Ringraziamo Jonna Ellis per l&#39;assistenza editoriale. Il progetto descritto è stato sostenuto da Number Award R01MH079751 (PI: F. Peruzzi), dal National Institute of Mental Health. Il contenuto è di esclusiva responsabilità degli autori e non necessariamente rappresentano le opinioni ufficiali dell&#39;Istituto Nazionale di Salute Mentale o il National Institutes of Health.

1. Poly-D-lisina (PDL): Preparazione <ol><li> Aggiungere 5 ml di soluzione sterile ddh 2 O a 5 mg di PDL per ottenere una soluzione stock di 1 mg / ml. </li><li> Mescolare soluzione madre pipettando più volte. </li><li> Utilizzare immediatamente o memorizzare Poly-D-lisina soluzione a 2-8 ° C. </li></ol> 2. Poly-D-lisina (PDL): rivestimento in plastica Piatti di coltura cellulare <ol><li> Diluire la soluzione stock con PDL sterile ddh 2 O alla concentrazione finale di 10 mcg / ml. </li><li> Pipettare soluzione sufficiente in piastre da 60 mm a coprire superficie cultura (3 ml per piastre da 60 mm). </li><li> Oscillare delicatamente per assicurare anche il rivestimento della superficie di coltura. </li><li> Incubare le piastre rivestite a temperatura ambiente (RT) per una notte. </li><li> Il giorno successivo, di solito il giorno di dissezione, rimuovere il Poly-D-lisina soluzione mediante aspirazione e lavare brevemente con 3 ml di sterile ddh 2 O. Ripetere questo passaggio. Dopo il secondo lavaggio, rimuovere completamente l&#39;acqua per aspirazione. </li><li>Le piastre possono essere conservati a 4 ° C fino a tre settimane. </li></ol> 3. Poly-D-lisina (PDL) e laminina: Preparazione e rivestimento di vetro a due camere Slides <ol><li> Soluzioni madri Mix PDL (1 mg / ml) e laminina (1 mg / ml) in sterile ddh 2 O alla concentrazione finale di 10 e 5 pg / ml, rispettivamente. </li><li> Pipettare soluzione sufficiente in pozzetti di un vetro a due camere di scorrimento per coprire la superficie coltura (1 ml per ogni pozzetto di un bicchiere 2 ben due camera di scorrimento). </li><li> Oscillare delicatamente per assicurare anche il rivestimento della superficie di coltura. </li><li> Incubare le piastre rivestite a temperatura ambiente tutta la notte. </li><li> Il giorno successivo, rimuovere il Poly-D-lisina-laminina soluzione di rivestimento mediante aspirazione e lavare brevemente due volte con 1 ml di soluzione sterile ddh 2 O. Dopo il secondo lavaggio, rimuovere completamente l&#39;acqua per aspirazione. </li><li> Camera diapositive possono essere conservati a 4 ° C per un massimo di tre settimane. </li></ol> Nota: qualsiasi diapositiva camera di vetro possono essere rivestiti following questo protocollo. Spesso utilizzare i due vetrini camera perché ogni vetrino fornisce il controllo test sperimentali impostazione (ad esempio non trattati rispetto trattati, non trasfettate rispetto trasfettate). 4. Dissezione neuronale e Cultura <ol><li> Riscaldare i seguenti reagenti in un bagno di acqua 37 ° C: <ul type="disc"><li> Triplo espresso sulla sua natura originale flacone da 100 ml. </li><li> Neurobasal/B27 terreno completo (vedi tabella I). Il volume riscaldato dipende dal numero di piatti per essere placcato (ad esempio 30 ml per dieci 60 piatti mm placcato). </li></ul></li><li> Aggiungere 3 ml di soluzione di Hibernate freddo E a quattro piatti da 60 mm e 13 ml di coltura in un tubo di 15 ml chiarezza BD polipropilene ad alta Falcon conica. </li><li> Aggiungere 25-30 ml di mezzo di dissezione a freddo (vedi tabella II, il dottor Meucci Olimpia, comunicazione personale) a ciascuno dei tre piatti da 100 mm cultura. Queste lastre, contenenti un grande volume di terreno, saranno utilizzati per lavare gli embrioni immediatamente dopo la lorola rimozione dei sacchi amniotici (punti 4.7 e 4.8). </li><li> Euthanize uno E17 femmine di ratto gravide a tempo di CO 2 in conformità con la Politica Servizi pubblici Salute sulle cure Humane ed uso di animali da laboratorio e sotto la cura degli animali istituzionalmente riconosciuto e usare il protocollo. </li><li> Spruzzare basso addome con il 70% EtOH e tagliare medialmente attraverso la pelle ed i muscoli con un paio di forbici che espongono l&#39;utero e gli embrioni. </li><li> Rimuovere tutti i feti e metterli in un contenitore sterile da 100 mm piatto contenente un eccesso di media dissezione a freddo (25-30 ml, vedere il punto 4.3). </li><li> Tagliare gli embrioni con un paio di piccole forbici dal sacco amniotico e metterli nella seconda 100-mm piatto contenente mezzo freddo dissezione. </li><li> Lavare gli embrioni a temperatura ambiente con delicatezza inclinando il piatto da 100 mm per 5-10 secondi. Poi, trasferire gli embrioni lavati al terzo piatto 100 mm contenente medie dissezione. Due lavaggi in eccesso di terreno sono in genere sufficienti per rimuovere tutte le tracce di sangue. Tuttavia, se ncario, lavare ancora una volta con un nuovo 100-mm piatto contenente 25-30 ml di mezzo di dissezione a freddo. </li><li> Utilizzando una pinza stereomicroscopio e curve, estrarre il cervello, ogni embrione di topo tirando la pelle e il cranio. Mettere intero cervello in uno dei 60 mm (in genere i piatti, con non più di 5 cervelli per piatto) con freddo Hibernate E. Tenere queste piastre su ghiaccio. </li><li> Prendete un piatto alla volta e, sotto un microscopio da dissezione, separare gli emisferi e isolare le cortecce cerebrali rimozione del mesencefalo e meningi. </li><li> Optional: cervelli taglio lungo la linea mediana, estrarre ippocampo, e seguire la procedura descritta di seguito per isolare i neuroni dell&#39;ippocampo. </li><li> Raccogli tutte le cortecce sezionato in 15 ml di tubo trasparente conica contenente 13 ml di freddo Hibernate E. Lasciare le cortecce cerebrali sul ghiaccio fino a quando tutte le dissezioni sono state completate. A causa delle dimensioni ridotte, sezionato ippocampi può essere raccolto in un 1,5 ml provetta Eppendorf invece di un 15-ml tubo. Se lo si desidera, a questo passo o cortecciaippocampi può essere collocato in una fiala cryotube contenente 1 ml di sospensione E + 2% B27 + gentamicina (50 ug / ml) + Fungizone (250 ng / ml) nella proporzione di 2-4 o 2-4 cortecce ippocampo per flacone. Tessuto cerebrale può essere conservato a 4 ° C al buio per una settimana (dopo tempi non sono ancora in prova). Quando necessario, utilizzare una pinza sottile per trasferire il tessuto cerebrale in una provetta contenente 15 ml E Hibernate e poi seguire il protocollo seguito per isolare i neuroni. </li><li> Trasferire il tubo ad una cappa coltura tissutale. Lasciare le cortecce di depositarsi sul fondo della provetta e poi accuratamente rimuovere il supernatante. </li><li> Aggiungere 13 ml di fresco E Hibernate a 15-ml tubo conico, consentono cortecce depositano sul fondo del tubo e rimuovere il supernatante. Ripetere questa operazione altre 2 volte e, dopo l&#39;ultimo lavaggio, rimuovere con cura tutti i media. </li><li> Enzimaticamente digerire cortecce cerebrali aggiungendo 1-2 ml (a seconda del numero delle cortecce, utilizzare meno per l&#39;isolamento ippocampo) di Tr caldiypLE Express. Sigillare il tappo del tubo con parafilm e galleggiano la provetta in un bagno di acqua 37 ° C per 10 minuti. </li><li> Spruzzare il tubo con etanolo al 70% prima di aprire il tappo e aggiungere 10 ml di Hibernate E. Lasciare le cortecce di stabilirsi sul fondo della provetta e rimuovere il surnatante. Ripetere questa operazione tre volte per lavare Triplo Express. In ultima fase, rimuovere con cura tutti i media. </li><li> Delicatamente triturare (4-5 volte) le cortecce in 2 ml di mezzo completo Neurobasal/B27 utilizzando un fuoco vetro lucido Pasteur (circa 1 mm di diametro). Fare attenzione a evitare le bolle. </li><li> Ripetere un&#39;altra 4-5 volte con una soluzione sterile di vetro minore pipetta Pasteur di diametro (cioè una pipetta circa 1/2-3/4 millimetri di diametro). Non usare una pipetta Pasteur inferiore a questo o distrugge le cellule. </li><li> Lasciare i pezzi rimanenti di tessuto (generalmente pochissimi, se del caso) per risolvere. </li><li> Trasferire il superiore singola sospensione cellulare a un nuovo tubo da 15 ml, lasciando dietro di sé pezzi di tessuto si insediarono. Pellicciather diluire la sospensione cellulare fino a 10-12 ml con Neurobasal/B27 mezzo completo. </li><li> Mescolare bene e diluire le cellule per il conteggio con l&#39;aggiunta di 10 microlitri di sospensione cellulare a 490 ml di soluzione di conteggio 50x (vedi tabella III) in una provetta Eppendorf 1,5 ml. </li><li> Cellule a piastre di PDL piastre rivestite alla densità di 5,0 x 10 4 / cm 2. Se nucleofection deve essere eseguita, si consiglia piastrando le cellule ad una concentrazione più elevata (8-10 x 10 4 / cm 2). </li><li> Normalmente, sezionare 9-10 feti per esperimento, come circa 13 x 10 6 neuroni sono derivati ​​da ogni feto E17. Se più embrioni sono necessari, assicurarsi che l&#39;intera procedura non dura più di due ore. </li><li> Se desiderato, 24 ore dopo l&#39;isolamento, 10 pM di citosina-β-D-arabinofuranoside (AraC) può essere aggiunto a ogni piatto, al fine di prevenire la proliferazione gliale. Tuttavia, questo passaggio non è necessario dal momento che Neurobasal/B27 medie inibisce la proliferazione gliale, Secondo le raccomandazioni del produttore (Invitrogen / Gibco). </li><li> I neuroni possono essere utilizzati per gli esperimenti successivi 4-5 giorni in vitro, anche se il tempo esatto dipende stadio di differenziazione desiderata. Abbiamo neuroni in coltura fino a 4 settimane senza una diminuzione significativa nella sopravvivenza (Figura 1). </li><li> Per la coltura prolungata, sostituire la coltura media ogni settimana con preparata Neurobasal/B27 terreno completo. </li></ol> 5. Risultati rappresentativi Neuroni coltivati ​​su vetrini di vetro camera può essere sottoposto a immunocitochimica. Figura 1 mostra una tipica immagine di un neurone corticale fissata dopo cinque giorni in coltura e immunolabeled con anti-MAP-2 anticorpo per mostrare processi neuronali. La figura 2 mostra un&#39;immagine rappresentativa di un neurone dell&#39;ippocampo ratto dopo 3 settimane in coltura. La morfologia neuronale di una cella completamente differenziata è evidenziata dal MAP-2 immunolabeling (MAP-2 marcatore neuronale, anticorpo monoclonale di topo clone AP-20, Gene Tex, Irvine, CA), a seguito di una procedura standard come descritto in precedenza 1. Le immagini sono state visualizzate con la Nikon Eclipse E400 microscopio a fluorescenza in posizione verticale dotato di fotocamera EXI aqua (Qimaging), motorizzato Z, e SlideBook5 acquisizione / deconvoluzione software (Intelligent Imaging Innovations, Inc., Denver, CO). Una serie di immagini tridimensionali di ogni singola foto sono state deconvoluted ad una immagine bidimensionale e risolto regolando il segnale di cut-off a vicino intensità massima per aumentare la risoluzione. La figura 3 mostra la purezza di colture neuronali. Lisati sono stati ottenuti da proteine ​​DIV7 colture neuronali di ratto (CTX) e da un caso di glioblastoma umano (GBM). Come previsto, il lisato neuronale è fortemente positiva per la proteina neuronale MAP-2 e negativo per la GFAP marcatore astrociti, mentre il lisato proteina GBM f è negativao MAP-2 e positiva per la GFAP. Anche se nel nostro protocollo abbiamo usato Hibernate E per diversi anni come la dissezione e risciacquo media, di recente abbiamo esplorato un utilizzo ulteriore e molto pratico per conservare i tessuti cerebrali per un ulteriore uso. La figura 4 mostra un paio di giorni in vitro 5 (DIV5) cultura di neuroni corticali del ratto isolati da cortecce conservati a 4 ° C per una settimana in Hibernate E + B27 dopo la dissezione originale degli embrioni. I neuroni sono stati piastrati su un vetro due camera di scorrimento rivestito con PDL e laminina come precedentemente descritto. L&#39;immagine è stata acquisita deconvoluted usando SlideBook5 acquisizione / software deconvoluzione come sopra descritto (Figura 2). <img alt="Figura 1" src="/files/ftp_upload/3965/3965fig1.jpg" /> Figura 1. Immagine rappresentativa di un neurone corticale nucleofected con pmaxGFP (Amaxa, Lonza, Walkersville, MD) e immunolabeled con MAP-2 Anticorpi, in rosso. Ingrandimento originale 100x. <img alt="Figura 2" src="/files/ftp_upload/3965/3965fig2.jpg" /> Figura 2. Immagine rappresentativa che mostra MAP-2 immunomarcatura, in rosso, dei neuroni dell&#39;ippocampo dopo 3 settimane in coltura. Colorazione DAPI, in blu, presenta nuclei cellulari. Original ingrandimento 40x. <img alt="Figura 3" src="/files/ftp_upload/3965/3965fig3.jpg" /> Figura 3. Western blot mostra la purezza di colture di cellule neuronali. 30 microgrammi di lisati GBM di ratto e umano proteine ​​neuronali sono stati separati mediante elettroforesi e sottoposto ad analisi Western blot seguendo procedure standard 1. Anti-MAP-2 era un coniglio policlonale di segnalazione cellulare (Danvers, MA), anti-GFAP anticorpo monoclonale di topo era da Chemicon (Millipore, Billerica, MA), e l&#39;anticorpo monoclonale murino anti-GRB2 anticorpo era da Transduction Laboratories (BD Sparks, MD). GRB2 è stato usato come controllo di caricamento. </p&gt; <img alt="Figura 4" src="/files/ftp_upload/3965/3965fig4.jpg" /> Immagini rappresentative Figura 4. Di 5 giorni in vitro (DIV5) neuroni corticali del ratto ottenuti da cortecce lasciate in Hibernate E + B27 a 4 ° C per una settimana dopo la loro dissezione. A) Il contrasto di fase di neuroni coltivati ​​su un vetro a due camere diapositiva. Original ingrandimento 20X. B) immunofluorescenza mostrano espressione di MAP-2 nei processi neuronali, in verde, la cultura era negativo per il marcatore astrociti GFAP. Colorazione DAPI, in blu, indica nuclei cellulari. Original ingrandimento 40x.

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Il metodo di dissezione e cultura di neuroni ippocampali di ratto e corticale qui descritto permette di eseguire esperimenti utilizzando colture neuronali quasi purezza coltivate in un mezzo chimicamente definito (Figura 3). Anche se i protocolli per i neuroni coltura quasi pura nel siero senza supporti sono stati precedentemente descritti 2,3,4, ci sono importanti cambiamenti nel nostro metodo. Diverso da protocolli tradizionali (vale a dire Banker et al.) 5, abbiamo sostituito tr...

<table border="1"><tbody><tr><td> Reagente </td><td> Concentrazione </td></tr><tr><td> Neurobasal </td><td> 98% </td></tr><tr><td> B27 </td><td> 2% </td></tr><tr><td> Glutamax </td><td> 0,5 mM </td></tr></tbody></table> Tabella I. Neurobasal/B27 mezzo completo. <table border="1"><tbody><tr><td> Reagente </td><td> Concentrazione </td></tr><tr><td> Glucosio </td><td> 16 mM </td></tr><tr><td> Saccarosio </td><td> 22 mm </td></tr><tr><td> HEPES </td><td> 10 mM </td></tr><tr><td> NaCl </td><td> 160 mM </td></tr><tr><td> KCl </td><td> 5 mM </td></tr><tr><td> Na 2 HPO 4 </td><td> 1 mM </td></tr><tr><td> KH 2 PO 2 </td><td> 0,22 mm </td></tr><tr><td> Gentamicina </td><td> 50 ug / ml </td></tr&gt; <tr><td> Fungizone </td><td> 250 ng / ml </td></tr><tr><td> pH </td><td> 7,4 </td></tr><tr><td> Osmolarità </td><td> 320-330 mOsm </td></tr></tbody></table> Tabella II. Dissezione mezzo. <table border="1"><tbody><tr><td> Reagente </td><td> Volume (pl) </td></tr><tr><td> Neurobasal/B27 terreno completo </td><td> 240 </td></tr><tr><td> Trypan Blue Stain 0,4% </td><td> 250 </td></tr><tr><td> Totale </td><td> 490 </td></tr></tbody></table> Tabella III. 50x conteggio soluzione. <table border="1"><tbody><tr><td> Reagente </td><td> Azienda </td><td> Cat. numero </td></tr><tr><td> E Hibernate </td><td> Brainbits </td><td> 767171 </td></tr><tr><td> Neurobasal </td><td> Gibco, Invitrogen </td><td> 21103-049 </td></tr><tr><td> B27 </td><td> Gibco, Invitrogen </td><td> 17504-044 </td></tr><tr><td> Fungizone </td><td> Gibco, Invitrogen </td><td> 15290-018 </td></tr><tr><td> Gentamicina solfato </td><td> Sigma Aldrich </td><td> G1264 </td></tr><tr><td> Glutamax 200 mM </td><td> Gibco, Invitrogen </td><td> 35050 </td></tr><tr><td> Triplo espresso w / o rosso fenolo </td><td> Gibco, Invitrogen </td><td> 12604 </td></tr><tr><td> Citosina-β-D-arabinofuranoside cloridrato </td><td> Sigma Aldrich </td><td> C6645 </td></tr><tr><td> Poly-D-lisina </td><td> Sigma Aldrich </td><td> P6407 </td></tr><tr><td> Laminina 1 mg / ml </td><td> Millipore </td><td> CC095 </td></tr><tr><td> HEPES </td><td> Sigma Aldrich </td><td> H3375 </td></tr><tr><td> Trypan Blue Stain 0,4% </td><td> Gibco, Invitrogen </td><td> 15250 </td></tr></tbody></table&gt; Tabella IV. Reagenti specifici. <table border="1"><tbody><tr><td> Attrezzatura </td><td> Azienda </td><td> Cat. numero </td></tr><tr><td> Microscopio Stereo </td><td> Olimpo </td><td> SZ61 </td></tr><tr><td> Pinze di grandi dimensioni </td><td> FST </td><td> 11022-14 </td></tr><tr><td> Punta fine pinze </td><td> Moria </td><td> MC40B </td></tr><tr><td> Micro punta fine pinze </td><td> Moria </td><td> MC31 </td></tr><tr><td> Affilate le forbici </td><td> Roboz </td><td> RS-6820 </td></tr><tr><td> Micro forbici per dissezione </td><td> FST </td><td> 91460-11 </td></tr><tr><td> Micro dissezione forbici curve </td><td> FST </td><td> 14067-11 </td></tr><tr><td> Vetro 2-camera vetrini </td><td> Lab-Tek </td><td> 154461 </td></tr><tr><td> 60 piatti mm </td&gt; <td> BD Falcon </td><td> 353002 </td></tr><tr><td> Dischi da 100 mm </td><td> Corning </td><td> 430167 </td></tr><tr><td> 15 ml provette </td><td> BD Falcon </td><td> 352099 </td></tr><tr><td> 1,5 ml crio-tube vial </td><td> Nunc </td><td> 375353 </td></tr></tbody></table> Attrezzature Tabella V. specifico.

isolation and culture of rat embryonic neural cells: a quick protocol

Stiamo descrivendo un metodo rapido di dissociarsi e di cultura neuroni dell&#39;ippocampo o corticale da E15-17 embrioni di ratto. La procedura può essere applicata con successo per l&#39;isolamento del mouse e neuroni umani primari e progenitori neurali. Neuroni dissociati sono mantenuti in terreno privo di siero fino a diverse settimane. Queste colture possono essere utilizzati per nucleofection, immunocitochimica, acidi nucleici preparazione, nonché elettrofisiologia. Vecchi colture neuronali possono anche essere trasfettate con un tasso di buona efficienza di trasduzione lentivirale e, meno efficiente, con fosfato di calcio o base di lipidi metodi come lipofectamina.

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Isolation and Culture of Rat Embryonic Neural Cells: A Quick Protocol

Noi descriviamo una metodologia rapida per isolare e cultura neuroni dell&#39;ippocampo e corticale da embrioni di roditori. Questo protocollo permette di effettuare esperimenti in cui sono richieste colture neuronali quasi puri.

Isolamento e la coltura di cellule embrionali neuronali di ratto: un protocollo veloce

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied ...

isolation-and-culture-of-rat-embryonic-neural-cells-a-quick-protocol

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38.0K Views.  LSU Health Sciences Center - New Orleans. Noi descriviamo una metodologia rapida per isolare e cultura neuroni dell'ippocampo e corticale da embrioni di roditori. Questo protocollo permette di effettuare esperimenti in cui sono richieste colture neuronali quasi puri.

Video: Isolation and Culture of Rat Embryonic Neural Cells: A Quick Protocol

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

Dissezione e la cultura di neuroni embrionali commissurali da midollo spinale

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

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Neuroscienze

Isolation and Expansion of the Adult Mouse Neural Stem Cells Using the Neurosphere Assay

Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a chemically defined serum-free culture system known as the neurosphere assay (NSA). In this assay, the majority of differentiated cell types die within a few days of culture but a small population of growth factor responsive precursor cells undergo active proliferation in the presence of epidermal growth factor (EGF) and/ basic fibroblastic growth factor (bFGF). These cells form colonies of undifferentiated cells called neurospheres, which in turn can be subcultured to expand the pool of neural stem cells. Moreover, the cells can be induced to differentiate, generating the three major cell types of the CNS i.e. neurons, astrocytes, and oligodendrocytes. This assay provides an invaluable tool to supply a consistent, renewable source of undifferentiated CNS precursors, which could be used for in vitro studies and also for therapeutic purposes.
This video demonstrates the NSA method to generate and expand NSCs from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures. The procedure includes harvesting the brain from the adult mouse, micro-dissection of the periventricular region, tissue preparation and culture in the NSA. The harvested tissue is first chemically digested using trypsin-EDTA and then mechanically dissociated in NSC medium to achieve a single cell suspension and finally plated in the NSA. After 7-10 days in culture, the resulting primary neurospheres are ready for subculture to reach the amount of cells required for future experiments.

This video protocol demonstrates the neurosphere assay method to generate and expand neural stem cells from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures.

Isolamento ed espansione della cellule staminali neurali del mouse utilizzando il saggio neurosfere

Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a ...

Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life span of the animal. They can potentially replace cells that are lost in the aging process or in the process of injury and disease. The existence of neural stem cells (NSCs) was first described by Reynolds and Weiss (1992) in the adult mammalian central nervous system (CNS) using a novel serum&#8208;free culture system, the neurosphere assay (NSA). Using this assay, it is also feasible to isolate and expand NSCs from different regions of the embryonic CNS. These in vitro expanded NSCs are multipotent and can give rise to the three major cell types of the CNS. While the NSA seems relatively simple to perform, attention to the procedures demonstrated here is required in order to achieve reliable and consistent results. This video practically demonstrates NSA to generate and expand NSCs from embryonic day 14-mouse brain tissue and provides technical details so one can achieve reproducible neurosphere cultures. The procedure includes harvesting E14 mouse embryos, brain microdissection to harvest the ganglionic eminences, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in NSA culture. After 5-7 days in culture, the resulting primary neurospheres are passaged to further expand the number of the NSCs for future experiments.

This video protocol demonstrates the application of the neurosphere assay for the isolation and expansion of neural stem cells from the ganglionic eminences of embryonic day 14-mouse brain.

Stabilire embrionali di topo Neural Stem Cell Culture del saggio di neurosfere

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life ...

Isolation and Culture of Neural Crest Stem Cells from Human Hair Follicles

Hair follicles undergo lifelong growth and hair cycle is a well-controlled process involving stem cell proliferation and quiescence. Hair bulge is a well-characterized niche for adult stem cells1. This segment of the outer root sheath contains a number of different types of stem cells, including epithelial stem cells2, melanocyte stem cells3 and neural crest like stem cells4-7. Hair follicles represent an accessible and rich source for different types of human stem cells. We and others have isolated neural crest stem cells (NCSCs) from human fetal and adult hair follicles4,5. These human stem cells are label-retaining cells and are capable of self-renewal through asymmetric cell division in vitro. They express immature neural crest cell markers but not differentiation markers. Our expression profiling study showed that they share a similar gene expression pattern with murine skin immature neural crest cells. They exhibit clonal multipotency that can give rise to myogenic, melanocytic, and neuronal cell lineages after in vitro clonal single cell culture. Differentiated cells not only acquire lineage-specific markers but also demonstrate appropriate functions in ex vivo conditions. In addition, these NCSCs show differentiation potential toward mesenchymal lineages. Differentiated neuronal cells can persist in mouse brain and retain neuronal differentiation markers. It has been shown that hair follicle derived NCSCs can help nerve regrowth, and they improve motor function in mice transplanted with these stem cells following transecting spinal cord injury8. Furthermore, peripheral nerves have been repaired with stem cell grafts9, and implantation of skin-derived precursor cells adjacent to crushed sciatic nerves has resulted in remyelination10. Therefore, the hair follicle/skin derived NCSCs have already shown promising results for regenerative therapy in preclinical models.
Somatic cell reprogramming to induced pluripotent stem (iPS) cells has shown enormous potential for regenerative medicine. However, there are still many issues with iPS cells, particularly the long term effect of oncogene/virus integration and potential tumorigenicity of pluripotent stem cells have not been adequately addressed. There are still many hurdles to be overcome before iPS cells can be used for regenerative medicine. Whereas the adult stem cells are known to be safe and they have been used clinically for many years, such as bone marrow transplant. Many patients have already benefited from the treatment. Autologous adult stem cells are still preferred cells for transplantation. Therefore, the readily accessible and expandable adult stem cells in human skin/hair follicles are a valuable source for regenerative medicine.

This article presents a robust protocol for isolation and culture of neural crest stem cells from human hair follicles.

L&#39;isolamento e la coltura di cellule staminali neurali Crest di follicoli dei capelli umani

Hair follicles undergo lifelong growth and hair cycle is a well-controlled process involving stem cell proliferation and quiescence. Hair bulge is a ...

Lectin-based Isolation and Culture of Mouse Embryonic Motoneurons

Spinal motoneurons develop towards postmitotic stages through early embryonic nervous system development and subsequently grow out dendrites and axons. Neuroepithelial cells of the neural tube that express Nkx6.1 are the unique precursor cells for spinal motoneurons1. Though postmitotic motoneurons move towards their final position and organize themselves into columns along the spinal tract2,3. More than 90% of all these differentiated and positioned motoneurons express the transcription factors Islet 1/2. They innervate the muscles of the limbs as well as those of the body and the inner organs. Among others, motoneurons typically express the high affinity receptors for brain derived neurotrophic factor (BDNF) and Neurotrophin-3 (NT-3), the tropomyosin-related kinase B and C (TrkB, TrkC). They do not express the tropomyosin-related kinase A (TrkA)4. Beside the two high affinity receptors, motoneurons do express the low affinity neurotrophin receptor p75NTR. The p75NTR can bind all neurotrophins with similar but lower affinity to all neurotrophins than the high affinity receptors would bind the mature neurotrophins. Within the embryonic spinal cord, the p75NTR is exclusively expressed by the spinal motoneurons5. This has been used to develop motoneuron isolation techniques to purify the cells from the vast majority of surrounding cells6. Isolating motoneurons with the help of specific antibodies (panning) against the extracellular domains of p75NTR has turned out to be an expensive method as the amount of antibody used for a single experiment is high due to the size of the plate used for panning. A much more economical alternative is the use of lectin. Lectin has been shown to specifically bind to p75NTR as well7. The following method describes an alternative technique using wheat germ agglutinin for a preplating procedure instead of the p75NTR antibody. The lectin is an extremely inexpensive alternative to the p75NTR antibody and the purification grades using lectin are comparable to that of the p75NTR antibody. Motoneurons from the embryonic spinal cord can be isolated by this method, survive and grow out neurites.

An alternative way of isolating mouse embryonic motoneurons from the spinal cord is described. The method takes into account the fact that lectin can bind to the low affinity nerve growth factor receptor p75NTR. This lectin-based preplating allows a purification similar to that with a specific antibody against the p75NTR.

Lectina a base di isolamento e la coltura del mouse motoneuroni embrionali

Spinal motoneurons develop towards postmitotic stages through early embryonic nervous system development and subsequently grow out dendrites and axons. ...

Isolation and Culture of Human Fungiform Taste Papillae Cells

Taste cells are highly specialized, with unique histological, molecular and physiological characteristics that permit detection of a wide range of simple stimuli and complex chemical molecules contained in foods. In human, individual fungiform papillae contain from zero to as many as 20 taste buds. There is no established protocol for culturing human taste cells, although the ability to maintain taste papillae cells in culture for multiple cell cycles would be of considerable utility for characterizing the molecular, regenerative, and functional properties of these unique sensory cells. Earlier studies of taste cells have been done using freshly isolated cells in primary culture, explant cultures from rodents, or semi-intact taste buds in tissue slices1,2,3,4. Although each of these preparations has advantages, the development of long-term cultures would have provided significant benefits, particularly for studies of taste cell proliferation and differentiation. Several groups, including ours, have been interested in the development and establishment of taste cell culture models. Most attempts to culture taste cells have reported limited viability, with cells typically not lasting beyond 3-5 d5,6,7,8. We recently reported on a successful method for the extended culture of rodent taste cells9. We here report for the first time the establishment of an in vitro culture system for isolated human fungiform taste papillae cells. Cells from human fungiform papillae obtained by biopsy were successfully maintained in culture for more than eight passages (12 months) without loss of viability. Cells displayed many molecular and physiological features characteristic of mature taste cells. Gustducin and phospholipase C &beta;2, (PLC-&beta;2) mRNA were detected in many cells by reverse transcriptase-polymerase chain reaction and confirmed by sequencing. Immunocytochemistry analysis demonstrated the presence of gustducin and PLC-&beta;2 expression in cultured taste cells. Cultured human fungiform cells also exhibited increases in intracellular calcium in response to appropriate concentrations of several taste stimuli indicating that taste receptors and at least some of the signalling pathways were present. These results sufficient indicate that taste cells from adult humans can be generated and maintained for at least eight passages. Many of the cells retain physiological and biochemical characteristics of acutely isolated cells from the adult taste epithelium to support their use as a model taste system. This system will enable further studies of the processes involved in proliferation, differentiation and function of mammalian taste receptor cells in an in vitro preparation.
Human fungiform taste papillae used for establishing human fungiform cell culture were donated for research following proper informed consent under research protocols that were reviewed and approved by the IRB committee. The protocol (#0934) was approved by Schulman Associates Institutional Review Board Inc., Cincinnati, OH. Written protocol below is based on published parameters reported by Ozdener et al. 201110.

We aimed to develop a reproducible protocol for isolating and maintaining long-term cultures of human fungiform taste papillae cells. Cells from human fungiform papillae obtained by biopsy were successfully maintained in culture for more than eight passages (12 months) without loss of viability.

Isolamento e la coltura di cellule umane Gusto papille fungiformi

Taste cells are highly specialized, with unique histological, molecular and physiological characteristics that permit detection of a wide range of simple ...

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types 1-3. NC also has the unique ability to influence the differentiation and maturation of target organs4-6. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. 
NC multipotency was first described from explants of the avian neural tube7-9. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo10-13. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. 
To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors11,14-20, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties13,21,22. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors11,13,14,17. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter23. 
The method presented here is adapted from protocols optimized for the culture of rat NC11,13. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC2,24-28.

Isolation of embryonic neural crest from the neural tube facilitates the use of in vitro methods for studying migration, self-renewal, and multipotency of neural crest.

Isolamento e la coltura delle cellule della cresta neurale da Embryonic Murine tubo neurale

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

The process by which the anterior region of the neural plate gives rise to the vertebrate retina continues to be a major focus of both clinical and basic research. In addition to the obvious medical relevance for understanding and treating retinal disease, the development of the vertebrate retina continues to serve as an important and elegant model system for understanding neuronal cell type determination and differentiation1-16. The neural retina consists of six discrete cell types (ganglion, amacrine, horizontal, photoreceptors, bipolar cells, and M&uuml;ller glial cells) arranged in stereotypical layers, a pattern that is largely conserved among all vertebrates 12,14-18.
While studying the retina in the intact developing embryo is clearly required for understanding how this complex organ develops from a protrusion of the forebrain into a layered structure, there are many questions that benefit from employing approaches using primary cell culture of presumptive retinal cells 7,19-23. For example, analyzing cells from tissues removed and dissociated at different stages allows one to discern the state of specification of individual cells at different developmental stages, that is, the fate of the cells in the absence of interactions with neighboring tissues 8,19-22,24-33. Primary cell culture also allows the investigator to treat the culture with specific reagents and analyze the results on a single cell level 5,8,21,24,27-30,33-39. Xenopus laevis, a classic model system for the study of early neural development 19,27,29,31-32,40-42, serves as a particularly suitable system for retinal primary cell culture 10,38,43-45.
Presumptive retinal tissue is accessible from the earliest stages of development, immediately following neural induction 25,38,43. In addition, given that each cell in the embryo contains a supply of yolk, retinal cells can be cultured in a very simple defined media consisting of a buffered salt solution, thus removing the confounding effects of incubation or other sera-based products 10,24,44-45.
However, the isolation of the retinal tissue from surrounding tissues and the subsequent processing is challenging. Here, we present a method for the dissection and dissociation of retinal cells in Xenopus laevis that will be used to prepare primary cell cultures that will, in turn, be analyzed for calcium activity and gene expression at the resolution of single cells. While the topic presented in this paper is the analysis of spontaneous calcium transients, the technique is broadly applicable to a wide array of research questions and approaches (Figure 1).

Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

Xenopus laevis provides an ideal model system for studying cell fate specification and physiological function of individual retinal cells in primary cell culture. Here we present a technique for dissecting retinal tissues and generating primary cell cultures that are imaged for calcium activity and analyzed by in situ hybridization.

Dissezione, Cultura, e analisi di<em&gt; Xenopus laevis</em&gt; Tessuto embrionale della retina

The process by which  the anterior region of the neural plate gives rise to the vertebrate retina  continues to be a major focus of both clinical and ...

Isolation and Culture of Endothelial Cells from the Embryonic Forebrain

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two vascular networks of the embryonic forebrain, pial and periventricular, are spatially distinctive and have different origins and growth patterns. Endothelial cells from the pial and periventricular vascular networks have unique gene expression profiles and functions. Here we present a step-by-step protocol for isolation, culture, and verification of pure populations of endothelial cells from the periventricular vascular network (PVECs) of the embryonic forebrain (telencephalon). In this approach, telencephalon devoid of pial membrane obtained from embryonic day 15 mice is minced, digested with collagenase/dispase, and dispersed mechanically into a single cell suspension. PVECs are purified from cell suspension using positive selection with anti-CD-31/PECAM-1 antibody conjugated to MicroBeads using a strong magnetic separation method. Purified cells are cultured on collagen 1&#160;coated culture dishes in endothelial cell culture medium until they become confluent and further subcultured. PVECs obtained with this protocol exhibit cobblestone and spindle shaped phenotypes, as visualized by phase-contrast light microscopy and fluorescence microscopy. Purity of PVEC cultures was established with endothelial cell markers. In our hands, this method reliably and consistently yields pure populations of PVECs. This protocol will benefit studies aimed at gaining mechanistic insights into forebrain angiogenesis, understanding PVEC interactions, and cross-talks with neuronal cell types and holds tremendous potential for therapeutic angiogenesis.

This video demonstrates an easy and reliable strategy for preparation of pure cultures of endothelial cells from the embryonic forebrain within 10-12 days and will be useful for research focused on many aspects of cerebral angiogenesis.

Isolamento e coltura di cellule endoteliali dal embrionali Forebrain

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two ...

One Mouse, Two Cultures: Isolation and Culture of Adult Neural Stem Cells from the Two Neurogenic Zones of Individual Mice

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult neural stem cells in vitro. These assays can be used to compare the precursor potential of cells isolated from genetically different or differentially treated animals&#160;to determine the effects of exogenous factors on neural precursor cell proliferation and differentiation and to generate neural precursor cell lines that can be assayed over continuous passages. The neurosphere assay is traditionally used for the post-hoc identification of stem cells, primarily due to the lack of definitive markers with which they can be isolated from primary tissue&#160;and has the major advantage of giving a quick estimate of precursor cell numbers in brain tissue derived from individual animals. Adherent monolayer cultures, in contrast, are not traditionally used to compare proliferation between individual animals, as each culture is generally initiated from the combined tissue of between 5-8 animals. However, they have the major advantage that, unlike neurospheres, they consist of a mostly homogeneous population of precursor cells and are useful for following the differentiation process in single cells. Here, we describe, in detail, the generation of neurosphere cultures and, for the first time, adherent cultures from individual animals. This has many important implications including paired analysis of proliferation and/or differentiation potential in both the subventricular zone (SVZ) and dentate gyrus (DG) of treated or genetically different mouse lines, as well as a significant reduction in animal usage.

Here we describe a detailed protocol for the simultaneous generation of neural precursor cell cultures, as either adherent monolayers or neurospheres, from the subventricular zone and dentate gyrus of individual adult mice.

Un mouse, due culture: isolamento e la coltura di Adult Neural Stem Cells dalle due zone neurogena Mice individuale

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult ...

Methods Article

Isolamento e la coltura di cellule embrionali neuronali di ratto: un protocollo veloce