Hinweis: Um sterile Kultur Bedingungen aufrechtzuerhalten, werden alle Verfahren in diesem Protokoll unter Verwendung von sterilen Laborpraxis durchgeführt und führte unter einer Laminar-Flow-Haube. Vor dem Start, sicherzustellen, dass alle Medien äquilibriert bis 37 ° C und entsprechend vergast. Vorbereitung Geltrex-beschichteten Kulturschalen Hinweis: siehe Anhang für die Nutzung von CELLstart beschichteten Kulturschalen <ol><li> Thaw eine Tube Geltrex (1 mL) langsam bei 2-8 ° C und verdünnt 1:100 in 99 ml Knockout D-MEM/F-12. Mischen Sie die Lösung vorsichtig. Hinweis: Einige iPS-Zelllinien kann eine andere Geltrex Verdünnung für ein optimales Wachstum benötigen. Siehe Anhang für alternative Verdünnungen. </li><li> Abdeckung der gesamten Oberfläche jeder Kulturschale mit dem Geltrex-Lösung (1 mL für eine 35-mm-Schale, 1,5 mL für eine 60-mm-Schale). </li><li> Seal jedes Gericht mit Parafilm zum Trocknen zu verhindern und inkubieren Sie die Gerichte für 1 Stunde bei 37 ° C. Hinweis: An dieser Stelle können Sie die Geltrex-beschichteten Kulturschalen bei 4 ° C lagern bis zu 1 Monat. Seal jedes Gericht mit Parafilm die Geltrex vor dem Austrocknen zu verhindern. </li><li> Vor dem Einsatz, übertragen Sie die Geltrex-beschichtete Platten zu einem Laminarströmungshaube und es ihnen ermöglichen, auf Raumtemperatur (ca. 1 Stunde) ausgleichen. </li></ol> Vorbereitung Komplette KnockOut SR Feeder-freiem Medium <ol><li> Zur Vorbereitung 1 mL von 10 pg / mL Grundlegende FGF-Lösung, aseptisch mischen die unten aufgeführten Komponenten. Aliquot der Lösung und bei -20 ° C bis zu 6 Monaten. <table border="0"><tbody><tr><td> Grundlegende FGF </td><td> 10 ug </td></tr><tr><td> D-PBS </td><td> 990 ul </td></tr><tr><td> 10% BSA </td><td> 10 l </td></tr></tbody></table> Hinweis: BSA mit HSA oder Knockout SR in der gleichen Konzentration ersetzt werden. </li><li> Zur Vorbereitung 50 ml 2 mg / mL Dispase-Lösung, aseptisch mischen die unten aufgeführten Komponenten. Filter sterilisieren der Lösung und lagern bei 4 ° C für bis zu 2 Wochen. <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> Zu 100 ml komplette KnockOut SR Feeder-Free (KSR-FF) Medium aseptisch verbinden die Komponenten in der folgenden Tabelle aufgeführten vorzubereiten. <table border="1"><tbody><tr><td> Komponente </td><td> Lager-Konzentration </td><td> Endkonzentration </td><td> Volumen </td></tr><tr><td> Knockout DMEM/F12 (Kat.-Nr. 12660-012) </td><td> - </td><td> 1X </td><td> 76,8 mL </td></tr><tr><td> Glutamax-I (Kat. Nr. 35050-061) </td><td> 200 mM </td><td> 2 mM </td><td> 1 mL </td></tr><tr><td> KnockOut SR (Kat.-Nr. 10828-028) </td><td> - </td><td> 20% </td><td> 20 mL </td></tr><tr><td> KnockOut SR-GFC (Kat.-Nr. A10580-01) </td><td> 50X </td><td> 1X </td><td> 2 mL </td></tr><tr><td> bFGF (Kat.-Nr. PHG0024) </td><td> 10 ug / mL </td><td> 20 ng / mL </td><td> 200 ul </td></tr></tbody></table> Sie können die KSR-FF Medium bei 2-8 ° C lagern bis zu einer Woche. </li><li> Kurz vor pre-Gleichgewicht der gesamten mittel-bis Temperatur und Gase, aseptisch fügen Sie die erforderlichen Volumen von 2 Mercaptoethanol (55 mM Lager-Konzentration) für eine 0,1 mM Endkonzentration. Zum Beispiel auf 100 ml KSR-FF Medium herzustellen add 182 uL 55 mM 2-Mercaptoethanol (1:550 Verdünnung) Alternativ kann die 2-Mercaptoethanol zum 1X fertig zugesetzt werden und bei 2-8 ° C für bis zu einer Woche. </li></ol> Anpassung IPSCs zu KSR-FF Medium <ol><li> Vor Beginn der Pre-Gleichgewicht Ihrer Geltrex beschichtete Platten auf Raumtemperatur in der Motorhaube. </li><li> Kultur der IPSCs am MEF Feeder-Zellen, bis sie 70-80% konfluent. Bitte beachten Sie die GIBCO embryonalen Maus-Fibroblasten-Handbuch für MEF-basierte Kultur-Protokolle. </li&gt; <li> Pre-warm das erforderliche Volumen der Dispase in einem 37 ° C Wasserbad. Siehe Tabelle 1 unten für Details auf den Datenträgern erforderlich. </li><li> Pre-Gleichgewicht das erforderliche Volumen des KSR-FF in einem 37 ° C Wasserbad for15 min. Siehe Tabelle 1 unten für Details auf den Datenträgern erforderlich. </li><li> Saugen Sie das Medium von den Kulturschalen, und fügen Sie eine entsprechende Menge an Dispase-Lösung. Inkubieren Sie die Speisen bei 37 ° C für 3-5 Minuten. </li><li> Saugen Sie das Dispase-Lösung aus jeder Kultur Gefäß und waschen Sie die MEF Feeder-Zellen vorsichtig mit D-PBS (2 bis 3 mal). </li><li> Fügen Sie einen angemessenen Betrag von kompletten KnockOut SR-Medium pro Kulturgefäß. Verwenden Sie eine Zelle Schaber oder einer 5-ml-Pipette vorsichtig kratzen die Zellen von der Oberfläche des Kulturgefäßes. Hinweis: siehe Anhang alternative Anpassung Protokolle für schwer zu iPS-Zelllinien anpassen, </li><li> Sammeln Sie die Zellsuspension aus jeder Kulturschale in verschiedene 15 ml konische Röhrchen. Spülen Sie jedes Kulturgefäß mit einer entsprechenden Menge von kompletten KnockOut SR Medium, und fügen Sie die D-PBS spülen Medium in den 15 ml konische Röhrchen mit der Zellsuspension. Seien Sie vorsichtig, nicht auf die Zellklumpen in einzelne Zellen zu brechen </li><li> Zentrifugieren Sie die 15 ml konische Röhrchen bei 200 xg für 5 Minuten auf dem Pellet IPSCs. </li><li> Saugen Sie den Überstand aus der iPSC Pellet. Das Pellet in einer entsprechenden Menge an KSR-FF-Medium nach dem Split-Verhältnis (Tabelle 1). Zerbrechen Sie das Zellklumpen auf eine kleinere Größe, da die kleineren Klumpen nicht gut befestigen zu tun, um die Oberfläche. Hinweis: Wir empfehlen eine Split-Verhältnis von 1:2 für die ersten 3 Stellen nach dem IPSCs wurden direkt aus dem iPSC MEF Kulturmedium KSR-FF Medium passagiert. Normalerweise wird ein Split-Verhältnis von 1.03 bis 01.05 angebracht, aber die Passage bei 1:2 sichert die höhere Dichte der Zellen benötigt bei der Anpassung in ein Feeder-freien Kultur. </li><li> Vor dem Ausplattieren der IPSCs auf Geltrex beschichtete Platten, absaugen Rest Geltrex Lösung aus dem vorbeschichteten Schüssel geben und langsam eine entsprechende Menge an Zellsuspension jeder Kulturschale. Hinweis: nicht ausspülen Gerichte vor dem Beschichten. </li><li> Bewegen Sie die Kulturschale hin und her und hin und her mehrere Male, um die Zellen auf der Oberfläche der Schale zu verteilen. Sanft statt der Kulturschale in einem 37 ° C Inkubator mit einer angefeuchteten Atmosphäre von 4 bis 6% CO 2 in Luft. Ersetzen Sie die verbrauchte Medium mit KSR-FF jeden Tag. </li></ol> Passagierung menschlichen iPS-Zellen mit KSR-FF <ol start="13"><li> Beachten Sie die menschliche IPSCs wachsen komplette KSR-FF unter dem Mikroskop, um zu bestätigen, dass die Zellen 70-80% konfluent und bereit, subkultiviert werden. Siehe Abbildung 1. Hinweis: Wenn Kolonien zu dicht oder zu groß werden, tritt stärkere Differenzierung. </li><li> Schneiden Sie und entfernen Sie alle differenzierten iPSC Kolonien vor Passage der Kultur. </li><li> Pre-warm das erforderliche Volumen der Dispase in einem 37 ° C Wasserbad. Siehe Tabelle 1 unten für Details auf den Datenträgern erforderlich. </li><li> Pre-Gleichgewicht das erforderliche Volumen des KSR-FF in einem 37 ° C Wasserbad for15 min. Siehe Tabelle 1 unten für Details auf den Datenträgern erforderlich. </li><li> Saugen Sie das verbrauchte Medium aus dem Kulturgefäß mit einer Pipette, und spülen Sie die Zellen zweimal mit D-PBS. </li><li> Vorsichtig fügen vorgewärmten Dispase-Lösung, um die Kultur Gefäß (z. B. 1 ml Dispase-Lösung pro 60-mm Kulturschale). Swirl das Kulturgefäß zu beschichten die gesamte Zelloberfläche. </li><li> Inkubieren Sie die Behälter bei 37 ° C für 3 Minuten. </li><li> Entfernen Sie das Gefäß aus dem Inkubator, absaugen Dispase-Lösung und vorsichtig waschen der Zellen mit D-PBS. </li><li> Vorsichtig kratzen die Zellen von der Oberfläche der Kulturschale mit einem Zellschaber, und übertragen Sie die Zellen in ein steriles 15 ml Zentrifugenröhrchen. </li><li> Spülen Sie die Kulturschale zweimal mit KSR-FF, sanft &quot;Abspritzen&quot; alle Zellen, die nicht getrennt haben. Pool der Spül-Medium mit den Zellen in der 15 ml Tube. </li><li> Zentrifugieren Sie die Röhrchen bei 200 xg für 5 Minuten bei Raumtemperatur die Zellen zu pelletieren. </li><li> Vorsichtig absaugen der Überstand ohne die Zellpellet und entsorgen Sie sie. </li><li> Gently Flick das Rohr vollständig verdrängen das Zellpellet aus dem Rohrboden. </li><li> Vorsichtig die Zellen in voräquilibriert KSR-FF mit einem 5 ml serologischen Pipette. Nicht verreiben. Hinweis: Es ist entscheidend, um den Schritt in die sanft resuspendieren ohne Anwendung von Gewalt, um Schäden zu vermeiden. </li><li> Übertragen Sie die Zellen in ein frisches 60-mm Geltrex-beschichtete Schale an der gewünschten Teilungsverhältnis und bewegen Sie die Kulturschale hin und her und hin und her mehrere Male, um die Zellen auf ihrer Oberfläche zu verteilen. </li><li> Legen Sie die Kulturschale in einem 37 ° C Inkubator mit einer angefeuchteten Atmosphäre von4 bis 6% CO 2 in Luft. </li><li> Am nächsten Tag sanft ersetzen das verbrauchte Medium mit KSR-FF, um Zelltrümmer zu entfernen. Ersetzen Sie die verbrauchte Medium Alltag danach. Beachten Sie die iPS-Zellen täglich und Passage sie nach Bedarf (ca. alle 4-5 Tage). Passagieren wird empfohlen, wenn die Zellen 70-80% Konfluenz erreichen. Für iPS-Zellen die Kryokonservierung und Auftauen zu unserem Protokoll beziehen sich unter dem Titel &quot;Kryokonservierung und Wiederherstellen von Human iPS-Zellen unter Verwendung Komplette KnockOut Serum Ersatz Feeder-freiem Medium&quot;. </li></ol> Erwartete Ergebnisse <img src="/files/ftp_upload/2236/2236fig1.jpg" alt="Abbildung 1" /> Abbildung 1. Die Phasenkontrast-Abbildung unten zeigt IPSCs auf Geltrex-beschichteten Kulturschalen mit kompletten KnockOut Serum Ersatz Feeder-Free-Medium. Die IPSCs weisen Morphologie ähnlich hESCs, von großen Kernen und wenig Zytoplasma charakterisiert. (40fache Vergrößerung) <table border="1"><tbody><tr><td> Komponente </td><td> 35mm Dish </td><td> 60mm Dish </td><td> 100mm Dish </td></tr><tr valign="top"><td> Komplette KnockOut SR Medium </td><td> 2 mL </td><td> 4 mL </td><td> 10 mL </td></tr><tr valign="top"><td> Geltrex Lösung </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 für das Spülen </td><td> 2 mL </td><td> 4 mL </td><td> 10 mL </td></tr></tbody></table> Tabelle 1. Empfohlene Volumes <a href="http://www.jove.com/files/ftp_upload/2236/2236appendix.pdf">Bitte klicken Sie hier, um die Anlage zu sehen</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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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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<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

Die Entdeckung im Jahr 2006, dass Mensch und Maus-Fibroblasten umprogrammiert könnte zu iPS-Zellen 1-3 mit Qualitäten bemerkenswert ähnlich wie embryonale Stammzellen zu erzeugen ist eine wertvolle neue Quelle für pluripotente Zellen für die Wirkstoffforschung, Zell-Therapie und Grundlagenforschung geschaffen. GIBCO Medien und Reagenzien sind an der Spitze der pluripotenten Stammzellen seit Jahren. Knockout DMEM mit Knockout Serum Replacement ergänzt wird das Medium der Wahl für die embryonalen Stammzellen das Wachstum und nun iPS Zellkultur 3-9. Dieser Goldstandard Medien kann nun für Feeder-freien Kultur mit der Zugabe von Knockout SR Growth Factor Cocktail verwendet werden. Traditionelle menschliche ES-und iPS-Zellkultur-Methoden erfordern den Einsatz von Maus oder humanen Fibroblasten Feederschichten oder Feeder-konditioniertem Medium. Diese Kultur Methoden sind arbeitsintensiv, hart zu skalieren und es ist schwierig, Hüften Zellen undifferenziert wegen der undefinierten Zustand zu erhalten. Invitrogen hat Knockout SR Growth Factor Cocktail entwickelt, damit Sie auf einfache Weise Übergang Hüfte Zellkulturen Feeder-free unter Beibehaltung Ihrer Benutzung der Knockout SR.

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

Das folgende Protokoll enthält Anweisungen für die Anpassung menschlichen induzierten pluripotenten Stammzellen (iPS-Zellen) zu Feeder-freien Kultur mit kompletten KnockOut Serum Ersatz Feeder-freiem Medium (KSR-FF). Einmal angepasst, Anweisungen für die kontinuierliche Wartung sind ebenfalls vorhanden.

Feeder-freie Adaption, Kultur und Passagierung der Menschenrechte iPS-Zellen mit Komplett KnockOut Serum Ersatz Feeder-freiem 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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Serie: 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.

Passagierung FARBEN humanen embryonalen Stammzellen-Linien mit 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 ...

Forschung

Biologie

Human ES cells: Starting Culture from Frozen Cells

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

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

Humane ES-Zellen: Starting Kultur aus eingefrorenen Zellen

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.

Passagierung humaner neuraler Stammzellen

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.

Kultur und Wartung von humanen embryonalen Stammzellen

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.

Kryokonservierung und Wiederherstellen von Human iPS-Zellen unter Verwendung Komplette KnockOut Serum Ersatz Feeder-freiem Medium

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.

Generation of Human Cardiomyocytes: Eine Differenzierung Protokoll von Feeder-freien menschlichen induzierten pluripotenten Stammzellen

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.

Isolierung von Kleine-kodierenden RNAs von 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 ...

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.

MicroRNA Expressionsprofile der Menschen iPS-Zellen, Retinapigmentepithel Abgeleitet aus iPS und Fetal Retinapigmentepithel

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.

Effiziente iPS Zellgeneration aus Blut Mit Episomen und HDAC-Inhibitoren

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.

Vergleichende Analyse von Human Growth Hormone im Serum Mit SPRi, Nano-SPRi und 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 ...