We thank Mr. Hajime Ichijo for video recording and helpful advice concerning editing the video. We also thank the Osumi lab members for animal care. This work is supported by a Grant-in-Aid for Scientific Research on Innovative Area, Neural Diversity and Neocortical Organization, from MEXT of Japan (to N. O.). T. K. was supported by a Research Fellowship of JSPS for Young Scientists.

ملاحظة: تم إجراء التجارب على الحيوانات وفقا للمعاهد الوطنية للصحة دليل لرعاية واستخدام الحيوانات المختبرية. وافقت اللجنة لتجارب على الحيوانات في مدرسة جامعة توهوكو للطب الإجراءات التجريبية الموصوفة هنا. 1. التخدير وفتح البطن <ol style="margin-left: 40px;;text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> لجمع الدم، واستخدام الذكور الفئران معينة خالية من مسببات الأمراض سبراغ داولي. صيام الفئران لمدة 18 ساعة على الأقل مع توفير المياه. استخدام الفئران مربي متقاعد الذكور في 6-8 أشهر من العمر (550-650 ز) لجمع الدم. </li><li style=";text-align:right;direction:rtl"> تدفق 2،5-4،0٪ isoflurane و، وهو مخدر الاستنشاق، في مربع متصلا جهاز التخدير لإدخال التخدير في 2،0-3،5 لتر / دقيقة لمدة 10 دقيقة. نقل الفئران في خانة لإدخال التخدير. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تأكيد التخدير عن طريق التحقق من فقدان ردا على التحفيز على الكفوف بالملقط. تغطية الأنف من واي الفئرانال قناع متصلة جهاز التخدير لتخدير عميق الفئران خلال جمع الدم. يتم الحفاظ على تركيز وتدفق في 2،5-4،0٪ و2،0-3،5 لتر / دقيقة، على التوالي. ملاحظة: هالوثان لا ينبغي أن تستخدم للتخدير استنشاق لأن الأجنة مثقف في المصل التي تم تجميعها من الفئران تخدير مع هذا كاشف المعرض تأخير في التطور الجنيني مقارنة مع الأجنة مثقف في المصل تم الحصول عليها من الفئران تخدير مع isoflurane و22. </li></ol></li><li style=";text-align:right;direction:rtl"> لتجنب التلوث من الفراء، وتطهير الجلد عن طريق سكب الايثانول 70٪ على البطن من الفئران تخدير. التقاط الجلد تطهيرها وجدار البطن في المنطقة السفلية مع زوج من ملقط وقطع هذه الطبقات في وقت واحد تجاه المنطقة الصدر مع زوج من مقص كبير لفضح الأعضاء الداخلية. تعكس الأمعاء خارج تجويف البطن مع زوج من الملقط. </li><li style=";text-align:right;direction:rtl"> قطع الجلد وجدار البطن في اتجاه أطرافه الخلفية معزوج من مقص كبير لمواصلة فضح منطقة البطن الخلفي. تعكس الدهون الزائدة خارج تجويف البطن مع زوج من الملقط. </li><li style=";text-align:right;direction:rtl"> التقاط الدهون الحشوية في خط الوسط باستخدام اثنين من أزواج من ملقط وتقسيم الدهون بعناية في اتجاه مواز لفضح الشريان الأورطي البطني. كرر هذا الإجراء لتقسيم الدهون الحشوية في الاتجاه الطولي لتحديد بسهولة والتشعب من الشريان الأورطي البطني. بالإضافة إلى ذلك، الشريان الأورطي هو نابض ويمكن تمييزها من الوريد الأجوف المجاورة باستخدام هذه الخاصية. </li><li