Development of this protocol was funded by private donations directed to the Parkinson's Institute Brain Donation Program.

Beyin bağış kaydı bağışlamak için kendi niyet tescil ile belgeleri içerir. yasaların izin verdiği şekilde doku alımı için otopsi izni yakın akraba tarafından sağlanmaktadır. Toplanan otopsi örnekleri kullanarak araştırma çalışmaları Sağlık Sigortası Taşınabilirlik ve Sorumluluk Yasası (HIPAA) düzenlemelerine uyum sağlamak için kurumsal inceleme kurulu (KİK) tarafından gözden geçirilmektedir. NOT: leptomeninkslerde örnekleri beyin diseksiyonu sırasında beyin dissektör veya nöropatolog ile toplanır ve 25-30 ml Meninksler büyüme ortamı içeren 50 ml konik tüpler saklanır. Numune, numune hazırlama kadar 4 ° C&#39;de saklanır. hücre yaşamı sürdürme kabiliyeti zaman otopsi azalır gibi işlem mümkün olan en kısa sürede yapılmalıdır. 1. Ayar leptomeninkslerde Diseksiyon başlayarak önce NOT: - 1.3 biyogüvenlik kabini içinde yapılmalıdır olan 1.1 adımlar. <ol><li> 375 mL Dulbecco Modifiye Eagle Ortamı içinde birleştirerek zarları büyüme ortamının hazırlanması, fetal sığır serumu, 100 mL, 5 mL 10 mM esansiyel olmayan amino asitler, 5 ml 200 mM L-alanil-L-glutamin, 5 ml 100 mM sodyum piruvat, 5 ml 10,000 U / ml penisilin / streptomisin ve 5 mL, 250 ug / ml Amfoterisin B 500 mL filtre ünitesinin. Filtre ve karıştırın. kadar dört hafta ortam kullanın. </li><li> diseksiyon başlamadan önce biyogüvenlik kabini tüm malzemeleri koyun: 4x Petri kapları, 2x 6-kuyu plakaları, 2x neşter, 15-20 sterilize 15 mm kapak fişleri, pipetler aspire fosfat tampon salin (PBS), serolojik pipetler. </li><li> 6 gözlü bir plakanın her bir oyuğuna steril otoklava% 0.1 jelatin çözeltisi 1 ml. 30-60 dakika boyunca bir kenara plaka ayarlayın. 2x 6-kuyu tabak hazırlayın. </li></ol> Leptomeninkslerde Numune 2. Hazırlık NOT: - 2.3 biyogüvenlik kabini içinde yapılmalıdır olan 2.1 adımlar. <ol><li> PBS yaklaşık 10 ml ekleyinsteril 10-cm doku kültürü çanak ve çanak meninks numuneyi yerleştirin. forseps kullanarak, yavaşça kan kaldırmak için PBS ile leptomeningial yıkayın. </li><li> Taze 10 ml PBS ihtiva eden yeni bir 10 cm&#39;lik bir doku kültürü kabı içine meninksleri aktarın ve yıkama devam edin. mümkün olduğunca çok kan çıkarmak için gerektiği kadar tekrarlayın. </li><li> yaklaşık 6 cm x 6 cm büyük parça halinde dokuya leptomeninkslerde kesilmiş iki neşter, kullanma. </li><li> yatay laminer akış kaputu bir mikroskop bir kültür çanak ve transfer yerleştirin kapağı. </li></ol> Leptomeninkslerde Doku 3. Diseksiyon NOT: - 3.3 diseksiyon mikroskobu kullanılarak yatay laminer akış kaputu içinde yapılmalıdır 3,1 adımlar. <ol><li> en az 20-25 3 mm x 3 mm parçaları oluşturmak için yeterince büyük leptomeninkslerde parça (adım 2.3) bir bölgeden kan damarlarını çıkarın. bir neşter ile yerde leptomeningial tutun ve diğer, nazik ve sığ kazıma mo kullanıntion damarları ayırmak. </li><li> Hazırlanan bölgeden doku küçük kareler kesin. İlk büyük parçaları yapma ve daha küçük bölümlere olanlar dilimleme kademeli süreçte kesin. </li><li> Sallanan hareketi ile doku kesmek için yerinde doku ve başka neşter tutmak için bir neşter kullanabilirsiniz. doku kıvamına bağlı, birbirine karşı neşter slayt zordur. Bu sadece ayrı doku gözyaşı ve düzensiz kenarları sonuçlanır olacaktır. </li><li> 20-25 3 mm x 3 mm parçalar vardır kadar yarısında parçaları kesme devam edin. </li></ol> Doku kültürü plakaları üzerine Dissected zarları Adet 4. Transferi Not: Aşama 4, bir biyo-güvenlik kabini içinde yapılacak olan. <ol><li> 6-çukurlu plaka (adım 1.3) aspire jelatin. Her bir oyuğa zarları büyüme ortamı 1 ml. Steril forseps kullanarak, her kuyuya 3-4 biyopsi parçaları yerleştirin. </li></ol> M üzerinde Kapak Slips 5. YerleştirmeEninges adet NOT: - 5.2 diseksiyon mikroskobu kullanılarak yatay laminer akış kaputu içinde yapılmalıdır 5.1 adımlar. <ol><li> kuyu başına 1-2 steril 15 mm yuvarlak lamelleri ile parçaları örtün. </li><li> adet plakanın alt dokunmadan emin olmak için forseps ile sıkıca bastırın. Bu iyi altına, doku parçalarının bağlanmasına izin vermektedir. </li><li> 37 ° C,% 5 CO2 inkübatör içine 6-plaka. </li></ol> 6. Hücre kültürü Bakım NOT: - 6.3 biyogüvenlik kabini içinde yapılmalıdır olan 6.1 adımlar. lamelleri taşımak ve meninks parçaları çıkarmak değil gerektiği gibi özenle kültür kaplarına anlaştım. <ol><li> kültür içinde 2 gün sonra, her bir oyuğa taze zarları büyüme ortamı ilave 1 ml. </li><li> 7. Günde dikkatle medya aspire ve her iyi taze medya 2 mL ekleyin. </li><li> Gün 9 sonra büyüme ortamı her değişmeye devamkültür kadar geçen gün birleşen. </li></ol> 7. Pasajlanması NOT: Tüm adımlar biyogüvenlik kabini içinde yapılmalıdır vardır. Meningeal fibroblastlar Meningeal doku parçaları dışarı göç ve büyük kültür kabına menengial fibroblastlar, kültür geminin kenarlarına doğru büyümeye genişletmek başlar sonra. <ol><li> 15-30 dakika süreyle% 1 jelatin ile Pasajlanması, ceket kültür çanak başlamadan önce. </li><li> Aspire 6-iyi çanak medya ve 2 ml PBS ile yıkayın hücreleri. </li><li> Her kuyucuğa tripsin / EDTA 1 ml ilave edilir ve 5-10 dakika boyunca 37 ° C&#39;de inkübe edin. Kapak slipi veya bu aşama için zarı doku kaldırmak için gerekli değildir. </li><li> Hücreler yuvarlak ve, ayırmak kültür ortamı 2 mL ekleyin başladığınızda. </li><li> Pipet hücreleri yukarı ve tamamen tüm kümeleri bozmak ve büyük kültür çanak hücre çözümü transfer aşağı. </li><li> 1 ila 3: 1 bir oran için 4 üç 6 oyuklu tabaklar hücreleri birleştirmekbir T75 kültür çanak. </li><li> Meningeal fibroblastlar hücreleri T75 kültür şişelerinde birleştirilebilmeli sonra, hücreleri trypsinize ve 1x10 6 hücre / ml şişe başına en kriyoprezervasyon medya cryopreserve. konfluan meningeal fibroblastlar Tek T75 kültürü şişesi yaklaşık 5-7 milyon hücre içeriyor. </li></ol> Immün Meningial Fibroblast 8. Karakterizasyonu Not: prosedürü başlamadan önce, PBS (bloke tamponu) içinde% 4 paraformaldehit çözeltisi,% 5 normal keçi serumu hazırlamak% 0.3 Triton X-100, PBS (permeabilizasyon tamponu), primer ve sekonder antikor çözeltileri ve 1 ug / mL Hoechst 33342, 10 mg / ml stok PBS içinde seyreltilmiştir. <ol><li> 8-iyi odasının slayt Tohum 15.000 30.000 meningeal fibroblastlar% 0,1 jelatin ile kaplı. </li><li> Hücreler eklendiğinde 24 saat sonra, oda sıcaklığında 10 dakika boyunca oyuk başına 100 uL% 4 paraformaldehid içinde hücrelerin tespit. 1x PBS ile 3 kez yıkayın. </li><li> Oda sıcaklığında 5 dakika boyunca% 0.3 Triton X-100, 150 uL hücreler geçirgenliği. 1x PBS ile 3 kez yıkayın. </li><li> Oda sıcaklığında 1 st için bloke etme çözeltisi 200 uL (PBS +% 5 normal keçi serumu) ekleyin. </li><li> Engelleme çözüm aspire ve çözümü engelleme seyreltilmiş istenen birincil antikor 150 mcL ekleyin. çift ​​boyama için, aşağıdaki gibi, buz üzerinde ayrı bir santrifüj tüplerine birincil antikor çalışma dilüsyonları hazırlayın: <ol><li> Anti-Fibronektin + 1 300 seyreltme: çözümü engelleme, anti-Nestin 200 seyreltme 1 hazırlayın. Anti-SERPINH1 + 1 250 seyreltme: çözümü engelleme, anti-Tuj1 1000 seyreltme 1 hazırlayın. 1 hazırlayın: Anti-SERPINH1 250 seyreltme + 1: çözümü engelleme, anti-Sox2 100 seyreltme </li></ol></li><li> gece boyunca 4 ° C&#39;de inkübe edin. </li><li> 1x PBS ile 3 kez yıkayın. </li><li> Bir çalışan 1 hazırlayın: ikincil antikorlar anti-tavşan keçi floresan etiketli 400 seyreltme (yeşil; Ex / Em 2 </sYukarıya&gt; 495/519 nm) ve keçi anti-fare (kırmızı Ex / Em 2 590/617 nm) bloke çözeltisi içinde ve her bir kuyucuğa 100 uL ekleyin. Oda sıcaklığında, karanlıkta, 1 saat boyunca inkübe edilir. Daha sonra 1 x PBS ile bir kez kuyu yıkayın. </li><li> Her bir çözüm de ve 3 dakika süreyle karanlıkta inkübe edilir; / ml Hoechst 1 ug ile 100 uL 33342 (Örnek / Em 2 358/461 nm mavi) ekleyin. iyi son 1x PBS yıkama bırakarak, 1x PBS ile 3 kez yıkayın ve alüminyum folyo ile slayt kapsamaktadır. </li><li> floresan mikroskop altında hücrelerin analiz. </li></ol>

<ol>
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The authors have nothing to disclose.

Bu protokol, beyin bağışı ile birlikte toplanan insan postmortem leptomeninks bir meningeal fibroblast kültürü elde etmek için basit ve sağlam bir protokol tanımlamaktadır. postmortem insan malzemeden hücre kültürleri elde etmek için protokoller çok az açıklamaları vardır. İki çalışma, Cilt türevli, fibroblast kültürler 7, 8, 9, bir çalışma dura numuneler 10 tarif eder ve bi...

dura mater, araknoid ve pia mater: zarları beyni korumak üç membranların oluşur. Daha yakın zamanlarda, meninksler da beyin gelişimi ve beyin homeostazında 1 önemli bir rol oynadığı kabul edilmiştir. Zarları mezenşimal ve nöral tepe türetilmiş hücrelerden elde edilir ve ilginç bir şekilde, in vitro ve in vivo, 2, 3, 4 ve transplantasyon sonrası nöronlar ortaya çıkmasına neden olabilir meninks ikamet eden progenitör hücreler gösterilmiştir. Bunlar dopaminerjik nöronlar 5 embriyonik kök hücre farklılaşması için stromal hücre türevli indükleyici aktivite gösterirler zarları kültürleri de başarılı bir şekilde, besleyici tabakalar olarak kullanılmıştır. Ayrıca, leptomeninkslerde doğrudan iskemik koşullarda 6 altında nöronlar, astrositler ve oligodendrositlere içine ayırt etme potansiyeline sahiptir. Bu protokol için, insan postmortem zarları örnekleri topluca, araknoid ve pia mater toplanan leptomeningial denilen ve araştırma amaçlı bir insan beyninin bağışı parçası olarak temin edilir edilir. beyin Diseksiyon ölüm 24 saat içinde gerçekleştirilir, bu protokolde burada gösterildiği gibi leptomeninkslerde Örnek daha sonra 6-8 saat içinde daha fazla işlem için soğuk büyüme ortamı içinde yerleştirilir. Bu protokol, hastaya primer leptomeninkslerde hücre kültürü gelişimi için diseksiyon ve insan beyin zarı numunelerin hazırlanmasını tarif etmektedir. Doku, yaklaşık 3 mm x 3 mm kare 25-30 parçalar halinde kesilir. Üç adet her biri 6-kuyu yerleştirilir jelatin kaplı iyi ve yuvarlak cam lamelleri aşağı düzenledi. zarları diseksiyon yaklaşık 25-35 dakika sürer. Bu kültürün ana zorluk beyin tedarik, taşıma ve diseksiyon olarak sterilite genellikle steril koşullar altında yapılır edilmemektedir. Therefore, penisilin, streptomisin, ve amfoterisin B&#39;nin bir kokteyl ile kültür ortamını desteklemek ve ayrı ayrı kültür doku parçaları çok iyi yemekler kullanmak önemlidir. Meningeal fibroblast akıbet genellikle ilk hafta içinde ortaya çıkar. Hücreler birleştiklerinde kadar Ortam, her iki ya da üç günde bir değiştirilir ve hücreler, enzimatik olarak geçirilir. Meningeal fibroblastlar dondurulması medyada ml / şişe başına 1 Milyon hücre Kriyoprezerve bulunmaktadır. Bu protokol ile, 20-30.000.000 meningeal fibroblastlar dondurulması için 6-8 hafta içinde elde edilebilir. Bu Meningeal fibroblastların Mansap uygulamaları hastalık araştırması, doğrudan nöronal farklılaşma ya da hastalık mekanizmalarının anlaşılması için ve ilaç geliştirme leptomeninks gelen uyarılmış pluripotent kök hücrelerin derivasyon için birincil kültürleri vardır.

<table border="0" cellpadding="0" cellspacing="0" width="1256">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:459px;">Corning Petri dishes</td>
			<td style="width:187px;">Fisher Scientific</td>
			<td style="width:127px;">351029</td>
			<td style="width:485px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nunc 6-well plate</td>
			<td>Fisher Scientific</td>
			<td>14-832-11</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">15-mm cover slips</td>
			<td>Fisher Scientific</td>
			<td colspan="2">12-545-83 15CIR-1D</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Scalpels, sterile blade, No. 15</td>
			<td>Miltex</td>
			<td>4-415</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Curved precision tip forceps</td>
			<td>Fisher Scientific</td>
			<td>16-100-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Serological pipettes</td>
			<td>Fisher Scientific</td>
			<td>13-678-11E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pasteur pipettes</td>
			<td>Fisher Scientific</td>
			<td>22-230490</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gelatin</td>
			<td>Sigma</td>
			<td>G1890-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate Buffer Solution</td>
			<td>Fisher Scientific</td>
			<td>SH30264.02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Corning 500 mL filter unit</td>
			<td>Fisher Scientific</td>
			<td>430770</td>
			<td>Combine media components and filter.</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Nunc Cell Culture Treated Flasks with Filter Caps, T175 cm2&#160;</td>
			<td>Thermo Scientific</td>
			<td>178883</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Growth Media</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Hyclone DMEM</td>
			<td>Fisher Scientific</td>
			<td>SH30081.02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hyclone FBS</td>
			<td>Fisher Scientific</td>
			<td>SH30910.03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MEM Non-Essential Amino Acids Solution (100x)</td>
			<td>Thermo Fisher</td>
			<td>11140-050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GlutaMAX Supplement (100x)</td>
			<td>Thermo Fisher</td>
			<td>35050-061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Pyruvate (100 mM)</td>
			<td>Thermo Fisher</td>
			<td>11360-070</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Thermo Fisher</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Amphotericin B (Yellow Solution/250 &#181;g/mL)</td>
			<td>Fisher Scientific</td>
			<td>BP264520</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bambanker Freeze 120 mL</td>
			<td>Fisher Scientific</td>
			<td>NC9582225</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Fibronectin Staining</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">8 well chamber slides&#160;</td>
			<td>Fisher Scientific</td>
			<td>1256518</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">20% paraformaldehyde&#160;</td>
			<td>Electron Microscopy Sciences</td>
			<td>15713</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triton X-100&#160;</td>
			<td>Sigma</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100% Glycerol&#160;</td>
			<td>BioRad</td>
			<td>9455</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100% normal goat serum&#160;</td>
			<td>Fisher Scientific</td>
			<td>101098-382</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-Fibronectin antibody [F1]</td>
			<td>Abcam</td>
			<td>ab32419</td>
			<td>1:300 dilution in blocking solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-SERPINH1</td>
			<td>Sigma</td>
			<td>S5950-200ul</td>
			<td>1:250 dilution in blocking solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-SOX2</td>
			<td>Millipore</td>
			<td>MAB4343</td>
			<td>1:100 dilution in blocking solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-Nestin</td>
			<td>Millipore</td>
			<td>MAB5326</td>
			<td>1:200 dilution in blocking solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-TUJ1</td>
			<td>Covance</td>
			<td>MMS-435P</td>
			<td>1:1000 dilution in blocking solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 488 anti-rabbit</td>
			<td>Thermo Fisher</td>
			<td>A11029</td>
			<td>1:400 dilution in blocking solution; (green channel; Ex/Em2 495/519 nm)&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 555 anti-mouse</td>
			<td>Thermo Fisher</td>
			<td>A21424</td>
			<td>1:400 dilution in blocking solution; (red channel; Ex/Em2 590/617 nm)&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hoechst 33342 stain</td>
			<td>Thermo Fisher</td>
			<td>H3570</td>
			<td>dilute to a final concentration of 1.0 &#956;g/ml; (blue channel; Ex/Em2 358/461 nm)&#160;</td>
		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;">Suppliers are suggestions, similar products from alternative vendors can be used as well.</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

derivation of leptomeninges explant cultures from postmortem human brain donors

Büyük ilerleme Parkinson hastalığının klinik karakterizasyonu yapılmış olsa bile, bazı çalışmalar Parkinson hastalığı tanısı patolojik klinik olarak teşhis Parkinson hastalığı% 25 kadar teyit olmadığını bildirmektedir. Bu nedenle, doku yanlış tanı yüksek oranda olabilir idiyopatik Parkinson hastalığı olan klinik tanısı alan hastaların toplanan; Bir klinik öncesi bir model beyhude olabilir gibi dolayısıyla bu tür dokulardan in vitro çalışmalar Parkinson hastalığı incelemek için. Parkinson hastalığının bir teyit nöropatolojik tanısı ile postmortem insan leptomeningial toplama ve nigrostriatal hücre kaybı ve Lewy cisimcikleri denilen hücre içi protein kapanım ile karakterize tarafından, bir klinik gözlenen parkinsonizm altta yatan başka bir hastalık süreci (örneğin tümör, damar sertliği) neden olmadığından emin olabilirsiniz. Bu protokol, miyodiseksiyon ve meningeal fibroblast kültürü türetilmesi için postmortem insan leptomeninks hazırlanması ts. Bu prosedür sağlam ve yüksek bir başarı oranına sahiptir. Beyin tedarik genellikle steril koşullar altında yapılır değil gibi kültürün meydan kısırlık olduğunu. Nedenle, penisilin, streptomisin ve amfoterisin B bir kokteyli ile kültür ortamı tamamlanması önemlidir Parkinson hastalığı olan otopsi doğrulanmış vakaların gelen Meningeal fibroblast türetme Parkinson hastalığı in vitro modelleme için temel sağlar. Meningeal fibroblastlar 3-9 gün numune hazırlandıktan sonra ortaya çıkar ve yaklaşık 20-30 milyon hücre 6-8 hafta içinde dondurularak saklanabilir. Meningeal fibroblast kültürü homojen ve hücreler fibronektin, meninksler tanımlamak için yaygın olarak kullanılan bir işaretleyici ifade eder.

leptomeninkslerde işleme protokolü başarılı olmuştur bu beyin otopsi aralığının uzunluğuna bağlıdır rağmen, meningeal fibroblast akıbet, üç ile dokuz gün diseksiyonu sonrası gözlenen ilk edilir. Şekil 1, dört farklı donörlerin meningeal fibroblast kültürleri göstermektedir. Şekil 1A 10 saat 20 dk otopsi aralığına sahip 88 yaşındaki bir donörden işlendikten sonra doku dört gün etrafında bir cam kapak slip (koyu çapr...

Watch this Scientific Journal Video about Derivation of Leptomeninges Explant Cultures from Postmortem Human Brain Donors at JoVE.com

Derivation of Leptomeninges Explant Cultures from Postmortem Human Brain Donors

İnsan postmortem beyin leptomeninkslerde eksplant kültür protokolü 6-8 hafta içinde fibronektin pozitif meningeal fibroblastlar elde ve yaklaşık 20-30 mikrogram milyon hücre cryopreserve için teknik sağlam ve basit bir yoludur.

Otopsi İnsan beyni Donörleri gelen leptomeninkslerde Eksplantasyon Kültürlerin türetilmesi

Even though great progress has been made in the clinical characterization of Parkinson&#39;s disease, several studies report that the diagnosis of ...

derivation-leptomeninges-explant-cultures-from-postmortem-human-brain

Research

JoVE Journal

Neuroscience

8.2K Görüntüleme.  Parkinson’s Institute and Clinical Center.  İnsan postmortem beyin leptomeninkslerde eksplant kültür protokolü 6-8 hafta içinde fibronektin pozitif meningeal fibroblastlar elde ve yaklaşık 20-30 mikrogram milyon hücre cryopreserve için teknik sağlam ve basit bir yoludur. 

Video: Derivation of Leptomeninges Explant Cultures from Postmortem Human Brain Donors

DiOLISTIC Labeling of Neurons from Rodent and Non-human Primate Brain Slices

DiOLISTIC staining uses the gene gun to introduce fluorescent dyes, such as DiI, into neurons of brain slices (Gan et al., 2009; O'Brien and Lummis, 2007; Gan et al., 2000). Here we provide a detailed description of each step required together with exemplary images of good and bad outcomes that will help when setting up the technique. In our experience, a few steps proved critical for the successful application of DiOLISTICS. These considerations include the quality of the DiI-coated bullets, the extent of fixative exposure, and the concentration of detergent used in the incubation solutions. Tips and solutions for common problems are provided. This is a versatile labeling technique that can be applied to multiple animal species at a wide range of ages. Unlike other fluorescent labeling techniques that are limited to preparations from young animals or restricted to mice because they rely on the expression of a fluorescent transgene, DiOLISTIC labeling can be applied to animals of all ages, species and genotypes and it can be used in combination with immunostaining to identify a specific subpopulation of cells. Here we demonstrate the use of DiOLISTICS to label neurons in brain slices from adult mice and adult non-human primates with the purpose of quantifying dendrite branching and dendritic spine morphology.

We demonstrate the use of the gene gun to introduce fluorescent dyes, such as DiI, into neurons in brain slices from rodents and non-human primates of different ages. In this particular case, we use adult mice (3-6 months old) and adult cynomologus monkeys (9-15 years old). This technique, originally described by the laboratory of Dr. Lichtman (Gan et al., 2000), is well suited for the study of dendritic branching and dendritic spine morphology and can be combined with traditional immunostaining, if detergents are kept at a low concentration.

Kemirgen Nöronlar ve Sigara insan Primate Beyin Slices DiOLISTIC Etiketleme

DiOLISTIC staining uses the gene gun to introduce fluorescent dyes, such as DiI, into neurons of brain slices (Gan et al., 2009; O'Brien and Lummis, 2007; ...

Nörobilim

Multi-electrode Array Recordings of Neuronal Avalanches in Organotypic Cultures

The cortex is spontaneously active, even in the absence of any particular input or motor output. During development, this activity is important for the migration and differentiation of cortex cell types and the formation of neuronal connections1. In the mature animal, ongoing activity reflects the past and the present state of an animal into which sensory stimuli are seamlessly integrated to compute future actions. Thus, a clear understanding of the organization of ongoing i.e. spontaneous activity is a prerequisite to understand cortex function. 
Numerous recording techniques revealed that ongoing activity in cortex is comprised of many neurons whose individual activities transiently sum to larger events that can be detected in the local field potential (LFP) with extracellular microelectrodes, or in the electroencephalogram (EEG), the magnetoencephalogram (MEG), and the BOLD signal from functional magnetic resonance imaging (fMRI). The LFP is currently the method of choice when studying neuronal population activity with high temporal and spatial resolution at the mesoscopic scale (several thousands of neurons). At the extracellular microelectrode, locally synchronized activities of spatially neighbored neurons result in rapid deflections in the LFP up to several hundreds of microvolts. When using an array of microelectrodes, the organizations of such deflections can be conveniently monitored in space and time. 
Neuronal avalanches describe the scale-invariant spatiotemporal organization of ongoing neuronal activity in the brain2,3. They are specific to the superficial layers of cortex as established in vitro4,5, in vivo in the anesthetized rat 6, and in the awake monkey7. Importantly, both theoretical and empirical studies2,8-10 suggest that neuronal avalanches indicate an exquisitely balanced critical state dynamics of cortex that optimizes information transfer and information processing.
In order to study the mechanisms of neuronal avalanche development, maintenance, and regulation, in vitro preparations are highly beneficial, as they allow for stable recordings of avalanche activity under precisely controlled conditions. The current protocol describes how to study neuronal avalanches in vitro by taking advantage of superficial layer development in organotypic cortex cultures, i.e. slice cultures, grown on planar, integrated microelectrode arrays (MEA; see also 11-14).

A robust way to study neuronal avalanches, i.e. scale-invariant spatio-temporal activity bursts, indicative of critical state dynamics in cortex. Avalanches emerge spontaneously in developing superficial layers of cultured cortex which allows for long-term measurements of the activity with planar integrated multi-electrode arrays (MEA) under precisely controlled conditions.

Organotipik Kültürleri nöronal Çığ Çok elektrot Dizi Kayıtlar

The cortex is spontaneously active, even in the absence of any particular input or motor output.  During development, this activity is important for the ...

Efficient Derivation of Human Neuronal Progenitors and Neurons from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system (CNS) structure and circuitry in today's healthcare industry. Cell-based therapies hold great promise to restore the lost nerve tissue and function for CNS disorders. However, cell therapies based on CNS-derived neural stem cells have encountered supply restriction and difficulty to use in the clinical setting due to their limited expansion ability in culture and failing plasticity after extensive passaging1-3. Despite some beneficial outcomes, the CNS-derived human neural stem cells (hNSCs) appear to exert their therapeutic effects primarily by their non-neuronal progenies through producing trophic and neuroprotective molecules to rescue the endogenous cells1-3. Alternatively, pluripotent human embryonic stem cells (hESCs) proffer cures for a wide range of neurological disorders by supplying the diversity of human neuronal cell types in the developing CNS for regeneration1,4-7. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity7-10. In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic11-13. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules14 (please see a schematic in Fig. 1). Retinoic acid (RA) does not induce neuronal differentiation of undifferentiated hESCs maintained on feeders1, 14. And unlike mouse ESCs, treating hESC-differentiated embryoid bodies (EBs) only slightly increases the low yield of neurons1, 14, 15. However, after screening a variety of small molecules and growth factors, we found that such defined conditions rendered retinoic acid (RA) sufficient to induce the specification of neuroectoderm direct from pluripotent hESCs that further progressed to neuroblasts that generated human neuronal progenitors and neurons in the developing CNS with high efficiency (Fig. 2). We defined conditions for induction of neuroblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human neuronal cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of neuroblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human neuronal progenitors and neuronal cell types in the developing CNS for neural repair.

Küçük Molekül İndüksiyon Pluripotent İnsan Embriyonik Kök Hücreleri İnsan Nöronal atalarıdır ve Nöronlar Verimli türetilmesi

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system ...

Derivation of Enriched Oligodendrocyte Cultures and Oligodendrocyte/Neuron Myelinating Co-cultures from Post-natal Murine Tissues

Identifying the molecular mechanisms underlying OL development is not only critical to furthering our knowledge of OL biology, but also has implications for understanding the pathogenesis of demyelinating diseases such as Multiple Sclerosis (MS). Cellular development is commonly studied with primary cell culture models. Primary cell culture facilitates the evaluation of a given cell type by providing a controlled environment, free of the extraneous variables that are present in vivo. While OL cultures derived from rats have provided a vast amount of insight into OL biology, similar efforts at establishing OL cultures from mice has been met with major obstacles. Developing methods to culture murine primary OLs is imperative in order to take advantage of the available transgenic mouse lines. 
Multiple methods for extraction of OPCs from rodent tissue have been described, ranging from neurosphere derivation, differential adhesion purification and immunopurification 1-3. While many methods offer success, most require extensive culture times and/or costly equipment/reagents. To circumvent this, purifying OPCs from murine tissue with an adaptation of the method originally described by McCarthy &amp; 
de Vellis 2 is preferred. This method involves physically separating OPCs from a mixed glial culture derived from neonatal rodent cortices. The result is a purified OPC population that can be differentiated into an OL-enriched culture. This approach is appealing due to its relatively short culture time and the unnecessary requirement for growth factors or immunopanning antibodies. 
While exploring the mechanisms of OL development in a purified culture is informative, it does not provide the most physiologically relevant environment for assessing myelin sheath formation. Co-culturing OLs with neurons would lend insight into the molecular underpinnings regulating OL-mediated myelination of axons. For many OL/neuron co-culture studies, dorsal root ganglion neurons (DRGNs) have proven to be the neuron type of choice. They are ideal for co-culture with OLs due to their ease of extraction, minimal amount of contaminating cells, and formation of dense neurite beds. While studies using rat/mouse myelinating xenocultures have been published 4-6, a method for the derivation of such OL/DRGN myelinating co-cultures from post-natal murine tissue has not been described. Here we present detailed methods on how to effectively produce such cultures, along with examples of expected results. These methods are useful for addressing questions relevant to OL development/myelinating function, and are useful tools in the field of neuroscience.

This article describes methods to derive enriched populations of murine oligodendrocyte precursor cells (OPCs) in primary culture, which differentiate to produce mature oligodendrocytes (OLs). In addition, this report describes techniques to produce murine myelinating co-cultures by seeding mouse OPCs onto a neurite bed of mouse dorsal root ganglion neurons (DRGNs).

Post-natal murin Dokular Co-kültürler Myelinating zenginleştirilmiş oligodendrosit Kültürleri ve oligodendrosit / Nöron türetilmesi

Identifying the molecular mechanisms underlying OL development is not only critical to furthering our knowledge of OL biology, but also has implications ...

Derivation of Glial Restricted Precursors from E13 mice

This is a protocol for derivation of glial restricted precursor (GRP) cells from the spinal cord of E13 mouse fetuses. These cells are early precursors within the oligodendrocytic cell lineage. Recently, these cells have been studied as potential source for restorative therapies in white matter diseases. Periventricular leukomalacia (PVL) is the leading cause of non-genetic white matter disease in childhood and affects up to 50% of extremely premature infants. The data suggest a heightened susceptibility of the developing brain to hypoxia-ischemia, oxidative stress and excitotoxicity that selectively targets nascent white matter. Glial restricted precursors (GRP), oligodendrocyte progenitor cells (OPC) and immature oligodendrocytes (preOL) seem to be key players in the development of PVL and are the subject of continuing studies. Furthermore, previous studies have identified a subset of CNS tissue that has increased susceptibility to glutamate excitotoxicity as well as a developmental pattern to this susceptibility. Our laboratory is currently investigating the role of oligodendrocyte progenitors in PVL and use cells at the GRP stage of development. We utilize these derived GRP cells in several experimental paradigms to test their response to select stresses consistent with PVL. GRP cells can be manipulated in vitro into OPCs and preOL for transplantation experiments with mouse PVL models and in vitro models of PVL-like insults including hypoxia-ischemia. By using cultured cells and in vitro studies there would be reduced variability between experiments which facilitates interpretation of the data. Cultured cells also allows for enrichment of the GRP population while minimizing the impact of contaminating cells of non-GRP phenotype.

This protocol outlines the derivation of Glial Restricted Precursors from fetal spinal cords and maintained in vitro either for transplantation or for the study of oligodendrocytic lineage.

E13 farelerin Glial Yasak Öncüleri türetilmesi

This is a protocol for derivation of glial restricted precursor (GRP) cells from the spinal cord of E13 mouse fetuses. These cells are early precursors ...

Neural Explant Cultures from Xenopus laevis

The complex process of axon guidance is largely driven by the growth cone, which is the dynamic motile structure at the tip of the growing axon. During axon outgrowth, the growth cone must integrate multiple sources of guidance cue information to modulate its cytoskeleton in order to propel the growth cone forward and accurately navigate to find its specific targets1. How this integration occurs at the cytoskeletal level is still emerging, and examination of cytoskeletal protein and effector dynamics within the growth cone can allow the elucidation of these mechanisms. Xenopus laevis growth cones are large enough (10-30 microns in diameter) to perform high-resolution live imaging of cytoskeletal dynamics (e.g.2-4 ) and are easy to isolate and manipulate in a lab setting compared to other vertebrates. The frog is a classic model system for developmental neurobiology studies, and important early insights into growth cone microtubule dynamics were initially found using this system5-7 . In this method8, eggs are collected and fertilized in vitro, injected with RNA encoding fluorescently tagged cytoskeletal fusion proteins or other constructs to manipulate gene expression, and then allowed to develop to the neural tube stage. Neural tubes are isolated by dissection and then are cultured, and growth cones on outgrowing neurites are imaged. In this article, we describe how to perform this method, the goal of which is to culture Xenopus laevis growth cones for subsequent high-resolution image analysis. While we provide the example of +TIP fusion protein EB1-GFP, this method can be applied to any number of proteins to elucidate their behaviors within the growth cone.

Neural Explant Cultures from Xenopus laevis

Culturing neural explants from dissected Xenopus laevis embryos that express fluorescent fusion proteins allows for imaging of growth cone cytoskeletal dynamics.

Dan Sinir Eksplantasyon Kültürleri<em&gt; Xenopus laevis</em

The complex process of axon guidance is largely driven by the growth cone, which is the dynamic motile structure at the tip of the growing axon. During ...

Progenitor-derived Oligodendrocyte Culture System from Human Fetal Brain

Differentiation of human neural progenitors into neuronal and glial cell types offers a model to study and compare molecular regulation of neural cell lineage development. In vitro expansion of neural progenitors from fetal CNS tissue has been well characterized. Despite the identification and isolation of glial progenitors from adult human sub-cortical white matter and development of various culture conditions to direct differentiation of fetal neural progenitors into myelin producing oligodendrocytes, acquiring sufficient human oligodendrocytes for in vitro experimentation remains difficult. Differentiation of galactocerebroside+ (GalC) and O4+ oligodendrocyte precursor or progenitor cells (OPC) from neural precursor cells has been reported using second trimester fetal brain. However, these cells do not proliferate in the absence of support cells including astrocytes and neurons, and are lost quickly over time in culture. The need remains for a culture system to produce cells of the oligodendrocyte lineage suitable for in vitro experimentation.
Culture of primary human oligodendrocytes could, for example, be a useful model to study the pathogenesis of neurotropic infectious agents like the human polyomavirus, JCV, that in vivo infects those cells. These cultured cells could also provide models of other demyelinating diseases of the central nervous system (CNS). Primary, human fetal brain-derived, multipotential neural progenitor cells proliferate in vitro while maintaining the capacity to differentiate into neurons (progenitor-derived neurons, PDN) and astrocytes (progenitor-derived astrocytes, PDA) This study shows that neural progenitors can be induced to differentiate through many of the stages of oligodendrocytic lineage development (progenitor-derived oligodendrocytes, PDO). We culture neural progenitor cells in DMEM-F12 serum-free media supplemented with basic fibroblast growth factor (bFGF), platelet derived growth factor (PDGF-AA), Sonic hedgehog (Shh), neurotrophic factor 3 (NT-3), N-2 and triiodothyronine (T3). The cultured cells are passaged at 2.5e6 cells per 75cm flasks approximately every seven days. Using these conditions, the majority of the cells in culture maintain a morphology characterized by few processes and express markers of pre-oligodendrocyte cells, such as A2B5 and O-4. When we remove the four growth factors (GF) (bFGF, PDGF-AA, Shh, NT-3) and add conditioned media from PDN, the cells start to acquire more processes and express markers specific of oligodendrocyte differentiation, such as GalC and myelin basic protein (MBP). We performed phenotypic characterization using multicolor flow cytometry to identify unique markers of oligodendrocyte.

Primary, human fetal brain-derived, multipotential progenitor cells proliferate in vitro while maintaining the capacity to differentiate into neurons and astrocytes. This work shows that neural progenitors can be induced to differentiate through stages of the oligodendrocytic lineage by conditioning with select growth factors.

İnsan Fetal Beyin gelen Progenitör-türetilmiş Oligodendrosit Kültür Sistemi

Differentiation of human neural progenitors into neuronal and glial cell types offers a model to study and compare molecular regulation of neural cell ...

Feeder-free Derivation of Neural Crest Progenitor Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a source of transplantable cells for regenerative applications after disease or accidents. Neural crest (NC) cells are the precursors for a large variety of adult somatic cells, such as cells from the peripheral nervous system and glia, melanocytes and mesenchymal cells. They are a valuable source of cells to study aspects of human embryonic development, including cell fate specification and migration. Further differentiation of NC progenitor cells into terminally differentiated cell types offers the possibility to model human diseases in vitro, investigate disease mechanisms and generate cells for regenerative medicine. This article presents the adaptation of a currently available in vitro differentiation protocol for the derivation of NC cells from hPSCs. This new protocol requires 18 days of differentiation, is feeder-free, easily scalable and highly reproducible among human embryonic stem cell (hESC) lines as well as human induced pluripotent stem cell (hiPSC) lines. Both old and new protocols yield NC cells of equal identity.

Neural crest (NC) cells derived from human pluripotent stem cells (hPSC) have great potential for modeling human development and disease and for cell replacement therapies. Here, a feeder-free adaptation of the currently widely used in vitro differentiation protocol for the derivation of NC cells from hPSCs is presented.

İnsan Pluripotent dan Sinir Crest Progenitör Hücreler besleyici ücretsiz Derivasyon Kök Hücre

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a ...

High-resolution Structural Magnetic Resonance Imaging of the Human Subcortex In Vivo and Postmortem

The focus of this study was to test the resolution limits of structural MRI of a postmortem brain compared to living human brains. The resolution of structural MRI in vivo is ultimately limited by physiological noise, including pulsation, respiration and head movement. Although imaging hardware continues to improve, it is still difficult to resolve structures on the millimeter scale. For example, the primary visual sensory pathways synapse at the lateral geniculate nucleus (LGN), a visual relay and control nucleus in the thalamus that normally is organized into six interleaved monocular layers. Neuroimaging studies have not been able to reliably distinguish these layers due their small size that are less than 1 mm thick.
The resolving limit of structural MRI, in a postmortem brain was tested using multiple images averaged over a long duration (~24 h). The purpose was to test whether it was possible to resolve the individual layers of the LGN in the absence of physiological noise. A proton density (PD)1 weighted pulse sequence was used with varying resolution and other parameters to determine the minimum number of images necessary to be registered and averaged to reliably distinguish the LGN and other subcortical regions. The results were also compared to images acquired in living human brains. In vivo subjects were scanned in order to determine the additional effects of physiological noise on the minimum number of PD scans needed to differentiate subcortical structures, useful in clinical applications.

High-resolution Structural Magnetic Resonance Imaging of the Human Subcortex In Vivo and Postmortem

Here we present a protocol to determine the minimum number images that needed to be registered and averaged to resolve subcortical structures and test whether the individual layers of the LGN could be resolved in the absence of physiological noise.

İnsan Subcortex yüksek çözünürlüklü yapısal Manyetik Rezonans Görüntüleme<I&gt; İn Vivo</I&gt; Ve Postmortem

The focus of this study was to test the resolution limits of structural MRI of a postmortem brain compared to living human brains. The resolution of ...

Isolation of Cerebral Capillaries from Fresh Human Brain Tissue

Understanding blood-brain barrier function under physiological and pathophysiological conditions is critical for the development of new therapeutic strategies that hold the promise to enhance brain drug delivery, improve brain protection, and treat brain disorders. However, studying the human blood-brain barrier function is challenging. Thus, there is a critical need for appropriate models. In this regard, brain capillaries isolated from human brain tissue represent a unique tool to study barrier function as close to the human in vivo situation as possible. Here, we describe an optimized protocol to isolate capillaries from human brain tissue at a high yield and with consistent quality and purity. Capillaries are isolated from fresh human brain tissue using mechanical homogenization, density-gradient centrifugation, and filtration. After the isolation, the human brain capillaries can be used for various applications including leakage assays, live cell imaging, and immune-based assays to study protein expression and function, enzyme activity, or intracellular signaling. Isolated human brain capillaries are a unique model to elucidate the regulation of the human blood-brain barrier function. This model can provide insights into central nervous system (CNS) pathogenesis, which will help the development of therapeutic strategies for treating CNS disorders.

Isolated brain capillaries from human brain tissue can be used as a preclinical model to study barrier function under physiological and pathophysiological conditions. Here, we present an optimized protocol to isolate brain capillaries from fresh human brain tissue.

Yalıtım taze insan beyin dokusundan beyin kapiller

Understanding blood-brain barrier function under physiological and pathophysiological conditions is critical for the development of new therapeutic ...

Otopsi İnsan beyni Donörleri gelen leptomeninkslerde Eksplantasyon Kültürlerin türetilmesi

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