Name: intrasplenic transplantation of hepatocytes after partial hepatectomy in nod.scid mice
Uploaded: 2018-02-10T13:00:00.000+00:00
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Description: Watch this Scientific Journal Video about Intrasplenic Transplantation of Hepatocytes After Partial Hepatectomy in NOD.SCID Mice at JoVE.com

Este trabalho foi financiado pela subvenção núcleo recebida do departamento de biotecnologia, governo da Índia para o Instituto Nacional de Imunologia, Nova Deli. Endereço atual do Dr. Bhattacharjee é a divisão de Gastroenterologia, Hepatologia e nutrição, Hospital Los Angeles das crianças.

Os procedimentos apresentados neste protocolo foram aprovados pelo Comitê de ética institucional Animal do Instituto Nacional de Imunologia, Nova Deli. O número de série de referência da aprovação é IAEC #319/13.
Nota: Existem excelentes recursos em procedimentos de cirurgia geral20 e protocolos específicos para a cirurgia do roedor21. Para aqueles que fazem a cirurgia animal pela primeira vez, é aconselhável extensivamente em prática procedimentos cirúrgicos em bonecos antes de operar em animais.
1. preparação
<ol><li>Antes do experimento, manter estéril tampão fosfato salino (PBS) ou salino pronto.</li>
	<li>Monte um kit de cirurgia contendo tesouras, pinças serrilhadas, fórceps do tecido, algodão, cotonetes, fios de nylon e porta agulhas micro diferentes. O kit de cirurgia de autoclave. Cuidado especial deve ser tomado se um aceno imunológico comprometido. Rato SCID é incluído no protocolo.</li>
	<li>Executar todo o procedimento experimental, desde a preparação para o fim da cirurgia, da bio-segurança classe I do armário.</li>
	<li>Pese um aceno. Rato SCID de 6-8 semanas antes da cirurgia. Ratos com peso entre 14-18 g são utilizados neste estudo.</li>
	<li>Barba superior central e hipocondríaco região abdominal do mouse com o aparador de cabelo. Aplica o cabelo removendo o creme uniformemente em toda a região com uma espátula para remover completamente os pelos aparados. Remova os pelos suavemente com a ajuda de um pedaço de algodão estéril após 2-5 min.</li>
	<li>Posicione o mouse na câmara de isoflurano e desbloquear a válvula do cilindro de oxigênio. Manter o fluxo de oxigênio à taxa de 4 L/min e a vaporização de isoflurano em 4% para induzir anestesia.<ol>
<li>Certifique-se de que o mouse tem sido anestesiado corretamente beliscando suave do dedo do pé.</li>
	</ol>
</li>
	<li>Coloque uma placa de cirurgia dentro uma biossegurança do armário. Coloque o animal no quadro de cirurgia, tal que a porção ventral do mouse é voltada para cima e a porção anterior do rato é colocada dentro do cone de nariz ligado à alimentação de isoflurano e oxigênio.</li>
	<li>Reduzir a vaporização de isoflurano para 2% e mantê-lo durante todo o procedimento cirúrgico.</li>
	<li>Desinfectar a pele do rato e esterilize-limpando com algodão estéril embebida de etanol a 70%.</li>
</ol>2. ato cirúrgico
<ol><li>
Hepatectomy parcial
	<ol>
<li>Fazer uma incisão transversa de cerca de 1 cm na pele logo abaixo do esterno, perpendicular ao processo xifoide e paralelo da caixa torácica, com a ajuda de uma tesoura reta de funcionamento.</li>
		<li>Separe suavemente a pele anexada com a camada muscular abdominal nas imediações da zona da incisão com pinça ou dicas de algodão umedecido estéril para distinguir entre a pele e a camada muscular abdominal. Molhe a região intradérmica com PBS usando dicas de algodão estéril para evitar a dessecação.</li>
		<li>Expor a área do lobo esquerdo suavemente, fazendo uma incisão transversal através da camada peritoneal logo abaixo do xifoide. Use duas dicas de algodão umedecido para expor e levantar o lobo esquerdo do fígado.</li>
		<li>Coloque uma das pontas do algodão do lado abdominal do corte e coloque a outra ponta de algodão do lado do diafragma. Delicadamente pressione a ponta colocada para o diafragma e dar um empurrão deslizando pela outra ponta para levantar o lobo esquerdo do fígado.</li>
		<li>Passar um fio de nylon com um loop através do lóbulo esquerdo levantado e deslize o loop em direção à base do lobo esquerdo perto do hilo, com a ajuda das dicas de microfórceps ou algodão. Empurre para baixo o loop de fio de nylon na base do lobo esquerdo.</li>
		<li>Amarre duas extremidades do fio de nylon por cima do lobo esquerdo usando um porta-agulha microcirurgia e o micro fórceps. Faça dois nós adicionais do outro lado.</li>
		<li>Disse para fora o lóbulo amarrado com a ajuda de uma tesoura. Não tente cortar muito perto o thread. No caso do procedimento dura mais de 5 min, mantenha a cavidade peritoneal e órgãos úmido com PBS estéril para evitar a desidratação devido à perda de líquidos.</li>
		<li>Costure o peritônio por sutura contínua usando uma sutura de Catgut 4-0. Subsequentemente, feche a pele por sutura descontínuos tão rapidamente quanto possível.</li>
	</ol>
</li>
	<li>
Transplante de células Nota: Os hepatócitos expressando GFP foram isolados de ratos transgénicos GFP (C57BL/6-Tg (UBC-GFP) 30 Scha/J), de acordo com o procedimento descrito por Lee et al. 22 e Shen et al. 23 hepatócitos células (NeoHep) de origem humana diferenciada de monócitos24 também foram utilizadas para transplante. No entanto, as células derivadas de outras fontes podem também ser utilizadas no protocolo.<ol>
<li>Suspenda a 0,2 milhões de células viáveis em cerca de 50 µ l de modificado Dulbecco do Iscove médio (IMDM) e Aspire-lo em uma seringa de insulina 1 mL com agulha 30 G. Não deixar a seringa, colocando-o no gelo.</li>
		<li>Coloque o mouse em uma forma que a parte lateral esquerda enfrenta até em direção a pessoa que realizar a cirurgia. Identificar a área esplênica e transversalmente dissecar a pele perto da região hipocondríaca, seguida por uma incisão pequena através da camada peritoneal apenas para expor o baço.</li>
		<li>Delicadamente Levante o baço e mantenha que fora da cavidade com a ajuda de dois PBS umedecido com pontas de algodão.</li>
		<li>Segure o baço cuidadosamente com duas pontas de algodão com uma mão e coloque a agulha da seringa exatamente vertical para o baço. Delicadamente, perfuram o baço e empurra a agulha muito lentamente por dentro; a agulha não deve ter mais que 2 mm.</li>
		<li>Empurre o êmbolo da seringa lentamente para injetar as células no baço. Após transplante, manter a agulha da seringa estável e retire lentamente o baço para evitar sangramento ou perda de células.</li>
		<li>Depois de colocar o baço volta para a cavidade peritoneal com as pontas de algodão, feche a camada peritoneal por sutura contínua com uma sutura de Catgut 4-0. Costure a pele descontinuamente com a sutura do mesma. Evite usar clipes de ferida para o fechamento da pele; em vez disso, feche-a uma sutura de 4-0. Os clipes de ferida restringem o movimento natural do mouse, e muitas vezes clipes afrouxar-se e sair rapidamente.</li>
	</ol>
</li>
</ol>3. no pós-operatório cuidados
<ol><li>Depois de fechar a pele, limpe os arredores de ambos os sites de sutura com solução de iodo (betadine) usando uma ponta de algodão estéril.</li>
	<li>Injetar uma dose de antibiótica cefotaxima como 600 mg/kg de peso (tipicamente 12 mg cefotaxima em 100 µ l de solução salina/rato) de corpo intraperitonealmente com uma seringa de 1 mL.</li>
	<li>Dar doses diárias de analgésico Meloxicam como 1 mg/kg peso corporal (tipicamente 12 µ g Meloxicam em 100 µ l de solução salina/rato) ao animal intraperitonealmente, por até três dias após a cirurgia.</li>
	<li>Após a conclusão da cirurgia, interromper o fluxo de gás de isoflurano e posicione o mouse de volta na gaiola ventilada individualmente.</li>
</ol>4. eutanásia e caracterizações
<ol><li>Após os pontos experimentais (1 dia e 10 dias pós cirurgia), eutanásia os ratos de acordo com as orientações institucionais de ética animal.</li>
	<li>Colete sangue por terminal sangria dos animais, puncionando o plexo terminal ocular.</li>
	<li>Isole o soro do sangue.</li>
</ol>

