Este proyecto fue financiado por una beca de la Sociedad de Esclerosis Múltiple de Canadá a RKRWO es beneficiario de una beca de la Sociedad de Esclerosis Múltiple de Canadá. El DEG es un receptor de becas post-doctorales de la Sociedad de Esclerosis Múltiple de Canadá y de Institutos Canadienses de Investigación en Salud.

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	<li>Avellana-Adalid, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expansion+of+rat+oligodendrocyte+progenitors+into+proliferative+"oligospheres"+that+retain+differentiation+potential.">Expansion of rat oligodendrocyte progenitors into proliferative "oligospheres" that retain differentiation potential.</a> J Neurosci Res. 45, 558-570 (1996).</li><li>McCarthy, K. D., de Vellis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+separate+astroglial+and+oligodendroglial+cell+cultures+from+rat+cerebral+tissue.">Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue.</a> J Cell Biol. 85, 890-902 (1980).</li><li>Barres, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+death+and+control+of+cell+survival+in+the+oligodendrocyte+lineage.">Cell death and control of cell survival in the oligodendrocyte lineage.</a> Cell. 70, 31-46 (1992).</li><li>Chan, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NGF+controls+axonal+receptivity+to+myelination+by+Schwann+cells+or+oligodendrocytes.">NGF controls axonal receptivity to myelination by Schwann cells or oligodendrocytes.</a> Neuron. 43, 183-1891 (2004).</li><li>Camara, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrin-mediated+axoglial+interactions+initiate+myelination+in+the+central+nervous+system.">Integrin-mediated axoglial interactions initiate myelination in the central nervous system.</a> J Cell Biol. 185, 699-712 (2009).</li><li>Ishibashi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocytes+promote+myelination+in+response+to+electrical+impulses.">Astrocytes promote myelination in response to electrical impulses.</a> Neuron. 49, 823-832 (2006).</li><li>Chen, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+rat+and+mouse+oligodendrocyte+precursor+cells.">Isolation and culture of rat and mouse oligodendrocyte precursor cells.</a> Nat Protoc. 2, 1044-1051 (2007).</li><li>Pedraza, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production,+characterization,+and+efficient+transfection+of+highly+pure+oligodendrocyte+precursor+cultures+from+mouse+embryonic+neural+progenitors.">Production, characterization, and efficient transfection of highly pure oligodendrocyte precursor cultures from mouse embryonic neural progenitors.</a> Glia. 56, 1339-1352 (2008).</li><li>Lewin, G. R., Ritter, A. M., Mendell, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+role+of+nerve+growth+factor+in+the+development+of+myelinated+nociceptors.">On the role of nerve growth factor in the development of myelinated nociceptors.</a> J Neurosci. 12, 1896-1905 (1992).</li><li>Greene, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+in+vitro+studies+on+the+nerve+growth+factor+(NGF)+requirement+of+neurons.+II.+Sensory+neurons.">Quantitative in vitro studies on the nerve growth factor (NGF) requirement of neurons. II. Sensory neurons.</a> Dev Biol. 58, 106-113 (1977).</li></ol>

No hay conflictos de interés declarado.

Este informe describe un método para aislar OPC murino para la diferenciación de las culturas OL-enriquecido o OL / DRGN co-culturas. Cuando se cultiva solo, el OPC se diferencian en MBP + LB ve, la producción de mielina, como las hojas membranosas. Cuando se añade a DRGN camas neuritas, LB envuelven las neuritas DRGN con MBP + membrana ve. Este modelo beneficia a la investigación de los complejos fundamentos que rigen OL mediada ensheathment axonal. Aunque de gran valor, el establecimiento de estas culturas es un reto técnico. En particular, los aspectos difíciles incluyen la digestión del tejido eficiente / disociación, el mantenimiento de la cultura equilibrada pH del medio, y los cambios DRGN los medios de comunicación. Es importante tener en cuenta que la longitud de la digestión, la cantidad de tejido que se digieren y la cantidad de trituración afecta el resultado de la eficacia y al final de la disociación del tejido. No es inusual para los investigadores con experiencia para obtener bajos rendimientos celulares de disociar los tejidos del sistema nervioso. Además, los OPC murino tienden a ser sensibles a los cambios en el pH del medio de cultivo, especialmente bajo condiciones alcalinas. El mantenimiento de los cultivos en el 8,5% de CO 2 tiene por objeto prevenir esto, ya que los OPC parecen tolerar mejor las condiciones más básicas ligeramente ácido. En cuanto a DRGNs alimentación, cambios en los medios debe realizarse rápidamente ya que no para desecar las neuronas, sin embargo, debe ser suave como para no interrumpir la cama neuritas en desarrollo. Cambios bruscos de medios puede desplazar la cama neuritas del sustrato, y el resultado probable de su disociación completa de la cubreobjetos. El mérito potencial de este modelo de sistema enormemente eclipsa su naturaleza exigente técnicamente. Una de las ventajas de este sistema es el uso de post-natal para la derivación de los ratones de cultivo celular, evitando la necesidad de sacrificar las hembras reproductoras de la cosecha del tejido embrionario. Otra ventaja es la falta de exigencia de los factores de crecimiento (FC) para la expansión de las OPC. Mezcla de culturas gliales ofrecen un entorno que apoye la difusión de las OPC, presumiblemente debido a la presencia de factores tróficos astrocitos derivados. Otros métodos, como la derivación a través de neuroesferas 7,8, se basan en las propiedades mitogénicas de FC como el factor de crecimiento de fibroblastos básico (bFGF), factor de crecimiento epidérmico (EGF) y el derivado de plaquetas factor de crecimiento (PDGF) para la expansión de OPC. Del mismo modo, el uso postnatal (P5-10) de los ratones DRGNs evita la necesidad de complementar los medios de cultivo con el factor de crecimiento nervioso (NGF), un factor neurotrófico necesarios para la supervivencia in vitro de embriones DRGNs 9, 10. Es de interés para evitar el uso de NGF, ya que influye negativamente en la capacidad de myelinating LB cuando se cultivan con DRGNs 4. Evitar el uso de GF-complementado los medios de comunicación también tiene beneficios económicos, ya que estos reactivos ser costoso cuando se utiliza a gran escala. Tal vez el beneficio más importante de este modelo de cultivo es su derivación de ratón sólo los tejidos, proporcionando así las oportunidades para obtener los OPC y DRGNs de la amplia variedad de líneas de ratones transgénicos. Esto permite el estudio de ambos DRGN y / o OPC propiedades específicas que rigen la mielinización. Esto será especialmente importante para dilucidar las interacciones receptor / ligando que regulan OL mediada por la mielinización de los axones. En total, esta técnica es de gran valor en cuanto a investigación en neurociencias, debido a sus aplicaciones a la comprensión de las señales moleculares que subyacen en la mielinización.

<html><table border="1"><tbody><tr><td> Nombre del producto </td><td> Empresa </td><td> Número de producto </td></tr><tr><td> Dulbecco Eagle modificado (DMEM) </td><td> Multicelda </td><td> 319 a 005-CL </td></tr><tr><td> Solución salina equilibrada de Hank (HBSS) </td><td> Invitrogen </td><td> 14170-112 </td></tr><tr><td> Mínimo de los medios de comunicación esencial (MEM) </td><td> GIBCO </td><td> 12360-038 </td></tr><tr><td> De suero fetal bovino (FBS) </td><td> GIBCO </td><td> 10091-148 </td></tr><tr><td> Penicilina-estreptomicina (penicilina / estreptomicina) </td><td> GIBCO </td><td> 15140-122 </td></tr><tr><td> Glutamax </td><td> Invitrogen </td><td> 35050-061 </td></tr><tr><td> Poli-L-lisina </td><td> Sigma-Aldrich </td><td> P2636 </td></tr><tr><td> Albúmina de suero bovino (BSA) </td><td> Sigma-Aldrich </td><td> A4503 </td></tr><tr><td> Humanos merosina proteico purificado (LN2) </td><td> Millipore </td><td> CC085 </td></tr><tr><td> El factor recombinante de rata neurotrófico ciliar (CNTF) </td><td> PeproTech </td><td> 450-50 </td></tr><tr><td> L-tiroxina </td><td> Biochemika </td><td> 89430 </td></tr><tr><td> Holo-transferrina </td><td> Sigma-Aldrich </td><td> T0665 </td></tr><tr><td> Suplemento B27 </td><td> GIBCO </td><td> 0080085-SA </td></tr><tr><td> Insulina bovina </td><td> Sigma-Aldrich </td><td> I6634 </td></tr><tr><td> 3,3 &#39;,5-triyodo-L-tironina </td><td> Sigma-Aldrich </td><td> I6634 </td></tr><tr><td> Progesterona </td><td> Sigma-Aldrich </td><td> P8783 </td></tr><tr><td> Putrescina </td><td> Sigma-Aldrich </td><td> P7505 </td></tr><tr><td> Selenita de sodio </td><td> Sigma-Aldrich </td><td> S5261 </td></tr><tr><td> 5-fluoro-2&#39;-desoxiuridina (FUDR) </td><td> Sigma-Aldrich </td><td> F0503 </td></tr><tr><td> La papaína solución </td><td> Worthington </td><td> LS003126 </td></tr><tr><td> DNaseI </td><td> ROCHE </td><td> 1010159001 </td></tr><tr><td> L-cisteína </td><td> Sigma-Aldrich </td><td> C7352 </td></tr><tr><td> 24 y los platos de cultivo de tejidos </td><td> Cellstar </td><td> 662-160 </td></tr><tr><td> Frascos de cultivo de tejidos-T25 con la tapa del respiradero </td><td> Corning </td><td> 430639 </td></tr><tr><td> 10 cm platos de cultivo de tejidos </td><td> Corning </td><td> 430167 </td></tr><tr><td> 10 cm de una caja de petri </td><td> Fisher Scientific </td><td> 0875713 </td></tr><tr><td> Colagenasa A </td><td> ROCHE </td><td> 103578 </td></tr><tr><td> CellTrics 50 micras filtro (opcional) </td><td> PARTEC </td><td> 04-004-2327 </td></tr><tr><td> Proteína básica de mielina (MBP) Anticuerpo </td><td> AbD Serotec </td><td> MCA409S </td></tr><tr><td> NG2 anticuerpos </td><td> Millipore </td><td> AB5320 </td></tr><tr><td> Mielina glicoproteína asociada (MAG) de anticuerpos </td><td> Millipore </td><td> MAB1567 </td></tr><tr><td> 2 &#39;, 3&#39;-cíclico de nucleótido 3&#39;-fosfodiesterasa (CNP) de anticuerpos </td><td> Covance </td><td> SMI-91R-100 </td></tr><tr><td> Actina pan Ab-5 de anticuerpos </td><td> Fitzgerald </td><td> 10R-A106AX </td></tr><tr><td> Neurofilamentos-200 (NF) de anticuerpos </td><td> Sigma-Aldrich </td><td> N4142 </td></tr><tr><td> Beta-3 de cadena tubulina (Tuj1) de anticuerpos </td><td> Millipore </td><td> MAB5544 </td></tr><tr><td> Alexa Fluor 488 cabra anti-conejo IgG (H + L) anticuerpo secundario </td><td> Invitrogen </td><td> A11008 </td></tr><tr><td> Alexa Fluor 555 cabra anti-ratón IgG (H + L) anticuerpo secundario </td><td> Invitrogen </td><td> A21422 </td></tr><tr><td> Alexa Fluor 647 cabra anti-rata (IgG) (H + L) anticuerpo secundario </td><td> Invitrogen </td><td> A21247 </td></tr><tr><td> Cabra anti-ratón IgG (H + L)-HRP anticuerpo secundario conjugado </td><td> BioRad </td><td> 170-6516 </td></tr><tr><td> De cabra anti-IgG de rata (H + L)-HRP anticuerpo secundario conjugado </td><td> Santa Cruz de Biotecnología </td><td> SC-2065 </td></tr><tr><td> 4 &#39;,6-diamidino-2-phenylindole (DAPI) </td><td> Sigma-Aldrich </td><td> D9542 </td></tr></tbody></table> Los medios de comunicación Recetas OL-100X Suplemento * <table border="1"><tbody><tr><td> Ingrediente </td><td> Cantidad a añadir </td></tr><tr><td> DMEM </td><td> 100 ml </td></tr><tr><td> BSA </td><td> 1,02 g </td></tr><tr><td> Progesterona </td><td> 0,6 mg </td></tr><tr><td> Putrescina </td><td> 161 mg </td></tr><tr><td> Selenita de sodio </td><td> 0,05 mg </td></tr><tr><td> 3,3 &#39;,5-triyodo-L-tironina </td><td> 4 mg </td></tr></tbody></table> * Almacene a -80 ° C en 250 alícuotas t &quot;&gt; los medios de comunicación OL <table border="1"><tbody><tr><td> Ingrediente </td><td> Cantidad a añadir </td></tr><tr><td> DMEM </td><td> 23,75 ml </td></tr><tr><td> OL-100X Suplemento </td><td> 250 l </td></tr><tr><td> Insulina bovina (de 1 mg / ml) </td><td> 125 l </td></tr><tr><td> Glutamax </td><td> 250 l </td></tr><tr><td> Holo-transferrina (de 33 mg / ml) </td><td> 37,5 l </td></tr><tr><td> B27 Suplemento </td><td> 500 l </td></tr><tr><td> FBS </td><td> 125 l </td></tr><tr><td> CNTF (de 50 ng / mL de archivo) </td><td> 25 l </td></tr></tbody></table> Mezcla de medios de cultivo gliales (compuesta en DMEM) <table border="1"><tbody><tr><td> Ingrediente </td><td> La concentración final </td></tr><tr><td> FBS </td><td> 10% </td></tr><tr><td> Penicilina / estreptomicina (0,33% del stock) </td><td> 33 unidades / ml de penicilina y estreptomicina 33 mg / ml </td></tr><tr><td> Glutamax </td><td> 1% </td></tr></tbody></table> DRGN los medios de comunicación (hecho en DMEM) <table border="1"><tbody><tr><td> Ingrediente </td><td> La concentración final </td></tr><tr><td> FBS </td><td> 10% </td></tr><tr><td> Penicilina / estreptomicina (1% del stock) </td><td> 100 unidades / ml de penicilina y estreptomicina 100 mg / ml </td></tr></tbody></table> Recetas de la digestión de solución: OPC papaína solución (compuesto en el MEM) <table border="1"><tbody><tr><td> Ingrediente </td><td> La concentración final </td></tr><tr><td> La papaína solución </td><td> 1,54 mg / mL </td></tr><tr><td> L-cisteína </td><td> 360 mg / ml </td></tr><tr><td> DNaseI </td><td> 60 mg / ml </td></tr></tbody></table> DRG papaína solución (hecho en HBSS) <table border="1"><tbody><tr><td> Ingrediente </td><td> La concentración final </td></tr><tr><td> Papaína </td><td> 1,54 mg / mL </td></tr><tr><td> L-cisteína </td><td> 360 mg / ml </td></tr></tbody></table> DRG colagenasa Una solución (hecho en HBSS) <table border="1"><tbody><tr><td> Ingrediente </td><td> La concentración final </td></tr><tr><td> Colagenasa A </td><td> 4 mg / mL </td></tr></tbody></table>

derivation of enriched oligodendrocyte cultures and oligodendrocyte/neuron myelinating co-cultures from post-natal murine tissues

Identificar los mecanismos moleculares que subyacen al desarrollo OL no sólo es fundamental para ampliar nuestro conocimiento de la biología OL, pero también tiene implicaciones para la comprensión de la patogénesis de las enfermedades desmielinizantes como la esclerosis múltiple (EM). Desarrollo celular es comúnmente estudiadas con modelos de cultivos celulares primarios. Cultivo de células primarias facilita la evaluación de un tipo de célula, proporcionando un ambiente controlado y libre de las variables externas que están presentes en vivo. Mientras que las culturas OL derivadas de ratas han proporcionado una gran cantidad de conocimiento de la biología OL, esfuerzos similares en el establecimiento de cultivos de OL de los ratones se ha encontrado con obstáculos importantes. El desarrollo de métodos de cultivo primario LB murino es imprescindible a fin de tomar ventaja de las líneas de ratones transgénicos disponibles. Múltiples métodos para la extracción de OPC a partir de tejido de roedores se han descrito, desde la derivación neuroesfera, purificación y adhesión diferencial inmunopurificación 1-3. Mientras que muchos métodos ofrecen el éxito de la mayoría, requieren tiempos de amplia cultura y / o equipos costosos / reactivos. Para evitar esto, purificación de OPC a partir de tejido murino con una adaptación del método descrito originalmente por McCarthy y de Vellis 2 es preferido. Este método consiste en separar físicamente los OPC a partir de un cultivo mixto gliales derivadas de cortezas de roedores recién nacidos. El resultado es una población purificada OPC que pueden diferenciarse en una cultura OL-enriquecido. Este enfoque es atractivo debido a su tiempo de cultivo relativamente corto y el requisito innecesario para los factores de crecimiento o anticuerpos immunopanning. Mientras explora los mecanismos de desarrollo OL en una cultura purificada es informativo, no proporciona el entorno más fisiológicamente relevantes para la evaluación de la formación de la vaina de mielina. Co-cultivo LB con las neuronas que ayudan a comprender la las bases moleculares que regulan la OL mediada por la mielinización de los axones. Para muchos OL / co-cultivo de neuronas estudios, las neuronas ganglionares de la raíz dorsal (DRGNs) han demostrado ser el tipo de neurona de la elección. Son ideales para el co-cultivo con LB, debido a su facilidad de extracción, cantidad mínima de células contaminantes, y la formación de neuritas camas densas. Mientras que los estudios con ratas / ratones myelinating xenocultures han sido publicados por 4-6, un método para la obtención de tales OL / DRGN myelinating co-cultivos de post-natal tejido murino no se ha descrito. Aquí se presentan los métodos detallados sobre la manera de producir eficazmente estos cultivos, junto con ejemplos de los resultados esperados. Estos métodos son útiles para abordar cuestiones relacionadas con el desarrollo OL / myelinating función, y son herramientas útiles en el campo de la neurociencia.

Watch this Scientific Journal Video about Derivation of Enriched Oligodendrocyte Cultures and Oligodendrocyte/Neuron Myelinating Co-cultures from Post-natal Murine Tissues at JoVE.com

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

En este artículo se describen los métodos para obtener poblaciones enriquecidas de células precursoras de oligodendrocitos murinas (OPC) en cultivo primario, que se diferencian para producir oligodendrocitos maduros (MCO). Además, este informe se describen las técnicas para producir murino myelinating co-cultivos de siembra de los OPC del ratón sobre un lecho de neuritas de neuronas de ratón ganglio de la raíz dorsal (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 ...

derivation-enriched-oligodendrocyte-cultures-oligodendrocyteneuron

Research

JoVE Journal

Neuroscience

36.0K vistas.  Ottawa Hospital Research Institute. En este artículo se describen los métodos para obtener poblaciones enriquecidas de células precursoras de oligodendrocitos murinas (OPC) en cultivo primario, que se diferencian para producir oligodendrocitos maduros (MCO). Además, este informe se describen las técnicas para producir murino myelinating co-cultivos de siembra de los OPC del ratón sobre un lecho de neuritas de neuronas de ratón ganglio de la raíz dorsal (DRGNs).

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

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

In this study, we outline a standardized protocol for the successful cryopreservation and thawing of cortical brain tissue blocks to generate highly enriched neuronal cultures. For this protocol the freezing medium used is 10% dimethyl sulfoxide (DMSO) diluted in Hank's Buffered Salt Solution (HBSS). Blocks of cortical tissue are transferred to cryovials containing the freezing medium and slowly frozen at -1&deg;C/min in a rate-controlled freezing container. Post-thaw processing and dissociation of frozen tissue blocks consistently produced neuronal-enriched cultures which exhibited rapid neuritic growth during the first 5 days in culture and significant expansion of the neuronal network within 10 days. Immunocytochemical staining with the astrocytic marker glial fibrillary acidic protein (GFAP) and the neuronal marker beta-tubulin class III, revealed high numbers of neurons and astrocytes in the cultures. Generation of neural precursor cell cultures after tissue block dissociation resulted in rapidly expanding neurospheres, which produced large numbers of neurons and astrocytes under differentiating conditions. This simple cryopreservation protocol allows for the rapid, efficient, and inexpensive preservation of cortical brain tissue blocks, which grants increased flexibility for later generation of neuronal, astrocyte, and neuronal precursor cell cultures.

Here, we describe a method for efficient cryopreservation and thawing of cortical brain tissue blocks to generate highly enriched neuronal cultures. This simple protocol provides flexibility for later generation of neuronal, astrocyte, and neuronal precursor cell cultures.

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 ...

Investigación

Neurociencias

Derivation of Glial Restricted Precursors from E13 mice

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

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

Derivación de los precursores gliales restringidas de los ratones E13

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

Progenitor-derived Oligodendrocyte Culture System from Human Fetal Brain

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

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

Progenitor derivado de sistema de cultivo de oligodendrocitos de cerebro fetal humano

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

Simultaneous Pre- and Post-synaptic Electrophysiological Recording from Xenopus Nerve-muscle Co-cultures

Much information about the coupling of presynaptic ionic currents with the release of neurotransmitter has been obtained from invertebrate preparations, most notably the squid giant synapse1. However, except for the preparation described here, few vertebrate preparations exist in which it is possible to make simultaneous measurements of neurotransmitter release and presynaptic ionic currents. Embryonic Xenopus motoneurons and muscle cells can be grown together in simple culture medium at room temperature; they will form functional synapses within twelve to twenty-four hours, and can be used to study nerve and muscle cell development and synaptic interactions for several days (until overgrowth occurs). Some advantages of these co-cultures over other vertebrate preparations include the simplicity of preparation, the ability to maintain the cultures and work at room temperature, and the ready accessibility of the synapses formed2-4. The preparation has been used widely to study the biophysical properties of presynaptic ion channels and the regulation of transmitter release5-8. In addition, the preparation has lent itself to other uses including the study of neurite outgrowth and synaptogenesis9-12, molecular mechanisms of neurotransmitter release13-15, the role of diffusible messengers in neuromodulation16,17, and in vitro synaptic plasticity18-19.

Simultaneous Pre- and Post-synaptic Electrophysiological Recording from Xenopus Nerve-muscle Co-cultures

This video demonstrates the procedures used to grow primary cultures of embryonic Xenopus nerve and muscle cells and the usefulness of this preparation for making simultaneous pre- and post-synaptic patch clamp recordings.

Simultánea pre-y post-sináptica de registro electrofisiológico<em&gt; Xenopus</em&gt; Músculo Nervio-Co-culturas

Much information about the coupling of  presynaptic ionic currents with the release of neurotransmitter has been obtained  from invertebrate preparations, ...

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 ...

