Questo progetto è stato finanziato da una sovvenzione della Multiple Sclerosis Society of Canada a RKRWO è un destinatario di una borsa di studio dalla Multiple Sclerosis Society of Canada. SDR è un destinatario di Post-Doctoral Fellowships dalla Multiple Sclerosis Society of Canada e Canadian Institutes of Health Research.

<ol>
	<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>

Nessun conflitto di interessi dichiarati.

Questo rapporto descrive un metodo per isolare OPC murini per la differenziazione nelle culture OL-arricchito o OL / DRGN co-culture. Quando colta da solo, il OPC differenziarsi in MBP + OL ve, la produzione di mielina come fogli membranosa. Quando aggiunto a DRGN letti neuriti, OL avvolgono i neuriti DRGN con MBP + membrana ve. Questo modello benefici l&#39;indagine dei fondamenti complessi che regolano OL-mediata ensheathment assonale. Anche se di grande valore, l&#39;istituzione di tali culture è tecnicamente impegnativo. In particolare, gli aspetti impegnativi includono la digestione efficiente dei tessuti / dissociazione, il mantenimento del pH equilibrato terreni di coltura, e DRGN cambiamenti dei media. E &#39;importante considerare che la lunghezza della digestione, la quantità di tessuto essere digerito e la quantità di triturazione influenza il risultato efficacia e la fine della dissociazione dei tessuti. Non è insolito per i ricercatori esperti di ottenere basse rese cellulari dissociato da tessuti del sistema nervoso. Inoltre, OPC murino tendono ad essere sensibili alle variazioni del pH dei terreni di coltura, in particolare in ambiente alcalino. Il mantenimento di culture a 8,5% CO 2 mira a prevenire questo, dal momento che OPC sembrano tollerare condizioni migliori sul leggermente acido base. Per quanto riguarda DRGNs alimentazione, i cambiamenti dei media deve essere eseguita velocemente per non essiccare i neuroni, tuttavia, deve essere gentile come non perturbare il letto neuriti via di sviluppo. Bruschi cambiamenti dei media può rimuovere il letto neuriti dal substrato, e il risultato probabilmente nella sua completa dissociazione dalla coprioggetto. Il merito potenzialità di questo sistema modello oscura notevolmente la sua natura tecnicamente impegnativo. Un vantaggio di questo sistema è l&#39;uso di post-natale topi per la derivazione di coltura cellulare di eludere la necessità di sacrificare femmine riproduttrici per la raccolta dei tessuti embrionali. Un altro vantaggio è la mancanza di requisiti per i fattori di crescita (GF) per l&#39;espansione della OPC. Misti culture gliali forniscono un ambiente che supporta la propagazione della OPC, presumibilmente a causa della presenza di astrociti derivati ​​da fattori trofici. Altri metodi, come la derivazione via neurosfere 7,8, si basano sulle proprietà di GF mitogenica come fattore di crescita dei fibroblasti (bFGF), fattore di crescita epidermico (EGF) e delle piastrine fattore di crescita derivato dalle piastrine (PDGF) per l&#39;espansione OPC. Allo stesso modo, usando postnatale (P5-10) per topi DRGNs evita la necessità di integrare i terreni di coltura con fattore di crescita nervosa (NGF), un fattore neurotrofico necessario per la sopravvivenza in vitro di embrioni DRGNs 9, 10. E &#39;di interesse per evitare l&#39;uso di NGF poichè influenza negativamente la capacità di mielinizzanti OL quando coltivate con DRGNs 4. Evitare l&#39;uso di GF-integrato dei media ha anche benefici economici, in quanto questi reagenti diventare costoso se utilizzato su larga scala. Forse il vantaggio più importante di questo modello la cultura è la sua derivazione da mouse solo tessuti, fornendo così l&#39;opportunità di derivare sia OPC e DRGNs dalla grande varietà di linee di topi transgenici. Questo permette per lo studio di entrambi e DRGN / o OPC proprietà specifiche che regolano la mielinizzazione. Questo sarà particolarmente importante per chiarire il recettore / ligando che regolano le interazioni OL-mediata mielinizzazione degli assoni. In tutto, questa tecnica è di grande valore per quanto riguarda la ricerca delle neuroscienze per le sue applicazioni verso la comprensione i segnali molecolari alla base della mielinizzazione.

<html><table border="1"><tbody><tr><td> Nome prodotto </td><td> Azienda </td><td> Codice </td></tr><tr><td> Dulbecco modificato (DMEM) </td><td> Multicell </td><td> 319-005-CL </td></tr><tr><td> Sale di Hank soluzione equilibrata (HBSS) </td><td> Invitrogen </td><td> 14170-112 </td></tr><tr><td> Minimo Essential Media (MEM) </td><td> GIBCO </td><td> 12360-038 </td></tr><tr><td> Siero fetale bovino (FBS) </td><td> GIBCO </td><td> 10091-148 </td></tr><tr><td> Penicillina-streptomicina (Pen / Strep) </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> Sieroalbumina bovina (BSA) </td><td> Sigma-Aldrich </td><td> A4503 </td></tr><tr><td> Umano merosina purificata proteine ​​(LN2) </td><td> Millipore </td><td> CC085 </td></tr><tr><td> Ricombinante di ratto fattore neurotrofico ciliare (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> B27 supplemento </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-Triiodo-L-thyronine </td><td> Sigma-Aldrich </td><td> I6634 </td></tr><tr><td> Progesterone </td><td> Sigma-Aldrich </td><td> P8783 </td></tr><tr><td> Putrescina </td><td> Sigma-Aldrich </td><td> P7505 </td></tr><tr><td> Sodio Selenite </td><td> Sigma-Aldrich </td><td> S5261 </td></tr><tr><td> 5-Fluoro-2&#39;-deossiuridina (FuDR) </td><td> Sigma-Aldrich </td><td> F0503 </td></tr><tr><td> Papaina soluzione </td><td> Worthington </td><td> LS003126 </td></tr><tr><td> DNaseI </td><td> ROCHE </td><td> 1010159001 </td></tr><tr><td> L-cisteina </td><td> Sigma-Aldrich </td><td> C7352 </td></tr><tr><td> 24-bene di coltura tissutale piatti </td><td> Cellstar </td><td> 662-160 </td></tr><tr><td> T25-tessuto cultura fiaschi con tappo di sfiato </td><td> Corning </td><td> 430639 </td></tr><tr><td> 10 centimetri di tessuto cultura piatti </td><td> Corning </td><td> 430167 </td></tr><tr><td> 10 centimetri scatola di Petri </td><td> Fisher Scientific </td><td> 0875713 </td></tr><tr><td> Collagenasi A </td><td> ROCHE </td><td> 103578 </td></tr><tr><td> CellTrics 50 micron filtro (opzionale) </td><td> PARTEC </td><td> 04-004-2327 </td></tr><tr><td> Proteina basica della mielina (MBP) Anticorpo </td><td> ABD Serotec </td><td> MCA409S </td></tr><tr><td> NG2 anticorpi </td><td> Millipore </td><td> AB5320 </td></tr><tr><td> Glicoproteina associata alla mielina (MAG) anticorpi </td><td> Millipore </td><td> MAB1567 </td></tr><tr><td> 2 &#39;, 3&#39;-ciclico-nucleotide 3&#39;-fosfodiesterasi (CNP) anticorpi </td><td> Covance </td><td> SMI-91R-100 </td></tr><tr><td> Actina pan Ab-5 anticorpi </td><td> Fitzgerald </td><td> 10R-A106AX </td></tr><tr><td> Neurofilamenti-200 (NF) anticorpi </td><td> Sigma-Aldrich </td><td> N4142 </td></tr><tr><td> Tubulina beta-3 a catena (Tuj1) anticorpi </td><td> Millipore </td><td> MAB5544 </td></tr><tr><td> Alexa Fluor 488 di capra anti-IgG di coniglio (H + L) anticorpo secondario </td><td> Invitrogen </td><td> A11008 </td></tr><tr><td> Alexa Fluor 555 capra anti-topo IgG (H + L) anticorpo secondario </td><td> Invitrogen </td><td> A21422 </td></tr><tr><td> Alexa Fluor 647 di capra anti-topo (IgG) (H + L) anticorpo secondario </td><td> Invitrogen </td><td> A21247 </td></tr><tr><td> Capra anti-topo IgG (H + L)-HRP coniugato secondario </td><td> BioRad </td><td> 170-6516 </td></tr><tr><td> Capra anti-topo IgG (H + L)-HRP coniugato secondario </td><td> Santa Cruz Biotechnology </td><td> SC-2065 </td></tr><tr><td> 4 &#39;,6-diamidino-2-fenilindolo (DAPI) </td><td> Sigma-Aldrich </td><td> D9542 </td></tr></tbody></table> Ricette dei media OL-100X Supplemento * <table border="1"><tbody><tr><td> Ingrediente </td><td> Importo da aggiungere </td></tr><tr><td> DMEM </td><td> 100 ml </td></tr><tr><td> BSA </td><td> 1,02 g </td></tr><tr><td> Progesterone </td><td> 0,6 mg </td></tr><tr><td> Putrescina </td><td> 161 mg </td></tr><tr><td> Sodio Selenite </td><td> 0,05 mg </td></tr><tr><td> 3,3 &#39;,5-Triiodo-L-thyronine </td><td> 4 mg </td></tr></tbody></table> * Conservare a -80 ° C in aliquote da 250 microlitri t &quot;&gt; media OL <table border="1"><tbody><tr><td> Ingrediente </td><td> Importo da aggiungere </td></tr><tr><td> DMEM </td><td> 23,75 mL </td></tr><tr><td> OL-100X Supplemento </td><td> 250 microlitri </td></tr><tr><td> Insulina bovina (da 1 mg / ml stock) </td><td> 125 microlitri </td></tr><tr><td> GlutaMAX </td><td> 250 microlitri </td></tr><tr><td> Holo-transferrina (da 33 mg / ml stock) </td><td> 37,5 microlitri </td></tr><tr><td> B27 Supplemento </td><td> 500 microlitri </td></tr><tr><td> FBS </td><td> 125 microlitri </td></tr><tr><td> CNTF (da 50 ng / mL stock) </td><td> 25 microlitri </td></tr></tbody></table> Tecnica mista cultura gliali (costituita in DMEM) <table border="1"><tbody><tr><td> Ingrediente </td><td> Concentrazione finale </td></tr><tr><td> FBS </td><td> 10% </td></tr><tr><td> Pen / strep (0,33% dal magazzino) </td><td> 33 unità / ml di penicillina e 33 mg / ml di streptomicina </td></tr><tr><td> GlutaMAX </td><td> 1% </td></tr></tbody></table> DRGN media (composta in DMEM) <table border="1"><tbody><tr><td> Ingrediente </td><td> Concentrazione finale </td></tr><tr><td> FBS </td><td> 10% </td></tr><tr><td> Pen / Strep (1% dal magazzino) </td><td> 100 unità / ml di penicillina e 100 mg / ml di streptomicina </td></tr></tbody></table> Digestione Soluzione Ricette: OPC papaina soluzione (costituita nel MEM) <table border="1"><tbody><tr><td> Ingrediente </td><td> Concentrazione finale </td></tr><tr><td> Papaina soluzione </td><td> 1,54 mg / mL </td></tr><tr><td> L-cisteina </td><td> 360 mg / ml </td></tr><tr><td> DNaseI </td><td> 60 mg / ml </td></tr></tbody></table> DRG papaina soluzione (costituita in HBSS) <table border="1"><tbody><tr><td> Ingrediente </td><td> Concentrazione finale </td></tr><tr><td> Papaina </td><td> 1,54 mg / mL </td></tr><tr><td> L-cisteina </td><td> 360 mg / ml </td></tr></tbody></table> DRG Collagenasi Una soluzione (costituita in HBSS) <table border="1"><tbody><tr><td> Ingrediente </td><td> Concentrazione finale </td></tr><tr><td> Collagenasi 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

Identificare i meccanismi molecolari alla base dello sviluppo OL non solo è fondamentale per approfondire la nostra conoscenza della biologia OL, ma ha anche implicazioni per la comprensione della patogenesi delle malattie demielinizzanti come la sclerosi multipla (SM). Sviluppo cellulare è comunemente studiate con modelli di colture cellulari primarie. Colture cellulari primarie facilita la valutazione di un dato tipo di cellula, fornendo un ambiente controllato, privo di variabili estranee che sono presenti in vivo. Mentre le culture OL derivati ​​da ratti hanno fornito una grande quantità di conoscenza nella biologia OL, iniziative analoghe a stabilire culture OL dai topi è stata accolta con grandi ostacoli. Lo sviluppo di metodi di cultura OL murino primario è indispensabile al fine di sfruttare le linee disponibili nel topo transgenico. Diversi metodi per l&#39;estrazione di OPC da tessuto roditori sono state descritte, che vanno dalla derivazione neurosfere, adesione purificazione differenziale e immunopurification 1-3. Mentre molti metodi offrire il successo, la maggior parte richiedono tempi di cultura vasta e / o attrezzature costose / reagenti. Per aggirare questo, purificando OPC da tessuti murini con un adattamento del metodo originariamente descritto da McCarthy &amp; de Vellis 2 è preferito. Questo metodo comporta fisicamente OPC separazione da una coltura mista gliali derivati ​​da cortecce roditori neonatale. Il risultato è una popolazione purificata OPC che possono essere differenziati in un OL-arricchito la cultura. Questo approccio è attraente per la sua cultura tempo relativamente breve e il requisito necessario per fattori di crescita o anticorpi immunopanning. Durante l&#39;esplorazione dei meccanismi di sviluppo OL in una cultura purificato è informativa, non fornisce l&#39;ambiente più fisiologicamente rilevanti per valutare la formazione di guaina mielinica. Co-coltura OLS con i neuroni darebbe comprensione delle basi molecolari che regolano OL-mediata mielinizzazione degli assoni. Per molti OL / neuroni co-coltura studi, i neuroni dei gangli della radice dorsale (DRGNs) hanno dimostrato di essere il tipo neurone di scelta. Sono ideali per co-coltura con OLS per la loro facilità di estrazione, quantità minima di cellule contaminanti, e la formazione di posti letto dei neuriti denso. Mentre gli studi utilizzando ratto / topo mielinizzanti xenocultures sono stati pubblicati 4-6, un metodo per la derivazione di tale OL / DRGN mielinizzanti co-culture post-natale dei tessuti murino non è stata descritta. Qui vi presentiamo metodi dettagliate su come produrre in modo efficace tali colture, insieme a esempi di risultati attesi. Questi metodi sono utili per affrontare questioni rilevanti per lo sviluppo OL / funzione mielinizzanti, e sono strumenti utili nel campo delle neuroscienze.

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

Questo articolo descrive i metodi per ricavare le popolazioni murine arricchito di cellule precursori degli oligodendrociti (OPC) in colture primarie, che differenziano per produrre oligodendrociti maturi (OLS). Inoltre, questo rapporto descrive le tecniche per produrre murino mielinizzanti co-colture da semina OPC del mouse su un letto neurite dei neuroni dei gangli spinali del mouse (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 ...

derivation-enriched-oligodendrocyte-cultures-oligodendrocyteneuron

Research

JoVE Journal

Neuroscience

36.0K Views.  Ottawa Hospital Research Institute. Questo articolo descrive i metodi per ricavare le popolazioni murine arricchito di cellule precursori degli oligodendrociti (OPC) in colture primarie, che differenziano per produrre oligodendrociti maturi (OLS). Inoltre, questo rapporto descrive le tecniche per produrre murino mielinizzanti co-colture da semina OPC del mouse su un letto neurite dei neuroni dei gangli spinali del mouse (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.

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 ...

Ricerca

Neuroscienze

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.

Derivazione di precursori gliali limitati dalla E13 topi

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.

Progenitore di derivazione Sistema Cultura Oligodendrocyte dal cervello umano fetale

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.

Simultanea di pre-e post-sinaptica registrazione elettrofisiologica da<em&gt; Xenopus</em&gt; Nervo-muscolo Co-colture

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.

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 ...

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.

Culture Organotipica Slice per studiare Oligodendrocyte Dynamics e mielinizzazione

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.

Indagare rigenerazione funzionale in organotipiche midollo spinale Co-culture Grown in array multi-elettrodo

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.

Derivazione delle Culture leptomeningi Explant da Postmortem cervello umano donatori

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.

Neurone-macrofago co-colture per attivare i macrofagi che secernono fattori molecolari con attività di neuriti

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.

Preparazione e Immunostaining di su colture organotipiche Myelinating fetta cerebellare

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