Questa ricerca è stata sostenuta da finanziamenti del Programma triennale Opportunità di ricerca presso UCI (AR e SP) e le sovvenzioni da parte dello Stato della California Iniziativa malattia di Alzheimer e il National Institutes of Health non concede. HD38466, e il morbo di Alzheimer Research Center non concede. AG16573 (JB)

1. Crioconservazione di tessuto corticale blocchi Materiale di preparazione <ol><li> Preparare la soluzione salina tampone 1X Hank (HBSS) diluendo in soluzione madre 10X in acqua sterile (1:9). HBSS conservare a 4 ° C. </li><li> Preparare il 10% medio congelamento DMSO diluendo DMSO in HBSS (1:9). Il mezzo di congelamento dovrebbe essere fresca prima di crioconservazione e conservato a 4 ° C fino a freddo. </li><li> Una lama di rasoio in acciaio viene utilizzato per il taglio sistematico del tessuto. Prima dell&#39;uso, la lama è sterilizzata da immersione in etanolo al 70% per 2 ore. Immediatamente prima della lavorazione dei tessuti, sciacquare la lametta con acqua sterile 3 volte. Evitare di lasciare il rasoio in acqua sterile per una quantità eccessiva di tempo, in quanto è soggetto a ossidazione. </li><li> Un contenitore di congelamento Nalgene viene caricato con cryovials (2,5 ml) e poi portato in un armadio sterile biologica. 1 ml di acqua refrigerata media congelamento è aggiunto ad ogni flacone. Il contenitore congelamento è quindi posto a 4 ° C per almeno 2 ore. </li></ol> Pulizia, Tagliere, e congelamento <ol><li> Il tessuto cerebrale essere congelati viene pulita di membrana meningea e vasi sanguigni. Utilizzare punte ago sterile per rimuovere i detriti con attenzione dal tessuto durante il lavoro in cima ad un impacco di ghiaccio. Tessuti lavabili devono essere risciacquati leggermente con HBSS freddo e trasferito in un nuovo piatto di Petri per tritare. </li><li> Lavorare su di un impacco di ghiaccio, utilizzare una lama di rasoio sterile di tagliare rapidamente il tessuto di circa 1 mm in 3 blocchi. Lavorare con piccole porzioni di tessuto pulito in un momento risultati in un migliore controllo della procedura di taglio, con conseguente blocchi di dimensioni più uniforme. </li><li> Aggiungere lentamente il tessuto tagliato in 50 ml di HBSS in un tubo da 50 ml. Lavare delicatamente la piastra Petri con HBSS al fine di raccogliere tutti i blocchi del tessuto residuo. Lasciare i blocchi di tessuto a scendere sul fondo del tubo, creando un poco compresso tessuto blocco pellet. </li><li> Mentre il tessuto è assestamento, portare il contenitore refrigerato precedentemente congelamento e cryovials nella cabina di sicurezza biologica. Stappare tutti cryovials per accelerare il processo di aggiunta dei tessuti. </li><li> Dopo che tutti i blocchi di tessuto si sono stabiliti, dolcemente aspirare il HBSS in eccesso, lasciando uno strato molto sottile di media al di sopra del pellet. Centrifugazione è sconsigliato in quanto causerà i blocchi del tessuto di aderire gli uni agli altri, diminuendo in modo significativo l&#39;efficienza di congelamento. </li><li> Lentamente raccogliere 200 ul dalla parte inferiore del piede libero tessuto blocco pellet utilizzando punta della pipetta con orifizi larga (taglio punta con forbici sterili). Trasferire il materiale ad una provetta Microbank ™ e passare a quella successiva, il tessuto ancora raccogliere dal fondo del pellet. E &#39;essenziale che l&#39;intera procedura di assegnazione dei tessuti non prende più di 3-4 minuti per contenitore di congelamento. Questo assicura che il tessuto è esposto a DMSO solo per un breve periodo di tempo prima del congelamento. Dopo aver trasferito il tessuto per le fiale, porre il recipiente freddo-80 ° C in un freezer per almeno 4 ore. In alternativa, il contenitore di congelamento può essere lasciato una notte a -80 ° C. Ripetere la procedura per eventuali contenitori congelamento rimanenti. </li><li> Trasferire il cryovials dal contenitore congelamento (s) ad un cryobox e posto in un serbatoio di azoto liquido per la conservazione a lungo termine. </li></ol> 2. Scongelamento e coltura di blocchi congelati di tessuto corticale Materiale di preparazione <ol><li> Un giorno prima di campioni di tessuto scongelamento, poli-L-lisina rivestito Petri / coprioggetto sono preparati. In generale, per i neuroni corticali prepariamo piatti rivestiti con 500 mg / ml o 1 mg / mL. Aggiungi sufficiente poli-L-lisina soluzione (made in tampone borato) per coprire completamente il fondo del piatto. Se coprioggetto di vetro sono necessari, in modo che siano completamente sommersi sotto la poli-L-lisina soluzione. Incubare per almeno 12 ore. Prima dell&#39;uso, sciacquare i piatti con un buon quantitativo di acqua sterile per 3 volte, 5 minuti ciascuno. </li><li> HBSS calda è usata per pulire il tessuto scongelato e per dissociare il tessuto in sospensioni cellulari. Il volume di HBSS necessari variano a seconda della quantità di tessuto per essere scongelati, ma tipicamente 1 provetta Microbank ™ saranno diluiti in 50 mL di HBSS e sarà dissociato in 10 ml di HBSS. </li><li> Dulbecco Modified Eagle con medie del 10% di ferro integrato bovino siero bovino (EBS) e 1% antibiotici / antimicotici (AA) miscela (DMEM (++)) dovrebbe essere collocato in un bagno d&#39;acqua a 37 ° C. </li><li> Medio placcatura (NB (+++)) dovrebbero essere fresco prima dell&#39;uso. Questo terreno è preparato con l&#39;aggiunta di 1% antibiotico / antimicotico più B27 e integratori N2. </li></ol> Nota: i nomi media con l&#39;aggiunta di croci (+) indicano l&#39;aggiunta di additivi per la composizione di base dei media. In questo testo, DMEM (+ +) indica DMEM più 10% più 1% EBS AA, mentre NB (+++) denota NB plnoi l&#39;1% AA più B27 più N2. Scongelamento e coltura <ol><li> Rimuovere il cryovials dal serbatoio di azoto liquido e scongelare rapidamente a 37 ° C a bagnomaria fino a quando solo una piccola pallina di ghiaccio si osserva all&#39;interno del contenuto del flaconcino. </li><li> Delicatamente trasferire il contenuto della fiala da 50 mL HBSS caldo in un tubo conico. Utilizzare una punta larga orifizio per rimuovere delicatamente eventuali blocchi di tessuto residuo bloccato in provetta Microbank ™. Invertire il tubo di 3-4 volte e permettere al tessuto di raccogliere lentamente sul fondo. Tessuto deve risolvere in fretta, ma se i blocchi sono troppo piccoli non possono verificarsi e centrifugazione della luce (~ 200 g) è raccomandato. </li><li> Aspirare HBSS eccesso. </li><li> Aggiungere 10 ml di HBSS caldo al precipitato di tessuto e 300 ml di tripsina 0,25% e 50 ml di DNAase. Incubare in un bagno d&#39;acqua a 37 ° C per 5 minuti, poi posto in un agitatore orbitale impostato a 80 giri e 37 ° C per altri 5 min. </li><li> Dopo questo periodo, portare i blocchi di tessuto in una cappa di sicurezza biologica e utilizzare una pipetta 10 ml per agitare con cura i blocchi dei tessuti fino a una sospensione di cellule nuvoloso è formato. Disattivare la tripsina aggiungendo 10 ml di DMEM caldo (+ +) alla sospensione cellulare. </li><li> Centrifugare le cellule dissociate a 1200 x g per 5 min. Aspirare ed eliminare il surnatante, risospendere il pellet cellulare in 10 ml di DMEM caldo (+ +) e quantificare il numero delle cellule utilizzando un emocitometro. </li><li> Cellule piastra sui piatti rivestiti di poli-L-lisina. In genere, una di 60 mm piatto sarà placcato con 1x10 6 cellule. </li><li> Permettono alle cellule di allegare al piatto / coprioggetto per circa 1 ora nel tessuto culturale incubatore. Si raccomanda di monitorare i progressi di attaccamento in un piatto ogni 10 minuti fino a quando l&#39;attaccamento effettivo. Poi, gentilmente sostituire il mezzo con fresca DMEM (++). </li><li> Dopo 24 ore, sostituire la DMEM (+ +) con caldo NB (+++). Variazioni medie parziali (50%) sono effettuate ogni 5 giorni con fresca NB (+++). Culture sano di solito mostrano segni di differenziazione e di crescita dei processi dopo 24 ore. In queste condizioni, ed effettuando cambiamenti medie parziali ogni 4-5 giorni, le culture può essere mantenuta per periodi di tempo prolungati fino a 4-6 settimane. </li><li> Per la generazione di cellule precursore neuronali (NPC), un protocollo simile è usato con l&#39;eccezione che dopo la centrifugazione, DMEM senza EBS viene utilizzato. Le cellule sono placcati in T-25 palloni DMEM/F12 (1:3) integrata con B27 e EGF (20 ng / ml) per permettere la formazione di neuropheres. Per differenziare i PNG, neurosfere sono placcati in laminina (10 ug / mL) coprioggetto rivestito in DMEM/F12 (1:3) di media integrato con N2. </li></ol> 3. Rappresentante Risultati Culture sani mostrano una significativa crescita e differenziazione dopo 5 giorni dopo lo scongelamento e di solito stabilizzarsi nella sua crescita da 10 giorni (Figura 1). Immuncocytochemical colorazione di queste culture astrociti rivela numerosi (Figura 2A) e neuroni (Figura 2B) per le colture primarie umane e di ratto neuronale. Questo protocollo è adatto anche per la generazione di NPC come free-floating neurosfere (Figura 3A), che in condizioni di differenziazione, risultato in colture miste di alta qualità (Figura 3 B, C). <img src="/files/ftp_upload/2384/2384fig1.jpg" alt="Figura 1" /> Figura 1. Le colture cellulari di tessuti umani congelati corticale. Blocchi congelati di tessuto corticale umano sono scongelati e placcato su vetrini rivestiti di poli-lisina e cresciuto per 10 giorni. Dal giorno 5 di espansione delle cellule è evidente e da giorno 10 confluenza è raggiunto. Scala grafica: 100 micron. <img src="/files/ftp_upload/2384/2384fig2.jpg" alt="Figura 2" /> Figura 2. Colorazione immunocitochimica di colture cellulari. (A) Le culture sono state colorate con la proteina marker gliale fibrillare acida gliale (GFAP, frecce di spessore). (B) I neuroni sono stati rilevati con il marcatore neuronale beta-tubulina III (TIII, frecce sottili). (C) Sia e delle culture umane nel ratto mostrano glia abbondante e neuroni dopo 10 giorni in coltura. Anticorpi primari: topo anti-GFAP (1:1000) e di coniglio anti-TIII (1:1000). Anticorpi secondari: anti-topo Alexa 594 (1:500) e anti-coniglio Alexa 488 (1:500). Nuclei colorazione: blu Hoestch (1:1000). Scala grafica: 50 micron. <img src="/files/ftp_upload/2384/2384fig3.jpg" alt="Figura 3" /> Figura 3. Generazione di neurosfere. (A) tessuto congelato è stato elaborato come descritto in precedenza e ha permesso di propagare come neurosfere. (B) neurosfere sono stati placcati in coprioggetto di vetro in condizioni di differenziazione e cresciuto per 10 giorni, con conseguente migrazione delle cellule dalla neurosfere. (C) Sia i neuroni e astrociti sono presenti ai margini in espansione di un neurosfere di differenziazione. Controcolorazione nucleare, gli anticorpi primari e secondari utilizzati come in Fig. 2.

<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>

Nessun conflitto di interessi dichiarati.

Crioconservazione offre l&#39;opportunità di campioni di tessuto cerebrale preziosa banca per un uso futuro. Qui si descrive un protocollo semplice ma efficace per generare neuroni sia arricchito di culture e le cellule precursore neuronali da blocchi congelati tessuto cerebrale. Questa procedura evita i costi economici delle tecniche di crioconservazione tradizionali che utilizzano più costosi velocità controllata congelatori. Il protocollo permette la generazione di validi culture neuronali post-scongelamento, fornendo un mezzo rapido ma efficace per congelare i blocchi dei tessuti. L&#39;intero processo di congelamento può prendere così piccolo quanto 20 minuti. In aggiunta alle colture primarie neuronali, utilizzando questo metodo effettua il blocco del tessuto può anche essere scongelati per generare NPC cresciuta come libero neurosfere galleggiante. A questo proposito, la mancanza di siero nel nostro medio congelamento assicura che le cellule sono conservate in uno stato indifferenziato. In condizioni di differenziazione, neurosfere prodotta dall&#39;espansione surgelati mostrano tassi di differenziazione dei tessuti e del tutto paragonabile a neurosfere generate dal tessuto fresco.

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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

In questo studio, abbiamo delineare un protocollo standardizzato per la crioconservazione di successo e scongelamento dei blocchi cortical tessuto cerebrale di generare altamente arricchito culture neuronali. Per questo protocollo di congelamento del mezzo utilizzato è del 10% dimetilsolfossido (DMSO) diluito in soluzione salina tampone di Hank (HBSS). Blocchi di tessuto corticale vengono trasferiti cryovials contenenti il ​​mezzo di congelamento e lentamente congelati a -1 ° C / min in un tasso di controllo contenitore di congelamento. Disgelo post-elaborazione e la dissociazione dei blocchi di tessuti congelati costantemente prodotto neuronale arricchito di culture che hanno evidenziato crescita rapida neuritiche durante i primi 5 giorni di cultura e di significativa espansione della rete neuronale entro 10 giorni. Colorazione immunocitochimica con il astrociti proteina marker gliale fibrillare acida (GFAP) e il marcatore neuronale beta-tubulina di classe III, ha rivelato un alto numero di neuroni e astrociti nelle culture. Generazione di colture di cellule neurali progenitrici del tessuto dopo blocco dissociazione portato a rapida espansione neurosfere, che ha prodotto un gran numero di neuroni e astrociti in condizioni di differenziazione. Questo protocollo permette la crioconservazione semplice per una rapida, conservazione efficiente e poco costoso di blocchi di tessuto cerebrale corticale, che concede una maggiore flessibilità per la generazione successiva di culture precursore neuronali, astrociti e cellule neuronali.

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

Qui, descriviamo un metodo efficiente per la crioconservazione e lo scongelamento dei blocchi di tessuto cerebrale corticale per generare altamente arricchito culture neuronali. Questo semplice protocollo fornisce la flessibilità per la generazione successiva di culture precursore neuronali, astrociti e cellule neuronali.

Crioconservazione di tessuto corticale blocchi per la generazione di altamente arricchito Culture neuronale

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 Views.  University of California, Irvine. Qui, descriviamo un metodo efficiente per la crioconservazione e lo scongelamento dei blocchi di tessuto cerebrale corticale per generare altamente arricchito culture neuronali. Questo semplice protocollo fornisce la flessibilità per la generazione successiva di culture precursore neuronali, astrociti e cellule neuronali.

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.

Generazione di cellule staminali neurali da scartate tessuto fetale umano corticale

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

Ricerca

Neuroscienze

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).

Derivazione di Arricchito Culture oligodendrociti e degli oligodendrociti / Neuron mielinizzanti co-culture di tessuti murini post-natale

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.

Un sistema completamente automatizzato ed altamente versatile per test multi-funzioni cognitive e di registrare le attività neuronali in roditori

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.

Una procedura per l&#39;impianto Array organizzata di microfili per Recordings singola unità di Awake, Comportarsi Animali

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.

Preparazione della neuronali Co-culture con precisione singola cella

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.

L&#39;incubazione prolungata di acuta neuronale tessuto per Elettrofisiologia e Calcio imaging

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.

Aumento del tempo della risposta alla rete alla stimolazione delle colture cellulari neuronali sulle matrici di microelettrodi

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.

Generazione priva di OGM di cellule neuronali derivate dal sangue

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.

Culture arricchite di coni dalla retina degli embrioni di pollo per studiare le interazioni cellulari tra bastone e cono

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.

Generazione robusta e altamente riproducibile di organoidi cerebrali corticali per la modellazione della senescenza neuronale cerebrale in vitro

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