Organotypic Slice Cultures to Study Oligodendrocyte Dynamics and Myelination

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During embryonic and postnatal development they actively proliferate and generate myelinating oligodendrocytes. These cells have commonly been studied in primary dissociated cultures, neuron cocultures, and in fixed tissue. Using newly available transgenic mouse lines slice culture systems can be used to investigate proliferation and differentiation of oligodendrocyte lineage cells in both gray and white matter regions of the forebrain and cerebellum. Slice cultures are prepared from early postnatal mice and are kept in culture for up to 1 month. These slices can be imaged multiple times over the culture period to investigate cellular behavior and interactions. This method allows visualization of NG2 cell division and the steps leading to oligodendrocyte differentiation while enabling detailed analysis of region-dependent NG2 cell and oligodendrocyte functional heterogeneity. This is a powerful technique that can be used to investigate the intrinsic and extrinsic signals influencing these cells over time in a cellular environment that closely resembles that found in vivo.

A technique to study NG2 cells and oligodendrocytes using a slice culture system of the forebrain and cerebellum is described. This method allows examination of the dynamics of proliferation and differentiation of cells within the oligodendrocyte lineage where the extracellular environment can be easily manipulated while maintaining tissue cytoarchitecture.

Culturas Organotípicos Slice para Estudiar Oligodendrocyte Dinámica y mielinización

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During ...

Investigating Functional Regeneration in Organotypic Spinal Cord Co-cultures Grown on Multi-electrode Arrays

Adult higher vertebrates have a limited potential to recover from spinal cord injury. Recently, evidence emerged that propriospinal connections are a promising target for intervention to improve functional regeneration. So far, no in vitro model exists that grants the possibility to examine functional recovery of propriospinal fibers. Therefore, a representative model that is based on two organotypic spinal cord sections of embryonic rat, cultured next to each other on multi-electrode arrays (MEAs) was developed. These slices grow and, within a few days in vitro, fuse along the sides facing each other. The design of the used MEAs permits the performance of lesions with a scalpel blade through this fusion site without inflicting damage on the MEAs. The slices show spontaneous activity, usually organized in network activity bursts, and spatial and temporal activity parameters such as the location of burst origins, speed and direction of their propagation and latencies between bursts can be characterized. Using these features, it is also possible to assess functional connection of the slices by calculating the amount of synchronized bursts between the two sides. Furthermore, the slices can be morphologically analyzed by performing immunohistochemical stainings after the recordings. 
Several advantages of the used techniques are combined in this model: the slices largely preserve the original tissue architecture with intact local synaptic circuitry, the tissue is easily and repeatedly accessible and neuronal activity can be detected simultaneously and non-invasively in a large number of spots at high temporal resolution. These features allow the investigation of functional regeneration of intraspinal connections in isolation in vitro in a sophisticated and efficient way.

Here we present a protocol that is based on spinal cord slices cultured on multi-electrode arrays to study functional regeneration of propriospinal connections in vitro.

La investigación de la regeneración funcional en organotípicos médula espinal Co-culturas cultivadas en Arrays Multi-electrodos

Adult higher vertebrates have a limited potential to recover from spinal cord injury. Recently, evidence emerged that propriospinal connections are a ...

Derivation of Leptomeninges Explant Cultures from Postmortem Human Brain Donors

Even though great progress has been made in the clinical characterization of Parkinson&#39;s disease, several studies report that the diagnosis of Parkinson&#39;s disease is not pathologically confirmed in up to 25% of clinically diagnosed Parkinson&#39;s disease. Therefore, tissue collected from clinically diagnosed patients with idiopathic Parkinson&#39;s disease can have a high rate of misdiagnosis; hence in vitro studies from such tissues to study Parkinson&#39;s disease as a preclinical model can become futile.
By collecting postmortem human leptomeninges with a confirmed neuropathological diagnosis of Parkinson&#39;s disease and characterized by nigrostriatal cell loss and intracellular protein inclusions called Lewy bodies, one can be certain that clinically observed parkinsonism is not caused by another underlying disease process (e.g. tumor, arteriosclerosis).
This protocol presents the dissection and preparation of postmortem human leptomeninges for derivation of a meningeal fibroblast culture. This procedure is robust and has a high success rate. The challenge of the culture is sterility as the brain procurement is generally not performed under sterile conditions. Therefore, it is important to supplement the culture media with a cocktail of penicillin, streptomycin, and amphotericin B.
The derivation of meningeal fibroblasts from autopsy-confirmed cases with Parkinson&#39;s disease provides the foundation for in vitro modeling of Parkinson&#39;s disease. Meningeal fibroblasts appear 3-9 days after sample preparation and about 20-30 million cells can be cryopreserved in 6-8 weeks. The meningeal fibroblast culture is homogenous and the cells express fibronectin, a commonly used marker to identify meninges.

The leptomeninges explant culture protocol from human postmortem brain is a technically robust and simple way to derive fibronectin-positive meningeal fibroblasts within 6-8 weeks and cryopreserve approximately 20-30 million cells.

Derivación de las leptomeninges de explantes de culturas postmortem Humanos Los donantes de cerebro

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

Neuron-Macrophage Co-cultures to Activate Macrophages Secreting Molecular Factors with Neurite Outgrowth Activity

There is strong evidence that macrophages can participate in the regeneration or repair of injured nervous system. Here, we describe a protocol in which macrophages are induced to produce conditioned medium (CM) that promotes neurite outgrowth. Adult dorsal root ganglion (DRG) neurons are acutely dissociated and plated. After the neurons are stably attached, peritoneal macrophages are co-cultured on a cell culture insert overlaid on the same well. Dibutyryl cyclic AMP (db-cAMP) is applied to the co-cultures for 24 h, after which the cell culture insert containing the macrophages is moved to another well to collect CM for 72 h. The CM from the co-cultures treated with db-cAMP, when applied to a separate adult DRG neuron culture, exhibits robust neurite outgrowth activity. The CM obtained from the db-cAMP-treated cultures consisting of single cell type alone, either DRG neuron or peritoneal macrophage, did not exhibit neurite outgrowth activity. This indicates that the interaction between neurons and macrophages is indispensable for the activation of macrophages secreting molecular factors with neurite outgrowth activity into CM. Thus, our co-culture paradigm will also be useful to study intercellular signaling in the neuron-macrophage interaction to stimulate the macrophages to be endowed with a pro-regenerative phenotype.

The current protocol presents experimental procedures to stimulate cultured macrophages to be endowed with capacity to release molecular factors that promote neurite outgrowth. Treatment of cAMP to the neuron-macrophage co-cultures induces the macrophages to produce conditioned medium that possesses strong neurite outgrowth activity.

Co-cultivos de neurona y macrófagos para activar macrófagos secretando factores moleculares con actividad Neurite consecuencia

There is strong evidence that macrophages can participate in the regeneration or repair of injured nervous system. Here, we describe a protocol in which ...

Preparation and Immunostaining of Myelinating Organotypic Cerebellar Slice Cultures

In the nervous system, myelin is a complex membrane structure generated by myelinating glial cells, which ensheathes axons and facilitates fast electrical conduction. Myelin alteration has been shown to occur in various neurological diseases, where it is associated with functional deficits. Here, we provide a detailed description of an ex vivo model consisting of mouse organotypic cerebellar slices, which can be maintained in culture for several weeks and further be labeled to visualize myelin.

Here we present a method to prepare organotypic slice cultures from mouse cerebellum and myelin sheath staining by immunohistochemistry suitable for investigating mechanisms of myelination and remyelination in the central nervous system.

Preparación y el Immunostaining de Myelinating Organotypic rebanada cerebelosa culturas

In the nervous system, myelin is a complex membrane structure generated by myelinating glial cells, which ensheathes axons and facilitates fast electrical ...