Yazarlar Sindirim Moleküler Araştırmalar UPenn NIDDK Merkezi Moleküler Patoloji ve Görüntüleme Çekirdek ve görüntüleme yardım için Karaciğer Hastalıkları (P30 DK50306) müteşekkiriz. Bu çalışma (RGW için) Ulusal Sağlık Enstitüleri (R01 DK-092111) gelen ve Fred ve Suzanne Biesecker Pediatrik Karaciğer Merkezi&#39;nden hibe ile ve (SK) Çocukluk Karaciğer Hastalıkları Araştırma ve Eğitim Ağı bir burs ile desteklenmiştir.

Aksi belirtilmediği sürece, tüm işlem, oda sıcaklığında gerçekleştirilir. Tüm hayvan çalışmaları yerel Kurumsal Hayvan Bakım ve Kullanım Komitesi (IACUC) tarafından onaylanan bir protokol çerçevesinde insani şartlarda yapılmalıdır. 1.. Ekipmanları hazırlanması ve Çözümler <ol><li> Doku kültürü kaputu ve inkübatör (Şekil 1A) yakın fare cerrahi tablo oluşturdu. Not: Gerekli donanımları bir diseksiyon mikroskobu, bir ışık kaynağı, 12.5 cm uzunluğunda düz iris makası, ince ipuçları ile 6 inç olmayan tırtıklı kavisli bir forseps ve 4 inç kavisli uçları (Şekil 1B) ile tırtıklı forseps içerir. </li><li> Cerrahi masaya% 70 alkol, bir cam beher içinde sterilize aletleri yerleştirin. </li><li> Steril izolasyon medya (Tablo 1) ile üç steril 60 mm Petri kapları doldurun; cerrahi masada iki ve kaput biri, buz üzerinde tüm yerleştirin. </li><li> Sıçan kuyruğu kollajen yerleştirin,10x steril fosfat tamponlu tuzlu su (PBS), steril su, steril 1 N NaOH ve doku kültürü kaputu safra epitel hücre (BEC) ortamı (Tablo 1). Not: kollajen buz üzerinde olmalıdır. </li></ol> 2.. Safra Kanalı İzolasyon ve Kültür <ol><li> Bilinçsiz IACUC kılavuzlar (Şekil 1C) başına servikal dislokasyon ile takip kadar karbon dioksite maruz bırakılarak, yaşam 0-3 gün arasında yeni doğan farelerin Euthanize. Not: eski fareler spesifik deneysel gereksinimlerine bağlı olarak kullanılabilir. </li><li> Sırtüstü pozisyonda hayvan yerleştirin ve pelvis bölgesinde, orta hattı geçen bir küçük yatay bir kesi yapmak. U şekilli flep oluşturulması, yanal ve yukarı karın her iki tarafını bu kesi uzatın. Karın organları maruz kapağını açın. Karın açıldığında, dikkatle karaciğer ve safra kanalının daha iyi görselleştirme için hayvanın sağ tarafına doğru karın boşluğundan dışarı bağırsak hareket. Not: Karaciğer iyi kimlik elde etmek için göğüs kafesi içine yanal kesiler uzatmak için gerekli olabilir. </li><li> Diseksiyon mikroskobu kullanılarak, ortak safra kanalı, karaciğer, bağırsak ve tanımlamak. Yapıları çok hassas olduğu gibi, dikkatli kullanarak, ilk ortak safra kanalı (Şekil 1D) tanımlayın. Tırtıklı olmayan dişli forseps ile, titizlikle temiz ve safra çevreleyen yağ dahil olmak üzere bağ dokusu koparmak. Kanal ve temizlemek için diğer tutmak için bir forseps kullanın. </li><li> Safra kesesini temizlemek için benzer bir tekniği kullanın. </li><li> Buz üzerinde izolasyon medyanın ilk yemeğin içine temizlenmiş kanallarını ve safra kesesini yerleştirin. Medyada daha fazla bağ dokusu kaldırmak ve safra kesesi safra çıkartırken hafifçe olmayan dişli forseps ile yapıları masaj. </li><li> Izolasyon medyanın ikinci çanak kanallarını ve safra kesesini aktarın. </li><li> 5 hayvanlardan elde edilen kanal ve safra kesesi izolasyonundan sonra, aktarmakaputu üçüncü Petri kabı parçaları. Not: hücreler, en fazla 5 hayvanlardan izole edilecekse, 5 hayvandan oluşan her grup için yeni çözeltiler kullanılarak, gruplar halinde var. </li><li> Doku kültürü kaputu kalın kollajen jeller hazırlayın: Not: herhangi bir boyut çanak kullanılabilir. 5 yeni doğan her bir grup için 3 ml kolajen bir son hacme sahip 60 mm kültür çanağı iyi çalışır. Bu taze izole edilmiş kanalları gömme anda taze hazırlanmalıdır. <ol><li> Buz üzerinde fare kuyruk kolajeni, 10x steril fosfat tamponlu tuzlu su (PBS 10x), steril dH 2 O ve steril 1 N NaOH yerleştirin. </li><li> Kollajen 0.023 katı hacmine eşittir 1 N NaOH hacmi kullanılarak, dH 2 0, 10x PBS, NaOH ve kollajen 1x PBS içinde 2 mg / ml kolajen nihai konsantrasyonuna sahip jeller için gerekli olan miktarlar hesaplayın ilave edildi. Not: kollajen alternatif kaynak kullanılırsa, nötralizasyon ve jel oluşumu için üreticinin yönergelerini izleyin. </li><li> Birlikte dH 2 0, 1 mix0 x PBS, ve 1 N NaOH, daha sonra da karıştırma sağlamak için kolajen ve pipet kuyu ekleyin. </li></ol></li><li> Kolajen çözeltisi, oda sıcaklığında benzeri bir kıvam jel kalınlaştırılmış sonra, hemen kollajen kanalları gömmek. </li><li> % 5 CO2 30 dakika boyunca katılaşması için kolajen sağlamak için, 37 ° C&#39;de inkübatör yerleştirin. </li><li> Jel (beyaz açık renk değişiklikleri), BEC medya (Tablo 1) 3.5 ml ile kaplamayı katılaşmış ve 37 ° C&#39;de inkübe edildikten sonra,% 5 CO 2. </li><li> BEC medya haftada 3 kez kaldırmak ve 3.5 ml taze ortam ile değiştirin. </li></ol> Kalın Kollajen Jeller 3. Isolating İlköğretim Cholangiocytes Not: 3 hafta içinde, cholangiocytes (büyük çekirdekler gömülü kanallarından doğrudan uzanan hücreler) yaprak kalın kollajen görünür olacaktır. Çanak fibroblastların çok sayıda vardır, bu atılmalıdır. Yemeğin bir kaç bölge varsafibroblast büyüme, bu bir bistüriyle kesilip ve önceki cholangiocytes bölme için çıkarılabilir. <ol><li> Ince kollajen jeller hazırlayın: <ol><li> Steril PBS ile 1 mg / ml &#39;lik bir konsantrasyona kadar kollajen seyreltin. Not: 100 mm tabak için 1 ml kullanmak; Diğer boyut yemekler için buna göre ayarlayın. </li><li> Bir hücre dağıtıcı (büyük yemekleri) veya pipet ucu (küçük yemekler) ile eşit şekilde yayılmış kolajen çözeltisi, daha sonra 10 dakika için oda sıcaklığında bırakın. </li><li> DMEM/F12 plaka kapak ve 37 ° C, 10 dakika boyunca% 5 CO2 ile inkübe edilir. </li><li> Inkübatör plakasını çıkarın ve 1x PBS ile yıkayın. Hemen 1x PBS aspire. </li><li> Plakası döner veya bir çalkalayıcı levha üzerinde yayma kollajen yerleştirerek ikinci bir ince tabaka ile kaplayın. 37 ° C&#39;de, 10 dakika boyunca% 5 CO2 ile inkübe edin. </li><li> Kollajen katılaşan sonra, kapak DMEM/F12 plakası ve 37 ° C, 30 dakika boyunca% 5 CO2 ile inkübe edilir. </li></ol></li><li> Th hücreleri bölmeTran kollajen jeller: <ol><li> Bu yüzer böylece hücre kültürünün bir spatula ya da hücre kazıyıcı ile, plaka kapalı yavaşça kollajen jel kazıyın. </li><li> DMEM/F12 ile 10 mg / ml Tip XI kolajenaz (maddeler listesine bakınız) seyreltilerek kolajenaz çözeltisi hazırlayın. De ve steril filtre karıştırın. </li><li> Kalın kollajen hücrelerin 60 mm tabak başına kolajenaz çözeltisi (yukarıdaki) 1 ml ilave edilir. (3 ml olmalıdır) BEC ortamı çıkarmayın. 37 ° C&#39;de 30 dakika boyunca inkübe çanak,% 5 CO2. </li><li> Hücreleri içeren çözüm çıkarın ve 15 ml konik bir tüp içinde yer. </li><li> 5 dakika boyunca 2.200 xg&#39;de aşağı spin. Süpernatantı atın ve DMEM/F12 benzer hacminde pelet askıya yeniden. </li><li> 5 dakika boyunca 2,200 x g hızında tekrar çözelti Spin. Süpernatantı. </li><li> 3,% 0.5 tripsin ml ve 37 ° C&#39;de 5 dakika boyunca,% 5 CO2 inkübe içinde süspanse pelet. Inkübasyondan sonra, çözüm yukarı pipet ve aşağı cholangiocytes bir yaprak kırmak için. BEC medya a 5 ml ekleyin5 dakika boyunca 1500 xg nd sıkma çözüm. </li><li> Daha sonra 5 dakika boyunca 1500 x g&#39;de çözeltisi spin DMEM/F12 süpernatant ve pelet tekrar süspansiyon çıkarın. </li><li> Süpernatantı ve BEC medya (Tablo 1) pelet askıya yeniden. Önceden yapılmış ince kollajen jeller üzerinde plaka hücreleri. </li></ol></li></ol> Hücreler 4. Passage ve Depolama Not: Gerektiğinde ince kollajen jeller üzerinde hücreler bölünebilir. <ol><li> Önce 10-15 dakika boyunca 37 ° C&#39;de% 0.25 tripsin kuluçka için steril PBS ile plakaları yıkayın. </li><li> BEC ortamının bir eşit hacmi ile nötralize ve 5 dakika boyunca 1500 x g&#39;de topak. DMEM/F12 pelet durulayın, daha sonra 5 dakika boyunca 1500 x g&#39;de topak. </li><li> Kollajen kaplı hücre kültürü yemekleri dağıtımı için BEC medyada süspanse edin hücreleri. Not: Alternatif olarak, hücreler, depolama ortamı içinde yeniden süspansiyon haline getirilmiş ve -80 ° C veya sıvı azot dondurucu (Tablo 1) saklanabilir. </li></ol>

<ol>
	<li>Paradis, K., Sharp, 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=In+vitro+duct-like+structure+formation+after+isolation+of+bile+ductular+cells+from+a+murine+model.">In vitro duct-like structure formation after isolation of bile ductular cells from a murine model.</a> J. Lab. Clin. Med. 113 (6), 689-694 (1989).</li><li>Vroman, B., LaRusso, N. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+characterization+of+polarized+primary+cultures+of+rat+intrahepatic+bile+duct+epithelial+cells.">Development and characterization of polarized primary cultures of rat intrahepatic bile duct epithelial cells.</a> Lab. Invest. 74 (1), 303-313 (1996).</li><li>Kumar, U., Jordan, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+biliary+epithelial+cells+from+the+biliary+tract+fraction+of+normal+rats.">Isolation and culture of biliary epithelial cells from the biliary tract fraction of normal rats.</a> Liver. 6 (6), 369-378 (1986).</li><li>Ishii, M., Vromen, B., LaRusso, N. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+morphologic+characterization+of+bile+duct+epithelial+cells+from+normal+rat+liver.">Isolation and morphologic characterization of bile duct epithelial cells from normal rat liver.</a> Gastroenterology. 97 (5), 1236-1247 (1989).</li><li>Strazzabosco, M., Fabris, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+the+bile+ducts:+Essentials+for+the+clinical+hepatologist.">Development of the bile ducts: Essentials for the clinical hepatologist.</a> J. Hepatol. 56 (5), 1159-1170 (2012).</li><li>Carpentier, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+ductal+plate+cells+give+rise+to+cholangiocytes,+periportal+hepatocytes,+and+adult+liver+progenitor+cells.">Embryonic ductal plate cells give rise to cholangiocytes, periportal hepatocytes, and adult liver progenitor cells.</a> Gastroenterology. 141 (4), 1432-1438 (2011).</li><li>Cho, W. K., Mennon, A., Boyer, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+functional+polarized+bile+duct+units+from+mouse+liver.">Isolation of functional polarized bile duct units from mouse liver.</a> Am. J. Physiol. Gastrointest. Liver Physiol. 280 (2), (2001).</li><li>Karjoo, S., Hand, N. J., Loarca, L., Russo, P. A., Friedman, J. R., Wells, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extra-hepatic+cholangiocyte+cilia+are+abnormal+in+biliary+atresia.">Extra-hepatic cholangiocyte cilia are abnormal in biliary atresia.</a> J. Pediatr. Gastroenterol. Nutr. 57 (1), 96-101 (2013).</li><li>Sutton, M. E., op den Dries, S., Koster, M. H., Lisman, T., Gouw, A. S., Porte, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+of+human+extrahepatic+biliary+epithelium:+the+peribiliary+glands+as+progenitor+cell+compartment.">Regeneration of human extrahepatic biliary epithelium: the peribiliary glands as progenitor cell compartment.</a> Liver Int. 32 (4), 554-559 (2012).</li><li>Cardinale, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multipotent+stem/progenitor+cells+in+human+biliary+tree+give+rise+to+hepatocytes,+cholangiocytes,+and+pancreatic+islets.">Multipotent stem/progenitor cells in human biliary tree give rise to hepatocytes, cholangiocytes, and pancreatic islets.</a> Hepatology. 54 (6), 2159-2172 (2011).</li></ol>

Yazarlar, herhangi bir çıkar çatışması olmadığını beyan ederim.

Burada açıklanan yenidoğan dahil her yaştan farelerin farelerin ekstrahepatik safra kanallarında saf cholangiocytes izole etmek için bir tekniktir. Teknik ekstrahepatik cholangiocytes intrahepatik cholangiocytes ayrı ele alınabilir ki avantaj sunuyor, ve hücrelerin bu toplumlar arasında önemli farklılıklar tespit çalışmaları kolaylaştırabilir. Biz son zamanlarda bir çalışma demonstrating bu yöntemle izole ve al yanaklı rotavirüs 8 ile enfekte ekstrahepatik cholangiocytes içinde kirpi...

Intra ve ekstrahepatik cholangiocytes kökeni ve gelişim belirgin farklıdır. Karaciğer ventral foregut endoderm 5 bir divertikül gelişir. Kranial bölge intrahepatik safra yolları 5 üretirken divertikül kaudal bölge, ekstrahepatik safra yolları oluşturur. Intrahepatik kanal cholangiocytes peri portal bölgelerde 6&#39;da duktal plaka çevresinde projenitör hücrelerinden türetilmiştir. Bu hücreler hepatositler veya cholangiocytes 6 ya da içine ayırt etmek yeteneğine sahip. Bu cholangiopathies özellikle cholangiocytes bir kategori hedef olabileceğini verilen önemli klinik etkileri vardır. Alagille Sendromu intrahepatik kanalları etkiler Örneğin, biliyer atrezi başlangıçta, yenidoğanın ekstrahepatik kanalları etkiler. Fare ve sıçan cholangiocytes intrahepatik safra kanalı ve birimlerin tecrit çok açıklamaları vardır. Içindevestigators kanal izolasyon ve sonraki akıbet tarafından tek hücreleri izole veya karaciğer sindirim ve antikor spesifik hücre yüzey belirteçleri 1-4 ifade cholangiocytes aşağı çekme var. Fonksiyonel ve polarize safra kanalı birimleri karaciğer sindirim ve boyut filtrasyon 7 tarafından izole edilmiştir. Safra kanalı tane izole cholangiocytes vitro 1,2 kanalsal yapıların geliştirilmesi için gösterilmiştir ve, uyarı salgılama ve sıvı salgı 7 göstermek için yanıt yeteneğine sahiptir. Bu yöntemlerden Sınırlamalar sindirim enzimleri ile karaciğer perfüzyon için uzman teknik uzmanlık ve özel ekipman ihtiyacı bulunmaktadır. Buna ek olarak, mezenkimal hücreler 3 ile kirlenme riski vardır. Özel olarak neonatal ekstrahepatik safra kanallarının ikinci, ekstrahepatik safra kanalı cholangiocytes izole edilmesi için bir yöntem olup, daha önce tarif edilmemiştir. Bu yazıda yenidoğan a izole etmek için basitleştirilmiş bir teknik özetliyoriyi saflık yüksek seviyelerde yetişkin ekstrahepatik safra kanalı hücreleri olarak var. Bu teknik, intra ve ekstrahepatik cholangiocytes ve ekstrahepatik kanalları dahil safra atrezi gibi hastalıkların mekanizmaları araştırma arasındaki farkların çalışma kolaylaştıracaktır.

<table border="0" cellpadding="0" cellspacing="0" style="width:943px;" width="942">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Name of Material/Equipment</td>
			<td style="width:323px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">DMEM/F12 (1:1)</td>
			<td style="width:323px;">Gibco/ Life technologies</td>
			<td style="width:119px;">11320-033</td>
			<td>500ml, used in BEC media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">FBS</td>
			<td style="width:323px;">Atlanta Biologicals</td>
			<td style="width:119px;">S11150</td>
			<td>25 ml, used in BEC media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">MEM with non-essential amino acids</td>
			<td style="width:323px;">Gibco/ Life technologies</td>
			<td style="width:119px;">11140-019</td>
			<td>5 ml, used in BEC media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Insulin-transferrin-selenium</td>
			<td style="width:323px;">Gibco/ Life technologies</td>
			<td style="width:119px;">51300-044</td>
			<td>5 ml, used in BEC media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Na Pyruvate</td>
			<td style="width:323px;">Cellgro</td>
			<td style="width:119px;">25-000-CL</td>
			<td>5ml, used in BEC media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Chemically-defined lipid concentrate</td>
			<td style="width:323px;">Gibco/ Life technologies</td>
			<td style="width:119px;">11905-031</td>
			<td>5ml, used in BEC media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Penicillin-Streptomycin</td>
			<td style="width:323px;">Cellgro</td>
			<td style="width:119px;">30-002-CI</td>
			<td>5ml, used in BEC media and 500 ul in the isolation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Gentamicin</td>
			<td style="width:323px;">Gibco/ Life technologies</td>
			<td style="width:119px;">15750-060</td>
			<td>0.2ml, used in BEC media and 500 ul in the isolation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Ethanolamine</td>
			<td style="width:323px;">Sigma Aldrich</td>
			<td style="width:119px;">E9508-100ml</td>
			<td>0.13ml, used in BEC media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">MEM vitamin solution</td>
			<td style="width:323px;">Gibco/ Life technologies</td>
			<td style="width:119px;">11120-052</td>
			<td>5ml, used in BEC media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Soybean trypsin inhibitor</td>
			<td style="width:323px;">Biowhittaker</td>
			<td style="width:119px;">17-605E</td>
			<td>5ml, used in BEC media. Solvent is PBS, mix to 5mg/ml stock concentration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">L-glutamine</td>
			<td style="width:323px;">Cellgro</td>
			<td style="width:119px;">25-005-CL</td>
			<td>5ml, used in BEC media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Bovine pituitary extract</td>
			<td style="width:323px;">Gemini</td>
			<td style="width:119px;">500-102</td>
			<td>1.1ml, used in BEC media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Dexamethasone</td>
			<td style="width:323px;">Sigma Aldrich</td>
			<td style="width:119px;">D4902</td>
			<td>0.5ml, used in BEC media, Stock conc 393ug/ml dilute with ethanol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">3 3&#39;,5-triiodo-L-thyronine</td>
			<td style="width:323px;">Sigma Aldrich</td>
			<td style="width:119px;">T6397</td>
			<td>0.5ml, used in BEC media, Stock conc 3.4 mg/ml dilute with ethanol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Epidermal growth factor</td>
			<td style="width:323px;">millipore</td>
			<td style="width:119px;">01-101</td>
			<td>0.5ml, used in BEC media, 25ug/ml dilute with DMEM F12+1%BSA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Forskolin</td>
			<td style="width:323px;">Sigma Aldrich</td>
			<td style="width:119px;">F6886</td>
			<td>5ml, used in BEC media, use at stock concentration of 0.411mg/ml and dilute with DMSO</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Fungizone</td>
			<td style="width:323px;">Gibco/ Life technologies</td>
			<td style="width:119px;">15290-018</td>
			<td>1ml, used in BEC media and 500 ul in the isolation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Rat-tail collagen</td>
			<td style="width:323px;">BD Biosciences</td>
			<td align="right" style="width:119px;">354236</td>
			<td>variable depending on concentration of collagen</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">PBS 10X</td>
			<td style="width:323px;">USB Corporation</td>
			<td align="right" style="width:119px;">75889</td>
			<td>use at 10x, sterlie, used to make collagen, amount used depends on collagen concentration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">dH2O</td>
			<td style="width:323px;">N/A</td>
			<td style="width:119px;">N/A</td>
			<td>sterile, used to make collagen, amount used depends on collagen concentration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">NaOH 10N</td>
			<td style="width:323px;">Fischer Scientific</td>
			<td style="width:119px;">ss255-1</td>
			<td>Dilute to 1N, sterile, used to make collagen, amount used depends on collagen concentration</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:335px;">collagenase type XI from Clostridium histolyticum</td>
			<td style="width:323px;">Sigma Aldrich</td>
			<td style="width:119px;">C7657</td>
			<td>dilute in DMEM and sterile filter before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">trypsin-EDTA (1x) 0.25%</td>
			<td style="width:323px;">Gibco/ Life technologies</td>
			<td style="width:119px;">25200-056</td>
			<td>3 ml, incubate max 10 minutes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">trypsin-EDTA (10x) 0.5%</td>
			<td style="width:323px;">Gibco/ Life technologies</td>
			<td style="width:119px;">15400-054</td>
			<td>3 ml, incubate max 10 minutes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;"></td>
			<td style="width:323px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Dissecting microscope</td>
			<td style="width:323px;">Nikon</td>
			<td style="width:119px;">SMZ645</td>
			<td>Other models acceptable</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:335px;">Light source (fiberoptic illuminator)</td>
			<td style="width:323px;">Schott-Fostec</td>
			<td style="width:119px;">Ace EKE LR 92240</td>
			<td>Other models acceptable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">12.5 cm straight iris scissors</td>
			<td style="width:323px;">Kent Scientific</td>
			<td style="width:119px;"></td>
			<td>Other models acceptable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">6&#34; non-serrated curved forceps with fine tips</td>
			<td style="width:323px;">Electron Microscopy Sciences</td>
			<td style="width:119px;"></td>
			<td>Other models acceptable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">4&#34; serrated stainless forceps with fine tips</td>
			<td style="width:323px;">Electron Microscopy Sciences</td>
			<td style="width:119px;"></td>
			<td>Other models acceptable</td>
		</tr>
	</tbody>
</table>

isolation of neonatal extrahepatic cholangiocytes

Karaciğer içi ve ekstrahepatik safra kanalları gelişimsel farklıdır ve farklı şekilde bazı hastalıklar etkilenebilir. Ancak, intra ve ekstrahepatik cholangiocytes ve yenidoğan ve yetişkin hücreleri arasındaki arasındaki farklar, iyi anlaşılmış değildir. Intrahepatik safra kanallarının ikinci cholangiocytes izolasyonu için yöntemler de 1-4 kurulmuştur. Bu ayrı cholangiocyte popülasyonları arasındaki farklılıkları anlamak ve ekstrahepatik kanalları hedef gibi görünür, safra atrezi gibi hastalıklara okuyan büyük fayda olacağını, ancak özellikle Yenidoğandan ekstrahepatik duktal hücrelerin izolasyonu, henüz tarif edilmemiştir. Neonatal ve yetişkin fare hem de ekstra-hepatik safra kanalı hücreleri izole etmek için optimize edilmiş bir teknik aşağıda tarif edilmiştir. Bu teknik, fibroblastlar gibi mezenkimal hücrelerden en az kirlenme ile saf bir hücre popülasyonu elde edilir. Bu metod titiz diseksiyon ve yağ ve fibroblast tabakaları kaldırmak için kazıma tarafından takip ekstrahepatik kanalları ve safra kesesi, çıkarılması dayanmaktadır. Yapılar deneysel kullanım için daha sonra tripsinize edilebilir ve kaplama yeniden tek katmanlarda cholangiocytes arasında gelişimini sağlamak için yaklaşık 3 hafta boyunca kollajen ve kültüre kalın tabakalar gömülür.

Neonatal fare ekstrahepatik mükemmel saflıkta cholangiocytes, K19 immünofloresan boyama ile gösterildiği gibi (Şekil 2A ve 2B) bir nüfus izolasyonunda bu protokol sonuçlarının kullanılması; erişkin farelerin hücrelerini izole benzer sonuçlar elde. Bu kalın kolojen jeller üstünde tek tabakaları oluşturacak şekilde taze olarak izole edilen safra kanalları hücrelerin boyunca 3 hafta sürer olduğunu gözlemledik. Cholangiocytes kanallarından izole edilmiş hücre tabakaların oluşt...

Watch this Scientific Journal Video about Isolation of Neonatal Extrahepatic Cholangiocytes at JoVE.com

Isolation of Neonatal Extrahepatic Cholangiocytes

Yenidoğan farelerin ekstrahepatik safra kanallarından cholangiocytes izole etmek için bir teknik tarif edilir. Kanallar titizlikle kesilir, ve daha sonra hücreler, kalın kolojen jeller büyümesinin ile izole edilir. Bu yöntem, ekstrahepatik safra kanalı geliştirme ve patolojiyi eğitim için yararlı bir araç sağlar.

Yenidoğan Ekstrahepatik Cholangiocytes İzolasyonu

The intra and extrahepatic bile ducts of the liver are developmentally distinct, and may be differentially affected by certain diseases. However, ...

isolation-of-neonatal-extrahepatic-cholangiocytes

Research

JoVE Journal

Biology

10.2K Görüntüleme.  The Children's Hospital of Philadelphia.  Yenidoğan farelerin ekstrahepatik safra kanallarından cholangiocytes izole etmek için bir teknik tarif edilir. Kanallar titizlikle kesilir, ve daha sonra hücreler, kalın kolojen jeller büyümesinin ile izole edilir. Bu yöntem, ekstrahepatik safra kanalı geliştirme ve patolojiyi eğitim için yararlı bir araç sağlar. 

Video: Isolation of Neonatal Extrahepatic Cholangiocytes

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 ...

Biyoloji

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 of Drosophila melanogaster Testes

The testes of Drosophila melanogaster provide an important model for the study of stem cell maintenance and differentiation, meiosis, and soma-germline interactions. Testes are typically isolated from adult males 0-3 days after eclosion from the pupal case. The testes of wild-type flies are easily distinguished from other tissues because they are yellow, but the testes of white mutant flies, a common genetic background for laboratory experiments are similar in both shape and color to the fly gut. Performing dissection on a glass microscope slide with a black background makes identifying the testes considerably easier. Testes are removed from the flies using dissecting needles. Compared to protocols that use forceps for testes dissection, our method is far quicker, allowing a well-practiced individual to dissect testes from 200-300 wild-type flies per hour, yielding 400-600 testes. Testes from white flies or from mutants that reduce testes size are harder to dissect and typically yield 200-400 testes per hour.

Isolation of Drosophila melanogaster Testes

Drosophila melanogaster testes can be rapidly and efficiently isolated from adult males using dissecting needles. With practice, one can readily isolate in one or two days an amount of testes sufficient for the analysis of DNA or RNA by high throughput sequencing or more traditional molecular biology methods or of protein for antibody- or enzyme-based assays.

İzolasyonu Drosophila melanogaster Testisler

The testes of Drosophila melanogaster provide an important model for the study of stem cell maintenance and differentiation, meiosis, and soma-germline ...

Isolation and Culture of Neonatal Mouse Cardiomyocytes

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

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

İzolasyon ve Kültür Yenidoğan Fare kardiyomiyositlerin

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

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 of Pulmonary Artery Smooth Muscle Cells from Neonatal Mice

Pulmonary hypertension is a significant cause of morbidity and mortality in infants. Historically, there has been significant study of the signaling pathways involved in vascular smooth muscle contraction in PASMC from fetal sheep. While sheep make an excellent model of term pulmonary hypertension, they are very expensive and lack the advantage of genetic manipulation found in mice. Conversely, the inability to isolate PASMC from mice was a significant limitation of that system. Here we described the isolation of primary cultures of mouse PASMC from P7, P14, and P21 mice using a variation of the previously described technique of Marshall et al.26 that was previously used to isolate rat PASMC. These murine PASMC represent a novel tool for the study of signaling pathways in the neonatal period. Briefly, a slurry of 0.5% (w/v) agarose + 0.5% iron particles in M199 media is infused into the pulmonary vascular bed via the right ventricle (RV). The iron particles are 0.2 &#956;M in diameter and cannot pass through the pulmonary capillary bed. Thus, the iron lodges in the small pulmonary arteries (PA). The lungs are inflated with agarose, removed and dissociated. The iron-containing vessels are pulled down with a magnet. After collagenase (80 U/ml) treatment and further dissociation, the vessels are put into a tissue culture dish in M199 media containing 20% fetal bovine serum (FBS), and antibiotics (M199 complete media) to allow cell migration onto the culture dish. This initial plate of cells is a 50-50 mixture of fibroblasts and PASMC. Thus, the pull down procedure is repeated multiple times to achieve a more pure PASMC population and remove any residual iron. Smooth muscle cell identity is confirmed by immunostaining for smooth muscle myosin and desmin.

We have developed a novel and reproducible technique to isolate primary cultures of pulmonary artery smooth muscle cells (PASMC) from mice as young as P7, thereby allowing better study of the signaling pathways involved in neonatal smooth muscle cell contraction and relaxation.

Yenidoğan fareler ile Pulmoner Arter Düz Kas Hücre İzolasyonu

Pulmonary hypertension is a significant cause of morbidity and mortality in infants. Historically, there has been significant study of the signaling ...

Live Cell Imaging of Primary Rat Neonatal Cardiomyocytes Following Adenoviral and Lentiviral Transduction Using Confocal Spinning Disk Microscopy

Primary rat neonatal cardiomyocytes are useful in basic in vitro cardiovascular research because they can be easily isolated in large numbers in a single procedure. Due to advances in microscope technology it is relatively easy to capture live cell images for the purpose of investigating cellular events in real time with minimal concern regarding phototoxicity to the cells. This protocol describes how to take live cell timelapse images of primary rat neonatal cardiomyocytes using a confocal spinning disk microscope following lentiviral and adenoviral transduction to modulate properties of the cell. The application of two different types of viruses makes it easier to achieve an appropriate transduction rate and expression levels for two different genes. Well focused live cell images can be obtained using the microscope&#8217;s autofocus system, which maintains stable focus for long time periods. Applying this method, the functions of exogenously engineered proteins expressed in cultured primary cells can be analyzed. Additionally, this system can be used to examine the functions of genes through the use of siRNAs as well as of chemical modulators.

This protocol describes a method of live cell imaging using primary rat neonatal cardiomyocytes following lentiviral and adenoviral transduction using confocal spinning disk microscopy. This enables detailed observations of cellular processes in living cardiomyocytes.

İlköğretim Rat Yenidoğan kardiyomiyositlerin Canlı Hücre Görüntüleme Adenoviral izlenerek ve Lentiviral İletimi Confocal Disk Mikroskopi Spinning kullanma

Primary rat neonatal cardiomyocytes are useful in basic in vitro cardiovascular research because they can be easily isolated in large numbers in a single ...

Isolation of Murine Valve Endothelial Cells

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and surrounded by a monolayer of valve endothelial cells (VECs). VECs play essential roles in establishing the valve structures during embryonic development, and are important for maintaining life-long valve integrity and function. In contrast to a continuous endothelium over the surface of healthy valve leaflets, VEC disruption is commonly observed in malfunctioning valves and is associated with pathological processes that promote valve disease and dysfunction. Despite the clinical relevance, focused studies determining the contribution of VECs to development and disease processes are limited. The isolation of VECs from animal models would allow for cell-specific experimentation. VECs have been isolated from large animal adult models but due to their small population size, fragileness, and lack of specific markers, no reports of VEC isolations in embryos or adult small animal models have been reported. Here we describe a novel method that allows for the direct isolation of VECs from mice at embryonic and adult stages. Utilizing the Tie2-GFP reporter model that labels all endothelial cells with Green Fluorescent Protein (GFP), we have been successful in isolating GFP-positive (and negative) cells from the semilunar and atrioventricular valve regions using fluorescence activated cell sorting (FACS). Isolated GFP-positive VECs are enriched for endothelial markers, including CD31 and von Willebrand Factor (vWF), and retain endothelial cell expression when cultured; while, GFP-negative cells exhibit molecular profiles and cell shapes consistent with VIC phenotypes. The ability to isolate embryonic and adult murine VECs allows for previously unattainable molecular and functional studies to be carried out on a specific valve cell population, which will greatly improve our understanding of valve development and disease mechanisms.

The ability to isolate heart valve endothelial cells (VECs) is critical for understanding mechanisms of valve development, maintenance, and disease. Here we describe the isolation of VECs from embryonic and adult Tie2-GFP mice using FACS that will allow for studies determining the contribution of VECs in developmental and disease processes.

Murin Vana Endotel Hücreleri İzolasyon

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and ...

Intravenous Injections in Neonatal Mice

Intravenous injection is a clinically applicable manner to deliver therapeutics. For adult rodents and larger animals, intravenous injections are technically feasible and routine. However, some mouse models can have early onset of disease with a rapid progression that makes administration of potential therapies difficult. The temporal (or facial) vein is just anterior to the ear bud in mice and is clearly visible for the first two days after birth on either side of the head using a dissecting microscope. During this window, the temporal vein can be injected with volumes up to 50 &#956;l. The injection is safe and well tolerated by both the pups and the dams. A typical injection procedure is completed within 1-2 min, after which the pup is returned to the home cage. By the third postnatal day the vein is difficult to visualize and the injection procedure becomes technically unreliable. This technique has been used for delivery of adeno-associated virus (AAV) vectors, which in turn can provide almost body-wide, stable transgene expression for the life of the animal depending on the viral serotype chosen.

Animal models of pediatric disease can experience early onset and aggressive disease progression. Clinically relevant therapy delivery to young mouse models can be technically difficult. This protocol describes a non-invasive intravenous injection method for newborn mice within the first two postnatal days of life.

Yenidoğan Farelerde intravenöz enjeksiyonlar

Intravenous injection is a clinically applicable manner to deliver therapeutics. For adult rodents and larger animals, intravenous injections are ...

Isolation and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin defense by forming an intact skin barrier against environmental insults, such as UVB irradiation or pathogens, and also by initiating an inflammatory response upon those insults. Here we describe methods to isolate KCs from neonatal mouse skin and from adult mouse tail skin. We also describe culturing conditions using defined growth supplements (dGS) in comparison to chelexed fetal bovine serum (cFBS). Functionally, we show that both neonatal and adult KCs are highly responsive to high calcium-induced terminal differentiation, tight junction formation and stratification. Additionally, cultured adult KCs are susceptible to UVB-triggered cell death and can release large amounts of TNF upon UVB irradiation. Together, the methods described here will be useful to researchers for the setup of in vitro models to study epidermal biology in the neonatal mouse and/or the adult mouse.

Epidermal keratinocytes form a functional skin barrier and are positioned at the front line of host defense against external environmental insults. Here we describe methods for isolation and primary culture of epidermal keratinocytes from neonatal and adult mouse skin, and induction of terminal differentiation and UVB-triggered inflammatory response from keratinocytes.

Yenidoğan ve Erişkin Fare Ciltlerindeki Primer Fare Keratinositlerin İzolasyonu ve Kültürü

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin ...