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.

REMARQUE: Les expérimentations animales ont été menées en conformité avec les National Institutes of Health Guide pour le soin et l&#39;utilisation des animaux de laboratoire. Le Comité pour l&#39;expérimentation animale de l&#39;École de médecine de l&#39;Université du Tohoku a approuvé les procédures expérimentales décrites ici. 1. Anesthésie et laparotomie <ol style="margin-left: 40px;"><li> Pour la collecte de sang, utiliser des rats mâles spécifiques exemptes d&#39;agents pathogènes Sprague-Dawley. Jeûner les rats pendant au moins 18 heures tout en fournissant de l&#39;eau. Utilisez rats mâles reproducteurs retraités à 6-8 mois (550-650 g) pour la collecte de sang. </li><li> Débit de 2,5 à 4,0% d&#39;isoflurane, qui est un anesthésique par inhalation, dans une boîte reliée à un appareil d&#39;anesthésie pour introduire anesthésie à 2,0 à 3,5 L / min pendant 10 min. Transfert des rats dans la boîte à introduire l&#39;anesthésie. <ol><li> Confirmer l&#39;anesthésie par la vérification de la perte de la réponse à la stimulation sur les pattes avec une pince. Couvrir le nez du rat with un masque relié à un appareil d&#39;anesthésie anesthésier profondément les rats lors de la collecte de sang. La concentration et le débit est maintenu à 2,5 à 4,0% et de 2,0 à 3,5 L / min, respectivement. NOTE: L&#39;halothane doit pas être utilisé pour l&#39;anesthésie par inhalation car embryons mis en culture dans le sérum qui est recueillie à partir de rats anesthésiés avec ce réactif présentent un retard dans le développement embryonnaire par rapport aux embryons cultivés dans le sérum obtenu à partir de rats anesthésiés avec de l&#39;isoflurane 22. </li></ol></li><li> Pour éviter la contamination de la fourrure, à désinfecter la peau en versant 70% d&#39;éthanol sur l&#39;abdomen du rat anesthésié. Choisir la peau et la paroi abdominale désinfecté à la partie inférieure avec une paire de forceps et couper ces couches simultanément en direction de la région du thorax d&#39;une paire de grands ciseaux pour exposer les organes internes. Tenir compte de l&#39;intestin à l&#39;extérieur de la cavité abdominale avec une paire de forceps. </li><li> Coupez la peau et la paroi abdominale vers les membres postérieurs avec unpaire de grands ciseaux pour exposer davantage la région abdominale postérieure. Refléter la graisse supplémentaire en dehors de la cavité abdominale avec une paire de pinces. </li><li> Choisir la graisse viscérale sur la ligne médiane à l&#39;aide de deux paires de pinces et de diviser la matière grasse avec soin dans une direction parallèle à exposer l&#39;aorte abdominale. Répéter ce procédé pour diviser la graisse viscérale dans une direction longitudinale pour identifier facilement la bifurcation