Bu eser Bölümü Biyoteknoloji, Hindistan hükümeti Ulusal Enstitüsü İmmünoloji, Yeni Delhi için alınan çekirdek grant tarafından desteklenmiştir. Dr. Bhattacharjee'nın şu anki adresim mı bölümü Gastroenteroloji, Hepatoloji ve beslenme, Çocuk Hastanesi Los Angeles.

Bu protokol için sunulan yordamları kurumsal hayvan etik komite tarafından Ulusal Enstitüsü İmmünoloji, Yeni Delhi onaylanmış olması. IAEC #319/13. onayı seri referans numarasıdır
Not: Genel cerrahi prosedürler20 ve kemirgen cerrahi21belirli iletişim kuralları üzerinde mükemmel kaynakları vardır. O hayvan ameliyatı: ilk kez, bu kapsamlı bir şekilde pratik cerrahi mankenleri üzerinde hayvanlar üzerinde çalıştırmadan önce tavsiye edilir.
1. hazırlık
<ol><li>Deney öncesinde, steril fosfat tamponlu tuz (PBS) veya serum fizyolojik hazır tutun.</li>
	<li>Makas, tırtıklı forseps, doku forseps, pamuk, pamuk tomurcukları, naylon konuları ve farklı mikro iğne tutucular içeren bir cerrahi seti bir araya getirin. Otoklav cerrahi seti. Özel bir bağışıklık şaibeli NOD Eğer özen gösterilmelidir. SCID fare iletişim kuralında dahildir.</li>
	<li>Deneysel yordamın tamamı, ameliyat sonu için hazırlık gerçekleştirmek, biyo güvenlik sınıf ı kabine.</li>
	<li>Bir NOD tartın. 6-8 hafta ameliyat öncesi eski SCID fare. Fareler arasında 14-18 g ağırlığındaki bu çalışmada kullanılır.</li>
	<li>Fare üst orta ve hastalık karın bölgesi ile saç düzeltici tıraş. Saç kremi bölgenin tümünde eşit bir spatula ile tamamen kesilmiş kıllar kaldırmak için kaldırma uygulanır. 2-5 dakika sonra bir parça steril ıslak pamuk yardımıyla hafifçe tüyleri.</li>
	<li>Fareyi isoflurane odasında getirin ve oksijen silindir kapak kilidini. Oksijen akışı 4 L/dk hızında ve anestezi ikna etmek için % 4 isoflurane buharlaşma korumak.<ol>
<li>Fare düzgün nazik parmak pinching tarafından anestezi ki emin olun.</li>
	</ol>
</li>
	<li>Bir ameliyat kartını bir biyogüvenlik içinde kabine yerleştirin. Hayvan cerrahi gemide, öyle ki fare ventral kısmı yukarı gelecek şekilde ve fare ön kısmını isoflurane ve oksijen kaynağına bağlı burun konisi içinde yerleştirilir yerleştirin.</li>
	<li>% 2 isoflurane buharlaşma azaltmak ve cerrahi işlem sürdürmek.</li>
	<li>Fare cilt dezenfekte ve steril % 70 etanol batırılmış pamuk ile silerek sterilize.</li>
</ol>2. cerrahi müdahale
<ol><li>
Kısmi Hepatectomy
	<ol>
<li>Xiphoid süreci ve göğüs kafesi, paralel düz işletim makas yardımıyla yaklaşık 1 cm enine bir kesi derinin hemen altında göğüs kemiği, dik olun.</li>
		<li>Yavaşça forseps veya cilt ve karın kas katmanı birbirinden ayırmak için steril ıslatılmış pamuk ipuçları ile kazıma alanı çevresinde karın kas tabaka ile bağlı cilt ayırın. İntradermal bölge kuruma kaçınmak için steril pamuklu ipuçları kullanma PBS ile ıslatın.</li>
		<li>Sol lob alan sorunsuz hemen altındaki xiphoid Periton katmanı yoluyla enine bir kesi yaparak ortaya çıkarmak. İki ıslatılmış pamuk ipuçları ortaya çıkarmak ve karaciğer sol lob kaldırmak için kullanın.</li>
		<li>Kesme karın tarafında pamuk uçlarından birini yerleştirin ve başka pamuk uç diyafram tarafında yer. Yavaşça doğru diyafram yerleştirilen bastırın ve karaciğer sol lob kaldırmak için sürgülü diğer ucunu tarafından ayır.</li>
		<li>Naylon iplik kaldırdı sol lob bir döngü ile kayma ve döngü hilus yakın sol lob Bankası mikro-forseps veya pamuk ipuçları yardımıyla doğru kaydırın. Yavaşça sol lob tabanına naylon iplik döngü itin.</li>
		<li>Mikrocerrahi iğne bağı ve mikro forseps kullanarak sol lob üst üzerinde naylon iplik iki ucu kravat. İki ek knot diğer tarafında yapmak.</li>
		<li>Bir makas yardımı ile bağlı LOB incelemek. Çok yakın iplik kesmeye çalışmayın. Yordamı daha fazla 5 dakika sürer, Periton boşluğu ve organları ile kuruma sıvı kaybı nedeniyle önlemek için steril PBS nemli tutun.</li>
		<li>Karın zarını tarafından sürekli dikiş kullanarak 4-0 akorduna dikiş dikmek. Daha sonra cilt tarafından sürekli dikiş kısa zamanda kapatın.</li>
	</ol>
</li>
	<li>
Hücre transplantasyonu Not: GFP ifade tetkikine transgenik GFP fareler izole edildi (C57BL/6-Tg (UBC-GFP) 30 Scha/J), Lee ve arktarafından açıklanan yordamı göre. 22 ve Shen ve ark. 23 Hepatosit benzeri hücreler (NeoHep) insan kaynaklı monosit24 ayrıştırılan Ayrıca ekimi için kullanılmıştır. Ancak, herhangi bir diğer kaynaklardan elde hücreleri de protokol kullanılabilir.<ol>
<li>0,2 milyon hücrelerin yaklaşık 50 µL Iscove'nın modifiye Dulbecco'nın orta (IMDM) içinde askıya alma ve 1 mL insülin şırınga 30 G iğne ile şapkalı içinde Aspire edin. Şırınga buza koyarak soğuk tutmak.</li>
		<li>Fare sol yanal bölümü ameliyatı gerçekleştiren kişi doğru yukarı dönük bir şekilde yerleştirin. Dalak alanını belirlemek ve kemiği cilt sadece dalak ortaya çıkarmak için Periton katmanı üzerinden kısa bir kesi ardından evhamlı bölge yakınındaki incelemek.</li>
		<li>Yavaşça dalak kaldırın ve dışarı kavite iki PBS yardımı ile ıslatılmış pamuk ipuçları tutun.</li>
		<li>Dalak dikkatle iki pamuk ipuçları ile bir elinde tutmak ve şırınga iğne tam olarak dalak için dikey yerleştirin. Yavaşça dalak işlemek ve iğne itmek çok yavaş içinde; İğneyi 2 mm'den daha derin almak gerekir.</li>
		<li>Şırınga piston yavaş yavaş hücreleri dalak enjekte için itin. Ekimi sonra iğne şırınga sabit tutmak ve yavaş yavaş kanama önlemek için dalak veya hücrelerinin kaybı kaldırma.</li>
		<li>Dalak pamuk ipuçları ile Periton boşluğu içine geri koyduktan sonra Periton tabakası sürekli ile 4-0 akorduna Dikiş dikiş tarafından kapatın. Cilt aralıklı ile aynı dikiş dikmek. Cilt kapanış için yara klip kullanmaktan kaçının; Bunun yerine, 4-0 dikiş tarafından kapatın. Yara klip fare doğal hareket kısıtlayabilir ve kez klipleri gevşek olmak ve hızlı bir şekilde ortaya çıkıyor.</li>
	</ol>
</li>
</ol>3. ameliyat sonrası bakım
<ol><li>Cilt kapattıktan sonra her iki dikiş site steril pamuklu ipucu kullanarak iyot solüsyonu (betadin) çevresi silin.</li>
	<li>600 mg/kg vücut ağırlığına (genellikle 100 µL serum/fare içinde 12 mg sefotaksim) intraperitoneally gibi bir doz antibiyotik sefotaksim enjekte 1 mL şırınga kullanarak.</li>
	<li>Analjezik Meloksikam günlük doz 1 mg/kg hayvanın vücut ağırlığı (genellikle 12 µg Meloksikam 100 µL serum/fare içinde) intraperitoneally, ameliyattan sonra üç güne kadar ver.</li>
	<li>Ameliyat tamamlanması sonra isoflurane gaz akışını durdurmak ve tek tek havalandırılmış kafesine döndü fareyi getirin.</li>
</ol>4. euthanization ve karakterizasyonu
<ol><li>Deneysel endpoints sonra (1 gün ve 10 gün sonrası cerrahi), kurumsal hayvan etik kurallarına göre fareler ötenazi.</li>
	<li>Kan ölümcül oküler terminal pleksus delinme hayvanlar, kanama tarafından toplamak.</li>
	<li>Serum kandan yalıtmak.</li>
</ol>

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Yazarlar ifşa gerek yok.

Kısmi hepatectomy soruşturma karaciğer rejenerasyonu için kurulan bir tekniktir ve aşırı hepatectomy AHF modeli taklit etmek için bildirilir. Hayvan AHF arasında kemirgenler, özellikle fareler, en araştırılmış modeli modellerdir. % 70 hepatectomy kadar farelerde karaciğer yaralanma modeli elde etmek için bir iyi hayatta kalma oranı25,26' bildirilmiştir. Ancak çıplak ve diğer immünyetmezligi fare, % 70 hepatectomy ölümcül olarak bildirilm...

Şu anda, Hepatosit bütün organ nakli şiddetli karaciğer bozukluğu olan hastaları tedavi için bir alternatif olarak teklif edilir. Bu hastaların tüm organ nakli1köprü olabilir inanılıyor. Ek olarak allojenik tetkikine2, xenogenic tetkikine3 ve kök hücre4 ' ten türetilmiş tetkikine hayvan modellerinde da soruşturuluyor. Bu bağlamda, posta ve engraftment potansiyel alıcı transplante hücrelerinin temel hücre tedavisi için önemli bir kriter akut hepatik yetmezliği (AHF) var.
Hepatositlerin veya Hepatosit benzeri hücreler5nakli soruşturma için AHF hayvan modelinde cerrahi6 veya farmakolojik7 yordamlar, hücre nakli tarafından takip tarafından oluşturulur. Bir AHF hayvan modeli farmakolojik reaktifler, d-galactozamin8, asetaminofen9, karbon tetraklorür10, thioacetamide11, Concanavalin A12, lipopolysaccharide13 gibi birçok hepatotoxins tarafından yapmak için , vs., kullanılmıştır. Bu listeden her reaktif AHF için benzersiz bir özellik kümesi oluşturur, ancak ne yazık ki yok tek reaktif insan AHF taklit eder. Ayrıca, hepatotoxins tarafından indüklenen AHF hayvanlar kronik stres altında koyar, uzun bir zaman alır ve tekrarlanabilir sonuçlar elde etmek zordur.
Öte yandan, kısmi hepatectomy (PHx) cerrahi işlem beceri bağımlı ve tekrarlanabilir sonuçlar gerekli becerileri geliştirme sonra elde etmek kolaydır. Cerrahi müdahale yalnız AHF ikna etmek için daha--dan %70 karaciğer rezeksiyonu gereklidir; Ancak, bir % 70 hepatectomy hala engraftment ve yaygın eğitim için yararlı olabilir daha az tedavi kapasitelerini karaciğer hasarı14sırasında analiz etmek için karaciğer hücrelerinde nakledilmektedir. Hepatositlerin nakli karın zarını15, kuyruk damar16, hepatik ven17veya18dalak aracılığıyla gerçekleştirilen yazı hepatectomy olmuştur. Yeniden oluşturmak daha kolay olduğu gibi şu anda, hepatik ven infüzyon ve tetkikine intrasplenic nakli tercih edilen, işlemlerdir.
Bu yazıda, bir yordam için % 30 kısmi hepatectomy düğümde anlatmıştık. SCID (BAŞINI SALLAMAK. CB17-Prkdcscid/J) içinde karaciğer sol lob eksize fareler. Bu nakli 0,2 milyon GFP ifade fare (C57BL/6-Tg (UBC-GFP) 30Scha/J) tetkikine hem de insan kaynaklı NeoHep19 dalak içinde izler. Bu yordamı karaciğer nakledilen hücrelerinin engraftment yol açar. Bu yordam az invaziv ve en az acı veren bir teknik olacaktır.

<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

Kısmi hepatectomy karaciğer rejenerasyonu ve değişik patolojik koşullarda hücre dayalı tedavi etkisini incelemek için çok yönlü ve tekrarlanabilir bir yöntemdir. Kısmi hepatectomy da artan engraftment ve Transplante hücrelerin çoğalması hızlanan neovaskülarizasyon ve hücre göç doğru karaciğer tarafından kolaylaştırır. Burada, bir obez olmayan şeker hastası/şiddetli, dalak performans % 30 hepatectomy ve hücre nakli kombine immünyetmezligi başını SALLAMAK için basit bir protokol açıklayın. SCID (BAŞINI SALLAMAK. CB17-Prkdcscid/J) fare.
Bu yordam, iki küçük insizyon yapılır. Belgili tanımlık ilk kesme ortaya çıkarmak ve karaciğer sol lob çıkarabileceğim etmektir ve başka bir küçük kesi dalak hücre intrasplenic nakli için ortaya çıkarmak için yapılır. Bu yordam herhangi bir özel cerrahi beceri gerektirmez ve daha az stres ve ağrı, hızlı iyileşme ve daha iyi hayatta kalma ile 5-7 dakika içinde tamamlanabilir. Fare (transgenik C57BL/6-Tg (UBC-GFP) 30Scha/J) yanı sıra Hepatosit hücreler kısmen hepatectomized başını SALLAMAK içinde insan kaynaklı (NeoHep) gibi ifade bir floresan yeşil protein (GFP) dan izole tetkikine nakli göstermiştir. SCID fareler.

% 30 kısmi hepatectomy sonra Hepatosit nükleer silahların yayılmasına karşı: Yayılması içinde kalan hepatositlerin % 30 hepatectomy sonra karaciğer Hücre proliferasyonu avans, Ki-67 boyama immunohistokimyasal (IHC) tarafından incelenmiştir. Bir gün sonrası hepatectomy, fareler ötenazi, kalan karaciğer lobları eksize ve parafin kesitler elde. Bölümleri ile Ki-67 antikor, horseradish peroksidaz (HRP) konjuge ikincil antikor ile etiketleme tarafından takip lekeli. 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

Kısmi hepatectomy (PHx) ve hücre transplantasyonu dalak ile NOD gerçekleştirmek için bir protokol anlatmıştık. SCID (BAŞINI SALLAMAK. CB17-Prkdcscid/J) fareler. Bu protokol için ortaya çıkarmak ve hücre intrasplenic nakli için başka bir kesi ardından karaciğer sol lob çıkarabileceğim için bir kesi yapılır.

Hepatositlerin intrasplenic nakli düğümde kısmi Hepatectomy sonra. SCID fareler

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 Görüntüleme.  National Institute of Immunology. Kısmi hepatectomy (PHx) ve hücre transplantasyonu dalak ile NOD gerçekleştirmek için bir protokol anlatmıştık. SCID (BAŞINI SALLAMAK. CB17-Prkdcscid/J) fareler. Bu protokol için ortaya çıkarmak ve hücre intrasplenic nakli için başka bir kesi ardından karaciğer sol lob çıkarabileceğim için bir kesi yapılır.

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).

Hepatik İnflamasyon ve Fibrozis Ortamında Ortaya Çıkan Hepatosellüler Kanserin Onkojenik Hepatositkaynaklı Ortotopik Fare Modeli

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

Kanser Araştırmaları

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.

Bağışıklık yetersizliği olan farelere subkutan olarak ve intrahepatik insan hepatoselüler karsinoma ksenograftlarının Üretimi

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

Tıp

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.

Kısmi Lobular Hepatectomy: Morfolojik karaciğer rejenerasyonu için cerrahi bir Model

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

Gelişim Biyolojisi

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.

İndüklenmiş Pluripotent kök hücre kaynaklı tetkikine kullanarak ailesel hiperkolesterolemi insan karaciğer Chimeric fare modeli

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.

İnsan Karaciğer Kök Hücreler kullanma Insanlaşmış Fare Karaciğer Üretimi

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.

HIV ile ilişkili Karaciğer Patogenezi için Çift İnsanize TK-NOG Fare Modelinin Oluşturulması

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

İmmünoloji ve Enfeksiyon

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.

Dalak hücre biyoloji ve nakli bağışıklık eğitimi için vascularized heterotopik dalak transplantasyonunun fare modeli

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.

Farelerde İntrahepatik Adacık Transplantasyonu Sırasında İnfüzyon Sonrası Portal Ven Kanamasının En Aza İndirgenmesi

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.

Farelerde Parsiyel Hepatektomi Sonrası Rejenere Hepatositlerin İzolasyonu

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

Farede Karaciğer Transplantasyonu ve Allogreft Açıklığının İncelenmesi

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.

Yazar Spotlight: Murin Modellerinde MikroCT Anjiyografi Yoluyla Transplantasyon Araştırmalarının Geliştirilmesi 

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.

Mikrobilgisayarlı Tomografi Anjiyografi Kullanan Farelerde Başarılı Ortotopik Karaciğer Nakli

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