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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M., Campbell, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Local+factors+regulating+growth+and+function+of+human+thyroid+cells+in+vitro+and+in+vivo.">Local factors regulating growth and function of human thyroid cells in vitro and in vivo.</a> 213, 47-58 (2003).</li><li>Antonica, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+functional+thyroid+from+embryonic+stem+cells.">Generation of functional thyroid from embryonic stem cells.</a> Nature. 491, 66-71 (2012).</li><li>van Eyll, J. M., Pierreux, C. E., Lemaigre, F. P., Rousseau, G. 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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.

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		<tr height="21">
			<td height="21" style="height:21px;width:327px;">&#181;-slide 8 well, ibiTreat, tissue culture treated, sterile</td>
			<td style="width:85px;">proxylab</td>
			<td style="width:119px;">80826</td>
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Collagen Type I, rat tail</td>
			<td style="width:85px;">Millipore</td>
			<td>08-115</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fibronectin from human plasma</td>
			<td style="width:85px;">Invitrogen</td>
			<td># 33010-018</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">M199</td>
			<td style="width:85px;">Invitrogen</td>
			<td>31150-022</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">HBSS</td>
			<td style="width:85px;">Invitrogen</td>
			<td>14025-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Tungsten wire</td>
			<td style="width:85px;">Goodfellow</td>
			<td style="width:119px;">LS237450</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">culture plate insert (12 mm diameter)</td>
			<td style="width:85px;">Millipore</td>
			<td>PICM01250</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:327px;">GlycoBlue</td>
			<td style="width:85px;">Invitrogen</td>
			<td style="width:119px;">AM9516</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:327px;">E-cadherin</td>
			<td style="width:85px;">BD Biosciences</td>
			<td style="width:119px;">610182</td>
			<td>1/1000</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:327px;">Ezrin</td>
			<td style="width:85px;">Thermo Scientific</td>
			<td style="width:119px;">MS-661-P1</td>
			<td>1/400</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:327px;">PECAM</td>
			<td style="width:85px;">BD Biosciences</td>
			<td style="width:119px;">550274</td>
			<td>1/100</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Hoechst</td>
			<td style="width:85px;">Sigma</td>
			<td style="width:119px;">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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11.4K 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

Method for Culture of Early Chick Embryos ex vivo (New Culture)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  the formation and patterning of brain, neural tube, somite and heart primordia. Applications of chick embryo culture include electroporation of DNA or RNA constructs in order to analyze gene function, grafts of growth factor coated beads such as FGFs and BMPs , as well as whole mount in situ hybridization and immunohistochemistry. This video demonstrates the different steps in chick embryo culture; First, the embryo is explanted in saline. Then, the embryo is centered on a glass ring. The membranes surrounding the embryo are lifted along the walls of the ring. The ring is then placed in a culture dish containing a pool of albumine. The culture dish is sealed and placed in a humid chamber, where the embryo is cultured for up to 24 hrs. Finally, the embryo is removed from the ring, fixed and processed for further applications. A troubleshooting guide is also presented.

Method for Culture of Early Chick Embryos ex vivo New Culture

This video demonstrates New culture, a method by which chick embryos are cultured outside the egg for up to 24 hr. This method enables one to study early development (primitive streak to 14 som.), a period corresponding to E7-9 in mouse. Applications of this technique include electroporation, in situ hybridization and immunohistochemistry.

طريقة لثقافة الفرخ المبكر فيفو السابقين الأجنة (الثقافة الجديدة)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications

Understanding the relationships between genetic and microenvironmental factors that drive normal and malformed embryonic development is fundamental for discovering new therapeutic strategies. Advancements in imaging technology have enabled quantitative investigation of the organization and maturing of the body plan, but later stage embryonic morphogenesis is less clear. Chicken embryos are an attractive vertebrate animal model system for this application because of its ease of culture and surgical manipulation. Early embryos can be cultured for a short time on filter paper rings, which enables complete optical access for cell patterning and fate studies1,2. Studying advanced developmental processes such as cardiac morphogenesis are traditionally performed through a window of the eggshell3-5, but this technique limits optical access due to window size. We previously developed a simple method to culture whole embryos ex-ovo on hexagonal weigh boats for up to 10 days, which enabled high resolution imaging via ultrasonography6,7. These cultures were difficult to transport, limiting the types of imaging tools available for live experiments. We here present an improved shell-less culture system with a cost-effective, portable environmental chamber. Eggs were cracked onto a hammock created by a polyurethane membrane (cling wrap) affixed circumferentially to a plastic cup partially filled with sterile water. The dimensions of the circumference and depth of the hammock were both critical to maintain surface tension, while the mechanics of the hammock and water beneath helped dampen vibrations induced by transportation. A small footprint circulating water bath was also developed to enable continuous temperature control during experimentation. We demonstrate the ability to culture embryos in this way for at least 14 days without morphogenic defect or delay and employ this system in several microsurgical and imaging applications.

An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications

In this article, we present a simple methodology to enable long-term ex-ovo avian embryo culture. This technique is ideal for longitudinal experimentation requiring complete optical accessibility and/or sterile transportation in avian embryos.

و السابقين البيضة الثقافة الأجنة النظام الدجاج مناسبة للتطبيقات التصوير والجراحة المجهرية

Understanding the relationships between genetic and microenvironmental factors that drive normal and malformed embryonic development is fundamental for ...

Closed System Cell Culture Protocol Using HYPERStack Vessels with Gas Permeable Material Technology

Large volume adherent cell culture is currently standardized on stacked plate cell growth products when microcarrier beads are not an optimal choice. HYPERStack vessels allow closed system scale up from the current stacked plate products and delivers >2.5X more cells in the same volumetric footprint. The HYPERStack vessels function via gas permeable material which allows gas exchange to occur, therefore eliminating the need for internal headspace within a vessel. The elimination of headspace allows the compartment where cell growth occurs to be minimized to reduce space, allowing more layers of cell growth surface area within the same volumetric footprint.
For many applications such as cell therapy or vaccine production, a closed system is required for cell growth and harvesting. The HYPERStack vessel allows cell and reagent addition and removal via tubing from media bags or other methods.
This protocol will explain the technology behind the gas permeable material used in the HYPERStack vessels, gas diffusion results to meet the metabolic needs of cells, closed system cell growth protocols, and various harvesting methods.

An Introduction into the technology, protocol and handling of the Corning HYPERStack Vessels and accessories used for high yield adherent cell culture. The protocol will show how to use the closed system vessels for increasing cell harvesting over current stacked plate products.

مغلق الثقافة خلية بروتوكول النظام باستخدام السفن HYPERStack مع تقنية الغاز مادة نافذ

Large volume adherent cell culture is currently standardized on stacked plate cell growth products when microcarrier beads are not an optimal choice.   ...

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

Epithelial ovarian cancer is a leading cause of female cancer mortality in the United States. In contrast to other women-specific cancers, like breast and uterine carcinomas, where death rates have fallen in recent years, ovarian cancer cure rates have remained relatively unchanged over the past two decades 1. This is largely due to the lack of appropriate screening tools for detection of early stage disease where surgery and chemotherapy are most effective 2, 3. As a result, most patients present with advanced stage disease and diffuse abdominal involvement. This is further complicated by the fact that ovarian cancer is a heterogeneous disease with multiple histologic subtypes 4, 5. Serous ovarian carcinoma (SOC) is the most common and aggressive subtype and the form most often associated with mutations in the BRCA genes. Current experimental models in this field involve the use of cancer cell lines and mouse models to better understand the initiating genetic events and pathogenesis of disease 6, 7. Recently, the fallopian tube has emerged as a novel site for the origin of SOC, with the fallopian tube (FT) secretory epithelial cell (FTSEC) as the proposed cell of origin 8, 9. There are currently no cell lines or culture systems available to study the FT epithelium or the FTSEC. Here we describe a novel ex vivo culture system where primary human FT epithelial cells are cultured in a manner that preserves their architecture, polarity, immunophenotype, and response to physiologic and genotoxic stressors. This ex vivo model provides a useful tool for the study of SOC, allowing a better understanding of how tumors can arise from this tissue, and the mechanisms involved in tumor initiation and progression.

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

The fallopian tube (FT) is emerging as an alternative site of origin for serous ovarian carcinoma (SOC). This protocol describes a novel method for the isolation and ex vivo culture of fallopian tube epithelial cells. This system recapitulates the in vivo epithelium and allows the study of SOC pathogenesis.

