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> 990μL </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> Zur Herstellung von 100 ml komplette KnockOut SR Feeder-Free (KSR-FF) aseptisch verbinden die Komponenten in der Tabelle unten aufgeführt. <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. Um zum Beispiel die Zubereitung von 100 ml KSR-FF medium 182 ul 55 mM 2-Mercaptoethanol (1:550 Verdünnung) Alternativ kann die 2-Mercaptoethanol zum 1X fertig zugesetzt werden und bei 2-8 ° C bis zu einer Woche. </li></ol> Vorbereitung Freezing / Kryokonservierungsmedium Die Kryokonservierung Medium besteht aus zwei Medien; Freezing Medium A und Einfriermedium B. Sie sind, um die Zellen zu unterschiedlichen Zeiten aufgenommen und müssen getrennt bleiben. Sie sind jeweils 50% des Gesamtvolumens der Kryokonservierung Medium benötigt. Das Gesamtvolumen der Kryokonservierung Medium benötigt wird 1 ml pro Fläschchen eingefroren werden. A 90% konfluent 60mm Teller hESC kann zu Bank 2 Fläschchen hESC in Kryokonservierung Medien eingesetzt werden. Bereiten Sie genügend Volumen von jeweils Freezing Medium mit einer zusätzlichen 2-5ml bis overage zu gewährleisten. <table border="1"><tbody><tr><td> Freezing Medium A (50% Endvolumen): </td><td> 50% DMEM/F12 </td><td> 50% KSR </td></tr><tr><td> Freezing Medium B (50% Endvolumen): </td><td> 80% DMEM/F12 </td><td> 20% DMSO </td></tr></tbody></table> Beispiel: Wenn Sie das Einfrieren 20 Ampullen von Zellen sind, müssen Sie 20 mL der Kryokonservierung Medium. Add 4mL für overage für eine Gesamtmenge von 24ml Kryokonservierung Medium. Das heißt, Sie müssen 12ml von Freezing Medium A (50% 24ml) und 12ml der Freezing Medium B (50% 24ml). <table border="1"><tbody><tr><td> Freezing Medium A (12 ml): </td><td> 6 mL DMEM/F12 </td><td> 6 mL KSR </td></tr><tr><td> Freezing Medium B (12 ml): </td><td> 9,6 ml DMEM/F12 </td><td> 2,4 ml DMSO </td></tr></tbody></table> Die endgültige Zusammensetzung der Kryokonservierung Medium beträgt 65% DMEM/F12, 25% KSR, und 10% DMSO. Kryokonservierung IPSCs <ol><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 1below 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> Bereiten Sie eine ausreichende Menge an Kryoröhrchen. Sobald die Zellen in Kontakt mit DMSO sind, sollten sie aliquotiert und eingefroren werden innerhalb von 2-3 Minuten. </li><li> Gently Flick das Rohr voll zu verdrängen das Zellpellet aus dem Rohrboden und wieder die Zellen in Einfriermedium A. [durch vorsichtiges Auf-und Abpipettieren mit einer 5-ml-serologischen Pipette]. Nach gleichmäßige Suspension von Klumpen, fügen gleichen Volumen Einfriermedium B, um es in einem tropfenweise Weise, unter leichtem Schwenken der Zellsuspension zu mischen. Hinweis: An dieser Stelle werden die Zellen in Kontakt mit DMSO, und die Arbeit muss effizient durchgeführt werden. Sobald Zellen in Kontakt mit DMSO sind, sollten sie aliquotiert und eingefroren werden innerhalb von 2-3 Minuten. </li><li> Aliquot von 1 ml der Zellsuspension in jedes Kryoröhrchen. </li><li> Raschen Platzierung der Röhrchen in einem Mr. Frosty [schnell einfrieren] und überträgt es auf -80 ° C über Nacht. </li><li> Nach Lagerung über Nacht bei -80 ° C, übertragen Sie die Zellen, um eine flüssige Stickstoff-Tank für die langfristige Lagerung. </li></ol> iPSC Fläschchen auftauen und die Erholung der Zellen Hinweis: KSR-FF kann mit KSR-haltigen MEF-konditioniertem Medium oder KSR Medium für den Einsatz mit Zubringern ersetzt werden. Siehe Anhang finden Sie Anweisungen für die mediale Aufbereitung. Pre-warmen 10 mL KSR-FF-Medium in einem 50 ml Tube. <ol><li> Äquilibrieren der entsprechenden Menge Geltrex-beschichtete Platten auf Raumtemperatur und saugen jede Flüssigkeit aus der Gerichte nur vor dem Plattieren Sier-Zellen. </li><li> Entfernen Sie vorsichtig die hESC (oder IPSC) Fläschchen aus flüssigem Stickstoff Storage Tank. </li><li> Schnell Tauwetter gefrorenen Fläschchen von Zellen in einer 37 ° C Wasserbad, bis nur ein kleiner gefrorener Klumpen bleibt in der Flasche. </li><li> Spray Flasche mit 70% Isopropanol zu dekontaminieren und überträgt es auf die sterile Gewebekulturen Kapuze. </li><li> Überführen Sie den gesamten Inhalt des Fläschchens in einen 15 mL konischen Rohr mit einer 5-ml-Pipette. </li><li> Spülen Sie die Flasche mit 1 mL der KSR-FF und stell &#39;das zu den 15 mL