Soutenu par la Fondation de bourses d&#39;études supérieures à Fazit SS, le ministère fédéral de l&#39;Education et de la Recherche (BMBF 01 EO 0803) à Ko et la Commission européenne FP7 Grant PIRG08-GA-2010-276989, Neurex, et la fondation allemande de recherche Grant SCHA 1442 / 3-1 à CS Les auteurs n&#39;ont pas de conflits d&#39;intérêts financiers.

1. Isolement et placage de Mixed cellules corticales Mixte isolement des cellules corticales des cultures d&#39;astrocytes peuvent être effectuées à l&#39;aide de P1 à P4 souriceaux. Afin de parvenir à un bon densité des astrocytes, il est nécessaire d&#39;utiliser 4 cortex de souris chiot par T75 flacon de culture tissulaire. Par conséquent, les volumes dans le protocole suivant sont calculés pour une préparation de cellules en utilisant 4 souriceaux. <ol><li> Avant de commencer la procédure de dissection, préchauffer 30 ml de milieu de culture d&#39;astrocytes (DMEM, taux élevé de glucose + 10% de sérum de sérum de veau foetal + 1% de pénicilline / streptomycine; voir tableau) à 37 ° C. Revêtez un flacon T75 avec 20 ml de poly-D-lysine (PDL) à une concentration de 50 pg / ml dans l&#39;eau de culture de cellules de qualité pendant 1 heure à 37 ° C dans l&#39;incubateur CO 2. </li><li> Pour la procédure de dissection, préparer tous les réactifs et le matériel nécessaires. Vous aurez besoin: ciseaux chirurgicaux, pinces fines lisses, pinces à bout plat, serviettes en papier, sac à déchets, éthanol à 70% d&#39;und 2 plats de dissection (3,5 cm de diamètre) sur la glace remplie de 2 ml de HBSS chacun. </li><li> Tenir délicatement et pulvériser la tête et du cou de la souris chiot avec de l&#39;éthanol à 70%. L&#39;animal est sacrifié par décapitation à l&#39;aide des ciseaux. </li><li> Effectuer une incision médiane, arrière en avant, le long du cuir chevelu pour révéler le crâne. </li><li> Couper le crâne soigneusement à partir du cou vers le nez. Deux coupes supplémentaires sont effectués pour permettre un accès plus vers le cerveau: La première coupe est faite antérieure des bulbes olfactifs, l&#39;autre inférieure du cervelet de débrancher le crâne de la base du crâne. </li><li> En utilisant la pince à bout plat, les volets crâniens sont doucement basculé sur le côté et le cerveau est prélevé et placé dans le premier plat dissection rempli avec du HBSS. Placer le plat de retour sur la glace et continuer à récolter les 4 cerveaux. </li><li> Le reste de la procédure de dissection est effectuée sous une loupe binoculaire. Tout d&#39;abord, les bulbes olfactifs et le cervelet sont éliminés en utilisant l&#39;amendePince à dissection (figure 1B). </li><li> Afin de récupérer les cortex, saisir l&#39;extrémité postérieure du cerveau avec les pinces fines, effectuer une incision médiane entre les deux hémisphères, insérez un second jeu de pinces pour le bosquet créé et décoller la structure en forme de plaque du cortex de la cerveau. </li><li> Disséquer soigneusement les méninges des hémisphères du cortex en tirant avec une pince fine (figure 1D). Cette étape permet d&#39;éviter la contamination de la culture finale des astrocytes par les cellules méningées et les fibroblastes. Transférez les hémisphères du cortex préparés dans le deuxième plat rempli avec du HBSS et le retourner sur la glace. Continuer en conséquence avec les 4 corticales. </li><li> Enfin, couper chaque hémisphère du cortex en petits morceaux à l&#39;aide des lames tranchantes (environ 4 à 8 fois). </li><li> Dans des conditions stériles, des morceaux du cortex transfert dans un tube Falcon de 50 ml et ajouter HBSS pour un volume total de 22,5 ml. </li><li> Ajouter 2,5 ml de trypsine, mélange 2,5% et incuberle tissu dans le bain d&#39;eau à 37 ° C pendant 30 min. Mélanger en agitant de temps toutes les 10 min. </li><li> Centrifuger pendant 5 min à 300 xg à granulés de morceaux de tissus de cortex. </li><li> Retirez délicatement le surnageant par décantation. Afin d&#39;éviter de perdre le culot de tissus vous pouvez retenir mécaniquement à l&#39;aide d&#39;une pipette. Dissocier le tissu dans une suspension cellulaire unique en ajoutant 10 ml Milieu de pipetage astrocytes et vigoureuse à l&#39;aide d&#39;une pipette en plastique 10 ml jusqu&#39;à morceaux de tissu sont dissociées en cellules individuelles (20 à 30 fois). Réglez le volume à 20 ml en utilisant un milieu placage astrocytes. Vous pouvez la preuve de la dissociation des tissus du cortex dans des cellules individuelles en comptant l&#39;aide d&#39;un hématocytomètre. Une préparation de 4 cortex de souris chiot doit rendement de 10-15 x 10 6 cellules dissociées simples. </li><li> Aspirer PDL du flacon T75 culture, plaque de la suspension de cellules dissociées unique et incuber à 37 ° C dans l&#39;incubateur CO 2. </li></ol> 2. L&#39;obtention d&#39;un Enriched Culture Astrocyte <ol><li> Changer les 2 moyennes jours après l&#39;étalement des cellules corticales mixtes et les 3 jours suivants. </li><li> Après 7 à 8 jours, alors que les astrocytes sont les microglies confluentes et superposition s&#39;asseoir exposé sur la couche d&#39;astrocytes ou sont déjà détachés de la couche d&#39;astrocytes (figure 2), agiter le flacon T75 à 180 rpm pendant 30 min sur un agitateur orbital pour enlever la microglie. Jeter le surnageant contenant la microglie ou si vous souhaitez à la culture et à examiner les microglies, il ralentit ou plaque de culture 28,29. </li><li> Ajouter 20 ml de milieu de culture frais astrocytes et continuer en agitant à 240 tours par minute pendant 6 heures pour éliminer les cellules précurseurs d&#39;oligodendrocytes (OPC). Étant donné que certains pays producteurs de pétrole ne sera pas complètement se détacher de la couche d&#39;astrocytes, continuer à agiter vigoureusement à la main pendant 1 minute afin d&#39;éviter la contamination OPC. Jeter le surnageant ou le tourner vers le bas et plat, si vous le souhaitez à la culture OPC 29. </li><li> Rincez le reste confluent couche d&#39;astrocytes deux fois avec du PBS, aspirer le PBS, ajouter 5 ml de trypsine-EDTA et incuber dans l&#39;incubateur CO 2 à 37 ° C. Vérifiez détachement des astrocytes toutes les 5 min et appliquer détachement des astrocytes en frappant le ballon dans la paume de votre main (2-3 fois). </li><li> Après astrocytes sont détachées du flacon de culture, ajouter 5 ml de milieu de culture des astrocytes, cellules de spin à 180 xg pendant 5 minutes, aspirer le surnageant et ajouter 40 ml de milieu frais placage astrocytes. Un flacon de culture tissulaire T75 devrait produire environ 1x10 6 cellules après la scission première cellule. Cellules de la plaque dans deux flacons T75 culture et incuber à 37 ° C dans l&#39;incubateur CO 2. Changer le milieu tous les 2 à 3 jours. </li><li> 12-14 jours après les astrocytes première division sont étalées dans la concentration cellulaire appropriée 24-48 heures avant de réaliser l&#39;expérience. Un flacon de culture tissulaire T75 devrait produire environ 1,5 à 2 x 10 6 cellules après la scission deuxième cellule. </li></ol>

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Aucun conflit d&#39;intérêt déclaré.

La méthode décrite ici est basée sur la préparation des cultures d&#39;astrocytes à partir de cerveaux de rongeurs nouveau-nés, initialement décrite par McCarthy et de Vellis en 1980 27. La méthode modifiée de l&#39;isolement et la culture des astrocytes corticaux de P1 à P4 postnatal du cerveau de souris présentée ici est rapide, rendements pures astrocytes primaires et est hautement reproductible. Cette technique peut facilement être transférée à isoler les astrocytes à partir d&#39;autres ...

Les astrocytes sont un type cellulaire très abondante dans le système nerveux central (SNC). Le ratio des astrocytes aux neurones est de 1:3 dans le cortex des souris et des rats, alors il ya 1,4 astrocytes par neurone dans le cortex humain 1. L&#39;intérêt pour la fonction des astrocytes a augmenté de façon spectaculaire ces dernières années. Une des principales fonctions des astrocytes est leur rôle dans le soutien structural et métabolique des neurones 2,3. Rôles récemment découverts pour astrocytes couvrent un large éventail de fonctions. Il s&#39;agit notamment de guidage de la migration des axones et des neuroblastes développement au cours du développement de certaines 4-6, fonctions de la transmission synaptique, la force des synapses et de traitement d&#39;informations par des circuits neuronaux 7-9, les rôles de barrière hémato-encéphalique (BHE) formation 10 et 11-13 intégrité et la réglementation des 14 tonus cérébrovasculaire. Une autre caractéristique importante des astrocytes est leur réponse à une blessure. Sous astrocyt conditions pathologiqueses devenue plus réactive et augmentent l&#39;expression de la protéine intermédiaire filament gliale fibrillaire acide (GFAP) et inhibiteurs de la matrice extracellulaire (ECM) 15,16 protéines. Astrocytes réactifs délimiter le site de la lésion des tissus sains en formant une cicatrice gliale, qui se compose principalement d&#39;astrocytes protéines de la MEC sécrétées de la chondroïtine sulfate protéoglycanes (GCEP) de la famille, les principaux facteurs qui inhibent la régénération axonale après une lésion du SNC 15-17. Les astrocytes proviennent gliales radiales (RG), les cellules lors de l&#39;embryogenèse fin et au début de la vie postnatale. Après la spécification des astrocytes a eu lieu, précurseurs astrocytaires migrer vers leurs positions finales, où ils commencent le processus de différenciation terminale. In vivo, les astrocytes semblent être mature trois à quatre semaines après la naissance, comme indiqué par leur morphologie typique 18,19. Une sous-population de cellules RG convertir en zone sous-ventriculaire (astrocytes de type cellules B). Both, RG et les cellules de type B fonctionnent comme des astrocytes de type cellules souches neurales (NSC) au cours du développement et chez l&#39;adulte, respectivement. Comme les astrocytes, cellules RG et de type B expriment également le transporteur du glutamate astrocytes spécifique (GLAST), le cerveau des lipides-binding protein (BLBP), et de la GFAP, ce qui indique que ces marqueurs ne peuvent pas être exclusivement utilisé pour marquer spécifiquement les astrocytes adultes. Contrairement aux adultes, les astrocytes parenchymateux, qui ne se divisent pas dans le cerveau, RG sain et cellules de type B présentent un potentiel de cellules souches telles que la capacité d&#39;auto-renouvellement. Le dérèglement des astrocytes a été impliquée dans de nombreuses pathologies, y compris la maladie d&#39;Alzheimer 20,21, la maladie de Huntington 22, la maladie de Parkinson 23, le syndrome de Rett 24 et la maladie d&#39;Alexander 25. Par ailleurs, les astrocytes réagir à toutes les injures du système nerveux central, conduisant à l&#39;activation des astrocytes et astrocytaire formation cicatrice gliale 16,26. La cicatrice gliale astrocytaire qui forme tr cerveau suivantelésions de la moelle épinière ou auma est considéré comme le principal obstacle empêchant la régénération neuronale 15. Le développement de méthodes fiables d&#39;isoler et de maintenir des populations purifiées de cellules a été essentielle à notre compréhension du système nerveux. Travaux pionniers de McCarthy et de Vellis permet aux enquêteurs à ce jour pour préparer des cultures d&#39;astrocytes presque purs de tissu de rat nouveau-né 27. On a beaucoup appris sur la biologie des astrocytes en utilisant cette méthode, qui est présentée ici sous une forme légèrement modifiée pour isoler les astrocytes corticaux de souris. En complément des études in vivo, les astrocytes, ainsi que le milieu conditionné obtenu en utilisant le décrit la culture in vitro, sont des outils précieux pour mieux avoir un aperçu des fonctions astrocytes.

<table border="1"><tbody><tr><td> Nom de la solution de travail </td><td> Entreprise </td><td> Numéro de catalogue </td><td> La concentration finale </td></tr><tr><td></td><td></td><td></td><td> Milieux de culture d&#39;astrocytes </td></tr><tr><td> DMEM, glycémie élevée </td><td> Life Technologies </td><td> 31966-021 </td><td></td></tr><tr><td> FBS, inactivé à la chaleur </td><td> Life Technologies </td><td> 10082-147 </td><td> Concentration finale: 10% </td></tr><tr><td> Pénicilline-streptomycine </td><td> Life Technologies </td><td> 15140-122 </td><td> Concentration finale: 1% </td></tr><tr><td></td><td></td><td></td><td> Solution pour creuser le tissu cérébralestion </td></tr><tr><td> HBSS </td><td> Life Technologies </td><td> 14170-088 </td><td></td></tr><tr><td> La trypsine 2,5% </td><td> Life Technologies </td><td> 15090-046 </td><td> Concentration finale: 0,25% </td></tr><tr><td></td><td></td><td></td><td> Autre </td></tr><tr><td> 70% (volume / volume) d&#39;éthanol </td><td> Roth </td><td> 9065.2 </td><td></td></tr><tr><td> Poly-D-Lysine </td><td> Millipore </td><td> A-003-E </td><td> 50 pg / ml </td></tr><tr><td> Eau </td><td> AAP </td><td> S15-012 </td><td> de qualité culture cellulaire </td></tr><tr><td> PBS </td><td> AAP </td><td> H15-002 </td><td> de qualité culture cellulaire </td></tr><tr><td> Essayez de 0,05%PSIN-EDTA </td><td> Life Technologies </td><td> 25300-062 </td><td></td></tr><tr><td> Filtre de 0,45 um stérile </td><td> Sartorius </td><td> 16555 </td><td></td></tr><tr><td> 3,5 cm boîte de Pétri </td><td> BD Falcon </td><td> 353001 </td><td></td></tr><tr><td> Falcon de 15 ml Tube </td><td> BD Falcon </td><td> 352096 </td><td></td></tr><tr><td> Falcon de 50 ml Tube </td><td> BD Falcon </td><td> 352070 </td><td></td></tr><tr><td> 75 cm 2 flacon de culture tissulaire </td><td> BD Falcon </td><td> 353136 </td><td></td></tr><tr><td> Forceps, bien </td><td> Dumont </td><td> 2-1032, 2-1033 </td><td> # 3c; # 5 </td></tr><tr><td> Forceps, embout plat </td><td> KLS Martin </td><td> 12-120-11 </td><td></td></tr><tr><td> 13 cm ciseaux chirurgicaux </td><td> Aesculap </td><td> BC-140-R </td><td></td></tr><tr><td> Stéréomicroscope </td><td> Leica </td><td></td><td> MZ7.5 </td></tr><tr><td> Stéréomicroscope + Caméra </td><td> Leica </td><td></td><td> MZ16F; DFC320 </td></tr><tr><td> Microscope + Caméra </td><td> Zeiss; Canon </td><td></td><td> Primo Vert; PowerShot A650 IS </td></tr><tr><td> Centrifuger </td><td> Eppendorf </td><td> 5805000.017 </td><td> Centrifuge5804R </td></tr><tr><td> Agitateur orbital </td><td> Thermo Scientific </td><td> SHKE 4450-1CE </td><td> MaxQ 4450 </td></tr><tr><td> Bain d&#39;eau </td><td> Julabo </td><td></td><td> SW20; 37 ° C </td></tr></tbody></table>

isolation and culture of mouse cortical astrocytes

Les astrocytes sont un type de cellule abondant dans le cerveau des mammifères, mais il reste beaucoup à apprendre sur leurs caractéristiques moléculaires et fonctionnelles. Astrocytes in vitro systèmes de culture cellulaire peut être utilisé pour étudier les fonctions biologiques de ces cellules gliales dans le détail. Ce protocole vidéo montre comment obtenir les astrocytes pures par l&#39;isolement et la culture des cellules corticales mixtes de souriceaux. La méthode est basée sur l&#39;absence de neurones viables et la séparation des astrocytes, les oligodendrocytes et les microglies, les trois principales populations de cellules gliales du système nerveux central, de la culture. Images représentatifs au cours des premiers jours de culture en évidence la présence d&#39;une population cellulaire mixte et indiquer le point temporel, lorsque les astrocytes deviennent confluentes et ne doit être séparé de la microglie et les oligodendrocytes. De plus, nous démontrons la pureté et de la morphologie des astrocytes astrocytes en culture en utilisant des colorations immunocytochimiques pour bien établies etnouvellement décrit marqueurs astrocytaires. Ce système de culture peut être facilement utilisé pour obtenir des astrocytes de souris pures et astrocytes conditionné moyen pour étudier divers aspects de la biologie des astrocytes.

Après isolement du cerveau de la souris complète (figure 1A), le cervelet et des bulbes olfactifs doivent être éliminés (figure 1B). Les cortex sont pelées de la tige de cerveau de souris (figure 1C) et les méninges du cortex individuel (figure 1D ») sont soigneusement retirés (figure 1E). Méninges sont évidents par le système de l&#39;artère méningée et les résultats élimination incomplète de la contamination de la cu...

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

Isolation and Culture of Mouse Cortical Astrocytes

Les astrocytes ont été reconnus pour être des cellules polyvalentes qui participent au processus biologiques fondamentaux qui sont essentiels pour le développement normal du cerveau et la fonction, et réparation du système nerveux central. Nous présentons ici une procédure rapide afin d&#39;obtenir des cultures pures astrocytes de souris pour étudier la biologie de cette classe importante de cellules du système nerveux central.

Isolement et culture des astrocytes corticaux de souris

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

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Neuroscience

90.8K vues.  University of Freiburg. Les astrocytes ont été reconnus pour être des cellules polyvalentes qui participent au processus biologiques fondamentaux qui sont essentiels pour le développement normal du cerveau et la fonction, et réparation du système nerveux central. Nous présentons ici une procédure rapide afin d'obtenir des cultures pures astrocytes de souris pour étudier la biologie de cette classe importante de cellules du système nerveux central.

Vidéo: 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.

Nucléofection et de la culture primaire de neurones embryonnaires de souris hippocampe et cortex

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

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Neurosciences

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.

Lectine à base d&#39;isolement et de la culture des motoneurones embryonnaires de la souris

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.

Didermique co-culture de neurones de rat corticale primaire et des cellules gliales

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.

L&#39;isolement et la culture de neurones de l&#39;hippocampe de souris pendant la grossesse

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.

Isolement et culture de cellules humaines fongiformes papilles gustatives

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.

Modélisation astrocytome pathogenèse<em&gt; In Vitro</em&gt; Et<em&gt; In Vivo</em&gt; Utilisation corticale astrocytes ou cellules souches neurales à partir de, souris génétiquement modifiées conditionnelle

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.

Différences entre les sexes dans la souris hippocampiques astrocytes après<em&gt; In-Vitro</em&gt; ischémie

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.

Isolement et culture des adultes cellules souches neurales de la Subcallosal Zone Souris

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.

Différenciation des cellules souches embryonnaires de souris en précurseurs des interneurones corticaux

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.