Nous remercions le Dr Steven Austad de nous fournir la première version de ce protocole. Ce travail a été soutenu par des subventions du NIH et le Ellison Medical Foundation à VG et AS

1. Avant de partir <ol><li> Stériliser des ciseaux et des pinces à l&#39;éthanol 70%. </li><li> Placer les petits agitateur magnétique dans un bécher 30 mL, couvrir avec deux couches de papier d&#39;aluminium, et autoclave. </li><li> Préparer un 28 unités Wunsch / ml de solution stock Libérase Blendzyme 3 dans l&#39;eau stérile. Faire aliquotes de 0,5 ml et congeler à -20 ° C. Dégeler une aliquote de nouvelles avant chaque utilisation. La solution peut paraître nuageux après décongélation. Vortex la solution jusqu&#39;à ce qu&#39;elle devienne claire. </li><li> Réchauffez les milieux de culture cellulaire. </li></ol> 2. Préparation des échantillons d&#39;animaux Attention: les animaux sauvages peuvent contenir des agents pathogènes tels que virus de la rage. Soyez toujours conscient de dièses ouverte. <ol><li> Euthanasier l&#39;animal et le lieu de la carcasse à 4 ° C. Il est préférable d&#39;utiliser la carcasse immédiatement, toutefois, si cela n&#39;est pas pratique, comme si les animaux sont recueillies sur le terrain, les carcasses peuvent être utilisés dans les 24 heures. Après 24 heures, les baisses de rendement des cellules. </li><li> Disséquer l&#39;animal dans le bloc opératoire ou dans hotte chimique (et non dans la hotte de culture de tissus). </li><li> Aisselles zone est un site pratique pour recueillir des échantillons de peau comme la peau des aisselles est plus mince, contient moins de gras, et la fourrure est moins dense. Nettoyez le site d&#39;incision avec de l&#39;éthanol à 70%. Assurez-vous que la fourrure est imbibé d&#39;éthanol. </li><li> Rasez les poils autour du site d&#39;incision avec un scalpel tranchant. Rasage une zone plus vaste que le site d&#39;incision désirée. Essayez de minimiser les coupures de la peau. Vaporiser la zone avec 70% d&#39;éthanol et laisser sécher. </li><li> Pour recueillir un échantillon de peau d&#39;accise d&#39;un fragment d&#39;environ 1 cm 2. Pincer la peau avec une pince de tissu et couper avec des ciseaux. Essayez de ne pas couper la couche de graisse avec la peau, sous forme de graisse interfère avec la digestion par la collagénase. Pour recueillir les échantillons pulmonaires, lavez la région de la poitrine avec de l&#39;éthanol à 70%, et ouvert la peau sur la poitrine avec des ciseaux en faisant une incision en forme de T et en tirant en dehors des volets de la peau. Laver la zone musculaire ouvert avec 70% d&#39;éthanol et laisser sécher. Couper la cage thoracique à l&#39;aide des pinces stériles et les ciseaux (utilisation tailleurs d&#39;os pour les gros animaux). Utiliser des techniques stériles pour éviter de contaminer les organes internes. Ne pas toucher les organes internes avec des instruments qui a touché la fourrure animale. Coupez les fragments pulmonaires d&#39;environ 1 cm 2 à l&#39;aide des pinces stériles et des ciseaux. </li><li> Fragments de tissus place dans des tubes de 50 ml avec du PBS stérile pour éviter le dessèchement. </li><li> Laver l&#39;extérieur de l&#39;tubes de 50 ml avec des fragments de tissu avec de l&#39;éthanol, et les prendre à la hotte de culture de tissus. </li><li> Débarrassez-vous correctement des carcasses d&#39;animaux. </li></ol> 3. Cellules Extraction <ol><li> Transfert des fragments de tissu dans un plat de 10 cm de culture de tissu à l&#39;aide scalpel stérile. Ne transférez pas trop de PBS avec l&#39;échantillon. </li><li> Coupez le tissu dans ~ 1 mm en utilisant deux morceaux scalpels. Utilisez deux lames de ciseaux en utilisant une action à partir du centre et en tirant à part. Gardez le tissu en boule, ne pas couper morceau par morceau. Quand la coupe est suffisamment la peau ressemble à du mastic, il ne sera pas séparée en morceaux, mais s&#39;étendra mince. Le tissu pulmonaire est plus facile à couper, et il va séparer en petits morceaux. </li><li> Avec un scalpel, le transfert du tissu découpé en 30 stériles bécher mL avec un barreau d&#39;agitation stérile. Laver la plaque utilisée pour la découpe de tissus avec 10 ml de DMEM/F12 des médias avec les unités Wunsch 0,14 / mL Libérase Blendzyme 3, et 1X antibiotique / antimycotique, et ajouter la solution dans le bécher de 30 mL avec des fragments de tissus. </li><li> Couvrir le bécher avec du papier stérile, et incuber à 37 ° C, en remuant doucement, pendant 30 à 90 minutes. La longueur de l&#39;incubation dépend du type d&#39;espèces et de tissus. Prenez soin de ne pas trop digérer les tissus. Meilleurs rendements sont obtenus lorsque des fragments de tissus sont encore présentes à la fin de la digestion. Peau prend plus de temps à digérer que le poumon. Peau d&#39;animaux de grande taille prend plus de temps à digérer. Vérifiez la digestion après 30 minutes, puis toutes les 10 min. Lorsque la digestion est terminée la peau, les médias devient trouble et de fragments de peau séparés les uns des autres, et les bords des morceaux devenus &quot;flou&quot;. La digestion du poumon doit être arrêté lorsque la couleur change pulmonaires fragments du rouge au blanc et commencer à former des fibres collante. </li><li> Pipeter la solution contenant des fragments de tissus et pour briser les mottes. Transférer la solution dans le tube de 50 ml stérile. Si les fragments se déplacer facilement à travers la coupe 10 ml pipette et la digestion a été bien fait. Rincer le bécher 3 fois avec 10 ml d&#39;eau tiède DMEM/F12 médias avec 15% de FBS, 1X antibiotique / antimycotique et ajouter le support sur le tube de 50 ml avec des fragments de tissus. Fermer le tube de 50 ml et mélanger par inversion à quelques reprises. Le FBS dans les médias cesseront de digestion Libérase. </li><li> Centrifuger à 524 g dans une centrifugeuse de culture tissulaire seau balancer pendant 5 min. Enlever le surnageant. Reprendre le culot dans 10 ml d&#39;eau tiède DMEM/F12 médias avec 15% de FBS, 1X antibiotique / antimycotique. Pipet la suspension avecforce maximale pour briser les morceaux de tissu. </li><li> Ajouter un autre de 30 ml de DMEM/F12 médias avec 15% de FBS, 1X antibiotique / antimycotique, mélanger et centrifuger à 524 g dans une centrifugeuse de culture tissulaire seau balancer pendant 5 min. Répéter une fois de plus pour éliminer les traces d&#39;Libérase. </li><li> Reprendre le culot dans 10 ml de DMEM/F12 médias avec 15% de FBS, 1X antibiotique / antimycotique et transférer dans un plat de 10 cm de culture de tissu et placer dans un incubateur de culture tissulaire à 37 ° C, 5% de CO 2, 3% O 2 . </li><li> Vérifiez les plaques tous les jours pour les fibroblastes et la couleur des médias. Si l&#39;isolement a été un succès, les fibroblastes ramper hors des fragments de tissu et attacher à la plaque (figure 1). Les fibroblastes commencent à quitter les fragments de tissus dans les 2-5 jours. </li><li> Si les changements de supports de la couleur au jaune, cela indique une contamination potentielle ou le surpeuplement des cellules. Examiner les plaques sous la loupe à fort grossissement. Si les bactéries, les champignons ou les vers sont présents jeter les plaques (ce qui est rarement un problème avec les animaux de laboratoire, mais peut se produire lorsque les échantillons sont prélevés dans la nature). Si aucune contamination est présente, mais les médias ont changé de couleur, cela est causé soit par trop de cellules ou de fragments de tissus trop nombreux placés dans le même plat. Si plus de 60% de la plaque est recouverte par les fibroblastes joint, changer le support sur la plaque et le transfert des fragments de tissu d&#39;une nouvelle plaque avec les nouveaux médias. Si ce n&#39;est pas fibroblastes de nombreux attaché à la plaque, le changement des médias et de diviser les morceaux de tissu à un 2-4 plaques. </li><li> Après 7 jours, si les médias n&#39;avaient pas été changés plus tôt, changer le support et le transfert des fragments de tissu d&#39;une nouvelle plaque avec les nouveaux médias. </li><li> Incuber les cellules et de fragments de tissus pour 7 jours supplémentaires. Par tous les jours 14 fibroblastes viables ont quitté les fragments de tissus. </li><li> Après 14 jours depuis le début de l&#39;isolement cellulaire, jeter le vieux médias et des fragments de tissus, la récolte des cellules et la plaque entre eux sur une nouvelle plaque à 5x10 5 cellules / plaque avec EMEM FBS 15%, 1X pénicilline / streptomycine, acides aminés non essentiels , et de sodium pyruvate. Les médias EMEM viendra soutenir la croissance des fibroblastes types de cellules et d&#39;autres ne vont mourir ou d&#39;arrêter la prolifération. </li><li> Après que les cellules atteignent 80-90% de confluence, de geler une partie aliquote de cellules pour une utilisation future. </li><li> Poursuivre la culture des cellules en les découpant à 5x10 5 cellules / plaque lorsque les cellules atteignent 80-90% de confluence. </li></ol> 4. Les résultats représentatifs Fibroblastes normaux sont de grosses cellules avec des protubérances de premier plan (lamellipodes) (figure 2). Fibroblastes poussent en monocouche. Une culture de croissance sain contient des cellules 1-10% dans la phase M, reconnu comme arrondi cellules élevées sur la surface de la plaque, mais pas détaché de la plaque (figure 3). Typiquement, un plat de 10 cm ensemencé avec 5x10 5 cellules confluentes devient en 3-4 jours. Le temps de doublement varie considérablement entre les espèces, et peut être plus élevé pour certaines des espèces de rongeurs à long terme 1. Lorsque les cellules de remplir une plaque qu&#39;ils arrêtent la prolifération en phase G1. Plaque d&#39;confluent de fibroblastes contient une couche de cellules serrées (figure 4). Lorsque les cellules atteignent la confluence de 90% qu&#39;ils sont prêts pour le fractionnement. Si désiré, les cellules peuvent être maintenues sur une plaque arrêtés confluentes pendant de longues périodes de temps avec les changements de supports réguliers (une ou deux fois par semaine). <img src="/files/ftp_upload/2033/2033fig1.jpg" alt="Figure 1" /> Figure 1. L&#39;isolement des fibroblastes de peau de souris (A) et du poumon (B). Les médias a été changé pour éliminer les cellules seules et les débris avant de prendre les photos au jour 7. <img src="/files/ftp_upload/2033/2033fig2.jpg" alt="Figure 2" /> Figure 2. Fibroblastes pulmonaires de la souris. <img src="/files/ftp_upload/2033/2033fig3.jpg" alt="Figure 3" /> Figure 3. Un champ de fibroblastes de souris contenant des cellules en M-scène, photographiée à un grossissement de 10X. <img src="/files/ftp_upload/2033/2033fig4.jpg" alt="Figure 4" /> Figure 4. Une plaque confluent de fibroblastes de souris, photographiées à un grossissement de 10X.

<ol>
	<li>Seluanov, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+tumor+suppressor+mechanisms+evolve+in+rodent+species+that+differ+in+size+and+lifespan.+Aging+.">Distinct tumor suppressor mechanisms evolve in rodent species that differ in size and lifespan. Aging .</a> Cell. 7, 813-823 (2008).</li><li>Parrinello, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxygen+sensitivity+severely+limits+the+replicative+lifespan+of+murine+fibroblasts.">Oxygen sensitivity severely limits the replicative lifespan of murine fibroblasts.</a> Nat Cell Biol. 5, 741-747 (2003).</li></ol>

Normale fibroblastes primaires fournissent une excellente alternative aux établie lignées cellulaires de cancer dans la recherche biologique. Un avantage important des fibroblastes est qu&#39;ils ne portent des mutations dans les oncogènes et suppresseurs de tumeur et de maintenir intactes les points de contrôle du cycle cellulaire. Cela rend les fibroblastes normaux d&#39;un système privilégié pour les études de la régulation du cycle cellulaire, la réparation de l&#39;ADN, et l&#39;apoptose. Le protocole dé...

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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>DMEM/F12 media</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11330-032</td>
<td>&nbsp;</td>
<tr>
<td>Fetal Bovine Serum (FBS), Qualified</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>01437-036</td>
<td>The Qualified serum had been pre-tested to provide good growth support for primary fibroblasts.</td>
</tr>
<tr>
<td>Antibiotic/Antimycotic</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15420-096</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin/Streptomycin</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15140-122</td>
<td>&nbsp;</td>
<tr>
<td>Liberase TM Research Grade</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>05401127001</td>
<td>Replacement enzyme.</td>
<tr>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>A note from the authors:
Since Roche discontinued Liberase Blendzyme 3 (11814184001), they recommend using Liberase TM Research Grade medium Thermolysin (Cat. no. 05401119001 - 10 mg, Cat. no. 05401127001 - 100mg) instead. We have switched to this enzyme successfully with no issues.</td>
<tr>
<td>EMEM media</td>
<td>&nbsp;</td>
<td>ATCC</td>
<td>30-203</td>
<td>The EMEM media from ATCC already contains nonessential amino acids and sodium pyruvate.</td>
</tr>
<tr>
<td>Feathered #21 disposable, sterile scalpel</td>
<td>&nbsp;</td>
<td>Multiple suppliers</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Three Gas Control incubator</td>
<td>&nbsp;</td>
<td>Forma or Heraeus</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

establishing primary adult fibroblast cultures from rodents

L&#39;importance de l&#39;utilisation de cellules primaires, plutôt que de lignées cellulaires de cancer, pour des études biologiques est de plus largement reconnu. Les cellules primaires sont préférés dans les études sur le contrôle du cycle cellulaire, l&#39;apoptose, et la réparation de l&#39;ADN, les cellules cancéreuses sont porteurs de mutations dans les gènes impliqués dans ces processus. Les cellules primaires ne peuvent pas être cultivées indéfiniment en raison de l&#39;apparition de la sénescence réplicative ou aneuploidization. Ainsi, de nouvelles cultures doivent être établis régulièrement. La procédure d&#39;isolement de fibroblastes embryonnaires de rongeurs bien établie, mais d&#39;isoler des cultures de fibroblastes adultes représente souvent un défi. Fibroblastes de rongeurs adultes isolés de modèles murins de maladies humaines peut être un contrôle préféré quand on les compare à des fibroblastes provenant de patients humains. Par ailleurs, les fibroblastes adultes sont les seuls matériaux disponibles lorsque vous travaillez avec des rongeurs sauvages où les femmes enceintes ne peuvent pas être obtenus facilement. Ici nous fournissons un protocole pour l&#39;isolement et la culture de fibroblastes adultes de la peau des rongeurs et des poumons. Nous avons utilisé cette procédure avec succès pour isoler les fibroblastes de plus de vingt espèces de rongeurs à partir des souris de laboratoire et des rats à des rongeurs sauvages tels que le castor, le porc-épic et l&#39;écureuil.

Watch this Scientific Journal Video about Establishing Primary Adult Fibroblast Cultures From Rodents at JoVE.com

Establishing Primary Adult Fibroblast Cultures From Rodents

Cet article décrit un protocole pour l&#39;isolement et l&#39;entretien des cultures de fibroblastes primaires de la peau et les tissus pulmonaires de rongeurs sauvages.

Établir des cultures primaires de fibroblastes adultes à partir de rongeurs

The importance of using primary cells, rather than cancer cell lines, for biological studies is becoming widely recognized.  Primary cells are preferred ...

establishing-primary-adult-fibroblast-cultures-from-rodents

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JoVE Journal

Biology

49.0K vues.  University of Rochester. Cet article décrit un protocole pour l'isolement et l'entretien des cultures de fibroblastes primaires de la peau et les tissus pulmonaires de rongeurs sauvages.

Vidéo: Establishing Primary Adult Fibroblast Cultures From Rodents

Primary Neuronal Cultures from the Brains of Late Stage Drosophila Pupae

In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. The procedure begins with the removal of brains from animals at 70-78 hrs after puparium formation. The isolated brains are shown after brief incubation in papain followed by several washes in serum-free growth medium. The process of mechanical dissociation of each brain in a 5 ul drop of media on a coverslip is illustrated. The axons and dendrites of the post-mitotic neurons are sheered off near the soma during dissociation but the neurons begin to regenerate processes within a few hours of plating. Images show live cultures at 2 days. Neurons continue to elaborate processes during the first week in culture. Specific neuronal populations can be identified in culture using GAL4 lines to drive tissue specific expression of fluorescent markers such as  GFP or RFP. Whole cell recordings have demonstrated the cultured neurons form functional, spontaneously active cholinergic and GABAergic synapses. A short video segment illustrates calcium dynamics in the cultured neurons using Fura-2 as a calcium indicator dye to monitor spontaneous calcium transients and nicotine evoked calcium responses in a dish of cultured neurons. These pupal brain cultures are a useful model system in which genetic and pharmacological tools can be used to identify intrinsic and extrinsic factors that influence formation and function of central synapses.

This video demonstrates the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. Views of live cultures show neurite outgrowth and imaging of calcium levels using Fura-2.

Cultures neuronales primaires du cerveau des drosophiles stade tardif pupes

In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. The procedure begins with the ...

Recherche

Biologie

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 ...

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

La préparation des cultures primaires de cellules hématopoïétiques de la moelle osseuse murine pour l&#39;électroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Primary Culture of Adult Rat Heart Myocytes

Cultured primary adult rodent heart cells are an important model system for cardiovascular research. Nevertheless, establishment of robust, viable cultured adult myocytes can be a technically challenging, rate-limiting step for many researchers. Here we described a protocol to obtain a high yield of adult rat heart myocytes that remain viable in culture for several days. The heart is isolated and perfused with collagenase and protease under low Ca2+ conditions to recover single myocytes. Ca2+-tolerant cells are obtained by stepwise increases in extracellular Ca2+ concentration in three subsequent wash steps. Cells are filtered, resuspended in culture medium, and plated on laminin coated slips. Cultured myocytes obtained using this protocol are viable for up to four days and are suitable for most experiments including electrophysiology, biochemistry, imaging and molecular biology.

In this paper, we described a typical way to isolate and culture adult rat heart myocytes. Collagenase and protease are used to digest and isolate single myocytes. Myocytes cultured follow this protocol meet most experiment requirements.

Culture primaire des adultes myocytes cœur de rat

Cultured primary adult rodent heart cells are an important model system for cardiovascular research.  Nevertheless, establishment of robust, viable ...

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 ...

Rapid Fibroblast Removal from High Density Human Embryonic Stem Cell Cultures

Mouse embryonic fibroblasts (MEFs) were used to establish human embryonic stem cells (hESCs) cultures after blastocyst isolation1. This feeder system maintains hESCs from undergoing spontaneous differentiation during cell expansion. However, this co-culture method is labor intensive, requires highly trained personnel, and yields low hESC purity4. Many laboratories have attempted to minimize the number of feeder cells in hESC cultures (i.e. incorporating matrix-coated dishes or other feeder cell types5-8). These modified culture systems have shown some promise, but have not supplanted the standard method for culturing hESCs with mitomycin C-treated mouse embyronic fibroblasts in order to retard unwanted spontaneous differentiation of the hESC cultures. Therefore, the feeder cells used in hESC expansion should be removed during differentiation experiments. Although several techniques are available for purifying the hESC colonies (FACS, MACS, or use of drug resistant vectors) from feeders, these techniques are labor intensive, costly and/or destructive to the hESC. The aim of this project was to invent a method of purification that enables the harvesting of a purer population of hESCs. We have observed that in a confluent hESC culture, the MEF population can be removed using a simple and rapid aspiration of the MEF sheet. This removal is dependent on several factors, including lateral cell-to-cell binding of MEFs that have a lower binding affinity to the styrene culture dish, and the ability of the stem cell colonies to push the fibroblasts outward during the generation of their own "niche". The hESC were then examined for SSEA-4, Oct3/4 and Tra 1-81 expression up to 10 days after MEF removal to ensure maintenance of pluripotency. Moreover, hESC colonies were able to continue growing from into larger formations after MEF removal, providing an additional level of hESC expansion.

Despite ongoing efforts to transition cultures to feeder-free conditions, the derivation and culture of human embryonic stem cells (hESC) remain largely dependent on co-cultures with mouse embryonic feeders (MEFs). Here, we show a novel methodology for rapidly removing feeders from hESC cultures prior to experimentation.

Suppression des fibroblastes rapide de haute densité embryonnaires humaines cultures de cellules souches

Mouse embryonic fibroblasts (MEFs) were used to establish human embryonic stem cells (hESCs) cultures after blastocyst isolation1. This feeder system ...

Na&iuml;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

Over the last decade, several adult stem cell populations have been identified in human skin 1-4. The isolation of multipotent adult dermal precursors was first reported by Miller F. D laboratory 5, 6. These early studies described a multipotent precursor cell population from adult mammalian dermis 5. These cells--termed SKPs, for skin-derived precursors-- were isolated and expanded from rodent and human skin and differentiated into both neural and mesodermal progeny, including cell types never found in skin, such as neurons 5. Immunocytochemical studies on cultured SKPs revealed that cells expressed vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors, in addition to fibronectin and multipotent stem cell markers 6. Until now, the adult stem cells population SKPs have been isolated from freshly collected mammalian skin biopsies.
Recently, we have established and reported that a population of skin derived precursor cells could remain present in primary fibroblast cultures established from skin biopsies 7. The assumption that a few somatic stem cells might reside in primary fibroblast cultures at early population doublings was based upon the following observations: (1) SKPs and primary fibroblast cultures are derived from the dermis, and therefore a small number of SKP cells could remain present in primary dermal fibroblast cultures and (2) primary fibroblast cultures grown from frozen aliquots that have been subjected to unfavorable temperature during storage or transfer contained a small number of cells that remained viable 7. These rare cells were able to expand and could be passaged several times. This observation suggested that a small number of cells with high proliferation potency and resistance to stress were present in human fibroblast cultures 7.
We took advantage of these findings to establish a protocol for rapid isolation of adult stem cells from primary fibroblast cultures that are readily available from tissue banks around the world (Figure 1). This method has important significance as it allows the isolation of precursor cells when skin samples are not accessible while fibroblast cultures may be available from tissue banks, thus, opening new opportunities to dissect the molecular mechanisms underlying rare genetic diseases as well as modeling diseases in a dish.

Na&amp;#239;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

We report a method to isolate na&#239;ve multipotent skin-derived precursor (SKP) cells from primary human fibroblast cultures. We show that these SKPs derived from fibroblast cultures share similar stem cell properties to the ones derived directly from human skin biopsies. These cells express the neural crest marker, nestin, in addition to the multipotent markers such as OCT4 and Nanog.

Naïve cellules souches adultes Isolation de cultures de fibroblastes humains primaires

Over the last decade, several adult stem cell  populations have been identified in human skin 1-4.  The isolation of multipotent adult dermal precursors ...

Preparation of Primary Myogenic Precursor Cell/Myoblast Cultures from Basal Vertebrate Lineages

Due to the inherent difficulty and time involved with studying the myogenic program in vivo, primary culture systems derived from the resident adult stem cells of skeletal muscle, the myogenic precursor cells (MPCs), have proven indispensible to our understanding of mammalian skeletal muscle development and growth. Particularly among the basal taxa of Vertebrata, however, data are limited describing the molecular mechanisms controlling the self-renewal, proliferation, and differentiation of MPCs. Of particular interest are potential mechanisms that underlie the ability of basal vertebrates to undergo considerable postlarval skeletal myofiber hyperplasia (i.e.&#160;teleost fish) and full regeneration following appendage loss (i.e.&#160;urodele amphibians). Additionally, the use of cultured myoblasts could aid in the understanding of regeneration and the recapitulation of the myogenic program and the differences between them. To this end, we describe in detail a robust and efficient protocol (and variations therein) for isolating and maintaining MPCs and their progeny, myoblasts and immature myotubes, in cell culture as a platform for understanding the evolution of the myogenic program, beginning with the more basal vertebrates. Capitalizing on the model organism status of the zebrafish (Danio rerio), we report on the application of this protocol to small fishes of the cyprinid clade Danioninae. In tandem, this protocol can be utilized to realize a broader comparative approach by isolating MPCs from the Mexican axolotl (Ambystomamexicanum) and even laboratory rodents. This protocol is now widely used in studying myogenesis in several fish species, including rainbow trout, salmon, and sea bream1-4.

In vitro culture systems have proven indispensible to our understanding of vertebrate myogenesis. However, much remains to be learned about nonmammalian skeletal muscle development and growth, particularly in basal taxa. An efficient and robust protocol for isolating the adult stem cells of this tissue, the myogenic precursor cells (MPCs), and maintaining their self-renewal, proliferation, and differentiation in a primary culture setting allows for the identification of conserved and divergent regulatory mechanisms throughout the vertebrate lineages.

Préparation du primaire Myogenic précurseurs cellulaires / cultures de myoblastes de basales vertébrés lignées

Due to the inherent difficulty and time involved with studying the myogenic program in vivo, primary culture systems derived from the resident adult stem ...

Application of Retinoic Acid to Obtain Osteocytes Cultures from Primary Mouse Osteoblasts

The need for osteocyte cultures is well known to the community of bone researchers; isolation of primary osteocytes is difficult and produces low cell numbers. Therefore, the most widely used cellular system is the osteocyte-like MLO-Y4 cell line.
The method here described refers to the use of retinoic acid to generate a homogeneous population of ramified cells with morphological and molecular osteocyte features.
After isolation of osteoblasts from mouse calvaria, all-trans retinoic acid (ATRA) is added to cell medium, and cell monitoring is conducted daily under an inverted microscope. First morphological changes are detectable after 2 days of treatment and differentiation is generally complete in 5 days, with progressive development of dendrites, loss of the ability to produce extracellular matrix, down-regulation of osteoblast markers and up-regulation of osteocyte-specific molecules.
Daily cell monitoring is needed because of the inherent variability of primary cells, and the protocol can be adapted with minimal variation to cells obtained from different mouse strains and applied to transgenic models. 
The method is easy to perform and does not require special instrumentation, it is highly reproducible, and rapidly generates a mature osteocyte population in complete absence of extracellular matrix, allowing the use of these cells for unlimited biological applications.

Treatment of primary mouse osteoblasts with retinoic acid produces a homogeneous population of ramified cells bearing morphological and molecular features of osteocytes. The method overcomes the difficulty of obtaining and maintaining primary osteocytes in culture, and can be advantageous to study cells derived from transgenic models.&#160;

Application de l&#39;acide rétinoïque pour obtenir des cultures de ostéocytes de primaires ostéoblastes de souris

The need for osteocyte cultures is well known to the community of bone researchers; isolation of primary osteocytes is difficult and produces low cell ...

Isolation and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin defense by forming an intact skin barrier against environmental insults, such as UVB irradiation or pathogens, and also by initiating an inflammatory response upon those insults. Here we describe methods to isolate KCs from neonatal mouse skin and from adult mouse tail skin. We also describe culturing conditions using defined growth supplements (dGS) in comparison to chelexed fetal bovine serum (cFBS). Functionally, we show that both neonatal and adult KCs are highly responsive to high calcium-induced terminal differentiation, tight junction formation and stratification. Additionally, cultured adult KCs are susceptible to UVB-triggered cell death and can release large amounts of TNF upon UVB irradiation. Together, the methods described here will be useful to researchers for the setup of in vitro models to study epidermal biology in the neonatal mouse and/or the adult mouse.

Epidermal keratinocytes form a functional skin barrier and are positioned at the front line of host defense against external environmental insults. Here we describe methods for isolation and primary culture of epidermal keratinocytes from neonatal and adult mouse skin, and induction of terminal differentiation and UVB-triggered inflammatory response from keratinocytes.

Isolation et culture des kératinocytes de la souris primaire de la peau de souris néonatale et adulte

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin ...