Supportato dalla Fondazione Fazit Graduate borsa di studio per SS, il Ministero federale dell&#39;Istruzione e della ricerca (BMBF 01 EO 0803) a KB e della Commissione europea sovvenzione 7 ° PQ PIRG08-GA-2010-276989, Neurex, e il tedesco Research Foundation Grant SCHA 1442 / 3-1 per CS Gli autori non hanno conflitti di interesse finanziari.

1. L&#39;isolamento e la placcatura di misti cellule corticali Mixed isolamento cellulare corticale per colture astrociti può essere eseguita utilizzando P1 a P4 cuccioli di topo. Al fine di ottenere la giusta densità astrociti è necessario utilizzare 4 cortecce topo pup per T75 pallone di coltura tissutale. Pertanto, volumi nel seguente protocollo sono calcolati per un preparato cellulare utilizzando 4 cuccioli di topo. <ol><li> Prima di avviare la procedura di dissezione, preriscaldata 30 ml di mezzo di coltura di astrociti (DMEM, glucosio + 10% di siero fetale di siero bovino + 1% di penicillina / streptomicina, vedi tabella) a 37 ° C. Coat uno T75 pallone con 20 ml di poli-D-lisina (PDL) ad una concentrazione di 50 ug / ml in acqua di grado coltura cellulare per 1 ora a 37 ° C in incubatore CO 2. </li><li> Per la procedura di dissezione, preparare tutti i reagenti necessari e materiali. Avrete bisogno di: forbici chirurgiche, lisce una pinza sottile, pinze punta piatta, asciugamani di carta, sacchetto di rifiuti, il 70% di etanolo und 2 piatti dissezione (3.5 cm di diametro) sul ghiaccio riempito con 2 HBSS ml ciascuno. </li><li> Premere delicatamente e spruzzare la testa e il collo del mouse cucciolo con il 70% di etanolo. L&#39;animale viene sacrificato per decapitazione usando le forbici. </li><li> Eseguire una incisione mediana, posteriore a anteriore, lungo il cuoio capelluto per rivelare il cranio. </li><li> Tagliare il cranio con cura dal collo al naso. Due ulteriori tagli vengono eseguiti per permettere ulteriore accesso al cervello: Il primo taglio è fatto anteriore dei bulbi olfattivi, l&#39;altra inferiore del cervelletto di scollegare il cranio dalla base del cranio. </li><li> Utilizzando le pinze punta piatta, i lembi cranici sono gentilmente girato di lato e il cervello viene estratto e inserito nel primo piatto dissezione pieno di HBSS. Porre la capsula posteriore su ghiaccio e continuare a raccogliere tutti e 4 i cervelli. </li><li> Il resto della procedura di dissezione è eseguita sotto uno stereomicroscopio. In primo luogo, i bulbi olfattivi e il cervelletto vengono rimossi con l&#39;ammendadissettore (Figura 1B). </li><li> Al fine di recuperare le cortecce, afferrare l&#39;estremità posteriore del cervello con le pinza sottile, eseguire una incisione mediana tra i due emisferi, inserire una seconda serie di pinze al boschetto creato e rimuovere la piastra-come la struttura della corteccia dal cervello. </li><li> Attenzione sezionare le meningi dagli emisferi della corteccia tirando con la pinza fini (Figura 1D &#39;). Questo passaggio consente di evitare la contaminazione della cultura finale astrociti da cellule meningee e fibroblasti. Trasferire gli emisferi della corteccia preparati nel secondo piatto pieno di HBSS e restituirlo su ghiaccio. Continuare quindi con tutti e 4 cortecce. </li><li> Infine, tagliare ogni emisfero corteccia in piccoli pezzi con lame affilate (circa 4 a 8 volte). </li><li> In condizioni sterili, pezzi di corteccia di trasferimento in un unico tubo da 50 ml Falcon e aggiungere HBSS ad un volume totale di 22,5 ml. </li><li> Aggiungere 2,5 ml di 2,5% tripsina, mescolare e incubareil tessuto nel bagno d&#39;acqua a 37 ° C per 30 min. Mescolare occasionale agitando ogni 10 min. </li><li> Centrifugare per 5 minuti a 300 xg per pellet pezzi di tessuto della corteccia. </li><li> Rimuovere con attenzione il surnatante per decantazione. Al fine di evitare di perdere il tessuto pellet si può trattenere meccanicamente utilizzando una pipetta. Dissociare il tessuto in una sospensione cellulare singola aggiungendo 10 ml di mezzo di placcatura astrociti e vigorosa pipettaggio utilizzando una pipetta di plastica da 10 ml fino pezzi di tessuto sono dissociate in singole cellule (20 a 30 volte). Regolare il volume a 20 ml con media placcatura astrociti. La possibilità di controllare la dissociazione del tessuto corticale in cellule singole contando utilizzando un hematocytometer. Una preparazione di 4 cortecce topo cucciolo dovrebbe produrre 10-15 x10 6 cellule dissociate singole. </li><li> Aspirare PDL dalla fiasca T75 cultura, piastra sospensione dissociato singola cella e incubare a 37 ° C in incubatore CO 2. </li></ol> 2. Come ottenere un Enriched Cultura astrociti <ol><li> Modificare le medie 2 giorni dopo la placcatura delle cellule miste corticali e tutti i 3 giorni successivi. </li><li> Dopo 7-8 giorni, quando microglia astrociti sono confluenti e sovrapposizione sedersi esposta sullo strato astrociti o sono già staccati dallo strato astrociti (Figura 2), agitare il matraccio T75 a 180 rpm per 30 minuti su un agitatore orbitale per rimuovere microglia. Eliminare il supernatante contenente microglia o se si desidera esaminare la cultura e la microglia, che spin down e piastra per la cultura 28,29. </li><li> Aggiungere 20 ml di mezzo fresco cultura astrociti e proseguire con agitando a 240 rpm per 6 ore per rimuovere le cellule precursori degli oligodendrociti (OPC). Poiché alcuni OPC non si stacchi dallo strato di astrociti, continuano ad agitare vigorosamente per 1 minuto, al fine di prevenire la contaminazione OPC. Eliminare il surnatante o girare verso il basso e piatto, se si desidera la cultura OPC 29. </li><li> Sciacquare il restante confluestrato nt astrociti due volte con PBS, aspirare il PBS, aggiungere 5 ml di tripsina-EDTA e incubare in 2 CO incubatore a 37 ° C. Controllare distacco di astrociti ogni 5 min e far rispettare il distacco di astrociti colpendo il pallone contro il palmo della mano (2-3 volte). </li><li> Dopo astrociti sono staccati dal pallone cultura, aggiungere 5 ml di terreno di coltura astrociti, cellule di spin a 180 xg per 5 minuti, aspirare il surnatante e aggiungere 40 ml di terreno fresco placcatura astrociti. Un pallone di coltura di tessuti T75 dovrebbe produrre circa 1x10 6 cellule dopo la scissione prima cella. Celle a piastre in due fiasche T75 e incubare a 37 ° C in 2 CO incubatore. Cambiare il mezzo ogni 2 o 3 giorni. </li><li> 12-14 giorni dopo il primo split astrociti sono placcati in un apposito concentrazione cellulare 24-48 ore prima di eseguire l&#39;esperimento. Un pallone di coltura di tessuti T75 dovrebbe produrre circa 1,5-2 x10 6 cellule dopo la divisione seconda cella. </li></ol>

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Il metodo qui descritto si basa sulla preparazione cultura astrociti dal cervello di roditori neonatali, originariamente descritti da McCarthy e de Vellis nel 1980 27. Il metodo modificato di isolamento e la coltura di astrociti corticali da P1 postnatale al cervello P4 del mouse qui presentato è veloce, rese astrociti primari puri ed è altamente riproducibile. Questa tecnica può essere facilmente trasferiti per isolare astrociti da altre specie, come di ratto o di maiale e da altre regioni del cervello, c...

Astrociti sono un tipo cellulare molto abbondante nel sistema nervoso centrale (CNS). Il rapporto tra astrociti ai neuroni è di 1:3 nella corteccia di topi e ratti, mentre ci sono 1,4 astrociti per neurone nella corteccia umana 1. L&#39;interesse per la funzione degli astrociti è aumentato drammaticamente negli ultimi anni. Una funzione chiave degli astrociti è il loro ruolo di supporto strutturale e metabolico ai neuroni 2,3. Ruoli di recente scoperta per gli astrociti coprono un ampio spettro di funzioni. Questi includono guidare la migrazione degli assoni di sviluppo e alcuni neuroblasti durante lo sviluppo 4-6, funzioni nella trasmissione sinaptica, forza sinapsi e di elaborazione delle informazioni da circuiti neurali 7-9, ruoli nella barriera ematoencefalica (BBB) ​​formazione 10 e 11-13 integrità e la regolamentazione del 14 tono cerebrovascolare. Un&#39;altra caratteristica importante di astrociti è la loro risposta al danno. In condizioni patologiche astrocytes diventano reattive e upregulate ulteriormente l&#39;espressione della proteina di filamento intermedio acida fibrillare gliale (GFAP) e inibitori della matrice extracellulare (ECM) proteine ​​15,16. Astrociti reattivi delimitare il luogo di ferita da tessuto sano formando una cicatrice gliale, che consiste principalmente di proteine ​​secrete astrociti ECM del condroitin solfato proteoglicani (CSPG) famiglia, i principali fattori che inibiscono la rigenerazione assonale dopo lesione del SNC 15-17. Gli astrociti sono originari di radiali gliali (RG), le cellule durante l&#39;embriogenesi e all&#39;inizio della vita post-natale. Secondo le specifiche astrociti si è verificato, precursori degli astrociti migrare verso le loro posizioni finali, dove iniziare il processo di differenziazione terminale. In vivo, astrociti sembrano essere maturi tre o quattro settimane dopo la nascita come indicato dalla loro morfologia tipica 18,19. Una sottopopolazione di cellule RG convertire in zona subventricolare astrociti (cellule di tipo B). Both, RG e le cellule di tipo B funzionano come astrociti-come le cellule staminali neurali (NSC) durante lo sviluppo e nell&#39;adulto, rispettivamente. Come astrociti, RG e le cellule di tipo B anche esprimere la astrociti specifico trasportatore del glutammato (GLAST), lipidi del cervello-binding protein (BLBP), e GFAP, indicando che questi indicatori non possono essere utilizzati esclusivamente per etichettare specificamente astrociti adulti. In contrasto adulti astrociti parenchimali, che non si dividono nel cervello sano, RG e B tipo cellule mostra potenziale di cellule staminali, come la capacità di auto-rinnovarsi. Disregolazione di astrociti è stato implicato in numerose patologie, compreso il morbo di Alzheimer 20,21, malattia di Huntington 22, morbo di Parkinson 23, 24 Rett sindrome e malattia di Alexander 25. Inoltre, astrociti reagire a tutte insulti del SNC, portando all&#39;attivazione astrociti e astrocitica formazione della cicatrice gliale 16,26. La cicatrice che si forma astrocitari gliale tr seguente cervelloAUMA midollo spinale o lesioni è pensato per essere la barriera principale impedire la rigenerazione neuronale 15. Lo sviluppo di metodi affidabili per isolare e mantenere popolazioni purificate di cellule è stato fondamentale per la nostra comprensione del sistema nervoso. Lavoro pionieristico da McCarthy e de Vellis consente agli investigatori di data per preparare colture quasi pure di astrociti da tessuti di ratto neonato 27. Molto è stato appreso sulla biologia astrociti utilizzando questo metodo, che viene qui presentata in una forma leggermente modificata per isolare gli astrociti corticali di topo. Integrando studi in vivo, astrociti nonché mezzo condizionato ottenuto utilizzando la coltura in vitro descritto, sono strumenti utili per ottenere ulteriori approfondimenti funzioni astrociti.

<table border="1"><tbody><tr><td> Nome della soluzione di lavoro </td><td> Azienda </td><td> Numero di catalogo </td><td> Concentrazione finale </td></tr><tr><td></td><td></td><td></td><td> Terreni di coltura astrociti </td></tr><tr><td> DMEM, glucosio </td><td> Life Technologies </td><td> 31966-021 </td><td></td></tr><tr><td> FBS, inattivato al calore </td><td> Life Technologies </td><td> 10082-147 </td><td> Concentrazione finale: 10% </td></tr><tr><td> Penicillina-Streptomicina </td><td> Life Technologies </td><td> 15140-122 </td><td> Concentrazione finale: 1% </td></tr><tr><td></td><td></td><td></td><td> Soluzione per scavare tessuto cerebraleestion </td></tr><tr><td> HBSS </td><td> Life Technologies </td><td> 14170-088 </td><td></td></tr><tr><td> 2,5% tripsina </td><td> Life Technologies </td><td> 15090-046 </td><td> Concentrazione finale: 0,25% </td></tr><tr><td></td><td></td><td></td><td> Altro </td></tr><tr><td> 70% (vol / vol) di etanolo </td><td> Roth </td><td> 9.065,2 </td><td></td></tr><tr><td> Poli-D-lisina </td><td> Millipore </td><td> A-003-E </td><td> 50 ug / ml </td></tr><tr><td> Acqua </td><td> PAA </td><td> S15-012 </td><td> coltura cellulare grade </td></tr><tr><td> PBS </td><td> PAA </td><td> H15-002 </td><td> coltura cellulare grade </td></tr><tr><td> 0,05% ProvaPSIN-EDTA </td><td> Life Technologies </td><td> 25300-062 </td><td></td></tr><tr><td> Filtro da 0,45 micron sterile </td><td> Sartorius </td><td> 16555 </td><td></td></tr><tr><td> 3,5 centimetri Petri </td><td> BD Falcon </td><td> 353001 </td><td></td></tr><tr><td> 15 ml Falcon tubo </td><td> BD Falcon </td><td> 352096 </td><td></td></tr><tr><td> 50 ml Falcon tubo </td><td> BD Falcon </td><td> 352070 </td><td></td></tr><tr><td> 75 centimetri 2 pallone di coltura tissutale </td><td> BD Falcon </td><td> 353136 </td><td></td></tr><tr><td> Pinze, fine </td><td> Dumont </td><td> 2-1032, 2-1033 </td><td> # 3c; # 5 </td></tr><tr><td> Pinze, punta piatta </td><td> KLS Martin </td><td> 12-120-11 </td><td></td></tr><tr><td> 13 centimetri forbici chirurgiche </td><td> Aesculap </td><td> BC-140-R </td><td></td></tr><tr><td> Stereomicroscopio </td><td> Leica </td><td></td><td> MZ7.5 </td></tr><tr><td> Stereomicroscopio + Fotocamera </td><td> Leica </td><td></td><td> MZ16F; DFC320 </td></tr><tr><td> Microscopio + Fotocamera </td><td> Zeiss, Canon </td><td></td><td> Primo Vert; PowerShot A650 IS </td></tr><tr><td> Centrifuga </td><td> Eppendorf </td><td> 5805000.017 </td><td> Centrifuge5804R </td></tr><tr><td> Orbital Shaker </td><td> Thermo Scientific </td><td> SHKE 4450-1CE </td><td> MaxQ 4450 </td></tr><tr><td> Bagnomaria </td><td> Julabo </td><td></td><td> SW20, 37 ° C </td></tr></tbody></table>

isolation and culture of mouse cortical astrocytes

Gli astrociti sono un tipo di cellula abbondante nel cervello dei mammiferi, ma resta ancora molto da imparare sulle loro caratteristiche molecolari e funzionali. Nei sistemi di coltura in vitro di cellule astrociti possono essere utilizzati per studiare le funzioni biologiche di queste cellule gliali in dettaglio. Questo protocollo video mostra come ottenere astrociti puri di isolamento e la coltura di cellule corticali miste di cuccioli di topo. Il metodo si basa sulla mancanza di neuroni vitali e la separazione di astrociti, oligodendrociti e microglia, le tre popolazioni principali cellule gliali del sistema nervoso centrale, in coltura. Immagini rappresentative durante i primi giorni di cultura dimostrare la presenza di una popolazione cellulare mista e indicare il timepoint, quando astrociti diventano confluenti e devono essere separati da microglia e gli oligodendrociti. Inoltre, abbiamo dimostrato la purezza e la morfologia degli astrociti di astrociti in coltura utilizzando colorazioni immunocitochimiche per la consolidata eappena descritto marcatori astrociti. Questo sistema di coltura può essere facilmente utilizzato per ottenere astrociti topo puri e astrociti terreno condizionato per studiare vari aspetti della biologia astrociti.

Sull&#39;isolamento del cervello di topo completa (Figura 1A), il cervelletto e bulbi olfattivi devono essere rimossi (Figura 1B). Le cortecce sono sbucciati del tronco cerebrale mouse (Figura 1C) e meningi della corteccia individuale (Figura 1D &#39;) sono attentamente rimossi (Figura 1E). Meningi sono evidenti dal sistema arteria meningea e risultati incompleti rimozione della contaminazione della cultura finale astrociti da cellule m...

Watch this Scientific Journal Video about Isolation and Culture of Mouse Cortical Astrocytes at JoVE.com

Isolation and Culture of Mouse Cortical Astrocytes

Gli astrociti sono stati riconosciuti per essere versatili cellule che partecipano a processi biologici fondamentali che sono essenziali per il normale sviluppo del cervello e la funzione, e la riparazione del sistema nervoso centrale. Qui vi presentiamo una procedura rapida per ottenere colture pure astrociti topo per studiare la biologia di questa classe importante di cellule del sistema nervoso centrale.

Isolamento e la coltura di astrociti corticali mouse

Astrocytes  are an abundant cell type in the mammalian brain, yet much remains to be  learned about their molecular and functional characteristics. In ...

isolation-and-culture-of-mouse-cortical-astrocytes

Research

JoVE Journal

Neuroscience

90.6K Views.  University of Freiburg. Gli astrociti sono stati riconosciuti per essere versatili cellule che partecipano a processi biologici fondamentali che sono essenziali per il normale sviluppo del cervello e la funzione, e la riparazione del sistema nervoso centrale. Qui vi presentiamo una procedura rapida per ottenere colture pure astrociti topo per studiare la biologia di questa classe importante di cellule del sistema nervoso centrale.

Video: Isolation and Culture of Mouse Cortical Astrocytes

Nucleofection and Primary Culture of Embryonic Mouse Hippocampal and Cortical Neurons

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and synapse formation and function. An advantage of culturing these neurons is that they readily polarize, forming distinctive axons and dendrites, on a two dimensional substrate at very low densities. This property has made them extremely useful for determining many aspects of neuronal development. Furthermore, by providing glial conditioning for these neurons they will continue to develop, forming functional synaptic connections and surviving for several months in culture. In this protocol we outline a technique to dissect, culture and transfect embryonic mouse hippocampal and cortical neurons. Transfection is accomplished by electroporating DNA into the neurons before plating via nucleofection. This protocol has the advantage of expressing fluorescently-tagged fusion proteins early in development (~4-8hrs after plating) to study the dynamics and function of proteins during polarization, axon outgrowth and branching. We have also discovered that this single transfection before plating maintains fluorescently-tagged fusion protein expression at levels appropriate for imaging throughout the lifetime of the neuron (&gt; 2 months in culture). Thus, this methodology is useful for studying protein localization and function throughout CNS development with little or no disruption of neuronal function.

This protocol outlines the steps required to dissect, transfect via electroporation and culture mouse hippocampal and cortical neurons. Short-term cultures may be used for studies of axon outgrowth and guidance, while long-term cultures can be used for studies of synaptogenesis and dendritic spine analysis.

Nucleofection e cultura primaria di neuroni embrionali di topo ippocampale e corticale

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and ...

Ricerca

Neuroscienze

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. ...

Bilaminar Co-culture of Primary Rat Cortical Neurons and Glia

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or co-culture model. This system is suitable for a variety of experimental needs requiring either a glass or plastic growth substrate and can also be used for culture of other types of neurons.
Rat cortical neurons obtained from the late embryonic stage (E17) are plated on glass coverslips or tissue culture dishes facing a feeder layer of glia grown on dishes or plastic coverslips (known as Thermanox), respectively. The choice between the two configurations depends on the specific experimental technique used, which may require, or not, that neurons are grown on glass (e.g. calcium imaging versus Western blot). The glial feeder layer, an astroglia-enriched secondary culture of mixed glia, is separately prepared from the cortices of newborn rat pups (P2-4) prior to the neuronal dissection.
A major advantage of this culture system as compared to a culture of neurons only is the support of neuronal growth, survival, and differentiation provided by trophic factors secreted from the glial feeder layer, which more accurately resembles the brain environment in vivo. Furthermore, the co-culture can be used to study neuronal-glial interactions1.
At the same time, glia contamination in the neuronal layer is prevented by different means (low density culture, addition of mitotic inhibitors, lack of serum and use of optimized culture medium) leading to a virtually pure neuronal layer, comparable to other established methods1-3. Neurons can be easily separated from the glial layer at any time during culture and used for different experimental applications ranging from electrophysiology4, cellular and molecular biology5-8, biochemistry5, imaging and microscopy4,6,7,9,10. The primary neurons extend axons and dendrites to form functional synapses11, a process which is not observed in neuronal cell lines, although some cell lines do extend processes.
A detailed protocol of culturing rat hippocampal neurons using this co-culture system has been described previously4,12,13. Here we detail a modified protocol suited for cortical neurons. As approximately 20x106 cells are recovered from each rat embryo, this method is particularly useful for experiments requiring large numbers of neurons (but not concerned about a highly homogenous neuronal population). The preparation of neurons and glia needs to be planned in a time-specific manner. We will provide the step-by-step protocol for culturing rat cortical neurons as well as culturing glial cells to support the neurons.

Here we provide a protocol for culturing rat cortical neurons in the presence of a glial feeder layer. The cultured neurons establish polarity and create synapses, and can be separated from the glia for use in various applications, such as electrophysiology, calcium imaging, cell survival assays, immunocytochemistry, and RNA/DNA/protein isolation.

Bilaminar co-coltura di neuroni corticali primari di ratto e Glia

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or ...

Isolation and Culture of Hippocampal Neurons from Prenatal Mice

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual neurons, researchers are able to analyze properties related to cellular trafficking, cellular structure and individual protein localization using a variety of biochemical techniques. Results from such experiments are critical for testing theories addressing the neural basis of memory and learning. However, unambiguous results from these forms of experiments are predicated on the ability to grow neuronal cultures with minimum contamination by other brain cell types. In this protocol, we use specific media designed for neuron growth and careful dissection of embryonic hippocampal tissue to optimize growth of healthy neurons while minimizing contaminating cell types (i.e. astrocytes). Embryonic mouse hippocampal tissue can be more difficult to isolate than similar rodent tissue due to the size of the sample for dissection. We show detailed dissection techniques of hippocampus from embryonic day 19 (E19) mouse pups. Once hippocampal tissue is isolated, gentle dissociation of neuronal cells is achieved with a dilute concentration of trypsin and mechanical disruption designed to separate cells from connective tissue while providing minimum damage to individual cells. A detailed description of how to prepare pipettes to be used in the disruption is included. Optimal plating densities are provided for immuno-fluorescence protocols to maximize successful cell culture. The protocol provides a fast (approximately 2 hr) and efficient technique for the culture of neuronal cells from mouse hippocampal tissue.

We provide a protocol for the culture of highly purified hippocampal neurons from prenatal mouse brains without the use of a feeder glial cell layer.

Isolamento e la coltura di neuroni ippocampali da topi prenatali

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual ...

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 ...

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease pathogenesis and their utility for preclinical drug development. In order to design a better model system for these applications, phenotypically wild-type cortical astrocytes and neural stem cells (NSC) from conditional, genetically engineered mice (GEM) that harbor various combinations of floxed oncogenic alleles were harvested and grown in culture. Genetic recombination was induced in vitro using adenoviral Cre-mediated recombination, resulting in expression of mutated oncogenes and deletion of tumor suppressor genes. The phenotypic consequences of these mutations were defined by measuring proliferation, transformation, and drug response in vitro. Orthotopic allograft models, whereby transformed cells are stereotactically injected into the brains of immune-competent, syngeneic littermates, were developed to define the role of oncogenic mutations and cell type on tumorigenesis in vivo. Unlike most established human glioblastoma cell line xenografts, injection of transformed GEM-derived cortical astrocytes into the brains of immune-competent littermates produced astrocytomas, including the most aggressive subtype, glioblastoma, that recapitulated the histopathological hallmarks of human astrocytomas, including diffuse invasion of normal brain parenchyma. Bioluminescence imaging of orthotopic allografts from transformed astrocytes engineered to express luciferase was utilized to monitor in vivo tumor growth over time. Thus, astrocytoma models using astrocytes and NSC harvested from GEM with conditional oncogenic alleles provide an integrated system to study the genetics and cell biology of astrocytoma pathogenesis in vitro and in vivo and may be useful in preclinical drug development for these devastating diseases.

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Phenotypically wild-type astrocytes and neural stem cells harvested from mice engineered with floxed, conditional oncogenic alleles and transformed via viral Cre-mediated recombination can be used to model astrocytoma pathogenesis in vitro and in vivo by orthotopic injection of transformed cells into brains of syngeneic, immune-competent littermates.

Modellazione Astrocitoma Patogenesi<em&gt; In Vitro</em&gt; E<em&gt; In Vivo</em&gt; Utilizzo corticale astrociti o cellule staminali neurali da condizionale, topi geneticamente ingegnerizzati

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease ...

Sex Differences in Mouse Hippocampal Astrocytes after In-Vitro Ischemia

Astrogliosis following hypoxia/ischemia (HI)-related brain injury plays a role in increased morbidity and mortality in neonates. Recent clinical studies indicate that the severity of brain injury appear to be sex dependent, and that the male neonates are more susceptible to the effects of HI-related brain injury, resulting in more severe neurological outcomes as compared to females with comparable brain injuries. The development of reliable methods to isolate and maintain highly enriched populations of sexed hippocampal astrocytes is essential to understand the cellular basis of sex differences in the pathological consequences of neonatal HI. In this study, we describe a method for creating sex specific hippocampal astrocyte cultures that are subjected to a model of in-vitro ischemia, oxygen-glucose deprivation, followed by reoxygenation. Subsequent reactive astrogliosis was examined by immunostaining for the Glial Fibrillary Acidic Protein (GFAP) and S100B. This method provides a useful tool to study the role of male and female hippocampal astrocytes following neonatal HI, separately.

Sex Differences in Mouse Hippocampal Astrocytes after In-Vitro Ischemia

Astrocytes are one of the most important key players in the central nervous system (CNS). Here, we are reporting a practical method of sexed hippocampal astrocyte culture protocol in order to study the mechanisms underlying the astrocyte function in male and female neonate pups after in-vitro ischemia.

Le differenze di sesso in ippocampali di topo astrociti dopo<em&gt; In-Vitro</em&gt; Ischemia

Astrogliosis following hypoxia/ischemia (HI)-related brain injury plays a role in increased morbidity and mortality in neonates. Recent clinical studies ...

Isolation and Culture of Adult Neural Stem Cells from the Mouse Subcallosal Zone

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation into three types of cells: neurons, astrocytes, and oligodendrocytes. Identifying aNSC-derived regions and characterizing the aNSC properties are critical for the potential use of aNSCs and for the elucidation of their role in neural regeneration. The subcallosal zone (SCZ), located between white matter and the hippocampus, has recently been reported to contain aNSCs and continuously give rise to neuroblasts. A low percentage of aNSCs from the SCZ is differentiated into neurons; most cells are differentiated into glial cells, such as oligodendrocytes and astrocytes. These cells are suggested to have a therapeutic potential for traumatic cortical injury. This protocol describes in detail the process to generate SCZ-aNSCs from an adult mouse brain. A brain matrix with intervals of 1 mm is used to obtain the SCZ-containing coronal slices and to precisely dissect the SCZ from the whole brain. The SCZ sections are initially subjected to a neurosphere culture. A well-developed culture system allows for the verification of their characteristics and can increase research on NSCs. A neurosphere culture system provides a useful tool for determining proliferation and collecting the genuine NSCs. A monolayer culture is also an in vitro system to assay proliferation and differentiation. Significantly, this culture system provides a more homogenous environment for NSCs than the neurosphere culture system. Thus, using a discrete brain region, these culture systems will be helpful for expanding our knowledge about aNSCs and their applications for therapeutic uses.

Establishing culture systems for the expansion of adult neural stem cells (aNSCs) allows for the examination and application of aNSCs for therapy. The subcallosal zone (SCZ) has recently been recognized as a novel neuroblast-forming region in adult mice. Here, methods for the isolation, expansion, and differentiation of SCZ-aNSCs are described.

Isolamento e la coltura di adulti neurali cellule staminali dal Subcallosal Zona mouse

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation ...

Differentiation of Mouse Embryonic Stem Cells into Cortical Interneuron Precursors

GABAergic cortical interneurons are a heterogeneous population of cells that play critical roles in regulating the output of excitatory pyramidal neurons as well as synchronizing the outputs of pyramidal neuron ensembles. Deficits in interneuron function have been implicated in a variety of neuropsychiatric disorders, including schizophrenia, autism, and epilepsy. The derivation of cortical interneurons from embryonic stem cells not only allows for the study of their development and function, but provides insight into the molecular mechanisms underlying the pathogenesis of cortical interneuron-related disorders. Interneurons also have the remarkable capacity to survive, migrate, and integrate into host cortical circuitry post-transplantation, making them ideal candidates for use in cell-based therapies. Here, we present a scalable, highly efficient, modified embryoid body-to-monolayer method for the derivation of Nkx2.1-expressing interneuron progenitors and their progeny from mouse embryonic stem cells (mESCs). Using a Nkx2.1::mCherry:Lhx6::GFP dual reporter mESC line, Nkx2.1 progenitors or their Lhx6-expressing post-mitotic progeny can be isolated via fluorescence-activated cell sorting (FACS) and subsequently used in a number of downstream applications. We also provide methods to enrich for parvalbumin (PV) or somatostatin (SST) interneuron subgroups, which may be helpful for studying aspects of fate determination or for use in therapeutic applications that would benefit from interneuron subgroup-enriched transplantations.

This protocol describes a method for generating cortical interneuron progenitors and post-mitotic interneuron precursors from mouse embryonic stem cells using a modified embryoid body-to-monolayer method. These progenitors/precursors can be used in vitro or fluorescently sorted and transplanted into neonatal neocortex for studying fate determination, or used in therapeutic applications.

Differenziazione delle cellule staminali embrionali di topo in precursori Interneurone corticale

GABAergic cortical interneurons are a heterogeneous population of cells that play critical roles in regulating the output of excitatory pyramidal neurons ...

Isolation and Culture of Embryonic Mouse Neural Stem Cells

Neural stem cells (NSCs) are multipotent and can give rise to the three major cell types of the central nervous system (CNS). In vitro culture and expansion of NSCs provide a suitable source of cells for neuroscientists to study the function of neurons and glial cells along with their interactions. There are several reported techniques for the isolation of neural stem cells from adult or embryo mammalian brains. During the microsurgical operation to isolate NSCs from different regions of the embryonic CNS, it is very important to reduce the damage to the brain cells to obtain the highest ratio of live and expandable stem cells. A possible technique for stress reduction during isolation of these cells from the mouse embryo brain is the reduction of surgical time. Here, we demonstrate a developed technique for rapid isolation of these cells from the E13 mouse embryo ganglionic eminence. Surgical procedures include harvesting E13 mouse embryos from the uterus, cutting the frontal fontanelle of the embryo with a bent needle tip, extracting the brain from the skull, microdissection of the isolated brain to harvest the ganglionic eminence, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in suspension culture to generate neurospheres.

Here, we introduce a novel microsurgical technique for the isolation of neural stem cells from the E13 mouse embryo ganglionic eminence.