الثقافة السابقين فيفو من خلايا الإنسان الأولية قناة فالوب طلائي

Epithelial ovarian cancer is a leading cause of female cancer mortality in the United States. In contrast to other women-specific cancers, like breast and ...

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 ...

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. Thus, protocols to isolate and culture individual organs of interest are essential to provide a method of both visualizing changes in development and allowing novel treatment strategies. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel. This substrate provides enough support to maintain the three-dimensional structure but is flexible enough to allow continued contraction. In brief, hearts are excised from the embryo and placed in a mixture of cold Matrigel diluted 1:1 with growth medium. After the diluted Matrigel solidifies, growth medium is added to the culture dish. Hearts excised as late as embryonic day 16.5 were viable for four days post-dissection. Analysis of the coronary plexus shows that this method does not disrupt coronary vascular development. Thus, we present a novel method for long-term culture of embryonic hearts.

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel.

رواية<em&gt; خارج الحي</em&gt; الطريقة الثقافة لالجنينية القلب

Developmental studies in the mouse are hampered  by the inaccessibility of the embryo during gestation. Thus, protocols to  isolate and culture individual ...

Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

Most morphogenetic processes in the fetal intestine have been inferred from thin sections of fixed tissues, providing snapshots of changes over developmental stages. Three-dimensional information from thin serial sections can be challenging to interpret because of the difficulty of reconstructing serial sections perfectly and maintaining proper orientation of the tissue over serial sections. Recent findings by Grosse et al., 2011 highlight the importance of three- dimensional information in understanding morphogenesis of the developing villi of the intestine1. Three-dimensional reconstruction of singly labeled intestinal cells demonstrated that the majority of the intestinal epithelial cells contact both the apical and basal surfaces. Furthermore, three-dimensional reconstruction of the actin cytoskeleton at the apical surface of the epithelium demonstrated that the intestinal lumen is continuous and that secondary lumens are an artifact of sectioning. Those two points, along with the demonstration of interkinetic nuclear migration in the intestinal epithelium, defined the developing intestinal epithelium as a pseudostratified epithelium and not stratified as previously thought1. The ability to observe the epithelium three-dimensionally was seminal to demonstrating this point and redefining epithelial morphogenesis in the fetal intestine. With the evolution of multi-photon imaging technology and three-dimensional reconstruction software, the ability to visualize intact, developing organs is rapidly improving. Two-photon excitation allows less damaging penetration deeper into tissues with high resolution. Two-photon imaging and 3D reconstruction of the whole fetal mouse intestines in Walton et al., 2012 helped to define the pattern of villus outgrowth2. Here we describe a whole organ culture system that allows ex vivo development of villi and extensions of that culture system to allow the intestines to be three-dimensionally imaged during their development.

Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

Improved imaging technology is allowing three-dimensional imaging of organs during development. Here we describe a whole organ culture system that allows live imaging of the developing villi in the fetal mouse intestine.

نظام الثقافة الماوس الجنين الأمعاء الجامعة ل<em&gt; فيفو السابقين</em&gt; التلاعب في مسارات إشارات وتصوير لايف ثلاثي الأبعاد التنمية الزغبة

Most morphogenetic processes in the fetal intestine have been inferred from thin sections of fixed tissues, providing snapshots of changes over ...

Development of an Insert Co-culture System of Two Cellular Types in the Absence of Cell-Cell Contact

The role of secreted soluble factors in the modification of cellular responses is a recurrent theme in the study of all tissues and systems. In an attempt to make straightforward the very complex relationships between the several cellular subtypes that compose multicellular organisms, in vitro techniques have been developed to help researchers acquire a detailed understanding of single cell populations. One of these techniques uses inserts with a permeable membrane allowing secreted soluble factors to diffuse. Thus, a population of cells grown in inserts can be co-cultured in a well or dish containing a different cell type for evaluating cellular changes following paracrine signaling in the absence of cell-cell contact. Such insert co-culture systems offer various advantages over other co-culture techniques, namely bidirectional signaling, conserved cell polarity and population-specific detection of cellular changes. In addition to being utilized in the field of inflammation, cancer, angiogenesis and differentiation, these co-culture systems are of prime importance in the study of the intricate relationships that exist between the different cellular subtypes present in the central nervous system, particularly in the context of neuroinflammation. This article offers general methodological guidelines in order to set up an experiment in order to evaluating cellular changes mediated by secreted soluble factors using an insert co-culture system. Moreover, a specific protocol to measure the neuroinflammatory effects of cytokines secreted by lipopolysaccharide-activated N9 microglia on neuronal PC12 cells will be detailed, offering a concrete understanding of insert co-culture methodology.

