The authors would like to acknowledge Hans de Graaf and Tomas Veenendaal for their technical assistance in culturing the ECs.

Etik bildirimi: Bu çalışmada kullanılan Kan damarları taze köpek kadavra elde edilen artı malzemesi olarak hasat edildi (n = 4), diğer ilişkisiz araştırma (Üniversite 3R politikası) için ötenazi sağlıklı köpeklerden. Anormal kan damarları (intra ve ekstrahepatik portosistemik şantlar, n = 1 adet) Utrecht Üniversitesi Companion Animals Üniversite Kliniği sunulan köpekler sahiplerinin bilgilendirilmiş onam sonra otopsi hasat edilmiştir. 1. İzolasyon ve Kültür İlköğretim Köpek Endotel Hücreleri <ol><li> 2 ml / göz% 0.5 ağırlık / hacim jelatin ile ön-kat 6 oyuklu plakalar, 37 ° C&#39;de% 5 CO2 bulunan nemli bir atmosferde bir kuluçka makinesi içinde 2 saat süre ile bırakın. önce birincil hücrelerin ekim aşırı jelatin çözüm çıkarın. </li><li> Aseptik taze köpek kadavra (Şekil 1A) kan damarı (ler) ilgi (örneğin, aort, vena kava, vena porta) çıkarın. damar sisteminin anatomisini korumakÇevrede kabın her iki ucunda, düz forseps koyarak. endotel hücrelerine zarar görmesini önlemek için forseps ile doku gereksiz manipülasyon kaçının. <ol><li> cerrahi makas ile her iki ucunda kenetlenmiş gemi kesin ve kadavra kaldırmak. buz üzerinde Hank Dengeli Tuz Çözeltisi (HBSS) taşınması. Not: yaklaşık olarak 5 sm bir tekne uzunluğu, bu prosedür için tercih edilir, ancak 1 cm boyutlarında o da kültür için yeterli hücreleri sağlar. </li></ol></li><li> buz soğukluğunda HBSS ile dolu bir Petri kabı, kan damarı aktarın. Cerrahi makas kullanılarak, damar kendisi sağlam (Şekil 1B) tutmak kabın dışından yapışık doku ve yağ çıkarmak. Optimal görünüm için bir kelepçe veya forseps ile kenara çevredeki dokuyu çekin: keserken emin damar her zaman açıkça görünür olduğundan emin olun. <ol><li> Bitişik harfler ile her türlü tekne dalları kapatın (örneğin, 3-0 polyglactin) ve daha sonra bunları kaldırmakcerrahi makas veya neşter ile. </li></ol></li><li> Dikkatle, kavisli bir Halsted sivrisinek forseps ile bir gemi ucunu girmek geminin diğer ucundaki forseps ucu kelepçe ve dışarı (Şekil 1C-G) tamamen içine kadar sonra yavaş yavaş gemiyi tersini, geri çekin. dış artık endotel hücre tabakasını içermektedir. herhangi bir zorluk gemi tersini üzerine karşılaşılırsa, sürtünmeyi azaltmak için tekrar HBSS daldırın. </li><li> Tamamen kapatmak için ve sindirim prosedürü (Şekil 1H) herhangi olmayan endotelyal damar dokusu maruz kalmasını önlemek için geminin her iki ucunda purse-string sütür koyun. ters gemi işlemek için bağ uçlarını kullanın. </li><li> sindirim ve kültür daha sonra yapılırsa, sindirim protokolü (adım 1.7) öncesinde ters gemi cryopreserve. <ol><li> kriyotüpe içinde ters gemi yerleştirin ve hücre kültür ortamı dondurma ile doldurun. Bir dondurma co kullanarak aşağı -80 ° C&#39;ye kadar Freezentainer. -80 ° C&#39;de saklayın gemiler bir hafta içinde kullanılacak ise. Uzun süreli depolama, -180 ° C&#39;de yer cryovials için. AT izolasyon gerçekleştirirken, bir su banyosu (37 ° C) hızla cryovials çözülme hemen buz soğukluğunda HBSS yerleştirin. 1.7 belirtildiği gibi devam edin. Not: izolasyondan sonra EC toplam verimi sayesinde dondurma işlemi sırasında canlılığı kaybına daha düşük olacağı dikkate alacaktır. </li></ol></li><li> 50 ml&#39;lik bir tüp gemi aktarın ve (Şekil 1I) (çözülme durumunda veya artık dondurma orta) eritrositler kaldırmak HBSS iki kez yıkayın. </li><li> Aralıklı yumuşak olan 37 ° C &#39;de 1 saat boyunca köpek Endotel Hücrelerinde 30 mi kolajenaz tip II (0.15 U / ml) ve dispase (0.15 U / ml) içeren bir çözeltiye Gelişim Ortamı (CECGM) olan bir 50 ml tüp içinde damar Digest çalkalama. </li><li> g tüpten gemi çıkarın ve x 250 5 dakika boyunca hücre süspansiyonu santrifüj. </li><li> CECGM a hücre pelletinind tohum 2 ml hücre süspansiyonu oyuk başına (1-3 kuyu damar boyutuna bağlı olarak). Kültür hava içinde% 5 CO2 ile nemli bir atmosferde 37 ° C &#39;de hücreleri ve haftada iki kez kültür ortamı değiştirin. </li><li> % 80 ulaşıldığında - 70 bir confluency Passage hücreleri. <ol><li> Ölü ya da olmayan bağlanmış hücreler uzaklaştırılmıştır önceden ısıtılmış HBSS hücreleri yıkayın. Her bir oyuğa 200 ul rekombinant hücre-ayrılma enzimi (1x) ilave edin ve 5 dakika boyunca ya da tüm hücrelerin müstakil kadar tekrar inkübatör yerleştirin. </li><li> 15 ml&#39;lik bir tüpe transfer hücreleri ve% 10 cenin buzağı serumu (FCS) içeren kültür ortamı 10 ml (CEGM) eklenerek tripsinizasyon durdurun. 250 x g hızında 5 dakika süreyle santrifüj. Jelatin önceden kaplanmış plaka veya şişede kültür devam edin. </li></ol></li></ol> 2. Karakterizasyonu <ol><li> Kültür Köpek aort endotel hücreleri (CnAOECs) ve bir mikroskop ile haftada iki kez birincil ve ticari hücre hatlarının morfolojisi karşılaştırın. NOT: TO, tipik&gt;% 70 konfluansa ulaştığında iyi görülmektedir yamalar EC desen büyüyen. <ol><li> % 0.5 ağırlık / hacim jelatin önceden kaplanmış T75 şişesinin üzerine CECGM Kültür CnAOECs. haftada bir kez Passage hücreleri confluency 70 -% 80. <ol><li> geçirilmesi için önceden ısıtılmış bir kez HBSS ile yıkama hücreleri, 1 ml yeniden birleştirici hücre ayrışma enzimi (1x) ekleyin. 5 dakika boyunca veya tüm hücreler müstakil kadar inkübatör geri şişeyi yerleştirin. % 10 FCS içeren 10 ml kültür ortamı (CEGM) eklenerek tripsin inaktive. 