Ce travail a été soutenu par NIH EB007278.

Faire un CBF BFC sont réalisés par moulage polydiméthylsiloxane (PDMS) sur une 4 &quot;plaquette master silicium. Faire une plaquette master <ol><li> Commencez par déshydratation les plaquettes de Si pendant 30 min à 130 ° C. </li><li> Placer la galette sur un spin-coucheuse, versez SU8-2050 (Microchem) sur la tranche, d&#39;une rampe de 300 rpm / s à 3000-4000 rpm, et de spin pendant 30 s pour obtenir la hauteur caractéristique de 40-30 mm, respectivement. </li><li> Après le filage, placer la galette sur une plaque de cuisson à 65 ° C, immédiatement montée en puissance de la température à 95 ° C pendant 5-6 min, puis il rampe à 65 ° C. </li><li> Exposer les gaufrettes à une dose d&#39;UV de 200 mJ / cm 2 sur un aligneur de contact à l&#39;aide d&#39;un masque en champ sombre. </li><li> Placer les tranches sur une plaque de cuisson à 65 ° C, immédiatement montée en puissance de la température à 95 ° C pendant 4-5 min, puis il rampe à 65 ° C. </li><li> Développer les plaquettes dans l&#39;acétate de PM et d&#39;isopropanol. </li><li> Silaniser les plaquettes pendant 30 min à l&#39;aide hexaméthyldisiloxane (Shin-Etsu MicroSi), ou (tridécafluoro-1 ,1,2,2-tetrahydrooctyl)-1-trichlorosilane (t2492-KG, Spécialités UCT, Bristol, PA), pour éviter de PDMS adhérant à la tranche principale de silicium. </li></ol> Moulage puces <ol><li> Mélanger PDMS (Dow Corning, le Sylgard 184) dans un ratio de 10:1, le verser sur la plaquette master Si (~ 15 g par tranche), et laissez sécher durant la nuit à température ambiante. </li><li> Peler les guérir PDMS hors la plaquette de Si et découper chaque puce en utilisant une lame de rasoir. </li><li> Bond chaque puce d&#39;une lame de microscope cm coupé 1x1 pour une manipulation facile. </li></ol> Préparation de la BFC pour une utilisation <ol><li> Faire tremper la nuit dans du PBS BFC afin d&#39;empêcher l&#39;absorption du support dans le PDMS pendant l&#39;expérience. </li><li> Sécher les puces avec un Kimwipe (Kimtech Science). </li><li> Mettre une goutte de 7,5% albumine sérique bovine (BSA) (~ 200 ml) sur la surface à motifs de la CBF, et puis le gratter avec un embout de pipette pour disperser la BSA et de supprimer toutes les bulles dans les puits. </li><li> Laissez le BSA sur la surface BFC pour au moins 0,5 heures. Idéalement agiter la BSA toutes les 15 min pour empêcher la formation de croûtes. </li><li> Pour monter la CBF dans un plat 35x10 mm EPTC (Falcon, 35 à 3001), couper les bords d&#39;un plat à mi-chemin EPTC bas afin que les ¾ &quot;clips liant (Office Depot) ira parfaitement sur le rebord plat pour serrer les puces et vaisselle ensemble. </li><li> Coupez un joint entretoise (en forme de cadre avec des bords 20&#39;20 mm extérieur, 15&#39;15 bord mm intérieur, et de 250 à 500 mm d&#39;épaisseur) à partir d&#39;une feuille de PDMS (Specialty Products en silicone) ou de silicone d&#39;autres, et l&#39;appliquer à l&#39;antenne en utilisant une pince à épiler. </li></ol> Modélisation des cellules avec un BFC <ol><li> Stériliser le plat, joint, revêtu de BSA à puce, et 2 pinces pour reliure sous lumière UV pendant 1 minute. </li><li> Aspirer le BSA et rincer la BFC deux fois avec 200 ml de PBS. </li><li> Ajouter la gélatine à l&#39;antenne EPTC couper si nécessaire pour votre lignée cellulaire. Sinon, mouiller la surface EPTC avec certains médias avant de l&#39;appliquer à la puce (voir ci-dessous). Cela empêche la formation de bulles dans la chambre. </li><li> Après aspiration du PBS, une pipette de 200 mL de la solution de la cellule (~ 1 × 10 6 cellules / ml) sur la surface BFC. Laissez les cellules se contenter de 5-10 min. </li><li> Inclinez la CBF d&#39;un coin à ~ 15 ° d&#39;angle et lentement la pipette la solution de cellules avec une pipette de 200 ml. Ensuite, placez le plat BFC et ajouter 100 ml de PBS ou de médias vierges à l&#39;angle CBF opposé. Rincer la BFC de cette manière une fois 2-4 supplémentaires, au besoin, pour effacer toutes les cellules de l&#39;inter-régions bien. </li><li> Pipeter 200 ml de support sur la surface à puce. Aspirer gélatine / médias de l&#39;EPTC. Puis inverser le plat pré-mouillé et poussez lentement le haut BFC, dans le plat. </li><li> Appliquer un liant clip de chacun, aux deux côtés de la chambre pour la sceller. Retirez les broches métalliques de la reliure des clips pour ce plat reste au niveau où capoté. </li><li> Enfin, parcourir rapidement la configuration plus, tout en évitant tout mouvement inutile, et le placer dans l&#39;incubateur. </li></ol>

<ol>
	<li>Rosenthal, A., Macdonald, A., Voldman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+Patterning+Chip+for+Controlling+the+Stem+Cell+Microenvironment.">Cell Patterning Chip for Controlling the Stem Cell Microenvironment.</a> Biomaterials. 28, 3208-3216 (2007).</li></ol>

Bien que le protocole et vidéo présentés ici décrivent en utilisant l&#39;une BFC le modèle des cellules souches embryonnaires de souris (mESCs), BFC peuvent être utilisés pour motif de pratiquement n&#39;importe quel type de cellule d&#39;intérêt. Le seul changement on peut avoir besoin de faire est de modifier la taille des micropuits. Dans notre expérience, à utiliser la CBF pour les cellules de modèle unique d&#39;un autre type de cellule, d&#39;un diamètre et la hauteur micropuits égal à 10 microns de diamètr...

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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>PDMS</td>
<td>&nbsp;</td>
<td>Dw Corning</td>
<td>Sylgard 184</td>
<td>&nbsp;</td>
<tr>
<td>Silicon master mould</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Tissue culture dishes</td>
<td>&nbsp;</td>
<td>Falcon</td>
<td>35-3001</td>
<td>35 mm</td>
</tr>
<tr>
<td>3/4" binder clips</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>PBS</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>hexamethyldisiloxane</td>
<td>&nbsp;</td>
<td>Shin-Etsu MicroSi</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>New Item</td>
<td>&nbsp;</td>
<td>Microchem</td>
<td>SU8-2050</td>
<td>&nbsp;</td>
<tr>
<td>PM acetate</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Isopropanol</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>BSA</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>7.5% Bovine Serum Albumin </td>
</tr>		</tbody>
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patterning of embryonic stem cells using the bio flip chip

Interactions cellule-cellule composée de signalisation diffusibles et contact cellule-cellule (juxtacrine signalisation) sont importants dans de nombreux processus biologiques tels que la croissance tumorale, la différenciation des cellules souches et de cellules souches auto-renouvellement. Un certain nombre de méthodes existent actuellement pour moduler la signalisation cellulaire in vitro. Une méthode de moduler le montant total de la signalisation diffusible est de faire varier la densité de semis de cellules pendant la culture. En raison de la nature aléatoire de l&#39;ensemencement des cellules, cela se traduit par une variation considérable dans la cellule-cellule réelle d&#39;espacement et la quantité de contact cellule-cellule, et ne peut pas prescrire de l&#39;environnement local. Une approche plus spécifique pour la signalisation cellulaire modulant est d&#39;utiliser des inhibiteurs moléculaires ou des approches génétiques pour abattre spécifiques des protéines de signalisation, mais ces deux méthodes sont les mieux adaptés à la manipulation de petits nombres de molécules. Ici, nous démontrons une nouvelle approche de la modulation des cellules de la signalisation cellulaire qui module l&#39;environnement local d&#39;un amas de cellules en plaçant des nombres différents de cellules à des endroits désirés sur un substrat. Cette méthode fournit un moyen complémentaire pour contrôler la signalisation locale diffusibles et juxtacrine entre les cellules. Notre méthode permet l&#39;utilisation de la puce Bio Flip (BFC), une puce de silicone contenant des centaines microfabriqué à des milliers de micropuits, chacun dimensionné pour contenir soit une seule cellule ou un petit nombre de cellules. Nous charger la puce avec des cellules simplement en les pipetage sur le réseau de puits et le lavage des cellules déchargées du tableau. La puce est alors retourné sur un substrat, par lequel les cellules tombent du puits et sur le substrat, le maintien de leur structuration. Après que les cellules ont attaché, la puce peut être retiré (ou à gauche sur). Cette approche de la structuration cellulaire est unique en ce qu&#39;il: 1) ne modifie pas la chimie du substrat, permettant ainsi aux cellules de proliférer et à migrer; 2) permet la structuration sur n&#39;importe quel support, y compris la culture de tissus en polystyrène, verre, matrigel, et même chargeur de couches de cellules, et 3) est compatible avec l&#39;impression par microcontact traditionnelle, permettant la création d&#39;îles de la matrice extracellulaire des cellules placées à l&#39;intérieur des îles. Dans cette vidéo, nous démontrons la structuration des cellules souches embryonnaires de souris sur la culture de tissus en polystyrène en utilisant la CBF.

Watch this Scientific Journal Video about Patterning of Embryonic Stem Cells Using the Bio Flip Chip at JoVE.com

Patterning of Embryonic Stem Cells Using the Bio Flip Chip

Nous démontrons une méthode simple pour placer les cellules à des endroits désirés sur un substrat. Cette méthode schémas cellules en retournant une puce de silicone contenant des micropuits remplis de cellules sur le substrat. Cette méthode offre une nouvelle façon de moduler la signalisation diffusibles et juxtacrine entre les cellules.

Modélisation des cellules souches embryonnaires utilisant la puce Bio Retourner

Cell-cell interactions consisting of diffusible signaling and cell-cell contact (juxtacrine signaling) are important in numerous biological processes such ...

patterning-of-embryonic-stem-cells-using-the-bio-flip-chip

Research

JoVE Journal

Biology

9.4K vues.  MIT - Massachusetts Institute of Technology. Nous démontrons une méthode simple pour placer les cellules à des endroits désirés sur un substrat. Cette méthode schémas cellules en retournant une puce de silicone contenant des micropuits remplis de cellules sur le substrat. Cette méthode offre une nouvelle façon de moduler la signalisation diffusibles et juxtacrine entre les cellules.

Vidéo: Patterning of Embryonic Stem Cells Using the Bio Flip Chip

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

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Biologie

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are derived from the blastocyst of the early embryo and are pluripotent in differentiative ability.  Their vast differentiative potential has made them the focus of much research centered on deducing how to coax them to generate clinically useful cell types.  The successful derivation of hematopoietic stem cells (HSC) from mouse ESC has recently been accomplished and can be visualized in this video protocol.  HSC, arguably the most clinically exploited cell population, are used to treat a myriad of hematopoietic malignancies and disorders.  However, many patients that might benefit from HSC therapy lack access to suitable donors.  ESC could provide an alternative source of HSC for these patients.  The following protocol establishes a baseline from which ESC-HSC can be studied and inform efforts to isolate HSC from human ESC.  In this protocol, ESC are differentiated as embryoid bodies (EBs) for 6 days in commercially available serum pre-screened for optimal hematopoietic differentiation.  EBs are then dissociated and infected with retroviral HoxB4.  Infected EB-derived cells are plated on OP9 stroma, a bone marrow stromal cell line derived from the calvaria of  M-CSF-/- mice, and co-cultured in the presence of hematopoiesis promoting cytokines for ten days.  During this co-culture, the infected cells expand greatly, resulting in the generation a heterogeneous pool of 100s of millions of cells.  These cells can then be used to rescue and reconstitute lethally irradiated mice.

This protocol details the derivation of transplantable hematopoietic stem cells from mouse embryonic stem cells (ESC) and their subsequent injection into lethally irradiated recipient mice. Briefly, ESC are differentiated as embryoid bodies, which are then infected with retroviral HoxB4 and co-cultured with OP9 stromal cells and hematopoietic cytokines.

Dérivation de cellules souches hématopoïétiques de cellules souches embryonnaires murines

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are ...

CD4+ T-Lymphocyte Capture Using a Disposable Microfluidic Chip for HIV

T CD4 + des lymphocytes capturer en utilisant une puce jetable microfluidiques pour le VIH

RNA Extraction from Neuroprecursor Cells Using the Bio-Rad Total RNA Kit

Extraction d&#39;ARN à partir de cellules Neuroprecursor Utiliser le Bio-Rad ARN total Kit

Derivation of Human Embryonic Stem Cells by Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass. 


The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass.

Dérivation des cellules souches embryonnaires humaines par Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both ...

Chromatin Immunoprecipitation from Human Embryonic Stem Cells

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are characterized by two fundamental properties: self-renewal and multipotency that allows a stem cell to differentiate into virtually any cell type 1. The progression stem cell to differentiated cell is characterized by loss of multipotency, structural and morphological changes and the hierarchic activity of transcription factors and signaling molecules, whose activities establish and maintain cell-type specific gene expression patterns. At the molecular level, cell differentiation involves dynamic changes of the structure and composition of chromatin and the detection of those dynamic changes can provide valuable insights into the functional features of stem cells and the cell differentiation process 2,3. Chromatin is a highly compacted DNA-protein complex that forms when cells package chromosomal DNA with proteins, mainly histones 4. Stemcellness and cell differentiation has been correlated with the presence of specific arrays of regulatory proteins such as epigenetic factors, histone variants, and transcription factors 2,3,5.
 Chromatin immunoprecipitation (ChIP) provides a valuable method to monitor the presence of RNA, proteins, and protein modifications in chromatin 6,7. The comparison of chromatin from different cell types can elucidate dynamic changes in protein-chromatin associations that occur during cell differentiation.
Chromatin immunoprecipitation involves the purification of in vivo cross-linked chromatin. The isolated chromatin is reduced to smaller fragments by enzymatic digestion or mechanical force. Chromatin fragments are precipitated using specific antibodies to target proteins or protein and DNA modifications. The precipitated DNA or RNA is purified and used as a template for PCR or DNA microarray based assays. Prerequisites for a successful ChIP are high quality antibodies to the desired antigen and the availability of chromatin from control cells that do not express the target molecule. ChIP can correlate the presence of proteins, protein and RNA modifications, and RNA with specific target DNA, and depending on the choice of outread tool, detects the association of target molecules at specific target genes or in the context of an entire genome. The comparison of the distribution of proteins in the chromatin of differentiating cells can elucidate the dynamic changes of chromatin composition that coincide with the progression of cells along a cell lineage.

The differentiation of ESC coincides with cell-type specific changes in the structure and composition of chromatin. The detection of those changes provides valuable insights into the mechanisms that define stemcellness and cell differentiation. Chromatin immunoprecipitation (ChIP) represents a valuable method to dissect the molecular mechanisms underlying stem cell differentiation.

Immunoprécipitation de la chromatine des cellules souches embryonnaires humaines

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are ...

In vitro Differentiation of Mouse Embryonic Stem (mES) Cells Using the Hanging Drop Method

Stem cells have the remarkable potential to develop into many different cell types. When a stem cell divides, each new cell has the potential to either remain a stem cell or become another type of cell with a more specialized function, This promising of science is leading scientists to investigate the possibility of cell-based therapies to treat disease.

When culture in suspension without antidifferentiation factors, embryonic stem cells spontaneously differentiate and form three-dimensional multicellular aggregates. These cell aggregates are called embryoid bodies(EB). Hanging drop culture is a widely used EB formation induction method. The rounded bottom of hanging drop allows the aggregation of ES cells which can provide mES cells a good environment for forming EBs. The number of ES cells aggregatied in a hanging drop can be controlled by varying the number of cells in the initial cell suspension to be hung as a drop from the lid of Petri dish. Using this method we can reproducibly form homogeneous EBs from a predetermined number of ES cells. 

In vitro Differentiation of Mouse Embryonic Stem mES Cells Using the Hanging Drop Method

This video demonstrates how to conduct in vitro differentiation of mouse embryonic stem cells to embryoid bodies using the hanging drop method.

Différenciation in vitro de souris embryonnaires souches (MES) Cellules utilisant la méthode de goutte suspendue

Stem cells have the remarkable potential to develop into many different cell types. When a stem cell divides, each new cell has the potential to either ...

Culture and Maintenance of Human Embryonic Stem Cells  

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be  fed  every day by performing a complete medium change to replenish lost nutrients and to keep the cultures free of unwanted differentiation factors. Although a small amount of differentiation is normal and expected in stem cell cultures, the culture should be routinely cleaned up by manually removing, or "picking" differentiated areas. Identifying and removing excess differentiation from hES cell cultures are essential techniques in the maintenance of a healthy population of cells.

Culture and Maintenance of Human Embryonic Stem Cells

This protocol demonstrates how to maintain healthy, undifferentiated human embryonic stem (ES) cells.

Culture et entretien des cellules souches embryonnaires humaines

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be ...

Freezing and Thawing Human Embryonic Stem Cells

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

Congélation et de décongélation cellules souches embryonnaires humaines

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells ...

Isolation and Derivation of Mouse Embryonic Germinal Cells

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum (dpc) mouse embryos.  Developing gonadal ridges from mouse embryos (C57BL6J) were dissociated by mechanical disruption with collagenase, then plated in a mouse embryo fibroblast feeder layer (MEF-CF1) that was previously mitotically inactivated with mitomycin C in the presence of knockout media and supplemented with Leukemia Inhibitor Factor (LIF), basic Fibroblast Growth Factor (bFGF), and Stem Cell Factor (SCF).  Using these optimized methods for PCG identification, isolation, and establishment of culture conditions permits long term cultures of EG cells for more than 40 days.  The embryonic germinal cell lines showed embryonic phenotype and expression of common used markers of the pluripotent state.  Isolation and derivation of germinal cells in culture provide a tool to understand their development in vitro and offer the opportunity to monitor cumulative damage at genetic and epigenetic levels after exposure to oxidative stress.

The ability of embryonic germinal cells to differentiate into primordial germinal cells during early development stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum mouse embryos.

L&#39;isolement et la dérivation de cellules germinales embryonnaires de souris

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental ...