Araştırma ve Retina Araştırma Vakfı Clayton Vakfı tarafından bu proje için finansman sağlanmıştır.

1. Retinoblastom tumorspheres hazırlanması <ol><li> TC Sağlık Bakanlığı onaylı protokol kapsamında retinoblastom tümör örnek alın. </li><li> Taze tanımlanmış kök hücre ortamı hazırlayın (Neurobasal-medya, 1X B-27 takviyesi eksi A vitamini, 1X elzem olmayan bir amino asit çözeltisi, 1X L-glutamin, 20 ng / ml EGF, 10 ng / ml bFGF). </li><li> Steril bir, dokuda bir çapraz kesim tekniği kullanarak bir neşter kültür-tedavi 6-plaka ile tümör dokusu mekanik parçalara ayırma. Hemen doku tanımlanmış kök hücre medya 100 mcL ekleyin ve hafifçe neşter kullanılarak kıyılmış doku yayıldı. Iyi tanımlanmış kök hücre medya 5 ml ekleyin. </li><li> 37 ° ° C&#39;de,% 5 ile nemlendirilmiş bir inkübatör CO 2 ve günlük olarak kontrol edin . Bazı tumorspheres 2-4 gün içerisinde giderek artan sayıda, hemen bozulur tümör dokusu ortaya çıkmalıdır. </li><li> Haftalık 300xg az 5 dakika santrifüj tumorspheres, taze hazırlanmış tanımlanmış kök hücre medyada tekrar süspansiyon ve pipetlemeyin yukarı ve aşağı 10 kat daha büyük, merkezi nekrotik küreler bozabilir ve yeni, sağlıklı bir küre oluşturacak şekilde izin vermek. </li></ol> 2. Tümör hücrelerinin enjeksiyon için hazırlanması <ol><li> Eğer 15-ml konik bir tüp içine yavaşça kültürlü tumorspheres, pipetlemeyin küreler enjekte. Serolojik pipetlemeyin küreler bozmaya 10-15 kez yavaşça pipetlemeyin yukarı ve aşağı. </li><li> Hücreleri saymak bir hemasitometre ve kısım enjeksiyon başına en az 50.000 hücreleri kullanarak yeni bir tüp içine enjekte edilir. Kullanılabilir hücre sayısı mümkün olduğunca çok sayıda enjeksiyonlar için hazırlayın. </li><li> 5 dakika 300xg santrifüjleyin. 5 ml PBS dikkatlice supernatant ve tekrar süspansiyon aspirat. Santrifüj ve aspirasyon tekrarlayın ve enjeksiyon başına 10 mcL PBS içinde tekrar süspansiyon haline getirin. </li></ol> 3. Hayvanların hazırlanması <ol><li> Bir insülin enjektörü kullanarak, kemirgen anestezi kokteyl intraperitoneal (ketamin 37.6 mg / ml, ksilizin 1.92 mg / ml, acepromazine 0.38 mg / ml) gram vücut ağırlığı başına 1 mcL (GBW). Yönetmek Sedasyon olmak için hayvan için 5 dakika bekleyin. Kalp atışı olup olmadığını kontrol edin ve hayvan ısıtılmış bir yastık yerleştirin. Kurtarma kadar her zaman ısıtılmış bir pad üzerinde hayvanları koruyun. </li><li> Sedasyon sonra, sağ göz proparacaine HCl 1 damla yönetmek. Isıtmalı pad üzerinde hayvan değiştirin ve 1 dakika bekleyin. </li><li> Sağ göz HCl fenilefrin 1 damla yönetin. Isıtılmış pad üzerinde hayvan değiştirin ve 5 dakika bekleyin. Pupilla dilatasyonu oluştu varsa, 1 damla yeniden yönetmek ve başka bir 5 dakika bekleyin. </li><li> &gt; 30 dakika sonrası sedasyon oluşabilir, hareket ya da seğirmesi işaretleri, hayvan izleyin. Hareketin ilk işaretleri, 1 mcL / GBW ketamin HCl (10 mg / ml PBS içinde seyreltilmiş) yönetmek ve 5 dakika bekleyin. </li></ol> 4. Tümör hücrelerinin enjeksiyonu <ol><li> Yukarı bakacak şekilde sağ göz ve baş gazlı bez üzerine oturtulmuş ve mikroskop ile gözlenen sağ göz fundus (öğrenci şu anda dilate edilmelidir) kırmızı bir refleks elde etmek için merkezli, kendi tarafında mikroskop altında anestezi hayvan koyun . </li><li> Hafifçe göz