Mitral 07CVD04: Bu araştırma, Amerikan Kalp Derneği (Scientist Kalkınma Hibe # 0830384N) ve Vakıf Leducq Hartwell Vakfı tarafından desteklenmektedir.

1. Hazırlık <ol><li> Bereketli bıldırcın veya aşamasında 14 tavuk yumurtası inkübe - veya 25 (2 gün) (4,5 gün) yaklaşık% 60 nem ve 37 ° C. </li><li> Steril Earl Dengeli Tuz Çözeltisi hazırlayın (EBSS) <ol><li> 100 ml, 600 ml 18 M su 10X EBSS </li><li> 2.2 g sodyum bikarbonat </li><li> Çözeltinin pH 7.2 &#39;ye ayarlayın. </li><li> 1000 ml kadar çözüm getirmek </li><li> 0.2 mikron filtre geçerek çözüm sterilize </li></ol></li><li> Steril M199 kültür ortamı hazırlayın. <ol><li> M199 tozu 1 paket 700 ml 18 M su ekleyin. </li><li> 2.2 g sodyum bikarbonat </li><li> PH 7.2 &#39;ye çözüm ayarlayın. </li><li> 10 ml (% 1) Penisilin-Streptomisin </li><li> 1000 ml kadar çözüm getirmek </li><li> 0.2 mikron filtre geçerek çözüm sterilize </li><li> 1 ml steril 100X İnsülin Transferrin-Selenyum-G ek Ekle </li><li> 10 ml steril tavuk serumu (% 1) </li></ol></li><li> 1.5 mg / ml kollajen konsantrasyonu üç boyutlu kolajen jeller hazırlayın. 1.5 mg / ml &#39;den fazla bir kollajen konsantrasyonunu hücreler arasında hareket etmek için çok biyomekanik sert bir matris sağladığı laboratuarımızda Önceki çalışma göstermiştir. Mg / ml hücre tutam hücre üreten bir ada, yerel lifler parçalamak için bu kadar az kolajen liflerinin 1.5 kollajen jel konsantrasyonları daha düşüktür. <ol><li> Aşağıdaki buz reaktifler amacıyla 15 mL steril bir santrifüj tüpüne ekleyin: <ul><li> 3X M199: = toplam hacmi gerekli / 3 Cilt </li><li> Steril 18 M su: Cilt gerekli = Toplam hacmi (M199 hacmi + tavuk serum hacmi + 0.1 M NaOH hacmi + kolajen hacmi) </li><li> Steril tavuk serumu: Hacim = toplam hacmi gerekli * 0,01 </li><li> Sıçan kuyruk kollajen I: Hacim = (* toplam hacmi gerekli kollajen jel konsantrasyonu) / kollajen stok konsantrasyonu </li><li> Steril 0,1 M NaOH: Hacim = kollajen hacmi * 0.2 Not: su miktarı, 0.1 M NaOH ve kollajen, kollajen stok solüsyonun konsantrasyonuna bağlı olarak değişir ekledi. </li></ul></li><li> Kollajen solüsyon, steril bir pipet ile karıştırın. </li><li> 0.3 mL kollajen çözüm hemen 1.9 cm 2 büyüme alanı kuyu içine aktarın. Bu, tamamen iyi kapsayacak ve yaklaşık 3 mm kalınlığında bir jel sağlamak için yeterli kollajen bir çözümdür. Jel en az 250 mm kalınlığı cep işgali çalışmaları için gerekli. </li><li> 37 en az 1 saat jel kollajen çözüm İzin ° C,% 5 CO 2 ve% 100 nem. </li></ol></li></ol> 2. Ön-EMT Endokardial Hücre İzolasyonu ve Kültür <ol><li> Embriyo izolasyon <ol><li> Yumurta büyük ucunu üzerinde laboratuvar bant inç uzunluğunda bir parça yerleştirin. Bu hava hücresinin nerede. Kaset, kırılgan bıldırcın yumurta kabuğu dengeler. </li><li> Yavaşça ponksiyon sterilize kavisli makas ucu ile hava hücre yumurta bantlanmış alan. </li><li> Bantlanmış kısmı yumurta sarısı geçmesine izin vermek için yeterince büyük bir delik kesin. Ponksiyon delik başlayın ve dışa doğru bir spiral şeklinde kesmek. Dış zarından kesmek, ama sarısı piercing önlemek için emin olun. </li><li> Kabuğunda delik tamamlandığında, bıldırcın embriyo görselleştirmek. Embriyonun normal yumurta sarısı üst kısmında yer almaktadır. </li><li> Tek elle, yumurta delik aracılığıyla sarısı dökmek için başlayın. Öte yandan # 55 cımbız tutun. Yumurta sarısı üst kısmında embriyo yumurta çıkar gibi, yavaşça embriyo altında # 55 cımbız slayt ve yumurta sarısı çıkarın. Not: tavuk yumurtası için, embriyo izolasyonu, keskin, temiz bir yüzey üzerine tavuk yumurta çatlama ve 100 mm Petri kabı içine yumurta kırarak içerir. Embriyo, yumurta sarısı üst tarafında yer almalıdır. # 55 cımbız embriyo altında yavaşça kaydırın ve yumurta sarısı çıkarın. </li><li> Embriyo buz gibi soğuk, steril EBSS içeren bir 100 mm Petri kabı içine yerleştirin. </li></ol></li><li> Hamburger ve Hamilton 1 kriterlerine göre, embriyolar Sahne . Embriyolar aşamada 14 - Geçmiş aşamada 13, ancak henüz 20-21 Somitlerin ve bir kuyruk tomurcuk aşamasında 15,. 14 aşamada kilit özelliği - embriyolar vücut için 90 ° &#39;den biraz daha fazla kafa açısı. </li><li> Aşamada 14 bıldırcın embriyoları kalpleri Kaldır - AV kanalı ve OFT göğüs duvarı ile temas kurmaya enine keserek # 55 cımbız. Petri kabı bir tarafta kalpleri bir araya koyun. </li><li> , Steril bir transfer pipet yardımıyla izole kalpleri toplayın ve steril, buz EBSS ile dolu yeni bir 100 mm Petri kabı içine yerleştirin. </li><html><li> # 55 cımbız ile ventriküller OFTs ve AV kanallar enine kesin. </li><li> Bölüm kalan AV kanallar ve / veya boyuna çıkış yolları (OFTs). </li><li> 20 uL hacmi bir pipet ayarlayın. AV kanallar ve OFTs uzunlamasına kesilmiş altı aspire bu pipet kullanın. </li><li> AV kanallar ve kollajen jel üzerine OFTs boşaltınız. Jel yüzeyinden herhangi bir fazla EBSS aspire. </li><li> Düz şekilde yönlendirmek eksplantlar # 55 cımbız kullanın ve böylece lümen tarafında kollajen jel ile karşı karşıyadır. Cımbız ile ponksiyon kollajen jel deneyin. </li><li> 37 az 2 saat sonra ° C ve% 5 CO 2, # 55 cımbız ile eksplantlar miyokardın kaldırmak. Endokardiyal hücreler, kollajen jel yüzey geride bırakılmalıdır. Alternatif olarak, miyokardın eksplant üzerinde bırakılabilir. Miyokardın çıkarıldığında müdahalesi olmadan, değil ile endokardiyal hücreler EMT tabi. Miyokard varken endokardiyal hücrelerinin bir alt kümesi mezenşimin haline dönüştürmek, ancak dönüşüm derecesi dış faktörler ile modüle edilebilir. Not: orta eklenmeden önce miyokard kalacak olsa bile, eksplant jel eklemek için en az 2 saat olmalıdır. </li><li> De içeren her eksplantlar 0.4 mL M199 orta ekleyin. </li><li> Kültür 37 hücreleri ° C,% 5 CO 2 ve% 100 nem. </li></ol> 3. Post-EMT AV Yastık Mezenkimal Hücre İzolasyonu ve Kültür <ol><li> Crack keskin, temiz bir yüzey üzerinde tavuk yumurtası ve 100 mm Petri kabı içine yumurta kırmak. Embriyo, yumurta sarısı üst tarafında yer almalıdır. # 5 cımbız ile embriyo embriyo çevreleyen zarların kapma Pick up ve embriyo sonra buz gibi soğuk, steril EBSS ile dolu bir 100 mm Petri kabı içine yerleştirin. </li><li> Hamburger ve Hamilton 1 kriterlerine göre, embriyolar Sahne . Bir sahne 25 embriyo özellikleri farklı dirsek ve diz eklemleri ve hafif göz pigmentasyon içerir. </li><li> HH25 embriyolar kalpleri izole etmek ve kalpleri birbirine Petri kabı bir tarafta. </li><li> , Steril bir transfer pipet yardımıyla izole kalpleri toplayın ve steril, buz EBSS ile dolu yeni bir 100 mm Petri kabı içine yerleştirin. </li><li> Bir kerede tüm kalpleri AV bölgede izole edin. Petri kabı bir tarafta birlikte AVS yerleştirin. AV bölgedeki tüm kan, hücre canlılığını etkileyebilir hafifçe kaldırmak için ajitasyon. </li><li> Izole AVS, steril bir transfer pipet yardımıyla toplayın ve steril, buz EBSS ile dolu yeni bir 100 mm Petri kabı içine yerleştirin. </li><li> Sırayla AV yastıklar izole eder. AV minderler üzerinde hiçbir miyokardın mevcut olduğundan emin olun. </li><li> Izole yastıklar, steril bir transfer pipet yardımıyla toplayın ve 15 ml steril bir santrifüj tüpü içine koyun. </li><li> 3 dakika 15 xg&#39;de santrifüj tüpüne altındaki minderler dönerler. </li><li> Yastıklar rahatsız etmeden, mümkün olduğu EBSS süpernatant kadar çok kaldırmak için 1000 ml steril bir ucu kullanın. </li><li> 37 önceden ısıtılmış olan 1 ml% 0.25 &#39;lik tripsin-EDTA ° C İzin yastıkları tripsin içinde tamamen eriyene kadar inkübe, normalde 3-5 dakika. </li><li> Tripsin gidermek için santrifüj tüpüne tavuk serum 100 mcL ekleyin. </li><li> Pelet 5 dakika boyunca 160 xg&#39;de hücreleri. Steril, 1000 mcL ucu ile süpernatantı. </li><li> M199 orta ve pipetleme karışım 2 mL hücrelerin yeniden süspanse edin. Hemasitometre sayısı ve izole edilmiştir hücrelerinin toplam sayısını belirlemek için hücre süspansiyonu 10 mcL </li><li> Kollajen jeller izole hücrelerinin sayısına göre yapılacaktır kaç belirleyin. Hücreler 250 mcL jel hacmi ile 2 x 10 5 hücre / mL yoğunluğu kaplı olması gerekir. </li><li> Pelet 5 dakika boyunca 160 xg&#39;de hücreleri. Steril, 1000 mcL ucu ile süpernatantı. </li><li> Yukarıda listelenen protokolünü kullanarak, hücre pelet, bu sırayla 3X M199, su, tavuk serumu, kollajen, ve 0.1 M NaOH, eklemek. Nazik pipetleme ile karıştırın. </li><li> Her 1.9 cm 2 büyüme alanı 250 mcL ekleyin. </li><li> 37 en az 1 saat jel kollajen çözüm İzin ° C,% 5 CO 2 ve% 100 nem. </li><li> 0.4 ml kültür ortamı ekleyin ve gece inkübe edin. </li><li> Stressiz koşullarda kültür jeller devam etmek için, 200 mcL steril bir pipet kullanarak kültür taraftan jeller kurtardı. Mekanik kısıtlı kültürleri kültür taraf için de bağlı kalır jeller. </li></ol> 4. Temsilcisi Sonuçlar <img src="/files/ftp_upload/2159/2159fig1.jpg" alt="Şekil 1" /> Şekil 1: embriyo örnek olarak, (A) HH14-bıldırcın embriyo ve (B) HH25 tavuk embriyo burada gösterilmektedir. fig2.jpg &quot;alt =&quot; Şekil 2 &quot;/&gt; Şekil 2: 2 saat sonra kültür HH14-valf endokardiyal eksplantlar miyokardın varken (A) veya (B) miyokard kaldırıldı. <img src="/files/ftp_upload/2159/2159fig3.jpg" alt="Şekil 3" /> Şekil 3: HH14-valf endokardiyal eksplantlar (A) miyokardın mevcut hücrelerin çoğu mezenkimal dönüşüm endokardiyal geçmesi ve 48 saat sonra kültür iğ şeklinde, mezenkimal fenotip var. (B) miyokard tüm hücreleri kaldırıldığı zaman, 48 saat sonra kültür bir arnavut kaldırımı şeklinde, endokardiyal fenotip korumak. <img src="/files/ftp_upload/2159/2159fig4.jpg" alt="Şekil 4"/> Şekil 4: kollajen jel HH25 valf mezenkimal hücreler Hücreler hemen tohumlama sonra yuvarlanır. <img src="/files/ftp_upload/2159/2159fig5.jpg" alt="Şekil 5"/> Şekil 5: kültür HH25 mezenkimal hücreler (A), kısıtlı bir kollajen jel 48 saat sonra kültür HH25 mezenkimal hücreler yayılmaya başlıyor . (B), özgürlüğüne kavuştuktan sonra tohumlama ve 6 gün sonra 7 gün stressiz bir kollajen jel HH25 hücreleri orijinal alanının yaklaşık% 50 jel sıkıştırılmış.

