The work was supported by grants from the Universit&#233; catholique de Louvain (Action de recherch&#233; concert&#233;es) and the Fund for Scientific Medical Research (F.R.S.-FNRS, Belgium). A.-S.D. is a doctoral fellow from T&#233;l&#233;vie, A.-C.H. held a fellowship from the Fonds pour la formation &#224; la Recherche dans l'Industrie et dans l'Agriculture (Belgium), M.V. is supported by the de Duve Institute and C.E.P. is a senior research associate of the F.R.S.-FNRS (Belgium).

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The authors have nothing to disclose.

وتصف هذه الورقة طريقة لتشريح وزراعة إإكسبلنتس الغدة الدرقية (E12.5) أو فصوص (E14.5) من أجل دراسة وفهم أفضل للأحداث المعقدة التي تفضي إلى تشكيل الغدة الدرقية. ويرجع ذلك إلى الأصل المزدوج للANLAGEN الغدة الدرقية (بدأة خط الوسط واثنين من الهيئات التالية للخيشومية الجانبية)، وصغر...

الغدة الدرقية هي عبارة عن مجموعة من المجالات الظهارية مستقلة، ودعا بصيلات، وتحيط بها شبكة كثيفة من الشعيرات الدموية البطانية. هذه المنظمة تسمح وظيفة الغدة الدرقية: توفير الشعيرات الدموية البطانية thyrocytes مع اليود، اللازمة لT3 و T4 هرمون التوليف، وتوزيع هذه الأخيرة إلى الجسم كله. منتشرة في بين بصيلات والشعيرات الدموية، C خلايا إنتاج هرمون الكالسيتونين نقص كلس الدم 1. على الرغم من أن العمارة الغدة الدرقية الكبار وظائف معروفة، الآليات الخلوية والجزيئية المشاركة في التطور الجنيني الغدة الدرقية (تشكيل المسام والتمايز) بعيدة كل البعد عن أن يكون مفهوما. خلال مرحلة التطور الجنيني، thyrocytes السلف كما تنبع سماكة (بدأة خط الوسط) من الجدار البطني من الأديم الباطن المعى الأمامي في اليوم الجنينية (ه) 8.5 في جنين الفأر، في حين C خلايا الأسلاف تنشأ في E11.5 كما نتوءات على شكل قطرات ( بو التالية للخيشوميةيموت) من الحقائب البلعوم الرابع 2-6. برعم خط الوسط ثم يفصل من الأديم الباطن، ويوسع ثنائيا لتندمج في E13.5 مع الهيئات التالية للخيشومية على كل جانب من القصبة الهوائية. أخيرا، thyrocytes تنظيم في بصيلات وخلايا C-التفريق. المعرفة الحالية على تشكيل الغدة الدرقية يأتي أساسا من التحليل النسيجي لأنسجة ثابتة، ولكن الأحداث التخلق المشاركة في تشكيل الغدة الدرقية هي ديناميكية للغاية وتنطوي على الاتصالات والتفاعلات بين أنواع مختلفة من الخلايا ومع المصفوفة خارج الخلية. وأظهرت الأعمال الأخيرة التي الأسلاف thyrocyte تنتج مستويات عالية من VEGF لتجنيد الخلايا البطانية إلى الغدة الدرقية النامية، وبدوره بتجنيد الخلايا البطانية تشجيع تشكيل بصيلات وخلايا C التمايز 7. وقد أجريت معظم الدراسات المجراة سابقا على الغدة الدرقية على الخلايا المشتقة من الغدة الدرقية الكبار معزولة، ونمت إما على 2D البلاستيك زراعة الأنسجة ديتضاءل المجموع المذكور أعلاه أو في المصفوفات 3D. باستخدام هذه الأنواع من الثقافات، والخلايا الجريبي متباينة إما البقاء الاستقطاب وتنظيمها في بصيلات أو إعادة الاستحواذ على منظمة 3D 8-10. ومع ذلك، وهذه الخلايا الظهارية نقية، explanted من بيئتهم الفسيولوجية، والمثقف في 2D، تجاهل التفاعل مع المصفوفة خارج الخلية، السيتوكينات وعوامل النمو ومع أنواع الخلايا