Zentrifugenröhrchen. </li><li> Langsam, 4 ml KSR-FF, um Zellen in der 15 ml konischen Rohr tropfenweise. Beim Hinzufügen des Mediums, sanft bewegen Sie den Schlauch hin und her, um die Zellen zu mischen. (Dies reduziert osmotischen Schock der Zellen). </li><li> Zentrifugieren Sie die 15 ml Tube mit der Zellsuspension bei 1000rpm (200 x g) für 2 Minuten bei Raumtemperatur. </li><li> Vorsichtig absaugen und den Überstand verwerfen, ohne das Zellpellet. </li><li> Re-suspend das Zellpellet in vorgewärmten KSR-FF (zB 4mL/60 mm-Schale) mit einer 5-ml-Pipette und sanft Pipette die Zellen auf und ab, bis Zellpellet vollständig dispergiert ist. Ein Lebensfähigkeit von mehr als 70% zu erwarten ist, wenn die Lebensfähigkeit von weniger als 70%, kann es länger dauern, bis die Zellen bis zur Konfluenz zu erreichen. </li><li> Mit der gleichen Pipette die Zellsuspension auf Geltrex-beschichtete Kulturschale voräquilibriert auf Raumtemperatur tropfenweise. </li><li> Legen Sie die Kulturschale in einem 37 ° C Inkubator mit einer angefeuchteten Atmosphäre von 4 bis 6% CO 2 in Luft. Vorsichtig schwenken Schiff in einem von Norden nach Süden, von Osten nach Westen Muster zur gleichmäßigen Verteilung der Zellen. </li><li> Gently Fluid-Änderung der Kulturschale 24 Stunden nach dem Auftauen und danach täglich um Zelltrümmer zu entfernen und an die frische Nährstoffe liefern, bis das Gericht ist etwa 70-80% konfluent. Für die weitere Kultur und Passagierung der iPS-Zellen, zu unserem Protokoll mit dem Titel &quot;Feeder-Free Culture und Passagieren der menschlichen iPS-Zellen unter Verwendung Komplette KnockOut Serum Ersatz Feeder-freiem Medium&quot; beziehen </li></ol> Erwartete Ergebnisse Die Zellen sollten in etwa 70-80% konfluent vor Kryokonservierung werden. Dispase Verdauung Ergebnisse in Einrollen und Falzen der Kolonien entlang der Kolonie Margen. Nach der Ernte der Zellen werden sie als kleine Klumpen im Kryokonservierung Medien eingefroren. Sobald die Flasche wird aufgetaut, wieder Zellen und heften sich an die vorbeschichteten Gericht als kleine Klumpen. Sie wachsen schnell, was zu einer konfluenten Schale in 5-6 Tagen. Tabelle 1 - Empfohlene Volumes <table border="1"><tbody><tr><td> Komponente </td><td> 35mm Dish </td><td> 60mm Dish </td><td> 100mm Dish </td></tr><tr><td> Komplette KnockOut SR Medium </td><td> 2 mL </td><td> 4 mL </td><td> 10 mL </td></tr><tr><td> Geltrex Lösung </td><td> 1 mL </td><td> 1,5 ml </td><td> 4-5 ml </td></tr><tr><td> Dispase </td><td> 0,5 ml </td><td> 1 mL </td><td> 3-4 mL </td></tr><tr><td> D-PBS für das Spülen </td><td> 2 mL </td><td> 4 mL </td><td> 10 mL </td></tr></tbody></table> Bitte <a href="http://www.jove.com/files/ftp_upload/2237/2237_appendix.pdf">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>
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		<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>12660-012</td>
<td>Note: see appendix for the use of alternative DMEM products GlutaMAX-I (Cat. No. 35050-061)</td>
</tr>
<tr>
<td>KnockOut Serum Replacement</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>10828-028</td>
<td>&nbsp;</td>
<tr>
<td>KnockOut Serum Replacement Growth Factor Cocktail</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>A10580-01</td>
<td>&nbsp;</td>
<tr>
<td>FGF-basic, human recombinant protein, 10 &mu;g</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>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>21985-023</td>
<td>&nbsp;</td>
<tr>
<td>Dispase</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>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>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>14190-144</td>
<td>&nbsp;</td>
<tr>
<td>DMSO, 500mL</td>
<td>&nbsp;</td>
<td>JT Baker</td>
<td>JT9224-1</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>60 mm Tissue Culture treated dishes</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Liquid nitrogen storage tank</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Isopropanol freezing containers</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>1.5 mL Cryovials</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
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cryopreserving and recovering 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 und hES-Zell-Kulturen zu Feeder-free unter Beibehaltung Ihrer Benutzung der Knockout SR.