<ol>
	<li>Hamburger, V., Hamilton, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+series+of+normal+stages+in+the+development+of+the+chick+embryo.">A series of normal stages in the development of the chick embryo.</a> J Morphol. 88, 49-92 (1951).</li><li>Runyan, R. B., Markwald, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invasion+of+mesenchyme+into+three-dimensional+collagen+gels:+A+regional+and+temporal+analysis+of+interaction+in+embryonic+heart+tissue.">Invasion of mesenchyme into three-dimensional collagen gels: A regional and temporal analysis of interaction in embryonic heart tissue.</a> Developmental Biology. 95, 108-108 (1983).</li><li>Butcher, J. T., Norris, R. A., Hoffman, S., Mjaatvedt, C. H., Markwald, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Periostin+promotes+atrioventricular+mesenchyme+matrix+invasion+and+remodeling+mediated+by+integrin+signaling+through+Rho/PI+3-kinase.">Periostin promotes atrioventricular mesenchyme matrix invasion and remodeling mediated by integrin signaling through Rho/PI 3-kinase.</a> Developmental Biology. 302, 256-256 (2007).</li></ol>

Sahne HH14 endokardiyal hücreler izole yöntemi - Runyan ve Markwald tarafından geliştirilen kalpleri başlatmak ve embriyonik EMT 2 modüle faktörleri incelemek için in vitro ortamda, kontrollü bir sağlar. HH14 - miyokardın olmadan kültür endokardiyal eksplantlar biyomekanik veya biyokimyasal müdahalesi olmadan EMT uğramaz. Miyokardın eksplant bırakılırsa endokardiyal hücrelerinin bir alt kümesi mezenkimal dönüşüme işaret edecektir, ancak dış faktörler...

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<td>Extra fine Bonn scissors, curved</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>14085-08</td>
<td>&nbsp;</td>
<tr>
<td>Dumont tweezers #55</td>
<td>&nbsp;</td>
<td>World Precision Instruments, Inc.</td>
<td>14099</td>
<td>&nbsp;</td>
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<td>Dumont tweezers #5</td>
<td>&nbsp;</td>
<td>World Precision Instruments, Inc.</td>
<td>14098</td>
<td>&nbsp;</td>
<tr>
<td>Sterile transfer pipettes</td>
<td>&nbsp;</td>
<td>Samco Scientific </td>
<td>2041S</td>
<td>&nbsp;</td>
<tr>
<td>4-well culture plates</td>
<td>&nbsp;</td>
<td>Nunc</td>
<td>176740</td>
<td>&nbsp;</td>
<tr>
<td>Sterile disposable filter units, PES, 0.2 &mu;m pore size membrane</td>
<td>&nbsp;</td>
<td>Nalgene</td>
<td>566-0020</td>
<td>&nbsp;</td>
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<td>Sterile 15 mL centrifuge tubes</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>430828</td>
<td>&nbsp;</td>
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<td>Sterile petri dishes, 100 mm x 15 mm</td>
<td>&nbsp;</td>
<td>VWR International</td>
<td>25384-342</td>
<td>&nbsp;</td>
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<td>Laboratory tape</td>
<td>&nbsp;</td>
<td>VWR International</td>
<td>89097-920</td>
<td>&nbsp;</td>
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<td>Rat-tail collagen I</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>354236</td>
<td>&nbsp;</td>
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<td>10X Earl's Balanced Salt Solution</td>
<td>&nbsp;</td>
<td>Quality Biological, Inc.</td>
<td>119-064-131</td>
<td>&nbsp;</td>
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<td>M199 powder</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>31100-035</td>
<td>&nbsp;</td>
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<td>Penicillin-Streptomycin</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15140-122</td>
<td>&nbsp;</td>
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<td>Insulin-Transferrin-Selenium-G supplement (100X)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>41400-045</td>
<td>&nbsp;</td>
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<td>Chicken serum</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>16110-082</td>
<td>&nbsp;</td>
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<td>0.25% Trypsin-EDTA</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>25200-056</td>
<td>&nbsp;</td>
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<td>Sodium bicarbonate</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>S5761</td>
<td>&nbsp;</td>
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<td>Sodium hydroxide solution, 1.0 N</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>S2770</td>
<td>&nbsp;</td>
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<td>Fertile quail eggs (Coturnix coturnix)</td>
<td>&nbsp;</td>
<td>Lake Cumberland Game Bird Farm</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Fertile chicken eggs (Gallus gallus)</td>
<td>&nbsp;</td>
<td>Cornell University Poultry Farm</td>
<td>&nbsp;</td>
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isolation and culture of avian embryonic valvular progenitor cells

