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.

HINWEIS: Tierversuche wurden in Übereinstimmung mit den National Institutes of Health Guide für die Pflege und Verwendung von Labortieren durchgeführt. Der Ausschuss für Tierversuche der Tohoku University School of Medicine hat den hier beschriebenen experimentellen Verfahren. 1. Anästhesie und Laparotomie <ol style="margin-left: 40px;"><li> Für die Blutentnahme, verwenden Sie spezifischen Pathogen-freien männlichen Sprague-Dawley Ratten. Schnell die Ratten für mindestens 18 Stunden, während die Bereitstellung von Wasser. Verwenden Sie im Ruhestand männlichen Ratten Züchter bei 6-8 Monaten (550-650 g) zum Sammeln von Blut. </li><li> Durchfluss von 2,5 bis 4,0% Isofluran, die ein Inhalationsnarkosemittel ist, in eine Box zu einem Narkosegerät angeschlossen, um Anästhesie bei 2,0 bis 3,5 l / min für 10 Minuten vorstellen. Übertragen Ratten in der Box auf die Anästhesie einzuführen. <ol><li> Betäubung zu bestätigen, indem der Verlust der Reaktion auf die Stimulation an den Pfoten mit einer Pinzette. Decken Sie die Nase des Ratten with eine Maske auf ein Narkosegerät, um während der Blutabnahme tief betäuben die Ratten verbunden. Die Konzentration und Strömungs werden bei 2,5-4,0% gehalten und 2,0-3,5 L / min. HINWEIS: Halothan sollte nicht zur Inhalationsanästhesie verwendet, weil Embryonen im Serum, die von Ratten, die mit diesem Reagens weisen eine Verzögerung in der embryonalen Entwicklung im Vergleich zu Embryonen in der von Ratten mit Isofluran betäubt 22 erhaltene Serum kultiviert betäubt gesammelt, kultiviert werden. </li></ol></li><li> Um eine Kontamination des Fells zu vermeiden, die Haut desinfiziert durch Eingießen 70% Ethanol auf den Bauch der narkotisierten Ratte. Pick-up die Haut desinfiziert und Bauchwand am unteren Bereich mit einer Pinzette und gleichzeitig geschnitten, diese Schichten in Richtung der Thorax-Bereich mit einem Paar große Schere, um die inneren Organe aus. Reflect den Darm außerhalb der Bauchhöhle mit einer Pinzette. </li><li> Schneiden Sie die Haut und Bauchwand in Richtung der Hinterbeine mit einerPaar große Schere, um die hinteren Bauchbereich weiter aus. Reflektieren Sie das Extra-Fett außerhalb der Bauchhöhle mit einer Pinzette. </li><li> Nehmen Sie das viszerale Fett an der Mittellinie mit zwei Pinzetten und teilen das Fett vorsichtig in einer Richtung parallel zu der Bauchschlagader freizulegen. Wiederholen Sie diesen Vorgang, um das viszerale Fett in Längsrichtung geteilt, um leicht die Gabelung der Bauchaorta. Zusätzlich ist die Aorta pulsatilen und kann von der benachbarten Hohlvene mit diesen charakteristischen unterscheiden. </li><li> Nehmen Sie das Fett um die Bauchaorta und die Bifurkation vorsichtig mit zwei Pinzetten und trennen Sie das Fett auf der Bauchaorta. </li></ol> 2. Blutentnahme <ol style="margin-left: 40px;"><li> Vorsichtig die Spitze einer 21 G Nadel in eine 20 ml Spritze unmittelbar kranial der Bifurkation der Aorta, mit dem Kegel nach unten verbunden. Halten Sie die Spritze mit einemHand, und ziehen Sie die Spritze langsam zu 15 ml Blut aus einer männlichen Ratte zu sammeln. </li><li> Entfernen Sie die Nadel aus der Spritze, und gießen Sie das Blut in zwei eiskalte 10-ml sterilen Röhrchen (Abbildung 1A). Halten Sie die Röhrchen auf Eis, bis Zentrifugation, um die Bildung der Blutgerinnsel verzögern. Reduzieren Sie die Zeit Zentrifugation durch Zentrifugieren mehrere Rohre auf einmal. </li><li> Euthanize die narkotisierten Ratten durch Thorakotomie und Schneiden des Herzens oder Enthauptung. </li></ol> 3. Rat Serum Vorbereitung <ol style="margin-left: 40px;"><li> Zentrifugieren des gesammelten Blutes für 5 min bei 1.200 × g und Raumtemperatur (RT), um das Blut in einen oberen und unteren Lagen (1C) zu trennen. </li><li> Halten Sie die Röhrchen bei 4 ° C für 1,5-2 Stunden, um die Fibringerinnsel beheben. </li><li> Holen das Fibringerinnsel in der oberen Schicht unter Verwendung eines Paars von gebogenen Pinzette und quetschen das Fibringerinnsel, das Serum zu trennen. Drücken Sie dann die Fibringerinnsel mit den gebogenen Pinzette gegen downwards nahe der Grenzfläche zwischen den beiden Schichten (Fig. 1D). </li><li> Wiederholen Sie Schritt 3.3 weiter lösen das Fibringerinnsel. </li><li> Zentrifugieren Sie die Röhrchen für 5 min bei 1.200 xg und RT (1E). </li><li> Das Serum vorsichtig in einem 15 ml sterilen Röhrchen übertragen mit einer sterilen Pipette in einer Laminar-Air-Flow-Gehäuse. </li><li> Zentrifugieren des gesammelten Rattenserum für 5 min bei 1.200 × g und Raumtemperatur, um jegliche verbleibenden roten Blutkörperchen zu entfernen. </li><li> Pool Serum vorsichtig in ein neues 15 ml sterilisierten Röhrchen unter Verwendung einer sterilen Pipette, um eine Kontamination durch restliche Erythrozyten am Boden des Rohres (1F) zu vermeiden. </li><li> Inkubieren des Serums in einem Wasserbad bei 56 ° C für 30 min, um das Komplementsystem zu inaktivieren. </li><li> Kühlen Sie die Rohre aus Rattenserum auf RT im laminaren Luftstrom Schrank. Das Serum sollte auf 10 ml, einzeln steril Röhrchen aliquotiert werden. Die Röhrchen bei -20 ° C bis zur Verwendung (1H). </li></ol>

