This work was supported by funding from American Heart Association 12BGIA12040477, NC State University Chancellor&#8217;s Faculty Excellence Program, and National Natural Science Foundation of China H020381370216.

Aşağıdaki protokol, yenidoğan sıçan yavrularından (10-14 yavrular) bir çöp den, kardiyomiyositler izolasyonu için tasarlanmıştır. Çöp boyutu önemli ölçüde farklı ise, prosedür telafi etmek ölçekli gerekebilir. yavrular etrafında 48 ± 6 saat eski olmalıdır. Tüm işlemler Burada North Carolina State Üniversitesi Kurumsal Hayvan Bakım ve Kullanım Komitesi (IACUC) tarafından onaylanmıştır. 1. Hücre İzolasyonu hazırlanması NOT: izolasyon önce aşağıdaki adımları uygulayın gün. <ol><li> Büyük makas, küçük makas, küçük keskin forseps, büyük forseps: Aşağıdaki otoklavlayın. 121 ° C, en az 30 dakikalık bir kuruma süresi 60 dakika boyunca yerçekimi döngüsü kullanın. </li><li> / N O çözülme buzdolabı içinde% 0.1 tripsin solüsyonu 40 ml yerleştirin. Buzdolabında Hank&#39;in Dengeli Tuzları Çözüm (HBSS) zaten soğuk değilse. % 20 fetal sığır serumu (FBS), hisse senedi ortam çözeltisi hazırlayın. <ol><li> Isc&#39;den 400 ml&#39;likOve&#39;nin Modifiye Dulbecco ortamı (IMDM) içinde 100 ml FBS, 5 mi, L-glutamin (200 mM), 2.5 mi gentamisin (10 mg ml-1) ve 0.9 ml 2-merkaptoetanol ekleyin. Vakum filtre kullanarak sterilize. Mağaza kadar 3 hafta boyunca 4 ° C&#39;de medya ve bu küçük veya daha büyük hacimler için ölçeklenebilir. % 10 FBS NRCM medya üretmek için IMDM ile 1:% 20 FBS stok Ortamı 1 seyreltin. Sadece tekrarlanan ısıtma ve medya soğumasını önlemek için küçük gruplar halinde olun. </li></ol></li></ol> 2. Hücre İzolasyonu 1. Gün <ol><li> Numunelerin kirlenmesini önlemek için kültür kaputu aşağıdaki çalışmaları yapınız. Bir O / N inkübasyon orada beri en iyi zamanlama için öğleden sonra bu işe başlamak. </li><li> Steril bir kültür kaput içinde çalışma alanını hazırlayın. Kültür kaputu tezgah yastık yerleştirin. % 70 etanol ile iki adet 250 ml&#39;lik beher doldurun ve onları cerrahi aletleri yerleştirin. Kaputu buz küçük kova yerleştirin. Birkaç tüpleri ve 150 mm çanak tutmak için yeterince büyük olduğundan emin olun. Fo kaputu sterilize UVR 10 dk. Buz üzerinde 40 mi% 0.1 tripsin yerleştirin. </li><li> Anneden yenidoğan sıçan yavrular alın. Kullanılıncaya kadar sıcak hayvanları tutmak için, böyle sıcak tutmak için bir tezgah pad sarılmış bir buz kovası olarak yalıtılmış bir kap koyun. </li><li> 25 mi HBSS ile 150 mm kültür çanağı doldurun ve buz üzerine yerleştirin. Unwrap 2-3 gazlı bez ve çalışma alanında yer. Boyun arkasındaki cilt tarafından yavru tutarak, kaputun içinde% 70 etanol ve yer ile sprey. NOT: En iyi sterilite korumak için, bu daha sonra kaputu temizlenmiş yavru yerleştirir başka bir kişi tarafından yapılabilir olduğunu. </li><li> Boyun arkasındaki cilt tarafından yavru tutun. Sonra başları kesilerek euthanize bir hızlı, güçlü kesim, büyük makas kullanarak. Gazlı bez ile kan kurulayın. NOT: IACUC gereksinimleri değişimlerine, bir etanol ile yavru temizlemek için daha uygun olabilir nedeniyle onu püskürtme yerine silin. Bu adım için kendi kurallarına gibi üniversite IACUC ile kontrol ettiğinizden emin