Remarque: Pour maintenir des conditions de culture stérile, toutes les procédures dans ce protocole sont réalisées en utilisant des pratiques de laboratoire stérile et réalisée sous hotte à flux laminaire. Avant de commencer, s&#39;assurer que tout média est équilibré à 37 ° C et de manière appropriée gazés. Préparation des boîtes de culture Geltrex enduits Note: voir l&#39;annexe pour l&#39;utilisation de plats CELLstart culture revêtues <ol><li> Dégeler un tube de Geltrex (1 ml) lentement à 2-8 ° C et diluer 1:100 c&#39;est dans 99 ml de Knockout D-MEM/F-12. Mélanger délicatement la solution. Remarque: Certaines des lignées de cellules iPS peuvent nécessiter une dilution différente Geltrex pour une croissance optimale. Voir l&#39;annexe pour des dilutions alternatives. </li><li> Couvrir toute la surface de chaque boîte de culture avec la solution Geltrex (1 ml pour un plat de 35 mm, 1,5 ml pour un plat de 60 mm). </li><li> Sceau chaque plat avec du Parafilm pour éviter le dessèchement et incuber les plats pendant 1 heure à 37 ° C. Remarque: À ce stade, vous pouvez stocker les boîtes de culture Geltrex enduits à 4 ° C jusqu&#39;à un mois. Sceau chaque plat avec du Parafilm pour empêcher la Geltrex de se dessécher. </li><li> Avant d&#39;utiliser, de transférer les plats Geltrex enduits d&#39;une hotte à flux laminaire et de leur permettre de s&#39;équilibrer à température ambiante (environ 1 heure). </li></ol> Préparation complète d&#39;alimentation sans SR KnockOut Medium <ol><li> Pour préparer 1 mL de 10 ug / ml solution FGF basique, de façon aseptique mélanger les composants énumérés ci-dessous. Aliquoter la solution et conserver à -20 ° C jusqu&#39;à 6 mois. <table border="0"><tbody><tr><td> FGF basique </td><td> 10 ug </td></tr><tr><td> D-PBS </td><td> 990 uL </td></tr><tr><td> 10% de BSA </td><td> 10 uL </td></tr></tbody></table> Remarque: BSA peut être substitué avec HSA ou SR Knockout à la même concentration. </li><li> Pour préparer 50 ml de solution 2 Dispase mg / mL, de façon aseptique mélanger les composants énumérés ci-dessous. Filtre stériliser la solution et conserver à 4 ° C pendant 2 semaines. <table border="0"><tbody><tr><td> Dispase </td><td> 100 mg </td></tr><tr><td> D-PBS </td><td> 50 ml </td></tr></tbody></table></li><li> Pour préparer 100 ml de SR KnockOut complète Feeder-Free (KSR-FF) moyennes aseptiquement combiner les composants répertoriés dans le tableau ci-dessous. <table border="1"><tbody><tr><td> Composante </td><td> Concentration Stock </td><td> Concentration finale </td><td> Volume </td></tr><tr><td> Knockout DMEM/F12 (Cat. no. 12660-012) </td><td> - </td><td> 1X </td><td> 76,8 ml </td></tr><tr><td> Glutamax-I (cat. n ° 35050-061) </td><td> 200 mM </td><td> 2 mM </td><td> 1 ml </td></tr><tr><td> KnockOut SR (Cat. no. 10828-028) </td><td> - </td><td> 20% </td><td> 20 ml </td></tr><tr><td> KnockOut SR-GFC (Cat. no. A10580-01) </td><td> 50X </td><td> 1X </td><td> 2 ml </td></tr><tr><td> bFGF (Cat. n. PHG0024) </td><td> 10 ug / ml </td><td> 20 ng / mL </td><td> 200 uL </td></tr></tbody></table> Vous pouvez ranger le support KSR-FF à 2-8 ° C jusqu&#39;à une semaine. </li><li> Juste avant la pré-équilibration du milieu complet à la température et de gaz, ajouter aseptiquement le volume requis de 2 mercaptoéthanol (55 mM de concentration des stocks) pour une concentration de 0,1 mM final. Par exemple, pour préparer 100 ml de KSR-FF moyennes ajoutez 182 uL de 55 mM de 2-mercaptoéthanol (1:550 dilution) Alternativement, le 2-mercaptoéthanol peuvent être ajoutés au milieu de 1X terminé et stocké à 2-8 ° C pour jusqu&#39;à une semaine. </li></ol> S&#39;adapter aux iPSCs KSR-FF Medium <ol><li> Avant de commencer, pré-équilibrer votre vaisselle Geltrex revêtement à la température ambiante dans la hotte. </li><li> Culture du iPSCs sur des cellules nourricières MEF jusqu&#39;à ce qu&#39;elles soient confluentes à 70-80%. S&#39;il vous plaît se référer au manuel de fibroblastes de souris embryonnaires GIBCO pour les protocoles de culture à base MEF. </li&gt; <li> Pré-chauffer le volume requis de Dispase dans un bain d&#39;eau à 37 ° C. Se reporter au Tableau 1 ci-dessous pour plus de détails sur les volumes requis. </li><li> Pré-équilibrer le volume requis de KSR-FF dans un bain à 37 ° C pendant 15 min de l&#39;eau. Se reporter au Tableau 1 ci-dessous pour plus de détails sur les volumes requis. </li><li> Aspirer le support des boîtes de culture, et d&#39;ajouter une quantité appropriée de solution Dispase. Incuber les boîtes à 37 ° C pendant 3-5 minutes. </li><li> Aspirer la solution Dispase de chaque navire de la culture et de laver les cellules nourricières MEF doucement avec du D-PBS (2 à 3 fois). </li><li> Ajouter une quantité appropriée de milieu KnockOut SR complets pour chaque navire de la culture. Utilisez un grattoir cellulaire ou une pipette de 5 ml à gratter délicatement les cellules de la surface du récipient de culture. Note: voir l&#39;annexe pour l&#39;adaptation des protocoles alternatifs pour les lignes dures cellule de s&#39;adapter iPS, </li><li> Recueillir la suspension de cellules de chaque boîte de culture en tubes séparés 15 ml conique. Rincer chaque récipient de culture avec une quantité appropriée de milieu KnockOut SR complets, et ajouter le D-PBS rincez moyenne dans les 15 ml tubes coniques contenant la suspension cellulaire. Soyez prudent de ne pas briser les mottes de cellules dans des cellules individuelles </li><li> Centrifuger les tubes coniques 15 ml à 200 xg pendant 5 minutes pour culotter les iPSCs. </li><li> Aspirer le surnageant du culot IPSC. Reprendre le culot dans une quantité appropriée de KSR-FF moyennes selon le rapport de split (tableau 1). Ne pas casser les mottes de cellules d&#39;une taille plus petite, parce que les amas plus petits de ne pas attacher ainsi à la surface. Remarque: Nous recommandons un rapport de division de 1:2 pour les 3 premiers passages après la iPSCs ont été repiquées directement du milieu iPSC Culture MEF à KSR-FF moyenne. Normalement, un rapport de split 1:03-1:05 est approprié, mais au repiquage 01h02 assure la plus grande densité de cellules nécessaires lors de l&#39;adaptation à une culture nourricier libre. </li><li> Avant de plaquer les iPSCs sur les plats Geltrex revêtement, aspirer résiduelle solution Geltrex de l&#39;antenne de pré-enduit, et ajoutez lentement une quantité appropriée de suspension cellulaire à chaque boîte de culture. Remarque: ne pas rincer la vaisselle avant le placage. </li><li> Déplacer la boîte de culture avant en arrière et côté à l&#39;autre à plusieurs reprises pour disperser les cellules sur la surface du plat. Placez délicatement la boîte de culture dans un incubateur à 37 ° C avec une atmosphère humidifiée de 4 à 6% de CO 2 dans l&#39;air. Remplacez le support passé avec KSR-FF tous les jours. </li></ol> Le repiquage de cellules iPS humaines utilisant KSR-FF <ol start="13"><li> Respecter les iPSCs humaine de plus en plus complète KSR-FF sous le microscope pour confirmer que les cellules sont confluentes à 70-80% et prêt à être repiquées. Se reporter à la figure 1. Remarque: Si les colonies deviennent trop dense ou trop grande, une différenciation accrue survient. </li><li> Découper et retirer toutes les colonies différenciées iPSC avant repiquage de la culture. </li><li> Pré-chauffer le volume requis de Dispase dans un bain d&#39;eau à 37 ° C. Se reporter au Tableau 1 ci-dessous pour plus de détails sur les volumes requis. </li><li> Pré-équilibrer le volume requis de KSR-FF dans un bain à 37 ° C pendant 15 min de l&#39;eau. Se reporter au Tableau 1 ci-dessous pour plus de détails sur les volumes requis. </li><li> Aspirer le milieu épuisé du récipient de culture à l&#39;aide d&#39;une pipette, et rincer les cellules deux fois avec du D-PBS. </li><li> Ajouter délicatement solution de dispase préchauffée à la cuve de culture (par exemple, 1 ml de solution de dispase par boîte de culture de 60 mm). Agiter le récipient de culture pour enrober la surface des cellules entières. </li><li> Incuber le récipient de culture à 37 ° C pendant 3 minutes. </li><li> Retirez le récipient de l&#39;incubateur, aspirer la solution Dispase, et laver délicatement les cellules avec du D-PBS. </li><li> Gratter délicatement les cellules de la surface de la boîte de culture cellulaire à l&#39;aide d&#39;un grattoir, et le transfert des cellules dans un tube de centrifugeuse de 15 ml stérile. </li><li> Rincer la boîte de culture à deux reprises avec KSR-FF, doucement &quot;pulvériser off&quot; toutes les cellules qui n&#39;ont pas détaché. Piscine de rinçage moyenne avec des cellules dans le tube de 15ml. </li><li> Centrifuger le tube à 200 xg pendant 5 minutes à température ambiante pour un culot cellulaire. </li><li> Aspirer délicatement le surnageant sans perturber le culot cellulaire et le jeter. </li><li> Tapotez doucement le tube pour bien déloger le culot cellulaire du fond du tube. </li><li> Doucement remettre les cellules en pré-équilibrée KSR-FF aide d&#39;une pipette 5mL sérologique. Ne pas triturer. Remarque: il est essentiel à l&#39;étape pour remettre les cellules en douceur, sans utiliser la force pour éviter tout dommage. </li><li> Transférer les cellules d&#39;une nouvelle de 60 mm Geltrex revêtement plat à rapport de division désiré et déplacer la boîte de culture avant en arrière et côté à l&#39;autre à plusieurs reprises pour disperser les cellules à travers sa surface. </li><li> Placez la boîte de culture dans un incubateur à 37 ° C avec une atmosphère humidifiée de4 à 6% de CO 2 dans l&#39;air. </li><li> Le lendemain, doucement remplacer le milieu passé avec KSR-FF pour éliminer les débris cellulaires. Remplacez le support passé tous les jours par la suite. Observez les cellules iPS quotidienne et le passage au besoin (environ tous les 4-5 jours). Le repiquage est recommandé lorsque les cellules atteignent 70-80% de confluence. Pour la cryoconservation de cellules iPS et de dégel, reportez-vous à notre protocole intitulé «cryoconservation et récupération des cellules iPS humaines à l&#39;aide complète d&#39;alimentation sans remplacement KnockOut Sérum Medium&quot;. </li></ol> Résultats attendus <img src="/files/ftp_upload/2236/2236fig1.jpg" alt="Figure 1" /> Figure 1. L&#39;image en contraste de phase ci-dessous montre iPSCs cultivés sur des boîtes de culture Geltrex revêtement à l&#39;aide complète d&#39;alimentation sans remplacement KnockOut Sérum moyenne. Les CISP présentent une morphologie similaire aux CSEh, caractérisé par de gros noyaux et cytoplasme. (Grossissement 40x) <table border="1"><tbody><tr><td> Composante </td><td> Plat 35mm </td><td> Plat 60mm </td><td> Plat 100mm </td></tr><tr valign="top"><td> Remplissez KnockOut SR moyennes </td><td> 2 ml </td><td> 4 ml </td><td> 10 ml </td></tr><tr valign="top"><td> Solution Geltrex </td><td> 1 ml </td><td> 1,5 ml </td><td> 4-5 ml </td></tr><tr valign="top"><td> Dispase </td><td> 0,5 ml </td><td> 1 ml </td><td> 3-4 ml </td></tr><tr valign="top"><td> D-PBS pour le rinçage </td><td> 2 ml </td><td> 4 ml </td><td> 10 ml </td></tr></tbody></table> Tableau 1. Volumes recommandés <a href="http://www.jove.com/files/ftp_upload/2236/2236appendix.pdf">S&#39;il vous plaît cliquer ici pour voir l&#39;appendice</a> .

<ol>
	<li>Takahashi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+pluripotent+stem+cells+from+mouse+embryonic+and+adult+fibroblast+cultures+by+defined+factors.">Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.</a> Cell. 126, 663-676 (2006).</li><li>Takahashi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+pluripotent+stem+cells+from+adult+human+fibroblasts+by+defined+factors.">Induction of pluripotent stem cells from adult human fibroblasts by defined factors.</a> Cell. 131, 861-872 (2007).</li><li>Yu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induced+pluripotent+stem+cell+lines+derived+from+human+somatic+cells.">Induced pluripotent stem cell lines derived from human somatic cells.</a> Science. 318, 1917-1920 (2007).</li><li>Maherali, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guidelines+and+techniques+for+the+generation+of+induced+pluripotent+stem+cells.">Guidelines and techniques for the generation of induced pluripotent stem cells.</a> Cell Stem Cell. 3, 595-605 (2008).</li><li>Li, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+rat+and+human+induced+pluripotent+stem+cells+by+combining+genetic+reprogramming+and+chemical+inhibitors.">Generation of rat and human induced pluripotent stem cells by combining genetic reprogramming and chemical inhibitors.</a> Cell Stem Cell. 4, 16-19 (2009).</li><li>Liao, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+induced+pluripotent+stem+cell+lines+from+adult+rat+cells.">Generation of induced pluripotent stem cell lines from adult rat cells.</a> Cell Stem Cell. 4, 11-15 (2009).</li><li>Dimos, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induced+pluripotent+stem+cells+generated+from+patients+with+ALS+can+be+differentiated+into+motor+neurons.">Induced pluripotent stem cells generated from patients with ALS can be differentiated into motor neurons.</a> Science. 321, 1218-1221 (2008).</li><li>Aasen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+and+rapid+generation+of+induced+pluripotent+stem+cells+from+human+keratinocytes.">Efficient and rapid generation of induced pluripotent stem cells from human keratinocytes.</a> Nat Biotechnli. 26, 1276-1284 (2008).</li><li>Park, I. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+human-induced+pluripotent+stem+cells.">Generation of human-induced pluripotent stem cells.</a> Nat Protoc. 3, 1180-1186 (2008).</li></ol>

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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>Knockout DMEM/F12</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Cat. no. 12660-012</td>
<td>Note: see appendix for the use of alternative DMEM products</td>
</tr>
<tr>
<td>GlutaMAX-I</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Cat. No. 35050-061</td>
<td>&nbsp;</td>
<tr>
<td>KnockOut Serum Replacement</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>KnockOut SR, Cat. no. 10828-028</td>
<td>&nbsp;</td>
<tr>
<td>KnockOut Serum Replacement Growth Factor Cocktail</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>KnockOut SR-GFC, Cat. no. A10580-01</td>
<td>&nbsp;</td>
<tr>
<td>FGF-basic, human recombinant protein, 10 &mu;g</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>bFGF, Cat. no. PHG0024</td>
<td>Note: see appendix for alternative bFGF pack sizes.</td>
</tr>
<tr>
<td>2-Mercaptoethanol</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Cat. no. 21985-023</td>
<td>&nbsp;</td>
<tr>
<td>Dispase</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Cat. no. 17105-041</td>
<td>Note: see appendix for alternative passaging methods.</td>
</tr>
<tr>
<td>Geltrex hESC-Qualified Reduced Growth Factor Basement Matrix, 1 mL</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Cat. no. A10480-01</td>
<td>&nbsp;</td>
<tr>
<td>Dulbecco&#x2019;s Phosphate Buffered Saline (D-PBS) without calcium and magnesium </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Cat. no. 14190-144</td>
<td>&nbsp;</td>
<tr>
<td>Sterile Tissue Culture Hood</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Incubator set at 37&deg;C</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Pipette-Aid</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Water Bath set at 37&deg;C</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Sterile serological pipettes (5 mL, 10 mL)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Centrifuge</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>15 mL centrifuge tubes</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>60mm Tissue Culture treated dishes</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

feeder-free adaptation, culture and passaging of human ips cells using complete knockout serum replacement feeder-free medium

La découverte en 2006 que les fibroblastes humains et de souris pouvaient être reprogrammées pour produire des cellules iPS 1-3 avec des qualités remarquablement similaires aux cellules souches embryonnaires a créé une nouvelle source précieuse de cellules pluripotentes pour la découverte de médicaments, la thérapie cellulaire, et la recherche fondamentale. Les médias et les réactifs GIBCO ont été à la pointe de la recherche de cellules souches pluripotentes pendant des années. Knockout DMEM supplémenté avec remplacement Knockout sérum est le média de choix pour la croissance des cellules souches embryonnaires et maintenant la culture de cellules iPS 3-9. Cet or média standard du système peut maintenant être utilisé pour le chargeur sans la culture avec l&#39;ajout de SR Knockout facteur de croissance cocktail. Traditionnelle ES humaines et des méthodes de culture de cellules iPS nécessite l&#39;utilisation de la souris ou humains couches nourricières de fibroblastes, ou le chargeur milieu conditionné. Ces méthodes de culture sont une main-d&#39;œuvre, dur à l&#39;échelle et il est difficile de maintenir les cellules indifférenciées hanches à cause des conditions indéfini. Invitrogen a développé SR Knockout facteur de croissance cocktail pour vous permettre de passer facilement vos cultures hanches cellule d&#39;alimentation sans tout en conservant votre utilisation du SR Knockout.

Watch this Scientific Journal Video about Feeder-Free Adaptation, Culture and Passaging of Human IPS Cells using Complete KnockOut Serum Replacement Feeder-Free Medium at JoVE.com

Feeder-Free Adaptation, Culture and Passaging of Human IPS Cells using Complete KnockOut Serum Replacement Feeder-Free Medium

Le protocole suivant fournit des instructions pour l&#39;adaptation humaine souches pluripotentes induites (iPS) à la desserte de culture sans l&#39;aide complète d&#39;alimentation sans remplacement KnockOut Sérum moyennes (KSR-FF). Une fois adaptée, des instructions pour l&#39;entretien continu sont également fournis.

Chargeur sans adaptation, de la Culture et de repiquage des cellules iPS humaines à l&#39;aide complète d&#39;alimentation sans remplacement KnockOut Sérum Medium

The discovery in 2006 that human and mouse fibroblasts could be reprogrammed to generate iPS cells 1-3 with qualities remarkably similar to embryonic stem ...

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Vidéo: Feeder-Free Adaptation, Culture and Passaging of Human IPS Cells using Complete KnockOut Serum Replacement Feeder-Free Medium

Passaging HuES Human Embryonic Stem Cell-lines with Trypsin.

In this video we demonstrate how our lab routinely passages HuES human embryonic stem cell lines with trypsin.  Human embryonic stem cells are artifacts of cell culture, and tend to acquire karyotypic abnormalities with high population doublings.  Proper passaging is essential for maintaining a healthy, undifferentiated, karyotypically normal HuES human embryonic stem cell culture.  First, an expanding culture is washed in PBS to remove residual media and cell debris, then cells are overlaid with a minimal volume of warm 0.05% Trypsin-EDTA.  Trypsin is left on the cells for up to five minutes, then cells are gently dislodged with a 2mL serological pipette.  The cell suspension is collected and mixed with a large volume of HuES media, then cells are collected by gentle centrifugation.  The inactivated trypsin media mixture is removed, and cells resuspended in pre-warmed HuES media.  An appropriate split ratio is calculated (generally 1:10 to 1:20), and cells re-plated onto a 1-2 day old plate containing a monolayer of irradiated mouse embryonic fibroblast feeder cells.  The newly seeded HuES culture plate is left undisturbed for 48 hrs, then media is changed every day thereafter.  It is important not to trpsinize down to a single cell suspension, as this increases the risk of introducing karyotypic abnormalities.

In this video we demonstrate how our lab routinely passages HuES human embryonic stem cell lines with trypsin. Brought to you by JoVE.

HUES repiquage souches embryonnaires humaines lignées cellulaires à la trypsine

In this video we demonstrate how our lab routinely passages HuES human embryonic stem cell lines with trypsin.  Human embryonic stem cells are artifacts ...

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Human ES cells: Starting Culture from Frozen Cells

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

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

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

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

Passaging Human Neural Stem Cells

The ability to manipulate human neural stem/precursor cells (hNSPCs) in vitro provides a means to investigate their utility as cell transplants for therapeutic purposes as well as to explore many fundamental processes of human neural development and pathology.  This protocol presents a simple method of culturing and passaging hNSPCs in hopes of standardizing this technique and increasing reproducibility of human stem cell research.  The hNSPCs we use were isolated from cadaveric postnatal brain cortices by the National Human Neural Stem Cell Resource and grown as adherent cultures on flasks coated with fibronectin (Palmer et al., 2001; Schwartz et al., 2003).  We culture our hNSPCs in a DMEM:F12 serum-free media supplemented with EGF, FGF, and PDGF and passage them 1:2 approximately every seven days.  Using these conditions, the majority of the cells in the culture maintain a bipolar morphology and express markers of undifferentiated neural stem cells (such as nestin and sox2).

The ability to manipulate human neural stem/precursor cells (hNSPCs) in vitro allows to investigate their utility as cell transplants for therapeutic purposes and to explore human neural development. This protocol presents a method of culturing and passaging hNSPCs in hopes of increasing reproducibility of human stem cell research.

Le repiquage des cellules souches humaines

The ability to manipulate human neural stem/precursor cells (hNSPCs) in vitro provides a means to investigate their utility as cell transplants for ...

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 ...

Cryopreserving and Recovering of Human iPS Cells using Complete KnockOut Serum Replacement Feeder-Free Medium

The discovery in 2006 that human and mouse fibroblasts could be reprogrammed to generate iPS cells 1-3 with qualities remarkably similar to embryonic stem cells has created a valuable new source of pluripotent cells for drug discovery, cell therapy, and basic research.
GIBCO media and reagents have been at the forefront of pluripotent stem cell research for years. Knockout DMEM supplemented with Knockout Serum Replacement is the media of choice for embryonic stem cell growth and now iPS cell culture 3-9. This gold standard media system can now be used for feeder-free culture with the addition of Knockout SR Growth Factor Cocktail.
Traditional human ES and iPS cell culture methods require the use of mouse or human fibroblast feeder layers, or feeder-conditioned medium. These culture methods are labor-intensive, hard to scale and it is difficult to maintain hiPS cells undifferentiated due to the undefined conditions. Invitrogen has developed Knockout SR Growth Factor Cocktail to allow you to easily transition your hiPS and hES cell cultures to feeder-free while still maintaining your use of Knockout SR.

This protocol describes the detailed procedure for cryopreserving human iPS cells in KnockOut SR cryopreservation medium and recovering these cells in complete KnockOut SR Feeder Free (KSR-FF) medium or feeder-based KnockOut SR medium.

Cryoconservation et la récupération des cellules iPS humaines utilisant KnockOut complète Chargeur sans sérum remplacement moyen

Generation of Human Cardiomyocytes: A Differentiation Protocol from Feeder-free Human Induced Pluripotent Stem Cells

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is essential first to generate functional human cardiomyocytes (CMs). The use of these cells in drug discovery and toxicology studies would also be highly beneficial, allowing new pharmacological molecules for the treatment of cardiac disorders to be validated pre-clinically on cells of human origin. Of the possible sources of CMs, induced pluripotent stem (iPS) cells are among the most promising, as they can be derived directly from readily accessible patient tissue and possess an intrinsic capacity to give rise to all cell types of the body 1. Several methods have been proposed for differentiating iPS cells into CMs, ranging from the classical embryoid bodies (EBs) aggregation approach to chemically defined protocols 2,3. In this article we propose an EBs-based protocol and show how this method can be employed to efficiently generate functional CM-like cells from feeder-free iPS cells.

Pluripotent stem cells, either embryonic or induced pluripotent stem (iPS) cells, constitute a valuable source of human differentiated cells, including cardiomyocytes. Here, we will focus on cardiac induction of iPS cells, showing how to use them to obtain functional human cardiomyocytes through an embryoid bodies-based protocol.

Génération de cardiomyocytes humains: Un protocole de différenciation des induits des cellules souches pluripotentes humaines chargeur sans

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is ...

Isolation of Small Noncoding RNAs from Human Serum

The analysis of RNA and its expression is a common feature in many laboratories. Of significance is the emergence of small RNAs like microRNAs, which are found in mammalian cells. These small RNAs are potent gene regulators controlling vital pathways such as growth, development and death and much interest has been directed at their expression in bodily fluids. This is due to their dysregulation in human diseases such as cancer and their potential application as serum biomarkers. However, the analysis of miRNA expression in serum may be problematic. In most cases the amount of serum is limiting and serum contains low amounts of total RNA, of which small RNAs only constitute 0.4-0.5%1. Thus the isolation of sufficient amounts of quality RNA from serum is a major challenge to researchers today. In this technical paper, we demonstrate a method which uses only 400 &#181;l of human serum to obtain sufficient RNA for either DNA arrays or qPCR analysis. The advantages of this method are its simplicity and ability to yield high quality RNA. It requires no specialized columns for purification of small RNAs and utilizes general reagents and hardware found in common laboratories. Our method utilizes a Phase Lock Gel to eliminate phenol contamination while at the same time yielding high quality RNA. We also introduce an additional step to further remove all contaminants during the isolation step. This protocol is very effective in isolating yields of total RNA of up to 100 ng/&#181;l from serum but can also be adapted for other biological tissues.

This protocol describes a method for extracting small RNAs from human serum. We have used this method to isolate microRNAs from cancer serum for use in DNA arrays and also singleplex quantitative PCR. The protocol utilizes phenol and guanidinium thiocyanate reagents with modifications to yield high quality RNA.

Isolation des petits ARN non codant de sérum humain

The analysis of RNA and its expression is a common feature in many laboratories. Of significance is the emergence of small RNAs like microRNAs, which are ...

MicroRNA Expression Profiles of Human iPS Cells, Retinal Pigment Epithelium Derived From iPS, and Fetal Retinal Pigment Epithelium

The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, retinal pigment epithelium (RPE) derived from human iPS cells (iPS-RPE), and fetal RPE. The protocols include collection of RNA for analysis by microarray, and the analysis of microarray data to identify miRNAs that are differentially expressed among three cell types. The methods for culture of iPS cells and fetal RPE are explained. The protocol used for differentiation of RPE from human iPS is also described. The RNA extraction technique we describe was selected to allow maximal recovery of very small RNA for use in a miRNA microarray. Finally, cellular pathway and network analysis of microarray data is explained. These techniques will facilitate the comparison of the miRNA profiles of three different cell types.

The microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, retinal pigment epithelium (RPE) derived from human induced-pluripotent stem (iPS) cells (iPS-RPE), and fetal RPE, were compared.

Profils MicroRNA d&#39;expression de cellules iPS humaines, l&#39;épithélium pigmentaire rétinien Derived From iPS, et fœtale épithélium pigmentaire rétinien

The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, ...

Efficient iPS Cell Generation from Blood Using Episomes and HDAC Inhibitors

This manuscript illustrates a protocol for efficiently creating integration-free human induced pluripotent stem cells (iPSCs) from peripheral blood using episomal plasmids and histone deacetylase (HDAC) inhibitors. The advantages of this approach include: (1) the use of a minimal amount of peripheral blood as a source material; (2) nonintegrating reprogramming vectors; (3) a cost effective method for generating vector free iPSCs; (4) a single transfection; and (5) the use of small molecules to facilitate epigenetic reprogramming. Briefly, peripheral blood mononuclear cells (PBMCs) are isolated from routine phlebotomy samples and then cultured in defined growth factors to yield a highly proliferative erythrocyte progenitor cell population that is remarkably amenable to reprogramming. Nonintegrating, nontransmissible episomal plasmids expressing OCT4, SOX2, KLF4, MYCL, LIN28A, and a p53 short hairpin (sh)RNA are introduced into the derived erythroblasts via a single nucleofection. Cotransfection of an episome that expresses enhanced green fluorescent protein (eGFP) allows for easy identification of transfected cells. A separate replication-deficient plasmid expressing Epstein-Barr nuclear antigen 1 (EBNA1) is also added to the reaction mixture for increased expression of episomal proteins. Transfected cells are then plated onto a layer of irradiated mouse embryonic fibroblasts (iMEFs) for continued reprogramming. As soon as iPSC-like colonies appear at about twelve days after nucleofection, HDAC inhibitors are added to the medium to facilitate epigenetic remodeling. We have found that the inclusion of HDAC inhibitors routinely increases the generation of fully reprogrammed iPSC colonies by 2 fold. Once iPSC colonies exhibit typical human embryonic stem cell (hESC) morphology, they are gently transferred to individual iMEF-coated tissue culture plates for continued growth and expansion.

Here we describe a protocol for generating human induced pluripotent stem cells from peripheral blood using an episome based reprogramming strategy and histone deacetylase inhibitors.

Efficace sur les Cellules iPS génération de sang à l&#39;aide qu&#39;épisomes et les inhibiteurs de HDAC

This manuscript illustrates a protocol for efficiently creating integration-free human induced pluripotent stem cells (iPSCs) from peripheral blood using ...

Comparative Analysis of Human Growth Hormone in Serum Using SPRi, Nano-SPRi and ELISA Assays

Sensitive and selective methods for the detection of human growth hormone (hGH) over a wide range of concentrations (high levels of 50-100 ng ml&#8722;1 and minimum levels of 0.03 ng ml&#8722;1) in circulating blood are essential as variable levels may indicate altered physiology. For example, growth disorders occurring in childhood can be diagnosed by measuring levels of hGH in blood. Also, the misuse of recombinant hGH in sports not only poses an ethical issue it also presents serious health threats to the abuser. One popular strategy for measuring hGH misuse, relies on the detection of the ratio of 22 kDa hGH to total hGH, as non-22 kDa endogenous levels drop after exogenous recombinant hGH (rhGH) administration. Surface plasmon resonance imaging (SPRi) is an analytical tool that allows direct (label-free) monitoring and visualization of biomolecular interactions by recording changes of the refractive index adjacent to the sensor surface in real time. In contrast, the most frequently used colorimetric method, enzyme-linked immunosorbent assay (ELISA) uses enzyme labeled detection antibodies to indirectly measure analyte concentration after the addition of a substrate that induces a color change. To increase detection sensitivity, amplified SPRi uses a sandwich assay format and near infrared quantum dots (QDs) to increase signal strength. After direct SPRi detection of recombinant rhGH in spiked human serum, the SPRi signal is amplified by the sequential injection of detection antibody coated with near-infrared QDs (Nano-SPRi). In this study, the diagnostic potential of direct and amplified SPRi was assessed for measuring rhGH spiked in human serum and compared directly with the capabilities of a commercially available ELISA kit.

The proposed work assesses the diagnostic potential of direct and amplified surface plasmon resonance imaging (SPRi) assays, particularly for the detection of recombinant human growth hormone in spiked human serum, by comparing SPRi results directly with commercially available enzyme-linked immunosorbent assay (ELISA) kit.

Analyse comparative de l&#39;hormone de croissance dans le sérum en utilisant SPRi, Nano-SPRi et dosages ELISA

Sensitive and selective methods for the detection of human growth hormone (hGH) over a wide range of concentrations (high levels of 50-100 ng ml&#8722;1 ...