Watch this Scientific Journal Video about Cryopreserving and Recovering of Human iPS Cells using Complete KnockOut Serum Replacement Feeder-Free Medium at JoVE.com

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

Dieses Protokoll beschreibt die Einzelheiten des Verfahrens zur Kryokonservierung humanen iPS-Zellen in KnockOut SR Kryokonservierungsmedium und Gewinnung dieser Zellen in völliger KnockOut SR Feeder Free (KSR-FF) mittel-oder Feeder-basierte KnockOut SR Medium.

Kryokonservierung und Wiederherstellen von Human iPS-Zellen unter Verwendung Komplette 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 ...

cryopreserving-recovering-human-ips-cells-using-complete-knockout

Research

JoVE Journal

Biology

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

Predicting the Effectiveness of Population Replacement Strategy Using Mathematical Modeling

Charles Taylor and John Marshall explain the utility of mathematical modeling for evaluating the effectiveness of population replacement strategy.  Insight is given into how computational models can provide information on the population dynamics of mosquitoes and the spread of transposable elements through A. gambiae subspecies.  The ethical considerations of releasing genetically modified mosquitoes into the wild are discussed.

Charles Taylor and John Marshall explain the utility of mathematical modeling for evaluating the effectiveness of population replacement strategy. Insight is given into how computational models can provide information on the population dynamics of mosquitoes and the spread of transposable elements through A. gambiae subspecies. The ethical considerations of releasing genetically modified mosquitoes into the wild are discussed.

Die Vorhersage der Wirksamkeit der Bevölkerung Replacement-Strategie mittels mathematischer Modellierung

Charles Taylor and John Marshall explain the utility of mathematical modeling for evaluating the effectiveness of population replacement strategy.  ...

Forschung

Biologie

Generating iPS Cells from MEFS through Forced Expression of Sox-2, Oct-4, c-Myc, and Klf4

Pluripotency can be induced in differentiated murine by viral transduction of Oct4, Sox2, Klf4, and c-Myc (Takahashi and Yamanaka, 2006; Wernig, et al., 2007; Okita, et al., 2007; Maherali, et al., 2007). We have devised a reprogramming strategy in which these four transcription factors are expressed from doxycycline (dox)-inducible lentiviral vectors (Brambrink et al., 2008). Using these inducible constructs, we can derive induced pluripotent stem (iPS) cells from mouse embryonic fibroblasts (MEFs). In this video, we demonstrate the procedure for the generation of inducible lentiviruses that express the four transcription factors and show how to infect MEFs with these viruses in order to produce iPS cells. By using inducible lentiviruses, the expression of the four factors in controlled by the addition of doxycyline to the culture medium. The advantage of this system over the traditional retroviral infection is the ability to turn the genes on and off so that the kinetics of reprogramming and gene expression requirements can be analyzed in detail.

This video shows the procedure for generating induced pluripotent stem cells using inducible lentivirus that express Oct4, Sox2, c-Myc and Klf4.

Generieren von iPS-Zellen aus MEFS durch Forced Expression von Sox-2, Oct-4, c-Myc und Klf4

Pluripotency can be induced in differentiated murine by viral transduction of Oct4, Sox2, Klf4, and c-Myc (Takahashi and Yamanaka, 2006;  Wernig, et al., ...

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells (MSCs) and Endothelial Colony Forming Progenitor Cells (ECFCs)

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal stem cells, MSCs) and endothelial colony forming progenitor cells (ECFCs). Both cell types are key players in maintaining the integrity of tissue and are probably also involved in regenerative processes and tumor formation.
To study their biology and function in a comparative manner it is important to have both cells types available from the same donor. It may also be beneficial for regenerative purposes to derive MSCs and ECFCs from the same tissue.
Because cellular therapeutics should eventually find their way from bench to bedside we established a new method to isolate and further expand progenitor cells without the use of animal protein. Pooled human platelet lysate (pHPL) replaced fetal bovine serum in all steps of our protocol to completely avoid contact of the cells to xenogeneic proteins.
This video demonstrates a methodology for the isolation and expansion of progenitor cells from one umbilical cord.
All materials and procedures will be described.

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells MSCs and Endothelial Colony Forming Progenitor Cells ECFCs

This protocol describes the isolation and subsequent expansion of mesenchymal stromal cells and endothelial colony forming cells without the use of animal serum to generate autologous pairs for experimental transplantation purposes.

Isolation und tierisches Serum Freie Expansion der menschlichen Nabelschnur Derived mesenchymale Stromazellen (MSCs) und Endothelial Colony Forming Progenitorzellen (ECFCs)

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal ...

Feeder-Free Adaptation, Culture and Passaging 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 cell cultures to feeder-free while still maintaining your use of Knockout SR.

The following protocol provides instruction for adapting human induced Pluripotent Stem (iPS) Cells to feeder-free culture using complete KnockOut Serum Replacement Feeder-Free medium (KSR-FF). Once adapted, instructions for continual maintenance are also provided.

Feeder-freie Adaption, Kultur und Passagierung der Menschenrechte iPS-Zellen mit Komplett 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 ...

In Situ Monitoring of Transiently Formed Molecular Chaperone Assemblies in Bacteria, Yeast, and Human Cells

J-domain proteins (JDPs) form the largest and the most diverse co-chaperone family in eukaryotic cells. Recent findings show that specific members of the JDP family could form transient heterocomplexes in eukaryotes to fine-tune substrate selection for the 70 kDa heat shock protein (Hsp70) chaperone-based protein disaggregases. The JDP complexes target acute/chronic stress induced aggregated proteins and presumably help assemble the disaggregases by recruiting multiple Hsp70s to the surface of protein aggregates. The extent of the protein quality control (PQC) network formed by these physically interacting JDPs remains largely uncharacterized in vivo. Here, we describe a microscopy-based in situ protein interaction assay named the proximity ligation assay (PLA), which is able to robustly capture&#160;these transiently formed chaperone complexes in distinct cellular compartments of eukaryotic cells. Our work expands the employment of PLA from human cells to yeast (Saccharomyces cerevisiae) and bacteria (Escherichia coli), thus rendering an important tool to monitor the dynamics of transiently formed protein assemblies in both prokaryotic and eukaryotic cells.

Cognate J-domain proteins cooperate with the Hsp70 chaperone to assist in a myriad of biological processes ranging from protein folding to degradation. Here, we describe an in situ proximity ligation assay, which allows the monitoring of these transiently formed chaperone machineries in bacterial, yeast and human cells.

In Situ-Überwachung von transient gebildeten molekularen Chaperon-Baugruppen in Bakterien, Hefe und menschlichen Zellen

J-domain proteins (JDPs) form the largest and the most diverse co-chaperone family in eukaryotic cells. Recent findings show that specific members of the ...