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.

NOT: Hayvan deneyleri Laboratuar Hayvanlarının Bakımı ve Kullanımı için Sağlık Rehberi, National Institutes göre yapılmıştır. Tıp Tohoku Üniversitesi Hayvan Deneyleri için Komite, burada açıklanan deneysel prosedürleri onayladı. 1.. Anestezi ve laparotomi <ol style="margin-left: 40px;"><li> Kan toplama için, spesifik patojensiz, erkek Sprague-Dawley fareleri kullanır. Su sağlarken, en az 18 saat boyunca fareler Hızlı. Kan toplamak için yaş (550-650 gr) 6-8 ayda emekli erkek damızlık sıçanlar kullanın. </li><li> 10 dakika boyunca 2.0-3.5 L / dk &#39;da anestezi tanıtmak için bir anestezi cihazına bağlanmış bir kutuya, bir anestezik% 2,5-4,0 olan izofluran, akış. Anestezi tanıtmak için kutunun içine sıçan aktarın. <ol><li> Forseps ile pençelerinde uyarılmasına tepki kaybı için kontrol ederek anesthetization onaylayın. Sıçanlar wi burun Kapakderinden kan toplama sırasında fareler uyutmak için bir anestezi cihazına bağlanmış bir maske inci. Konsantrasyon ve akış sırasıyla% 2.5-4.0 &#39;de muhafaza ve 2.0-3.5 L / dakika edilir. NOT: Bu embriyolar reaktif sergi 22 izofluran ile anestezi sıçanlardan elde edilen serum içinde kültüre embriyolara kıyasla embriyo gelişiminde önemli bir gecikme ile anestezi altına alınan sıçanlarda toplanan serum içinde kültürlendi için Halothane inhalasyon anestezisi için kullanılmamalıdır. </li></ol></li><li> Kürk kirlenmesini önlemek için, anestezi sıçan karın üzerine% 70 etanol dökerek cildi dezenfekte. Forseps bir çift ile alt bölgede dezenfekte deri ve karın duvarı Pick up ve iç organların maruz büyük bir makas ile toraks bölgeye yönelik eş zamanlı olarak bu katmanları kesti. Forseps bir çift ile karın boşluğu dışında gut yansıtacak. </li><li> Bir ile arka bacaklarda doğru cilt ve karın duvarı kestibüyük makas çifti daha posterior karın bölgesini açığa çıkarmak. Forseps bir çift ile karın boşluğu dışında ekstra yağ yansıtacak. </li><li> Forseps iki çift kullanarak orta hatta viseral yağ Pick up ve abdominal aorta maruz paralel bir yönde dikkatlice yağ bölünmüş. Kolayca abdominal aortun çatallanma belirlemek için uzunlamasına bir doğrultuda visseral yağ bölmek için bu prosedürü yineleyin. Buna ek olarak, aort pulsatil ve bu özelliği ile bitişik vena kava ayırt edilebilir. </li><li> Forseps iki çift ile dikkatlice abdominal aorta etrafında yağ ve çatallanma Pick up ve abdominal aort yağ ayrı. </li></ol> 2.. Kan Toplama <ol style="margin-left: 40px;"><li> Dikkatli bir şekilde aşağı doğru bir yönde konik aort çatallanma, hemen kafatası bir 20 ml şırınga bağlı bir 21 G iğne ucu yerleştirin. Bir şırınga ile tutunel ve bir erkek sıçan kan 15 ml toplamak için yavaş yavaş şırınga çekin. </li><li> Şırıngadan iğne çıkarın ve iki buz soğukluğunda 10 ml steril test tüpleri (Şekil 1A) içine kan dökün. Santrifüj kan pıhtısı oluşmasını geciktirmek için kadar buz üzerinde tutun tüpler. Bir anda birkaç tüpler santrifüj ile santrifüj zamanı azaltın. </li><li> Torakotomi ve kalp ya da işten çıkarma keserek anestezi sıçan Euthanize. </li></ol> 3.. Rat Serum hazırlanması <ol style="margin-left: 40px;"><li> Üst ve alt tabakalar (Şekil 1C) içine kan ayırmak için 1200 x g ve oda sıcaklığında (RT), 5 dakika boyunca toplanan kan santrifüjleyin. </li><li> Fibrin pıhtısı düzeltmek için, 1.5-2 saat boyunca 4 ° C&#39;de tüpler tutun. </li><li> Kavisli bir forseps bir çift kullanarak üst tabakasında fibrin pıhtısı Pick up ve serum ayırmak için fibrin pıhtısı sıkmak. Sonra, do bakan kavisli bir forseps ile fibrin pıhtısı basıniki tabaka (Şekil 1D) arasındaki ara yakın wnwards. </li><li> Bundan başka, fibrin pıhtısı ayırmak için bir adım 3.3 tekrarlayın. </li><li> 1,200 xg ve oda sıcaklığında (Şekil 1 E) 5 dakika boyunca, test tüpleri santrifüjleyin. </li><li> Dikkatli bir laminar hava akışı kabini içinde bir steril pipet kullanılarak 15 ml steril tüp içine serum aktarın. </li><li> Kalan kırmızı hücreleri çıkarmak için 1200 x g&#39;da ve oda sıcaklığında 5 dakika boyunca toplanmış sıçan serum santrifüjleyin. </li><li> Tüpün (Şekil 1F) altındaki edilen kırmızı hücreleri tarafından kirlenmesini önlemek için steril bir pipet kullanarak steril bir tüp yeni bir 15 ml&#39;lik dikkatle serum havuzu. </li><li> Kompleman sistemi inaktive edilmesi, 30 dakika boyunca 56 ° C&#39;de bir su banyosu içinde inkübe serum. </li><li> Laminer hava akışı kabini içinde oda sıcaklığına kadar sıçan serum tüpleri soğutun. Serum ayrı steril tüplerde 10 ml aliquoted olmalıdır. Kullanım (Şekil 1 H) kadar -20 ° C&#39;de tüpler saklayın. </li></ol>

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No conflicts of interest are declared.

DEK deneylerde yeniden üretilebilir sonuçlar, yüksek kaliteli IC sıçan serumu ve doğru bir embriyo diseksiyon yönteminin 3 kullanımına bağlıdır. Açlık kan glukoz konsantrasyonları standardize etmek gerekli olduğundan kan toplama önce açlık süresini kısaltmak etmeyin. Hormon düzeyleri östrus döngüsü göre dişi hayvanlarda değiştirmek için dişi sıçanlar serum hazırlanması için kullanılmamalıdır, estradiol ve hormonlar gibi değişikliklerin embriyo kültürleri için uygun...

Hayvan modellerinde çeşitli moleküler ve hücresel düzeyde gelişim mekanizmaları incelemek için gelişim biyolojisi kullanılır. Örneğin, amfibi ve kuş türleri yaygın olarak bu embriyolar, annenin dışında geliştirmek için embriyo doğrudan manipülasyon için uygun olan klasik bir model hayvanları olarak kullanılmıştır. Bu hayvanların aksine, memeli embriyolar annenin rahmine büyür ve daha sonraki aşamalarda büyüme rahim fonksiyonu üzerinde en önemlisi bağlıdır. Bu nedenle, doğrudan bu tür erken aşamalarında, fare ve sıçandan gibi memeli embriyolar işlemek için, tipik olarak zordur. 1960 yılında, Denis Yeni bir sürekli oksijen kaynağı ve ısı kontrolü 1 ile WEC cihazları kullanarak bir memeli bütün embriyo kültürü (WEC) tekniği kurulmuştur. WEC, fare ve sıçan embriyolar ex vivo, (yani, rahim dışında) büyüyebilir. DEK teknik, çoğu zaman, çeşitli chemic eklenerek teratolojide kullanılmış olmasına rağmenal, kültür ortamı içine bileşikler bu teknik aynı zamanda, memelilerde 2-4 benzersiz gelişim mekanizmaları incelemek için çeşitli gelişim biyoloji çalışmalarda kullanılmıştır. Örneğin, DEK floresan boya 5, hücre nakli 6 ve 7 lipofeksiyon ve elektroporasyon 8-13 aracılığıyla gen giriş kullanarak, vahşi tip ve mutant embriyolar, hücre etiketleme gibi diğer tekniklerle ile birleştirilmiştir. Son zamanlarda, utero manipülasyon sonraki aşamalarda kemirgen embriyo gelişim süreçlerini analiz etmek için kullanılır olmuştur ve elektroporasyon teknikleri 14-16 ile kombine edilmiştir. Ancak, bu teknikler erken aşamalarında embriyolara DNA solüsyonu doğru lokal enjeksiyonu elde zorluklar nedeniyle ve implantasyon sonrası midgestation embriyoların manipülasyonu için uygun değildir. Her ne kadar erken embriyo (içine ultrason eşliğinde hücre transplantasyonu ve viral vektörlerin enjeksiyon yani, E8.5-E-9.5 fare) rahimde daha önce 17,18, mükemmel becerileri yüksek bir başarı oranı ile bu deneyleri gerçekleştirmek için gerekli olan rapor edilmiştir. Bu nedenle, yüksek erişilebilirlik ex vivo DEK fare ve sıçan embriyo manipülasyonu ile ilgili avantajlara sahiptir. Erkek sıçanlarda hazırlanabilir hemen santrifüje (IC) sıçan serum genellikle DEK ortamı için kullanılır. Embriyolar (sıçan, fare veya E10.0 E8.0 in daha önce, yani,) implantasyon sonrası aşamasında kültürlendiği zaman, sentetik orta ve IC sıçan serum oluşturduğu bir karışıma genellikle DEK 19 için bir araç olarak kullanılır. Hiçbir mevcut alternatif medya sağlar çünkü Ancak, orta-gebelikte kültür embriyoların (yani., E8.0-12.5 fare embriyoları veya sıçan embriyolarında E10.0-E14.5 in),% 100 serum aracı olarak kullanılması gerektiğini en fazla 2 gün boyunca in vitro olarak normal büyümeye embriyolar. Yüksek kaliteli sıçan serum hazırlanmasıdırWEC deneylerde röprodüsibilitesini ulaşmak için kritik bir adımdır. Gecikmeli santrifüj ile karşılaştırıldığında, IC kırmızı kan hücrelerinin çoğu zaten fibrin pıhtısı ayrılmış çünkü serum fibrin pıhtısı sıkarak ulaşılınca hemoliz azaltma avantajına sahiptir. Hemolitik sıçan serum sıçan ve fare embriyo normal gelişimini desteklemek için başarısız olarak, hemen santrifüj kullanılarak serumun hazırlanması gecikmeli santrifüj kullanılarak hazırlanması tercih edilir. Bizim protokol diğer protokollere göre iki ek adımları içerir 18-20 (yani. Önce ilk santrifüj için buz üzerinde toplanan kan depolama ve ilk santrifüjlemeden sonra, 4 ° C &#39;de 2 saat boyunca, toplanan kan örnekleri bakımı). Eski adım kan pıhtısı oluşmasını geciktirebilir ve ikinci adım kolay sıkma için fibrin pıhtısının katılaşma teşvik etmektedir. Bu nedenle, bizim protokol başlayanlar tarafından kullanılabilir. Ancak, üreyen, kan toplama ve serumsadece protokol kitaplara bakarak doğru ekstraksiyon 19-21 derece zordur. Bu ekran yazıda, erkek farelerde ve santrifüj ile serum ekstraksiyon abdominal aort kan koleksiyonu içerir uygun IC sıçan serumu, hazırlanması için, standart protokol göstermektedir.

<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.

Şekil 1, kan hücreleri ve serumun ayrılması için açıklanan prosedürlere temsili sonuçlarını göstermektedir. Bu tipik olarak, emekli erkek fare (Şekil 1A) kan 15 ml elde edilir. Santrifüj ile, toplanan kan serumu ve bir okla belirtilir fibrin pıhtısı ve kan hücreleri (Şekil 1 C) ihtiva eden bir alt tabaka ihtiva eden bir üst tabaka halinde ayrılabilir. Önemli olarak, hemolitik kan örnekleri, serum ve kan tabakalar (Şekil 1B) için...

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.

Memeli Tüm Embriyo Kültürü için uygun Rat Serum hazırlanması

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 Görüntüleme.  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. 

E18 Rat Brain Organotipik Dilim Kültür

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

Biyoloji

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.

Gen Tabancası İşaretleri ve Dilim Kültür Nöronlar Biyolistik Transfeksiyon hazırlanması

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.

Terminal Farklılaşma incelenmesi için hazırlanması ve Rat Lens Epitel eksplantlar Kültür

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.

Bir Eski-ovo</emGörüntüleme ve Mikrocerrahi Uygulamaları için uygundur> Tavuk Embriyo Kültürü Sistemi

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.

Rotator-tipi Şişe Kültür Sistemi kullanılarak Kemirgen Tüm Embriyo Kültürü Yöntemi

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.

Biyomekanik ve Mechanobiological Araştırmalar Rat Kuyruk Tendonlar hazırlanması

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.

Kültürleme İnsan Embriyonik ve Bağlı Pluripotent Kök Hücrelerinin için uygundur Fare embriyonik fibroblast hücreleri hazırlanması

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.

Serumsuz Tüm Embriyo Kültür Sistemde Fare Embriyonik Development

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.

Tüm Dağı ile Zebra balığı Embriyo örnekler Vitray gözlenmesi ve Analiz Daire Dağı Hazırlık<em&gt; In situ</em&gt; Melezleme

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.

Enzimatik olmayan, Mammospheres spontan Üretimi için Ön-invaziv Meme Lezyonların Serum serbest Doku Kültürü

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

Memeli Tüm Embriyo Kültürü için uygun Rat Serum hazırlanması

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