style=";text-align:right;direction:rtl"> التقاط الدهون حول الشريان الأورطي البطني والتشعب بعناية مع اثنين من أزواج من ملقط وفصل الدهون في الشريان الأورطي البطني. </li></ol> 2. جمع الدم <ol style="margin-left: 40px;;text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إدراج بعناية غيض من الإبرة G 21 متصلة حقنة 20 مل الجمجمة على الفور إلى التشعب من الشريان الأورطي، مع شطبة في اتجاه نزولي. عقد حقنة واحدةجهة، وسحب المحقنة ببطء لجمع 15 مل من الدم من الفئران الذكور. </li><li style=";text-align:right;direction:rtl"> إزالة الإبرة من الحقنة، وتصب الدم في كل منهما 10 مل أنابيب الاختبار العقيمة الجليد الباردة (الشكل 1A). إبقاء الأنابيب على الجليد حتى الطرد المركزي لتأخير تشكيل تجلط الدم. تقليل الوقت الطرد المركزي بواسطة الطرد المركزي أنابيب عدة في وقت واحد. </li><li style=";text-align:right;direction:rtl"> الموت ببطء الفئران تخدير عن طريق بضع الصدر وقطع القلب أو قطع الرأس. </li></ol> 3. الجرذ سيروم إعداد <ol style="margin-left: 40px;;text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> أجهزة الطرد المركزي الدم التي تم جمعها لمدة 5 دقائق عند 1،200 x ج ودرجة حرارة الغرفة (RT) لفصل الدم إلى الطبقات العليا والسفلي (الشكل 1C). </li><li style=";text-align:right;direction:rtl"> إبقاء الأنابيب في 4 درجة مئوية لمدة ساعة 1.5-2 لإصلاح جلطة الفيبرين. </li><li style=";text-align:right;direction:rtl"> التقاط جلطة الليفين في الطبقة العليا باستخدام زوج من ملقط المنحني، والضغط على تجلط الليفين لفصل المصل. ثم، اضغط على تجلط الليفين مع ملقط المنحني التي تواجه تأليفwnwards بالقرب من واجهة بين طبقتين (1D الشكل). </li><li style=";text-align:right;direction:rtl"> كرر الخطوة 3.3 لمزيد من فصل جلطة الفيبرين. </li><li style=";text-align:right;direction:rtl"> أجهزة الطرد المركزي أنابيب الاختبار لمدة 5 دقائق عند 1،200 x ج وRT (الشكل 1E). </li><li style=";text-align:right;direction:rtl"> نقل بعناية المصل في أنبوب 15 مل العقيمة باستخدام ماصة معقمة في الصفحي مجلس الوزراء تدفق الهواء. </li><li style=";text-align:right;direction:rtl"> أجهزة الطرد المركزي في مصل الفئران التي تم جمعها لمدة 5 دقائق عند 1،200 x ج وRT لإزالة أي خلايا الدم الحمراء المتبقية. </li><li style=";text-align:right;direction:rtl"> تجميع المصل بعناية إلى الجديد 15 مل أنبوب تعقيم باستخدام ماصة معقمة لتجنب تلوث الخلايا الحمراء المتبقية في الجزء السفلي من الأنبوب (الشكل 1F). </li><li style=";text-align:right;direction:rtl"> احتضان المصل في حمام الماء عند 56 درجة مئوية لمدة 30 دقيقة إلى تعطيل نظام المتممة. </li><li style=";text-align:right;direction:rtl"> تبريد أنابيب المصل الفئران لRT في الصفحي مجلس الوزراء تدفق الهواء. وينبغي aliquoted المصل إلى 10 مل في أنابيب معقمة الفردية. تخزين الأنابيب في -20 درجة مئوية حتى استخدامها (الشكل 1H). </li></ol>