Uygun oluşumu ve işlevi embriyonik kalp kapakçıkları, gelişim ilerlemesi için kritik öneme sahiptir. Erken embriyonik kalp miyokard tarafından çevrili endokard U-şeklinde bir tüp. Miyokardın salgılar kardiyak jöle, atriyoventriküler (AV) kavşak ve çıkış yolu (OFT) lümen içine hyaluronan zengin jelatinimsi matriks. Aşamada HH14 valvulogenesis endokardiyal hücrelerinin bir alt kümesi, mezenkimal dönüşüm (EMT) endokardiyal miyokard gelen sinyalleri geçiren alırsınız ve kalp jöle işgal başlar. Sahne HH25, kapak yastıkları tam mesenchymalized ve sonunda kalp kapak ve septal aparat formları bu mezenşimin. EMT ve hücre farklılaşma süreci başlatmak ve modüle mekanizmaların anlaşılması, ciddi konjenital kalp kusurları bağlantısı nedeniyle önemlidir. Bu çalışmada, prevalvular yastık iki farklı hücre fenotipleri-EMT endokardiyal öncesi ve post-EMT mezenkimal hücreler izole yöntemleri sunuyoruz. Ön-EMT endokardiyal hücreleri ile veya miyokard olmadan kültür olabilir. Post-EMT AV yastık mezenkimal hücreler içinde mekanik kısıtlı ya da stressiz kollajen jeller yetiştirilebilir. Bu 3D in vitro modellerinde anahtar kapak morfojenik olayları taklit ve erken ve geç dönem valvulogenesis mekanizmaları yapısızlaştırdıklarını için yararlıdır.

Watch this Scientific Journal Video about Isolation and Culture of Avian Embryonic Valvular Progenitor Cells at JoVE.com

Isolation and Culture of Avian Embryonic Valvular Progenitor Cells

Bu makale, izole etmek için bir yöntem ve kültür bıldırcın veya tavuk HH14 sağlayacak<sup&gt; -</sup&gt; Endokardiyal hücreler vana ve vana yastık HH25 mezenkimal hücreler.

Kuş Embriyonik Kapak Progenitör Hücreler izolasyonu ve Kültür

Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube of ...

isolation-and-culture-of-avian-embryonic-valvular-progenitor-cells

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Biology

10.1K Görüntüleme.  Cornell University. Bu makale, izole etmek için bir yöntem ve kültür bıldırcın veya tavuk HH14 sağlayacak - Endokardiyal hücreler vana ve vana yastık HH25 mezenkimal hücreler.

Video: Isolation and Culture of Avian Embryonic Valvular Progenitor Cells

Isolation and Culture of Post-Natal Mouse Cerebellar Granule Neuron Progenitor Cells and Neurons 

The cerebellar cortex is a well described structure that provides unique opportunities for studying neuronal properties and development1,2. Of the cerebellar neuronal types (granule cells, Purkinje cells and inhibitory interneurons), granule neurons are by far the most numerous and are the most abundant type of neurons in the mammalian brain. In rodents, cerebellar granule neurons are generated during the first two post-natal weeks from progenitor cells in the outermost layer of the cerebellar cortex, the external granule layer (EGL). The protocol presented here describes techniques to enrich and culture granule neurons and their progenitor cells from post-natal mouse cerebellum. We will describe procedures to obtain cultures of increasing purity3,4, which can be used to study the differentiation of proliferating progenitor cells into granule neurons5,6. Once the progenitor cells differentiate, the cultures also provide a homogenous population of granule neurons for experimental manipulation and characterization of phenomena such as synaptogenesis, glutamate receptor function7, interaction with other purified cerebellar cells8,9 or cell death7.

Isolation and Culture of Post-Natal Mouse Cerebellar Granule Neuron Progenitor Cells and Neurons

Here we present a method to isolate and culture cerebellar granule neuron progenitor cells and cerebellar granule neurons from postnatal mouse.

Post-Natal Mouse Serebellar Granül Nöron Progenitör Hücreler ve Nöronlar İzolasyon ve Kültür

The cerebellar cortex is a well described structure that provides unique opportunities for studying neuronal properties and development1,2. Of the ...

Biyoloji

Culture and Maintenance of Human Embryonic Stem Cells  

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be  fed  every day by performing a complete medium change to replenish lost nutrients and to keep the cultures free of unwanted differentiation factors. Although a small amount of differentiation is normal and expected in stem cell cultures, the culture should be routinely cleaned up by manually removing, or "picking" differentiated areas. Identifying and removing excess differentiation from hES cell cultures are essential techniques in the maintenance of a healthy population of cells.

Culture and Maintenance of Human Embryonic Stem Cells

This protocol demonstrates how to maintain healthy, undifferentiated human embryonic stem (ES) cells.

İnsan Embriyonik Kök Hücre Kültürü ve Bakım

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be ...

Isolation and Derivation of Mouse Embryonic Germinal Cells

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum (dpc) mouse embryos.  Developing gonadal ridges from mouse embryos (C57BL6J) were dissociated by mechanical disruption with collagenase, then plated in a mouse embryo fibroblast feeder layer (MEF-CF1) that was previously mitotically inactivated with mitomycin C in the presence of knockout media and supplemented with Leukemia Inhibitor Factor (LIF), basic Fibroblast Growth Factor (bFGF), and Stem Cell Factor (SCF).  Using these optimized methods for PCG identification, isolation, and establishment of culture conditions permits long term cultures of EG cells for more than 40 days.  The embryonic germinal cell lines showed embryonic phenotype and expression of common used markers of the pluripotent state.  Isolation and derivation of germinal cells in culture provide a tool to understand their development in vitro and offer the opportunity to monitor cumulative damage at genetic and epigenetic levels after exposure to oxidative stress.

The ability of embryonic germinal cells to differentiate into primordial germinal cells during early development stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum mouse embryos.

Fare Embriyonik Germinal Hücreleri İzolasyon ve türetilmesi

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental ...

Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a robust and reliable method for isolating relatively large numbers of proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. The authors have used micro-dissected explant cultures to analyse the growth characteristics of skeletal muscle cells in wild-type and dystrophic muscles. Each of the components of tissue growth, namely cell survival, proliferation, senescence and differentiation can be analysed separately using the methods described here. The net effect of all components of growth can be established by means of measuring explant outgrowth rates. The micro-explant method can be used to establish primary cultures from a wide range of different muscle types and ages and, as described here, has been adapted by the authors to enable the isolation of embryonic skeletal muscle precursors.
Uniquely, micro-explant cultures have been used to derive clonal (single cell origin) skeletal muscle stem cell (SMSc) lines which can be expanded and used for in vivo transplantation. In vivo transplanted SMSc behave as functional, tissue-specific, satellite cells which contribute to skeletal muscle fibre regeneration but which are also retained (in the satellite cell niche) as a small pool of undifferentiated stem cells which can be re-isolated into culture using the micro-explant method.

The micro-dissected explants technique is a robust and reliable method for isolating proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. Uniquely, these cells have been clonally derived to produce skeletal muscle stem cell lines used for in vivo transplantation.

Yetişkin ve Embriyonik İskelet, Kas Microexplant Kültür ve İskelet Kası Kök Hücre İzolasyonu

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a ...

Isolation of Valvular Endothelial Cells

Heart valves are solely responsible for maintaining unidirectional blood flow through the cardiovascular system. These thin, fibrous tissues are subjected to significant mechanical stresses as they open and close several billion times over a lifespan. The incredible endurance of these tissues is due to the resident valvular endothelial (VEC) and interstitial cells (VIC) that constantly repair and remodel in response to local mechanical and biological signals. Only recently have we begun to understand the unique behaviors of these cells, for which in vitro experimentation has played a key role. Particularly challenging is the isolation and culture of VEC. Special care must be used from the moment the tissue is removed from the host through final plating. Here we present protocols for direct isolation, side specific isolation, culture, and verification of pure populations of VEC. We use enzymatic digestion followed by a gentle swab scraping technique to dislodge only surface cells. These cells are then collected into a tube and centrifuged into a pellet. The pellet is then resuspended and plated into culture flasks pre-coated with collagen I matrix. VEC phenotype is confirmed by contact inhibited growth and the expression of endothelial specific markers such as PECAM1 (CD31), Von Willebrand Factor (vWF), and negative expression of alpha-smooth muscle actin (&alpha;-SMA). The functional characteristics of VEC are associated with high levels of acetylated LDL. Unlike vascular endothelial cells, VEC have the unique capacity to transform into mesenchyme, which normally occurs during embryonic valve formation1. This can also occur during significantly prolonged post confluent in vitro culture, so care should be made to passage at or near confluence. After VEC isolation, pure populations of VIC can then be easily acquired.

We provide a method for isolating and culturing pure populations of heart valve endothelial cells (VEC). VEC can be isolated from either side of the cusp or leaflet and immediately following, underlying interstitial cell (VIC) isolation is straightforward.

Kapak Endotel Hücreleri İzolasyon

Heart valves are solely responsible for maintaining unidirectional blood flow through the cardiovascular system.  These thin, fibrous tissues are ...

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells (HUVECs) have shown to be convenient, easy to obtain and culture, and thus are the most widely studied endothelial cells. However, for research focused on processes like angiogenesis, permeability or many others, microvascular endothelial cells (ECs) are a much more physiologically relevant model to study 2. Furthermore, ECs isolated from knockout mice provide a useful tool for analysis of protein function ex vivo. Several approaches to isolate and culture microvascular ECs of different origin have been reported to date 3-7, but consistent isolation and culture of pure ECs is still a major technical problem in many laboratories. Here, we provide a step-by-step protocol on a reliable and relatively simple method of isolating and culturing mouse lung endothelial cells (MLECs). In this approach, lung tissue obtained from 6- to 8-day old pups is first cut into pieces, digested with collagenase/dispase (C/D) solution and dispersed mechanically into single-cell suspension. MLECS are purified from cell suspension using positive selection with anti-PECAM-1 antibody conjugated to Dynabeads using a Magnetic Particle Concentrator (MPC). Such purified cells are cultured on gelatin-coated tissue culture (TC) dishes until they become confluent. At that point, cells are further purified using Dynabeads coupled to anti-ICAM-2 antibody. MLECs obtained with this protocol exhibit a cobblestone phenotype, as visualized by phase-contrast light microscopy, and their endothelial phenotype has been confirmed using FACS analysis with anti-VE-cadherin 8 and anti-VEGFR2 9 antibodies and immunofluorescent staining of VE-cadherin. In our hands, this two-step isolation procedure consistently and reliably yields a pure population of MLECs, which can be further cultured. This method will enable researchers to take advantage of the growing number of knockout and transgenic mice to directly correlate in vivo studies with results of in vitro experiments performed on isolated MLECs and thus help to reveal molecular mechanisms of vascular phenotypes observed in vivo.

Here, we describe a protocol for isolation and culture of murine pulmonary endothelial cells. This method comprises mechanic and enzymatic lung tissue dissociation as well as a 2-step purification process using anti-PECAM-1 and anti-ICAM-2 antibodies conjugated to magnetic beads, which produces a pure endothelial cell population of mostly microvascular origin.

Yenidoğan Fareler Pulmoner Endotel Hücreleri İzolasyon ve Kültür

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells ...

