Bu araştırma, UCI (AR ve SP) Lisans Araştırma Fırsatlar Programı hibe ve hibe Devlet California Alzheimer Hastalığı Girişimi ve hayır, Ulusal Sağlık hibe Enstitüleri tarafından da desteklenmiştir. HD38466 ve Alzheimer Hastalığı Araştırma Merkezi hiçbir hibe. AG16573 (JB)

1. Kortikal Doku Blokları, Kriyoprezervasyon Malzeme Hazırlığı <ol><li> 10X stok solüsyonu steril su (1:9) seyreltilmesi 1X Hank Tamponlu Tuz Çözeltisi (HBSS) hazırlayın. 4oC saklayın HBSS. </li><li> HBSS (1:9) DMSO seyreltilmesi ile% 10 DMSO dondurma orta hazırlayın. Dondurma orta kriyoprezervasyon önce taze, soğutulmuş kadar yapılan ve 4oC saklanan olmalıdır. </li><li> Çelik tıraş bıçağı, doku sistematik doğramak için kullanılır. Kullanmadan önce, tıraş bıçağı, 2 saat için% 70 etanol içinde boğulma yolu ile sterilize edilmiştir. Doku işleme hemen önce tıraş bıçağı, 3 kez steril su ile durulayın. Oksidasyon eğilimli olduğu gibi, bir zaman aşırı miktarda steril su jilet bırakarak kaçının. </li><li> Bir Nalgene dondurma kabı cryovials (2.5 mL) ile yüklenir ve daha sonra steril bir biyolojik kabin içine getirilir. Her flakon 1 ml soğutulmuş dondurma orta eklenir. Dondurma kabı sonra en az 2 saat için 4oC yerleştirilir. </li></ol> Temizleme, Doğrama ve Donma <ol><li> Meningeal membran ve kan damarlarının donmuş gibi beyin dokusu temizlenir. Bir buz paketi üzerinde çalışırken, doku dikkatle enkaz kaldırmak için steril iğne ipuçlarını kullanın. Temizlenmiş doku hafifçe soğuk HBSS ile durulanır ve doğrama için yeni bir Petri kabı transfer edilmelidir. </li><li> Bir buz paketi üstünde çalışarak, yaklaşık 1 mm 3 blok halinde hızla doku chop steril bir jilet kullanın. Temizlenir doku küçük porsiyonlar, daha düzgün blok boyutları, kesme işlemi üzerinde daha iyi kontrol bir zaman sonuçları ile çalışma. </li><li> HBSS 50 ml 50 ml konik tüp içine doğranmış dokusu yavaş yavaş ekleyin. HBSS ile Petri kabında kalan herhangi bir doku blokları toplamak için nazikçe yıkayın. Gevşekçe paketlenmiş bir doku blok pelet oluşturmak doku blokları, tüpün dibine inmek için izin verin. </li><li> Doku yerleşme iken, biyolojik güvenlik kabininin içine önceden soğutulmuş dondurma kabı ve cryovials getirmek. Doku ekleme süreci hızlandırmak için bütün cryovials şapkasını çıkarmak. </li><li> Tüm doku blokları yerleştikten sonra, yavaşça, pelet yukarıda medya çok ince bir tabaka bırakarak aşırı HBSS aspirat. Doku blokları birbirine yapışır neden olacaktır Santrifüj, donma verimliliği önemli ölçüde azalan önerilmez. </li><li> 200 mcL geniş orifisleri (steril makas ile ucu kesim) pipet kullanarak gevşek doku blok pelet alttan yavaş yavaş topluyoruz. Cryovial bir malzeme transferi ve pelet alttan sonraki tekrar toplanması doku geçmek. Tüm doku tahsis usulü dondurma konteyner başına 3-4 dk daha fazla olmadığını esastır. Bu doku, dondurmadan önce zaman sadece kısa bir süre için DMSO maruz kaldığı sağlar. Şişeleri doku aktardıktan sonra, en az 4 saat süreyle bir-80oC dondurucu dondurma kabı yerleştirin. Alternatif olarak, dondurma kabı -80 oC bir gecede terk edilebilir. Kalan herhangi bir dondurma kapları için bu işlemi tekrarlayın. </li><li> Donma konteyner (ler), uzun süreli depolama için bir sıvı nitrojen tankı cryobox ve yer cryovials aktarın. </li></ol> 2. Dondurulmuş Kortikal Doku Blokları Çözülme ve ekimi Malzeme Hazırlığı <ol><li> Bir gün önce çözülme doku örnekleri, poli-L-lizin kaplı Petri kutularına / lamelleri hazırlanmıştır. Genel olarak, kortikal nöronların için 500 mcg / mL veya 1 mg / ml ile kaplı yemekleri hazırlamak. Tam çanak alt kapağı için yeterli poli-L-lisin çözüm (borat tamponu yapılan) ekleyin. Cam lamelleri gerekiyorsa, poli-L-lisin çözümü tamamen altında kalmış olduğundan emin olun. En az 12 saat süreyle inkübe edin. Kullanmadan önce iyice liberal bir miktarda steril su ile 3 kez, her biri 5 dakika bulaşıkları yıkayın. </li><li> Sıcak HBSS çözülmüş dokusunun yanı sıra doku hücre süspansiyonları ayırmak temizlemek için kullanılır. Gerekli HBSS hacmi çözülmüş olması doku miktarına bağlı olarak değişir, ancak tipik olarak 1 cryovial HBSS 50 ml seyreltik ve 10 ml HBSS ayrışmış olacak. </li><li> Dulbecco&#39;nun Modifiye Kartal Orta% 10 demir takviye sığır buzağı serumu (EBS) ve% 1 / antibiyotik antimikotik (AA) karışımı (37 ° C su banyosunda DMEM (++)) konulmalıdır. </li><li> Kaplama orta (NB (+++)) kullanımdan önce taze yapılmalıdır. Bu ortam, antibiyotik% 1 / antimikotik artı B27 ve N2 takviyeleri ekleyerek hazırlanır. </li></ol> Not: Medya isimleri çarpılar (+) ile eklenen katkı maddeleri de dahil medya baz bileşimi göstermektedir. Bu metinde, DMEM (+) gösterir DMEM artı 10% EBS artı% 1 AA ise NB (+++) gösterir NB plbize% 1 AA artı B27 artı N2. Çözülme ve Kültürleme <ol><li> Sıvı nitrojen tankı cryovials çıkarın ve hızlı bir şekilde 37 Çözülme ° C su banyosu, sadece küçük bir buz pelet kadar flakon içeriği içinde gözlenir. </li><li> Hafifçe konik bir tüp içerisinde 50 ml sıcak HBSS flakon içerik aktarmak. Cryovial sıkışıp kalan doku blokları nazikçe çıkarmak için geniş bir orifis ucu kullanın. Tüp 3-4 kez çevirin ve dokusu yavaş yavaş alt toplamak için izin verir. Doku hızlı bir şekilde çözmek gerekir, ama bloklar çok küçük, bu tavsiye edilir santrifüj (~ 200 x g) oluşur ve ışık olmayabilir. </li><li> Aşırı HBSS aspire. </li><li> Doku pelet ve 300% 0.25 tripsin ve DNAase 50 mcL mcL sıcak HBSS 10 ml ekleyin. Orbital çalkalayıcı 5 dakika sonra, yer için 37 ° C su banyosunda inkübe ek bir 5 dakika için 80 ° C rpm ve 37 olarak ayarlayın. </li><li> Bu dönemden sonra, biyolojik güvenlik kabini içine doku blokları getirmek ve bulutlu bir hücre süspansiyon oluşuncaya kadar dikkatlice doku blokları karıştırma 10 ml pipet kullanın. 10 ml sıcak DMEM ekleyerek tripsin devre dışı bırakın (+ +) hücre süspansiyonu. </li><li> 5 dakika boyunca 1200 x g ayrışmış hücreleri santrifüjleyin. Süpernatantı aspire ve atın, 10 ml sıcak DMEM hücre pelletini tekrar süspansiyon (+ +) ve hemasitometre kullanarak hücre sayısı belirlenir. </li><li> Poli-L-lisin kaplı yemekler Plaka hücreleri. Tipik olarak, bir 60 mm çanak 1x10 6 hücreleri ile kaplı olacaktır. </li><li> Hücrelerin doku kültürü inkübatörde yaklaşık 1 saat için çanak / lamelleri eklemek için izin ver. Bu etkili ek görülmektedir kadar her 10 dakikada bir tabak eki ilerlemeyi izlemek için tavsiye edilir. Sonra, yavaşça orta yerine taze DMEM (++). </li><li> 24 saat sonra, DMEM replace (+) sıcak NB (+++). Kısmi orta değişiklikler (% 50), taze NB (+++). ile her 5 günde bir yapılmaktadır Sağlıklı kültürler genellikle 24 saat sonra farklılaşma ve büyüme süreçleri belirtilerini gösteriyor. Bu koşullar altında, her 4-5 günde kısmi orta değişiklikleri gerçekleştirirken, kültürleri, 4-6 hafta kadar zaman uzun süre muhafaza edilebilir. </li><li> Nöronal prekürsör hücreler (NPC) üretimi için de benzer bir protokol santrifüj sonra, EBS olmadan DMEM olduğunu haricinde kullanılır. Hücreler neuropheres oluşumuna izin B27 ve EGF (20 ng / ml) ile desteklenmiş DMEM/F12 (1:3), T-25 şişeler kaplanmıştır. NPC&#39;ler ayırt etmek için, neurospheres laminin (10 ug / ml), DMEM/F12 (1:3) N2 ile desteklenmiş orta kaplamalı lamelleri kaplanmıştır. </li></ol> 3. Temsilcisi Sonuçlar Sağlıklı kültürler, 5 gün post-çözülme sonra, önemli bir büyüme ve farklılaşma gösterecek ve genellikle 10 gün (Şekil 1) büyüme stabilize edecek. Bu kültürlerin Immuncocytochemical boyanması ortaya koyan ve sayısız astrositler (Şekil 2A), hem insan ve fare primer nöronal kültürler için nöronlar (Şekil 2B). Bu protokol aynı zamanda serbest yüzen neurospheres (Şekil 3A), ayırt koşulları altında, yüksek kalitede karışık kültürler (Şekil 3B, C) sonuç olarak NPC&#39;ler üretimi için uygundur. <img src="/files/ftp_upload/2384/2384fig1.jpg" alt="Şekil 1" /> Şekil 1. Donmuş insan kortikal doku hücre kültürleri Dondurulmuş insan kortikal doku blokları çözülmüş ve poli-lizin kaplı lamelleri kaplama ve 10 gün boyunca yetişen. Geçen gün 5 hücre genişleme görülür ve gündüz 10 confluency elde edilir. Ölçek çubuğu: 100 mm. <img src="/files/ftp_upload/2384/2384fig2.jpg" alt="Şekil 2" /> Şekil 2. Hücre kültürlerinin İmmünositokimyasal boyama (A) Kültürler glial marker glial fibriller asidik protein (GFAP, kalın oklar) ile boyandı. (B) Nöronlar nöronal belirteç beta-tubulin III (TIII ince oklar) ile tespit edildi. (C) Her ikisi de insan ve sıçan kültürleri kültür 10 gün sonra bol glia ve nöronlar göstermektedir. İlköğretim antikorlar: fare anti-GFAP (1:1000) ve tavşan anti-TIII (1:1000). İkincil antikorlar anti-fare Alexa 594 (1:500) ve anti-tavşan Alexa 488 (1:500). Çekirdekler boyama: Hoestch mavi (1:1000). Ölçek çubuğu: 50 mm. <img src="/files/ftp_upload/2384/2384fig3.jpg" alt="Şekil 3" /> Şekil 3. Neurospheres nesil (A) Dondurulmuş doku, yukarıda açıklandığı ve neurospheres yaymak için izin olarak işlendi . (B) Neurospheres neurosphere uzak göç hücreleri, farklılaşan koşullar altında cam lamelleri kaplama ve 10 gün için yetiştirilir. (C), her ikisi de ayırt neurosphere genişleyen saçak nöronlar ve astrositler mevcut. Nükleer counterstaining, Şekil 2 olarak kullanılan birincil ve ikincil antikorlar.

<ol>
	<li>Robbins, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryopreservation+of+human+brain+tissue.">Cryopreservation of human brain tissue.</a> Exp Neurol. 107, 208-213 (1990).</li><li>Ware, C. B., Nelson, A. M., Blau, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled-rate+freezing+of+human+ES+cells.">Controlled-rate freezing of human ES cells.</a> Biotechniques. 38, 879-880 (2005).</li><li>Paynter, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+and+practical+issues+for+cryopreservation+of+nerve+cells.">Principles and practical issues for cryopreservation of nerve cells.</a> Brain Res Bull. 75, 1-14 (2008).</li><li>Thirumala, S., Gimble, J. M., Devireddy, R. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryopreservation+of+stromal+vascular+fraction+of+adipose+tissue+in+a+serum-free+freezing+medium.">Cryopreservation of stromal vascular fraction of adipose tissue in a serum-free freezing medium.</a> J Tissue Eng Regen Med. 4, 224-232 (2010).</li></ol>

Kriyoprezervasyon gelecekte kullanılmak üzere banka değerli beyin doku örnekleri için bir fırsat sunuyor. Burada nöron zengin kültürleri ve nöronal prekürsör hücreler dondurulmuş beyin dokusu blokları oluşturmak için basit ama etkili bir protokol açıklar. Bu ekonomik prosedürü hız kontrollü dondurucular daha pahalı kullanan geleneksel kriyoprezervasyon teknikleri maliyetlerini ortadan kaldırır. Hızlı ama etkili bir araç sağlayarak doku blokları donma-çözülme sonrası canlı nöronal kültürlerin üretimi için protokol sağlar. Tüm dondurma işlemi en az 20 dakika sürebilir. Primer nöronal kültürlerin yanı sıra, bu yöntemi doku blokları kullanarak serbest dalgalı neurospheres olarak yetiştirilen NPC&#39;ler oluşturmak için çözülmüş olabilir. Bu bağlamda, bizim dondurma orta serum eksikliği hücreleri farklılaşmamış bir devlet korunmuş olmasını sağlar. Neurospheres farklılaşan koşullar altında, dondurulmuş doku gösterisi genişleme ve taze doku elde neurospheres çok karşılaştırılabilir farklılaşma oranları üretti.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Dulbecco's Modified Eagle Medium (DMEM)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11995-073</td>
<td>High gluclose 1X</td>
</tr>
<tr>
<td>Bovine calf serum supplemented</td>
<td>&nbsp;</td>
<td>HYCLONE</td>
<td>SH30072.03</td>
<td>For culture medium </td>
</tr>
<tr>
<td>Neurobasal-A Medium (1X), liquid</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>1088-022</td>
<td>&nbsp;</td>
<tr>
<td>B27 Supplement</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>17504-044</td>
<td>Supplement for Neurobasal medium</td>
</tr>
<tr>
<td>N2 Supplement</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>17502-048</td>
<td>Supplement for Neurobasal medium</td>
</tr>
<tr>
<td>Trypsin (10X)</td>
<td>&nbsp;</td>
<td>Cellgro </td>
<td>25-054CI </td>
<td>Tissue dissociation</td>
</tr>
<tr>
<td>Deoxyribonuclease 1 from bovine pancreas (DNase)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>D4527-30KU</td>
<td>Tissue dissociation</td>
</tr>
<tr>
<td>Hanks Balanced Salt Solution (HBSS) (10X)</td>
<td>&nbsp;</td>
<td>GIBCO</td>
<td>14065</td>
<td>Cleaning tissue, washes, and freezing medium</td>
</tr>
<tr>
<td>Poly-L-Lysine- 500mg</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>P2636</td>
<td>Substrate for adhesion of neuronal cells</td>
</tr>
<tr>
<td>antibiotic-antimycotic (100X), liquid</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15240-062</td>
<td>To prevent contamination</td>
</tr>
<tr>
<td>Large orifice pipette Tips (1-200 ul)</td>
<td>&nbsp;</td>
<td>Fischer Scientific</td>
<td>02-681-141</td>
<td>To prevent shear stress to cells</td>
</tr>
<tr>
<td>Graduated pipette tips (101-1000 ul)</td>
<td>&nbsp;</td>
<td>USA Scientific</td>
<td>1111-2721</td>
<td>&nbsp;</td>
<tr>
<td>21 G1 precision guide needles</td>
<td>&nbsp;</td>
<td>Beckton Dickinson</td>
<td>305165</td>
<td>To clean tissue</td>
</tr>
<tr>
<td>10 ml pipette </td>
<td>&nbsp;</td>
<td>USA Scientific</td>
<td>1071-0810</td>
<td>Individually wrapped</td>
</tr>
<tr>
<td>50 ml tubes</td>
<td>&nbsp;</td>
<td>USA Scientific</td>
<td>926-9-04</td>
<td>&nbsp;</td>
<tr>
<td>Single edge razor blade</td>
<td>&nbsp;</td>
<td>Smith Brand</td>
<td>67-0238</td>
<td>To chop tissue</td>
</tr>
<tr>
<td>60 x 15 mm polystyrene petri dish </td>
<td>&nbsp;</td>
<td>USA Scientific</td>
<td>8609-0160</td>
<td>For general culture</td>
</tr>
<tr>
<td>100 x 15 mm polystyrene petri dish </td>
<td>&nbsp;</td>
<td>USA Scientific</td>
<td>8609-0010</td>
<td>For cleaning tissue</td>
</tr>
<tr>
<td>Cryogenic box </td>
<td>&nbsp;</td>
<td>NALGENE Labware</td>
<td>5026-1010</td>
<td>&nbsp;</td>
<tr>
<td>Freezing container</td>
<td>&nbsp;</td>
<td>NALGENE Labware</td>
<td>5100-0001</td>
<td>"Mr. Frosty"</td>
</tr>
<tr>
<td>2.0 ml cryogenic vials</td>
<td>&nbsp;</td>
<td>NALGENE Labware</td>
<td>5012-0020</td>
<td>&nbsp;</td>
<tr>
<td>DMSO</td>
<td>&nbsp;</td>
<td>Fischer Scientific</td>
<td>D128-500</td>
<td>For freezing medium</td>
</tr>
<tr>
<td>Ethanol 200 proof</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>E7023</td>
<td>For sterilizing razor blade</td>
</tr>		</tbody>
		</table>
		</div>

cryopreservation of cortical tissue blocks for the generation of highly enriched neuronal cultures

Bu çalışmada, biz, başarılı bir şekilde dondurulması ve zenginleştirilmiş nöronal kültürlerini oluşturmak için kortikal beyin dokusu blokları çözülme için standart bir protokol özetlemektedir. Bu protokolü için kullanılan donma orta Hank Tamponlu Tuz Çözeltisi (HBSS) seyreltilmiş% 10 dimetil sülfoksit (DMSO). Kortikal doku blokları, donma orta içeren cryovials transfer ve yavaş yavaş -1 ° C / dak hızı kontrollü bir dondurma kabı dondurulur. Post-çözülme işleme ve sürekli olarak 10 gün içinde ilk 5 günü kültür ve nöron ağının önemli bir genişleme sırasında hızlı nöritik büyüme sergiledi zenginleştirilmiş nöronal kültürlerin dondurulmuş doku blokları ayrılma. Astrositik marker glial fibriller asidik protein (GFAP) ve nöronal belirteç beta-tubulin sınıf III, İmmünositokimyasal boyanması kültürlerin astrositler nöronlar ve yüksek sayıda ortaya çıkardı. Doku blok disosiasyon sonra nöral prekürsör hücre kültürlerinde üretilmesi hızla farklılaşan koşullar altında çok sayıda nöron ve astrosit neurospheres genişleyen sonuçlandı. Bu basit kriyoprezervasyon protokolü kortikal beyin dokusu blokları, nöronal astrosit ve nöronal prekürsör hücre kültürlerinin sonraki nesil için daha fazla esneklik hibe hızlı, verimli ve ucuz korunması, sağlar.

Watch this Scientific Journal Video about Cryopreservation of Cortical Tissue Blocks for the Generation of Highly Enriched Neuronal Cultures at JoVE.com

Cryopreservation of Cortical Tissue Blocks for the Generation of Highly Enriched Neuronal Cultures

Burada, biz verimli dondurulması için bir yöntem tarif ve zenginleştirilmiş nöronal kültürlerini oluşturmak için kortikal beyin dokusu blokları çözülme. Bu basit protokol, nöronal astrosit ve nöronal prekürsör hücre kültürlerinin sonraki nesil için esneklik sağlar.

Yüksek zenginleştirilmiş Nöronal Kültürlerin Üretimi için Kortikal Doku Blokları, Kriyoprezervasyon

In this study, we outline a standardized protocol for the successful cryopreservation and thawing of cortical brain tissue blocks to generate highly ...

cryopreservation-cortical-tissue-blocks-for-generation-highly

Research

JoVE Journal

Neuroscience

13.5K Görüntüleme.  University of California, Irvine. Burada, biz verimli dondurulması için bir yöntem tarif ve zenginleştirilmiş nöronal kültürlerini oluşturmak için kortikal beyin dokusu blokları çözülme. Bu basit protokol, nöronal astrosit ve nöronal prekürsör hücre kültürlerinin sonraki nesil için esneklik sağlar.

Video: Cryopreservation of Cortical Tissue Blocks for the Generation of Highly Enriched Neuronal Cultures

Generation of Neural Stem Cells from Discarded Human Fetal Cortical Tissue

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ultimately give rise to intermediate progenitors and later, the various neuronal and glial cell subtypes that form the cerebral cortex. The capacity to generate and expand human NSCs (so called neurospheres) from discarded normal fetal tissue provides a means with which to directly study the functional aspects of normal human NSC development 1-5. This approach can also be directed toward the generation of NSCs from known neurological disorders, thereby affording the opportunity to identify disease processes that alter progenitor proliferation, migration and differentiation 6-9. We have focused on identifying pathological mechanisms in human Down syndrome NSCs that might contribute to the accelerated Alzheimer's disease phenotype 10,11. Neither in vivo nor in vitro mouse models can replicate the identical repertoire of genes located on human chromosome 21. 
Here we use a simple and reliable method to isolate Down syndrome NSCs from aborted human fetal cortices and grow them in culture. The methodology provides specific aspects of harvesting the tissue, dissection with limited anatomical landmarks, cell sorting, plating and passaging of human NSCs. We also provide some basic protocols for inducing differentiation of human NSCs into more selective cell subtypes.

A simple and reliable method on isolation and culture of neural stem cells from discarded human fetal cortical tissue is described. Cultures derived from known human neurological disorders can be used for characterization of pathological cellular and molecular processes, as well as provide a platform to assess pharmacological efficacy.

Atılan İnsan Fetal Kortikal Doku Sinir Kök Hücre Üretimi

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ...

Nörobilim

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 ...

A Fully Automated and Highly Versatile System for Testing Multi-cognitive Functions and Recording Neuronal Activities in Rodents

We have developed a fully automated system for operant behavior testing and neuronal activity recording by which multiple cognitive brain functions can be investigated in a single task sequence. The unique feature of this system is a custom-made, acoustically transparent chamber that eliminates many of the issues associated with auditory cue control in most commercially available chambers. The ease with which operant devices can be added or replaced makes this system quite versatile, allowing for the implementation of a variety of auditory, visual, and olfactory behavioral tasks. Automation of the system allows fine temporal (10 ms) control and precise time-stamping of each event in a predesigned behavioral sequence. When combined with a multi-channel electrophysiology recording system, multiple cognitive brain functions, such as motivation, attention, decision-making, patience, and rewards, can be examined sequentially or independently.

In this report, we present a fully automated and highly versatile system capable of simultaneously testing multi-cognitive behaviors and recording neuronal activities for rodents.

Multi-bilişsel işlevler Test ve Kemirgenler Nöronal Etkinlikler kayıt için Tam Otomatik ve Çok Yönlü Sistemi

We have developed a fully automated system for operant behavior testing and neuronal activity recording by which multiple cognitive brain functions can be ...

A Procedure for Implanting Organized Arrays of Microwires for Single-unit Recordings in Awake, Behaving Animals

In vivo electrophysiological recordings in the awake, behaving animal provide a powerful method for understanding neural signaling at the single-cell level. The technique allows experimenters to examine temporally and regionally specific firing patterns in order to correlate recorded action potentials with ongoing behavior. Moreover, single-unit recordings can be combined with a plethora of other techniques in order to produce comprehensive explanations of neural function. In this article, we describe the anesthesia and preparation for microwire implantation. Subsequently, we enumerate the necessary equipment and surgical steps to accurately insert a microwire array into a target structure. Lastly, we briefly describe the equipment used to record from each individual electrode in the array. The fixed microwire arrays described are well-suited for chronic implantation and allow for longitudinal recordings of neural data in almost any behavioral preparation. We discuss tracing electrode tracks to triangulate microwire positions as well as ways to combine microwire implantation with immunohistochemical techniques in order to increase the anatomical specificity of recorded results.

Implanting organized arrays of microwires for use in single-unit electrophysiological recordings presents a number of technical challenges.&#160;Methods for performing this technique and the equipment necessary are described.&#160;Also, the beneficial use of organized microwire arrays to record from distinct neural subregions with high spatial selectivity is discussed.

Hayvanlar davranmak, Awake Tek-birim Recordings Microwires Organize Dizileri implantasyon için Usulü

In vivo electrophysiological recordings in the awake, behaving animal provide a powerful method for understanding neural signaling at the single-cell ...

Preparation of Neuronal Co-cultures with Single Cell Precision

Microfluidic embodiments of the Campenot chamber have attracted great interest from the neuroscience community. These interconnected co-culture platforms can be used to investigate a variety of questions, spanning developmental and functional neurobiology to infection and disease propagation. However, conventional systems require significant cellular inputs (many thousands per compartment), inadequate for studying low abundance cells, such as primary dopaminergic substantia nigra, spiral ganglia, and Drosophilia melanogaster neurons, and impractical for high throughput experimentation. The dense cultures are also highly locally entangled, with few outgrowths (&#60;10%) interconnecting the two cultures. In this paper straightforward microfluidic and patterning protocols are described which address these challenges: (i) a microfluidic single neuron arraying method, and (ii) a water masking method for plasma patterning biomaterial coatings to register neurons and promote outgrowth between compartments. Minimalistic neuronal co-cultures were prepared with high-level (&#62;85%) intercompartment connectivity and can be used for high throughput neurobiology experiments with single cell precision.

Protocols for single neuron microfluidic arraying and water masking for the in-chip plasma patterning of biomaterial coatings are described. Highly interconnected co-cultures can be prepared using minimal cell inputs.

Tek Hücre Hassas Nevronik Co-kültürler hazırlanması

Microfluidic embodiments of the Campenot chamber have attracted great interest from the neuroscience community. These interconnected co-culture platforms ...

Prolonged Incubation of Acute Neuronal Tissue for Electrophysiology and Calcium-imaging

Acute neuronal tissue preparations, brain slices and retinal wholemount, can usually only be maintained for 6 - 8 h following dissection. This limits the experimental time, and increases the number of animals that are utilized per study. This limitation specifically impacts protocols such as calcium imaging that require prolonged pre-incubation with bath-applied dyes. Exponential bacterial growth within 3 - 4 h after slicing is tightly correlated with a decrease in tissue health. This study describes a method for&#160;limiting the proliferation of bacteria in acute preparations to maintain viable neuronal tissue for prolonged periods of time (&#62;24 h) without the need for antibiotics, sterile procedures, or tissue culture media containing growth factors. By cycling the extracellular fluid through UV irradiation and keeping the tissue in a custom holding chamber at 15 - 16 &#176;C, the tissue shows no difference in electrophysiological properties, or calcium signaling through intracellular calcium dyes at &#62;24 h postdissection. These methods will not only extend experimental time for those using acute neuronal tissue, but will reduce the number of animals required to complete experimental goals, and will set a gold standard for acute neuronal tissue incubation.

Once removed from the body, neuronal tissue is greatly affected by environmental conditions, leading to eventual degradation of the tissue after 6 - 8 h. Using a unique incubation method, which closely monitors and regulates the extracellular environment of the tissue, tissue viability can be significantly extended for &#62;24 h.

Elektrofizyoloji ve Kalsiyum-görüntüleme için akut nöronal Doku Uzamış İnkübasyon

Acute neuronal tissue preparations, brain slices and retinal wholemount, can usually only be maintained for 6 - 8 h following dissection. This limits the ...

Time-dependent Increase in the Network Response to the Stimulation of Neuronal Cell Cultures on Micro-electrode Arrays

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network dynamics in neuronal cultures. In contrast with more traditional methods, such as patch-clamping, which can only record activity from a single cell, MEAs can record simultaneously from multiple sites in a network, without requiring the arduous task of placing each electrode individually. Moreover, numerous control and stimulation configurations can be easily applied within the same experimental setup, allowing for a broad range of dynamics to be explored. One of the key dynamics of interest in these in vitro studies has been the extent to which cultured networks display properties indicative of learning. Mouse neuronal cells cultured on MEAs display an increase in response following training induced by electrical stimulation. This protocol demonstrates how to culture neuronal cells on MEAs; successfully record from over 95% of the plated dishes; establish a protocol to train the networks to respond to patterns of stimulation; and sort, plot, and interpret the results from such experiments. The use of a proprietary system for stimulating and recording neuronal cultures is demonstrated. Software packages are also used to sort neuronal units. A custom-designed graphical user interface is used to visualize post-stimulus time histograms, inter-burst intervals, and burst duration, as well as to compare the cellular response to stimulation before and after a training protocol. Finally, representative results and future directions of this research effort are discussed.

Mouse neuronal cells cultured on multi-electrode arrays display an increase in response following electrical stimulation. This protocol demonstrates how to culture neurons, how to record activity, and how to establish a protocol to train the networks to respond to patterns of stimulation.

Mikro Elektrod Dizilerinde Nöronal Hücre Kültürlerinin Uyarılmasına Ağ Yanıtına Zamana Bağlı Artış

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network ...

GM-Free Generation of Blood-Derived Neuronal Cells

Many human&#160;neurological disorders are caused by degeneration of neurons and glial cells in the brain. Due to limitations in pharmacological and other therapeutic strategies, there is currently no cure available for the injured or diseased brain. Cell replacement appears as a promising therapeutic strategy for neurodegenerative conditions. To this day, neural stem cells (NSCs) have been successfully generated from fetal tissues, human embryonic cells (ES) or induced pluripotent stem cells (iPSC). A process of dedifferentiation was initiated by activation of the novel human GPI-linked glycoprotein, which leads to generation of pluripotent stem cells. These blood-derived pluripotent stem cells (BD-PSCs) differentiate in vitro into cells with a neural phenotype as shown by brightfield and immunofluorescence microscopy. Ultrastructural analysis of these cells by means of electron microscopy confirms their primitive structure as well as neuronal-like morphology and subcellular characteristics.

We present a genetically modified-free (GM-free) method to obtain cells with a neuronal phenotype from reprogrammed peripheral blood cells. Activation of a signaling pathway linked to novel human GPI-linked protein reveals an efficient GM-free method for obtaining human pluripotent stem cells.

Kan Türevi Nöronal Hücrelerin GM'den Arındırılmış Üretimi

Many human&#160;neurological disorders are caused by degeneration of neurons and glial cells in the brain. Due to limitations in pharmacological and other ...

Cone-Enriched Cultures from the Retina of Chicken Embryos to Study Rod to Cone Cellular Interactions

Human daytime vision relies on the function of cone photoreceptors at the center of the retina, the fovea. Patients suffering from the most prevalent form of inherited retinal degeneration, retinitis pigmentosa, lose night vision because of mutation driven loss of rod photoreceptors, a phenomenon followed by a progressive loss of function and death of cones leading to blindness. Geneticists have identified many genes with mutations causing this disease, but the first mutations identified questioned the mechanisms of secondary cone degeneration and how a dominant mutation in the rhodopsin gene encoding for the visual pigment expressed exclusively in rods can trigger cone degeneration.
This result of transplantations in a genetic model of the disease led to the concept of cell interactions between rods and cones and of non-cell autonomous degeneration of cones in all genetic forms of retinitis pigmentosa.
Cones comprise 5% of all photoreceptors in humans and only 3% in the mouse, so their study is difficult in these species, but cones outnumber rods in bird species. We have adapted 96-well plates to culture retinal precursors from the retina of chicken embryos at stage 29 of their development. In these primary cultures, cones represent 80% of the cells after in vitro differentiation. The cells degenerate over a period of one week in the absence of serum. Here, we describe the methods and its standardization.
This cone-enriched culture system was used to identify the epithelium-derived cone viability factor (EdCVF) by high content screening of a rat retinal pigmented epithelium normalized cDNA library. Recombinant EdCVF prevents the degeneration of the cones.

We describe a method to obtain primary cultures of cone photoreceptors from the retina of chicken embryos and its use for high content screening.

Tavuk Embriyolarının Retinasından Koni Zenginleştirilmiş Kültürler Çubuktan Koni Hücresel Etkileşimlerine

Human daytime vision relies on the function of cone photoreceptors at the center of the retina, the fovea. Patients suffering from the most prevalent form ...

Robust and Highly Reproducible Generation of Cortical Brain Organoids for Modelling Brain Neuronal Senescence In Vitro

Brain organoids are three-dimensional models of the developing human brain and provide a compelling, cutting-edge platform for disease modeling and large-scale genomic and drug screening. Due to the self-organizing nature of cells in brain organoids and the growing range of available protocols for their generation, issues with heterogeneity and variability between organoids have been identified. In this protocol paper, we describe a robust and replicable protocol that largely overcomes these issues and generates cortical organoids from neuroectodermal progenitors within 1 month, and that can be maintained for more than 1 year. This highly reproducible protocol can be easily carried out in a standard tissue culture room and results in organoids with a rich diversity of cell types typically found in the developing human cortex. Despite their early developmental make-up, neurons and other human brain cell types will start to exhibit the typical signs of senescence in neuronal cells after prolonged in vitro culture, making them a valuable and useful platform for studying aging-related neuronal processes. This protocol also outlines a method for detecting such senescent cells in cortical brain organoids using senescence-associated beta-galactosidase staining.

Robust and Highly Reproducible Generation of Cortical Brain Organoids for Modelling Brain Neuronal Senescence In Vitro

In this study, we provide a detailed technique for a simple yet robust cortical organoid culture system using standard feeder-free hPSC cultures. This is a rapid, efficient, and reproducible protocol for generating organoids that model aspects of brain senescence in vitro.

Beyin Nöronal Yaşlanmasını Modellemek için Kortikal Beyin Organoidlerinin Sağlam ve Yüksek Oranda Yeniden Üretilebilir Üretimi In Vitro

Brain organoids are three-dimensional models of the developing human brain and provide a compelling, cutting-edge platform for disease modeling and ...