Name: in vitro culture of epicardial cells from mouse embryonic heart
Uploaded: 2016-04-27T14:00:00.000+00:00
Duration: 6 min 31 s
Description: Watch this Scientific Journal Video about In Vitro Culture of Epicardial Cells From Mouse Embryonic Heart at JoVE.com

Bu çalışma DUKE-NUS Lisans Tıp Fakültesi Singapur, Manvendra K. Singh Goh vakıf ve Singapur NRF bursu (NRF-NRFF2016-01) fon tarafından desteklenmiştir.

Tüm deneyler Duke-NUS Lisans Tıp Fakültesi Kurumsal Hayvan Bakım ve Kullanım Kurulu tarafından kabul edildi. 1. Embriyonik karıncıklar alınıyor <ol><li> karbondioksit gaz temini veya başka bir onaylanmış ötenazi yöntemi ile bir ötenazi odasını kullanarak istenen embriyonik aşamada (E11.5 veya E12.5) bir zaman aşımına hamile fare Kurban. </li><li> diseksiyon masaya sırtında fare yerleştirin. % 70 etanol ile kadın karın dezenfekte edin. karın üzerindeki deriyi kaldırın ve bir bıçak ile küçük bir kesi (1-2 cm) yapmak, sonra iki elinizle cildinizi tutun ve karın duvarı açığa çıkarmak için uzağa çekin. </li><li> Şimdi uterin boynuz maruz karın duvarı kesti. uterin horn yukarı kaldırmak için steril forseps kullanın ve dikkatli uterin boynuz bağlı yağ doku ve kan damarları kesip. serviks kesilmiş uterusu almak için. </li><li> Steril soğuk 1x Fosfat Buff içeren bir petri uterin horn koyun<html> Ered tuz (PBS) ve yavaşça çalkalayın. Buz üzerinde petri yerleştirin. </li><li> yumurta sarısının içinde hala ve plasenta yoluyla rahim duvarına bağlı embriyolar maruz (ters plasenta bulunduğu siteye) uterin horn orta hat boyunca kesmek için makas kullanın. </li><li> Steril forseps kullanarak, embriyo serbest embriyonik yolk sac açık kesti. </li><li> Steril soğuk 1x PBS içeren yeni bir petri embriyo yerleştirin. embriyo başını kesmek ve sırtında üzerine yerleştirin. kalp maruz göğüs duvarı açık kesti. </li><li> kalbi kaldırın ve göğüs duvarından kalbini kurtarmak için onun etrafında damarları kesip steril forseps kullanabilir. cerrahi aletler ile kalbin epikardiyumu zarar vermemek için özen gösterin. </li><li> çıkış yolunu hem atriyumları keserek. Soğuk 1x PBS ile başka bir çanak ventrikül aktarın. Buz üzerinde çanak yerleştirin. </li><li> Tekrarlayın tüm karıncık alınır kadar 1.6-1.9 adımları. </li></ol>le &quot;&gt; 2. Epikardiyal Eksplantasyon Kültür Not: Kültür cam haznesi slaytlar üzerinde ya da alt uygulamalar için kollajen jel üzerinde ya bu epikardiyal eksplantlar. <ol><li> Cam Odası Slaytlar <ol><li> Kültür ortamı hazırlamak Dulbecco tadil edilmiş Eagle ortamı (DMEM)% 10 fetal sığır serumu (FBS),% 1 penisilin / streptomisin ve 2 ng / ml yeniden