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No conflicts of interest are declared.

النتائج استنساخه في التجارب WEC تعتمد على استخدام ذات جودة عالية IC مصل الفئران ودقيقة تقنية الجنين تشريح 3. لا تقصير فترة الصيام قبل جمع الدم بسبب الصيام هو ضروري لتوحيد تركيزات الجلوكوز في الدم. لا ينبغي أن تستخدم إناث الفئران من أجل إعداد المصل لأن مستويات هرمو?...

وتستخدم مجموعة متنوعة من الحيوانات النموذج في البيولوجيا التطورية للتحقيق في الآليات التنموية على المستويين الجزيئي والخلوي. على سبيل المثال، استخدمت على نطاق واسع نوعا من البرمائيات والطيور كحيوانات النموذج الكلاسيكي التي هي مناسبة للتلاعب مباشرة من الأجنة لأن هذه الأجنة تطوير خارج الأم. وعلى النقيض من هذه الحيوانات، أجنة الثدييات ينمو في رحم الأم، والنمو في مراحل لاحقة تعتمد بشكل حاسم على وظيفة الرحم. وبالتالي، فمن الصعب عادة لمعالجة الأجنة مباشرة الثدييات مثل تلك التي من الماوس والفئران في مراحل مبكرة. في 1960s، التي أنشئت دينيس جديد الثدييات ثقافة الجنين كله (WEC) باستخدام تقنية الأجهزة WEC مع امدادات الاوكسجين المستمر ومراقبة الحرارة 1. في WEC، يمكن أن الماوس والفئران الأجنة تنمو خارج الجسم، (أي خارج الرحم). على الرغم من أن تقنية WEC كثيرا ما كان يستخدم في المسخ عن طريق إضافة مختلف كيميائيآل مركبات في مستنبت، كما تم استخدام هذه التقنية في مختلف الدراسات البيولوجيا التطورية لدراسة الآليات التنموية الفريدة في الثدييات 2-4. على سبيل المثال، يتم الجمع بين WEC مع تقنيات أخرى، مثل وضع العلامات الخلية، في البرية من نوع ومتحولة الأجنة باستخدام صبغة الفلورسنت 5، 6 زرع الخلايا، وإدخال الجين عبر lipofection 7 و electroporation 8-13. في الآونة الأخيرة، في التلاعب الرحم وقد استخدمت لتحليل العمليات التنموية في الأجنة القوارض في مراحل لاحقة، وقد تم دمجها مع تقنيات التثقيب الكهربائي 14-16. ولكن هذه التقنيات ليست مناسبة للتلاعب من الأجنة postimplantation وmidgestation بسبب صعوبات تحقيق الحقن المحلية دقيقة من الحل الحمض النووي في الأجنة في المراحل المبكرة. على الرغم من أن زرع الخلايا الموجهة بالأمواج فوق الصوتية وحقن النواقل الفيروسية في الأجنة في وقت مبكر (أي، تم الإبلاغ عن E8.5-E-9.5 في الماوس) في الرحم سابقا 17،18، يطلب من مهارات ممتازة لتنفيذ هذه التجارب مع نسبة نجاح عالية. وبالتالي، مع إمكانية الوصول WEC عالية فيفو السابقين مزاياه فيما يتعلق التلاعب الماوس والفئران الأجنة. وكثيرا ما يستخدم طرد فورا (IC) مصل الفئران المحضرة من الفئران الذكور لمتوسطة WEC. عند الأجنة هي مثقف في مرحلة postimplantation (أي، في وقت سابق من E8.0 في الماوس أو E10.0 في الفئران)، وغالبا ما يتم استخدام خليط من المتوسطة الاصطناعية وIC مصل الفئران كوسيلة للWEC 19. ولكن، لأجنة الثقافة في منتصف الحمل (أي.، E8.0-12.5 في أجنة الفئران أو E10.0-E14.5 في الأجنة الفئران)، ينبغي استخدام 100٪ مصل كوسيلة لأنه لا يوجد وسائل الإعلام البديلة المتاحة حاليا تسمح أجنة تنمو عادة في المختبر لأكثر من 2 أيام. إعداد عالية الجودة مصل الفئرانخطوة حاسمة لتحقيق استنساخ التجارب في WEC. مقارنة تأخر الطرد المركزي، IC له فائدة في الحد من انحلال الدم عندما يتم جمع الدم عن طريق الضغط على تجلط الليفين لأن معظم خلايا الدم الحمراء وقد تم بالفعل فصل من جلطة الفيبرين. كما فشل مصل الفئران الانحلالي لدعم النمو الطبيعي للأجنة الجرذان والفئران، وإعداد المصل باستخدام الطرد المركزي المباشر هو الأفضل لإعداد باستخدام تأخر الطرد المركزي. بروتوكول لدينا يحتوي على اثنين من خطوات إضافية مقارنة مع غيرها من البروتوكولات 18-20 (مثال. وتخزين الدم التي تم جمعها على الجليد قبل الطرد المركزي الأول والحفاظ على عينات الدم التي تم جمعها لمدة 2 ساعة في 4 درجات مئوية بعد الطرد المركزي الأول). الخطوة السابقة يمكن تأخير تشكيل تجلط الدم، والخطوة الأخيرة تعزز التصلب من جلطة الليفين لسهولة الضغط. لذلك، لدينا بروتوكول يمكن استخدامها من قبل المبتدئين. ومع ذلك، استنساخ جمع الدم والمصلاستخراج بدقة بمجرد اشارة الى الكتب البروتوكول هو في غاية الصعوبة 19-21. في هذه المقالة الفيديو، علينا أن نظهر بروتوكول لدينا معيار لإعداد الأمثل مصل الفئران IC، والذي يتضمن جمع الدم من الشريان الأورطي البطني من الفئران الذكور واستخراج المصل بواسطة الطرد المركزي.