الأخرى مثل الخلايا البطانية أو الأعصاب التي يواجهونها عادة في الجسم الحي. وصفت دراسة لطيفة جدا مؤخرا بروتوكول تمايز الخلايا ES في بصيلات الغدة الدرقية باستخدام الخطوة النهائية في الثقافة 3D matrigel 11. ومع ذلك، هذه الثقافات 3D تفتقر اتصال مع أنواع الخلايا الأخرى. بناء على الخبرة السابقة على البنكرياس والغدد اللعابية ثقافة الجهاز 12-14، طريقة لتشريح الفأر الجنينية ANLAGEN الغدة الدرقية وزراعة لل explants على مرشحات semiporous أو على شرائح البلاستيك المجهر وقد وضعت. عندماالعمل مع الأجنة E12.5، والأصل المزدوج للANLAGEN الغدة الدرقية (بدأة خط الوسط واثنين من الهيئات التالية للخيشومية الجانبي) فرضت تسليخ مجهري من جزء كبير من الأنسجة. يرد هذا القصبة الهوائية، ولكن ليس المريء، وامتدت من الشرايين البلعوم القوس حتى تورم الطرجهالي. عند تربيتها على مرشحات، وبدأة خط الوسط يمتد أفقيا على كل جانب من القصبة الهوائية، حيث تلتحم مع الهيئات التالية للخيشومية لتشكيل اثنين من فصوص الغدة الدرقية، لا تزال متصلة بواسطة برزخ ضيق. في الثقافة، وتتكاثر الخلايا الظهارية، وتنظيم في بصيلات والتمايز إلى خلايا thyrocytes وC، اعتمادا على أصلهم. الخلايا البطانية الواردة في الأنسجة microdissected أيضا تتكاثر وتغزو فصوص الغدة الدرقية لربط أخيرا بشكل وثيق مع هيكل مسامي الظهارية، بصرف النظر عن تدفق الدم أو عوامل المنتشرة. كما تطور لل explants يلخص بأمانة في الجسم الحي تطويرمنة، وهذا النظام هو الأمثل الثقافة لدراسة التخلق والتمايز الأحداث التي تحدث أثناء نمو الغدة الدرقية. ويمكن الحصول على أنسجة الغدة الدرقية من النوع البري، بالضربة القاضية أو الأجنة وراثيا الفلورسنت، ونظام الثقافة هي قابلة للخسارة والربح من وظيفة التجارب. أخيرا، يمكن استغلال الوقت الفاصل بين التصوير من إإكسبلنتس الغدة الدرقية fluorescently المسمى على أطباق البلاستيك المجهري للتحقيق أفضل حركية واستمرارية حركات التخلق التي تحدث في الجسم الحي. وقد تم بالفعل استخدام الوقت الفاصل بين التصوير لدراسة التشكل المتفرعة من البنكرياس 15،16 أو الحالبي برعم 17.

<table><tbody><tr><td>&amp;micro;-slide 8 well, ibiTreat, tissue culture treated, sterile</td><td>proxylab</td><td>80826</td><td></td></tr><tr><td>Collagen Type I, rat tail</td><td>Millipore</td><td>08-115</td><td></td></tr><tr><td>Fibronectin from human plasma</td><td>Invitrogen</td><td># 33010-018</td><td></td></tr><tr><td>M199</td><td>Invitrogen</td><td>31150-022</td><td></td></tr><tr><td>HBSS</td><td>Invitrogen</td><td>14025-100</td><td></td></tr><tr><td>Tungsten wire</td><td>Goodfellow</td><td>LS237450</td><td></td></tr><tr><td>culture plate insert (12 mm diameter)</td><td>Millipore</td><td>PICM01250</td><td></td></tr><tr><td>GlycoBlue</td><td>Invitrogen</td><td>AM9516</td><td></td></tr><tr><td>E-cadherin</td><td>BD Biosciences</td><td>610182</td><td>1/1,000</td></tr><tr><td>Ezrin</td><td>Thermo Scientific</td><td>MS-661-P1</td><td>1/400</td></tr><tr><td>PECAM</td><td>BD Biosciences</td><td>550274</td><td>1/100</td></tr><tr><td>Hoechst</td><td>Sigma</td><td>B2261</td><td></td></tr></tbody></table>