birleştirici fibroblast büyüme faktörü 2 (FGF2) ekleyin. laminer akış kaputu tüm adımları uygulayın. </li><li> 8 oyuklu bölmenin her bir kuyu için 500 ul kültür ortamı eklenir. </li><li> Bir kuyunun merkezinde her eksize ventrikül yerleştirin. iyi alt yüzeyinde aşağı disseke tarafı tutmak için ventrikül yönlendirmek. </li><li> Yavaşça 37 ° C,% 5 CO2, hücre kültürü inkübatöründe plaka koydu. </li><li> eksplantlar uymak için izin minimum rahatsızlık kültürünü korumak. Epikardiyal tek tabaka oluşumu 48-72 saat sonra gözlenebilir. </li><li> epicard ayrımındadüz kas hücreleri, kültür bir 6 gün farklılaşma medya epikardiyal tek tabakalı hücreler içine ial hücreleri. Farklılaşma ortam hazırlamak DMEM,% 10 FBS,% 1 penisilin / streptomisin ve 50 ng / ml yeniden birleştirici transforme edici büyüme faktörü beta 1 (TGF-β1) ekleyin. </li><li> diğer günlerde kültür ortamı değiştirin. </li></ol></li><li> kollajen jel <ol><li> imalatçının protokolüne göre, ticari bir 3 boyutlu Kollajen Kültür kiti kullanılarak bir 96-çukurlu plaka içinde kollajen jel hazırlayın. </li><li> kollajen jel istenilen hacmi hazırlamak için, DMEM 5x ve pipet yukarı ve aşağı kollajen çözüm uygun miktarda karıştırın. Çözelti sarı dönmelidir. </li><li> nötralizasyon solüsyonu gelen hacim ekleme ve yukarı ve aşağı pipetleme iyice hemen karıştırın. Çözelti pembe dönecek. </li><li> Pipet, 96 oyuklu plakanın her bir oyuğuna, bu karışımın 100 ul. Al, 30-60 dakika boyunca 37 ° C,% 5 CO2 inkübatörü içinde plakasınıdüşük jel polimerize etmek. </li><li> Her bir oyuğa, yukarıda tarif edilen kültür ortamı 200 ul ekle. </li><li> Bir kuyunun merkezinde her eksize ventrikül yerleştirin. Orient ventrikül (deneyci bakmalıdır ventrikül apeks) kollajen jel üzerinde disseke tarafı tutmak için. Yavaşça geri hücre kültürü inkübatör plaka koymak. </li><li> 3 gün sonra, ventriküllere kaldırın. başka bir 2 gün hücre kültürü kuvöz içine geri plaka koyun. </li><li> diğer günlerde kültür ortamı değiştirin. </li></ol></li></ol> 3. Fiksasyon ve Görselleştirme Not: Epikardiyal hücreler cam haznesi slaytlar veya kollajen jel ya da üzerinde görülebilir. <ol><li> kültür ortamı üzerinden aspire ve hücreleri yıkamak için steril 1x PBS eşit hacimde ekleyin. </li><li> hücreleri kaplamak için yeterli miktarda% 4 paraformaldehit (PFA) ekleyin. 4 ° C&#39;de 10 dakika boyunca hücreler sabitleyin. </li><li> PFA çıkarın ve 1x PBST (1x PBS ile eksplantlar yıkayın% 0.1 Triton X-100) hücreleri geçirgenliği. </li><li> İstenen antikor (örneğin, Alexa Fluor 488-falloidin, ZO-1, α-tubulin α-düz kas aktini) ile immunostain 7. </li></ol>

<ol>
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The authors declare that they have no competing financial interests.

Kalp gelişme ve rejenerasyon epikarda artan önemi hitap epikardiyumun çalışma kolaylaştıran teknikleri geliştirmek için kilit önem taşımaktadır. epikardiyal kültür sistemi epikardiyal araştırma için önemli avantajlar getirmektedir. Epikardiyal hücreleri izole etmek için alternatif bir yol floresans ile aktive edilen hücre tasnif (FACS) kullanmaktır. Bu yöntem, diğer soylarından epikardiyal hücreleri ayırmak için epikardiyal belirteçleri (veya floresan protein ep...

Deneysel verilerin bir servet epicardium kalp gelişiminde kritik adımlar etkilediğini gösterdik. Gelişimi sırasında, septum transversum proepicardium 1-4 şekilde bilinmektedir mezotelyal hücrelerinin bir yığın yol açar. proepicardium gelen hücreler, göç ve endokard oluşturucu miyokard zarf. Bunu takiben, epikardiyal hücrelerinin bir alt, daha sonra miyokard işgal epikardiyum türetilmiş hücreler (EPDCs) içindeki bir göç popülasyonuna EMT veren yükselmesine uğrar. Deney izleme genetik olarak retroviral nesilli EPDCs düz kas hücreleri, fibroblastlar, endotel hücreleri ve kardiyomiyositlerde (eğer varsa) dahil olmak üzere çeşitli soy ayırt olduğunu göstermiştir. Bu nedenle EPDCs koroner damar sistemi ve miyokard mimarisinin 1,2,4-9 gelişmesine önemli katkı sağlamaktadır. Ayrıca, epikardiyum ventriküler yoğun tabakaya 10-12 gelişimi için gereklidir. example Gittenberger-de Groot ve ark., proepicardium aşırı büyümesini inhibe etmek, örneğin ince bir miyokard, kalp ve anormal septum oluşumu eksikliği döngü gibi bir sonucu olarak, embriyo ölümüne 13 gibi kusurları bir dizi yol açtığı gösterilmiştir. embriyonik epicardium salgılanan parakrin faktörler kardiyomiyosit çoğalmasını ve farklılaşmasını modüle. Buna uygun olarak, bu tür retinoik asit (RA), fibroblast büyüme faktörleri (FGFs) ve Wnt / β-katenin olarak sinyal yollarının epikardiyum özgü silinmesi bozuk miyokardiyal büyüme ve embriyonik öldürücülüğü 14-16 sonuçlandı. Epicardium yetişkin kalpte sessiz olduğuna inanılan olmasına rağmen, son yıllarda yapılan çalışmalarda gelişimsel bir program kardiyak hasar 17,18 aşağıdaki epicardium olarak yeniden olduğunu göstermiştir. aktivasyonu üzerine, hücreler EPDC oluşumuna neden hızla çoğalması ve EMT geçer. Bu hücreler capacit sergileyenY, fibroblast ve düz kas hücrelerinin değil kardiyomiyositlerde ve endotel hücreleri 18 ayırt etmek. Buna ek olarak, EPDCs yaralı bölgenin damarlanma yardım ve böylece enfarktüs boyutunu azaltarak gelişmiş kardiyak fonksiyonu kolaylaştıran proanjiyogenik faktörler salgılar. Nedeniyle bu bulgulara, epicardium kardiyovasküler geliştirme, hastalık ve yenilenme çalışmasında ilgi kazanmıştır. Transgenik teknoloji 21. yüzyılda tıbbi araştırma devrim yarattı. Transgenik teknolojilerin yardımıyla, metabolik ve patofizyolojik insan durumunu taklit eden hastalıklı fare modelleri başarıyla geliştirilmiştir. Ancak, bu mutantlar epikardiyal hücre davranışını okuyan erken embriyonik öldürücülüğü kaynaklanmaktadır bir meydan okuma olmuştur. Epicardium kalp gelişim ve yenilenme oynadığı önemli rol göz önüne alındığında, biz fare epicardia için bir in vitro kültür sistemi kurdukL hücreleri. Bu yöntem, epikardiyal hücrelerin uzun süreli kültür sağlar ve endokard iki önemli özelliklerinin detaylı bir çalışma kolaylaştırır: göç eder ve ayırt etme yeteneği. fare eksize ventriküller geçiş deneyleri yürütmek için kullanılabilir kollajen jel üzerinde kültürlenebilir olabilir. Daha subepikardiyal tabakanın kollajen zengin hücre dışı matrisi çoğaltır, in vivo hücre fizyolojisi tekrarlar 3B matris içinde kültüre edildikten. Alternatif olarak daha sonra aşağı doğru çeşitli uygulamalar için kullanılabilir epikardiyal tek tabaka oluşturmak amacıyla oda slaytlar üzerinde kültürlenebilir. Bu tek-katmanın taşıma için çok önemlidir EMT geçmesi epikardiyumun yeteneği hakkında bilgiler sağlayacaktır sıkı bağlantı proteinleri için leke kullanılabilir. Buna ek olarak, farklılaşma deneyler de bu hücrelerde gerçekleştirilebilir. Bundan başka, gen ekspresyon profili hücrelerinden RNA ekstrakte ile analiz edilebilir vekantitatif polimeraz zincir reaksiyonu (qPCR) gerçekleştirmek. Son olarak, tek tabakalar, aynı zamanda potansiyel terapötikler test etmek için bir moleküler analiz, ardından maddelerle tedavi edilebilir. Birlikte koymak, bu epikardiyal kültür sistemi görselleştirmek ve epikardiyal gelişimi hakkında anlayışımızı furthers moleküler veri toplamak için fırsat sağlıyor. Bu yöntemin diğer bir istenen özelliği, basittir ve herhangi karmaşık ayar gerekli olmasıdır. Kısaca, embriyolar kalp eksize E11.5 veya E12.5 aşağıda hasat edilir. ventriküller sonra kollajen jel veya oda slaytlar ya üzerinde yetiştirilmiştir. Daha sonra, bu hücreler, aşağı doğru deneyler kullanılabilir.

<table><tbody><tr><td>Dulbecco&amp;rsquo;s modified Eagle&amp;rsquo;s medium (DMEM)</td><td>Life tech invitrogen&amp;nbsp;</td><td>11995065</td><td></td></tr><tr><td>Penicillin/streptomycin solution</td><td>Life tech invitrogen&amp;nbsp;</td><td>15140122</td><td></td></tr><tr><td>Fetal bovine serum (FBS)&amp;nbsp;</td><td>Life tech invitrogen&amp;nbsp;</td><td>10500064</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma</td><td>P6148-5KG</td><td></td></tr><tr><td>Recombinant fibroblast growth factor 2 (FGF2)</td><td>PeproTech</td><td>450-33</td><td></td></tr><tr><td>Recombinant transforming growth factor beta 1 (TGF-&amp;beta;1)</td><td>PeproTech</td><td>100-21</td><td></td></tr><tr><td>ZO-1 antibody</td><td>Life tech invitrogen&amp;nbsp;</td><td>40-2200</td><td></td></tr><tr><td>&amp;alpha;-Tubulin antibody</td><td>Sigma</td><td>T 6074</td><td></td></tr><tr><td>&amp;alpha;-smooth muscle actin (SMA) antibody</td><td>Sigma</td><td>A 2547</td><td></td></tr><tr><td>Phalloidin antibody</td><td>Life tech invitrogen&amp;nbsp;</td><td>A12379</td><td></td></tr><tr><td>3D Collagen Culture kit&amp;nbsp;</td><td>Millipore&amp;nbsp;</td><td>ECM 675</td><td></td></tr><tr><td>8-well chamber slide</td><td>Fisher Scientific</td><td>NNU 154534-PK</td><td></td></tr><tr><td>Trizol reagent</td><td>Life Technologies</td><td>15596-018</td><td></td></tr><tr><td>ViiA 7 Real-Time PCR System</td><td>Life Technologies</td><td>4453536</td><td></td></tr><tr><td>Superscript First Strand Synthesis kit</td><td>Life Technologies</td><td>11904-018</td><td></td></tr></tbody></table>

in vitro culture of epicardial cells from mouse embryonic heart

During embryogenesis, the epicardial contribution to coronary vasculature development has been very well established. Cells derived from the epicardium differentiate into smooth muscle cells, fibroblasts and endothelial cells that contribute to the formation of coronary vessels. Here we have established an in vitro culture method for embryonic epicardial cells. Using genetic labelling, we have demonstrated that the majority of the migrating cells in our explant culture are of epicardial origin. Epicardial explant cells also retain the expression of epicardial markers (Wt1 and Tbx18). Furthermore, we provide evidence that epicardial explant cells undergo epithelial to mesenchymal transition (EMT), migrate and differentiate into smooth muscle cells after Transforming growth factor beta 1 (TGF-&#946;1) treatment in a manner indistinguishable from that of epicardial cells in vivo. In conclusion, we provide a novel method for the culture of embryonic epicardial cells, which will help to explore the role of specific genes in epicardial cell biology.

Bu kültürün protokolü kullanılarak, primer epikardiyal hücreleri alt uygulamalar için yüksek saflıkta izole edilebilir. Kültürlü hücreler, EMT tabi göç ve epikardiyal hücreleri in vivo olduğu gibi ayırt edebiliyoruz. Birincil epikardiyal hücre kültürünün saflığını belirlemek için, Sema3d GFPCre / + elde epikardiyal eksplantlar analiz R26 Tom / ...

Watch this Scientific Journal Video about In Vitro Culture of Epicardial Cells From Mouse Embryonic Heart at JoVE.com

In Vitro Culture of Epicardial Cells From Mouse Embryonic Heart

Fare Embriyonik From The Heart Epikardiyal Hücrelerinin Vitro Kültür

The epicardium is an essential source of multipotent cardiovascular progenitor cells and paracrine factors that are required for cardiovascular development and regeneration. We describe here a method to culture mouse embryonic epicardial cells.

During embryogenesis, the epicardial contribution to coronary vasculature development has been very well established. Cells derived from the epicardium ...

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Research

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Engineering

In Vitro Culture of Epicardial Cells From Mouse Embryonic Heart

7.3K Görüntüleme.  Duke-NUS Graduate Medical School. The epicardium is an essential source of multipotent cardiovascular progenitor cells and paracrine factors that are required for cardiovascular development and regeneration. We describe here a method to culture mouse embryonic epicardial cells. 

Video: In Vitro Culture of Epicardial Cells From Mouse Embryonic Heart

Epicardial Outgrowth Culture Assay and Ex Vivo Assessment of Epicardial-derived Cell Migration

A single layer of epicardial cells lines the heart, providing paracrine factors that stimulate cardiomyocyte proliferation and directly contributing cardiovascular progenitors during development and disease. While a number of factors have been implicated in epicardium-derived cell (EPDC) mobilization, the mechanisms governing their subsequent migration and differentiation are poorly understood. Here, we present in vitro and ex vivo strategies to study EPDC motility and differentiation. First, we describe a method of obtaining primary epicardial cells by outgrowth culture from the embryonic mouse heart. We also introduce a detailed protocol to assess three-dimensional migration of labeled EPDC in an organ culture system. We provide evidence using these techniques that genetic deletion of myocardin-related transcription factors in the epicardium attenuates EPDC migration. This approach serves as a platform to evaluate candidate modifiers of EPDC biology and could be used to develop genetic or chemical screens to identify novel regulators of EPDC mobilization that might be useful for cardiac repair.

Epicardial Outgrowth Culture Assay and Ex Vivo Assessment of Epicardial-derived Cell Migration

Here, we describe methods for isolating primary mouse epicardial cells by an outgrowth culture assay and assessing the functional migration of epicardial-derived cells (EPDC) using an ex vivo heart culture system. These protocols are suitable for identifying genetic and chemical modulators of epicardial epithelial-to-mesenchymal transition (EMT) and motility.

Epikardiyal dışsal gelişimi Kültürü Deneyi ve<em&gt; Ex Vivo</em&gt; Epikardiyal türetilmiş Hücre Göç Değerlendirilmesi

A single layer of epicardial cells lines the heart, providing paracrine factors that stimulate cardiomyocyte proliferation and directly contributing ...

Gelişim Biyolojisi

The Isolation and Culture of Primary Epicardial Cells Derived from Human Adult and Fetal Heart Specimens

The epicardium, an epithelial cell layer covering the myocardium, has an essential role during cardiac development, as well as in the repair response of the heart after ischemic injury. When activated, epicardial cells undergo a process known as epithelial to mesenchymal transition (EMT) to provide cells to the regenerating myocardium. Furthermore, the epicardium contributes via secretion of essential paracrine factors. To fully appreciate the regenerative potential of the epicardium, a human cell model is required. Here we outline a novel cell culture model to derive primary epicardial derived cells (EPDCs) from human adult and fetal cardiac tissue. To isolate EPDCs, the epicardium is dissected from the outside of the heart specimen and processed into a single cell suspension. Next, EPDCs are plated and cultured in EPDC medium containing the ALK 5-kinase inhibitor SB431542 to maintain their epithelial phenotype. EMT is induced by stimulation with TGF&#946;. This method enables, for the first time, the study of the process of human epicardial EMT in a controlled setting, and facilitates gaining more insight in the secretome of EPDCs that may aid heart regeneration. Furthermore, this uniform approach allows for direct comparison of human adult and fetal epicardial behavior.

The epicardium plays a crucial role in the development and repair of the heart by providing cells and growth factors to the myocardial wall. Here, we describe a method to culture human primary epicardial cells that enables the study and comparison of their developmental and adult characteristics.

Yalıtım ve kültür insan yetişkin ve Fetal kalp örnekler elde edilen birincil Epicardial hücre

The epicardium, an epithelial cell layer covering the myocardium, has an essential role during cardiac development, as well as in the repair response of ...