<ol>
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No conflicts of interest are declared.

Reproduzierbare Ergebnisse in WEC Experimente sind abhängig von der Verwendung von hochwertigen IC Rattenserum und genaue Embryo Dissektionstechnik 3. Den Zeitraum des Fastens vor Blutentnahme nicht kürzen, weil das Fasten ist notwendig, um Glucose-Konzentrationen im Blut zu standardisieren. Weibliche Ratten, nicht zur Herstellung von Serum verwendet werden, da Hormonspiegel ändern bei weiblichen Tieren nach dem Östrus-Zyklus, und diese Änderungen in Hormone Östradiol für Embryokulturen geeignet sind. ...

Eine Vielzahl von Modelltiere sind in der Entwicklungsbiologie zur Entwicklungsmechanismen auf molekularer und zellulärer Ebene zu untersuchen. Zum Beispiel Amphibien-und Vogelspezies wurden weithin als klassische Modell Tiere, die für die direkte Behandlung der Embryonen sind, weil diese Embryonen entwickeln sich außerhalb der Mutter eingesetzt. Im Gegensatz zu diesen Tieren wachsen Säugetier-Embryonen in die Gebärmutter der Mutter, und das Wachstum in späteren Phasen hängt entscheidend von der Funktion der Gebärmutter. Daher ist es in der Regel schwierig, Säugetierembryos, wie sie aus der Maus und Ratte in einem frühen Stadium direkt zu manipulieren. In den 1960er Jahren wurde eine neue Denis ganze Säugetierembryokultur (WEC)-Technik mit WEC Geräte mit einer kontinuierlichen Sauerstoffzufuhr und Wärme ein. WEC können Maus-und Rattenembryos zu wachsen ex vivo (dh außerhalb des Uterus). Obwohl die WEC-Technik wurde oft in Teratologie durch Zugabe verschiedener chemic verwendetAl-Verbindungen in dem Kulturmedium wurde diese Technik auch in verschiedenen Entwicklungsbiologie Studien verwendet worden, um einzigartige Entwicklungsmechanismen in Säugetieren 2-4 zu prüfen. Zum Beispiel wird WEC mit anderen Techniken, wie z. B. Zellmarkierung, in Wildtyp-und Mutanten-Embryos unter Verwendung fluoreszierender Farbstoff 5, Zelltransplantation 6 und Gen-Einführung über 7 Lipofektion und Elektroporation 8-13 kombiniert. Kürzlich wurde in utero Manipulation verwendet wurde, um Entwicklungsprozesse in Nagetierembryonen in späteren Stadien zu analysieren und mit Elektroporationsverfahren 14-16 zusammengefasst. Jedoch sind diese Techniken nicht für die Handhabung von Postimplantationsembryonen und midgestation aufgrund von Schwierigkeiten Erzielung einer genauen lokalen Injektion der DNA-Lösung in Embryonen im Frühstadium. Obwohl Ultraschall-geführte Zelltransplantation und Injektion von viralen Vektoren in frühen Embryonen (dh, E8.5-E-9.5 in der Maus) in utero wurde berichtet, zuvor 17,18, sind sehr gute Kenntnisse erforderlich, um diese Experimente mit einer hohen Erfolgsquote durchzuführen. Daher WEC mit hoher Zugänglichkeit ex vivo hat Vorteile in Bezug auf die Manipulation der Maus-und Rattenembryos. Sofort zentrifugiert (IC) Rattenserum von männlichen Ratten vorbereitet wird oft für WEC Medium verwendet. Wenn Embryonen sind in der Postimplantationsstufe (dh früher als E8.0 im E10.0 Maus oder der Ratte) kultiviert wird eine Mischung aus synthetischen Mediums und IC Rattenserum häufig als Medium für WEC 19 verwendet. Doch die Kultur Embryonen in der Mitte der Schwangerschaft (dh., E8.0-12.5 in Maus-Embryonen oder E10.0-E14.5 in Rattenembryonen), 100% Serum sollte als Medium verwendet werden, da es derzeit keine alternative Medien ermöglicht Embryonen in vitro normalerweise länger als 2 Tage wachsen. Die Herstellung von hochwertigen RattenserumEin entscheidender Schritt zur Erreichung Reproduzierbarkeit WEC Experimenten. Verglichen mit verzögerter Zentrifugation hat den IC Vorteil der Reduzierung der Hämolyse, wenn das Serum wird durch Zusammendrücken des Fibringerinnsels gesammelt, da die meisten der roten Blutzellen bereits aus der Fibrin-Gerinnsel getrennt. Als hämolytischer Rattenserum nicht auf das normale Wachstum der Ratten-und Maus-Embryonen zu unterstützen, ist die Herstellung von Serum mit sofortiger Zentrifugation bevorzugt Zubereitung mit verzögerter Zentrifugation. Unser Protokoll enthält zwei zusätzliche Schritte im Vergleich zu anderen Protokollen 18-20 (dh., Speichern des gesammelten Bluts auf Eis vor der ersten Zentrifugation und Halten der gesammelten Blutproben 2 h bei 4 º C nach der ersten Zentrifugation). Die ersteren Stufe die Bildung des Blutgerinnsels zu verzögern, und der letztere Schritt fördert die Verfestigung des Fibringerinnsels für einfache Quetschen. Daher kann unser Protokoll von Anfängern verwendet werden. Allerdings reproduzieren Blutentnahme und SerumExtraktion genau durch einfaches Protokoll Bücher bezieht, ist extrem schwierig, 19-21. In diesem Video-Beitrag zeigen wir, unsere Standard-Protokoll für die Herstellung der optimalen IC Rattenserum, die Blutentnahme aus der Bauchaorta von männlichen Ratten und Gewinnung von Serum durch Zentrifugation enthält.

<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>
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		</tr>
	</tbody>
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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.

Abbildung 1 zeigt repräsentative Ergebnisse von den beschriebenen Verfahren zur Abtrennung von Blutzellen und Serum. Wir erhalten in der Regel 15 ml Blut aus einem pensionierten männlichen Ratte (Abbildung 1A). Durch Zentrifugieren, kann das gesammelte Blut in eine obere Schicht, die das Serum und das Fibringerinnsel, die mit einem Pfeil angedeutet ist, und eine untere Schicht, die die Blutzellen (Fig. 1C) getrennt werden. Wichtiger ist, hämolytische Blutproben nicht...

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.

Herstellung von Ratten Serum Geeignet für Säugetier 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 ...

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

Research

JoVE Journal

Biology

13.8K Aufrufe.  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.

Serie: 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. 

Organotypischen Kultur der E18 Gehirn von Ratten

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

Forschung

Biologie

Preparation of Gene Gun Bullets and Biolistic Transfection of Neurons in Slice Culture

Biolistic transfection is a physical means of transfecting cells by bombarding tissue with high velocity DNA coated particles. We provide a detailed protocol for biolistic transfection of rat hippocampal slices, from the initial preparation of DNA coated bullets to the final shooting of the organotypic slice cultures using a gene gun. Gene gun transfection is an efficient and easy means of transfecting neurons and is especially useful for fluorescently labeling a small subset of cells in tissue slice. In this video, we first outline the steps required to coat gold particles with DNA. We next demonstrate how to line the inside of plastic tubing with the gold/DNA bullets, and how to cut this tubing to obtain the plastic cartridges for loading into the gene gun. Finally, we perform biolistic transfection of rat hippocampal slice cultures, demonstrating handling of the Bio-Rad Helios gene gun, and offering trouble shooting advice to obtain healthy and optimally transfected tissue slices.  

We describe a method for preparing DNA coated gold bullets and demonstrate the use of such bullets to biolistically transfect neurons in cultured hippocampal slices.

Vorbereitung der Gene Gun und Aufzählungszeichen Biolistic Transfektion von Neuronen in Scheiben Kultur

Biolistic transfection is a physical means of transfecting cells by bombarding tissue with high velocity DNA coated particles. We provide a detailed ...

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.

Vorbereitung und Kultur der Rat Linsenepithelzellen Explantate für ein Studium der terminalen Differenzierung

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.

Ein Ex-ovo Chicken Embryo Culture-System Geeignet für Imaging und Mikrochirurgie Anwendungen

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.

Die Methode der Nager Whole Embryo Culture mit der Rotator-type Bottle Kultur-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 ...

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.

Vorbereitung der Rattenschwanzsehnen für Biomechanik und 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 ...

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.

Vorbereitung von embryonalen Maus-Fibroblasten-Zellen zum Kultivieren von humanen embryonalen und induzierten pluripotenten Stammzellen

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.

Maus embryonalen Entwicklung in einem Serum-freien 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 ...

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.

Wohnung Berge Vorbereitung für Beobachtung und Analyse der Zebrafisch-Embryo Proben von Bunt Ganze Berge<em&gt; In situ</em&gt; Hybridisierung

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.

Nicht-enzymatische, Serumfreie Zellkultur von Pre-invasive Brustläsionen für Spontane Erzeugung Mammospheres

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