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Hepatectomy parcial é uma técnica estabelecida para investigar regeneração hepática e hepatectomy excessiva é relatado para imitar o modelo de AHF. Entre modelos animais de AHF, roedores, sobretudo ratos, são o modelo mais pesquisado. Para obter um modelo de lesão hepática em ratos, até um 70% hepatectomy tem sido relatado com uma taxa de sobrevivência bom25,26. No entanto em nude e outro rato imunodeficientes, um hepatectomy 70% foi relatado como fata...

Atualmente, transplante de hepatócitos é proposto como uma alternativa ao transplante de órgão inteiro para tratar pacientes com distúrbios hepáticos graves. Acredita-se que ele pode ponte pacientes de transplante de órgão inteiro1. Além dos hepatócitos alogênico2, hepatócitos xenogénica3 e hepatócitos derivados de células-tronco4 também estão sendo investigados em modelos animais. Neste contexto, a orientação e enxertia potencial das células transplantadas no receptor é um critério importante para a terapia de célula com base na insuficiência hepática aguda (AHF).
Para investigar o transplante de hepatócitos ou células hepatócito5, AHF é criada em um modelo animal por cirúrgica6 ou procedimentos farmacológicos7 , seguidos de transplante de células. Tornar-se um modelo animal de AHF por reagentes farmacológicos, hepatotoxinas muitos como d-galactosamina8, paracetamol9, tetracloreto de carbono10, tioacetamida11, Concanavalina A12, lipopolissacarídeo13 , etc., têm sido utilizados. Desta lista, cada reagente gera um conjunto único de características para AHF, mas infelizmente não reagente único imita as AHF humana. Além disso, a AHF induzida por hepatotoxinas leva um longo tempo, o que coloca os animais sob estresse crônico, e obter resultados reprodutíveis são difíceis de obter.
Por outro lado, o procedimento cirúrgico de hepatectomy parcial (PHx) é dependente de habilidade, e obter resultados reprodutíveis são fáceis de obter depois de desenvolver as habilidades necessárias. Para induzir AHF por intervenção cirúrgica sozinha, ressecção de mais de 70% do fígado é necessária; no entanto, menos do que um hepatectomy 70% ainda pode ser utilizado para estudar a enxertia e proliferação de transplantadas células no fígado para analisar sua capacidade terapêutica durante dano hepático14. O transplante de hepatócitos foram realizados post hepatectomy através do peritônio15, cauda veia16, veia hepática17ou o baço18. Atualmente, infusão de veia hepática e transplante intrasplenic dos hepatócitos são os procedimentos preferenciais, porque são mais fáceis de reproduzir.
Neste trabalho, descrevemos um procedimento para uma hepatectomy parcial de 30% na NOD. SCID (ASSENTIMENTO. CB17-Prkdcscid/J) ratos em que o lobo esquerdo do fígado é extirpado. Ele é seguido por transplante de hepatócitos de rato (C57BL/6-Tg (UBC-GFP) 30Scha/J) expressando GFP 0,2 milhões, bem como origem humana NeoHep19 no baço. Este procedimento leva a enxertia das células transplantadas no fígado. Este procedimento é menos invasivo e uma técnica minimamente 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

Hepatectomy parcial é um método versátil e pode ser reproduzido para estudar a regeneração hepática e o efeito da terapêutica de célula com base em várias condições patológicas. Hepatectomy parcial também facilita a enxertia aumentada e proliferação das células transplantadas por neovascularização acelerada e migração de células para o fígado. Aqui, descrevemos um protocolo simples para executar hepatectomy 30% e o transplante de células no baço de um não-obesos diabéticos/grave combinado NOD imunodeficientes. SCID (ASSENTIMENTO. CB17-Prkdcscid/J) rato.
Neste procedimento, são feitas duas pequenas incisões. A primeira incisão é expor e fazer a ressecção do lobo esquerdo do fígado, e outra pequena incisão para expor o baço para o transplante intrasplenic de células. Este procedimento não requer nenhuma habilidade cirúrgica especializada, e pode ser completado em 5-7 minutos com menos estresse e dor, recuperação mais rápida e melhor sobrevivência. Nós demonstramos que o transplante de hepatócitos isolados de uma proteína verde fluorescente (GFP), expressando o rato (transgénico C57BL/6-Tg (UBC-GFP) 30Scha/J), bem como hepatócito como células de origem humana (NeoHep) em parcialmente hepatectomized NOD. Ratos SCID.

Proliferação hepatocyte após hepatectomy parcial de 30%: A proliferação dos hepatócitos nos restantes fígada após 30% hepatectomy foi examinada por imuno-histoquímica (IHC) mancha para um marcador de proliferação celular, Ki-67. Post um dia hepatectomy, os ratos foram sacrificados, os lóbulos de fígado restantes foram extirpados e cortes de parafina foram obtidos. As seções foram coradas com anticorpo Ki-67, seguido de rotulagem com anticorpo secundário conjugado de horseradish peroxidase...

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

Descrevemos um protocolo para a realização de hepatectomy parcial (PHx) e transplante de células através do baço em NOD. SCID (ASSENTIMENTO. CB17-Prkdcscid/J) ratos. Neste protocolo, uma incisão é feita para expor e fazer a ressecção do lobo esquerdo do fígado seguido por outra incisão para o transplante intrasplenic de células.

Intrasplenic transplante de hepatócitos após Hepatectomy parcial na NOD. Ratos 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

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

13.9K visualizações.  National Institute of Immunology. Descrevemos um protocolo para a realização de hepatectomy parcial (PHx) e transplante de células através do baço em NOD. SCID (ASSENTIMENTO. CB17-Prkdcscid/J) ratos. Neste protocolo, uma incisão é feita para expor e fazer a ressecção do lobo esquerdo do fígado seguido por outra incisão para o transplante intrasplenic de células.

Vídeo: 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).

Um modelo de camundongo de câncer hepatocelular induzido por hepatócitos oncogênico, decorrente do ajuste da inflamação e fibrose hepática

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

Pesquisa do câncer

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.

Geração de subcutânea e intra-hepática Humanos carcinoma hepatocelular Xenoenxertos em Immunodeficient Ratos

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.

Hepatectomy parcial de Lobular: Um modelo cirúrgico para regeneração hepática morfológica

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

Biologia do Desenvolvimento

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.

Um modelo de Mouse quimérico fígado humano de hipercolesterolemia familiar usando hepatócitos de derivadas de células-tronco pluripotentes induzidas

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.

Geração de um humanizado rato Liver Usando células-tronco 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.

Estabelecimento do modelo humanizado duplo do rato de TK-NOG para a patogénese HIV-associada do fígado

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

Imunologia e Infecção

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.

Um modelo de camundongo de transplante de baço heterotópico vascularizado para estudar a biologia celular do baço e a imunidade a transplantes

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.

Minimizando o sangramento da veia do portal pós-infusão durante o transplante de ilhotas intrahápticas em camundongos

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.

Isolamento de Hepatócitos Regeneradores após Hepatectomia Parcial em Camundongos

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

Transplante de fígado em camundongo e análise da perviedade do aloenxerto

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 em destaque: Aprimorando a pesquisa de transplante por meio de angiografia MicroCT em 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.

Transplante Ortotópico de Fígado em Camundongos Utilizando Angiotomografia Microcomputadorizada

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