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.

NOTA: Gli esperimenti sugli animali sono stati condotti in conformità con il National Institutes of Health Guide per la cura e l&#39;uso di animali da laboratorio. Il Comitato per la Sperimentazione Animale della Facoltà di Medicina di Tohoku University ha approvato le procedure sperimentali qui descritte. 1. Anestesia e laparotomia <ol style="margin-left: 40px;"><li> Per la raccolta di sangue, utilizzare specifici ratti maschi Sprague-Dawley esenti da organismi patogeni. Digiunare i ratti per almeno 18 ore, fornendo acqua. Utilizzare pensionati ratti allevatore maschi a 6-8 mesi di età (550-650 g) per la raccolta di sangue. </li><li> Portata 2.5-4.0% isoflurano, che è un anestetico inalatorio, in una scatola collegata ad un apparecchio anestesia introdurre anestesia a 2,0-3,5 L / min per 10 min. Trasferire i ratti nella casella di introdurre l&#39;anestesia. <ol><li> Conferma anesthetization controllando la perdita della risposta alla stimolazione delle zampe con pinze. Coprire il naso di ratti wi° Una maschera collegata ad un apparecchio di anestesia per anestetizzare profondamente i ratti durante il prelievo. La concentrazione e flusso sono mantenuti a 2.5-4.0% e 2.0-3.5 L / min, rispettivamente. NOTA: alotano non deve essere utilizzato per l&#39;anestesia per inalazione perché embrioni coltivati ​​nel siero che vengono raccolti da ratti anestetizzati con questa mostra reagente un ritardo nello sviluppo embrionale rispetto a embrioni coltivati ​​nel siero ottenuti da ratti anestetizzati con isoflurano 22. </li></ol></li><li> Per evitare la contaminazione del pelo, disinfettare la pelle versando il 70% di etanolo sul ventre di ratto anestetizzato. Sollevare la pelle disinfettata e parete addominale in corrispondenza della zona inferiore con un paio di pinze e tagliare questi strati contemporaneamente verso la regione torace con un paio di forbici grandi per esporre gli organi interni. Riflettere l&#39;intestino di fuori della cavità addominale con un paio di pinze. </li><li> Tagliare la pelle e la parete addominale verso gli arti posteriori conpaio di forbici grandi per esporre ulteriormente la regione addominale posteriore. Riflettere il grasso extra al di fuori della cavità addominale con un paio di pinze. </li><li> Sollevare il grasso viscerale sulla linea mediana utilizzando due coppie di pinze e dividere accuratamente il grasso in una direzione parallela esporre l&#39;aorta addominale. Ripetere questa procedura per dividere il grasso viscerale in una direzione longitudinale di identificare facilmente la biforcazione dell&#39;aorta addominale. Inoltre, l&#39;aorta è pulsatile e può essere distinto dalla vena cava adiacente utilizzando questa caratteristica. </li><li> Sollevare il grasso intorno l&#39;aorta addominale e la biforcazione accuratamente con due coppie di pinze e separare il grasso sulla aorta addominale. </li></ol> 2. Raccolta di sangue <ol style="margin-left: 40px;"><li> Inserire delicatamente la punta di un ago G 21 collegato ad una siringa da 20 ml subito cranialmente alla biforcazione dell&#39;aorta, con il bordo inclinato verso il basso. Tenere la siringa con una solamano, e tirare la siringa lentamente per raccogliere 15 ml di sangue da un ratto maschio. </li><li> Rimuovere l&#39;ago dalla siringa, e versare il sangue in due tubi ghiacciati 10 ml sterili test (Figura 1A). Conservare le provette in ghiaccio fino centrifugazione per ritardare la formazione del coagulo. Ridurre il tempo di centrifugazione mediante centrifugazione diversi tubi in una sola volta. </li><li> Euthanize il ratto anestetizzato da toracotomia e tagliando il cuore o decapitazione. </li></ol> 3. Rat Siero Preparazione <ol style="margin-left: 40px;"><li> Centrifugare il sangue raccolto per 5 minuti a 1200 xg e temperatura ambiente (RT) per separare il sangue in strati superiore e inferiore (Figura 1C). </li><li> Conservare le provette a 4 ° C per 1,5-2 ore per risolvere il coagulo di fibrina. </li><li> Sollevare il coagulo di fibrina nello strato superiore usando un paio di pinze curve, e spremere il coagulo di fibrina per separare il siero. Quindi, premere il coagulo di fibrina con le pinze curve fronte downwards prossimità dell&#39;interfaccia tra i due strati (Figura 1D). </li><li> Ripetere passo 3.3 per staccare ulteriormente il coagulo di fibrina. </li><li> Centrifugare le provette per 5 minuti a 1200 xg e RT (Figura 1E). </li><li> Trasferire accuratamente il siero in una provetta sterile da 15 ml con una pipetta sterile in un armadio flusso d&#39;aria laminare. </li><li> Centrifugare il siero di ratto raccolti per 5 minuti a 1200 xg e RT per eliminare cellule rosse rimanenti. </li><li> Pool attenzione il siero in un nuovo tubo 15 ml sterilizzati utilizzando una pipetta sterile per evitare la contaminazione da globuli rossi residue al fondo della provetta (figura 1F). </li><li> Incubare il siero in un bagno d&#39;acqua a 56 ° C per 30 minuti per inattivare il sistema del complemento. </li><li> Raffreddare le provette di siero di ratto a RT nell&#39;armadio aria a flusso laminare. Il siero deve essere frazionare a 10 ml in provette sterili individuali. Conservare le provette a -20 ° C fino al momento dell&#39;uso (Figura 1H). </li></ol>

<ol>
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No conflicts of interest are declared.

Risultati riproducibili in esperimenti WEC dipendono l&#39;uso di siero di ratto IC alta qualità e accurata tecnica embrione dissezione 3. Non accorciare il periodo di digiuno prima del prelievo del sangue, perché il digiuno è necessario standardizzare le concentrazioni di glucosio nel sangue. Ratti femmina non devono essere utilizzati per la preparazione di siero perché i livelli ormonali cambiano in femmine secondo il ciclo estrale, e tali cambiamenti di ormoni estradiolo non sono adatti per colture emb...

Una varietà di modelli animali sono utilizzati in biologia dello sviluppo per studiare meccanismi di sviluppo a livello molecolare e cellulare. Ad esempio, anfibi e aviarie sono stati ampiamente usati come animali modello classico che sono adatti per la manipolazione diretta di embrioni perché questi embrioni si sviluppano al di fuori della madre. In contrasto con questi animali, embrioni mammiferi crescono nell&#39;utero della madre, e la crescita in fasi successive è strettamente dipendente dalla funzione dell&#39;utero. Pertanto, è tipicamente difficile da manipolare direttamente embrioni di mammifero come quelli di topo e nel ratto nelle fasi iniziali. Nel 1960, Denis New istituito un mammifero intera coltura degli embrioni (WEC), tecnica che utilizza apparecchiature WEC con un apporto di ossigeno continuo e controllo di calore 1. In WEC, topo e ratto embrioni possono crescere ex vivo (cioè, al di fuori dell&#39;utero). Anche se la tecnica del WEC è stato spesso utilizzato in teratologia con l&#39;aggiunta di vari chemicAl composti nel terreno di coltura, questa tecnica è stata utilizzata anche in diversi studi di biologia dello sviluppo per esaminare unici meccanismi di sviluppo nei mammiferi 2-4. Ad esempio, WEC è combinata con altre tecniche, quali l&#39;etichettatura delle cellule, in wild-type e mutanti embrioni utilizzando colorante fluorescente 5, il trapianto di cellule 6, e l&#39;introduzione del gene via lipofezione 7 ed elettroporazione 8-13. Recentemente, in manipolazione utero è stato utilizzato per analizzare i processi di sviluppo in embrioni di roditori in fasi successive ed è stato combinato con tecniche di elettroporazione 14-16. Tuttavia, queste tecniche non sono adatti per la manipolazione di postimpianto e midgestation embrioni causa di difficoltà a raggiungere iniezione locale accurata della soluzione di DNA in embrioni nelle fasi iniziali. Sebbene il trapianto di cellule eco-guidata e l&#39;iniezione di vettori virali in embrioni precoci (cioè, Sono stati segnalati E8.5-E-9.5 nel topo) in utero precedentemente 17,18, ottime capacità sono necessarie per eseguire questi esperimenti con un alto tasso di successo. Pertanto, WEC con alta accessibilità ex vivo ha dei vantaggi rispetto alla manipolazione di topo e ratto embrioni. Subito centrifugati (IC) siero di ratto preparati da ratti maschi è spesso usato per le medie WEC. Quando gli embrioni sono coltivate nella fase post-impianto (cioè, prima del E8.0 nel topo o E10.0 nel ratto), una miscela di terreno sintetico e siero di ratto IC viene spesso usata come mezzo per WEC 19. Tuttavia, per gli embrioni di cultura a metà della gestazione (cioè., E8.0-12.5 in embrioni di topo o E10.0-E14.5 in embrioni di ratto), siero al 100% dovrebbe essere utilizzato come mezzo perché nessun media alternativi attualmente disponibili consentono embrioni a crescere normalmente in vitro per più di 2 giorni. La preparazione del siero di ratto di alta qualità èun passo fondamentale per il raggiungimento riproducibilità negli esperimenti WEC. Rispetto a centrifugazione differite, IC ha il vantaggio di ridurre emolisi quando il siero viene raccolto comprimendo il coagulo di fibrina perché la maggior parte dei globuli rossi sono stati già separato dal coagulo di fibrina. Come siero di ratto emolitica non riesce a sostenere la crescita normale di ratto e di topo embrioni, preparazione del siero utilizzando immediata centrifugazione è preferibile preparato utilizzando centrifugazione ritardato. Il nostro protocollo contiene due passaggi aggiuntivi rispetto ad altri protocolli 18-20 (es., Conservare il sangue raccolto in ghiaccio prima della prima centrifugazione e mantenere i campioni di sangue raccolti per 2 ore a 4 ° C dopo la prima centrifugazione). Il primo passo può ritardare la formazione del coagulo di sangue, e quest&#39;ultimo passo promuove la solidificazione del coagulo di fibrina per facile spremitura. Pertanto, il nostro protocollo può essere utilizzato da principianti. Tuttavia, la riproduzione di raccolta del sangue e sieroestrazione con precisione semplicemente facendo riferimento ai libri di protocollo è estremamente difficile 19-21. In questo articolo video, dimostriamo il nostro protocollo standard per la preparazione di siero ottimale ratto IC, che comprende la raccolta di sangue dall&#39;aorta addominale di ratti maschi e estrazione del siero mediante centrifugazione.

<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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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 Figura 1 mostra i risultati rappresentativi delle procedure descritte per la separazione delle cellule del sangue e siero. Abbiamo tipicamente ottiene 15 ml di sangue da un ratto maschio, in pensione (Figura 1A). Per centrifugazione, il sangue raccolto può essere separato in uno strato superiore contenente siero e il coagulo di fibrina, che è indicato con una freccia, e uno strato inferiore contenente i globuli (Figura 1C). È importante sottolineare che i campioni...

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.

Preparazione di Rat Siero Adatto per mammiferi tutta la cultura Embrione

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 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. 

Slice Cultura organotipica di E18 cervello dei topi

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

Ricerca

Biologia

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.

Preparazione di proiettili pistola Gene e Biolistic Transfection dei neuroni in cultura Slice

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.

La preparazione e la cultura di espianti Lens Rat epiteliali per lo Studio differenziazione 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.

Un Ex-ovo Sistema di embrioni di pollo Cultura Adatto per applicazioni di imaging e Microchirurgia

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.

Il metodo di coltura degli embrioni roditori intero utilizzando il Rotator tipo di sistema cultura Bottiglia

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.

Preparazione dei tendini coda di topo per studi di biomeccanica e Mechanobiological

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.

Preparazione di cellule di fibroblasti embrionali di topo adatto per colture di cellule pluripotenti umane embrionali e staminali indotta

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.

Embrionali di topo di sviluppo in un embrione di tutta la cultura di sistema privo di siero

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.

Preparazione di montaggio schermo per l&#39;osservazione e l&#39;analisi di zebrafish campioni macchiato da Whole Monte<em&gt; In situ</em&gt; Ibridazione

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.

Non enzimatica, siero-libero Tissue Culture delle lesioni mammarie pre-invasive per la generazione spontanea di mammospheres

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