Bu çalışma Şizofreni Araştırmaları Ulusal İttifak ve Depresyon (NARSAD) Genç Araştırmacı Ödülü ve Ulusal Sağlık Enstitüleri AV R01NS073635 hibe tarafından desteklenmiştir.

1.. Reaktifler ve Çözümleri hazırlanması <ol><li> 35 mm kültür çanağı Kaplama: Kolajen Tip 1 çözeltisi, 20 mM asetik asit (~ 100 mg protein / şişe) içinde bir sulu çözelti olarak temin edilir. Steril doku kültürü dereceli su kullanarak,% 0.01 &#39;lik bir konsantrasyona kadar çalışma kollajen çözeltisinin uygun bir hacmi ile seyreltilir. 1, oda sıcaklığında (RT) 3-4 saat boyunca kollajen çözeltisinin ml ya da 37 ° C arasında veya bir gece boyunca 2-8 ° C&#39;de kaplayın yemekler Kaplı çanak aşırı çözüm çıkarın ve bir gecede kurumasını bekleyin. Medya eklemeden önce doku kültürü dereceli su veya PBS ile çanak durulayın. Kaplı yemekler bir ay boyunca 4 ° C&#39;de saklanabilir. </li><li> Tam Dulbecco Modifiye Eagle Ortamı (DMEM) hazırlayın. (, 1 ml/100 ml konsantrasyonu 1x),% 10 FBS ve antibiyotik-antimikotik çözelti ile DMEM (500 mi) Ek. Tam DMEM iki ay boyunca 4 ° C&#39;de saklanabilir. </li><li> Bir kısım DNase I (0.001 mg / ml) ile birlikte DMEM (50 mi) Hazırlama, (D olarak anılacaktırMEM-DNase I). </li><li> Kolajenaz ve dispaz (DMEM-collagenase/dispase olarak anılacaktır) (1 mg / ml) ile birlikte DMEM bir kısım (10 ml) hazırlayın. </li><li> EGF (5 ug) ile takviye edilmiş endotel hücre kültür ortamı (ECCM, 500 mi), hazırlama, ECGS (100 mg) ve antibiyotik-antimikotik çözelti 5 ul. ECCM iki ay boyunca 4 ° C&#39;de saklanabilir. </li><li> Kullanmadan önce tüm medya Prewarm. </li><li> % 0.5 BSA ve 2 mM EDTA ile PBS, pH 7.2 Saflaştırma tamponu hazırlayın. Soğuk tampon Ol (2-8 ° C). </li></ol> 2. Telencephalon Embriyolar Dissekan Kaldırma Laboratuvar hayvanları kullanan tüm deneyler McLean Hastanesi hayvan bakım ve kullanım komiteleri tarafından onaylanan ve laboratuvar hayvanlarının bakımı ve kullanımı için NIH yönergelere uygun edilmektedir. <ol><li> Zamanlanmış hamile CD1 fareleri (embriyonik gün 15) kullanın. Vajinal fiş keşif günü embriyonik gün 0 (E0) olarak kabul edilir. CD1 barajlar genellikle büyük ibrelerin üretmek, yani 10-12 embriyos hamile baraj bekleniyor. </li><li> Histerotomi için, Ketamin (100 mg / kg) / ksilazin (5 mg / kg) intraperitonal enjeksiyonu ile anestezi hamile farelere. Farenin yaklaşık 20-30 g, bir 10 ml / kg Ketamin / Ksilazin anestetik çözeltisi 0,3 ml sunmak ve bu ağrı sağlar ve tehlikeli anestezik intraperitoneal enjeksiyonu sırasında az. Enjeksiyonlar için el kısıtlama kullanın. </li><li> Ayak tutam ile derin anestezi edin. Derin anestezi farelerden embriyolar çıkarın. Hemen anneden uzaklaştırılması üzerine her embriyo başını kesmek. Buz soğukluğunda PBS içinde steril Petri kaplarında embriyonik kafaları yerleştirin. </li><li> 2013 Sürümü: anestezik aşırı doz (130 mg / kg pentobarbital, ip), Hayvanların ötanazi AVMA Yönergeleri ile tutarlıdır ötenazi için bir yöntem ile hemen ötenazi histerektomi izleyin. </li><li> Stereo mikroskop altında, ince mikrotip makas ve ince forseps ile baş beyin kaldırmak. Not: Yüksek kaliteli bir teşrihiyon başarıyla embriyonik beyinden Pial membran kaldırmak için gereklidir. Bu uygulama ile elde edilir. Ince forseps ve mikrotip makas aynı zamanda iyi bir diseksiyon almak için önemli araçlardır. </li><li> Pial zardan soymak için beyin ve mikrotip makas bir tutuş elde etmek için forseps kullanabilir. Mesencephalon ve metencephalon çıkarın ve telencephalon izole. Buz içinde 2 ml PBS ile bir 35 mm kültür çanağı için her embriyolardan Pial içermeyen telencephalon aktarın. </li></ol> 3. Hücre İzolasyonu <ol><li> Bir neşter ile 1-2 mm parçalar halinde telencephalon kıyma ve tam DMEM 2 ml ihtiva eden bir 15 ml Falcon tüpüne doku toplamak. </li><li> Kümeleri kaybolur ve sütsü süspansiyon elde edilene kadar, 1 ml&#39;lik bir pipet ile yavaşça ancak iyice doku ayrıştırmaları. </li><li> Santrifüj, oda sıcaklığında 5 dakika boyunca 800-1.000 xg&#39;de hücreleri ayrışmış. </li><li> Dikkatli ce ayırmak yavaşça DMEM-DNase I süpernatant ve tekrar süspansiyon pelet çıkarınLLS tekrar oda sıcaklığında 5 dakika boyunca 800-1.000 xg&#39;de 1 ml pipet ve santrifüj kullanılarak. </li><li> Sıcak tam DMEM&#39;de pelletini. Steril bir 70 um naylon elekle filtre hücreleri. Süzülür hücreleri toplamak ve buz tutmak. </li><li> Meşin üzerinde tutulan hücreye bölümlerini toplamak ve oda sıcaklığında 1-2 dakika boyunca DMEM-collagenase/dispase (2 mi) ile daha da kolaylaştırırlar. Oda sıcaklığında 5 dakika boyunca 800-1.000 xg santrifüj yoluyla DMEM-DNase I hücreler 3 kez yıkayın. Adım 3.5, toplanan süzülmüş hücrelerle, bu hücreleri havuzda toplayın. Bitmiş DMEM ile bir kez toplam hücrelerin yıkayın. Hücre sayısı ve manyetik etiketleme adımları belirlemek için devam edin. Not: Bu manyetik etiketleme önce, tek bir hücre süspansiyonu elde etmek için önemlidir. </li></ol> 4. Manyetik Etiketleme <ol><li> Bu protokol, 10 7 hücrelerinin manyetik etiketleme göstermektedir. Hücre sayıları 10&#39;dan az 7, aynı reaktif hacmi kullanımı ve hücre sayıları 10&#39;dan büyük 7, tüm reaktif hacimlerini ve toplam birimler büyütmekbuna göre (örneğin 2 x 10 7 toplam hücre için, gösterilen tüm reaktiflerin iki hacmi kullanın). Buz içinde tüm reaktifler ve hücreler tutun. </li><li> Bir hemositometreyle ile hücre sayısını belirlemek. </li><li> Oda sıcaklığında 10 dakika boyunca 300 x g&#39;de santrifüjleyin hücre süspansiyonu. Aspire tamamen yüzer. </li><li> CD31 mikro, 10 ul ve ardından hücre pelletine arıtma tamponu 90 μl/10 7 hücreleri ekleyin. Üretici tarafından sağlanan mikro-anti-fare CD31 monoklonal antikoru konjuge edilir. </li><li> İyice karıştırın ve buzdolabında 15 dakika boyunca inkübe edin. Not: Manyetik etiketleme sırasında yüksek sıcaklıklar ve / veya daha uzun bir enkübasyon süresi spesifik olmayan bağlanma ile sonuçlanabilir. </li><li> 10 dakika boyunca 300 xg&#39;de arıtma tampon ve santrifüj 1-2 ml/10 7 hücreleri eklenerek hücreler yıkayın. Arıtma tampon 500 ul süpernatan ve hücreler tekrar süspansiyon çıkarın. Manyetik ayırma veya saflaştırma aşamasına geçin. </li></ol> 5. Puaralığında olacak şekilde yapılır <ol><li> MACS Ayırıcı manyetik alan MS sütun yerleştirin. </li><li> Arıtma tampon 500 ul ile sütun durulayın. </li><li> Kolona hücre süspansiyonu uygulanır. Yüksek bir hücre sayısı sütunu tıkayabilir olarak MS kolona hücrelerin uygun sayıda yükleyin. Dikkat: Her MS sütun daha sütunları kullanmak, daha yüksek bir hücre sayısı için 2 x 10 7 hücre sayısını ağırlayacak. </li><li> Toplayın ve etiketsiz hücreleri içeren flow-through atın. </li><li> Arıtma tampon 500 ul üç kez sütun yıkayın. Yıkama adımları sırasında, kolon rezervuar müteakip arıtma tampon bölüntülerin eklemeden önce boş olduğundan emin olun. </li><li> MACS ayırıcı gelen sütun çıkarın ve 15 ml Falcon tüp üzerine yerleştirin. </li><li> Bir piston MS sütunu ile birlikte temin edilir. Pipet MS kolona ve hemen arıtma tampon 1 ml sıkıca sütuna pistonu iterek manyetik etiketli hücreleri yıkayın. </li><li> Plaka celKollajen Tip 1 ile kaplanmış ve% 95 O 2 ve% 5 CO 2 ile 37 ° C&#39;ye ayarlanmış bir kuluçka ECCM hücreleri büyümek bir 35 mm kültür çanak ls. </li><li> Her dört gün orta değiştirin. Çanak konfluent olduğunda, 10-12 gün sonra deney ya da alt-kültür için PVECs kullanın. </li></ol> 6. PV ECS alt kültür <ol><li> ECCM çıkarın ve steril PBS içinde 1-2 ml PVECS olan tek tabaka durulayın. </li><li> Yavaşça hücre tek katmanının karşılamak için% 0.25 tripsin-EDTA çözeltisi 1,5 ml ekleyin. </li><li> Inkübatör tabak dönmek. 5-7 dakika içinde, hücreler tabak yüzeyinden ayırır. </li><li> Hücreler müstakil hemen sonra tripsin inhibitörü eşit hacimde ve birkaç kez çiğnemek. </li><li> , Steril bir 15 ml Falcon tüpüne hücreleri aktarın. 5 dakika boyunca 500 x g&#39;de santrifüj tüpü. </li><li> Süpernatantı ve prewarmed ECCM 1 ml pelletini. </li><li> Kültür, im genişletilmesi için hücreleri tohumyaşlanma ya da başka deneyler. </li></ol>

<ol>
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Çıkar çatışması ilan etti.

PVEC ait daha fazla fizyolojik olarak uygun nörovasküler etkileşimleri ilgili çalışma other doku kaynaklarından erişkin beyin endotel hücreleri ve EC &#39;den ve aynı zamanda tedavi edici potansiyele sahiptir. PVEC hazırlanması için, ölü hücrelerin CD31 mikro, için spesifik olmayan bağlamak olabilir çünkü iyi bir canlılığı elde etmek için hızlı çalışması için diseksiyon ile kritik bir başlangıçtır. Tek hücre süspansiyonu önce manyetik etiketleme aşamasından elde değilse, hücre...

Anjiyogenezis, nöron ve nöronal göç merkezi sinir sistemi (MSS) geliştirme, onarım ve rejenerasyon kritik olaylar. Çeşitli çalışmalar, zarif, endotel hücreleri, çözünebilir faktörleri serbest bırakılması yoluyla ve doğrudan temas ile nöronal proliferasyonu ve tersi uyardığını göstermiştir. Biz meraklı nöronal progenitorlar / nöral kök hücre, embriyonik beyninden izole ise bu çalışmaların çoğunda 1-3, bunlar yetişkin beyin, diğer yetişkin doku kaynakları, veya endotelyal hücre hatları ile endotel hücreleri ile kültürlenmiş olduğu bulundu. Bu kısmen embriyonik beyin endotel hücreleri popülasyonları saf izole edilmesi ve kültür ile ilgili teknik zorluklar nedeniyle olabilir. Ancak, anjiyogenez, nöron oluşumu, ve nöronal migrasyon, normal erişkin beyinde daha embriyonik beyinde daha sağlam büyüklükte emir meydana gelen eşzamanlı olaylardır. Embryoni ait periventriküler damar ağıc ön beyin (telencephalon) bazal ganglia primordiumunun içinde yer alan bir kaptan kaynaklanan ve embriyonik gün 11 (E11) 4,5 ile telencephalon dorsal ventral gelen düzenli bir gradyan şeklinde gelişir. Periventriküler damar ağı damarlarının Bu pleksus kökenleri, anatomik konumu, büyüme modelleri ve gelişim düzenlemesi 4,5 dayalı Pial gemiler farklıdır. Periventriküler anjiyogenez degrade yayılma yönü telencephalic enine nörogenetik degrade eşleşir. Telencephalon içinde, periventriküler anjiyogenez degrade ve göç GABA nöron degrade teğet yanı sıra mekansal 6 çakışıyor. Zamanlama ile ilgili olarak, anjiyojenez gradyan yaklaşık bir gün ile nörogenetik gradyanı ve GABA nöron gradyanı önceden olduğu. Böylece, periventriküler endotel hücreleri mekansal ve zamansal iyi telencephalic nörogenezi desteklemek için kritik ipuçları sağlamak için konumlandırılmış venöronal migrasyon 4,6. Bu nedenle, nöronal soylarının ve / veya nöronlarla kokültürü deneylerde embriyonik periventriküler endotel hücrelerinin kullanımı nörovasküler etkileşimlerin incelenmesinde ve nörodejeneratif hastalık veya iskemik / travmatik beyin hasarı tedavisi için yeni yollar geliştirmek için daha uygun bir modeli sağlayacaktır. Biz, epitel hücre bulaşma sınırlamak için değil, aynı zamanda moleküler ve periventriküler damar ağı 4,6 (Pial EC&#39;ler ayırmak adlandırılır PVECs) endotel hücrelerinden fonksiyonel olarak farklı olan Pial endotel hücreleri ayırmak için değil, sadece Pial membran kaldırma önemini vurgulamaktadır . Burada, biz rutin PVECs zengin ve saf verim elde etmek bizim laboratuvarımızda kullandığımız yöntem tarif. Bu endotel hücreleri, tek bir zaman ayarlı gebe fare izole edilmiş embriyonik forebrains hazırlanır. Onlar ileride kullanmak için başarılı bir şekilde, genişletilmiş alt kültürleri ve aşağı donmuş olabilir.

<table border="0" cellpadding="0" cellspacing="0" width="768">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">DNase I</td>
			<td style="width:183px;">Sigma</td>
			<td style="width:147px;">D-4527</td>
			<td style="width:167px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Collagen, Type 1 solution from rat tail </td>
			<td>Sigma</td>
			<td>C3867</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DPBS</td>
			<td>Quality Biologicals</td>
			<td style="width:147px;">114057-131</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EDTA</td>
			<td>Fisher Scientific</td>
			<td>M4055</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BSA</td>
			<td style="width:183px;">Sigma</td>
			<td>A2058</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MS column</td>
			<td>Miltenyi Biotech</td>
			<td>130-042-201</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CD31 microbeads</td>
			<td>Miltenyi Biotech</td>
			<td>130-097-418</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MACS separator</td>
			<td>Miltenyi Biotech</td>
			<td>130-042-102</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MACS multi stand</td>
			<td>Miltenyi Biotech</td>
			<td>130-042-303</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell strainer 70 &#181;m</td>
			<td style="width:183px;">BD Bioscience</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Antibiotic and antimycotic solution</td>
			<td>Sigma</td>
			<td>A5955</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FBS</td>
			<td>Sigma</td>
			<td>F4135</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Collagenase/Dispase</td>
			<td>Roche</td>
			<td>10269638001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">DMEM </td>
			<td style="width:183px;">Lonza</td>
			<td>12-604F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">35 mm culture dish</td>
			<td style="width:183px;">BD Bioscience</td>
			<td>353001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">15 ml falcon tube </td>
			<td style="width:183px;">BD Bioscience</td>
			<td>352097</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">50 ml falcon tube</td>
			<td style="width:183px;">BD Bioscience</td>
			<td>352098</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">ECCM kit</td>
			<td style="width:183px;">BD Bioscience</td>
			<td style="width:147px;">355054</td>
			<td>Kit Includes Endothelial cell growth supplement, EGF and Soybean Trypsin Inhibitor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Endothelial Cell Growth Supplement (ECGS)</td>
			<td style="width:183px;">BD Bioscience</td>
			<td style="width:147px;">354006</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RBECGM</td>
			<td style="width:183px;">Cell applications </td>
			<td style="width:147px;">R819-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM F12 </td>
			<td style="width:183px;">Life Technologies</td>
			<td style="width:147px;">10565-018</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">glutamax</td>
			<td style="width:183px;">Life Technologies</td>
			<td style="width:147px;">305050-061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">tissue culture grade water</td>
			<td style="width:183px;">Life Technologies</td>
			<td style="width:147px;">15230162</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">0.25% Trypsin</td>
			<td style="width:183px;">Life Technologies</td>
			<td style="width:147px;">15050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Soyabean trypsin inhibitor</td>
			<td style="width:183px;">BD Bioscience</td>
			<td style="width:147px;">5425</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Matrigel</td>
			<td style="width:183px;">BD Bioscience</td>
			<td style="width:147px;">354234</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Qtracker 655 Cell Labeling Kit</td>
			<td style="width:183px;">Life Technologies</td>
			<td style="width:147px;">Q25021</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:271px;">CellLight Plasma Membrane-RFP, BacMam 2.0</td>
			<td style="width:183px;">Life Technologies</td>
			<td style="width:147px;">C10608</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Biotinylated Isolectin B4 antibody</td>
			<td style="width:183px;">Sigma</td>
			<td style="width:147px;">L2140</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Anti-Von Willebrand factor</td>
			<td style="width:183px;">Sigma</td>
			<td style="width:147px;">F3520</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Anti-CD31/PECAM-1</td>
			<td style="width:183px;">BD Pharmingen</td>
			<td style="width:147px;">550274</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:271px;">Vectashield Hardset Mounting media with DAPI</td>
			<td style="width:183px;">Vector Laboratories</td>
			<td style="width:147px;">H-1500</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:271px;">Ketamine</td>
			<td style="width:183px;">Butler Schein Animal Health Supply</td>
			<td style="width:147px;">44028</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Xylazine</td>
			<td style="width:183px;">Lloyd Laboratories</td>
			<td style="width:147px;">1009</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Stereomicroscope</td>
			<td style="width:183px;">Motic</td>
			<td style="width:147px;">SMZ-168</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">hemocytometer</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td style="width:147px;">267110</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">inverted microscope</td>
			<td style="width:183px;">Olympus CK-40</td>
			<td style="width:147px;">CK-40</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Flouroscent microscope</td>
			<td style="width:183px;">Olympus FSX-100</td>
			<td style="width:147px;">FSX-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Fine forceps</td>
			<td style="width:183px;">Roboz surgical instrument</td>
			<td>7 inox</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Fine microtip scissors</td>
			<td style="width:183px;">Roboz surgical instrument</td>
			<td>RS5611</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation and culture of endothelial cells from the embryonic forebrain

Embriyonik beyin endotel hücreleri anjiyogenez ve nörovasküler gelişim ve etkileşim çalışmasında önemli bir araç olarak hizmet edebilir. Embriyonik ön beyin, PIAL ve periventriküler iki damar ağları, mekansal farklı ve farklı kökenleri ve büyüme desenler var. Pial ve periventriküler damar ağlarından endotel hücreleri benzersiz gen ekspresyon profillerini ve işlevleri vardır. Burada embriyonik ön beyin (telencephalon) periventriküler damar ağı (PVECs) endotel hücrelerinin saf popülasyonlarının izolasyonu, kültür ve doğrulama için bir adım adım protokol mevcut. Bu yaklaşımda, embriyonik gün 15 farenin elde pial membran yoksun telencephalon, doğrandı kolajenaz / dispaz ile sindirilir ve tek bir hücre süspansiyonu içine mekanik olarak dağıtılabilir. PVECs güçlü bir manyetik kullanılarak mikro, konjüge anti-CD-31/PECAM-1 antikoru ile pozitif seçim kullanılarak hücre süspansiyonundan saflaştınlırayırma yöntemi. Bunlar birbirine karıştırılmış ve bundan başka, alt-kültürlenmiş olana kadar saflaştırılmış hücreler, endotelyal hücre kültür ortamı içinde 1 kollajen kaplı kültür tabakları üzerinde kültürlenmiştir. Faz-kontrast ışık mikroskobu ve floresan mikroskobu ile görüntülenmiştir gibi PVECs, bu protokol sergi parkelerde ve iğ biçimli fenotipleri ile elde. PVEC kültürlerin saflığı endotel hücre belirleyicileri ile kurulmuştur. Bizim ellerde, bu yöntem güvenilir ve tutarlı PVECs saf popülasyonları verir. Bu protokol, ön beyin anjiyojenezde içine mekanik bakış açısı kazanıyor PVEC etkileşimleri ve nöronal hücre tipleri ile çapraz görüşmeleri anlayış ve terapötik anjiyogenez için muazzam bir potansiyele sahiptir hedefleyen çalışmaları yararlanacaktır.

Günde 1-12 dan PVECs fenotipik karakterizasyonu faz kontrast mikroskopisi ile (Şekil 2) ile gösterilir. Hücre bölünmesi (Şekil 2A) 1. gün gösteri morfolojisi karakteristik çanak bağlı hücreler. 5-8 gün arasında, taş taş PVECs geçiş endotel hücreleri ve (Şekil 2B ve 2C) ile in vivo duruma daha yakındır için tipik şekilli morfolojiye mili için. 12. günde kültür tarafından PVEC tam izdiham (Şekil 2B)...

Watch this Scientific Journal Video about Isolation and Culture of Endothelial Cells from the Embryonic Forebrain at JoVE.com

Isolation and Culture of Endothelial Cells from the Embryonic Forebrain

Bu video 10-12 gün içinde embriyonik ön beyin endotel hücreleri saf kültürlerin hazırlanması için kolay ve güvenilir bir strateji gösterir ve serebral damar birçok açıdan odaklanmış araştırma için yararlı olacaktır.

Embriyonik ön beyin gelen Endotel Hücreleri İzolasyon ve Kültürü

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two ...

isolation-culture-endothelial-cells-from-embryonic

Research

JoVE Journal

Neuroscience

15.3K Görüntüleme.  Harvard Medical School.  Bu video 10-12 gün içinde embriyonik ön beyin endotel hücreleri saf kültürlerin hazırlanması için kolay ve güvenilir bir strateji gösterir ve serebral damar birçok açıdan odaklanmış araştırma için yararlı olacaktır. 

Video: Isolation and Culture of Endothelial Cells from the Embryonic Forebrain

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

Embriyonik Omurilik Commissural Nöronlar Diseksiyon ve Kültür

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

Nörobilim

Isolation and Culture of Neural Crest Stem Cells from Human Hair Follicles

Hair follicles undergo lifelong growth and hair cycle is a well-controlled process involving stem cell proliferation and quiescence. Hair bulge is a well-characterized niche for adult stem cells1. This segment of the outer root sheath contains a number of different types of stem cells, including epithelial stem cells2, melanocyte stem cells3 and neural crest like stem cells4-7. Hair follicles represent an accessible and rich source for different types of human stem cells. We and others have isolated neural crest stem cells (NCSCs) from human fetal and adult hair follicles4,5. These human stem cells are label-retaining cells and are capable of self-renewal through asymmetric cell division in vitro. They express immature neural crest cell markers but not differentiation markers. Our expression profiling study showed that they share a similar gene expression pattern with murine skin immature neural crest cells. They exhibit clonal multipotency that can give rise to myogenic, melanocytic, and neuronal cell lineages after in vitro clonal single cell culture. Differentiated cells not only acquire lineage-specific markers but also demonstrate appropriate functions in ex vivo conditions. In addition, these NCSCs show differentiation potential toward mesenchymal lineages. Differentiated neuronal cells can persist in mouse brain and retain neuronal differentiation markers. It has been shown that hair follicle derived NCSCs can help nerve regrowth, and they improve motor function in mice transplanted with these stem cells following transecting spinal cord injury8. Furthermore, peripheral nerves have been repaired with stem cell grafts9, and implantation of skin-derived precursor cells adjacent to crushed sciatic nerves has resulted in remyelination10. Therefore, the hair follicle/skin derived NCSCs have already shown promising results for regenerative therapy in preclinical models.
Somatic cell reprogramming to induced pluripotent stem (iPS) cells has shown enormous potential for regenerative medicine. However, there are still many issues with iPS cells, particularly the long term effect of oncogene/virus integration and potential tumorigenicity of pluripotent stem cells have not been adequately addressed. There are still many hurdles to be overcome before iPS cells can be used for regenerative medicine. Whereas the adult stem cells are known to be safe and they have been used clinically for many years, such as bone marrow transplant. Many patients have already benefited from the treatment. Autologous adult stem cells are still preferred cells for transplantation. Therefore, the readily accessible and expandable adult stem cells in human skin/hair follicles are a valuable source for regenerative medicine.

This article presents a robust protocol for isolation and culture of neural crest stem cells from human hair follicles.

İnsan Saç köklerinin arasından Sinir Crest Kök Hücre İzolasyonu ve Kültür

Hair follicles undergo lifelong growth and hair cycle is a well-controlled process involving stem cell proliferation and quiescence. Hair bulge is a ...

Lectin-based Isolation and Culture of Mouse Embryonic Motoneurons

Spinal motoneurons develop towards postmitotic stages through early embryonic nervous system development and subsequently grow out dendrites and axons. Neuroepithelial cells of the neural tube that express Nkx6.1 are the unique precursor cells for spinal motoneurons1. Though postmitotic motoneurons move towards their final position and organize themselves into columns along the spinal tract2,3. More than 90% of all these differentiated and positioned motoneurons express the transcription factors Islet 1/2. They innervate the muscles of the limbs as well as those of the body and the inner organs. Among others, motoneurons typically express the high affinity receptors for brain derived neurotrophic factor (BDNF) and Neurotrophin-3 (NT-3), the tropomyosin-related kinase B and C (TrkB, TrkC). They do not express the tropomyosin-related kinase A (TrkA)4. Beside the two high affinity receptors, motoneurons do express the low affinity neurotrophin receptor p75NTR. The p75NTR can bind all neurotrophins with similar but lower affinity to all neurotrophins than the high affinity receptors would bind the mature neurotrophins. Within the embryonic spinal cord, the p75NTR is exclusively expressed by the spinal motoneurons5. This has been used to develop motoneuron isolation techniques to purify the cells from the vast majority of surrounding cells6. Isolating motoneurons with the help of specific antibodies (panning) against the extracellular domains of p75NTR has turned out to be an expensive method as the amount of antibody used for a single experiment is high due to the size of the plate used for panning. A much more economical alternative is the use of lectin. Lectin has been shown to specifically bind to p75NTR as well7. The following method describes an alternative technique using wheat germ agglutinin for a preplating procedure instead of the p75NTR antibody. The lectin is an extremely inexpensive alternative to the p75NTR antibody and the purification grades using lectin are comparable to that of the p75NTR antibody. Motoneurons from the embryonic spinal cord can be isolated by this method, survive and grow out neurites.

An alternative way of isolating mouse embryonic motoneurons from the spinal cord is described. The method takes into account the fact that lectin can bind to the low affinity nerve growth factor receptor p75NTR. This lectin-based preplating allows a purification similar to that with a specific antibody against the p75NTR.

Fare Embriyonik motor nöronlar lektin tabanlı İzolasyon ve Kültür

Spinal motoneurons develop towards postmitotic stages through early embryonic nervous system development and subsequently grow out dendrites and axons. ...

Isolation and Culture of Hippocampal Neurons from Prenatal Mice

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual neurons, researchers are able to analyze properties related to cellular trafficking, cellular structure and individual protein localization using a variety of biochemical techniques. Results from such experiments are critical for testing theories addressing the neural basis of memory and learning. However, unambiguous results from these forms of experiments are predicated on the ability to grow neuronal cultures with minimum contamination by other brain cell types. In this protocol, we use specific media designed for neuron growth and careful dissection of embryonic hippocampal tissue to optimize growth of healthy neurons while minimizing contaminating cell types (i.e. astrocytes). Embryonic mouse hippocampal tissue can be more difficult to isolate than similar rodent tissue due to the size of the sample for dissection. We show detailed dissection techniques of hippocampus from embryonic day 19 (E19) mouse pups. Once hippocampal tissue is isolated, gentle dissociation of neuronal cells is achieved with a dilute concentration of trypsin and mechanical disruption designed to separate cells from connective tissue while providing minimum damage to individual cells. A detailed description of how to prepare pipettes to be used in the disruption is included. Optimal plating densities are provided for immuno-fluorescence protocols to maximize successful cell culture. The protocol provides a fast (approximately 2 hr) and efficient technique for the culture of neuronal cells from mouse hippocampal tissue.

We provide a protocol for the culture of highly purified hippocampal neurons from prenatal mouse brains without the use of a feeder glial cell layer.

Prenatal Fareler gelen Hipokampal Nöronlar izolasyonu ve Kültürü

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual ...

Isolation and Culture of Human Fungiform Taste Papillae Cells

Taste cells are highly specialized, with unique histological, molecular and physiological characteristics that permit detection of a wide range of simple stimuli and complex chemical molecules contained in foods. In human, individual fungiform papillae contain from zero to as many as 20 taste buds. There is no established protocol for culturing human taste cells, although the ability to maintain taste papillae cells in culture for multiple cell cycles would be of considerable utility for characterizing the molecular, regenerative, and functional properties of these unique sensory cells. Earlier studies of taste cells have been done using freshly isolated cells in primary culture, explant cultures from rodents, or semi-intact taste buds in tissue slices1,2,3,4. Although each of these preparations has advantages, the development of long-term cultures would have provided significant benefits, particularly for studies of taste cell proliferation and differentiation. Several groups, including ours, have been interested in the development and establishment of taste cell culture models. Most attempts to culture taste cells have reported limited viability, with cells typically not lasting beyond 3-5 d5,6,7,8. We recently reported on a successful method for the extended culture of rodent taste cells9. We here report for the first time the establishment of an in vitro culture system for isolated human fungiform taste papillae cells. Cells from human fungiform papillae obtained by biopsy were successfully maintained in culture for more than eight passages (12 months) without loss of viability. Cells displayed many molecular and physiological features characteristic of mature taste cells. Gustducin and phospholipase C &beta;2, (PLC-&beta;2) mRNA were detected in many cells by reverse transcriptase-polymerase chain reaction and confirmed by sequencing. Immunocytochemistry analysis demonstrated the presence of gustducin and PLC-&beta;2 expression in cultured taste cells. Cultured human fungiform cells also exhibited increases in intracellular calcium in response to appropriate concentrations of several taste stimuli indicating that taste receptors and at least some of the signalling pathways were present. These results sufficient indicate that taste cells from adult humans can be generated and maintained for at least eight passages. Many of the cells retain physiological and biochemical characteristics of acutely isolated cells from the adult taste epithelium to support their use as a model taste system. This system will enable further studies of the processes involved in proliferation, differentiation and function of mammalian taste receptor cells in an in vitro preparation.
Human fungiform taste papillae used for establishing human fungiform cell culture were donated for research following proper informed consent under research protocols that were reviewed and approved by the IRB committee. The protocol (#0934) was approved by Schulman Associates Institutional Review Board Inc., Cincinnati, OH. Written protocol below is based on published parameters reported by Ozdener et al. 201110.

We aimed to develop a reproducible protocol for isolating and maintaining long-term cultures of human fungiform taste papillae cells. Cells from human fungiform papillae obtained by biopsy were successfully maintained in culture for more than eight passages (12 months) without loss of viability.

İnsan Mantar şeklinde Lezzet papilla hücrelerinin izolasyonu ve Kültürü

Taste cells are highly specialized, with unique histological, molecular and physiological characteristics that permit detection of a wide range of simple ...

Isolation and Culture of Rat Embryonic Neural Cells: A Quick Protocol

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied successfully to the isolation of mouse and human primary neurons and neural progenitors. Dissociated neurons are maintained in serum-free medium up to several weeks. These cultures can be used for nucleofection, immunocytochemistry, nucleic acids preparation, as well as electrophysiology. Older neuronal cultures can also be transfected with a good efficiency rate by lentiviral transduction and, less efficiently, with calcium phosphate or lipid-based methods such as lipofectamine.

We describe a rapid methodology to isolate and culture hippocampal and cortical neurons from rodent embryos. This protocol allows us to perform experiments in which nearly pure neuronal cultures are required.

Sıçan Embriyonik Nöral Hücre İzolasyonu ve Kültür: Bir Hızlı Protokol

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied ...

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types 1-3. NC also has the unique ability to influence the differentiation and maturation of target organs4-6. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. 
NC multipotency was first described from explants of the avian neural tube7-9. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo10-13. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. 
To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors11,14-20, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties13,21,22. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors11,13,14,17. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter23. 
The method presented here is adapted from protocols optimized for the culture of rat NC11,13. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC2,24-28.

Isolation of embryonic neural crest from the neural tube facilitates the use of in vitro methods for studying migration, self-renewal, and multipotency of neural crest.

Embriyonik Mürin Nöral Tüp çıkan sinirsel Crest Hücre İzolasyonu ve Kültür

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

Isolation of Primary Murine Brain Microvascular Endothelial Cells

The blood-brain-barrier is ultrastructurally assembled by a monolayer of brain microvascular endothelial cells (BMEC) interconnected by a junctional complex of tight and adherens junctions. Together with other cell-types such as astrocytes or pericytes, they form the neurovascular unit (NVU), which specifically regulates the interchange of fluids, molecules and cells between the peripheral blood and the CNS. Through this complex and dynamic system BMECs are involved in various processes maintaining the homeostasis of the CNS. A dysfunction of the BBB is observed as an essential step in the pathogenesis of many severe CNS diseases. However, specific and targeted therapies are very limited, as the underlying mechanisms are still far from being understood. 
Animal and in vitro models have been extensively used to gain in-depth understanding of complex physiological and pathophysiological processes. By reduction and simplification it is possible to focus the investigation on the subject of interest and to exclude a variety of confounding factors. However, comparability and transferability are also reduced in model systems, which have to be taken into account for evaluation. The most common animal models are based on mice, among other reasons, mainly due to the constantly increasing possibilities of methodology. In vitro studies of isolated murine BMECs might enable an in-depth analysis of their properties and of the blood-brain-barrier under physiological and pathophysiological conditions. Further insights into the complex mechanisms at the BBB potentially provide the basis for new therapeutic strategies.
This protocol describes a method to isolate primary murine microvascular endothelial cells by a sequence of physical and chemical purification steps. Special considerations for purity and cultivation of MBMECs as well as quality control, potential applications and limitations are discussed.

Brain microvascular endothelial cells (BMEC) are interconnected by specific junctional proteins forming a highly regulated barrier separating blood and the central nervous system (CNS), the so-called blood-brain-barrier (BBB). The isolation of primary murine brain microvascular endothelial cells, as discussed in this protocol, enables detailed in vitro studies of the BBB.

İlköğretim Kemirgen Beyin mikrovasküler endotel hücreleri İzolasyonu

The blood-brain-barrier is ultrastructurally assembled by a monolayer of brain microvascular endothelial cells (BMEC) interconnected by a junctional ...

Isolation, Culture and Long-Term Maintenance of Primary Mesencephalic Dopaminergic Neurons From Embryonic Rodent Brains

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes involved in physiological functions and vulnerability and death of these neurons is imparative to understanding the underlying causes and unraveling the cure for this common neurodegenerative disorder. Primary cultures of mesDA neurons provide a tool for investigation of the molecular, biochemical and electrophysiological properties, in order to understand the development, long-term survival and degeneration of these neurons during the course of disease. Here we present a detailed method for the isolation, culturing and maintenance of midbrain dopaminergic neurons from E12.5 mouse (or E14.5 rat) embryos. Optimized cell culture conditions in this protocol result in presence of axonal and dendritic projections, synaptic connections and other neuronal morphological properties, which make the cultures suitable for study of the physiological, cell biological and molecular characteristics of this neuronal population.

The causes of degeneration of midbrain dopaminergic neurons during Parkinson&#8217;s disease are not fully understood. Cellular culture systems provide an essential tool for study of the neurophysiological properties of these neurons. Here we present an optimized protocol, which can be utilized for in vitro modeling of neurodegeneration.

Embriyonik Kemirgen Brain itibaren İlköğretim Mesensefalik dopaminerjik Nöronlar İzolasyonu, Kültür ve Uzun Vadeli Bakım

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes ...

Isolation and Culture of Adult Neural Stem Cells from the Mouse Subcallosal Zone

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation into three types of cells: neurons, astrocytes, and oligodendrocytes. Identifying aNSC-derived regions and characterizing the aNSC properties are critical for the potential use of aNSCs and for the elucidation of their role in neural regeneration. The subcallosal zone (SCZ), located between white matter and the hippocampus, has recently been reported to contain aNSCs and continuously give rise to neuroblasts. A low percentage of aNSCs from the SCZ is differentiated into neurons; most cells are differentiated into glial cells, such as oligodendrocytes and astrocytes. These cells are suggested to have a therapeutic potential for traumatic cortical injury. This protocol describes in detail the process to generate SCZ-aNSCs from an adult mouse brain. A brain matrix with intervals of 1 mm is used to obtain the SCZ-containing coronal slices and to precisely dissect the SCZ from the whole brain. The SCZ sections are initially subjected to a neurosphere culture. A well-developed culture system allows for the verification of their characteristics and can increase research on NSCs. A neurosphere culture system provides a useful tool for determining proliferation and collecting the genuine NSCs. A monolayer culture is also an in vitro system to assay proliferation and differentiation. Significantly, this culture system provides a more homogenous environment for NSCs than the neurosphere culture system. Thus, using a discrete brain region, these culture systems will be helpful for expanding our knowledge about aNSCs and their applications for therapeutic uses.

Establishing culture systems for the expansion of adult neural stem cells (aNSCs) allows for the examination and application of aNSCs for therapy. The subcallosal zone (SCZ) has recently been recognized as a novel neuroblast-forming region in adult mice. Here, methods for the isolation, expansion, and differentiation of SCZ-aNSCs are described.

Fare Subcallosal Zone İzolasyon ve Yetişkin Nöral Kök Hücre Kültürü

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation ...