Cette recherche est soutenue par la Fondation Hartwell, l&#39;American Heart Association (Scientist Development Grant # 0830384N) et la Fondation Leducq: 07CVD04 mitrale.

1. Préparation <ol><li> Incuber cailles fertiles ou des oeufs de poule au stade 14 - (environ 2 jours) ou 25 (environ 4,5 jours) à 60% d&#39;humidité et 37 ° C. </li><li> Préparer la solution stérile de Earl saline équilibrée (EBSS) <ol><li> Ajouter 100 ml EBSS 10X à 600 ml d&#39;eau 18 MQ </li><li> Ajouter 2,2 g de bicarbonate de sodium </li><li> Ajuster le pH de la solution à 7,2 </li><li> Porter la solution jusqu&#39;à 1000 ml </li><li> Stériliser la solution en passant par un filtre de 0,2 um </li></ol></li><li> Préparer stériles M199 milieu de culture. <ol><li> Ajouter 1 paquet de poudre à M199 700 ml d&#39;eau 18 MQ. </li><li> Ajouter 2,2 g de bicarbonate de sodium </li><li> Ajuster le pH de la solution à 7,2. </li><li> Ajouter 10 ml (1%) de pénicilline-streptomycine </li><li> Porter la solution jusqu&#39;à 1000 ml </li><li> Stériliser la solution en passant par un filtre de 0,2 um </li><li> Ajouter 1 mL stérile 100X Insulin-transferrine-Selenium-G complément </li><li> Ajouter 10 ml (1%) de sérum de poulet stériles </li></ol></li><li> Préparer trois dimensions des gels de collagène à une concentration de collagène de 1,5 mg / ml. Des travaux antérieurs dans notre laboratoire a montré que la concentration en collagène de plus de 1,5 mg / ml fournit une matrice qui est trop rigide pour biomécaniquement les cellules se déplacer à travers. Les concentrations gel de collagène inférieure à 1,5 mg / mL ont des fibres de collagène si peu que le bouquet de cellules peuvent déchiqueter les fibres localement, produisant une île de cellules. <ol><li> Ajoutez les lignes suivantes glacée réactifs à un tube à centrifuger stérile de 15 ml dans l&#39;ordre: <ul><li> 3X M199: Volume = volume total nécessaire / 3 </li><li> Stérile 18 MQ eau: Volume = volume total nécessaire (M199 volume + volume de sérum de poulet + 0,1 M de NaOH volume de volume de collagène +) </li><li> Sérum de poulet stérile: Volume = volume total nécessaire * 0,01 </li><li> Collagène de queue de rat i: concentration des stocks = Volume (concentration du gel de collagène * volume total nécessaire) / collagène </li><li> Stérile NaOH 0,1 M: Volume du volume de collagène = 0,2 * Remarque: La quantité d&#39;eau, 0,1 M de NaOH, et de collagène ajoutée varie selon la concentration de la solution stock de collagène. </li></ul></li><li> Mélanger la solution de collagène bien avec une pipette stérile. </li><li> Immédiatement le transfert de 0,3 mL de la solution de collagène dans 1,9 cm 2 puits secteur de croissance. Ceci est une solution de collagène assez pour couvrir complètement le bien et de fournir un gel qui est d&#39;environ 3 mm d&#39;épaisseur. Une épaisseur de gel d&#39;au moins 250 pm est nécessaire pour les études de l&#39;invasion cellulaire. </li><li> Laisser la solution de collagène pour un gel d&#39;au moins 1 heure à 37 ° C, 5% de CO 2, et 100% d&#39;humidité. </li></ol></li></ol> 2. Pré-EMT isolement cellulaire endocardique et de la Culture <ol><li> L&#39;isolement des embryons <ol><li> Placer un morceau de ruban pouce de long en laboratoire au cours de la plus grande extrémité de l&#39;œuf. C&#39;est là que la cellule d&#39;air est située. La bande stabilise la coquille d&#39;oeuf de caille fragiles. </li><li> Doucement la zone de ponction scellés de l&#39;œuf dans la cellule d&#39;air avec la pointe de ciseaux courbes stérilisés. </li><li> Coupez un trou dans la partie enregistrée de l&#39;œuf qui est assez grande pour permettre le jaune de passer à travers. Démarrer le trou à la perforation et à l&#39;extérieur coupés en forme de spirale. Assurez-vous de couper à travers la membrane externe, mais éviter de percer le jaune. </li><li> Lorsque le trou dans la coquille est complète, de visualiser l&#39;embryon de caille. L&#39;embryon est normalement situé sur le côté supérieur du jaune. </li><li> Commencer à verser le jaune d&#39;œuf à travers le trou percé dans l&#39;œuf avec une seule main. Tenir une pince # 55 dans l&#39;autre main. Comme l&#39;embryon sort de l&#39;œuf sur le dessus du jaune, faites glisser doucement la pince à épiler # 55 sous l&#39;embryon et le retirer de la jaune. Note: Pour les œufs de poule, l&#39;isolement d&#39;embryon implique la fissuration de l&#39;œuf de poulet sur un pointu, surface propre et casser l&#39;oeuf dans un plat de Pétri de 100 mm. L&#39;embryon doit être situé sur le côté supérieur du jaune. Faites glisser doucement # 55 pinces sous l&#39;embryon et le retirer de la jaune. </li><li> Placez l&#39;embryon dans un plat de 100 mm de Petri contenant de la glace-froid, EBSS stérile. </li></ol></li><li> Stade des embryons, selon les critères de Hamburger et Hamilton 1. Les embryons au stade 14 - 13 sont passés scène, mais pas encore à l&#39;étape 15, avec 20 à 21 somites et un bourgeon queue. Une caractéristique clé de l&#39;étape 14 - embryons est l&#39;angle de la tête, ce qui est légèrement supérieur à 90 ° par rapport au corps. </li><li> Retirer le cœur à partir d&#39;embryons de caille au stade 14 - avec une pince à épiler # 55 en coupant transversalement où le canal AV et l&#39;OFT de prendre contact avec la paroi thoracique. Placez les coeurs ensemble sur un côté de la boîte de Pétri. </li><li> Recueillir les coeurs isolés avec une pipette de transfert stérile et les placer dans un nouveau plat de 100 mm de Pétri remplie de stériles, glacée EBSS. </li><html><li> Couper le OFT et les canaux AV transversalement des ventricules du # 55 pincettes. </li><li> Section des canaux restants AV et / ou voies d&#39;éjection (OFT) longitudinalement. </li><li> Régler une pipette pour un volume de 20 uL. Utilisez cette pipette pour aspirer six longitudinalement coupé canaux AV et OFT. </li><li> Expulser les canaux AV et OFT sur le gel de collagène. Aspirer toute EBSS excès de la surface du gel. </li><li> Utilisez # 55 pinces à orienter les explants de sorte qu&#39;ils sont à plat et que le côté lumineux face au gel de collagène. Essayez de ne pas percer le gel de collagène avec la pince à épiler. </li><li> Après 2 heures à 37 ° C et 5% de CO 2, retirez le myocarde des explants avec # 55 pincettes. Les cellules endocardiques doit être laissé à la surface du gel de collagène. Alternativement, le myocarde ne peut être laissé sur l&#39;explant. Avec l&#39;enlevés myocarde, les cellules endocardiques avec subissent pas EMT sans intervention. Avec le myocarde présentent un sous-ensemble de cellules endocardiques va se transformer en mésenchyme, mais le degré de transformation peut être modulée par des facteurs externes. Remarque: Même si le myocarde ne sera laissé sur le explant doit être autorisé au moins 2 heures à joindre à la moyenne gel avant est ajouté. </li><li> Ajouter 0,4 ml à chaque milieu M199 explants puits contenant. </li><li> Culture des cellules à 37 ° C, 5% de CO 2, et 100% d&#39;humidité. </li></ol> 3. Post-EMT Coussin AV cellule d&#39;isolement mésenchymateuses et de la Culture <ol><li> Crack l&#39;œuf de poulet sur une forte surface propre et casser l&#39;oeuf dans un plat de Pétri de 100 mm. L&#39;embryon doit être situé sur le côté supérieur du jaune. Ramassez l&#39;embryon avec # 5 pincettes en saisissant les membranes qui entourent l&#39;embryon, puis placer l&#39;embryon dans un plat de Pétri de 100 mm rempli de glace-froid, EBSS stérile. </li><li> Stade des embryons, selon les critères de Hamburger et Hamilton 1. Caractéristiques d&#39;un stade de 25 embryons comprennent coude distinctes et les articulations du genou et de la pigmentation des yeux légère. </li><li> Isoler les cœurs de tous les embryons HH25 et placer les coeurs ensemble sur un côté de la boîte de Pétri. </li><li> Recueillir les coeurs isolés avec une pipette de transfert stérile et les placer dans un nouveau plat de 100 mm de Pétri remplie de stériles, glacée EBSS. </li><li> Isoler la région AV de tous les cœurs à la fois. Placez l&#39;AVS ainsi que sur un côté de la boîte