In multicellular organisms, secreted soluble factors elicit responses from different cell types as a result of paracrine signaling. Insert co-culture systems offer a simple way to assess the changes mediated by secreted soluble factors in the absence of cell-cell contact.

تطوير نظام إدراج المشارك ثقافة نوعين الخلوية في غياب خلية خلية الاتصال

The role of secreted soluble factors in the modification of cellular responses is a recurrent theme in the study of all tissues and systems. In an attempt ...

Establishment of an Extracellular Acidic pH Culture System

Conditions of the tumor microenvironment, such as hypoxia or nutrient starvation, play critical roles in cancer progression and malignancy. However, the role of acidic extracellular pH in tumor aggressiveness and its underlying mechanism has not been extensively studied compared to hypoxic or nutrient starvation conditions. In addition, a well-defined culture method to mimic the acidic extracellular tumor microenvironment has not been fully reported.
Here we present a simple in vitro culture method to maintain acidic extracellular pH using reduced bicarbonate and increased lactate or HCl concentrations in the culture medium. The medium pH was sustained for at least 24 h and gradually decreased by 72 h following culture of PANC-1 and AsPC-1 pancreatic cancer cells. Three distinct acidic media conditions in this study highly upregulated pH-responsive genes such as MSMO1, INSIG1, and IDI1 compared to hypoxia or nutrient starvation. The upregulation of these genes can be used as a marker of acidic pH. These simple techniques are beneficial to elucidate underlying mechanisms of tumor malignancy under acidic tumor microenvironment. Therefore, our extracellular acidic pH culture system enables discovery of cellular acidic pH responses not only in cancer cells but also in primary cells, such as renal tubular cells, in relation to the other acidic disorders including, diabetic ketoacidosis, lactic acidosis, renal tubular acidosis, and respiratory acidosis.

The acidic tumor microenvironment plays critical roles in tumor progression. To assess the effects of acidic extracellular pH on cancer cells in vitro, we established simple acidic culture systems.

إنشاء درجة الحموضة خارج الخلية الحمضية منظومة الثقافة

Conditions of the tumor microenvironment, such as hypoxia or nutrient starvation, play critical roles in cancer progression and malignancy. However, the ...

An Ex Vivo Tissue Culture Model of Cartilage Remodeling in Bovine Knee Explants

Ex vivo culture systems cover a broad range of experiments dedicated to studying tissue and cellular function in a native setting. Cartilage is a unique tissue important for proper function of the synovial joint and is constituted by a dense extracellular matrix (ECM), rich in proteoglycan and type II collagen. Chondrocytes are the only cell type present within cartilage and are widespread and relatively low in number. Altered external stimuli and cellular signalling can lead to changes in ECM composition and deterioration, which are important pathological hallmarks in diseases such as osteoarthritis (OA) and rheumatoid arthritis.
Ex vivo cartilage models allow 1) profiling of chondrocyte mediated alterations of cartilage tissue turnover, 2) visualizing the cartilage ECM composition, and 3) chondrocyte rearrangement directly in the tissue. Profiling these alterations in response to stimuli or treatments are of high importance in various aspects of cartilage biology, and complement in vitro experiments in isolated chondrocytes, or more complex models in live animals where experimental conditions are more difficult to control.
Cartilage explants present a translational and easily accessible method for assessing tissue remodeling in the cartilage ECM in controllable settings. Here, we describe a protocol for isolating and culturing live bovine cartilage explants. The method uses tissue from the bovine knee, which is easily accessible from the local butchery. Both explants and conditioned culture medium can be analyzed to investigate tissue turnover, ECM composition, and chondrocyte function, thus profiling ECM modulation.

Here, we present a protocol describing isolation and culturing of cartilage explants from bovine knees. This method provides an easy and accessible tool to describe tissue changes in response to biological stimuli or novel therapeutics targeting the joint.

نموذج ثقافة الانسجه السابق فيفو من أعاده عرض الغضروف في الركبة البقري الشرح

Ex vivo culture systems cover a broad range of experiments dedicated to studying tissue and cellular function in a native setting. Cartilage is a unique ...