olun. </li><li> Ch ön tarafı aşağı kesim Devamgöğüs kafesi üzerinden est. Kalp görünür kadar göğüs kafesi açmak yardımcı olmak için boyun cildi sıkın. Küçük makas kullanarak, buz üzerinde HBSS kültür çanak kalp ve yer çıkarın. Diğer yavrular için tekrarlayın. </li><li> Örneklerden olmayan kalp dokusu çıkarın ve kalpleri yıkamak için hafifçe HBSS girdap. Taze HBSS içeren başka kültür çanak kalp dokusu aktarın ve daha doku yıkayın. Adet 1-3 mm 3 kadar daha doku kesin, ve sonra tripsin solüsyonu transfer. 4 ° C soğutma O / N rocker üzerinde tripsin çözüm yerleştirin. </li></ol> Hücre İzolasyonu 2. Gün Not: Aşağıdaki adımlarda aspire ederken, bir vakum hattı yerine bir pipet kullanın. Doku kolayca hareket, ve bir vakum hattı yapışmasına veya hücreleri içeren hala doku kaldıracaktır. Bunun yerine aspirasyon için bir pipet kullanmak en iyisidir. Doku pipet girerse, pipet çıkarmak için dışarı geri. <ol><li> T yap% 10 NRCM Medya ANTE&#39;lerin alikotları: 15 ml, 25 ml, bir ve iki. 37 ° C su banyosunda medyanın bir 25 ml&#39;lik tüp yerleştirin. 5 farklı 15 ml konik tüplere 4 mi HBSS ekleyin ve buz üzerinde her yerleştirin. Her 15 ml tüp # 1-5 Etiket. </li><li> 50 ml&#39;lik bir konik tüp, 40 ml HBSS ekleyin. HBSS 10 ml askıya, pipet kollajenaz ekle, ve 50 ml 1 mg ml -1 kollajenaz çözüm oluşturmak için çözümler birleştirir. 0.22 mikron vakum filtre kullanılarak sterilize ve oda sıcaklığında saklayın. </li><li> Kalp dokusu / tripsin tüp alın. Emin olun tripsin açıktır, ve doku kabarık görünür. Doku tüp ve aspirat köşesine kadar sıvı mümkün olduğunca yerleşmek için izin verin. </li><li> Elle tüp ve girdap 25 ml sıcak% 10 FBS NRCM medya ekleyin. Kap ve 2 dakika için 37 ° C su banyosu içinde döner. Yine aspire sıvı. Doku bu aşamada yüzer gerekir; eğer öyleyse, tüpün dibinden aspire. </li><li> 10 mi kolajenaz ekleyin. 2 dakika boyunca ya da çözelti, bulanık olana kadar su banyosunda döndürün. Quickly sıvı aspire. Doku içeren bir tüpe 10 ml taze kolajenaz ilave edin ve 2 dakika boyunca bir su banyosu içinde döner. Pipet, tüp # 1 transfer çözüm karıştırmak ve buz üzerinde yerleştirmek için. Bu sıvının kaldırmak mümkün olmayabilir. Dier üç 15 ml konik tüpler için 3,2-3,6 adımları. Tüp # 5 kalan sıvı aktarın. </li><li> Santrifüj 8 dakika 410 RCF 15 ml tüpler. Santrifüj birlikte, su banyosu içinde, 15 ml% 10 FBS NRCM ortam tüp yerleştirin. 40 mikron hücre 50 ml&#39;lik konik tüp üstünde süzgeç ve 2 ml soğuk HBSS ile ıslak yerleştirin. </li><li> Aşağı bükülmüş hücrelerden aspire süpernatant. Her bir tüp ve tekrar süspansiyon 6 ml soğuk HBSS ekleyin. 50 ml&#39;lik konik içine süzgecinden Pipet hücre süspansiyonları herhangi bir doku kaldırmak için. 3 ml soğuk HBSS her 15 ml tüp kenarlarını durulayın ve süzgeç ekleyin. 10 dakika boyunca 410 RCF Santrifüj hücreleri. Süpernatant aspire. T-75 şişeye 10 mi% 10 FBS NRCM ortam transfer içinde süspanse hücre topağı. 5ml medya ile konik durulayın ve fl eklemeksormak. </li><li> 37 ° C&#39;de 1 saat karıştırıldı ve% 5 CO2 inkübe edin. Su banyosu içine son 15 ml% 10 FBS NRCM medya tüpü yerleştirin. T-75, yeni bir T-175 balonuna içeriğini aktarın 15 mi ortam ile T-75 durulama ve T-175 ilave edin. 1 saat süre ile inkübe edilir. </li></ol> 4. Kriyoprezervasyon <ol><li> Buz medya dondurma yerleştirin. T-175-50 ml konik tüp hücre ortamı aktarın ve hücre Tripan mavisi kullanarak sayılması için hücre süspansiyonu 10 ul toplamak. 