kapaklarınızın üzerinde iki parmak ile aynı anda aşağı iterek ve prosedürün geri kalanı için sürekli bu konumda tutun, sağ kürenin Prop. </li><li> Delmek pars plana alan kornea limbus bitişik konjonktiva ve sklera (Şekil 1) ve vitreus boşluğuna koroid yanal, Luer-lock şırınga bağlı steril bir dünya kullanarak 30-gauge iğne. Açılış iğne çıkarın. </li><li> Hamilton iğnesi bir alkollü bez ile sterilize edin. Hamilton enjektöre hücre süspansiyonu çizin ve iğne iğne merceğin arkasında retinanın yakınındaki vitreus mikroskop aracılığıyla görülünceye kadar ağzına takın. Bu pozisyonda sürekli iğne tutarken ikinci bir kişinin yardımı ile, piston yavaşça bastırın. İğneyi çıkarın. Gerekirse, açılışından itibaren herhangi bir sıvı absorbe etmek için bir pamuklu bir bez kullanın. </li><li> Fare göz kapaklarını kapatmak için göz kapaklarının basıncı yavaşça bırakın. </li><li> Her bir göze 1 damla Hipromellöz Yönetici ve kurtarma için ısıtmalı yastık hayvan dönmek. </li></ol> 5. Enjekte edilen farelerin tümörler hasadı ve İzlenmesi <ol><li> Günlük, genellikle 2-4 hafta enjeksiyon sonrası belirgin hale gelir enjekte leukocoria için göz (beyaz papiller refleks) ve / veya perioküler distansiyon inceleyin. </li><li> Tümör büyümesi hayvan kurumsal kurallarına göre, euthanize algılanır. </li><li> Göz ve tümör göz kapaklarının kaplı ise, palpebral fissür dik iki kesiler yapmak ve cilt flep kaldırma neşter kullanabilirsiniz. </li><li> Stereoskopik mikroskop kullanarak görüntüleme altında, kornea ve lens çıkarmadan, limbus dairesel kesi yapmak ve dikkatle twee kullanarak diğer dokularda tümör kitlesi toplu teşrihzers. Steril RPMI-1640 medya tümör yerleştirin. </li><li> Açık küre, yavaş yavaş prizden çekmek için cımbız kullanın. Ekli bir optik sinir tümör invazyonu görüntülenmiştir böylece sağlamak için, sinir maruz kadar dünyanın çekin ve mümkün olduğunca uzun süre sinir kesmek için makas kullanın. Histolojik muayene için düzenli olarak işlenmesi için% 10 formalin yerleştirin. </li></ol> 6. Temsilcisi Sonuçlar: Retinoblastom tumorspheres hemen tümör kitlesi kurtuldu olarak ayrıştırılmış doku görünmeye başlayacaktır. 2-4 gün içinde, daha tumorspheres formu ve boyutu artacak başlayacak. Küreler, düzenli ve agrega (Şekil 2) çevreleyen iyi tanımlanmış, ikincil membran sergilemek eğilimindedir. Hayvan genellikle dünya ve tümör büyüdükçe enjeksiyonu 5-8 hafta sonra çevre dokulara distansiyon genişleme, enjeksiyon sonrası (Şekil 3b) 4 hafta içinde leukocoria ile sunar (Şekil 3c). <img src="/files/ftp_upload/2644/2644fig1.jpg" alt="Şekil 1"/> Şekil 1 protokol başvurulan özellikleri vurgulayarak fare göz kesit diyagramı. <img src="/files/ftp_upload/2644/2644fig2.jpg" alt="Şekil 2"/> Şekil 2 a) 4x in vitro insan retinoblastom hücrelerinin Kültür görüntülü ve b) objektif 10x büyütme. Retinoblastom primer tümör hücrelerinin düzenli bir sfero şekil ve net bir dış zar ile tumorspheres üretir. Ölçek bar a) 500 mikron temsil eder, b) 200 mikron . <img src="/files/ftp_upload/2644/2644fig3.jpg" alt="Şekil 3"/> Şekil 3. Göz Rag2 - / -, b) normal özellikleri gösteren fare) vitreusa bir tümör kitlesi göstergesidir leukocoria, ve c) büyük bir tümör kitlesi dünya ilişkili perioküler distansiyon, göz içi kanama ile doldurma.