an ex vivo culture system to study thyroid development

الغدة الدرقية هي غدة الصماء bilobated المترجمة في قاعدة العنق، وتنتج هرمونات الغدة الدرقية T3، T4، والكالسيتونين. ويتم إنتاج T3 و T4 من قبل thyrocytes المتباينة، التي نظمت في مجالات مغلقة تسمى المسام، في حين يتم تصنيعه بواسطة C الكالسيتونين الخلايا، ويتخلل بين بصيلات وشبكة كثيفة من الشعيرات الدموية. على الرغم من أن العمارة الغدة الدرقية الكبار وظائف وقد وصفت على نطاق واسع، ودرس، وتشكيل وحدات &quot;الوعاء الجريبي&quot;، وتوزيع خلايا C-في لحمة والاتصالات نظير الصماوي بين الظهارية والخلايا البطانية بعيد كل البعد عن أن يكون مفهوما. يصف هذا الأسلوب في خطوات متتابعة من الماوس الغدة الدرقية الجنينية ANLAGEN تشريح وثقافتها على مرشحات semiporous أو على شرائح البلاستيك المجهري. في غضون أربعة أيام، وهذا النظام الثقافة يلخص بأمانة في التنمية الغدة الدرقية الجسم الحي. في الواقع، (ط) مليارobation من الجهاز يحدث (لإإكسبلنتس E12.5)، (ب) تنظيم thyrocytes السلائف في بصيلات واستقطاب، (ج) C وthyrocytes الخلايا التفريق، و (iv) الخلايا البطانية موجودة في الأنسجة تتكاثر microdissected، تهاجر إلى فصوص الغدة الدرقية، وبشكل وثيق مع ربط الخلايا الظهارية، كما يفعلون في الجسم الحي. ويمكن الحصول على أنسجة الغدة الدرقية من النوع البري، بالضربة القاضية أو الأجنة وراثيا الفلورسنت. علاوة على ذلك، إإكسبلنتس الثقافة يمكن التلاعب بها عن طريق إضافة مثبطات، ومنع الأجسام المضادة، عوامل النمو، أو حتى الخلايا أو المتوسطة مكيفة. يمكن تحليلها خارج الحي التنمية في الوقت الحقيقي، أو في أي وقت من الثقافة التي كتبها المناعية وRT-QPCR. في الختام، والثقافة ازدراع الغدة الدرقية جنبا إلى جنب مع المصب كامل جبل أو على توفر أقسام التصوير والتعبير الجيني التنميط نظام قوي لمعالجة ودراسة التخلق والتمايز أحداث س الغدة الدرقيةrganogenesis.

يتم تشريح anlages الغدة الدرقية (خط الوسط وUB، جنبا إلى جنب مع الأنسجة المحيطة بها)، وفصوص الغدة الدرقية من أجنة الفئران في E12.5 وe13.5/e14.5، على التوالي (الشكل 1). بعد يوم واحد في الثقافة على مرشح، وبدأة خط الوسط مرئيا (الشكل 2A) باعتبارها نسيج ممدود تمتد على الج...

Watch this Scientific Journal Video about An Ex vivo Culture System to Study Thyroid Development at JoVE.com

An Ex vivo Culture System to Study Thyroid Development

This protocol describes dissection of mouse embryonic thyroid anlagen and the culture of explants on semiporous filters or on microscopy plastic slides. This system is ideal to study morphogenetic or differentiation events occurring during thyroid development of wild type or knockout embryos, and is amenable to gain- and loss-of-function experiments.

و<em&gt; خارج الحي</em&gt; نظام الثقافة لدراسة التنمية الغدة الدرقية

The thyroid is a bilobated endocrine gland localized at the base of the neck, producing the thyroid hormones T3, T4, and calcitonin. T3 and T4 are ...

