Nota: Per mantenere condizioni di coltura sterili, tutte le procedure descritte in questo protocollo sono effettuate utilizzando pratiche di laboratorio sterile e condotto sotto una cappa a flusso laminare. Prima di iniziare, assicurarsi che i media sono equilibrati a 37 ° C e opportunamente gasati. Preparazione Geltrex rivestita Piatti Cultura Nota: vedi appendice per l&#39;utilizzo di piatti della cultura CELLstart rivestiti <ol><li> Scongelare un tubo di Geltrex (1 ml) lentamente a 2-8 ° C e diluire 1:100 in 99 ml di Knockout D-MEM/F-12. Mescolare delicatamente la soluzione. Nota: alcune linee di cellule iPS possono richiedere una diversa diluizione Geltrex per una crescita ottimale. Vedi appendice per le diluizioni alternative. </li><li> Coprono l&#39;intera superficie di ogni piatto cultura con la soluzione Geltrex (1 mL per un piatto da 35 mm, 1,5 ml per un piatto di 60 mm). </li><li> Sigillare ogni piatto con Parafilm contro l&#39;essiccamento e incubare i piatti per 1 ora a 37 ° C. Nota: A questo punto si può memorizzare il Geltrex rivestite piatti di cultura a 4 ° C per un massimo di 1 mese. Sigillare ogni piatto con Parafilm per evitare che il Geltrex si secchi. </li><li> Prima di utilizzare, trasferire il Geltrex rivestite piatti di una cappa a flusso laminare e permettere loro di equilibrare a temperatura ambiente (circa 1 ora). </li></ol> Preparazione completa alimentatore-Free SR KnockOut Media <ol><li> Per preparare 1 mL di 10 mg / ml soluzione di base FGF, asettico miscelare i componenti elencati di seguito. Aliquota della soluzione e conservare a -20 ° C fino a 6 mesi. <table border="0"><tbody><tr><td> Di base FGF </td><td> 10 mcg </td></tr><tr><td> D-PBS </td><td> 990 microlitri </td></tr><tr><td> 10% di BSA </td><td> 10 L </td></tr></tbody></table> Nota: BSA può essere sostituito con HSA o SR Knockout alla stessa concentrazione. </li><li> Per preparare 50 ml di 2 mg / ml soluzione dispasi, asettico miscelare i componenti elencati di seguito. Filtrare la soluzione sterilizzare e conservare a 4 ° C per un massimo di 2 settimane. <table border="0"><tbody><tr><td> Dispasi </td><td> 100 mg </td></tr><tr><td> D-PBS </td><td> 50 mL </td></tr></tbody></table></li><li> Per preparare 100 mL di SR KnockOut completo alimentatore-Free (KSR-FF) medium asettico combinare i componenti elencati nella tabella sottostante. <table border="1"><tbody><tr><td> Componente </td><td> Concentrazione magazzino </td><td> Concentrazione finale </td><td> Volume </td></tr><tr><td> Knockout DMEM/F12 (n ° cat. 12660-012) </td><td> - </td><td> 1X </td><td> 76,8 mL </td></tr><tr><td> GlutaMAX-I (Cat. No. 35050-061) </td><td> 200 mM </td><td> 2 mM </td><td> 1 ml </td></tr><tr><td> KnockOut SR (n ° cat. 10828-028) </td><td> - </td><td> 20% </td><td> 20 ml </td></tr><tr><td> KnockOut SR-GFC (n ° cat. A10580-01) </td><td> 50X </td><td> 1X </td><td> 2 ml </td></tr><tr><td> bFGF (cat. no. PHG0024) </td><td> 10 mcg / ml </td><td> 20 ng / mL </td><td> 200 l </td></tr></tbody></table> Si può memorizzare il KSR-FF medio a 2-8 ° C per un massimo di una settimana. </li><li> Poco prima di pre-raggiunga l&#39;equilibrio completo a temperatura e gas, in modo asettico aggiungere il volume richiesto di 2 mercaptoetanolo (55 concentrazione magazzino mm) per una concentrazione di 0,1 mM finale. Ad esempio, per preparare 100 ml di KSR-FF medio aggiungere 182 microlitri di 55 mM di 2-mercaptoetanolo (diluizione 1:550) In alternativa, il 2-mercaptoetanolo possono essere aggiunti al mezzo di 1X completato e conservato a 2-8 ° C per fino ad una settimana. </li></ol> Adattamento iPSCs a KSR-FF Media <ol><li> Prima di iniziare, pre-equilibrare i vostri piatti Geltrex rivestito a temperatura ambiente nel cappuccio. </li><li> Cultura iPSCs sulle cellule alimentatore MEF fino a quando sono 70-80% confluenti. Fare riferimento al manuale GIBCO fibroblasti embrionali di topo per MEF protocolli basati su cultura. </li&gt; <li> Preriscaldare il volume richiesto di dispasi in un bagno d&#39;acqua a 37 ° C. Fare riferimento alla Tabella 1 riportata di seguito per i dettagli sui volumi richiesti. </li><li> Pre-equilibrare il volume di KSR-FF in A ° C 37 bagnomaria per 15 min. Fare riferimento alla Tabella 1 riportata di seguito per i dettagli sui volumi richiesti. </li><li> Aspirare il terreno dai piatti della cultura, e aggiungere una quantità appropriata di soluzione dispasi. Incubare i piatti a 37 ° C per 3-5 minuti. </li><li> Aspirare la soluzione dispasi da ciascun recipiente di coltura e lavare le cellule MEF alimentatore delicatamente con D-PBS (2 a 3 volte). </li><li> Aggiungere una quantità appropriata di terreno completo SR KnockOut a ciascun recipiente di coltura. Utilizzare un raschietto cella o un pipetta da 5 ml per raschiare delicatamente le cellule dalla superficie del recipiente di coltura. Nota: vedi appendice per i protocolli di adattamento alternativa è difficile adattare le linee di cellule iPS, </li><li> Raccogliere la sospensione cellulare da ogni piatto cultura in separata provette da 15 ml conica. Sciacquare ciascun recipiente di coltura con una quantità appropriata di terreno completo SR ad eliminazione diretta, e aggiungere il D-PBS risciacquare medio in provette da 15 ml contenente la sospensione cellulare. Essere cauti a non rompere i grumi di cellule in cellule singole </li><li> Centrifugare le provette da 15 ml a 200 xg per 5 minuti per far sedimentare il iPSCs. </li><li> Aspirare il surnatante dal pellet IPSC. Risospendere il precipitato in una quantità