<ol>
	<li>Ch&#232;vez-Barrios, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metastatic+and+Nonmetastatic+Models+of+Retinoblastoma.">Metastatic and Nonmetastatic Models of Retinoblastoma.</a> Am. J. Pathol. 157, 1405-1412 (2000).</li><li>Suber, M. L., Hurwitz, M. Y., Ch&#232;vez-Barrios, P., Hurwitz, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+consequences+of+intraocular+administration+of+modified+adenoviral+vectors.">Immune consequences of intraocular administration of modified adenoviral vectors.</a> Hum. Gene Ther. 12, 833-838 (2001).</li><li>Mallam, J. N., Hurwitz, M. Y., Mahoney, T., Ch&#232;vez-Barrios, P., Hurwitz, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+Gene+Transfer+into+Retinal+Cells+Using+Adenoviral+Vectors:+Dependence+on+Receptor+Expression.">Efficient Gene Transfer into Retinal Cells Using Adenoviral Vectors: Dependence on Receptor Expression.</a> Invest. Ophthalmol. Vis. Sci. 45, 1680-1687 (2004).</li></ol>

Baylor Tıp IACUC Komitesi tarafından belirlenen kurallara ve yönetmeliklere uygun olarak hayvanlar üzerinde deneyler yapıldı.

Burada açıklanan teknik yayılımı retinoblastom tümörleri, göz içi, intravitreal ortamda kolaylaştırır. Göziçi enjeksiyon tekniği retinoblastom kaynaklı hücre hatları 1 yanı sıra göz içi gen tedavisi 2,3 için viral vektörler sunmak için tümörler oluşturmak için geçmişte kullanılan olmuştur. Bu teknik, birincil tümör ve enjeksiyon tumorspheres xenograft tümörlerinin yanı sıra seri yayılma hücrelerinin doğrudan enjeksiyon yoluyla insan retinoblastom tümör başarıyla üretmek için kullanılmıştır. Tümör oluşumu (genellikle leukocoria) Görünür bir kanıt genellikle ilk yörünge çevreleyen dünya ve / veya dokuların göz içi kanama, şişme sonra 5-8 hafta içinde gelişir, 4 hafta içinde olduğunu kaydetti. Enjekte edilen farelerin bir azınlık, göz kapaklarının sürekli kapatılması önde gelen enjekte göz yaralayabilir. Bu durumlarda, distansiyon tümör oluşumu tek bir işaretidir. Kurulan xenograft tümörlerin yayılması son derece başarılı olmasına rağmen fare xenograft tümörlerin kurulması, tüm insan retinoblastom tümörlerinin başarılı olmamıştır. Bu gözlem, invazivlik düzeyi ve farklılaşma ya da başka bir bilinmeyen faktör olarak primer tümörün bazı özellikleri, bu tümörlerin fare oküler ortamda devam yeteneğini etkileyen olabileceğini düşündürmektedir. Bir insan retinoblastom tümör elde edilebilir doku miktarı oldukça küçük ve sınırlı bir yaşam süresi, solid tümör histolojisi kaybı ve hücresel fenotip değişiklikler gibi in vitro kültür insan retinoblastom hücrelerinin yeteneği üzerinde önemli sınırlamalar vardır. Bu protokol, insan tümör yaymak ve insan hastalığının bir fare modeli kurmak için nispeten basit bir yol sağlar. Bu in vitro ve in vivo deneyler retinoblastom ve hastanın tümör dışında uzun bakım biyoloji daha da sağlar .

<table border="1"><tbody><tr><th> Isim </th><th> Şirket </th><th> Katalog # </th><th> Yorumlar </th></tr><tr><td> HCl 2.5% Fenilefrin </td><td> Bausch &amp; Lomb </td><td> 053-11 </td><td> Oftalmik solüsyon </td></tr><tr><td> 30-ga İğne </td><td> BD </td><td> 305128 </td><td> Düzenli konik </td></tr><tr><td> 10-ml Luer-Lock Şırınga </td><td> BD </td><td> 309604 </td><td></td></tr><tr><td> 3/10-cc İnsülin Şırınga </td><td> BD </td><td> 328431 </td><td></td></tr><tr><td> Alkol bezlerden </td><td> BD </td><td> 326895 </td><td></td></tr><tr><td> 6-Peki Tabak, Doku Kültürü Boyalı </td><td> BD </td><td> 353046 </td><td></td></tr><tr><td> Proparacaine HCl% 0.5 </td><td> Butler AHS </td><td> 017239 </td><td> Oftalmik solüsyon </td></tr><tr><td> Ketamin HCl 100mg/mL </td><td> Fort Dodge </td><td> 4402A </td><td></td></tr><tr><td> 10-mcL Hamilton Şırınga </td><td> Hamilton Şirket </td><td> 7648-01 </td><td></td></tr><tr><td> 32-ga Hamilton İğne </td><td> Hamilton Şirket </td><td> 7803-04 </td><td> Özel uzunluğu - 0.5 &quot; </td></tr><tr><td> Neurobasal-Ortam </td><td> Invitrogen </td><td> 10888-022 </td><td></td></tr><tr><td> B-27 Ek Eksi A vitamini, 50X </td><td> Invitrogen </td><td> 12587-010 </td><td></td></tr><tr><td> RPMI-1640 Medya </td><td> Mediatech </td><td> 10-040-CV </td><td></td></tr><tr><td> Sigara esansiyel amino asit Çözüm, 100X </td><td> Mediatech </td><td> 25-025-CI </td><td></td></tr><tr><td> L-Glutamine, 100X </td><td> Mediatech </td><td> 25-005-CI </td><td></td></tr><tr><td> Tek kullanımlık # 11 bistüri </td><td> Miltex </td><td> 4-411 </td><td></td></tr><tr><td> Kemirgen Anestezi Combo </td><td> n / a </td><td> n / a </td><td> In-house eczane formülasyonu (ketamin 37.6 mg / ml, ksilizin 1.92 mg / ml, acepromazine 0.38 mg / ml) </td></tr><tr><td> Rekombinant İnsan Epidermal Büyüme Faktörü (EGF) </td><td> STEMCELL Teknolojileri </td><td> 02633 </td><td> 10 mcg / mL stok çözelti az sulandırın </td></tr><tr><td> Rekombinant İnsan Basic Fibroblast Büyüme Faktörü (bFGF) </td><td> STEMCELL Teknolojileri </td><td> 02634 </td><td> 10 mcg / mL stok çözelti az sulandırın </td></tr><tr><td> OMS-75 Operasyonu Mikroskop </td><td> Topcon Medikal Sistemleri </td><td> OMS-75 </td><td> Bu model kesildikten </td></tr><tr><td> % 10 formalin </td><td> VWR </td><td> 95042-908 </td><td></td></tr></tbody></table>

establishment and propagation of human retinoblastoma tumors in immune deficient mice

Tanımlanmış kök hücre medya kültür retinoblastom tümör hücreleri yalnızca sınırlı bir süre için yetiştirilen ve korunabilir birincil tumorspheres yol açmaktadır. Bu kültür tumorspheres, orijinal tümörlere kıyasla, belirgin bir şekilde farklı hücre fenotipleri gösterebilir. Kültür hücreleri yeni tümörler oluşturma yeteneğine sahip olduğunu gösteri bu kültür hücreleri modeli orijinal tümör biyolojisi sağlamak için önemlidir. / - Bağışıklık eksikliği olan farelere Burada Rag2 kullanarak in vivo insan retinoblastom tümör propaganda için bir protokol mevcut. Kültüre insan retinoblastom tumorspheres düşük geçit veya fare gözlerde vitreus boşluğuna doğrudan enjekte taze hasat insan retinoblastom tümörler elde edilen hücreler, 2-4 hafta içinde tümörleri oluşturur. Bu tümörler hasat ve ya daha ileri in vivo fare gözlerinin içine geçişli ya da in vitro tumorspheres olarak yetiştirilen olabilir . Yayılma, böylece daha fazla deneyler için insan retinoblastom dokusu sürekli bir kaynak oluşturulması en az üç geçişleri başarıyla yapılmıştır. Wesley S. Bond ve Lalita Wadhwa co-ilk yazarın.

Watch this Scientific Journal Video about Establishment and Propagation of Human Retinoblastoma Tumors in Immune Deficient Mice at JoVE.com

Establishment and Propagation of Human Retinoblastoma Tumors in Immune Deficient Mice

Insan retinoblastom farelerde tümörlerin yaymak için bir yöntem açıklanmıştır. Tümör hücreleri bağışıklık eksikliği olan farelerde gözlerinin içine doğrudan enjekte edilir. İkincil tümörlerin başarıyla doğrudan insan tümörleri ve kültürlü tumorspheres hasat hücreler kullanılarak kurulmuştur.