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An Ex vivo Culture System to Study Thyroid Development

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11.5K Views.  Université catholique de Louvain & de Duve Institute. This protocol describes dissection of mouse embryonic thyroid anlagen and the culture of explants on semiporous filters or on microscopy plastic slides. This system is ideal to study morphogenetic or differentiation events occurring during thyroid development of wild type or knockout embryos, and is amenable to gain- and loss-of-function experiments.

Video: An Ex vivo Culture System to Study Thyroid Development

3D Organotypic Co-culture Model Supporting Medullary Thymic Epithelial Cell Proliferation, Differentiation and Promiscuous Gene Expression

Intra-thymic T cell development requires an intricate three-dimensional meshwork composed of various stromal cells, i.e., non-T cells. Thymocytes traverse this scaffold in a highly coordinated temporal and spatial order while sequentially passing obligatory check points, i.e., T cell lineage commitment, followed by T cell receptor repertoire generation and selection prior to their export into the periphery. The two major resident cell types forming this scaffold are cortical (cTECs) and medullary thymic epithelial cells (mTECs). A key feature of mTECs is the so-called promiscuous expression of numerous tissue-restricted antigens. These tissue-restricted antigens are presented to immature thymocytes directly or indirectly by mTECs or thymic dendritic cells, respectively resulting in self-tolerance.
Suitable in vitro models emulating the developmental pathways and functions of cTECs and mTECs are currently lacking. This lack of adequate experimental models has for instance hampered the analysis of promiscuous gene expression, which is still poorly understood at the cellular and molecular level. We adapted a 3D organotypic co-culture model to culture ex vivo isolated mTECs. This model was originally devised to cultivate keratinocytes in such a way as to generate a skin equivalent in vitro. The 3D model preserved key functional features of mTEC biology: (i) proliferation and terminal differentiation of CD80lo, Aire-negative into CD80hi, Aire-positive mTECs, (ii) responsiveness to RANKL, and (iii) sustained expression of FoxN1, Aire and tissue-restricted genes in CD80hi mTECs.

Studying medullary thymic epithelial cells in vitro has been largely unsuccessful, as current 2D culture systems do not mimic the in vivo scenario. The 3D culture system described herein - a modified skin organotypic culture model - has proven superior in recapitulating mTEC proliferation, differentiation and maintenance of promiscuous gene expression.

3D عضوي النمط المشارك الثقافة نموذج دعم النخاع التوتة الظهارية تكاثر الخلايا، التمايز ومختلط التعبير الجيني

Intra-thymic T cell development requires an intricate three-dimensional meshwork composed of various stromal cells, i.e., non-T cells. Thymocytes traverse ...

Developmental Biology

Using Ex Vivo Upright Droplet Cultures of Whole Fetal Organs to Study Developmental Processes during Mouse Organogenesis

Investigating organogenesis in utero is a technically challenging process in placental mammals due to inaccessibility of reagents to embryos that develop within the uterus. A newly developed ex vivo upright droplet culture method provides an attractive alternative to studies performed in utero. The ex vivo droplet culture provides the ability to examine and manipulate cellular interactions and diverse signaling pathways through use of various blocking and activating compounds; additionally, the effects of various pharmacological reagents on the development of specific organs can be studied without unwanted side effects of systemic drug delivery in utero. As compared to other in vitro systems, the droplet culture not only allows for the ability to study three-dimensional morphogenesis and cell-cell interactions, which cannot be reproduced in mammalian cell lines, but also requires significantly less reagents than other ex vivo and in vitro protocols. This paper demonstrates proper mouse fetal organ dissection and upright droplet culture techniques, followed by whole organ immunofluorescence to demonstrate the effectiveness of the method. The ex vivo droplet culture method allows the formation of organ architecture comparable to what is observed in vivo and can be utilized to study otherwise difficult-to-study processes due to embryonic lethality in in vivo models. As a model application system, a small-molecule inhibitor will be utilized to probe the role of vascularization in testicular morphogenesis. This ex vivo droplet culture method is expandable to other fetal organ systems, such as lung and potentially others, although each organ must be extensively studied to determine any organ-specific modifications to the protocol. This organ culture system provides flexibility in experimentation with fetal organs, and results obtained using this technique will help researchers gain insights into fetal development.

Using Ex Vivo Upright Droplet Cultures of Whole Fetal Organs to Study Developmental Processes during Mouse Organogenesis

The&#160;ex vivo&#160;upright droplet culture is an alternative to current&#160;in vitro&#160;and&#160;in vivo&#160;experimental techniques. This protocol is easy to perform and requires smaller amounts of reagent, while permitting the ability to manipulate and study fetal vascularization, morphogenesis, and organogenesis.

باستخدام<em&gt; السابقين فيفو</em&gt; الثقافات تستقيم القطرة من الجامعة الجنين هيئات لدراسة العمليات التنموية خلال الماوس توالد

Investigating organogenesis in utero is a technically challenging process in placental mammals due to inaccessibility of reagents to embryos that develop ...

Preparation of 2-dGuo-Treated Thymus Organ Cultures

In the thymus, interactions between developing T-cell precursors and stromal cells that include cortical and medullary epithelial cells are known to play a key role in the development of a functionally competent T-cell pool. However, the complexity of T-cell development in the thymus in vivo can limit analysis of individual cellular components and particular stages of development. In vitro culture systems provide a readily accessible means to study multiple complex cellular processes. Thymus organ culture systems represent a widely used approach to study intrathymic development of T-cells under defined conditions in vitro. Here we describe a system in which mouse embryonic thymus lobes can be depleted of endogenous haemopoeitic elements by prior organ culture in 2-deoxyguanosine, a compound that is selectively toxic to haemopoeitic cells. As well as providing a readily accessible source of thymic stromal cells to investigate the role of thymic microenvironments in the development and selection of T-cells, this technique also underpins further experimental approaches that include the reconstitution of alymphoid thymus lobes in vitro with defined haemopoietic elements, the transplantation of alymphoid thymuses into recipient mice, and the formation of reaggregate thymus organ cultures. (This article is based on work first reported Methods in Molecular Biology 2007, Vol. 380 pages 185-196).

This video demonstrates the dissection and removal of the fetal thymus as well the preparation of ex vivo cultures of 2-dGuo-treated thymus.

إعداد 2 - dGuo المعاملة الثقافات الجهاز الغدة الصعترية

In the thymus, interactions between developing T-cell precursors and stromal cells that include cortical and medullary epithelial cells are known to play ...

An Orthotopic Mouse Model of Anaplastic Thyroid Carcinoma

Several types of animal models of human thyroid carcinomas have been established, including subcutaneous xenograft and orthotopic implantation of cancer cells into immunodeficient mice. Subcutaneous xenograft models have been valuable for preclinical screening and evaluation of new therapeutic treatments. There are a number of advantages to using a subcutaneous model; 1) rapid, 2) reproducible, and 3) tumor establishment, growth, and response to therapeutic agents may be monitored by visual inspection. However, substantial evidence has shed light on the short-comings of subcutaneous xenograft models1-3. For instance, medicinal treatments demonstrating curative properties in subcutaneous xenograft models often have no notable impact on the human disease. The microenvironment of the site of xenographic transplantation or injection lies at the heart of this dissimilarity.
Orthotopic tumor xenograft models provide a more biologically relevant context in which to study the disease. The advantages of implanting diseased cells or tissue into their anatomical origin equivalent within a host animal includes a suitable site for tumor-host interactions, development of disease-related metastases and the ability to examine site-specific influence on investigational therapeutic remedies. Therefore, orthotopic xenograft models harbor far more clinical value because they closely reproduce human disease. For these reasons, a number of groups have taken advantage of an orthotopic thyroid cancer model as a research tool4-7.
Here, we describe an approach that establishes an orthotopic model for the study of anaplastic thyroid carcinoma (ATC), which is highly invasive, resists treatment, and is virtually fatal in all diagnosed patients. Cultured ATC cells are prepared as a dissociated cellular suspension in a solution containing a basement membrane matrix. A small volume is slowly injected into the right thyroid gland. Overall appearance and health of the mice are monitored to ensure minimal post-operative complications and to gauge pathological penetrance of the cancer. Mice are sacrificed at 4 weeks, and tissue is collected for histological analysis. Animals may be taken at later time-points to examine more advance progression of the disease. Production of this orthotopic mouse model establishes a platform that accomplishes two objectives: 1) further our understanding of ATC pathology, and 2) screen current and future therapeutic agents for efficacy in combating ATC.

Generation of an orthotopic mouse model of anaplastic thyroid carcinoma is described here. This technique employs surgical placement of human anaplastic thyroid cancer cells into the thyroid of immunodeficient mice, thus creating a more clinically relevant setting to study disease progression as well as screen innovative therapeutic interventions.

ماوس نموذج مثلي من سرطان الغدة الدرقية كشمي

Several types of animal models of human thyroid carcinomas have been established, including subcutaneous xenograft and orthotopic implantation of cancer ...

Medicine

Two-step Approach to Explore Early- and Late-stages of Organ Formation in the Avian Model: The Thymus and Parathyroid Glands Organogenesis Paradigm

The avian embryo, as an experimental model, has been of utmost importance for seminal discoveries in developmental biology. Among several approaches, the formation of quail-chicken chimeras and the use of the chorioallantoic membrane (CAM) to sustain the development of ectopic tissues date back to the last century. Nowadays, the combination of these classical techniques with recent in vitro methodologies offers novel prospects to further explore organ formation.
Here we describe a two-step approach to study early- and late-stages of organogenesis. Briefly, the embryonic region containing the presumptive territory of the organ is isolated from quail embryos and grown in vitro in an organotypic system (up to 48 h). Cultured tissues are subsequently grafted onto the CAM of a chicken embryo. After 10 days of in ovo development, fully formed organs are obtained from grafted tissues. This method also allows the modulation of signaling pathways by the regular administration of pharmacological agents and tissue genetic manipulation throughout in vitro and in ovo developmental steps. Additionally, developing tissues can be collected at any time-window to analyze their gene-expression profile (using quantitative PCR (qPCR), microarrays, etc.) and morphology (assessed with conventional histology and immunochemistry).
The described experimental procedure can be used as a tool to follow organ formation outside the avian embryo, from the early stages of organogenesis to fully formed and functional organs.

This article provides an updated approach to the classical quail-chicken chimera system to study organ formation, by combining novel in vitro and in ovo experimental procedures.

نهج خطوتين لاستكشاف ومتأخرة-المراحل الأولى من تشكيل الهيئة في نموذج إنفلونزا الطيور: الغدة الصعترية ونموذج Organogenesis الغدد الجار درقية

The avian embryo, as an experimental model, has been of utmost importance for seminal discoveries in developmental biology. Among several approaches, the ...

High-Resolution Ultrasonography for the Analysis of Orthotopic ATC Tumors in a Genetically Engineered Mouse Model

Anaplastic thyroid carcinoma (ATC) is associated with a poor prognosis and short median survival time, but no effective treatment improves the outcomes significantly. Genetically engineered murine models that mimic ATC&#39;s progression may help researchers to study treatments for this disease. Crossing three different genotypes of mice, a TPO-cre/ERT2; BrafCA/wt; Trp53&#916;ex2-10/&#916;ex2-10&#160;transgenic ATC model was developed. The ATC murine model was induced by an intraperitoneal injection of tamoxifen with overexpression of BrafV600E and deletion of Trp53, and the tumors were generated within about 1 month. High-resolution ultrasound was applied to investigate the tumor initiation and progression, and the dynamic growth curve was obtained by measuring the tumor sizes. Compared to magnetic resonance imaging (MRI) and computed tomography scanning, ultrasound has advantages in observing the ATC murine model, such as being noninvasive, portable, in real-time, and without radiation exposure. High-resolution ultrasound is suitable for dynamic and multiple measurements. However, ultrasonographic examination of the thyroid in mice requires relevant anatomical knowledge and experience. This article provides a detailed procedure for utilizing high-resolution ultrasound to scan tumors in the transgenic ATC model. Meanwhile, ultrasonic parameter adjustment, ultrasound scanning skills, anesthesia and recovery of the animals, and other elements that need attention during the process are listed.

The present protocol describes high-frequency ultrasonography for visualizing the entire mouse thyroid gland and monitoring the growth of anaplastic thyroid carcinoma.

التصوير بالموجات فوق الصوتية عالية الدقة لتحليل أورام ATC التقويمية في نموذج فأر مهندس وراثيا

Anaplastic thyroid carcinoma (ATC) is associated with a poor prognosis and short median survival time, but no effective treatment improves the outcomes ...

Cancer Research

Preparation and Applications of Organotypic Thymic Slice Cultures

Thymic selection proceeds in a unique and highly organized thymic microenvironment resulting in the generation of a functional, self-tolerant T cell repertoire. In vitro models to study T lineage commitment and development have provided valuable insights into this process. However, these systems lack the complete three-dimensional thymic milieu necessary for T cell development and, therefore, are incomplete approximations of in vivo thymic selection. Some of the challenges related to modeling T cell development can be overcome by using in situ models that provide an intact thymic microenvironment that fully supports thymic selection of developing T cells. Thymic slice organotypic cultures complement existing in situ techniques. Thymic slices preserve the integrity of the thymic cortical and medullary regions and provide a platform to study development of overlaid thymocytes of a defined developmental stage or of endogenous T cells within a mature thymic microenvironment. Given the ability to generate ~20 slices per mouse, thymic slices present a unique advantage in terms of scalability for high throughput experiments. Further, the relative ease in generating thymic slices and potential to overlay different thymic subsets or other cell populations from diverse genetic backgrounds enhances the versatility of this method. Here we describe a protocol for the preparation of thymic slices, isolation and overlay of thymocytes, and dissociation of thymic slices for flow cytometric analysis. This system can also be adapted to study non-conventional T cell development as well as visualize thymocyte migration, thymocyte-stromal cell interactions, and TCR signals associated with thymic selection by two-photon microscopy.

We describe the preparation of thymic slices that, in combination with flow cytometry, can be used to model positive and negative selection of developing T cells. Thymic slices can also be adapted for the in situ analysis of thymocyte migration, localization, and signaling via immunofluorescence and two-photon microscopy.

إعداد وتطبيقات الثقافات عضوي التوتة شريحة

Thymic selection proceeds in a unique and highly organized thymic microenvironment resulting in the generation of a functional, self-tolerant T cell ...

Immunology and Infection

A System for ex vivo Culturing of Embryonic Pancreas

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels1. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation2. A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.
 During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing &beta;-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.
 Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.

A System for ex vivo Culturing of Embryonic Pancreas

Here, we describe a method for isolation, culture and manipulation of mouse embryonic pancreas. This represents an excellent ex vivo system for studying various aspects of pancreatic development, including morphogenesis, differentiation and growth. Pancreatic bud explants can be cultured for several days and used in a range of different applications, including whole-mount immunofluorescence and live imaging.

نظام ل<em&gt; خارج الجسم</em&gt; التثقيف البنكرياس الجنينية

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

Generation of a Mouse Spontaneous Autoimmune Thyroiditis Model

In recent years, Hashimoto's thyroiditis (HT) has become the most common autoimmune thyroid disease. It is characterized by lymphocyte infiltration and the detection of specific serum autoantibodies. Although the potential mechanism is still not clear, the risk of Hashimoto's thyroiditis is related to genetic and environmental factors. At present, there are several types of models of autoimmune thyroiditis, including experimental autoimmune thyroiditis (EAT) and spontaneous autoimmune thyroiditis (SAT).
EAT in mice is a common model for HT, which is immunized with lipopolysaccharide (LPS) combined with thyroglobulin (Tg) or supplemented with complete Freund's adjuvant (CFA). The EAT mouse model is widely established in many types of mice. However, the disease progression is more likely associated with the Tg antibody response, which may vary in different experiments.
SAT is also widely used in the study of HT in the NOD.H-2h4 mouse. The NOD.H2h4 mouse is a new strain obtained from the cross of the nonobese diabetic (NOD) mouse with the B10.A(4R), which is significantly induced for HT with or without feeding iodine. During the induction, the NOD.H-2h4 mouse has a high level of TgAb accompanied by lymphocyte infiltration in the thyroid follicular tissue. However, for this type of mouse model, there are few studies to comprehensively evaluate the pathological process during the induction of iodine.
A SAT mouse model for HT research is established in this study, and the pathologic changing process is evaluated after a long period of iodine induction. Through this model, researchers can better understand the pathological development of HT and screen new treatment methods for HT.

Several types of animal models of Hashimoto's thyroiditis have been established, as has spontaneous autoimmune thyroiditis in the NOD mouse. H-2h4 mice are a simple and reliable model for HT induction. This article describes this approach and evaluates the pathological process for a better understanding of the SAT murine model.

جيل من الفأر نموذج التهاب الغدة الدرقية المناعي الذاتي التلقائي

In recent years, Hashimoto's thyroiditis (HT) has become the most common autoimmune thyroid disease. It is characterized by lymphocyte infiltration and ...

تصوير الفئران مؤشر عمل هرمون الغدة الدرقية لتقييم اضطراب الغدد الصماء

In vivo Characterization of Endocrine Disrupting Chemical Effects via Thyroid Hormone Action Indicator Mouse

Thyroid hormones (TH) play a critical role in cell metabolism and tissue function. TH economy is susceptible to endocrine disrupting chemicals (EDCs) that can disturb hormone production or action. Many environmental pollutants are EDCs, representing an emerging threat to both human health and agricultural production. This has led to an increased demand for proper test systems to examine the effects of potential EDCs. However, current methodologies face challenges. Most test systems use endogenous markers regulated by multiple, often complex regulatory processes, making it difficult to distinguish direct and indirect effects. Moreover, in vitro test systems lack the physiological complexity of EDC metabolism and pharmacokinetics in mammals. Additionally, exposure to environmental EDCs usually involves a mixture of multiple compounds, including in vivo generated metabolites, so the possibility of interactions cannot be ignored. This complexity makes EDC characterization difficult. The Thyroid Hormone Action Indicator (THAI) mouse is a transgenic model that carries a TH-responsive luciferase reporter system, enabling the assessment of tissue-specific TH action. One can evaluate the tissue-specific effects of chemicals on local TH action by quantifying luciferase reporter expression in tissue samples. Furthermore, with in vivo imaging, the THAI mouse model allows for longitudinal studies on the effects of potential EDCs in live animals. This approach provides a powerful tool for testing long-term exposure, complex treatment structures, or withdrawal, as it enables the assessment of changes in local TH action over time in the same animal. This report describes the process of in vivo imaging measurements on THAI mice. The protocol discussed here focuses on developing and imaging hyper- and hypothyroid mice, which can serve as controls. Researchers can adapt or expand the treatments presented to meet their specific needs, offering a foundational approach for further investigation.

تسليط الضوء على المؤلف: في الجسم الحي تقييم اضطراب هرمون الغدة الدرقية باستخدام نموذج الماوس التايلاندي

The Thyroid Hormone Action Indicator mouse model was developed to enable tissue-specific quantification of local thyroid hormone action using its endogenous regulatory machinery. Recently, it has been shown that the model is suitable for characterizing endocrine-disrupting chemicals interacting with thyroid hormone economy, both by ex vivo and in vivo methodologies.

في الجسم الحي توصيف الآثار الكيميائية لتعطيل الغدد الصماء عن طريق هرمون الغدة الدرقية مؤشر عمل الماوس

Thyroid hormones (TH) play a critical role in cell metabolism and tissue function. TH economy is susceptible to endocrine disrupting chemicals (EDCs) that ...

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