appropriata di KSR-FF medio in base al rapporto di divisione (Tabella 1). Non rompere i grumi di cellule a una dimensione inferiore, perché le macchie più piccole non attaccano bene alla superficie. Nota: Si consiglia un rapporto di divisione di 1:2 per i primi 3 passaggi dopo il iPSCs sono stati diversi passaggi direttamente dal Media IPSC Cultura MEF per KSR-FF media. Normalmente, un rapporto di divisione 1:03-1:05 è appropriato, ma passaging a 1:2 garantisce la maggiore densità di cellule necessarie per l&#39;adattamento in un alimentatore senza cultura. </li><li> Prima di placcatura del iPSCs su piatti Geltrex patinata, aspirare residui soluzione Geltrex da preverniciato piatto, e aggiungere lentamente una quantità appropriata di sospensione cellulare per ogni piatto cultura. Nota: non lavare i piatti prima di placcatura. </li><li> Spostare il piatto della cultura avanti e indietro e di lato a lato diverse volte per disperdere le cellule sulla superficie del piatto. Delicatamente la capsula la cultura in un incubatore a 37 ° C con atmosfera umidificata da 4 a 6% di CO 2 in aria. Sostituire il medio trascorso con KSR-FF ogni giorno. </li></ol> Passaging cellule umane iPS usando KSR-FF <ol start="13"><li> Osservare le iPSCs umano cresce in completo KSR-FF sotto il microscopio per confermare che le cellule sono 70-80% confluenti e pronto per essere sottocoltura. Fare riferimento alla Figura 1. Nota: Se le colonie diventano troppo dense o troppo grande, si verifica una maggiore differenziazione. </li><li> Tagliare e rimuovere eventuali colonie differenziati IPSC prima passaging la cultura. </li><li> Preriscaldare il volume richiesto di dispasi in un bagno d&#39;acqua a 37 ° C. Fare riferimento alla Tabella 1 riportata di seguito per i dettagli sui volumi richiesti. </li><li> Pre-equilibrare il volume di KSR-FF in A ° C 37 bagnomaria per 15 min. Fare riferimento alla Tabella 1 riportata di seguito per i dettagli sui volumi richiesti. </li><li> Aspirare il terreno trascorso dalla nave cultura con una pipetta, e lavare le cellule due volte con D-PBS. </li><li> Aggiungere delicatamente preriscaldato soluzione dispasi alla nave cultura (ad esempio, 1 mL di soluzione dispasi per 60 mm piatto di cultura). Agitare il recipiente di coltura per ricoprire l&#39;intera superficie delle cellule. </li><li> Incubare il recipiente di coltura a 37 ° C per 3 minuti. </li><li> Rimuovere il recipiente dal termostato, aspirare la soluzione dispasi, e lavare delicatamente le cellule con D-PBS. </li><li> Raschiare delicatamente le cellule dalla superficie del piatto cultura utilizzando un raschietto cellula, e trasferire le cellule in una provetta sterile centrifuga 15ml. </li><li> Lavare il piatto cultura due volte con KSR-FF, dolcemente &quot;spruzzo off&quot; tutte le cellule che non si sono staccati. Piscina di risciacquo medio con le cellule nel tubo 15ml. </li><li> Centrifugare la provetta a 200 xg per 5 minuti a temperatura ambiente per agglomerare le cellule. </li><li> Con attenzione aspirare il sopranatante senza disturbare il pellet di cellule e smaltirlo. </li><li> Picchiettare lievemente la provetta per rimuovere completamente il pellet dal fondo del tubo. </li><li> Risospendere delicatamente le cellule in fase di pre-equilibrato KSR-FF con una pipetta 5 ml sierologico. Non triturare. Nota: è fondamentale nella fase di risospendere delicatamente le cellule senza usare la forza per evitare danni. </li><li> Trasferire le cellule in una nuova di 60 mm Geltrex rivestite piatto al rapporto di divisione desiderato e spostare il piatto della cultura avanti e indietro e di lato a lato diverse volte per disperdere le cellule in tutta la sua superficie. </li><li> Porre la capsula la cultura in un incubatore a 37 ° C con atmosfera umidificata al4 al 6% di CO 2 in aria. </li><li> Il giorno seguente, gentilmente sostituire il medio trascorso con KSR-FF per rimuovere i detriti cellulari. Sostituire il medio speso tutti i giorni dopo. Osservare le cellule iPS quotidiana e il passaggio in base alle esigenze (circa ogni 4-5 giorni). Passaging è consigliato quando le cellule raggiungono il 70-80% confluenza. Per la crioconservazione delle cellule iPS e disgelo, fare riferimento al nostro protocollo dal titolo &quot;Cryopreserving e recupero di cellule umane iPS usando completa alimentatore-Free siero sostituzione KnockOut Media&quot;. </li></ol> Risultati attesi <img src="/files/ftp_upload/2236/2236fig1.jpg" alt="Figura 1" /> Figura 1. L&#39;immagine a contrasto di fase che segue mostra iPSCs coltivate su Geltrex rivestite con piatti della cultura completa alimentatore-Free siero sostituzione KnockOut media. La mostra iPSCs morfologia simile alle hESC, caratterizzato da nuclei di grandi dimensioni e scarso citoplasma. (Ingrandimento 40x) <table border="1"><tbody><tr><td> Componente </td><td> 35 millimetri Piatto </td><td> Piatto 60 millimetri </td><td> Dish 100 millimetri </td></tr><tr valign="top"><td> Completa KnockOut SR media </td><td> 2 ml </td><td> 4 mL </td><td> 10 mL </td></tr><tr valign="top"><td> Geltrex Soluzione </td><td> 1 ml </td><td> 1,5 ml </td><td> 4-5 mL </td></tr><tr valign="top"><td> Dispasi </td><td> 0,5 ml </td><td> 1 ml </td><td> 3-4 ml </td></tr><tr valign="top"><td> D-PBS per il risciacquo </td><td> 2 ml </td><td> 4 mL </td><td> 10 mL </td></tr></tbody></table> Tabella 1. I volumi consigliati <a href="http://www.jove.com/files/ftp_upload/2236/2236appendix.pdf">Clicca qui per vedere 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>

Gli autori di questo articolo sono impiegati da Life Technologies che produce reagenti e strumenti utilizzati in questo articolo.

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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 scoperta nel 2006 che fibroblasti umani e mouse potrebbe essere riprogrammato per generare cellule iPS 1-3 con caratteristiche molto simili alle cellule staminali embrionali ha creato una nuova fonte preziosa di cellule pluripotenti per la scoperta di farmaci, terapia cellulare, e la ricerca di base. Mezzi di comunicazione GIBCO e reagenti sono stati in prima linea nella ricerca sulle cellule staminali pluripotenti da anni. Knockout DMEM integrato con sostituzione Knockout siero è il media di scelta per la crescita delle cellule staminali embrionali e ora di cellule iPS cultura 3-9. Il gold standard del sistema dei media può ora essere utilizzato per alimentazione priva di cultura con l&#39;aggiunta di Cocktail SR Factor Knockout crescita. Tradizionale ES umane e metodi di coltura delle cellule iPS richiedono l&#39;uso di mouse o strati alimentatore fibroblasti umani, o l&#39;alimentatore condizionata media. Questi metodi sono cultura alta intensità di lavoro, difficili da scalare ed è difficile mantenere anche le cellule indifferenziate a causa delle condizioni indefinito. Invitrogen ha sviluppato SR Knockout Cocktail fattore di crescita per consentire facilmente la tua transizione colture cellulari anche alla rinfusa, senza pur mantenendo l&#39;uso di 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

Il protocollo seguente fornisce istruzioni per l&#39;adattamento umano staminali pluripotenti indotte (iPS), cellule di alimentazione priva di cultura con completo alimentatore-Free siero sostituzione KnockOut medio (KSR-FF). Una volta adattato, istruzioni per la manutenzione costante sono anche previste.

Alimentatore-Free adattamento, la cultura e Passaging di cellule IPS utilizzando Completa alimentatore-Free siero sostituzione KnockOut Media

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 ...

feeder-free-adaptation-culture-passaging-human-ips-cells-using

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JoVE Journal

Biology

22.3K Views.  Life Technologies.  中文 日本語 

Video: 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.

RIFLESSI Passaging sulle cellule staminali embrionali umane-linee con tripsina

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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Biologia

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.

Le cellule umane ES: Cultura partire da cellule congelate

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.

Passaging cellule staminali neuronali

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.

Cultura e manutenzione di cellule staminali embrionali umane

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.

Cryopreserving e recupero di cellule umane iPS usando KnockOut Completa alimentatore-Free siero sostituzione Media

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.

Generazione di cardiomiociti umani: Un protocollo di differenziazione da alimentatore-Free umani cellule staminali pluripotenti indotte

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.

Isolamento dei Piccoli RNA non codificanti dal siero umano

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 Expression Profili di cellule iPS umane, dell&#39;epitelio pigmentato retinico derivate da iPS, e fetale dell&#39;epitelio pigmentato retinico

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.

Generazione efficiente delle cellule iPS da Blood Utilizzando episomi e HDAC inibitori

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.

Analisi comparativa di ormone umano della crescita nel siero Utilizzando SPRI, Nano-SPRI e saggi 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 ...