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			<td style="width:156px;">Company</td>
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			<td height="20" style="height:20px;width:216px;">Isoflurane</td>
			<td>Abbott</td>
			<td>B506</td>
			<td>For anesthesia of rats.</td>
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			<td height="20" style="height:20px;width:216px;">Large scissors</td>
			<td>Napox</td>
			<td>B-7H</td>
			<td>Stelized scissors for cutting the skin and muscle of rats.&#160;</td>
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			<td height="20" style="height:20px;">Curved forceps</td>
			<td>Napox</td>
			<td>A-3-2</td>
			<td>Stelized forceps for picking up the skin of rats and squeezing the fibrin clot.</td>
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			<td height="20" style="height:20px;">Sprague-Dawley (SD) rat</td>
			<td>Charles Rivers Laboratories</td>
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			<td>Retired male rats from colony in the lab or purchased retired male rats.</td>
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			<td>TERUMO</td>
			<td>SS-29ESZ</td>
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			<td height="20" style="height:20px;">Needle (21G x 5/8&#34;)</td>
			<td>TERUMO</td>
			<td>NN-2116R</td>
			<td></td>
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			<td height="20" style="height:20px;">Sterile test (spitz) tube (10 ml)</td>
			<td>ASIAKIZAI</td>
			<td style="width:119px;">1101C000B-10</td>
			<td>For collection of boold</td>
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			<td height="20" style="height:20px;">Sterile disposable pipette</td>
			<td>Eiken Chemical</td>
			<td>CD2000 No.4</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">Sterilie disposable tube (15 ml)&#160;</td>
			<td>Falcon</td>
			<td>2196</td>
			<td></td>
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preparation of rat serum suitable for mammalian whole embryo culture

Mammalian whole embryo culture (WEC) is a widely used technique for examining pharmacological toxicity in developing mouse and rat embryos and for investigating the mechanisms of developmental processes. Immediately centrifuged (IC) rat serum is commonly used for WEC and is essential for the growth and development of cultured mouse and rat embryos ex vivo. For the culture of midgestation embryos (i.e., E8.0-12.5 for the mouse, and E10.0-14.5 for the rat), 100% rat serum is the best media for supporting the growth of the embryo ex vivo. To prepare rat serum suitable for WEC, the collected blood should be centrifuged immediately to separate the blood cells from the plasma fraction. After centrifugation, the fibrin clot forms in the upper layer; this clot should be squeezed gently using a pair of sterile forceps and subsequently centrifuged to completely separate the blood cells from the serum. In this video article, we demonstrate our standard protocol for the preparation of optimal IC rat serum, including blood collection from the abdominal aorta of male rats and extraction of the serum by centrifugation.

ويبين الشكل 1 نتائج ممثل الإجراءات الموضحة لفصل خلايا الدم والمصل. نحن عادة الحصول على 15 مل من الدم من الفئران الذكور متقاعد (الشكل 1A). بواسطة الطرد المركزي، ويمكن فصل الدم التي تم جمعها إلى الطبقة العليا التي تحتوي على مصل وجلطة الليفين، والتي يشار ?...

Watch this Scientific Journal Video about Preparation of Rat Serum Suitable for Mammalian Whole Embryo Culture at JoVE.com

Preparation of Rat Serum Suitable for Mammalian Whole Embryo Culture

Mammalian whole embryo culture (WEC) is widely used in teratology and developmental biology. Immediately centrifuged rat serum is commonly provided as a medium for both mouse and rat WEC. In this video, we demonstrate our standard protocol for the preparation of high-quality rat serum suitable for mammalian WEC.

إعداد الجرذ مصل مناسب للالثدييات الجامع الثقافة الأجنة

Mammalian whole embryo culture (WEC) is a widely used technique for examining pharmacological toxicity in developing mouse and rat embryos and for ...

preparation-of-rat-serum-suitable-for-mammalian-whole-embryo-culture

Research

JoVE Journal

Biology

13.7K Views.  Jichi Medical University. Mammalian whole embryo culture (WEC) is widely used in teratology and developmental biology. Immediately centrifuged rat serum is commonly provided as a medium for both mouse and rat WEC. In this video, we demonstrate our standard protocol for the preparation of high-quality rat serum suitable for mammalian WEC.

Video: Preparation of Rat Serum Suitable for Mammalian Whole Embryo Culture

Organotypic Slice Culture of E18 Rat Brains

Organotypic slice cultures from embryonic rodent brains are widely used to study brain development. While there are often advantages to an in-vivo system, organotypic slice cultures allow one to perform a number of manipulations that are not presently feasible in-vivo. To date, organtotypic embryonic brain slice cultures have been used to follow individual cells using time-lapse microscopy, manipulate the expression of genes in the ganglionic emanances (a region that is hard to target by in-utero electroporation), as well as for pharmacological studies. In this video protocol we demonstrate how to make organotypic slice cultures from rat embryonic day 18 embryos. The protocol involves dissecting the embryos, embedding them on ice in low melt agarose, slicing the embedded brains on the vibratome, and finally plating the slices onto filters in culture dishes. This protocol is also applicable in its present form to making organotypic slice cultures from different embryonic ages for both rats and mice. 

عضوي النمط من الثقافة شريحة أدمغة الفئران E18

Organotypic slice cultures from embryonic rodent brains are widely used to study brain development. While there are often advantages to an in-vivo system, ...

Primary Culture of Adult Rat Heart Myocytes

Cultured primary adult rodent heart cells are an important model system for cardiovascular research. Nevertheless, establishment of robust, viable cultured adult myocytes can be a technically challenging, rate-limiting step for many researchers. Here we described a protocol to obtain a high yield of adult rat heart myocytes that remain viable in culture for several days. The heart is isolated and perfused with collagenase and protease under low Ca2+ conditions to recover single myocytes. Ca2+-tolerant cells are obtained by stepwise increases in extracellular Ca2+ concentration in three subsequent wash steps. Cells are filtered, resuspended in culture medium, and plated on laminin coated slips. Cultured myocytes obtained using this protocol are viable for up to four days and are suitable for most experiments including electrophysiology, biochemistry, imaging and molecular biology.

In this paper, we described a typical way to isolate and culture adult rat heart myocytes. Collagenase and protease are used to digest and isolate single myocytes. Myocytes cultured follow this protocol meet most experiment requirements.

الابتدائية ثقافة الكبار Myocytes قلب فأر

Cultured primary adult rodent heart cells are an important model system for cardiovascular research.  Nevertheless, establishment of robust, viable ...

Preparation and Culture of Rat Lens Epithelial Explants for Studying Terminal Differentiation

The anterior surface of the ocular lens is covered by a monolayer of epithelial cells, which proliferate in an annular zone underlying the ciliary body.  Following division, these cells migrate posteriorly, where FGF diffusing from the retina induces them to differentiate into a posterior array of elongated lens fiber cells, which compose the bulk of the lens.  Differentiation of lens epithelial cells into lens fibers can be induced in vitro by culturing explants of the central region of the anterior epithelium in the presence of FGF-2.  Explants are prepared from lenses of neonatal rats by removing the lens from the eye and grasping the lens capsule on the posterior side with dissecting tweezers.  The posterior capsule is then gently torn open and pressed down into the plastic bottom of a tissue culture dish. The peripheral regions of the explant are removed with a scalpel and the central area is then cultured in the presence of 100ng/ml FGF-2 for as long as 2-3 weeks, depending on the parameters to be studied.  Since epithelial cells in cultured explants differentiate in approximate synchrony over a period of days to weeks, the time course of signaling and gene expression can be determined using molecular, biochemical, and pharmacological techniques.  Immunofluorescence microscopy is a powerful adjunct to these methods as it demonstrates the subcellular localization of proteins of interest and can reveal the physiological consequences of experimental manipulations of signaling pathways.

Explants of the central region of rat lens epithelia differentiate synchronously when cultured in the presence of FGF-2. Immunofluorescence microscopy of such cultures can provides novel information about gene expression and signaling events associated with terminal differentiation.

إعداد وثقافة Explants لنس الجرذ طلائي لدراسة التمايز الطرفية

The anterior surface of the ocular lens is covered by a monolayer of epithelial cells, which proliferate in an annular zone underlying the ciliary body.  ...

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 ...

The Method of Rodent Whole Embryo Culture using the Rotator-type Bottle Culture System

Whole embryo culture (WEC) technique has been developed in 1950's by New and his colleagues, and applied for developmental biology 1. Although development and growth of mammalian embryos are critically dependent on the function of the placenta, WEC technique allows us to culture mouse and rat embryos ex vivo condition during limited periods corresponding to midgestation stages during embryonic day (E) 6.5-E12.5 in the mouse or E8.5-E14.5 in the rat 2, 3, 4. In WEC, we can directly target desired areas of embryos using fine glass capillaries because embryos can be manipulated under the microscope. Therefore, rodent WEC is very useful technique when we want to study dynamic developmental processes of postimplanted mammalian embryos. Up to date, several types of WEC systems have been developed 1. Among those, the rotator-type bottle culture system is most popular and suitable for long-term culture of embryos at midgestation, i.e., after E9.5 and E11.5 in the mouse and rat, respectively 1. In this video protocol, we demonstrate our standard procedures of rat WEC after E12.5 using a refined model of the original rotator system, which was designed by New and Cockroft 5, 6, and introduce various applications of WEC technique for studies in mammalian developmental biology.

Whole embryo culture technique allows us to culture mouse and rat embryos ex vivo condition during limited periods corresponding to midgestation stages. In this video protocol, we demonstrate our standard procedures of rat whole embryo culture after E12.5 using the rotator-type bottle culture system.

الطريقة الثقافة القوارض باستخدام الأجنة الجامع للثقافة الدوار زجاجة من نوع النظام

Whole embryo culture (WEC) technique has been developed in 1950's by New and his colleagues, and applied for developmental biology 1. Although development ...

Scale-Up of Mammalian Cell Culture using a New Multilayered Flask

A growing number of cell-based applications require large numbers of cells. Usage of single layer T-flasks, that are adequate during small-scale expansion, may become cumbersome, laborious and time-consuming when large numbers of cells are required. To address this need, the performance of a new multi-layered cell culture vessel to facilitate easy scale up of cells from single layered T-flasks will be discussed. The flasks tested are available in 3- and 5-layer format and enable culture and complete recovery of three and five times the number of cells respectively, compared to T-175 flasks. A key feature of the BD Multi-Flask is a mix/equilibration port that allows rapid in-vessel mixing as well as uniform distribution of cells and reagents within and 
between layers of each vessel and consistently produce cells that can be cultured in an environment that is congruent to T-175 flasks.
The design of these Multi-Flasks also allows for convenient pipette access for adding reagents and cells directly into the flasks as well as efficient recovery of valuable cells and reagents and reduces risk of contamination due to pouring. For applications where pouring is preferred over pipetting, the design allows for minimal residual liquid retention so as to reduce wastage of valuable cells and reagents.

Cells play an instrumental and increasing role in research, and the discovery and development of new therapeutics. With this increasing need for greater number of cells we need more efficient and effective ways for growing and harvesting attachment dependent cells. A Multilayered flask with the right features can serve this purpose.

مقياس الهاتفي للثقافة خلايا الثدييات باستخدام قارورة جديد متعدد الطبقات

A growing number of cell-based applications require large numbers of cells. Usage of single layer T-flasks, that are adequate during small-scale ...

Preparation of Mouse Embryonic Fibroblast Cells Suitable for Culturing Human Embryonic and Induced Pluripotent Stem Cells

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it is not easy to establish an effective system for culturing these cells. Since the culture conditions can influence gene expression that confers pluripotency in hESCs and hiPSCs, the optimization and standardization of the culture method is crucial.
The establishment of hESC lines was first described by using MEFs as feeder cells and fetal bovine serum (FBS)-containing culture medium2. Next, FBS was replaced with knockout serum replacement (KSR) and FGF2, which enhances proliferation of hESCs3. Finally, feeder-free culture systems enable culturing cells on Matrigel-coated plates in KSR-containing conditioned medium (medium conditioned by MEFs)4. Subsequently, hESCs culture conditions have moved towards feeder-free culture in chemically defined conditions5-7. Moreover, to avoid the potential contamination by pathogens and animal proteins culture methods using xeno-free components have been established8.
To obtain improved conditions mouse feeder cells have been replaced with human cell lines (e.g. fetal muscle and skin cells9, adult skin cells10, foreskin fibroblasts11-12, amniotic mesenchymal cells13). However, the efficiency of maintaining undifferentiated hESCs using human foreskin fibroblast-derived feeder layers is not as high as that from mouse feeder cells due to the lower level of secretion of Activin A14. Obviously, there is an evident difference in growth factor production by mouse and human feeder cells.
Analyses of the transcriptomes of mouse and human feeder cells revealed significant differences between supportive and non-supportive cells. Exogenous FGF2 is crucial for maintaining self-renewal of hESCs and hiPSCs, and has been identified as a key factor regulating the expression of Tgf&beta;1, Activin A and Gremlin (a BMP antagonist) in feeder cells. Activin A has been shown to induce the expression of OCT4, SOX2, and NANOG in hESCs15-16.
For long-term culture, hESCs and hiPSCs can be grown on mitotically inactivated MEFs or under feeder-free conditions in MEF-CM (MEF-Conditioned Medium) on Matrigel-coated plates to maintain their undifferentiated state. Success of both culture conditions fully depends on the quality of the feeder cells, since they directly affect the growth of hESCs.
Here, we present an optimized method for the isolation and culture of mouse embryonic fibroblasts (MEFs), preparation of conditioned medium (CM) and enzyme-linked immunosorbent assay (ELISA) to assess the levels of Activin A within the media.

The quality of mouse embryonic fibroblasts (MEFs) is dictated by the right strain of mouse such as CF-1. Pluripotency-supportive MEFs and conditioned media (CM) obtained from these should contain optimal concentrations of Activin A, Gremlin and Tgf&beta;1 needed for the Activin/Nodal and FGF pathways to co-operatively maintain self-renewal and pluripotency.

إعداد خلايا الفأر الجنينية الخلايا الليفية مناسبة للالإنسان التثقيف الجنينية والتي يسببها الخلايا الجذعية المحفزة

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it ...

Mouse Embryonic Development in a Serum-free Whole Embryo Culture System

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in an oxygenated rolling bottle culture system. Mouse embryos at E10.5 were carefully isolated from the uterus with intact yolk sac and in a process involving precise surgical maneuver the embryos were gently exteriorized from the yolk sac while maintaining the vascular continuity of the embryo with the yolk sac. Compared to embryos prepared with intact yolk sac or with the yolk sac removed, these embryos exhibited superior survival rate and developmental progression when cultured under similar conditions. We show that these mouse embryos, when cultured in a defined medium in an atmosphere of 95% O2 / 5% CO2 in a rolling bottle culture apparatus at 37 &#176;&#8203;C for 16-40 hr, exhibit morphological growth and development comparable to the embryos developing in utero. We believe this method will be useful for investigators needing to utilize whole embryo culture to study signaling interactions important in embryonic organogenesis.

Serum utilized in embryo cultures contains unknown components that can affect the outcome of experiments especially in studies involving signaling interactions. Here we utilized a serum-free oxygenated culture system and show that mid-gestation mouse embryos cultured for 16-40 hr&#160;exhibit morphological development comparable to embryos developing in utero.

الفأر الجنينية التنمية في الجامعة نظام الثقافة الأجنة الخالية من المصل

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in ...

Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount In situ Hybridization

The zebrafish embryo is now commonly used for basic and biomedical research to investigate the genetic control of developmental processes and to model congenital abnormalities. During the first day of life, the zebrafish embryo progresses through many developmental stages including fertilization, cleavage, gastrulation, segmentation, and the organogenesis of structures such as the kidney, heart, and central nervous system. The anatomy of a young zebrafish embryo presents several challenges for the visualization and analysis of the tissues involved in many of these events because the embryo develops in association with a round yolk mass. Thus, for accurate analysis and imaging of experimental phenotypes in fixed embryonic specimens between the tailbud and 20 somite stage (10 and 19 hours post fertilization (hpf), respectively), such as those stained using whole mount in situ hybridization (WISH), it is often desirable to remove the embryo from the yolk ball and to position it flat on a glass slide. However, performing a flat mount procedure can be tedious. Therefore, successful and efficient flat mount preparation is greatly facilitated through the visual demonstration of the dissection technique, and also helped by using reagents that assist in optimal tissue handling. Here, we provide our WISH protocol for one or two-color detection of gene expression in the zebrafish embryo, and demonstrate how the flat mounting procedure can be performed on this example of a stained fixed specimen. This flat mounting protocol is broadly applicable to the study of many embryonic structures that emerge during early zebrafish development, and can be implemented in conjunction with other staining methods performed on fixed embryo samples.

Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount In situ Hybridization

The zebrafish embryo is an excellent model for developmental biology research. During embryogenesis, zebrafish develop with a yolk mass, which presents three-dimensional challenges for sample observation and analysis. This protocol describes how to create two-dimensional flat mount preparations of whole mount in situ (WISH) stained zebrafish embryo specimens.

إعداد جبل مسطح لمراقبة وتحليل الأجنة الزرد عينات الملون بواسطة جبل الجامعة<em&gt; في الوضع الطبيعي</em&gt; التهجين

The zebrafish embryo is now commonly used for basic and biomedical research to investigate the genetic control of developmental processes and to model ...

Non-enzymatic, Serum-free Tissue Culture of Pre-invasive Breast Lesions for Spontaneous Generation of Mammospheres

Breast ductal carcinoma in situ (DCIS), by definition, is proliferation of neoplastic epithelial cells within the confines of the breast duct, without breaching the collagenous basement membrane. While DCIS is a non-obligate precursor to invasive breast cancers, the molecular mechanisms and cell populations that permit progression to invasive cancer are not fully known. To determine if progenitor cells capable of invasion existed within the DCIS cell population, we developed a methodology for collecting and culturing sterile human breast tissue at the time of surgery, without enzymatic disruption of tissue.
Sterile breast tissue containing ductal segments is harvested from surgically excised breast tissue following routine pathological examination. Tissue containing DCIS is placed in nutrient rich, antibiotic-containing, serum free medium, and transported to the tissue culture laboratory. The breast tissue is further dissected to isolate the calcified areas. Multiple breast tissue pieces (organoids) are placed in a minimal volume of serum free medium in a flask with a removable lid and cultured in a humidified CO2 incubator. Epithelial and fibroblast cell populations emerge from the organoid after 10 - 14 days. Mammospheres spontaneously form on and around the epithelial cell monolayer. Specific cell populations can be harvested directly from the flask without disrupting neighboring cells. Our non-enzymatic tissue culture system reliably reveals cytogenetically abnormal, invasive progenitor cells from fresh human DCIS lesions.

Primary cell culture using intact tissue organoids provides a model system that mimics the multi-cellular in vivo microenvironment. We developed a serum-free primary breast epithelium tissue culture model that perpetuates mixed cell culture lineages and exhibits differentiated morphology, without enzymatic tissue disruption. Breast organoids remain viable for &#62;6 months.

غير الأنزيمية، خالية من مصل زراعة الأنسجة من آفات الثدي ما قبل الغازية لتوليد عفوية من Mammospheres

Breast ductal carcinoma in situ (DCIS), by definition, is proliferation of neoplastic epithelial cells within the confines of the breast duct, without ...