5 dakika boyunca 410 RCF santrifüjleyin. Santrifüj sırasında, bir hücre sayacı kullanarak hücreleri saymak. </li><li> Ml başına 2-4000000 hücre konsantrasyonunda ortam donma aspire kültür ortamı, ve tekrar süspansiyon hücreleri. Kriyojenik şişe içinde hücre süspansiyonu 1 ml yerleştirin ve yer -80 ° C dondurucu O / N kontrollü soğutma kabı donma hızı ve yer şişeler. 1-2 gün sonra, sıvı azot içinde şişeleri aktarın. </li></ol> 5. Çözülme Hücreler <ol><li> 250 ul çözerek fibronektin içinde Coat çanak60; 9.75 ml su (nihai konsantrasyon 0.025 mg ml -1) içine fibronektin (stok konsantrasyonu 1 mg ml -1) olarak. Kültürü plakası kapağı ve en az 30 dakika boyunca 37 C &#39;de inkübe etmek yeterli bir çözüm ekleyin. <ol><li> 20% Medya DMSO ekleyerek aşağıdaki gibi dört 15 ml konik tüplerde, DMSO / medya karışımları yapmak. 500 ul DMSO ile (% 5) 9.5 ml ortam ekleyin. 250 ul DMSO ile (% 2.5), 9.75 mi ortam ekleyin. 10 ml medya (bu iki yapmak) ekleyin. </li></ol></li><li> 37 ° C su banyosu içinde yukarıda belirtilen ortam karışımları ısıtın. Sıvı azot hücreleri çıkarın ve kuru buz üzerinde yerleştirin. Medya karışımlar sıcak olduğunda sadece çözülmüş kadar, 37 ° C su banyosunda hücreleri eritin. Bu hücrelere zarar verebilir gibi çok uzun süre su banyosunda hücreleri bırakmayın. </li><li> % 5 DMSO / ortam tüpüne kriyojenik bir şişenin muhtevasının aktarın. 8 dakika boyunca 410 RCF santrifüjleyin. Hücre pelet bırakarak aspire süpernatant. % 2.5 DMSO / medya tüpünün içeriği pelet ve r hücre ekleesuspend. 8 dakika boyunca 410 RCF santrifüjleyin. Hücre pelet bırakarak aspire süpernatant. </li><li> Pelet ve tekrar süspansiyon hücre, bir 0% DMSO / medya tüpünün içeriğini ekleyin. 8 dakika boyunca 410 RCF santrifüjleyin. Hücre pelet bırakarak aspire süpernatant. </li><li> Hücre pelet ve tekrar süspansiyon ikinci 0% DMSO / medya içeriğini ekleyin. Hücre sayımı için küçük bir örnek alın. 8 dakika boyunca 410 RCF santrifüjleyin. Bir hematositometrede santrifüj sayısı hücreleri Tripan Blue kullanırken ölü hücreleri etiketlemek için. Tutanak hücre miktarı, yanı sıra canlılık. </li><li> Hücre pelet bırakarak aspire süpernatant. % 10 NRCM medyada süspanse (opsiyonel: 100 mcM bromodeoxyuridine (BrdU) ekleyin) fibronektin-kaplı yemekleri ve plaka hücreleri. </li></ol> 6. Hücre Kültürü <ol><li> Cm 2 başına yaklaşık 100k hücre Plaka hücreleri. Kültür kullanım amacına göre kaplama yoğunluğu ayarlayın. 1:10 oranında IMDM içerisinde% 20 Ortamı seyreltilmesi ile% 2 NRCM ortamı hazırlar. Aşağıdaki tipik bir kültürün bir taslaktır. &lt;/ Li&gt; <li> 1 gün, uzak, sıcak IMDM ile ölü hücreleri yıkayın% 10 NRCM (isteğe bağlı 100 mcM BrdU) ekleyin. 2 gün, uzak, sıcak IMDM ile ölü hücreleri yıkayın% 10 NRCM ekleyin. Gün 3-eklemek% 2 NRCM. 4 gün% 2 NRCM ekleyin. 5. günde hücreler belirsiz ve dayak olduğundan emin olun. Yaklaşık bir hafta boyunca kültür içinde hücreleri koruyun. Bu noktada fibroblastlar anlamlı NRCMs büyümek başlayabilir. </li></ol>

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The authors have nothing to disclose.

NRCMs, izole dondurulan ve çözülmüş olması için bu protokol sağlar. Hücrelerin çözülmesi prosedürünün çok önemli bir kısmıdır. DMSO Bir dizi sıvı yavaşça hücreler 14 DMSO&#39;yu çıkarmak için kullanılır. Bu DMSO çıkarma hücreleri çözüldükten sonra hemen ölen özellikle duyarlı hızlı bir şekilde yapılması önemlidir. Daha fazla veya daha az hücre eritme işlemi için gerekli olan, gerektiğinde, DMSO çözeltisi hacimleri ölçeklendirilebilir. <p class="jove_co...

Kardiyomiyositlerin Hücre kültürü, modern kardiyak araştırma kritik bir araçtır. Yenidoğan sıçan kardiyomiyositler (NRCMs) yaygın izolasyon beri kullanılan ve kültür yetişkin sıçan kardiyomiyositlerde 1 daha kolaydır. NRCM yöntemi hala çanak, uzun izolasyon prosedürü ve sınırlı hücre çoğalması dahil olmak üzere, çeşitli sınırlamalar vardır. En genel iş 2-6 4-48 saat gerektiren NRCMs izolasyonu için çok sayıda protokoller vardır. Buna ek olarak, hücreler sıklıkla 2 günlük sıçan yavrularından 2,4-7 1&#39;den izole edilir; Doğum zamanlaması laboratuarında diğer çalışmaları ile öngörülemeyen ve çatışma olabilir. hücrelerin sadece küçük bir miktar deneyler için gerekli olup olmadığını izolasyon israf olduğu olabilir. Izolasyon süresini azaltarak iş akışı odak iyileştirilmesi çoğu çabaları, henüz bu yavruların doğum zamanlama sorunları çözmez. Alternatif olarak, birçok laboratuvar kullanmakEmbriyonik kök hücreler (EKH) ya da uyarılmış pluripotent kök hücreler (iPSCs) türetilen kardiyomiyositlerde. Ancak, yeniden programlama ve / veya farklılaşma süreci çok zaman alıcı ve masraflı de olabilir. In vitro olarak, miyosit model olarak, bu hücreler kullanılarak başka problemler de olabilir. Hem Esc ve iPSC- türetilmiş kardiyomiyositlerde birincil kardiyomiyositlerde 8-10 elektrofizyoloji farklılıkları sergiledikleri gösterilmiştir. Disosiye NRCMs soğutma 11 kullanılarak birkaç gün depolanan yeteneğine sahip, henüz bu uzun süreli depolama için izin vermez. Sıvı azot tipik olarak belli bir süre ile hücreleri korumak için kullanılır, ancak dimetil sülfoksit (DMSO) gibi bir dondurarak saklama koruyucusu gibi gerektirir. Önceki araştırmalar dondurma medyada% 5-10 DMSO arasında ideal konsantrasyon NRCMs iyileştirilmesi için izin verdiğini göstermiştir, ancak o zaman bile canlılığı düşük 12 kalır. DMSO ücretsiz sırasında hücrelerin korunmasına yardımcı olur rağmenkuvvet, 1.5,% 13 üzerindeki konsantrasyonlarda hücreler için toksik olabilir. Daha önce yapılan çalışmalar yavaş hücrelerden DMSO kaldırma hücre canlılığı 14 iyileştirebileceğini göstermektedir. Bu ayrılmasından sonra cryopreserving hücreleri tarafından NRCM hücre bazlı deneylerde verimliliğini artırmak için çalışmıştır. Bu hücreler çözülür ve gerektiğinde, izolasyonları ve hayvan tüketimi sıklığının azaltılması kullanılacak sağlar. Bu yöntemi kullanarak, bu NRCMs dondurularak saklanabilmesi ve daha sonraki bir zamanda kullanılmak üzere çözülme mümkün olduğunu göstermektedir. Çözülme sonra hücreler kabul edilebilir canlılığını korumak ve kendiliğinden α-aktinin sarkomerik (α-SA) ve sözleşme için pozitif NRCM kültürleri üretmek.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>IMDM (+25mM HEPES +L-glutamine)</td>
			<td>Gibco</td>
			<td>12440-046</td>
			<td>With added 25mM HEPES and L-glutamine</td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Gibco</td>
			<td>25030-081</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Hyclone</td>
			<td>SH30070.03</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin</td>
			<td>Gibco</td>
			<td>15710-064</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Gibco</td>
			<td>21985-023</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS (+Ca +Mg)</td>
			<td>Corning</td>
			<td>21-020-CV</td>
			<td>With added calcium and magnesium, pH 7.1-7.4</td>
		</tr>
		<tr>
			<td>Trypsin 0.25%</td>
			<td>Gibco</td>
			<td>25300-056</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin 0.05%</td>
			<td>Gibco</td>
			<td>25300-054</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryostor CS5</td>
			<td>BioLife Solutions</td>
			<td>205102</td>
			<td>Freezing media</td>
		</tr>
		<tr>
			<td>Cryogenic Vial</td>
			<td>Corning</td>
			<td>430659</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase</td>
			<td>Sigma</td>
			<td>C1889-50MG</td>
			<td></td>
		</tr>
		<tr>
			<td>40&#956;m Cell Strainer</td>
			<td>Greiner Bio-One</td>
			<td>542040</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterilizing Vacuum Filter (0.22&#956;m)</td>
			<td>Corning</td>
			<td>431118</td>
			<td></td>
		</tr>
		<tr>
			<td>50mL Conical</td>
			<td>Corning</td>
			<td>430828</td>
			<td></td>
		</tr>
		<tr>
			<td>15mL Conical</td>
			<td>Corning</td>
			<td>430790</td>
			<td></td>
		</tr>
		<tr>
			<td>trypan blue</td>
			<td>Cellgro</td>
			<td>25-900-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Mr Frosty Freezing Container</td>
			<td>ThermoScientific</td>
			<td>5100-0001</td>
			<td></td>
		</tr>
		<tr>
			<td>Millicell EZ&#160;SLIDES</td>
			<td>Millipore</td>
			<td>PEZGS0416</td>
			<td></td>
		</tr>
		<tr>
			<td>&#945;-sarcomeric actinin antibody</td>
			<td>Sigma</td>
			<td>A7811</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibronectin</td>
			<td>Corning</td>
			<td>356008</td>
			<td></td>
		</tr>
		<tr>
			<td>Bromodeoxyuridine</td>
			<td>BD Biosciences</td>
			<td>51-7581KZ</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation and cryopreservation of neonatal rat cardiomyocytes