de Pétri. Agiter doucement la région AV à éliminer tout le sang, ce qui peut affecter la viabilité cellulaire. </li><li> Recueillir les isolés AV avec une pipette de transfert stérile et les placer dans un nouveau plat de 100 mm de Pétri remplie de stériles, glacée EBSS. </li><li> Séquentiellement isoler les coussins de l&#39;AV. Assurez-vous qu&#39;il n&#39;ya pas présents sur les coussins du myocarde AV. </li><li> Recueillir les coussins isolés avec une pipette de transfert stérile et les placer dans un tube à centrifuger stérile de 15 ml. </li><li> Faites tourner les coussins au fond du tube à centrifuger à 15 xg pendant 3 minutes. </li><li> Utiliser une pointe de 1000 ml stériles pour enlever autant de surnageant EBSS que possible sans déranger les coussins. </li><li> Ajouter 1 ml de 0,25% de trypsine-EDTA qui a été pré-chauffé à 37 ° C. Autoriser les coussins à incuber dans de la trypsine jusqu&#39;à ce qu&#39;ils soient complètement dissous, ce qui est normalement d&#39;environ 3-5 minutes. </li><li> Ajouter 100 ul de sérum de poulet pour le tube à centrifuger pour étancher la trypsine. </li><li> Pellet les cellules à 160 xg pendant 5 minutes. Enlever le surnageant avec une solution stérile, pointe uL 1000. </li><li> Resuspendre les cellules dans 2 ml de milieu M199 et mélanger par pipetage. Prenez 10 ul de la suspension cellulaire à compter sur hématimètre et déterminer le nombre total de cellules qui ont été isolées </li><li> Déterminer le nombre de gels de collagène sera effectué en fonction du nombre de cellules isolées. Les cellules ont besoin d&#39;être étalées à une densité de 2 x 10 5 cellules / mL avec un volume de 250 uL de gel. </li><li> Pellet les cellules à 160 xg pendant 5 minutes. Enlever le surnageant avec une solution stérile, pointe uL 1000. </li><li> En utilisant le protocole ci-dessus, ajouter 3X M199, l&#39;eau, du sérum de poulet, de collagène, et 0,1 M de NaOH, dans cet ordre, au culot cellulaire. Mélanger à la pipette en douceur. </li><li> Ajouter 250 uL de chaque zone de croissance de 1,9 cm 2 bien. </li><li> Laisser la solution de collagène pour un gel d&#39;au moins 1 heure à 37 ° C, 5% de CO 2, et 100% d&#39;humidité. </li><li> Ajouter 0,4 ml de milieux de culture et incuber pendant la nuit. </li><li> Pour continuer à cultiver les gels dans des conditions sans stress, libérer les gels sur les côtés de la culture ainsi l&#39;aide d&#39;une pipette stérile de 200 uL. Mécaniquement contraints cultures sont des gels qui restent attachées sur les côtés de la culture ainsi. </li></ol> 4. Les résultats représentatifs <img src="/files/ftp_upload/2159/2159fig1.jpg" alt="Figure 1" /> Figure 1:. Exemples d&#39;embryons (A) HH14-cailles embryon, et (B) HH25 embryon de poulet sont montrés ici. fig2.jpg &quot;alt =&quot; Figure 2 &quot;/&gt; Figure 2: HH14 soupapes explants endocardique après 2 heures de culture (A) avec le présent myocarde, ou (B) avec le myocarde enlevé.. <img src="/files/ftp_upload/2159/2159fig3.jpg" alt="Figure 3" /> Figure 3:. HH14 soupapes explants endocardique (A) Avec le présent myocarde, la plupart des cellules subissent endocardique à la transformation mésenchymateuses et ont une forme de fuseau, phénotype mésenchymateux après 48 heures de culture. (B) Lorsque le myocarde est enlevé toutes les cellules de maintenir un caillou en forme, le phénotype endocardique après 48 heures dans la culture. <img src="/files/ftp_upload/2159/2159fig4.jpg" alt="Figure 4"/> Figure 4:. Cellules HH25 cellules mésenchymateuses dans le robinet d&#39;un gel de collagène sont arrondis immédiatement après le semis. <img src="/files/ftp_upload/2159/2159fig5.jpg" alt="Figure 5"/> Figure 5:. HH25 cellules mésenchymateuses en culture (A) Après 48 heures de culture dans un gel de collagène de la contrainte HH25 cellules mésenchymateuses commencent à se répandre. (B) HH25 cellules dans un gel de collagène sans stress 7 jours après l&#39;ensemencement et 6 jours après la libération ont compacté le gel d&#39;environ 50% de la superficie d&#39;origine.

<ol>
	<li>Hamburger, V., Hamilton, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+series+of+normal+stages+in+the+development+of+the+chick+embryo.">A series of normal stages in the development of the chick embryo.</a> J Morphol. 88, 49-92 (1951).</li><li>Runyan, R. B., Markwald, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invasion+of+mesenchyme+into+three-dimensional+collagen+gels:+A+regional+and+temporal+analysis+of+interaction+in+embryonic+heart+tissue.">Invasion of mesenchyme into three-dimensional collagen gels: A regional and temporal analysis of interaction in embryonic heart tissue.</a> Developmental Biology. 95, 108-108 (1983).</li><li>Butcher, J. T., Norris, R. A., Hoffman, S., Mjaatvedt, C. H., Markwald, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Periostin+promotes+atrioventricular+mesenchyme+matrix+invasion+and+remodeling+mediated+by+integrin+signaling+through+Rho/PI+3-kinase.">Periostin promotes atrioventricular mesenchyme matrix invasion and remodeling mediated by integrin signaling through Rho/PI 3-kinase.</a> Developmental Biology. 302, 256-256 (2007).</li></ol>

La méthode pour isoler les cellules endocardiques du stade HH14 - coeurs initialement développé par Runyan et Markwald fournit un environnement contrôlé, dans un environnement in vitro pour étudier les facteurs qui déclenchent et modulent embryonnaire EMT 2. HH14 - explants endocardique cultivées sans myocarde ne subira pas d&#39;EMT sans intervention biomécaniques ou biochimiques. Si le myocarde est à gauche sur l&#39;explant il signalera un sous-ensemble de ...

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<td>Extra fine Bonn scissors, curved</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>14085-08</td>
<td>&nbsp;</td>
<tr>
<td>Dumont tweezers #55</td>
<td>&nbsp;</td>
<td>World Precision Instruments, Inc.</td>
<td>14099</td>
<td>&nbsp;</td>
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<td>Dumont tweezers #5</td>
<td>&nbsp;</td>
<td>World Precision Instruments, Inc.</td>
<td>14098</td>
<td>&nbsp;</td>
<tr>
<td>Sterile transfer pipettes</td>
<td>&nbsp;</td>
<td>Samco Scientific </td>
<td>2041S</td>
<td>&nbsp;</td>
<tr>
<td>4-well culture plates</td>
<td>&nbsp;</td>
<td>Nunc</td>
<td>176740</td>
<td>&nbsp;</td>
<tr>
<td>Sterile disposable filter units, PES, 0.2 &mu;m pore size membrane</td>
<td>&nbsp;</td>
<td>Nalgene</td>
<td>566-0020</td>
<td>&nbsp;</td>
<tr>
<td>Sterile 15 mL centrifuge tubes</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>430828</td>
<td>&nbsp;</td>
<tr>
<td>Sterile petri dishes, 100 mm x 15 mm</td>
<td>&nbsp;</td>
<td>VWR International</td>
<td>25384-342</td>
<td>&nbsp;</td>
<tr>
<td>Laboratory tape</td>
<td>&nbsp;</td>
<td>VWR International</td>
<td>89097-920</td>
<td>&nbsp;</td>
<tr>
<td>Rat-tail collagen I</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>354236</td>
<td>&nbsp;</td>
<tr>
<td>10X Earl's Balanced Salt Solution</td>
<td>&nbsp;</td>
<td>Quality Biological, Inc.</td>
<td>119-064-131</td>
<td>&nbsp;</td>
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<td>M199 powder</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>31100-035</td>
<td>&nbsp;</td>
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<td>Penicillin-Streptomycin</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15140-122</td>
<td>&nbsp;</td>
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<td>Insulin-Transferrin-Selenium-G supplement (100X)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>41400-045</td>
<td>&nbsp;</td>
<tr>
<td>Chicken serum</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>16110-082</td>
<td>&nbsp;</td>
<tr>
<td>0.25% Trypsin-EDTA</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>25200-056</td>
<td>&nbsp;</td>
<tr>
<td>Sodium bicarbonate</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>S5761</td>
<td>&nbsp;</td>
<tr>
<td>Sodium hydroxide solution, 1.0 N</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>S2770</td>
<td>&nbsp;</td>
<tr>
<td>Fertile quail eggs (Coturnix coturnix)</td>
<td>&nbsp;</td>
<td>Lake Cumberland Game Bird Farm</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Fertile chicken eggs (Gallus gallus)</td>
<td>&nbsp;</td>
<td>Cornell University Poultry Farm</td>
<td>&nbsp;</td>
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isolation and culture of avian embryonic valvular progenitor cells

Bonne formation et la fonction des valves cardiaques embryonnaires humaines est indispensable à la progression du développement. Le coeur embryonnaire précoce est un tube en U de l&#39;endocarde entouré par myocarde. Le myocarde sécrète cardiaques gelée, une matrice riche en acide hyaluronique gélatineux, dans le auriculo-ventriculaire (AV) de jonction et des voies d&#39;écoulement (OFT) lumen. Au stade HH14 valvulogenesis commence quand un sous-ensemble de cellules endocardiques recevoir des signaux du myocarde, l&#39;endocarde subissent à la transformation mésenchymateuse (TEM), et envahissent la cardiaques gelée. Au stade HH25 les coussins valvulaires sont entièrement mesenchymalized, et c&#39;est ce mésenchyme qui forme éventuellement l&#39;appareil valvulaire et septale du cœur. Comprendre les mécanismes qui déclenchent et modulent les processus de différenciation cellulaire et l&#39;EMT sont importantes en raison de leur connexion à de graves malformations cardiaques congénitales. Dans cette étude, nous présentons les méthodes pour isoler pré-EMT endocardique et post-EMT cellules mésenchymateuses, qui sont les deux phénotypes cellulaires différents du coussin prevalvular. Pré-EMT cellules endocardiques peuvent être cultivées avec ou sans le myocarde. Post-EMT cellules mésenchymateuses coussin AV peuvent être cultivées à l&#39;intérieur des gels de collagène mécaniquement contraint ou sans stress. Ces 3D dans des modèles in vitro imitent les principaux événements morphogéniques valvulaire et sont utiles pour déconstruire les mécanismes de valvulogenesis stade précoce et tardive.

Watch this Scientific Journal Video about Isolation and Culture of Avian Embryonic Valvular Progenitor Cells at JoVE.com

Isolation and Culture of Avian Embryonic Valvular Progenitor Cells

Cet article propose une méthode pour isoler et cultiver la caille ou de poulet HH14<sup&gt; -</sup&gt; Soupape de cellules endocardiques et HH25 cellules mésenchymateuses coussin de soupape.

Isolement et culture de cellules progénitrices embryonnaires aviaires valvulaire

Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube of ...

isolation-and-culture-of-avian-embryonic-valvular-progenitor-cells

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10.1K vues.  Cornell University. Cet article propose une méthode pour isoler et cultiver la caille ou de poulet HH14 - Soupape de cellules endocardiques et HH25 cellules mésenchymateuses coussin de soupape.

Vidéo: Isolation and Culture of Avian Embryonic Valvular Progenitor Cells

Isolation and Culture of Post-Natal Mouse Cerebellar Granule Neuron Progenitor Cells and Neurons 

The cerebellar cortex is a well described structure that provides unique opportunities for studying neuronal properties and development1,2. Of the cerebellar neuronal types (granule cells, Purkinje cells and inhibitory interneurons), granule neurons are by far the most numerous and are the most abundant type of neurons in the mammalian brain. In rodents, cerebellar granule neurons are generated during the first two post-natal weeks from progenitor cells in the outermost layer of the cerebellar cortex, the external granule layer (EGL). The protocol presented here describes techniques to enrich and culture granule neurons and their progenitor cells from post-natal mouse cerebellum. We will describe procedures to obtain cultures of increasing purity3,4, which can be used to study the differentiation of proliferating progenitor cells into granule neurons5,6. Once the progenitor cells differentiate, the cultures also provide a homogenous population of granule neurons for experimental manipulation and characterization of phenomena such as synaptogenesis, glutamate receptor function7, interaction with other purified cerebellar cells8,9 or cell death7.

Isolation and Culture of Post-Natal Mouse Cerebellar Granule Neuron Progenitor Cells and Neurons

Here we present a method to isolate and culture cerebellar granule neuron progenitor cells and cerebellar granule neurons from postnatal mouse.

Isolement et culture de la Post-Natal cellules granulaires du cervelet de la souris progénitrices Neuron et les neurones

The cerebellar cortex is a well described structure that provides unique opportunities for studying neuronal properties and development1,2. Of the ...

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Biologie

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 ...

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 ...

Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a robust and reliable method for isolating relatively large numbers of proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. The authors have used micro-dissected explant cultures to analyse the growth characteristics of skeletal muscle cells in wild-type and dystrophic muscles. Each of the components of tissue growth, namely cell survival, proliferation, senescence and differentiation can be analysed separately using the methods described here. The net effect of all components of growth can be established by means of measuring explant outgrowth rates. The micro-explant method can be used to establish primary cultures from a wide range of different muscle types and ages and, as described here, has been adapted by the authors to enable the isolation of embryonic skeletal muscle precursors.
Uniquely, micro-explant cultures have been used to derive clonal (single cell origin) skeletal muscle stem cell (SMSc) lines which can be expanded and used for in vivo transplantation. In vivo transplanted SMSc behave as functional, tissue-specific, satellite cells which contribute to skeletal muscle fibre regeneration but which are also retained (in the satellite cell niche) as a small pool of undifferentiated stem cells which can be re-isolated into culture using the micro-explant method.

The micro-dissected explants technique is a robust and reliable method for isolating proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. Uniquely, these cells have been clonally derived to produce skeletal muscle stem cell lines used for in vivo transplantation.

Adultes et embryonnaires Culture squelettique Microexplant musculaire et isolement de cellules souches du muscle squelettique

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a ...

Isolation of Valvular Endothelial Cells

Heart valves are solely responsible for maintaining unidirectional blood flow through the cardiovascular system. These thin, fibrous tissues are subjected to significant mechanical stresses as they open and close several billion times over a lifespan. The incredible endurance of these tissues is due to the resident valvular endothelial (VEC) and interstitial cells (VIC) that constantly repair and remodel in response to local mechanical and biological signals. Only recently have we begun to understand the unique behaviors of these cells, for which in vitro experimentation has played a key role. Particularly challenging is the isolation and culture of VEC. Special care must be used from the moment the tissue is removed from the host through final plating. Here we present protocols for direct isolation, side specific isolation, culture, and verification of pure populations of VEC. We use enzymatic digestion followed by a gentle swab scraping technique to dislodge only surface cells. These cells are then collected into a tube and centrifuged into a pellet. The pellet is then resuspended and plated into culture flasks pre-coated with collagen I matrix. VEC phenotype is confirmed by contact inhibited growth and the expression of endothelial specific markers such as PECAM1 (CD31), Von Willebrand Factor (vWF), and negative expression of alpha-smooth muscle actin (&alpha;-SMA). The functional characteristics of VEC are associated with high levels of acetylated LDL. Unlike vascular endothelial cells, VEC have the unique capacity to transform into mesenchyme, which normally occurs during embryonic valve formation1. This can also occur during significantly prolonged post confluent in vitro culture, so care should be made to passage at or near confluence. After VEC isolation, pure populations of VIC can then be easily acquired.