de l&#39;aorte abdominale. En outre, l&#39;aorte est pulsatile et peut être distinguée de la veine cave adjacente à l&#39;aide de cette caractéristique. </li><li> Choisir la matière grasse autour de l&#39;aorte abdominale et la bifurcation soigneusement avec deux paires de pinces et séparer la graisse de l&#39;aorte abdominale. </li></ol> 2. Collecte de sang <ol style="margin-left: 40px;"><li> Insérer avec précaution la pointe d&#39;une aiguille 21 G raccordée à une seringue de 20 ml immédiatement crânienne à la bifurcation de l&#39;aorte, avec le biseau vers le bas. Tenez la seringue avec unmain, et tirez lentement la seringue pour recueillir 15 ml de sang d&#39;un rat mâle. </li><li> Retirez l&#39;aiguille de la seringue, et verser le sang en deux 10 ml tubes à essai stériles glacées (figure 1A). Conserver les tubes sur de la glace jusqu&#39;à ce que la centrifugation pour retarder la formation du caillot de sang. Réduire le temps de centrifugation par centrifugation de plusieurs tubes à la fois. </li><li> Euthanasier le rat anesthésié par thoracotomie et couper le coeur ou la décapitation. </li></ol> 3. Sérum de rat Préparation <ol style="margin-left: 40px;"><li> Centrifuger le sang recueilli pendant 5 min à 1200 x g et à température ambiante (RT) pour séparer le sang en couches supérieure et inférieure (figure 1C). </li><li> Gardez les tubes à 4 ° C pendant 1,5-2 heures à fixer le caillot de fibrine. </li><li> Relever le caillot de fibrine dans la couche supérieure à l&#39;aide d&#39;une paire de forceps incurvés, et presser le caillot de fibrine pour séparer le sérum. Ensuite, appuyez sur le caillot de fibrine avec les pinces courbes face downwards proches de l&#39;interface entre les deux couches (Figure 1D). </li><li> Répétez l&#39;étape 3.3 de détacher davantage le caillot de fibrine. </li><li> Centrifuger les tubes à essai pendant 5 minutes à 1200 xg et RT (figure 1E). </li><li> Soigneusement transférer le sérum dans un tube stérile de 15 ml à l&#39;aide d&#39;une pipette stérile dans une armoire flux d&#39;air laminaire. </li><li> Centrifuger le sérum de rat recueillies pendant 5 min à 1200 xg et RT pour enlever les globules rouges restants. </li><li> Regrouper le sérum avec précaution dans un nouveau tube de 15 ml stérilisés à l&#39;aide d&#39;une pipette stérile pour éviter la contamination par les globules rouges résiduels dans le fond du tube (Figure 1F). </li><li> Faire incuber le sérum dans un bain d&#39;eau à 56 ° C pendant 30 minutes pour inactiver le système du complément. </li><li> Refroidir les tubes de sérum de rat à la température ambiante dans l&#39;armoire flux d&#39;air laminaire. Le sérum doit être aliquote de 10 ml dans des tubes stériles individuels. Conserver les tubes à -20 ° C jusqu&#39;à utilisation (figure 1H). </li></ol>