250 x g&#39;de 5 dakika boyunca bir hücre süspansiyonu santrifüj süpernatan atılır ve 1 ml kültür ortamında tekrar süspansiyon hücreleri. </li><li> Hücre süspansiyonu, 10 ul bir kısım almak ve 1 seyreltin:% 0.4 tripan mavisi ile 1. Otomatik hücre sayıcı kullanarak hücre sayımı ve yeni T75 şişesi içinde 4.0 x 10 5 canlı hücreleri plaka. % 5 CO2 ile nemli bir atmosferde 37 ° C&#39;de bir şişeye ve kültür önceden ısıtılmış CECGM 10 ml ekleyin. </li></ol></li></ol></li><li> Kültür CaPECs ve CnAOECs geçişi 1 RNA izole edin. <ol><li> Bir pelet gibi en az 1 x 10 3 hücreleri toplamak örnek hazırlama reaktifi (SPR) 20 ul ekle ve hücreleri lize etmek için 1 dakika için inkübe edilir. İnkübasyondan sonra, dikkatli bir şekilde -70 ° C&#39;de, toplam RNA ve mağaza ihtiva eden hücre lizatı toplar. </li></ol></li><li> Üreticinin talimatları izlenerek bir cDNA sentez kiti (örneğin, iScript) kullanılarak cDNA&#39;ya mRNA dönüştürün. qPCR gerçekleştirmek için hazır olana kadar uzun süre için 4 ° C&#39;de bir hafta kadar ya da -20 ° C&#39;de saklayın cDNA. </li><li> endotel hücre kaynağını teyit etmek için endotelyal işaretleyici CD31 gen ifadesini ölçün. MIQE Özetleyerek yönergeleri 10 kullanılarak deney düzeneği ve kantifikasyonunu gerçekleştirin. Normalleşmesi için, referans genler GAPDH, RPS19 ve B2MG 11 ifadesini ölçmek. <ol><li> nükleaz içermeyen su elde edilen cDNA bir 10-kat seyreltme hazırlayın. </li><li> hazırlamakStandart hattı için bir araya getirilmiş cDNA örnekleri bir 4-kat seyreltme ve şablon olmayan kontrol olarak nükleaz serbest su kullanın. qPCR reaksiyonları için 50 kat, toplam seyreltme ulaşmak için numune beş kez seyreltilir. 4 ul cDNA ve geri ve ileri doğru primer (Tablo 1) 20 pmol ile karıştırılmış fluorofor 6 ul kullanılarak 384 oyuklu bir formatta iki kez pipetle 10 ul reaksiyonları. </li><li> denatürasyon için 95 ° C de 10 saniye 39 döngü, ardından Taq polimeraz aktivasyonu için 5 dakika, tavlama ve uzama için Tm 30 saniye boyunca 95 ° C&#39;ye kadar bir program ayarlama. Her bir primer grubu için Tm Tablo 1 &#39;de gösterilmiştir. </li><li> erime eğrisi gerçekleştirin yükseltilir tek bir ürün sağlamak için her çalıştırmak aşağıdaki analiz eder. Referans genlerin ortalama bağıl miktarda kullanarak numunelerin ifade düzeylerini normale ve reaksiyon verimi% 95 ile% 105 arasında ise ACt hesaplayın. </li></ol></li><li> Bir Angi EC işlevselliğini değerlendirmekogenesis deneyi. <ol><li> önceden soğutulmuş bir anjiyojenez sürgünün her çukuruna hücre dışı matrisin 10 ul ilave edilip iyice yüzeyini kaplayan bir pipet ucu ile yayılır. buz üzerinde hücre dışı matris tutmak ve jele hava kabarcıklarının giriş önlemek. 30 dakika boyunca% 5 CO2 ile 37 ° C &#39;de, bir kuluçka makinesi içinde suda ıslatılmış kağıt havlu ile bir Petri kabındaki slayt koyarak jel katılaşmaya. </li><li> 1.0 x 10 4 birincil ekle EC 50 ul Endotel Gelişim Ortamı ile sabitleştirilir. % 5 CO2 ile 37 ° C &#39;de 6 saat süre ile slayt inkübe edin. </li><li> 6 saat sonra görüntüdeki tüm kuyu içerdiğinden emin yapma, 20X büyütme ile fotoğraf çekmek. </li></ol></li></ol>

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The authors have no competing financial interests.

Köpek EC odaklanan çalışmalarda CnAOEC birincil hat köpek 3, 12, 13 endotel soy modellemek için kullanılır. İnsan çalışmalarında, HUVEC kültürü hala altın standart olarak kabul edilmektedir. Açıkçası, göbek kordonu elde edilen EC üzerinde sadece odaklanan Kardiyovasküler araştırmalarda bir firma kısıtlama olduğunu. Endotel hücreleri arteriovenöz özelliklerinin belirlenmesiyle belirli bir gen ifade desen var. Doğum sonrası damarlarda bu farklılıkların hesaba katmak için bi...

Köpekler kardiyovasküler hastalık araştırmaları için büyük bir hayvan modeli olarak kullanılmaktadır ve aynı zamanda doğuştan (genetik) vasküler anormallikler 1, 2 muzdarip olabilir. Ticari endotel hücre hatları genellikle endotel hücre (EC) işlevselliğini değerlendirmek için kullanılan bu hastalıkların incelenmesi. köpekler için köpek aortundan alınan (CnAOEC) mevcuttur ticari bir endotelyal hücre hattı vardır. Bu hücre hattı çoğunlukla kontrol, normal EC 3-5 olarak çalışmalarda kullanılmaktadır. kan dolaşım araştırmada en yaygın olarak kullanılan endotel hücre çizgileri sırasıyla insan göbek kordonu damar ve arter türetilen insan Göbek Daman Endotel Hücreleri (HUVECs) ve İnsan Umbilikal Arter Endotel Hücreleri (HUAECs) vardır. HUVECler 1980&#39;lerden 6 yılından bu yana damar araştırmalarda altın standart olarak kullanılmıştır. Onlar endotel fonksiyonu ve hastalık adaptasyonunu incelemek için klasik model sistem olarak kabul edilmektedir. Farklı kan damarları izole endotel hücreleri appearanc değişirE ve mikroçevresinin 7 genetik arka plan ve maruz kalma nedeniyle işlevsellik. Buna ek olarak, HUVECler ve HUAECs, göbek kordonu şartlarına göre tamamen taklit yetişkin kan damarlarının onlar ve hastalığa yanıt maruz olmayabilir gelişimsel vasküler yapı elde edilir. Bu nedenle, genel olarak kardiyovasküler hastalık için HUVEC bulunan sonuçları ve HUAECs tercüme yetersizdir. adaptasyon ve yetişkin EC davranışını incelerken, ilgilenilen damarın birincil EC daha doğrudan bir yaklaşım olarak kullanılmalıdır. bu hücrelerin izole edilmesi için, çeşitli yöntemler bildirilmiştir. Ayrıca, HUVEC için kullanılan bir çok tarif edilen yöntem, enzimatik sindirim çözeltisi 8 kap yıkama edilir. Bu çoğu zaman, düz kas hücreleri, fibroblastlar ve 9 olmayan EC ile kontaminasyon sonuçlanır. izolasyon için yaygın olarak kullanılan bir yöntem, Floresan-takiben kıyılmış damar doku enzimatik sindirimi olanFarklılaşma (CD) 31 7, 8. FACS ayıklama endotelyal hücre belirteci küme ve daha sonra, hücre kültürü göre (FACS) aktif hücre hücrelerin nispeten büyük miktarlarda gerektirir ve bu nedenle ince kan damarlarından endotelyumun izolasyon için uygun değildir. Bu nedenle yüksek saflıkta çeşitli köpek kan damarlarından saf endotel hücre popülasyonunu izole etmek için yeni bir sağlam bir yöntem geliştirmeyi amaçladık. Yeni izolasyon yönteminin etkinliğini test etmek için, izole ve büyük ve küçük her ikisi de farklı köpek arterler ve venler, saf Canine Primer endotel hücre (CapeC) kültürleri elde etti. Bu yöntem aynı zamanda doğuştan içi veya dışı hepatik portosistemik şant, köpekler 2 ortak bir hastalık olarak hastalıklı ve / veya anormal damarlardan kaynaklanan endotel hücrelerinin kültürü sağlar. kabın en int olarak kalmaktadır, bu yöntem, vasküler düz kas hücreleri gibi aynı geminin alakalı ek hücre tiplerinin izolasyonu sağlarİşlem sırasında hareket ederler.

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			<td style="width:164px;">Cell Applications</td>
			<td style="width:211px;">Cn211-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">CnAOECs</td>
			<td style="width:164px;">Cell Applications</td>
			<td>Cn304-05</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Fetal Calf Serum (FCS)&#160;</td>
			<td style="width:164px;">GE Healthcare</td>
			<td style="width:211px;">16000-044</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TrypLE Express</td>
			<td style="width:164px;">Life Technologies</td>
			<td style="width:211px;">12604-013</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">SPR</td>
			<td style="width:164px;">Bio-Rad</td>
			<td>170-8898</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">iScript synthesis kit</td>
			<td style="width:164px;">Bio-Rad</td>
			<td style="width:211px;">170-8891</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">SYBR green super mix</td>
			<td style="width:164px;">Bio-Rad</td>
			<td style="width:211px;">170-8886</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Recovery Cell Freezing Medium</td>
			<td style="width:164px;">Gibco/Life Technologies</td>
			<td style="width:211px;">12648-010</td>
			<td>Keep on ice prior to use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Freezing container, Nalgene Mr. Frosty</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td style="width:211px;">C1562</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Gelatin</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td style="width:211px;">G1890</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Surgical scissors (Mayo or Metzenbaum)</td>
			<td style="width:164px;">B. Braun Medical</td>
			<td style="width:211px;">BC555R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mosquito forceps&#160;</td>
			<td style="width:164px;">B. Braun Medical</td>
			<td style="width:211px;">FB440R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Mosquito forceps curved</td>
			<td style="width:164px;">B. Braun Medical</td>
			<td style="width:211px;">FB441R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">polyglactin 3-0</td>
			<td style="width:164px;">Ethicon</td>
			<td style="width:211px;">VCP311H</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Trypan blue</td>
			<td style="width:164px;">Bio-Rad</td>
			<td style="width:211px;">145-0013</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Automated counting chamber</td>
			<td style="width:164px;">Bio-Rad</td>
			<td style="width:211px;">145-0102</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Counting Slides, Dual Chamber</td>
			<td style="width:164px;">Bio-Rad</td>
			<td style="width:211px;">145-0011</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Matrigel</td>
			<td style="width:164px;">BD Biosciences</td>
			<td style="width:211px;">BD356231</td>
			<td>Slowly thaw on ice</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">&#181;-Slide Angiogenesis</td>
			<td style="width:164px;">Ibidi</td>
			<td style="width:211px;">81501</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Endothelial Growth Medium</td>
			<td style="width:164px;">Lonza</td>
			<td style="width:211px;">CC-3156</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">EGM-2 SingleQuot Kit&#160;</td>
			<td style="width:164px;">Lonza</td>
			<td style="width:211px;">CC-4176</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation and culture of primary endothelial cells from canine arteries and veins

Cardiovascular disease is studied in both human and veterinary medicine. Endothelial cells have been used extensively as an in vitro model to study vasculogenesis, (tumor) angiogenesis, and atherosclerosis. The current standard for in vitro research on human endothelial cells (ECs) is the use of Human Umbilical Vein Endothelial Cells (HUVECs) and Human Umbilical Artery Endothelial Cells (HUAECs). For canine endothelial research, only one cell line (CnAOEC) is available, which is derived from canine aortic endothelium. Although currently not completely understood, there is a difference between ECs originating from either arteries or veins. For a more direct approach to in vitro functionality studies on ECs, we describe a new method for isolating Canine Primary Endothelial Cells (CaPECs) from a variety of vessels. This technique reduces the chance of contamination with fast-growing cells such as fibroblasts and smooth muscle cells, a problem that is common in standard isolation methods such as flushing the vessel with enzymatic solutions or mincing the vessel prior to digestion of the tissue containing all cells. The technique we describe was optimized for the canine model, but can easily be utilized in other species such as human.

Farklı kan damarları başarıyla açıklanan izolasyon protokolü (Şekil 2) tabi tutuldu. Teşrih ve sağlıklı köpeklerden aort, vena kava, vena porta ve koroner arter ters mümkün olmuştur (her köpekten tüm damarları, n = 4). Aynı yaklaşım EC iki doğumsal portosistemik şant (ekstrahepatik ve intrahepatik, n = 1 adet) izole edilmiştir ile. Aort kolayca ters olmasına rağmen, aort segmentleri abdominal aorta daha zorlu idi. göğüs segmentlerde aort sin...

Watch this Scientific Journal Video about Isolation and Culture of Primary Endothelial Cells from Canine Arteries and Veins at JoVE.com

Isolation and Culture of Primary Endothelial Cells from Canine Arteries and Veins

Novel isolation methods of primary endothelial cells from blood vessels are needed. This protocol describes a new technique that completely inverts blood vessels of interest, exposing only the endothelial side to enzymatic digestion. The resulting pure endothelial cell culture can be used to study cardiovascular diseases, disease modelling, and angiogenesis.

Köpek arter ve venlerin Primer Endotel Hücreleri İzolasyon ve Kültür

Cardiovascular disease is studied in both human and veterinary medicine. Endothelial cells have been used extensively as an in vitro model to study ...

isolation-culture-primary-endothelial-cells-from-canine-arteries

Research

JoVE Journal

Medicine

11.2K Görüntüleme.  Utrecht University. Novel isolation methods of primary endothelial cells from blood vessels are needed. This protocol describes a new technique that completely inverts blood vessels of interest, exposing only the endothelial side to enzymatic digestion. The resulting pure endothelial cell culture can be used to study cardiovascular diseases, disease modelling, and angiogenesis.

Video: Isolation and Culture of Primary Endothelial Cells from Canine Arteries and Veins

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

İnsan Diş Pulp Kök Hücre İzolasyonu, Karakterizasyonu ve Karşılaştırmalı Farklılaşma İki Farklı Yöntemler Kullanılarak Daimi Dişler türetilen

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Tıp

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

Hasta Türetilen Hücre Kültürü ve CD133 İzolasyonu<sup&gt; +</supMelanom dan&gt; sağlayan olası Kanser Kök Hücreleri

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

Primary Culture of Human Vestibular Schwannomas

Vestibular schwannomas (VSs) represent Schwann cell (SC) tumors of the vestibular nerve, compromising 10% of all intracranial neoplasms. VSs occur in either sporadic or familial (neurofibromatosis type 2, NF2) forms, both associated with inactivating defects in the NF2 tumor suppressor gene. Treatment for VSs is generally surgical resection or radiosurgery, however the morbidity of such procedures has driven investigations into less invasive treatments. Historically, lack of access to fresh tissue specimens and the fact that schwannoma cells are not immortalized have significantly hampered the use of primary cultures for investigation of schwannoma tumorigenesis. To overcome the limited supply of primary cultures, the immortalized HEI193 VS cell line was generated by transduction with HPV E6 and E7 oncogenes. This oncogenic transduction introduced significant molecular and phenotypic alterations to the cells, which limit their use as a model for human schwannoma tumors. We therefore illustrate a simplified, reproducible protocol for culture of primary human VS cells. This easily mastered technique allows for molecular and cellular investigations that more accurately recapitulate the complexity of VS disease.

Vestibular Schwannomas (VSs) are non-malignant tumors of Schwann cell (SC) origin, associated with mutations in the NF2 tumor suppressor gene. We report a reproducible, efficient protocol for primary human VS cell culture that allows for molecular and cellular experimental manipulation and analysis and recapitulates the heterogeneous nature of human disease.

İnsan Vestibüler Schwannomalar İlköğretim Kültürü

Vestibular schwannomas (VSs) represent Schwann cell (SC) tumors of the vestibular nerve, compromising 10% of all intracranial neoplasms. VSs occur in ...

Method for Obtaining Primary Ovarian Cancer Cells From Solid Specimens

Reliable tools for investigating ovarian cancer initiation and progression are urgently needed. While the use of ovarian cancer cell lines remains a valuable tool for understanding ovarian cancer, their use has many limitations. These include the lack of heterogeneity and the plethora of genetic alterations associated with extended in vitro passaging.&#160;Here we describe a method that allows for rapid establishment of primary ovarian cancer cells form solid clinical specimens collected at the time of surgery. The method consists of subjecting clinical specimens to enzymatic digestion for 30 min. The isolated cell suspension is allowed to grow and can be used for downstream application including drug screening. The advantage of primary ovarian cancer cell lines over established ovarian cancer cell lines is that they are representative of the original specific clinical specimens they are derived from and can be derived from different sites whether primary or metastatic ovarian cancer.

This study describes a detailed method for isolation and characterization of primary ovarian cancer cells from solid clinical specimens. Ovarian cancer clinical specimens are subjected to enzymatic digestion to obtain viable, fibroblast-free epithelial ovarian cancer (EOC) cells highly suitable for downstream applications.

Katı Örneklerinin From İlköğretim Yumurtalık Kanseri Hücreleri alma için yöntem

Reliable tools for investigating ovarian cancer initiation and progression are urgently needed. While the use of ovarian cancer cell lines remains a ...

Isolation, Cryopreservation and Culture of Human Amnion Epithelial Cells for Clinical Applications

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a potential source of cells for regenerative medicine. The reason for this interest is evidence that these cells have highly multipotent differentiation ability, low immunogenicity, and anti-inflammatory functions. These properties have prompted researchers to investigate the potential of hAECs to be used to treat a variety of diseases and disorders in pre-clinical animal studies with much success.
hAECs have found widespread application for the treatment of a range of diseases and disorders. Potential clinical applications of hAECs include the treatment of stroke, multiple sclerosis, liver disease, diabetes and chronic and acute lung diseases. Progressing from pre-clinical animal studies into clinical trials requires a higher standard of quality control and safety for cell therapy products. For safety and quality control considerations, it is preferred that cell isolation protocols use animal product-free reagents. 
We have developed protocols to allow researchers to isolate, cryopreserve and culture hAECs using animal product-free reagents. The advantage of this method is that these cells can be isolated, characterized, cryopreserved and cultured without the risk of delivering potentially harmful animal pathogens to humans, while maintaining suitable cell yields, viabilities and growth potential. For researchers moving from pre-clinical animal studies to clinical trials, these methodologies will greatly accelerate regulatory approval, decrease risks and improve the quality of their therapeutic cell population.

We describe a protocol to isolate and culture human amnion epithelial cells (hAECs) using animal product-free reagents in accordance with current good manufacturing practices (cGMP) guidelines.

İzolasyon, Kriyoprezervasyon ve Kültür Klinik Uygulamaları İnsan Amniyon Epitel Hücreleri

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a ...

Isolation of Human Lymphatic Endothelial Cells by Multi-parameter Fluorescence-activated Cell Sorting

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity but little is known about their biology. Isolating highly pure human lymphatic endothelial cells (LECs) from diseased and healthy tissue would facilitate studies of the lymphatic endothelium at genetic, molecular and cellular levels. It is anticipated that these investigations may reveal targets for new therapies that may change the clinical management of these conditions. A protocol describing the isolation of human foreskin LECs and lymphatic malformation lymphatic endothelial cells (LM LECs) is presented. To obtain a single cell suspension tissue was minced and enzymatically treated using dispase II and collagenase II. The resulting single cell suspension was then labelled with antibodies to cluster of differentiation (CD) markers CD34, CD31, Vascular Endothelial Growth Factor-3 (VEGFR-3) and PODOPLANIN. Stained viable cells were sorted on a fluorescently activated cell sorter (FACS) to separate the CD34LowCD31PosVEGFR-3PosPODOPLANINPos LM LEC population from other endothelial and non-endothelial cells. The sorted LM LECs were cultured and expanded on fibronectin-coated flasks for further experimental use.

The goal of this protocol is to isolate lymphatic endothelial cells lining human lymphatic malformation cyst-like vessels and foreskins using fluorescence-activated cell sorting (FACS). Subsequent cell culturing and expansion of these cells permits a new level of experimental sophistication for genetic, proteomic, functional and cell differentiation studies.

Çoklu-parametre floresans ile aktive edilen hücre sınıflandırma İnsan lenfatik endotel hücreleri İzolasyonu

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity ...

Isolation and Propagation of Circulating Tumor Cells from a Mouse Cancer Model

Cancer metastasis is the foremost cause of cancer-associated deaths. Recent studies have shown that circulating tumor cells (CTCs) are important in cancer metastasis. Indeed, the number of CTCs correlates with tumor size. Here, a detailed description is provided of a methodology for isolation and propagation of CTCs from a syngeneic mouse model of hepatocellular carcinoma (HCC) which allows for downstream analysis of potentially important molecular mechanisms of solid organ tumor metastasis. This method is efficient and reproducible. It is a non-invasive technique and, therefore, has potential to replace the invasive biopsy of tissues from humans which may be associated with complications. Therefore, the method discussed here allows for the isolation and propagation of CTCs from whole blood samples such that they can be examined and characterized. This has potential for future adaptation for clinical applications such as diagnosis, and personalized targeted therapy.

Circulating tumor cells (CTCs) have been shown to play an important role in tumor metastasis. Here, a method for the isolation and propagation of CTCs from the whole blood of a syngeneic mouse tumor model of hepatocellular carcinoma (HCC) metastasis is described.

Fare Kanser Modeli Tümör Hücreleri Sirkülasyon İzolasyonu ve Yayılım

Cancer metastasis is the foremost cause of cancer-associated deaths. Recent studies have shown that circulating tumor cells (CTCs) are important in cancer ...

Isolation and Culture Expansion of Tumor-specific Endothelial Cells

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model for developing new angiogenesis inhibitors for cancer. However, long-term in vitro expansion of murine endothelial cells (EC) is challenging due to phenotypic drift in culture (endothelial-to-mesenchymal transition) and contamination with non-EC. This is especially true for TEC which are readily outcompeted by co-purified fibroblasts or tumor cells in culture. Here, a high fidelity isolation method that takes advantage of immunomagnetic enrichment coupled with colony selection and in vitro expansion is described. This approach generates pure EC fractions that are entirely free of contaminating stromal or tumor cells. It is also shown that lineage-traced Cdh5cre:ZsGreenl/s/l reporter mice, used with the protocol described herein, are a valuable tool to verify cell purity as the isolated EC colonies from these mice show durable and brilliant ZsGreen fluorescence in culture.

We report a reliable method to isolate and culture primary tumor-specific endothelial cells from genetically engineered mouse models.

Tümör spesifik Endotel Hücreleri İzolasyon ve Kültürü Genişleme

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model ...

Isolation of Endothelial Progenitor Cells from Healthy Volunteers and Their Migratory Potential Influenced by Serum Samples After Cardiac Surgery

Endothelial progenitor cells (EPCs) are recruited from the bone marrow under pathological conditions like hypoxia and are crucially involved in the neovascularization of ischemic tissues. The origin, classification and characterization of EPCs are complex; notwithstanding, two prominent sub-types of EPCs have been established: so-called &#34;early&#34; EPCs (subsequently referred to as early-EPCs) and late-outgrowth EPCs (late-EPCs). They can be classified by biological properties as well as by their appearance during in vitro culture. While &#34;early&#34; EPCs appear in less than a week after culture of peripheral blood-derived mononuclear cells in EC-specific media, late-outgrowth EPCs can be found after 2-3 weeks. Late-outgrowth EPCs have been recognized to be directly involved in neovascularization, mainly through their ability to differentiate into mature endothelial cells, whereas &#34;early&#34; EPCs express various angiogenic factors as endogenous cargo to promote angiogenesis in a paracrine manner. During myocardial ischemia/reperfusion (I/R), various factors control the homing of EPCs to regions of blood vessel formation.
Macrophage migration inhibitory factor (MIF) is a chemokine-like pro-inflammatory and ubiquitously expressed cytokine and was recently described to function as key regulator of EPCs migration at physiological concentrations1. Interestingly, MIF is stored in intracellular pools and can rapidly be released into the blood stream after several stimuli (e.g. myocardial infarction). 
This protocol describes a method for the reliable isolation and culture of early-EPCs from adult human peripheral blood based on CD34-positive selection with subsequent culture in medium containing endothelial growth factors on fibronectin-coated plates for use in in vitro migration assays against serum samples of cardiac surgical patients. Furthermore, the migratory influence of MIF on chemotaxis of EPCs compared to other well-known angiogenesis-stimulating cytokines is demonstrated.

Endothelial progenitor cells (EPCs) are crucially involved in the neovascularization of ischemic tissues. This method describes the isolation of human EPCs from peripheral blood, as well as the identification of their migratory potential against serum samples of cardiac surgical patients.

Kalp Damar Cerrahisi sonra serum Örnekleri etkilenerek Sağlıklı Gönüllüler ve Bunların Göçmen Potansiyelden Endotel Ata Hücreleri İzolasyon

Endothelial progenitor cells (EPCs) are recruited from the bone marrow under pathological conditions like hypoxia and are crucially involved in the ...

A Cell Culture Model of Resistance Arteries

The myoendothelial junction (MEJ), a unique signaling microdomain in small diameter resistance arteries, exhibits localization of specific proteins and signaling processes that can control vascular tone and blood pressure. As it is a projection from either the endothelial or smooth muscle cell, and due to its small size (on average, an area of ~1 &#181;m2), the MEJ is difficult to study in isolation. However, we have developed a cell culture model called the vascular cell co-culture (VCCC) that allows for in vitro MEJ formation, endothelial cell polarization, and dissection of signaling proteins and processes in the vascular wall of resistance arteries. The VCCC has a multitude of applications and can be adapted to suit different cell types. The model consists of two cell types grown on opposite sides of a filter with 0.4 &#181;m pores in which the in vitro MEJs can form. Here we describe how to create the VCCC via plating of cells and isolation of endothelial, MEJ, and smooth muscle fractions, which can then be used for protein isolation or activity assays. The filter with intact cell layers can be fixed, embedded, and sectioned for immunofluorescent analysis. Importantly, many of the discoveries from this model have been confirmed using intact resistance arteries, underscoring its physiological relevance.

A cell culture model of resistance arteries is described, allowing for the dissection of signaling pathways in endothelium, smooth muscle, or between endothelium and smooth muscle (the myoendothelial junction). The selective application of agonists or protein isolation, electron microscopy, or immunofluorescence can be utilized using this cell culture model.

Hücre kültür manken direnç arter

The myoendothelial junction (MEJ), a unique signaling microdomain in small diameter resistance arteries, exhibits localization of specific proteins and ...