Cell culture has become increasingly important in cardiac research, but due to the limited proliferation of cardiomyocytes, culturing cardiomyocytes is difficult and time consuming. The most commonly used cells are neonatal rat cardiomyocytes (NRCMs), which require isolation every time cells are needed. The birth of the rats can be unpredictable. Cryopreservation is proposed to allow for cells to be stored until needed, yet freezing/thawing methods for primary cardiomyocytes are challenging due to the sensitivity of the cells. Using the proper cryoprotectant, dimethyl sulfoxide (DMSO), cryopreservation was achieved. By slowly extracting the DMSO while thawing the cells, cultures were obtained with viable NRCMs. NRCM phenotype was verified using immunocytochemistry staining for &#945;-sarcomeric actinin. In addition, cells also showed spontaneous contraction after several days in culture. Cell viability after thawing was acceptable at 40-60%. In spite of this, the methods outlined allow one to easily cryopreserve and thaw NRCMs. This gives researchers a greater amount of flexibility in planning experiments as well as reducing the use of animals.

Yenidoğan Sıçan kardiyomiyositlerinde izolasyonundan sonra, hücreler sıvı azot kadar dondurulur ve en az birkaç ay boyunca saklanabilir. Tipik haliyle eritme üzerine, Tripan mavisi analizi ile belirlenen canlılık% 40-60 arasında olacaktır. Bu doğru tohumlama ve kültürü ile, diğer hücre türlerine göre daha düşük olmasına rağmen hücreleri (Şekil 1) çoğalacak. Kasılma doğrulamak için, hücreler, bir faz kontrast mikroskobisi kullanılarak görüntülenebilir. Hücreler kendi...

Watch this Scientific Journal Video about Isolation and Cryopreservation of Neonatal Rat Cardiomyocytes at JoVE.com

Isolation and Cryopreservation of Neonatal Rat Cardiomyocytes

The isolation of neonatal rat cardiomyocytes is a time consuming and unpredictable procedure. This study describes methods for cryopreservation and thawing of neonatal rat cardiomyocytes that allows for more efficient use of cells. The thawed NRCMs can be used for various experiments without the need for performing isolations each time.

İzolasyon ve Neonatal Rat kardiyomiyositlerde Kryoprezervasyon

Cell culture has become increasingly important in cardiac research, but due to the limited proliferation of cardiomyocytes, culturing cardiomyocytes is ...

isolation-and-cryopreservation-of-neonatal-rat-cardiomyocytes

Research

JoVE Journal

Developmental Biology

12.4K Görüntüleme.  College of Veterinary Medicine, North Carolina State University. The isolation of neonatal rat cardiomyocytes is a time consuming and unpredictable procedure. This study describes methods for cryopreservation and thawing of neonatal rat cardiomyocytes that allows for more efficient use of cells. The thawed NRCMs can be used for various experiments without the need for performing isolations each time.

Video: Isolation and Cryopreservation of Neonatal Rat Cardiomyocytes

Isolation of Neural Stem/Progenitor Cells from the Periventricular Region of the Adult Rat and Human Spinal Cord

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of multipotent, self-renewing, and expandable neural stem cells. In serum conditions, these multipotent NSPCs will differentiate, generating neurons, astrocytes, and oligodendrocytes. The harvested tissue is enzymatically dissociated in a papain-EDTA solution and then mechanically dissociated and separated through a discontinuous density gradient to yield a single cell suspension which is plated in neurobasal medium supplemented with epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and heparin. Adult rat spinal cord NSPCs are cultured as free-floating neurospheres and adult human spinal cord NSPCs are grown as adherent cultures. Under these conditions, adult spinal cord NSPCs proliferate, express markers of precursor cells, and can be continuously expanded upon passage. These cells can be studied in vitro in response to various stimuli, and exogenous factors may be used to promote lineage restriction to examine neural stem cell differentiation. Multipotent NSPCs or their progeny can also be transplanted into various animal models to assess regenerative repair.

The adult mammalian spinal cord contains neural stem/progenitor cells (NSPCs) that can be isolated and expanded in culture. This protocol describes the harvesting, isolation, culture, and passaging of NSPCs generated from the periventricular region of the adult spinal cord from the rat and from human organ transplant donors.

Periventriküler Yetişkin Rat Bölgesinde ve İnsan Omurilik gelen Nöral Kök / Progenitör Hücreler izolasyonu

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of ...

Gelişim Biyolojisi

Isolation and Characterization of Satellite Cells from Rat Head Branchiomeric Muscles

Fibrosis and defective muscle regeneration can hamper the functional recovery of the soft palate muscles after cleft palate repair. This causes persistent problems in speech, swallowing, and sucking. In vitro culture systems that allow the study of satellite cells (myogenic stem cells) from head muscles are crucial to develop new therapies based on tissue engineering to promote muscle regeneration after surgery. These systems will offer new perspectives for the treatment of cleft palate patients. A protocol for the isolation, culture and differentiation of satellite cells from head muscles is presented. The isolation is based on enzymatic digestion and trituration to release the satellite cells. In addition, this protocol comprises an innovative method using extracellular matrix gel coatings of millimeter size, which requires only low numbers of satellite cells for differentiation assays.

This protocol describes the isolation of satellite cells from branchiomeric head muscles of a 9 week-old rat. The muscles originate from different branchial arches. Subsequently, the satellite cells are cultured on a spot coating of millimeter size to study their differentiation. This approach avoids the expansion and passaging of satellite cells.

Sıçan Başkanı Branchiomeric Kaslar gelen İzolasyon ve Uydu Hücrelerinin Karakterizasyonu

Fibrosis and defective muscle regeneration can hamper the functional recovery of the soft palate muscles after cleft palate repair. This causes persistent ...

Improved Generation of Induced Cardiomyocytes Using a Polycistronic Construct Expressing Optimal Ratio of Gata4, Mef2c and Tbx5

Direct conversion of cardiac fibroblasts (CFs) into induced cardiomyocytes (iCMs) holds great potential for regenerative medicine by offering alternative strategies for treatment of heart disease. This conversion has been achieved by forced expression of defined factors such as Gata4 (G), Mef2c (M) and Tbx5 (T). Traditionally, iCMs are generated by a cocktail of viruses expressing these individual factors. However, reprogramming efficiency is relatively low and most of the in vitro G,M,T-transduced fibroblasts do not become fully reprogrammed, making it difficult to study the reprogramming mechanisms. We recently have shown that the stoichiometry of G,M,T is crucial for efficient iCM reprogramming. An optimal stoichiometry of G,M,T with relative high level of M and low levels of G and T achieved by using our polycistronic MGT vector (hereafter referred to as MGT) significantly increased reprogramming efficiency and improved iCM quality in vitro. Here we provide a detailed description of the methodology used to generate iCMs with MGT construct from cardiac fibroblasts. Isolation of cardiac fibroblasts, generation of virus for reprogramming and evaluation of the reprogramming process are also included to provide a platform for efficient and reproducible generation of iCMs.

We describe here a protocol for the generation of iCMs using retrovirus-mediated delivery of Gata4, Tbx5 and Mef2c in a polycistronic construct. This protocol yields a relatively homogeneous population of reprogrammed cells with improved efficiency and quality and is valuable for future studies of iCM reprogramming.

Gata4, Mef2c ve Tbx5 Optimal Oranı ifade bir polisistronik Construct kullanma Kaynaklı kardiyomiyositlerde Geliştirilmiş Nesil

Direct conversion of cardiac fibroblasts (CFs) into induced cardiomyocytes (iCMs) holds great potential for regenerative medicine by offering alternative ...

Isolation of Murine Embryonic Hemogenic Endothelial Cells

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, and is necessary for the emergence of definitive HSPC from the murine extra embryonic yolk sac, placenta, umbilical vessels, and the embryonic aorta-gonad-mesonephros (AGM) region. The transient nature and small size of this cell population renders its reproducible isolation for careful quantification and experimental applications technically difficult. We have established a fluorescence-activated cell sorting (FACS)-based protocol for simultaneous isolation of hemogenic endothelial cells and HSPC during their peak generation times in the yolk sac and AGM. We demonstrate methods for dissection of yolk sac and AGM tissues from mouse embryos, and we present optimized tissue digestion and antibody conjugation conditions for maximal cell survival prior to identification and retrieval via FACS. Representative FACS analysis plots are shown that identify the hemogenic endothelial cell and HSPC phenotypes, and describe a methylcellulose-based assay for evaluating their blood forming potential on a clonal level.

Hematopoietic stem and progenitor cells (HSPC) derive from specialized (hemogenic) endothelial cells during development, yet little is known about the process by which some endothelial cells specify to become blood forming. We demonstrate a flow-cytometry based method allowing simultaneous isolation of hemogenic endothelial cells and HSPC from murine embryonic tissues.

Fare Embriyonik Hemogenic endotel hücrelerinin izolasyonu

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, ...

Effective Isolation of Functional Islets from Neonatal Mouse Pancreas

Perfusion-based islet-isolation protocols from large mammalian pancreata are well established. Such protocols are readily conducted in many laboratories due to the large size of the pancreatic duct that allows for ready collagenase injection and subsequent tissue perfusion. In contrast, islet isolation from small pancreata, like that of neonatal mice, is challenging because perfusion is not readily achievable in the small pancreata. Here we describe a detailed simple procedure that recovers substantial numbers of islets from newly born mice with visual assistance. Freshly dissected whole pancreata were digested with 0.5 mg/mL collagenase IV dissolved in Hanks&#39; Balanced Salt Solution (HBSS) at 37 &#176;C, in microcentrifuge tubes. Tubes were tapped regularly to aid tissue dispersal. When most of the tissue was dispersed to small clusters around 1 mm, lysates were washed three to four times with culture media with 10% fetal bovine serum (FBS). Islet clusters, devoid of recognizable acinar tissues, can then be recovered under dissecting stereoscope. This method recovers 20 - 80 small- to large-sized islets per pancreas of newly born mouse. These islets are suitable for most conceivable downstream assays, including insulin secretion, gene expression, and culture. An example of insulin secretion assay is presented to validate the isolation process. The genetic background and degree of digestion are the largest factors determining the yield. Freshly made collagenase solution with high activity is preferred, as it aids in endocrine-exocrine isolation. The presence of cations [calcium (Ca2+) and magnesium (Mg2+)] in all solutions and fetal bovine serum in the wash/picking media are necessary for good yield of islets with proper integrity. A dissecting scope with good contrast and magnification will also help.

We describe a protocol herein for isolating intact islets from neonatal mice. Pancreata were partially digested with collagenase, followed by washing and hand picking. 20 - 80 islet clusters can be obtained per pancreas from newly born mice, which are suitable for several islet studies.

Yenidoğan Fare Pankreas gelen Fonksiyonel Adacık etkili İzolasyonu

Perfusion-based islet-isolation protocols from large mammalian pancreata are well established. Such protocols are readily conducted in many laboratories ...

Visualization of Cell Cycle Variations and Determination of Nucleation in Postnatal Cardiomyocytes

Cardiomyocytes are prone to variations of the cell cycle, such as endoreduplication (continuing rounds of DNA synthesis without karyokinesis and cytokinesis) and acytokinetic mitosis (karyokinesis but no cytokinesis). Such atypical cell cycle variations result in polyploid and multinucleated cells rather than in cell division. Therefore, to determine cardiac turnover and regeneration, it is of crucial importance to correctly identify cardiomyocyte nuclei, the number of nuclei per cell, and their cell cycle status. This is especially true for the use of nuclear markers for identifying cell cycle activity, such as thymidine analogues Ki-67, PCNA, or pHH3. Here, we present methods for recognizing cardiomyocytes and their nuclearity and for determining their cell cycle activity. We use two published transgenic systems: the Myh6-H2B-mCh transgenic mouse line, for the unequivocal identification of cardiomyocyte nuclei, and the CAG-eGFP-anillin mouse line, for distinguishing cell division from cell cycle variations. Combined together, these two systems ease the study of cardiac regeneration and plasticity.

To distinguish cell division from cell cycle variations in cardiomyocytes, we present protocols using two transgenic mouse lines: Myh6-H2B-mCh transgenic mice, for the unequivocal identification of cardiomyocyte nuclei, and CAG-eGFP-anillin mice, for distinguishing cell division from cell cycle variations.

Doğum sonrası kardiyomiyositlerde Hücre Döngüsü Varyasyonları görselleştirme ve Çekirdeklenme Belirlenmesi

Cardiomyocytes are prone to variations of the cell cycle, such as endoreduplication (continuing rounds of DNA synthesis without karyokinesis and ...

Bottlenose Dolphin (Tursiops truncatus) Spermatozoa: Collection, Cryopreservation, and Heterologous In Vitro Fertilization

The use of cryopreserved dolphin spermatozoa facilitates the exchange of genetic material between aquatic parks and makes spermatozoa accessible to laboratories for studies to further our understanding of marine mammal reproduction. Heterologous IVF, a replacement for homologous IVF, could provide a means to test the sperm fertility potential; to study gamete physiology and early embryo development; and to avoid the use of valuable dolphin oocytes, which are difficult to obtain. Here, we present protocols that have been successfully used to collect and cryopreserve dolphin spermatozoa. The collection of semen is performed by manual stimulation on trained dolphins. Cryopreservation is accomplished using a TRIS egg-yolk based extender with glycerol. In addition, we present a protocol that describes heterologous IVF using dolphin spermatozoa and bovine oocytes and that verifies the hybrid nature of the resulting embryo using PCR. Heterologous fertilization raises questions on fertilization and can be used as a tool to study gamete physiology and early embryo development. In addition, the success of heterologous IVF demonstrates the potential of this technique to test dolphin sperm fertilizing capacity, which is worth further examination.

Bottlenose Dolphin Tursiops truncatus Spermatozoa: Collection, Cryopreservation, and Heterologous In Vitro Fertilization

Here, we present protocols that have been successfully used for dolphin spermatozoa collection, cryopreservation, and heterologous IVF performance using bovine oocytes.

Şişe burunlu yunus (Tursiops truncatus) sperm: koleksiyon, dondurma ve kapaklı In Vitro fertilizasyon

The use of cryopreserved dolphin spermatozoa facilitates the exchange of genetic material between aquatic parks and makes spermatozoa accessible to ...

Maturation of Human Stem Cell-derived Cardiomyocytes in Biowires Using Electrical Stimulation

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have been a promising cell source and have thus encouraged the investigation of their potential applications in cardiac research, including drug discovery, disease modeling, tissue engineering, and regenerative medicine. However, cells produced by existing protocols display a range of immaturity compared with native adult ventricular cardiomyocytes. Many efforts have been made to mature hPSC-CMs, with only moderate maturation attained thus far. Therefore, an engineered system, called biowire, has been devised by providing both physical and electrical cues to lead hPSC-CMs to a more mature state in vitro. The system uses a microfabricated platform to seed hPSC-CMs in collagen type I gel along a rigid template suture to assemble into aligned cardiac tissue (biowire), which is subjected to electrical field stimulation with a progressively increasing frequency. Compared to nonstimulated controls, stimulated biowired cardiomyocytes exhibit an enhanced degree of structural and electrophysiological maturation. Such changes are dependent upon the stimulation rate. This manuscript describes in detail the design and creation of biowires.

The cardiac biowire platform is an in vitro method used to mature human embryonic and induced pluripotent stem cell-derived cardiomyocytes (hPSC-CM) by combining three-dimensional cell cultivation with electrical stimulation. This manuscript presents the detailed setup of the cardiac biowire platform.

Elektriksel Uyarı Biowires İnsan Kök Hücre kaynaklı kardiyomiyositlerde Olgunlaşma

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have been a promising cell source and have thus encouraged the investigation of their ...

Isolation and Characterization of Primary Rat Valve Interstitial Cells: A New Model to Study Aortic Valve Calcification

Calcific aortic valve disease (CAVD) is characterized by the progressive thickening of the aortic valve leaflets. It is a condition frequently found in the elderly and end-stage renal disease (ESRD) patients, who commonly suffer from hyperphosphatemia and hypercalcemia. At present, there are no medication therapies that can stop its progression. The mechanisms that underlie this pathological process remain unclear. The aortic valve leaflet is composed of a thin layer of valve endothelial cells (VECs) on the outer surfaces of the aortic cusps, with valve interstitial cells (VICs) sandwiched between the VECs. The use of a rat model enables the in vitro study of ectopic calcification based on the in vivo physiopathological serum phosphate (Pi) and calcium (Ca) levels of patients who suffer from hyperphosphatemia and hypercalcemia. The described protocol details the isolation of a pure rat VIC population as shown by the expression of VIC markers: alpha-smooth muscle actin (&#945;-SMA) vimentin and tissue growth factor beta (TGF&#946;) 1 and 2, and the absence of cluster of differentiation (CD) 31, a VEC marker. By expanding these VICs, biochemical, genetic, and imaging studies can be performed to study and unravel the key mediators underpinning CAVD.

This protocol describes the isolation, culture, and calcification of rat-derived valve interstitial cells, a highly physiological in vitro model of calcific aortic valve disease (CAVD). Exploitation of this rat model facilitates CAVD research in exploring the cell and molecular mechanisms that underlie this complex pathological process.

Birincil fare malzemelerin yalıtım ve Vana interstisyel hücreleri: Aort Kapak kalsifikasyon eğitim için yeni bir Model

Calcific aortic valve disease (CAVD) is characterized by the progressive thickening of the aortic valve leaflets. It is a condition frequently found in ...

Handling and Treatment of Male European Eels (Anguilla anguilla) for Hormonal Maturation and Sperm Cryopreservation

During the last years, several research groups have been working on the development and improvement of new protocols for the European eel handling and maturation. As of yet, weekly injections of human chorionic gonadotropin (hCG) have proved to maturate males after just 5-6 weeks of treatment, producing high volumes of high-quality sperm during several weeks. In addition, sperm cryopreservation protocols using different extenders, cryoprotectants and cooling and thawing times have been previously described for European eel. Here, we show that Tanaka&#180;s extender solution can be directly used for fertilization or for cryopreservation, making unnecessary the usage of different types of solutions and dilutions. Furthermore, the use of methanol as a cryoprotectant makes this protocol easy to use as methanol has low toxicity and does not activate the sperm. The sperm does not need to be cryopreserved immediately after the addition of the cryoprotectant, and it can be used long after being thawed. Moreover, sperm motility is still high after thawing although it is lower than that of fresh sperm. The aim of this work is to show the best available protocol for European eel handling, maturation, and sperm cryopreservation.

Handling and Treatment of Male European Eels Anguilla anguilla for Hormonal Maturation and Sperm Cryopreservation

Protocols for European eel maturation and sperm cryopreservation have been improved over the last years. This article describes the best protocol available using human chorionic gonadotropin (hCG) for inducing maturation and methanol as cryoprotectant.

Kullanım ve tedavi, erkek Avrupa yılan balığı (Anguilla anguilla) Hormonal olgunlaşma ve Sperm dondurma için

During the last years, several research groups have been working on the development and improvement of new protocols for the European eel handling and ...