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No conflicts of interest are declared.

Des résultats reproductibles lors des expérimentations WEC dépendent de l&#39;utilisation de sérum de rat IC de haute qualité et de précision technique embryon de dissection 3. Ne pas raccourcir la période de jeûne avant la collecte de sang, car le jeûne est nécessaire de normaliser les concentrations de glucose dans le sang. Des rats femelles doivent pas être utilisés pour la préparation de sérum parce que les niveaux d&#39;hormones chez les animaux femelles changent en fonction du cycle oestra...

Une variété de modèles animaux sont utilisés en biologie du développement pour étudier les mécanismes de développement aux niveaux moléculaires et cellulaires. Par exemple, les espèces d&#39;amphibiens et d&#39;oiseaux ont été largement utilisés comme animaux modèles classiques qui sont appropriés pour la manipulation directe d&#39;embryons parce que ces embryons se développent en dehors de la mère. Contrairement à ces animaux, les embryons de mammifères se développent dans l&#39;utérus de la mère, et la croissance à des stades ultérieurs dépend essentiellement de la fonction de l&#39;utérus. Par conséquent, il est souvent difficile de manipuler directement les embryons de mammifères, tels que ceux de la souris et le rat à des stades précoces. Dans les années 1960, Denis New établi une culture d&#39;embryons total de mammifère (CME) de la technique utilisant des appareils WEC avec un apport d&#39;oxygène et d&#39;un contrôle de chaleur 1. En WEC, souris et rats embryons peuvent se développer ex vivo, (c&#39;est à dire, en dehors de l&#39;utérus). Bien que la technique du CME a été souvent utilisé en tératologie en ajoutant divers chemiccomposés al dans le milieu de culture, cette technique a également été utilisée dans plusieurs études de biologie du développement à examiner les mécanismes de développement uniques chez les mammifères 2-4. Par exemple, le CME est combiné avec d&#39;autres techniques, telles que le marquage cellulaire, dans le type sauvage et mutantes embryons en utilisant un colorant fluorescent 5, 6 transplantation de cellules, et l&#39;introduction de gène par l&#39;intermédiaire de lipofection et d&#39;électroporation 7 13.8. Récemment, dans la manipulation de l&#39;utérus a été utilisé pour analyser les processus de développement des embryons de rongeurs à des stades ultérieurs et a été combiné avec des techniques d&#39;électroporation 14-16. Cependant, ces techniques ne sont pas appropriées pour la manipulation de post-implantation d&#39;embryons et midgestation raison de la difficulté à atteindre injection locale précise de la solution d&#39;ADN dans des embryons à un stade précoce. Bien que la transplantation de cellules guidée par ultrasons et l&#39;injection de vecteurs viraux dans des embryons précoces (à savoir, E8.5-E-9.5 chez la souris) in utero ont été rapportés précédemment 17,18, d&#39;excellentes compétences sont nécessaires pour effectuer ces expériences avec un taux de réussite élevé. Par conséquent, le WEC avec une grande accessibilité ex vivo présente des avantages par rapport à la manipulation de la souris et du rat des embryons. Immédiatement centrifugé (IC) de sérum de rat préparée à partir de rats mâles est souvent utilisé pour les moyennes du CME. Lorsque les embryons sont mis en culture au stade de la post-implantation (par exemple, plus tôt que E8.0 chez la souris E10.0 ou chez le rat), d&#39;un mélange de milieu synthétique et du sérum de rat IC est souvent utilisé comme un moyen pour WEC 19. Cependant, les embryons de la culture à la mi-gestation (c&#39;est à dire., E8.0-12.5 dans des embryons de souris E10.0 ou E14.5-dans des embryons de rat), 100% de sérum doit être utilisé comme un moyen car aucune médias alternatifs actuellement disponibles permet embryons de se développer normalement in vitro depuis plus de 2 jours. La préparation de sérum de rat de haute qualité estune étape cruciale pour atteindre la reproductibilité des expériences WEC. Par rapport à une centrifugation retardée, IC a l&#39;avantage de réduire l&#39;hémolyse lorsque le sérum est recueilli en serrant le caillot de fibrine parce que la plupart des cellules rouges du sang ont déjà été séparé du caillot de fibrine. Comme le sérum de rat hémolytique ne parvient pas à soutenir la croissance normale des embryons de rats et de souris, la préparation du sérum par centrifugation immédiate est préférable de préparation en utilisant la centrifugation retardée. Notre protocole comprend deux étapes supplémentaires par rapport à d&#39;autres protocoles de 18 à 20 (c.-à-., Stocker le sang recueilli sur de la glace avant la première centrifugation et de maintenir les échantillons de sang prélevés pendant 2 heures à 4 ° C après la première centrifugation). L&#39;ex-étape peut retarder la formation du caillot de sang, et la dernière étape favorise la solidification du caillot de fibrine pour la compression simple. Par conséquent, notre protocole peut être utilisé par les débutants. Toutefois, la reproduction et la collecte de sérum sanguinextraction avec précision par simple référence aux livres de protocole est extrêmement difficile 19-21. Dans cet article, vidéo, nous démontrons notre protocole standard pour la préparation de sérum de rat IC optimal, qui comprend la collecte de sang à partir de l&#39;aorte abdominale de rats mâles et extraction du sérum par centrifugation.

<table border="0" cellpadding="0" cellspacing="0" width="892">
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:156px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:401px;">Comments/Description</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Isoflurane</td>
			<td>Abbott</td>
			<td>B506</td>
			<td>For anesthesia of rats.</td>
		</tr>
		<tr height="20">
			<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>
		</tr>
		<tr height="20">
			<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>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sprague-Dawley (SD) rat</td>
			<td>Charles Rivers Laboratories</td>
			<td></td>
			<td>Retired male rats from colony in the lab or purchased retired male rats.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Syringe (20 ml)</td>
			<td>TERUMO</td>
			<td>SS-29ESZ</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Needle (21G x 5/8&#34;)</td>
			<td>TERUMO</td>
			<td>NN-2116R</td>
			<td></td>
		</tr>
		<tr height="20">
			<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>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sterile disposable pipette</td>
			<td>Eiken Chemical</td>
			<td>CD2000 No.4</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sterilie disposable tube (15 ml)&#160;</td>
			<td>Falcon</td>
			<td>2196</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

La figure 1 montre les résultats représentatifs des modes opératoires décrits pour la séparation des cellules du sang et le sérum. On obtient typiquement 15 ml de sang d&#39;un rat mâle retiré (Figure 1A). Par centrifugation, le sang collecté peut être séparé en une couche supérieure contenant du sérum et du caillot de fibrine, ce qui est indiqué par une flèche, et une couche inférieure contenant les cellules sanguines (figure 1C). Fait important, les ?...

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.

Préparation du Rat Serum Convient pour la culture d&#39;embryons de mammifères entier

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 vues.  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.

Vidéo: 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. 

Culture organotypique des cerveaux de rat 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, ...

Recherche

Biologie

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.

Culture primaire des adultes myocytes cœur de rat

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.

Préparation et de la culture d&#39;explants Objectif Rat épithéliales pour l&#39;étude de la différenciation terminale

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.

Une Ex-ovo- Poulet embryonnaire du système de culture approprié pour les applications d&#39;imagerie et de microchirurgie

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.

La méthode de culture des embryons rongeurs entier en utilisant le système des rotateurs de type flacon de culture

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

Preparation of Rat Tail Tendons for Biomechanical and Mechanobiological Studies

Rat tail tendons (RTTs) are a common biological model used in experimental in vitro studies in the fields of tendon physiology and tendinopathy. Working with those tissues is challenging because they are very fragile, and until now there was no rigorously detailed protocol for their isolation.
Faced with these challenges, we have developed methods and instruments to facilitate manipulation of RTTs and control tissue viability, sterility and integrity. This article describes the experimental procedures used to prepare RTTs for biomechanical and mechanobiological studies. Our work is divided into four main steps: extraction, cross-sectional area measurement, rinsing and loading into the bioreactor chamber.
At each step, all procedures, materials and manipulations are presented in detail so that they can be easily reproduced. Moreover, the specific instruments developed are presented: a manipulation plate used to segregate RTTs, an optic micrometer to position the tissue during the cross-sectional area measurement and an anchoring system to attach the RTTs onto a bioreactor.
Finally, we describe the results obtained after multiple tests to validate our methods. The viability, sterility and integrity evaluations demonstrate that our procedures are sufficiently rigorous for manipulations of fragile tissues such as rat tail tendons.

This article describes the experimental procedures used to prepare rat tail tendons for biomechanical and mechanobiological studies. Several features of the main steps in preparation are demonstrated, beginning with extraction, cross-sectional area measurement, rinsing and loading into the bioreactor chamber.

Préparation des tendons de queue de rat pour les études biomécaniques et mécanobiologique

Rat tail tendons (RTTs) are a common biological model used in experimental in vitro studies in the fields of tendon physiology and tendinopathy. Working ...

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.

Scale-Up de la culture des cellules de mammifères en utilisant un nouveau flacon multicouches

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.

Préparation des cellules de souris embryonnaires fibroblastiques conviennent pour la culture embryonnaires humaines et cellules souches pluripotentes induites

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.

Souris développement embryonnaire dans tout un système de culture d&#39;embryons sans sérum

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.

Appartement Mont Préparation pour l&#39;observation et l&#39;analyse des échantillons embryon de poisson zèbre souillé par toute la montagne<em&gt; In situ</em&gt; Hybridation

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