In Vitro Generation of Heart Field-specific Cardiac Progenitor Cells

Pluripotent stem cells offer great potential for understanding heart development and disease and for regenerative medicine. While recent advances in developmental cardiology have led to generating cardiac cells from pluripotent stem cells, it is unclear if the two cardiac fields - the first and second heart fields (FHF and SHF) &#8212; are induced in pluripotent stem cells systems. To address this, we generated a protocol for in vitro specification and isolation of heart field-specific cardiac progenitor cells. We used embryonic stem cells lines carrying Hcn4-GFP and Tbx1-Cre; Rosa-RFP reporters of the FHF and the SHF, respectively, and live cell immunostaining of the cell membrane protein Cxcr4, a SHF marker. With this approach, we generated progenitor cells which recapitulate the functional properties and transcriptome of their in vivo counterparts. Our protocol can be utilized to study early specification and segregation of the two heart fields and to generate chamber-specific cardiac cells for heart disease modelling. Since this is an in vitro organoid system, it may not provide precise anatomical information. However, this system overcomes the poor accessibility of gastrulation-stage embryos and can be upscaled for high-throughput screens.

The purpose of this method is to generate heart field-specific cardiac progenitor cells in vitro in order to study the progenitor cell specification and functional properties, and to generate chamber specific cardiac cells for heart disease modelling.

In vitro Generation fare kalp alanı-spesifik kardiyak progenitor hücreler

Pluripotent stem cells offer great potential for understanding heart development and disease and for regenerative medicine. While recent advances in ...

Fare Kalplerinde Kardiyak Hücrelerin Elektroporasyona Dayalı Transgenezi

An Efficient Transgenesis Approach for Gene Delivery in the Mouse Embryonic Heart

The mammalian heart is a complex organ formed during development via highly diverse populations of progenitor cells. The origin, timing of recruitment, and fate of these progenitors are vital for the proper development of this organ. The molecular mechanisms that govern the morphogenesis of the heart are essential for understanding the pathogenesis of congenital heart diseases and embryonic cardiac regeneration. Classical approaches to investigate these mechanisms employed the generation of transgenic mice to assess the function of specific genes during cardiac development. However, mouse transgenesis is a complex, time-consuming process that often cannot be performed to assess the role of specific genes during heart development. To address this, we have developed a protocol for efficient electroporation and culture of mouse embryonic hearts, enabling transient transgenesis to rapidly assess the effect of gain- or loss-of-function of genes involved in cardiac development. Using this methodology, we successfully overexpressed Meis1 in the embryonic heart, with a preference for epicardial cell transfection, demonstrating the capabilities of the technique.

Yazar Spotlight: Hızlı Gen Taraması için Kardiyak Hücre Transgenezi

This protocol presents a detailed methodological framework for electroporation-based transgenesis of cardiac cells in developing mouse hearts. The video assets provided here will facilitate learning of this versatile technique.

Fare Embriyonik Kalbinde Gen İletimi için Etkili Bir Transgenez Yaklaşımı

The mammalian heart is a complex organ formed during development via highly diverse populations of progenitor cells. The origin, timing of recruitment, ...

Isolating Primary Melanocyte-like Cells from the Mouse Heart

We identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial arrhythmia triggers in mice. To investigate the electrical and biological properties of cardiac melanocytes we developed a procedure to isolate them from mouse hearts that we derived from those designed to isolate neonatal murine cardiomyocytes. In order to obtain healthier cardiac melanocytes suitable for more extensive patch clamp or biochemical studies, we developed a refined procedure for isolating and plating cardiac melanocytes based on those originally designed to isolate cutaneous melanocytes. The refined procedure is demonstrated in this review and produces larger numbers of healthy melanocyte-like cells that can be plated as a pure population or with cardiomyocytes.

In this protocol, we identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial arrhythmia triggers in mice.&#160;

Fare Kalpten İlköğretim Melanocyte gibi Hücreleri Ayırma

We identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial ...

Biyoloji

Isolation and Culture of Neonatal Mouse Cardiomyocytes

Cultured neonatal cardiomyocytes have long been used to study myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow for easy investigation and manipulation of biochemical pathways, and their effect on the biomechanical properties of spontaneously beating cardiomyocytes.	
The following 2-day protocol describes the isolation and culture of neonatal mouse cardiomyocytes. We show how to easily dissect hearts from neonates, dissociate the cardiac tissue and enrich cardiomyocytes from the cardiac cell-population. We discuss the usage of different enzyme mixes for cell-dissociation, and their effects on cell-viability. The isolated cardiomyocytes can be subsequently used for a variety of morphological, electrophysiological, biochemical, cell-biological or biomechanical assays. We optimized the protocol for robustness and reproducibility, by using only commercially available solutions and enzyme mixes that show little lot-to-lot variability. We also address common problems associated with the isolation and culture of cardiomyocytes, and offer a variety of options for the optimization of isolation and culture conditions.

Primary mouse cardiomyocyte cultures are one of the pivotal tools for the investigation of myofibrillar organization and function. The following protocol describes the isolation and culture of primary cardiomyocytes from neonatal mouse hearts. The resulting cardiomyocyte cultures may be subsequently used for a variety of biomechanical, biochemical and cell-biological assays.

İzolasyon ve Kültür Yenidoğan Fare kardiyomiyositlerin

Cultured neonatal cardiomyocytes have long been used to study  myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow  for easy ...

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. Thus, protocols to isolate and culture individual organs of interest are essential to provide a method of both visualizing changes in development and allowing novel treatment strategies. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel. This substrate provides enough support to maintain the three-dimensional structure but is flexible enough to allow continued contraction. In brief, hearts are excised from the embryo and placed in a mixture of cold Matrigel diluted 1:1 with growth medium. After the diluted Matrigel solidifies, growth medium is added to the culture dish. Hearts excised as late as embryonic day 16.5 were viable for four days post-dissection. Analysis of the coronary plexus shows that this method does not disrupt coronary vascular development. Thus, we present a novel method for long-term culture of embryonic hearts.

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel.

Yeni Bir<em&gt; Ex vivo</emEmbriyonik Fare Kalp için&gt; Kültür Yöntemi

Developmental studies in the mouse are hampered  by the inaccessibility of the embryo during gestation. Thus, protocols to  isolate and culture individual ...

Isolation of Embryonic Ventricular Endothelial Cells

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines offer significant advantages for their ease of use, these cell lines are unavailable for all potential cell types. By isolating primary cells from a specific region of interest, particularly from a transgenic mouse, more nuanced studies can be performed. The basic technique involves dissecting the organ or partial organ of interest (e.g. the heart or a specific region of the heart) and dissociating the organ to single cells. These cells are then incubated with magnetic beads conjugated to an antibody that recognizes the cell type of interest. The cells of interest can then be isolated with the use of a magnet, with a short trypsin incubation dissociating the cells from the beads. These isolated cells can then be cultured and analyzed as desired. This technique was originally designed for adult mouse organs but can be easily scaled down for use with embryonic organs, as demonstrated herein. Because our interest is in the developing coronary vasculature, we wanted to study this population of cells during specific embryonic stages. Thus, the original protocol had to be modified to be compatible with the small size of the embryonic ventricles and the low potential yield of endothelial cells at these developmental stages. Utilizing this scaled-down approach, we have assessed coronary plexus remodeling in transgenic embryonic ventricular endothelial cells.

Primary cell culture is a useful technique for analyzing specific populations of cells, particularly from transgenic mouse embryos at specific developmental stages. Herein, embryonic ventricles are dissected and dissociated, and antibody-conjugated beads recognize and separate out the endothelial cells for further analysis.

Embriyonik Ventriküler Endotel Hücre İzolasyonu

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines ...

Fare Embriyonik From The Heart Epikardiyal Hücrelerinin Vitro Kültür

Özet

Daha Fazla Video Keşfet

Bu videodaki bölümler

Epikardiyal dışsal gelişimi Kültürü Deneyi ve

Yalıtım ve kültür insan yetişkin ve Fetal kalp örnekler elde edilen birincil Epicardial hücre

In vitro Generation fare kalp alanı-spesifik kardiyak progenitor hücreler

Fare Embriyonik Kalbinde Gen İletimi için Etkili Bir Transgenez Yaklaşımı

Fare Embriyonik Kalbinde Gen İletimi için Etkili Bir Transgenez Yaklaşımı

Fare Embriyonik Kalbinde Gen İletimi için Etkili Bir Transgenez Yaklaşımı

Fare Kalpten İlköğretim Melanocyte gibi Hücreleri Ayırma

İzolasyon ve Kültür Yenidoğan Fare kardiyomiyositlerin

Yeni Bir

Embriyonik Ventriküler Endotel Hücre İzolasyonu