Esta investigación fue financiada por becas de la Investigación de Pregrado Programa de Oportunidades de la UCI (AR y SP) y las subvenciones del Estado de California Iniciativa de Enfermedades de Alzheimer y los Institutos Nacionales de Salud de subvención no. HD38466, y el Centro de Investigación de la Enfermedad de Alzheimer no se conceda ninguna. AG16573 (JB)

1. La criopreservación de tejido cortical bloques Preparación de materiales <ol><li> Preparar buffer 1X Hank solución de sal (HBSS) diluyendo en solución madre 10 veces en agua estéril (1:9). HBSS almacenar a 4 ° C. </li><li> Prepare un 10% medio de congelación DMSO mediante la dilución de DMSO en HBSS (1:9). El medio de congelación debe ser fresca antes de la crioconservación y se almacenan a 4 ° C hasta que se enfríe. </li><li> Una hoja de afeitar de acero se utiliza para la tala sistemática de los tejidos. Antes de su uso, la hoja de afeitar se esteriliza por inmersión en etanol al 70% durante 2 horas. Inmediatamente antes de la elaboración del tejido, enjuagar la cuchilla con agua estéril tres veces. Evite dejar la navaja en agua estéril para una cantidad excesiva de tiempo, ya que es propensa a la oxidación. </li><li> Un contenedor de congelación Nalgene se carga con crioviales (2,5 ml) y luego llevados a un gabinete biológicos estériles. 1 ml de medio de congelación frío se añade a cada vial. El contenedor de congelación se coloca a 4 ° C durante al menos 2 horas. </li></ol> Limpieza, picado, congelación y <ol><li> El tejido cerebral para ser congelados se limpia de la membrana de las meninges y los vasos sanguíneos. Use puntas aguja estéril para retirar cuidadosamente los restos de los tejidos mientras se trabaja en la parte superior de una bolsa de hielo. Tejido limpiados deben ser enjuagados con un poco de HBSS frío y trasladado a una nueva placa de Petri para picar. </li><li> Trabajo en la parte superior de una bolsa de hielo, use una hoja de afeitar esterilizada para cortar rápidamente los tejidos en aproximadamente 1 mm a 3 cuadras. Trabajar con pequeñas porciones de tejido limpiado en un tiempo resulta en un mejor control sobre el procedimiento de cortar, lo que resulta en tamaños de bloque más uniforme. </li><li> Agregue lentamente el tejido cortado en 50 ml de HBSS en un tubo cónico de 50 ml. Enjuague suavemente la placa de Petri con HBSS para recoger los bloques de tejido restante. Permita que los bloques de tejido para descender al fondo del tubo, la creación de un tejido muy apretadas bloque de pellets. </li><li> Mientras que el tejido es la solución, poner el contenedor de congelación previamente enfriada y crioviales en la cabina de seguridad biológica. Destapa todos los crioviales para acelerar el proceso de adición de tejido. </li><li> Después de todos los bloques de tejido se han asentado, Aspire con cuidado el exceso de HBSS, dejando una capa muy fina de los medios de comunicación por encima de la pastilla. Centrifugación se recomienda, ya que hará que los bloques de tejido se adhieran unas a otras, disminuyendo significativamente la eficiencia de la congelación. </li><li> Poco a poco recolectar 200 l de la parte inferior del bloque de pellets sueltos tejido mediante el uso de punta de la pipeta con orificios de ancho (corte la punta con unas tijeras estériles). Transferir el material a un criovial y pasar a los tejidos próximos, de nuevo, recogiendo la parte inferior de la pastilla. Es esencial que el procedimiento de asignación de todo el tejido no toma más de 3-4 minutos por cada contenedor de congelación. Esto asegura que el tejido se expone a DMSO por sólo un corto período de tiempo antes de la congelación. Después de transferir el tejido de los viales, coloque el recipiente de congelación en un congelador de-80oC durante al menos 4 horas. Por otra parte, el contenedor de congelación se puede dejar toda la noche a -80 oC. Repita este proceso para todos los contenedores de congelación restantes. </li><li> Transferir el crioviales desde el contenedor de congelación (s) a una Caja criogénica y el lugar en un tanque de nitrógeno líquido para su almacenamiento a largo plazo. </li></ol> 2. Descongelación y cultivo de bloques congelados de tejido cortical Preparación de materiales <ol><li> Un día antes de descongelar las muestras de tejido, poli-L-lisina recubierto Petri / cubreobjetos está preparado. En general, para las neuronas corticales que la preparación de platos recubiertos con 500 mg / ml o 1 mg / ml. Agregue suficiente ácido poli-L-lisina solución (hecho en tampón borato) para cubrir completamente el fondo del plato. Si cubreobjetos de vidrio es necesario, asegúrese de que estén completamente sumergidos en la solución de poli-L-lisina. Incubar durante al menos 12 horas. Antes de usarlo, enjuague los platos con una cantidad abundante de agua estéril 3 veces, 5 minutos cada uno. </li><li> HBSS caliente se utiliza para limpiar el tejido descongelado, así como para disociar el tejido en suspensiones celulares. El volumen de HBSS necesario variará en función de la cantidad de tejido para ser descongelados, pero por lo general un criovial va a diluir en 50 ml de HBSS y se disocia en 10 ml de HBSS. </li><li> Modificado por Dulbecco medio de Eagle con 10% suplementado con hierro de suero bovino ternero (EBS) y el 1% antibiótico / antimicótico (AA) mezcla (DMEM (++)) debe ser colocado en un baño de agua 37 ° C. </li><li> Medio chapado (NB (+++)) debe ser fresca antes de su uso. Este medio se prepara añadiendo 1% antibiótico / antimicótico más B27 y los suplementos de N2. </li></ol> Nota: Los nombres de los medios de comunicación adjunta con cruces (+) indican la inclusión de aditivos a la base de la composición de los medios de comunicación. En este texto, DMEM (+ +) denota DMEM más 10% de EBS más el 1% de AA, mientras que denota (+++) NB NB plnos del 1% de AA, además B27 además de N2. Descongelación y cultivo <ol><li> Retire la crioviales del tanque de nitrógeno líquido y descongelación rápida a 37 º C al baño maría hasta sólo una pequeña bolita de hielo es observado dentro de los contenidos vial. </li><li> Suavemente transferir el contenido del vial de 50 ml de HBSS caliente en un tubo cónico. Utilice una punta de orificio amplio para desalojar suavemente cualquier resto de los bloques de tejido atrapado en el criovial. Invierta el tubo 3-4 veces y permitir que el tejido de recoger poco a poco en la parte inferior. Tejidos deben resolver rápidamente, pero si los bloques son demasiado pequeños como este no puede ocurrir y la luz centrifugación (~ 200 x g) se recomienda. </li><li> Aspirar HBSS exceso. </li><li> Agregar 10 ml de HBSS caliente al precipitado de tejido y 300 L de 0,25% de tripsina y 50 L de ADNasa. Incubar en un baño de agua 37 ° C durante 5 minutos, luego coloque en un agitador orbital conjunto de 80 rpm y 37 ° C durante 5 min. </li><li> Después de este período, traer los bloques de tejido en una cabina de seguridad biológica y la utilización de una pipeta 10 ml de agitar cuidadosamente los bloques de tejido hasta obtener una suspensión de células se forma nubes. Desactivar la tripsina mediante la adición de 10 ml de agua tibia DMEM (+ +) a la suspensión celular. </li><li> Centrifugar las células disociadas a 1200 x g por 5 min. Aspirar y desechar el sobrenadante, resuspender el botón celular en 10 ml de agua tibia DMEM (+ +) y cuantificar el número de células con un hemocitómetro. </li><li> Células de la placa de poli-L-lisina platos recubiertos. Por lo general, un plato de 60 mm será plateado con 1x10 6 células. </li><li> Permitir que las células se adhieren a la placa / cubreobjetos durante aproximadamente 1 hora en el cultivo de tejidos incubadora. Se recomienda monitorear el progreso del archivo adjunto en una placa cada 10 minutos hasta que adjunto efectiva que se observa. Entonces, suavemente reemplazar el medio con DMEM fresco (++). </li><li> Después de 24 horas, cambie el DMEM (+ +) con agua tibia (+++). NB Cambios parciales de media (50%) se llevan a cabo cada 5 días con nuevas (+++). NB Culturas saludables por lo general muestran signos de diferenciación y el crecimiento de los procesos después de 24 hrs. En estas condiciones, y realizar cambios parciales de medio cada 4-5 días, los cultivos se pueden mantener por periodos prolongados de tiempo de hasta 4-6 semanas. </li><li> Para la generación de células precursoras neuronales (CPN), un protocolo similar se utiliza con la excepción de que después de la centrifugación, DMEM sin EBS se utiliza. Las células se colocan en el T-25 frascos en DMEM/F12 (01:03), complementado con B27 y EGF (20 ng / mL) para permitir la formación de neuropheres. Para diferenciar los NPCs, neuroesferas se colocan en laminina (10 ug / mL) en cubreobjetos recubiertos DMEM/F12 (1:3) suplementado con N2. </li></ol> 3. Resultados representante Culturas sano muestra un importante crecimiento y la diferenciación de los 5 días post-descongelación y por lo general se estabiliza en su crecimiento por 10 días (Figura 1). Tinción Immuncocytochemical de estas culturas revela numerosos astrocitos (Figura 2A) y neuronas (Figura 2B) para los humanos y de rata cultivos neuronales primarios. Este protocolo también es adecuado para la generación de NPCs como de libre flotación neuroesferas (Figura 3), que en condiciones de diferenciación, el resultado en las culturas mixtas de alta calidad (Figura 3 B, C). <img src="/files/ftp_upload/2384/2384fig1.jpg" alt="Figura 1" /> Figura 1. Los cultivos de células congeladas de tejido cortical humano. Bloques congelados de tejido cortical humano se descongelan y se sembraron en poli-lisina cubreobjetos recubiertos y crecido durante 10 días. El día 5 la expansión celular es evidente y el día 10 de confluencia se logra. Barra de escala: 100 micras. <img src="/files/ftp_upload/2384/2384fig2.jpg" alt="Figura 2" /> Figura 2. Inmuno tinción de los cultivos celulares. (A) los cultivos fueron teñidas con el marcador de proteína glial fibrilar glial ácida (GFAP, flechas gruesas). (B) Las neuronas se detectaron con el marcador neuronal beta-tubulina III (TIII, flechas delgadas). (C) Los dos humanos y las culturas de ratas muestran células gliales y las neuronas abundantes después de 10 días de cultivo. Anticuerpos primarios: el ratón anti-GFAP (1:1000) y el conejo anti-TIII (1:1000). Secundaria de anticuerpos: anti-ratón Alexa 594 (1:500) y anti-conejo Alexa 488 (1:500). Tinción de los núcleos: Hoestch azul (1:1000). Barra de escala: 50 micras. <img src="/files/ftp_upload/2384/2384fig3.jpg" alt="Figura 3" /> Figura 3. Generación de neuroesferas. (A) El tejido congelado fue procesada como se describió anteriormente y se permite que se propagan como neuroesferas. (B) neuroesferas se cultivaron en cubreobjetos de vidrio en condiciones de diferenciar y crecido durante 10 días, dando lugar a células que migran fuera de la neuroesfera. (C) Tanto las neuronas y los astrocitos están presentes en la periferia en expansión de una neuroesfera diferenciador. Contratinción nuclear, los anticuerpos primarios y secundarios utilizados como en la figura 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>

Criopreservación ofrece la oportunidad de banco preciosas muestras de tejido cerebral para uso futuro. Aquí se describe un protocolo sencillo pero eficaz para generar tanto neuronas enriquecido las culturas y las células precursoras neuronales a partir de bloques congelados de tejido cerebral. Este procedimiento evita los costos económicos de las técnicas de criopreservación tradicionales que utilizan más caro velocidad controlada congeladores. El protocolo permite la generación de cultivos neuronales viable después de la descongelación, proporcionando un medio rápido pero efectivo para congelar bloques de tejido. El proceso de congelación completa puede tomar tan poco como 20 minutos. Además de los cultivos primarios de neuronas, usando este método de bloques de tejido también se pueden descongelar para generar NPCs crecido como neuroesferas libre flotación. En este sentido, la falta de suero en nuestro medio de congelación asegura que las células se conservan en un estado indiferenciado. En condiciones de diferenciación, neuroesferas producido a partir de la expansión del tejido congelado y las tasas muestran una diferenciación muy comparable a la generada a partir de neuroesferas tejido 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

En este estudio, se describe un protocolo estandarizado para la criopreservación exitosa y descongelación de bloques de corteza de tejido cerebral para generar altamente enriquecido cultivos neuronales. Para este protocolo el medio de congelación utilizado es del 10% dimetilsulfóxido (DMSO) diluido en solución salina tamponada de Hank (HBSS). Los bloques de tejido cortical se transfieren a crioviales que contiene el medio de congelación lenta y congelados a -1 ° C / min en un recipiente de congelación de velocidad controlada. Después de la descongelación de procesamiento y la disociación de los bloques de tejido congelado producido constantemente neuronal enriquecido con las culturas que mostraron un crecimiento neurítico rápido durante los primeros 5 días de la cultura y la significativa expansión de la red neuronal dentro de 10 días. Tinción inmunocitoquímica con el marcador de astrocitos proteína glial fibrilar ácida (GFAP) y el marcador neuronal beta-tubulina III clase, reveló un gran número de neuronas y astrocitos en los cultivos. Generación de células precursoras neuronales culturas después de la disociación del tejido bloque resultó en rápida expansión neuroesferas, que produjo un gran número de neuronas y astrocitos en condiciones de diferenciar. Este protocolo de crioconservación simple permite la conservación es rápido, eficaz y de bajo costo de los bloques de tejido cerebral cortical, que otorga una mayor flexibilidad para la última generación de cultivos de células neuronales, astrocitos, y precursoras neuronales.

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

Aquí se describe un método eficiente para la criopreservación y descongelación de bloques de corteza de tejido cerebral para generar altamente enriquecido cultivos neuronales. Este sencillo protocolo proporciona flexibilidad para la generación posterior de cultivos de células neuronales, astrocitos, y precursoras neuronales.

La criopreservación de tejido cortical Bloques para la generación de cultivos neuronales altamente enriquecido

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 vistas.  University of California, Irvine. Aquí se describe un método eficiente para la criopreservación y descongelación de bloques de corteza de tejido cerebral para generar altamente enriquecido cultivos neuronales. Este sencillo protocolo proporciona flexibilidad para la generación posterior de cultivos de células neuronales, astrocitos, y precursoras neuronales.

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.

Generación de las células troncales nerviosas de desechados Humanos tejido cortical fetal

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

Investigación

Neurociencias

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.

Electrodos múltiples grabaciones matriz de avalanchas neuronal en cultivos organotípicos

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

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).

Derivación de las Culturas enriquecido oligodendrocitos y neuronas oligodendrocitos / myelinating Co-culturas de Post-natal tejidos murinos

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 totalmente automatizado y altamente versátil para las pruebas múltiples funciones cognitivas y el registro de las actividades neuronales en los roedores

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

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.

Preparación de Neuronales Co-culturas con precisión Single Cell

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.

La incubación prolongado de aguda neuronal de tejidos para la Sección de Electrofisiología y calcio de imágenes

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.

Incremento dependiente del tiempo en la respuesta de la red a la estimulación de cultivos de células neuronales en matrices de microelectrodos

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.

Generación Libre De Gm De Células Neuronales Derivadas De La Sangre

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.

Cultivos Enriquecidos con Conos De La Retina De Embriones De Pollo Para Estudiar Las Interacciones Celulares De Varilla A 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.

Generación robusta y altamente reproducible de organoides cerebrales corticales para modelar la senescencia neuronal cerebral in vitro

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