Isolation and Culture of Cells from the Nephrogenic Zone of the Embryonic Mouse Kidney

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and to gain understanding of the origins of congenital kidney disease. More recently, the possibility of steering na&iuml;ve embryonic stem cells toward nephrogenic fates has been explored in the emerging field of regenerative medicine. Genetic studies in the mouse have identified several pathways required for kidney development, and a global catalog of gene transcription in the organ has recently been generated <a href="http://www.gudmap.org/">http://www.gudmap.org/</a>, providing numerous candidate regulators of essential developmental functions. Organogenesis of the rodent kidney can be studied in organ culture, and many reports have used this approach to analyze outcomes of either applying candidate proteins or knocking down the expression of candidate genes using siRNA or morpholinos. However, the applicability of organ culture to the study of signaling that regulates stem/progenitor cell differentiation versus renewal in the developing kidney is limited as cultured organs contain a compact extracellular matrix limiting diffusion of macromolecules and virus particles. To study the cell signaling events that influence the stem/progenitor cell niche in the kidney we have developed a primary cell system that establishes the nephrogenic zone or progenitor cell niche of the developing kidney ex vivo in isolation from the epithelial inducer of differentiation. Using limited enzymatic digestion, nephrogenic zone cells can be selectively liberated from developing kidneys at E17.5. Following filtration, these cells can be cultured as an irregular monolayer using optimized conditions. Marker gene analysis demonstrates that these cultures contain a distribution of cell types characteristic of the nephrogenic zone in vivo, and that they maintain appropriate marker gene expression during the culture period. These cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which greatly facilitates the study of candidate stem/progenitor cell regulator effects. Basic cell biological parameters such as proliferation and cell death as well as changes in expression of molecular markers characteristic of nephron stem/progenitor cells in vivo can be successfully used as experimental outcomes. Ongoing work in our laboratory using this novel primary cell technique aims to uncover basic mechanisms governing the regulation of self-renewal versus differentiation in nephron stem/progenitor cells.

In this report we describe a method for the isolation and culture of the progenitor cell niche from the embryonic mouse kidney that can be used to study signaling pathways regulating stem/progenitor cells of the developing kidney. These cultured cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which allows efficient manipulation of candidate pathways.

Embriyonik Fare Böbrek Nefrojenik Zone Hücreleri İzolasyon ve Kültür

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and  to gain ...

Isolation and Primary Culture of Rat Hepatic Cells

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology and other related subjects. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 1 and, since then, has undergone many modifications. The most commonly used technique was described by Seglenin 1976 2. Essentially, hepatocytes are dissociated from anesthetized adult rats by a non-recirculating collagenase perfusion through the portal vein. The isolated cells are then filtered through a 100 &mu;m pore size mesh nylon filter, and cultured onto plates. After 4-hour culture, the medium is replaced with serum-containing or serum-free medium, e.g. HepatoZYME-SFM, for additional time to culture. These procedures require surgical and sterile culture steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol, which allow consistently in the generation of viable hepatocytes in large numbers.

Primary hepatocytes provide a valuable tool to evaluate biochemical, molecular, and metabolic functions in a physiologically relevant experimental system. We describe a reliable protocol for rat in situ liver perfusion, which consistently generates viable hepatocytes up to 1.0 &times; 108 cells per preparation with cell viability between 88 ~ 96%.

Sıçan Karaciğer Hücreleri İzolasyon ve İlköğretim Kültür

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

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 ...

Isolation and Culture of Mouse Primary Pancreatic Acinar Cells

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one week. More than 20 x 106 acinar cells can be obtained from a single murine pancreas. This protocol offers the possibility to independently process as many as 10 pancreases in parallel. Because it preserves acinar architecture, this model is well suited for studying the physiology of the exocrine pancreas in vitro in contrast to cell lines established from pancreatic tumors, which display many genetic alterations resulting in partial or total loss of their acinar differentiation.

In this publication, we describe a rapid and convenient procedure for isolating and culturing primary pancreatic acinar cells from the murine pancreas. This method constitutes a valuable approach to study the physiology of fresh primary normal/untransformed exocrine pancreatic cells.

Fare Primer Pankreas Asiner Hücreleri İzolasyon ve Kültür

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one ...