incisiones. La primera incisión es para exponer y resecar el lóbulo izquierdo del hígado, y otra pequeña incisión para exponer el bazo para el intrasplenic trasplante de células. Este procedimiento no requiere ninguna habilidad quirúrgica especializada y puede realizarse en 5-7 minutos con menos estrés y dolor, una recuperación más rápida y mejor supervivencia. Hemos demostrado el trasplante de hepatocitos aislados de una proteína fluorescente verde (GFP) expresando su ratón (transgénicos C57BL/6-Tg (UBC-GFP) 30Scha/J), así como los hepatocitos como células de origen humano (NeoHep) en hepatectomized parcialmente NOD. Ratones de SCID.

Proliferación de hepatocitos después de hepatectomy parcial 30%: La proliferación de hepatocitos en el resto de hígado después de hepatectomy 30% fue examinada por inmunohistoquímica (IHC) para un marcador de proliferación celular, Ki-67. Un día post hepatectomía, los ratones fueron sacrificados, los lóbulos de hígado restantes fueron suprimidos y se obtuvieron secciones de la parafina. Las secciones fueron teñidas con anticuerpos Ki-67, seguido por etiquetado con rábano peroxidasa (HRP) con...

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

Hemos descrito un protocolo para la realización de hepatectomía parcial (PHx) y trasplante de la célula vía bazo en cabeceo. SCID (CABECEO. CB17-Prkdcscid/j) ratones. En este protocolo, se realiza una incisión para exponer y resecar el lóbulo izquierdo del hígado, seguido por otra incisión para el intrasplenic trasplante de células.

Intrasplenic trasplante de hepatocitos después de Hepatectomy parcial en cabeceo. Ratones de SCID

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 vistas.  National Institute of Immunology. Hemos descrito un protocolo para la realización de hepatectomía parcial (PHx) y trasplante de la célula vía bazo en cabeceo. SCID (CABECEO. CB17-Prkdcscid/j) ratones. En este protocolo, se realiza una incisión para exponer y resecar el lóbulo izquierdo del hígado, seguido por otra incisión para el intrasplenic trasplante de células.

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).

Un modelo de ratón ortotópico inducido por hepatocitos oncogénicos que surge en el ajuste de la inflamación hepática y la fibrosis

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

Investigación

Investigación sobre el cáncer

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.

Generación de subcutánea e intrahepática hepatocelular carcinoma humano xenoinjertos en ratones inmunodeficientes

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

Medicina

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.

Hepatectomía parcial lobulado: Un modelo quirúrgico para regeneración hepática morfológico

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

Biología del Desarrollo

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.

Un modelo de ratón quimérico hígado humano de hipercolesterolemia familiar con hepatocitos derivados de células madre pluripotentes inducidas

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.

Generación de un ratón humanizado hígado uso de células madre hepáticas humanas

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.

Establecimiento del modelo de ratón TK-NOG humanizado dual para la patogénesis hepática asociada al VIH

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

Inmunología e Infección

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.

Un modelo de ratón del trasplante de bazo heterotópico vascularizado para estudiar biología de células del bazo y inmunidad al trasplante

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.

Minimizar el sangrado de la vena porta posterior a la infusión durante el trasplante intrahepático de islotes en ratones

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.

Aislamiento de hepatocitos regeneradores después de hepatectomía parcial en ratones

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

Trasplante hepático en ratón y análisis de la permeabilidad del aloinjerto

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.

Autor destacado: Mejora de la investigación de trasplantes a través de la angiografía por microTC en modelos murinos 

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.

Trasplante hepático ortotópico exitoso en ratones mediante angiografía por microtomografía computarizada

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