Bağışıklık Eksik Fare Kuruluş ve İnsan Retinoblastom Tümörler yayılımı

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited ...

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Research

JoVE Journal

Medicine

11.7K Görüntüleme.  Baylor College of Medicine. Insan retinoblastom farelerde tümörlerin yaymak için bir yöntem açıklanmıştır. Tümör hücreleri bağışıklık eksikliği olan farelerde gözlerinin içine doğrudan enjekte edilir. İkincil tümörlerin başarıyla doğrudan insan tümörleri ve kültürlü tumorspheres hasat hücreler kullanılarak kurulmuştur.

Video: Establishment and Propagation of Human Retinoblastoma Tumors in Immune Deficient Mice

Flexible Colonoscopy in Mice to Evaluate the Severity of Colitis and Colorectal Tumors Using a Validated Endoscopic Scoring System

The use of modern endoscopy for research purposes has greatly facilitated our understanding of gastrointestinal pathologies. In particular, experimental endoscopy has been highly useful for studies that require repeated assessments in a single laboratory animal, such as those evaluating mechanisms of chronic inflammatory bowel disease and the progression of colorectal cancer. However, the methods used across studies are highly variable. At least three endoscopic scoring systems have been published for murine colitis and published protocols for the assessment of colorectal tumors fail to address the presence of concomitant colonic inflammation. This study develops and validates a reproducible endoscopic scoring system that integrates evaluation of both inflammation and tumors simultaneously. This novel scoring system has three major components: 1) assessment of the extent and severity of colorectal inflammation (based on perianal findings, transparency of the wall, mucosal bleeding, and focal lesions), 2) quantitative recording of tumor lesions (grid map and bar graph), and 3) numerical sorting of clinical cases by their pathological and research relevance based on decimal units with assigned categories of observed lesions and endoscopic complications (decimal identifiers). The video and manuscript presented herein were prepared, following IACUC-approved protocols, to allow investigators to score their own experimental mice using a well-validated and highly reproducible endoscopic methodology, with the system option to differentiate distal from proximal endoscopic colitis (D-PECS).

Over the past few years, new generation endoscopes have emerged as important diagnostic research aids for evaluating murine colitis and colorectal tumors. We present herein a detailed protocol for endoscopic assessment of inflammation and colorectal tumors in mice, as well as a novel scoring system that uses decimal identifiers to document the endoscopic severity of colitis and colorectal tumors.

Bir Geçerliliği Endoskopik Puanlama Sisteminin Kullanılması Kolit ve Kolorektal Tümörlerin Şiddetini Değerlendirmek için Farelerde esnek Kolonoskopi

The use of modern endoscopy for research purposes has greatly facilitated our understanding of gastrointestinal pathologies. In particular, experimental ...

Tıp

Induction of Invasive Transitional Cell Bladder Carcinoma in Immune Intact Human MUC1 Transgenic Mice:  A Model for Immunotherapy Development

A preclinical model of invasive bladder cancer was developed in human mucin 1 (MUC1) transgenic (MUC1.Tg) mice for the purpose of evaluating immunotherapy and/or cytotoxic chemotherapy. To induce bladder cancer, C57BL/6 mice (MUC1.Tg and wild type) were treated orally with the carcinogen N-butyl-N-(4-hydroxybutyl)nitrosamine (OH-BBN) at 3.0 mg/day, 5 days/week for 12 weeks. To assess the effects of OH-BBN on serum cytokine profile during tumor development, whole blood was collected via submandibular bleeds prior to treatment and every four weeks. In addition, a MUC1-targeted peptide vaccine and placebo were administered to groups of mice weekly for eight weeks. Multiplex fluorometric microbead immunoanalyses of serum cytokines during tumor development and following vaccination were performed. At termination, interferon gamma (IFN-&#947;)/interleukin-4 (IL-4) ELISpot analysis for MUC1 specific T-cell immune response and histopathological evaluations of tumor type and grade were performed. The results showed that: (1) the incidence of bladder cancer in both MUC1.Tg and wild type mice was 67%; (2) transitional cell carcinomas (TCC) developed at a 2:1 ratio compared to squamous cell carcinomas (SCC); (3) inflammatory cytokines increased with time during tumor development; and (4) administration of the peptide vaccine induces a Th1-polarized serum cytokine profile and a MUC1 specific T-cell response. All tumors in MUC1.Tg mice were positive for MUC1 expression, and half of all tumors in MUC1.Tg and wild type mice were invasive. In conclusion, using a team approach through the coordination of the efforts of pharmacologists, immunologists, pathologists and molecular biologists, we have developed an immune intact transgenic mouse model of bladder cancer that expresses hMUC1.

An N-butyl-N-(4-hydroxybutyl)nitrosamine-induced bladder cancer model was developed in human mucin 1 (MUC1) transgenic mice for the purpose of testing MUC1-directed immunotherapy. After administering a MUC1-targeted peptide vaccine, a cytotoxic T lymphocyte response to MUC1 was confirmed by measuring serum cytokine levels and T-cell specific activity.

Bağışıklık Sağlam İnsan MUC1 Transgenik Farelerde İnvaziv Geçici Hücre Mesane Karsinomu indüksiyon: immünoterapi Kalkınma İçin Bir Model

A preclinical model of invasive bladder cancer was developed in human mucin 1 (MUC1) transgenic (MUC1.Tg) mice for the purpose of evaluating immunotherapy ...

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent marker. Mouse models (brain tumor and arthritis) were used to evaluate the usefulness of this method. Saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles tagged with CellVue Maroon (CVM) fluorophore were administered intravenously. Animals were then placed in the rotational holder (MARS) of the in vivo imaging system. Images were acquired in 10&#176; steps over 380&#176;. A rectangular&#160;region of interest (ROI) was placed across the full image width at the model disease site. Within the ROI, and for every image, mean fluorescence intensity was computed after background subtraction. In the mouse models studied, the labeled nanovesicles were taken up in both the orthotopic and transgenic brain tumors, and in the arthritic sites (toes and ankles). Curve analysis of the multi angle image ROIs determined the angle with the highest signal. Thus, the optimal angle for imaging each disease site was characterized. The MAROI method applied to imaging of fluorescent compounds is a noninvasive, economical, and precise tool for in vivo quantitative analysis of the disease states in the described mouse models.

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo quantitation of a fluorescent marker delivered by saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles. Employing mouse models of cancer and arthritis, we demonstrate how the MAROI signal curve analysis can be used for the precise mapping and biological characterization of disease processes.

Floresan SapC-DOPS Nanovesicles kullanma Beyin Tümörleri ve Artrit Vivo Optik Görüntüleme

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent ...

Femoral Bone Marrow Aspiration in Live Mice

Serial sampling of the cellular composition of bone marrow (BM) is a routine procedure critical to clinical hematology. This protocol describes a detailed step-by-step technical procedure for an analogous procedure in live mice which allows for serial characterization of cells present in the BM. This procedure facilitates studies aimed to detect the presence of exogenously administered cells within the BM of mice as would be done in xenograft studies for instance. Moreover, this procedure allows for the retrieval and characterization of cells enriched in the BM such as hematopoietic stem and progenitor cells (HSPCs) without sacrifice of mice. Given that the cellular composition of peripheral blood is not necessarily reflective of proportions and types of stem and progenitor cells present in the marrow, procedures which provide access to this compartment without requiring termination of the mice are very helpful. The use of femoral bone marrow aspiration is illustrated here for cytological analysis of marrow cells, flow cytometric characterization of the hematopoietic stem/progenitor compartment, and culture of sorted HSPCs obtained by femoral BM aspiration compared with conventional marrow harvest.

This protocol describes a procedure for serial sampling of femoral bone marrow (BM) without requiring the sacrifice of mice. This procedure facilitates longitudinal studies of the BM composition of mice over time and provides serial access to cells within the BM for ex vivo and transplantation studies.

Canlı Farelerde Femoral Kemik İliği Aspirasyon

Serial sampling of the cellular composition of bone marrow (BM) is a routine procedure critical to clinical hematology. This protocol describes a detailed ...

Creating Anatomically Accurate and Reproducible Intracranial Xenografts of Human Brain Tumors

Orthotopic tumor models are currently the best way to study the characteristics of a tumor type, with and without intervention, in the context of a live animal &#8211;&#160;particularly in sites with unique physiological and architectural qualities such as the brain. In vitro and ectopic models cannot account for features such as vasculature, blood brain barrier, metabolism, drug delivery and toxicity, and a host of other relevant factors. Orthotopic models have their limitations too, but with proper technique tumor cells of interest can be accurately engrafted into tissue that most closely mimics conditions in the human brain. By employing methods that deliver precisely measured volumes to accurately defined locations at a consistent rate and pressure, mouse models of human brain tumors with predictable growth rates can be reproducibly created and are suitable for reliable analysis of various interventions. The protocol described here focuses on the technical details of designing and preparing for an intracranial injection, performing the surgery, and ensuring successful and reproducible tumor growth and provides starting points for a variety of conditions that can be customized for a range of different brain tumor models.

The brain is a unique site with qualities that are not well represented by in vitro or ectopic analyses. Orthotopic mouse models with reproducible location and growth characteristics can be reliably created with intracranial injections using a stereotaxic fixation instrument and a low pressure syringe pump.

Oluşturma Anatomik Doğru ve İnsan Beyin Tümörleri Reproducible İntrakraniyal Ksenograftlar

Orthotopic tumor models are currently the best way to study the characteristics of a tumor type, with and without intervention, in the context of a live ...

Establishment of Human Epithelial Enteroids and Colonoids from Whole Tissue and Biopsy

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem cells (ISCs) and progeny that progressively migrate and differentiate toward the tip of the villi. These processes, essential for gastrointestinal homeostasis, have been extensively studied using multiple approaches. Ex vivo technologies, especially primary cell cultures have proven to be promising for understanding intestinal epithelial functions. A long-term primary culture system for mouse intestinal crypts has been established to generate 3-dimensional epithelial organoids. These epithelial structures contain crypt- and villus-like domains reminiscent of normal gut epithelium. Commonly, termed &#8220;enteroids&#8221; when derived from small intestine and &#8220;colonoids&#8221; when derived from colon, they are different from organoids that also contain mesenchyme tissue. Additionally, these enteroids/colonoids continuously produce all cell types found normally within the intestinal epithelium. This in vitro organ-like culture system is rapidly becoming the new gold standard for investigation of intestinal stem cell biology and epithelial cell physiology. This technology has been recently transferred to the study of human gut. The establishment of human derived epithelial enteroids and colonoids from small intestine and colon has been possible through the utilization of specific culture media that allow their growth and maintenance over time. Here, we describe a method to establish a small intestinal and colon crypt-derived system from human whole tissue or biopsies. We emphasize the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

We describe a method to establish human enteroids from small intestinal crypts and colonoids from colon crypts collected from both surgical tissue and biopsies. In this methodological article, we present the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

Tüm Doku ve Biyopsi İnsan Epitelyal Enteroids ve Colonoids kurulması

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem ...

Establishment of a Human Multiple Myeloma Xenograft Model in the Chicken to Study Tumor Growth, Invasion and Angiogenesis

Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the lack of the bone microenvironment and auto/paracrine growth factors human MM cells are difficult to cultivate. Therefore, there is an urgent need to establish proper in vitro and in vivo culture systems to study the action of novel therapeutics on human MM cells. Here we present a model to grow human multiple myeloma cells in a complex 3D environment in vitro and in vivo. MM cell lines OPM-2 and RPMI-8226 were transfected to express the transgene GFP and were cultivated in the presence of human mesenchymal cells and collagen type-I matrix as three-dimensional spheroids. In addition, spheroids were grafted on the chorioallantoic membrane (CAM) of chicken embryos and tumor growth was monitored by stereo fluorescence microscopy. Both models allow the study of novel therapeutic drugs in a complex 3D environment and the quantification of the tumor cell mass after homogenization of grafts in a transgene-specific GFP-ELISA. Moreover, angiogenic responses of the host and invasion of tumor cells into the subjacent host tissue can be monitored daily by a stereo microscope and analyzed by immunohistochemical staining against human tumor cells (Ki-67, CD138, Vimentin) or host mural cells covering blood vessels (desmin/ASMA).
In conclusion, the onplant system allows studying MM cell growth and angiogenesis in a complex 3D environment and enables screening for novel therapeutic compounds targeting survival and proliferation of MM cells.

Human multiple myeloma (MM) cells require the supportive microenvironment of mesenchymal cells and extracellular matrix components for survival and proliferation. We established an in vivo chicken embryo model with engrafted human myeloma and mesenchymal cells to study effects of cancer drugs on tumor growth, invasion and angiogenesis.

Chicken İnsan Multipl Miyelom Xenograftlarında Modeli Kurulması Tümör Büyüme, Invasion ve Anjiogenez Eğitim için

Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the ...

Induction of Accelerated Atherosclerosis in Mice: The "Wire-Injury" Model

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. Moreover, by rupture of the defective vascular wall, atherosclerosis induces occlusive thrombus formation, which represents the main cause of myocardial infarction or stroke and the most frequent cause of death. Despite the advances in the cardiovascular field, many questions remain unanswered, and additional basic research is essential to improve our understanding of the molecular mechanisms during atherosclerosis and its effects. Due to limited clinical studies, there is a need for representative animal models recreating atherosclerotic conditions such as neointima formation after stent implantation, balloon angioplasty, or endarterectomy. Since the mouse presents many advantages and is the most frequently used model for studying molecular processes, the current study proposes an invasive procedure of endothelial denudation, also known as the wire-injury model, which is representative of the human condition of neointima formation in arteries after revascularization procedures.

Induction of Accelerated Atherosclerosis in Mice: The &quot;Wire-Injury&quot; Model

This study describes an invasive procedure for the induction of accelerated atherosclerosis in mice. In comparison to other methods using electric- or cryo-induced injury, mechanical-induced injury mimics the human condition of restenosis after revascularization therapies and is ideal for the study of the molecular mechanisms involved.

Farelerde Hızlandırılmış Ateroskleroz Indüksiyonu: "Tel-Yaralanma" Modeli

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. ...

Characterization of Immune Cells in Human Adipose Tissue by Using Flow Cytometry

Infiltration of immune cells in the subcutaneous and visceral adipose tissue (AT) deposits leads to a low-grade inflammation contributing to the development of obesity-associated complications such as type 2 diabetes. To quantitatively and qualitatively investigate the immune cell subsets in human AT deposits, we have developed a flow cytometry approach. The stromal vascular fraction (SVF), containing the immune cells, is isolated from subcutaneous and visceral AT biopsies by collagenase digestion. Adipocytes are removed after centrifugation. The SVF cells are stained for multiple membrane-bound markers selected to differentiate between immune cell subsets and analyzed using flow cytometry. As a result of this approach, pro- and anti-inflammatory macrophage subsets, dendritic cells (DCs), B-cells, CD4+ and CD8+ T-cells, and NK cells can be detected and quantified. This method gives detailed information about immune cells in AT and the amount of each specific subset. Since there are numerous fluorescent antibodies available, our flow cytometry approach can be adjusted to measure various other cellular and intracellular markers of interest.

This article describes a method to analyze immune cell content of adipose tissue by isolation of immune cells from adipose tissue and subsequent analysis using flow cytometry.

Akış Sitometresi kullanarak insan yağ dokusu içinde bağışıklık hücreleri karakterizasyonu

Infiltration of immune cells in the subcutaneous and visceral adipose tissue (AT) deposits leads to a low-grade inflammation contributing to the ...

Induction of Atherosclerotic Plaques Through Activation of Mineralocorticoid Receptors in Apolipoprotein E-deficient Mice

Atherosclerosis is due to a chronic inflammatory response affecting vascular endothelium and is promoted by several factors such as hypertension, dyslipidemia, and diabetes. To date, there is evidence to support a role for circulating aldosterone as a risk factor for the development of cardiovascular disease. Transgenic mouse models have been generated to study cellular and molecular processes leading to atherosclerosis. In this manuscript, we describe a protocol that takes advantage of continuous infusion of aldosterone in ApoE-/- mice and generates atherosclerotic plaques in the aortic root after 4 weeks of treatment. We, therefore, illustrate a method for quantification and characterization of atherosclerotic lesions at the aortic root level. The added value of aldosterone infusion is represented by the generation of atherosclerotic lesions rich in lipid and inflammatory cells after 4 weeks of treatment. We describe in detail the staining procedures to quantify lipid and macrophage content within the plaque. Notably, in this protocol, we perform heart tissue-embedding in OCT in order to preserve the antigenicity of cardiac tissue and facilitate detectability of antigens of interest. Analysis of the plaque phenotype represents a valid approach to study the pathophysiology of atherosclerosis development and to identify novel pharmacological targets for the development of anti-atherogenic drugs.

We describe a protocol to induce atherosclerosis in the aortic root of ApoE-/- mice fed with an atherogenic diet, through a continuous release of aldosterone. Methods to characterize plaque composition are also described.