Etape 1 <ol><li> Plaque mitomycine C ggrowth-inhibée (10 pg / ml pendant 2,5 heures) MS5 cellules à une densité de 70.000 / cm2 sur la gélatine-couché (0,01% pour 30 minutes) 6 assiettes bien dans α-MEM médias. </li><li> Lorsque les cellules sont attachés et ont formé une monocouche (plus de la croissance de nuit), passer à SRM. </li><li> Manuellement isoler les colonies de cellules ES à partir des cultures de cellules ES à l&#39;aide d&#39;une seringue avec une aiguille de 27 G ½. </li><li> Tritrurate les colonies soigneusement avec une pointe de 1 ml bleu et plaque à faible densité sur les cellules MS5 (habituellement 2-3 colonies par plaque de 6 puits). </li></ol> Remarque: Les cellules ES peuvent également être prises à partir de propagation enzymatique utilisant la collagénase ou de trypsine. Etape 2 <ol><li> Cultiver les cellules ES sur la MS5 mangeoires pendant 14-21 jours, jusqu&#39;à rosette contenant former des colonies. </li></ol> Remarque: Les médias changent souvent. Les rosettes sont généralement à la périphérie des colonies et leur formation peut être grandement améliorée si 300-500 ng / ml Caboche est ajouté à la MRS (3). Dans certains protocoles de différenciation, les MRS peuvent être échangées avec N2-A médias et complétée par la croissance ou d&#39;autres facteurs afin de promouvoir la spécification des cellules (2-5). Étape 3 <ol><li> Isoler et ramasser les rosettes avec une aiguille de calibre 27 ½ sous un microscope. </li></ol> Remarque: Evitez de couper et ramasser l&#39;MS5 cellules stromales. Si les colonies sont emballés avec des rosettes, on peut également isoler la colonie entière. Etape 4 <ol><li> Aspirer les grappes de rosettes, les tritrurate soigneusement avec une pointe de 1 ml bleu, et la plaque eux sur la poly-L-ornithine (0,001%) et de la fibronectine (1 pg / ml) ou de la laminine (1 pg / ml) à revêtement 6 puits dans N2-Un média habituellement complétées avec bFGF (20 ng / ml). Les rosettes seront réforme et l&#39;expansion. </li></ol> Remarque: Pour améliorer la survie des cellules, on peut l&#39;assiette du petites grappes dans les gouttelettes d&#39;augmenter la densité cellulaire. Cette étape peut également être modifié en complétant la N2-Un média avec une croissance supplémentaires ou d&#39;autres facteurs afin de promouvoir la spécification des cellules (2-5).

<ol>
	<li>Kawasaki, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+midbrain+dopaminergic+neurons+from+ES+cells+by+stromal+cell-derived+inducing+activity.">Induction of midbrain dopaminergic neurons from ES cells by stromal cell-derived inducing activity.</a> Neuron. 28, 31-40 (2000).</li><li>Perrier, A. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivation+of+midbrain+dopamine+neurons+from+human+embryonic+stem+cells.">Derivation of midbrain dopamine neurons from human embryonic stem cells.</a> Proc Natl Acad Sci U S A. 101, 12543-12548 (2004).</li><li>Pruszak, J., Isacson, O., Sullivan, S., Cowan, C., Eggan, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+differentiation+of+human+embryonic+stem+cells+into+dopaminergic+neurons.">Directed differentiation of human embryonic stem cells into dopaminergic neurons.</a> Human Embryonic Stem Cells: The Practical Handbook. , (2007).</li><li>Pruszak, J., Sonntag, K. C., Aung, M. H., Sanchez-Pernaute, R., Isacson, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Markers+and+methods+for+cell+sorting+of+human+embryonic+stem+cell-derived+neural+cell+populations.">Markers and methods for cell sorting of human embryonic stem cell-derived neural cell populations.</a> Stem Cells. 25, 2257-2268 (2007).</li><li>Sonntag, K. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+yield+of+neuroepithelial+precursors+and+midbrain-like+dopaminergic+neurons+from+human+embryonic+stem+cells+using+the+bone+morphogenic+protein+antagonist+noggin.">Enhanced yield of neuroepithelial precursors and midbrain-like dopaminergic neurons from human embryonic stem cells using the bone morphogenic protein antagonist noggin.</a> Stem Cells. 25, 411-418 (2007).</li><li>Kawasaki, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+dopaminergic+neurons+and+pigmented+epithelia+from+primate+ES+cells+by+stromal+cell-derived+inducing+activity.">Generation of dopaminergic neurons and pigmented epithelia from primate ES cells by stromal cell-derived inducing activity.</a> Proc Natl Acad Sci U S A. 99, 1580-1585 (2002).</li><li>Ko, J. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+embryonic+stem+cell-derived+neural+precursors+as+a+continuous,+stable,+and+on-demand+source+for+human+dopamine+neurons.">Human embryonic stem cell-derived neural precursors as a continuous, stable, and on-demand source for human dopamine neurons.</a> J Neurochem. 103, 1417-1429 (2007).</li><li>Hong, S., Kang, U. J., Isacson, O., Kim, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+precursors+derived+from+human+embryonic+stem+cells+maintain+long-term+proliferation+without+losing+the+potential+to+differentiate+into+all+three+neural+lineages,+including+dopaminergic+neurons.">Neural precursors derived from human embryonic stem cells maintain long-term proliferation without losing the potential to differentiate into all three neural lineages, including dopaminergic neurons.</a> J Neurochem. , (2007).</li><li>Zhang, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+subtype+specification+from+embryonic+stem+cells.">Neural subtype specification from embryonic stem cells.</a> Brain Pathol. 16, 132-142 (2006).</li><li>Pankratz, M. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+neural+differentiation+of+human+embryonic+stem+cells+via+an+obligated+primitive+anterior+stage.">Directed neural differentiation of human embryonic stem cells via an obligated primitive anterior stage.</a> Stem Cells. 25, 1511-1520 (2007).</li><li>Lazzari, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+derivation+of+neural+rosettes+from+cloned+bovine+blastocysts:+a+model+of+early+neurulation+events+and+neural+crest+specification+in+vitro.">Direct derivation of neural rosettes from cloned bovine blastocysts: a model of early neurulation events and neural crest specification in vitro.</a> Stem Cells. 24, 2514-2521 (2006).</li><li>Pomp, O., Brokhman, I., Ben-Dor, I., Reubinoff, B., Goldstein, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+peripheral+sensory+and+sympathetic+neurons+and+neural+crest+cells+from+human+embryonic+stem+cells.">Generation of peripheral sensory and sympathetic neurons and neural crest cells from human embryonic stem cells.</a> Stem Cells. 23, 923-930 (2005).</li></ol>

The authors have nothing to disclose.

Ce protocole démontre les différentes étapes de production et d&#39;isoler les cellules neuro-épithéliales des cellules ES humaines à l&#39;aide de SDIA. L&#39;application de cette méthode est multiple et a été utilisé dans de nombreux protocoles à produire des neurones spécifiés (par exemple 1, 2, 5-9). Les rosettes sont pensé pour ressembler à des cellules du tube neural avec un phénotype antérieure (2, 5, 10) et contiennent aussi des progéniteurs de la crête neurale (11, 12). De plus, ils conservent un certain niveau d...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>L-glutamine</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>25030</td>
<td>&nbsp;</td>
<tr>
<td>alpha-MEM</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>12571</td>
<td>&nbsp;</td>
<tr>
<td>penicillin/streptomycin</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>15140</td>
<td>&nbsp;</td>
<tr>
<td>Knockout-DMEM</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>10829</td>
<td>&nbsp;</td>
<tr>
<td>Knockout serum replacement</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>10828</td>
<td>&nbsp;</td>
<tr>
<td>MEM non-essential amino acid solution</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>12383</td>
<td>&nbsp;</td>
<tr>
<td>DMEM/F12</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>11330</td>
<td>&nbsp;</td>
<tr>
<td>N2-A supplement</td>
<td>&nbsp;</td>
<td>Stem Cell Technologies</td>
<td>07152</td>
<td>&nbsp;</td>
<tr>
<td>mitomycin-C</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M0503</td>
<td>&nbsp;</td>
<tr>
<td>gelatine type-A</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G1890</td>
<td>&nbsp;</td>
<tr>
<td>poly-L-ornithine</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P4957</td>
<td>0.01 % solution </td>
</tr>
<tr>
<td>laminin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>L-2020</td>
<td>&nbsp;</td>
<tr>
<td>fibronectin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>F2006</td>
<td>&nbsp;</td>
<tr>
<td>basic fibroblast growth factor (bFGF)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>13256</td>
<td>&nbsp;</td>
<tr>
<td>1 ml syringe with 27 1/2 G needle</td>
<td>&nbsp;</td>
<td>Becton Dickinson</td>
<td>309623</td>
<td>&nbsp;</td>
<tr>
<td>N2-A media</td>
<td>medium</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>DMEM/F12 + 1% N2-A supplement </td>
</tr>
<tr>
<td>Serum replacement media (SRM)</td>
<td>medium</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Knockout-DMEM + 20 % Knockout serum replacement +1% MEM non-essential amino acid solution + 2 mM L-glutamine</td>
</tr>
<tr>
<td>&#x03B1;-MEM media</td>
<td>medium</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&#x03B1;-MEM + 10 % FBS + 2 mM L-glutamine + 1%penicillin/streptomycin</td>
</tr>
<tr>
<td>MS5</td>
<td>cell line</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>stromal cells </td>
</tr>
<tr>
<td>Microscope</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>6 well plates</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>for tissue culture</td>
</tr>		</tbody>
		</table>
		</div>
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es cell-derived neuroepithelial cell cultures

Les cellules ES ont le potentiel de se différencier en cellules de tous les feuillets embryonnaires, ce qui en fait un outil intéressant pour le développement de nouvelles thérapies. En général, la différenciation des cellules ES suit le concept d&#39;abord générer des cellules progénitrices immatures, qui peuvent ensuite être multipliées et différenciées en maturité phénotypes cellulaires. Ceci s&#39;applique également pour les dérivés de cellules ES neurogenèse, dans lequel le développement des cellules neurales suit deux grandes étapes: d&#39;abord, la dérivation et l&#39;expansion des précurseurs immatures neuroépithéliales et deuxièmement, leur différenciation en cellules matures neurones. Une méthode courante pour produire progéniteurs neuronaux à partir des cellules ES est basé sur le corps (EB) la formation embryoïdes, qui révèle la différenciation des cellules de tous les feuillets embryonnaires, y compris neuroectoderme. Une méthode alternative et plus efficace pour induire le développement des cellules stromales neuroépithéliales utilise l&#39;activité induisant des cellules dérivées (SDIA), qui peut être atteint par les cellules ES co-culture avec l&#39;os du crâne dérivées de la moelle des cellules stromales (1). Les deux, la formation EB et SDIA, révèlent le développement de la rosette de structures semblables, qui sont pensés pour ressembler à du tube neural et / ou de la crête neurale, comme géniteurs. Les précurseurs de neurones peuvent être isolés, étendus et différenciées en neurones spécifiques et les cellules gliales en utilisant des conditions de culture définie. Ici, nous décrivons la génération et l&#39;isolement de ces rosettes en co-culture des expériences avec la ligne de cellules stromales MS5 (2-5).

Watch this Scientific Journal Video about ES Cell-derived Neuroepithelial Cell Cultures at JoVE.com

ES Cell-derived Neuroepithelial Cell Cultures

Dérivation de précurseurs d&#39;neuroépithéliales souches embryonnaires (ES), les cellules stromales en utilisant l&#39;activité induisant des cellules dérivées (SDIA).

ES Cell dérivé des cultures cellulaires neuroépithéliales

ES cells have the potential to differentiate into cells from all germ layers, which makes them an attractive tool for the development of new therapies. In ...

es-cell-derived-neuroepithelial-cell-cultures

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13.5K vues.  Harvard Medical School. Dérivation de précurseurs d'neuroépithéliales souches embryonnaires (ES), les cellules stromales en utilisant l'activité induisant des cellules dérivées (SDIA).

Vidéo: ES Cell-derived Neuroepithelial Cell Cultures

Freezing Human ES Cells

Here we demonstrate how our lab freezes HuES human embryonic stem cell lines.  A healthy, exponentially expanding culture is washed with PBS to remove residual media that could otherwise quench the Trypsin reaction.  Warmed 0.05% Trypsin-EDTA is then added to cover the cells, and the plate allowed to incubate for up to 5 mins at room temperature.  During this time cells can be observed rounding, and colonies lifting off the plate surface.  Gentle repeated pipetting will remove cells and colonies from the plate surface.  Trypsinized cells are placed in a standard conical tube containing pre-warmed hES cell media to quench remaining trypsin, and then spun.  Cells are resuspended growth media at a concentration of approximately one million cells in one mL of media, a concentration such that one frozen aliquot is sufficient to resurrect a culture on a 10cm plate.  After cells are adequately resuspended, ice cold freezing media is added at equal volume.  Cell suspensions are mixed thoroughly, aliquoted into freezing vials, and allowed to slowly freeze to -80C over 24 hours.  Frozen cells can then moved to the vapor phase of liquid nitrogen for long term storage, or remain at -80 for approximately six months.

Here we demonstrate how our lab freezes HuES human embryonic stem cell lines.

La congélation des cellules ES humaines

Here we demonstrate how our lab freezes HuES human embryonic stem cell lines.  A healthy, exponentially expanding culture is washed with PBS to remove ...

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Biologie

Human ES cells: Starting Culture from Frozen Cells

Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock.  First, a one to two day old ten cm plate of  approximately one (to two) million irradiated mouse embryonic fibroblast feeder cells is rinsed with HuES media to remove residual serum and cell debris, and then HuES media added and left to equilibrate in the cell culture incubator.  A frozen vial of cells from long term liquid nitrogen storage or a -80C freezer is sourced and quickly submerged in a 37C water bath for quick thawing.  Cells in freezing media are then removed from the vial and placed in a large volume of HuES media.  The large volume of HuES media facilitates removal of excess serum and DMSO, which can cause HuES human embryonic stem cells to differentiate.  Cells are gently spun out of suspension, and then re-suspended in a small volume of fresh HuES media that is then used to seed the MEF plate.  It is considered important to seed the MEF plate by gently adding the HuES cells in a drop wise fashion to evenly disperse them throughout the plate.  The newly established HuES culture plate is returned to the incubator for 48 hrs before media is replaced, then is fed every 24 hours thereafter.

Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock.

Les cellules ES humaines: la culture à partir de cellules congelées

Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock.  First, a one to two day old ten cm plate of  ...

Propagation of Human Embryonic Stem (ES) Cells

Propagation of Human Embryonic Stem ES Cells

Yeah, so I'm Lauren Deon. I'm in charge of the Human Amgen stem cell co facility at MGH. Yeah, so I'm Lauren, I'm in charge of the Human AM stem cell co facility at MGH.So I've been working with human Amgen stem cells for years now since I was a postdoc in George De Lab before, from four, four years. And so right now we have two cell lines, H one and H nine, which are both from Y cell. And so the way we are expanding or maintaining these cells in culture is by, at least the way we are splitting these cells is by using collagenous.So what I usually do is for the first few passage after throwing the cells, I use a technical, the picking colonies. So in this case, I can check under the microscope which colonies are good and differentiate and nice shape, and I will collect only these colonies and I will cut them in few pieces and put them back in, in a fresh flat. And the, the colonies that look kind of differentiate it are just left them alone.The other way to pass the cells is by using trypsin. So I don't use this technique here. I think collagenase is a little bit safer because human being stem cells don't like to be individualized or they don't like to be alone.So if you do trypsin, if you use trypsin for a little bit too long, if you use it for like three to four minutes, which is the way we use strips in for other type of cells, human stem cells will be individualized and most of them will die or just differentiate or will not attach to the myth. So the idea is you have to keep the cells as clumps. You don't want them to be alone in the in, in the flask.So using collagenase, the advantage is to keep cells as plants. Using chipsy, you just select for cells that will survive this very, very harsh process of g opsonization. So these cells are usually very highly proliferative.So you get, you can adapt your cells to this tripsin process. The problem is that you have cells that they behave differently, they go much faster. And one risk is that you can get some commercial abnormality to, to this, to this very high division process.So you say that ization can cause selection of cells in culture for most proliferative cells.Right. More ative and probably also higher survival rates than other cells. So after proliferation, basically culture after proliferation in tripsin, after several passages become a different culture, these different properties.So it's, it's hard to figure out if they are exactly identical. If you do the test, the usual test, let's say you check markers, regular markers of end differentiated cells like OC four, nano one 60 SC four C, C3, all this molecule might still be expressed. If you do TER formation, this cells might still differentiate and be formed like al like normal cells.If you look at the karyotype, you might have some abnormality, but you might still have a normal karyotype. The, the, the selective advantage might be due to just a mutation that you cannot see by doing a karyotype. So the fact that they grow much faster is by itself I think a little bit worser.And if we want to use these cells for transplantation at some point, I would definitely not use this type of cells.Okay. So would you like to tell me what are the main technical problems with using this technique for, for passages of cells? So using S, yes.It's just a little bit longer and also it's, since the cells are not going as fast, it's just much longer to get a high number of cells to do some experiment. So it's just less convenient I would say. Would you like to tell me about few words about the goals of your experiment?How do you use CS cells? Okay, so my main project here is to get endothelial cells from human AB stem cells. And the idea is to be able to make blood.And we are working a lot with tissue bioengineering labs and one of their goal is to make tissue in vitro. But one of the limitation to this is the lack of blood vessels going through this tissues. So if we can bring endothelial cells or put endothelial cells in this tissue, we would have better growth of this tissue.So the idea few sources of cells are known for endothelial cells like cold blood or pressure blood, but it would be interesting to have another source of cells like human years. And the advantage is since human years can be expanded and most infinitely, you could modify these cells, make some inducible system and be able to use it. Then in your final product, your material cells, in this case.

Propagation des souches embryonnaires humaines (ES) Cellules

Preparation of Neuronal Cultures from Midgastrula Stage Drosophila Embryos

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos and their dechorionation using bleach are demonstrated. Using a glass pipet attached to a mouth suction tube, we illustrate the removal of all cells from single embryos. The method for dispersing cells from each embyro into a small (5 l) drop of medium on an uncoated glass coverslip is demonstrated. A view through the microscope at 1 hour after plating illustrates the preferred cell density. Most of the cells that survive when grown in defined medium are neuroblasts that divide one or more times in culture before extending neuritic processes by 12-24 hours. A view through the microscope illustrates the level of neurite outgrowth and branching expected in a healthy culture at 2 days in vitro. The cultures are grown in a simple bicarbonate based defined medium, in a 5% CO2 incubator at 22-24&deg;C. Neuritic processes continue to elaborate over the first week in culture and when they make contact with neurites from neighboring cells they often form functional synaptic connections. Neurons in these cultures express voltage-gated sodium, calcium, and potassium channels and are electrically excitable. This culture system is useful for studying molecular genetic and environmental factors that regulate neuronal differentiation, excitability, and synapse formation/function.

This video demonstrates the preparation of primary neuronal cultures from midgastrula stage Drosophila embryos. Views of live cultures show cells 1 hour after plating and differentiated neurons after 2 days of growth in a bicarbonate-based defined medium. The neurons are electrically excitable and form synaptic connections.

Préparation des cultures de neurones d&#39;embryons de drosophile Midgastrula scène

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos ...

Mouse Epidermal Neural Crest Stem Cell (EPI-NCSC) Cultures

EPI-NCSC are remnants of the embryonic neural crest in an adult location, the bulge of hair follicles. They are multipotent stem cells that have the physiological property to generate a wide array of differentiated cell types, including neurons, nerve supporting cells, smooth muscle cells, bone/cartilage cells and melanocytes. EPI-NCSC are easily accessible in the hairy skin and can be isolated as a highly pure population of stem cells. This video provides a detailed protocol for preparing mouse EPI-NCSC cultures from whisker follicles. The whisker pad of an adult mouse is removed, and whisker follicles dissected. The follicles are then cut longitudinally and subsequently transversely above and below the bulge region. The bulge is removed from the collagen capsule and placed in a culture plate. EPI-NCSC start to emigrate from the bulge explants 3 to 4 days later.

Mouse Epidermal Neural Crest Stem Cell EPI-NCSC Cultures

Here we show our method to isolate mouse epidermal neural crest stem cells (EPI-NCSC). Technique involves micro-dissecting whisker follicles, isolating the bulge and placeing it into tissue culture. EPI-NCSC start to emigrate from bulge explants onto the substratum within 3 - 4 days.

Souris épidermique Neural Stem Cell Crest (EPI-NCSC) cultures

EPI-NCSC are remnants of the embryonic neural crest in an adult location, the bulge of hair follicles. They are multipotent stem cells that have the ...

Primary Dissociated Midbrain Dopamine Cell Cultures from Rodent Neonates

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation from systemic input from elsewhere in the brain. In our lab, we use these neurons to assess dopamine release kinetics using carbon fiber amperometry, as well as expression levels of dopamine related genes and proteins using quantitative PCR and immunocytochemistry. In this video, we show you how we generate these cultures from rodent neonates.
The process involves several steps, including the plating of cortical glial astrocytes, the conditioning of neuronal cell culture media by the glial substrate, the dissection of the midbrain in neonates, the digestion, extraction and plating of dopamine neurons and the addition of neurotrophic factors to ensure cell survival.
The applications suitable for such a preparation include electrophysiology, immunocytochemistry, quantitative PCR, video microscopy (i.e., of real-time vesicular fusion with the plasma membrane), cell viability assays and other toxicological screens.

Primary dissociated midbrain dopamine cell cultures allow for the study of presynaptic characteristics of dopamine neurons. They can be used to monitor real-time dopamine release kinetics and protein/mRNA levels of regulators of dopamine exocytosis. Here, we show you how to generate these cultures from rodent neonates.

Primaire Dissocié cultures Mésencéphale dopamine cellulaire du nouveau-né contre les rongeurs

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation ...

Embryonic Stem Cell-Derived Endothelial Cells for Treatment of Hindlimb Ischemia

Peripheral arterial disease (PAD) results from narrowing of the peripheral arteries that supply oxygenated blood and nutrients to the legs and feet, This pathology causes symptoms such as intermittent claudication (pain with walking), painful ischemic ulcerations, or even limb-threatening gangrene. It is generally believed that the vascular endothelium, a monolayer of endothelial cells that invests the luminal surface of all blood and lymphatic vessels, plays a dominant role in vascular homeostasis and vascular regeneration. As a result, stem cell-based regeneration of the endothelium may be a promising approach for treating PAD.In this video, we demonstrate the transplantation of embryonic stem cell (ESC)-derived endothelial cells for treatment of unilateral hindimb ischemia as a model of PAD, followed by non-invasive tracking of cell homing and survival by bioluminescence imaging. The specific materials and procedures for cell delivery and imaging will be described. This protocol follows another publication in describing the induction of hindlimb ischemia by Niiyama et al.1

The surgical procedure for delivery of embryonic stem cell-derived endothelial cells to the ischemic hindlimb is demonstrated, with non-invasive tracking by bioluminescence imaging.

Les cellules souches embryonnaires des cellules dérivées endothéliales pour traitement de l&#39;ischémie des membres postérieurs

Peripheral arterial disease (PAD) results from narrowing of the peripheral arteries that supply oxygenated blood and nutrients to the legs and feet, This ...

Preparation of Aplysia Sensory-motor Neuronal Cell Cultures

The nervous system of the marine mollusk Aplysia californica is relatively simple, consisting of approximately 20,000 neurons. The neurons are large (up to 1 mm in diameter) and identifiable, with distinct sizes, shapes, positions and pigmentations, and the cell bodies are externally exposed in five paired ganglia distributed throughout the body of the animal. These properties have allowed investigators to delineate the circuitry underlying specific behaviors in the animal1. The monosynaptic connection between sensory and motor neurons is a central component of the gill-withdrawal reflex in the animal, a simple defensive reflex in which the animal withdraws its gill in response to tactile stimulation of the siphon. This reflex undergoes forms of non-associative and associative learning, including sensitization, habituation and classical conditioning. Of particular benefit to the study of synaptic plasticity, the sensory-motor synapse can be reconstituted in culture, where well-characterized stimuli elicit forms of plasticity that have direct correlates in the behavior of the animal2,3. Specifically, application of serotonin produces a synaptic strengthening that, depending on the application protocol, lasts for minutes (short-term facilitation), hours (intermediate-term facilitation) or days (long-term facilitation). In contrast, application of the peptide transmitter FMRFamide produces a synaptic weakening or depression that, depending on the application protocol, can last from minutes to days (long-term depression). The large size of the neurons allows for repeated sharp electrode recording of synaptic strength over periods of days together with microinjection of expression vectors, siRNAs and other compounds to target specific signaling cascades and molecules and thereby identify the molecular and cell biological steps that underlie the changes in synaptic efficacy.
An additional advantage of the Aplysia culture system comes from the fact that the neurons demonstrate synapse-specificity in culture4,5. Thus, sensory neurons do not form synapses with themselves (autapses) or with other sensory neurons, nor do they form synapses with non-target identified motor neurons in culture. The varicosities, sites of synaptic contact between sensory and motor neurons, are large enough (2-7 microns in diameter) to allow synapse formation (as well as changes in synaptic morphology) with target motor neurons to be studied at the light microscopic level.
In this video, we demonstrate each step of preparing sensory-motor neuron cultures, including anesthetizing adult and juvenile Aplysia, dissecting their ganglia, protease digestion of the ganglia, removal of the connective tissue by microdissection, identification of both sensory and motor neurons and removal of each cell type by microdissection, plating of the motor neuron, addition of the sensory neuron and manipulation of the sensory neurite to form contact with the cultured motor neuron.

Preparation of Aplysia Sensory-motor Neuronal Cell Cultures

Primary cultures of Aplysia sensory-motor neurons provide a model preparation for studying synapse formation and synaptic plasticity in vitro. This video demonstrates the identification and microdissection of sensory and motor neurons from Aplysia ganglia as well as the methods for establishing and maintaining sensory-motor neurons in culture.

Préparation des Aplysie Sensori-motrice cultures de cellules neuronales

The nervous system of the marine mollusk Aplysia californica is relatively simple, consisting of approximately 20,000 neurons.  The neurons are large (up ...

Primary Cell Cultures from Drosophila Gastrula Embryos

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos. In brief, a large amount of staged embryos from young and healthy flies are collected, sterilized, and then physically dissociated into a single cell suspension using a glass homogenizer. After being plated on culture plates or chamber slides at an appropriate density in culture medium, these cells can further differentiate into several morphologically-distinct cell types, which can be identified by their specific cell markers. Furthermore, we present conditions for treating these cells with double stranded (ds) RNAs to elicit gene knockdown. Efficient RNAi in Drosophila primary cells is accomplished by simply bathing the cells in dsRNA-containing culture medium. The ability to carry out effective RNAi perturbation, together with other molecular, biochemical, cell imaging analyses, will allow a variety of questions to be answered in Drosophila primary cells, especially those related to differentiated muscle and neuronal cells.

Primary Cell Cultures from Drosophila Gastrula Embryos

We provide a detailed protocol for preparing primary cells dissociated from Drosophila embryos. The ability to carry out the effective RNAi perturbation, together with other molecular, biochemical and cell imaging methods will allow a variety of questions to be addressed in Drosophila primary cells.

Cultures de cellules primaires à partir Drosophile Embryons Gastrula

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos.   In brief, a large amount of staged ...

Subcloning Plus Insertion (SPI) - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors

Gene targeting refers to the precise modification of a genetic locus using homologous recombination. The generation of novel cell lines and transgenic mouse models using this method necessitates the construction of a &#8216;targeting&#8217; vector, which contains homologous DNA sequences to the target gene, and has for many years been a limiting step in the process. Vector construction can be performed in vivo in Escherichia coli cells using homologous recombination mediated by phage recombinases using a technique termed recombineering. Recombineering is the preferred technique to subclone the long homology sequences (>4kb) and various targeting elements including selection markers that are required to mediate efficient allelic exchange between a targeting vector and its cognate genomic locus. Typical recombineering protocols follow an iterative scheme of step-wise integration of the targeting elements and require intermediate purification and transformation steps. Here, we present a novel recombineering methodology of vector assembly using a multiplex approach. Plasmid gap repair is performed by the simultaneous capture of genomic sequence from mouse Bacterial Artificial Chromosome libraries and the insertion of dual bacterial and mammalian selection markers. This subcloning plus insertion method is highly efficient and yields a majority of correct recombinants. We present data for the construction of different types of conditional gene knockout, or knock-in, vectors and BAC reporter vectors that have been constructed using this method. SPI vector construction greatly extends the repertoire of the recombineering toolbox and provides a simple, rapid and cost-effective method of constructing these highly complex vectors.

Subcloning Plus Insertion SPI - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors

Gene targeting methodologies can be used to generate transgenic mice with knockout, knock-in and tagged alleles. Here, we describe an improved method of recombineering in E. coli, that we term &#8216;subcloning plus insertion&#8217;, which can be used to generate custom gene targeting vectors rapidly.

Sous-clonage plus insertion (SPI) - Un Recombineering nouveau procédé pour la construction rapide de ciblage de gène Vecteurs

Gene targeting refers to the precise modification of a genetic locus using homologous recombination. The generation of novel cell lines and transgenic ...