We provide a method for isolating and culturing pure populations of heart valve endothelial cells (VEC). VEC can be isolated from either side of the cusp or leaflet and immediately following, underlying interstitial cell (VIC) isolation is straightforward.

L&#39;isolement des cellules endothéliales valvulaires

Heart valves are solely responsible for maintaining unidirectional blood flow through the cardiovascular system.  These thin, fibrous tissues are ...

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells (HUVECs) have shown to be convenient, easy to obtain and culture, and thus are the most widely studied endothelial cells. However, for research focused on processes like angiogenesis, permeability or many others, microvascular endothelial cells (ECs) are a much more physiologically relevant model to study 2. Furthermore, ECs isolated from knockout mice provide a useful tool for analysis of protein function ex vivo. Several approaches to isolate and culture microvascular ECs of different origin have been reported to date 3-7, but consistent isolation and culture of pure ECs is still a major technical problem in many laboratories. Here, we provide a step-by-step protocol on a reliable and relatively simple method of isolating and culturing mouse lung endothelial cells (MLECs). In this approach, lung tissue obtained from 6- to 8-day old pups is first cut into pieces, digested with collagenase/dispase (C/D) solution and dispersed mechanically into single-cell suspension. MLECS are purified from cell suspension using positive selection with anti-PECAM-1 antibody conjugated to Dynabeads using a Magnetic Particle Concentrator (MPC). Such purified cells are cultured on gelatin-coated tissue culture (TC) dishes until they become confluent. At that point, cells are further purified using Dynabeads coupled to anti-ICAM-2 antibody. MLECs obtained with this protocol exhibit a cobblestone phenotype, as visualized by phase-contrast light microscopy, and their endothelial phenotype has been confirmed using FACS analysis with anti-VE-cadherin 8 and anti-VEGFR2 9 antibodies and immunofluorescent staining of VE-cadherin. In our hands, this two-step isolation procedure consistently and reliably yields a pure population of MLECs, which can be further cultured. This method will enable researchers to take advantage of the growing number of knockout and transgenic mice to directly correlate in vivo studies with results of in vitro experiments performed on isolated MLECs and thus help to reveal molecular mechanisms of vascular phenotypes observed in vivo.

Here, we describe a protocol for isolation and culture of murine pulmonary endothelial cells. This method comprises mechanic and enzymatic lung tissue dissociation as well as a 2-step purification process using anti-PECAM-1 and anti-ICAM-2 antibodies conjugated to magnetic beads, which produces a pure endothelial cell population of mostly microvascular origin.

Isolement et culture de cellules endothéliales pulmonaires des souris néonatales

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells ...

Isolation and Culture of Cells from the Nephrogenic Zone of the Embryonic Mouse Kidney

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and to gain understanding of the origins of congenital kidney disease. More recently, the possibility of steering na&iuml;ve embryonic stem cells toward nephrogenic fates has been explored in the emerging field of regenerative medicine. Genetic studies in the mouse have identified several pathways required for kidney development, and a global catalog of gene transcription in the organ has recently been generated <a href="http://www.gudmap.org/">http://www.gudmap.org/</a>, providing numerous candidate regulators of essential developmental functions. Organogenesis of the rodent kidney can be studied in organ culture, and many reports have used this approach to analyze outcomes of either applying candidate proteins or knocking down the expression of candidate genes using siRNA or morpholinos. However, the applicability of organ culture to the study of signaling that regulates stem/progenitor cell differentiation versus renewal in the developing kidney is limited as cultured organs contain a compact extracellular matrix limiting diffusion of macromolecules and virus particles. To study the cell signaling events that influence the stem/progenitor cell niche in the kidney we have developed a primary cell system that establishes the nephrogenic zone or progenitor cell niche of the developing kidney ex vivo in isolation from the epithelial inducer of differentiation. Using limited enzymatic digestion, nephrogenic zone cells can be selectively liberated from developing kidneys at E17.5. Following filtration, these cells can be cultured as an irregular monolayer using optimized conditions. Marker gene analysis demonstrates that these cultures contain a distribution of cell types characteristic of the nephrogenic zone in vivo, and that they maintain appropriate marker gene expression during the culture period. These cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which greatly facilitates the study of candidate stem/progenitor cell regulator effects. Basic cell biological parameters such as proliferation and cell death as well as changes in expression of molecular markers characteristic of nephron stem/progenitor cells in vivo can be successfully used as experimental outcomes. Ongoing work in our laboratory using this novel primary cell technique aims to uncover basic mechanisms governing the regulation of self-renewal versus differentiation in nephron stem/progenitor cells.

In this report we describe a method for the isolation and culture of the progenitor cell niche from the embryonic mouse kidney that can be used to study signaling pathways regulating stem/progenitor cells of the developing kidney. These cultured cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which allows efficient manipulation of candidate pathways.

Isolement et culture de cellules de la zone néphrogénique du rein d&#39;embryon de souris

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and  to gain ...

Isolation and Primary Culture of Rat Hepatic Cells

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology and other related subjects. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 1 and, since then, has undergone many modifications. The most commonly used technique was described by Seglenin 1976 2. Essentially, hepatocytes are dissociated from anesthetized adult rats by a non-recirculating collagenase perfusion through the portal vein. The isolated cells are then filtered through a 100 &mu;m pore size mesh nylon filter, and cultured onto plates. After 4-hour culture, the medium is replaced with serum-containing or serum-free medium, e.g. HepatoZYME-SFM, for additional time to culture. These procedures require surgical and sterile culture steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol, which allow consistently in the generation of viable hepatocytes in large numbers.

Primary hepatocytes provide a valuable tool to evaluate biochemical, molecular, and metabolic functions in a physiologically relevant experimental system. We describe a reliable protocol for rat in situ liver perfusion, which consistently generates viable hepatocytes up to 1.0 &times; 108 cells per preparation with cell viability between 88 ~ 96%.

L&#39;isolement et la culture primaire de cellules de foie de rat

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

Isolation of Embryonic Ventricular Endothelial Cells

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines offer significant advantages for their ease of use, these cell lines are unavailable for all potential cell types. By isolating primary cells from a specific region of interest, particularly from a transgenic mouse, more nuanced studies can be performed. The basic technique involves dissecting the organ or partial organ of interest (e.g. the heart or a specific region of the heart) and dissociating the organ to single cells. These cells are then incubated with magnetic beads conjugated to an antibody that recognizes the cell type of interest. The cells of interest can then be isolated with the use of a magnet, with a short trypsin incubation dissociating the cells from the beads. These isolated cells can then be cultured and analyzed as desired. This technique was originally designed for adult mouse organs but can be easily scaled down for use with embryonic organs, as demonstrated herein. Because our interest is in the developing coronary vasculature, we wanted to study this population of cells during specific embryonic stages. Thus, the original protocol had to be modified to be compatible with the small size of the embryonic ventricles and the low potential yield of endothelial cells at these developmental stages. Utilizing this scaled-down approach, we have assessed coronary plexus remodeling in transgenic embryonic ventricular endothelial cells.

Primary cell culture is a useful technique for analyzing specific populations of cells, particularly from transgenic mouse embryos at specific developmental stages. Herein, embryonic ventricles are dissected and dissociated, and antibody-conjugated beads recognize and separate out the endothelial cells for further analysis.

Isolement des cellules endothéliales embryonnaires ventriculaires

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines ...

Isolation and Culture of Mouse Primary Pancreatic Acinar Cells

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one week. More than 20 x 106 acinar cells can be obtained from a single murine pancreas. This protocol offers the possibility to independently process as many as 10 pancreases in parallel. Because it preserves acinar architecture, this model is well suited for studying the physiology of the exocrine pancreas in vitro in contrast to cell lines established from pancreatic tumors, which display many genetic alterations resulting in partial or total loss of their acinar differentiation.

In this publication, we describe a rapid and convenient procedure for isolating and culturing primary pancreatic acinar cells from the murine pancreas. This method constitutes a valuable approach to study the physiology of fresh primary normal/untransformed exocrine pancreatic cells.

Isolement et culture de cellules primaires de souris acineuses du pancréas

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one ...