Name: culturing human pluripotent and neural stem cells in an enclosed cell culture system for basic and preclinical research
Uploaded: 2016-06-10T13:00:00.000+00:00
Duration: 8 min 5 s
Description: Watch this Scientific Journal Video about Culturing Human Pluripotent and Neural Stem Cells in an Enclosed Cell Culture System for Basic and Preclinical Research at JoVE.com

Gli autori vorrebbero riconoscere il personale Biospherix per il loro aiuto per imparare a utilizzare il sistema di coltura cellulare Xvivo chiusa, soprattutto Matt Freeman; il personale di Miles &amp; Kelley Construction Company, Inc. per il loro lavoro nella creazione delle infrastrutture di laboratorio, in particolare Russ Hughes; il personale dell&#39;ospedale dei bambini del dipartimento Orange County di Strutture e servizi di supporto per il loro lavoro nel coordinare la ristrutturazione di laboratorio, in particolare Adam Lukhard e Devin Hugie; il personale dell&#39;ospedale dei bambini del dipartimento Orange County di Sistemi Informativi per il loro aiuto nella creazione delle infrastrutture di gestione dei dati e l&#39;accesso remoto, in particolare Viet Tran; Ospedale dei Bambini di Orange County Executive Management team per il loro lunga sostegno del progetto, in particolare il Dr. Maria Minon e Brent Dethlefs. Questo lavoro è stato finanziato dal Children Hospital di Orange County e il California Institute for Regenerative Medicine attraverso la concessione TR3-05476 a PHS. Tutti gli autori hanno contribuito ugualmente a questo lavoro.

<ol>
	<li>Brick, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Autism+Spectrum+Disorders+Stem+Cell+Resource+at+Children's+Hospital+of+Orange+County:+Implications+for+Disease+Modeling+and+Drug+Discovery.">The Autism Spectrum Disorders Stem Cell Resource at Children's Hospital of Orange County: Implications for Disease Modeling and Drug Discovery.</a> Stem Cells Transl.Med. 3 (11), 1275-1286 (2014).</li><li>Nethercott, H. E., Brick, D. J., Schwartz, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivation+of+induced+pluripotent+stem+cells+by+lentiviral+transduction.">Derivation of induced pluripotent stem cells by lentiviral transduction.</a> Methods Mol.Biol. 767, 67-85 (2011).</li><li>Pistollato, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia+and+HIF1alpha+repress+the+differentiative+effects+of+BMPs+in+high-grade+glioma.">Hypoxia and HIF1alpha repress the differentiative effects of BMPs in high-grade glioma.</a> Stem Cells. 27 (1), 7-17 (2009).</li><li>Pistollato, F., Chen, H. L., Schwartz, P. H., Basso, G., Panchision, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxygen+tension+controls+the+expansion+of+human+CNS+precursors+and+the+generation+of+astrocytes+and+oligodendrocytes.">Oxygen tension controls the expansion of human CNS precursors and the generation of astrocytes and oligodendrocytes.</a> Mol.Cell Neurosci. 35, 424-435 (2007).</li><li>Raval, J. S., Koch, E., Donnenberg, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+monitoring+of+non-viable+airborne+particles+correlates+with+airborne+colonies+and+represents+an+acceptable+surrogate+for+daily+assessment+of+cell-processing+cleanroom+performance.">Real-time monitoring of non-viable airborne particles correlates with airborne colonies and represents an acceptable surrogate for daily assessment of cell-processing cleanroom performance.</a> Cytotherapy. 14 (9), 1144-1150 (2012).</li><li>Schwartz, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+potential+of+stem+cell+therapies+for+neurological+diseases.">The potential of stem cell therapies for neurological diseases.</a> Expert.Rev.Neurother. 6, 153-161 (2006).</li><li>Schwartz, P. H., Brick, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell+therapies+for+the+lysosomal+storage+diseases+-+the+quintessential+neurodegenerative+diseases.">Stem cell therapies for the lysosomal storage diseases - the quintessential neurodegenerative diseases.</a> Curr.Stem Cell Res.Ther. 3 (2), 88-98 (2008).</li><li>Schwartz, P. H., Brick, D. J., Nethercott, H. E., Stover, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Traditional+human+embryonic+stem+cell+culture.">Traditional human embryonic stem cell culture.</a> Methods Mol.Biol. 767, 107-123 (2011).</li><li>Stover, A. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Process-based+expansion+and+neural+differentiation+of+human+pluripotent+stem+cells+for+transplantation+and+disease+modeling.">Process-based expansion and neural differentiation of human pluripotent stem cells for transplantation and disease modeling.</a> J Neurosci.Res. 91 (10), 1247-1262 (2013).</li><li>Stover, A. E., Schwartz, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptation+of+human+pluripotent+stem+cells+to+feeder-free+conditions+in+chemically+defined+medium+with+enzymatic+single-cell+passaging.">Adaptation of human pluripotent stem cells to feeder-free conditions in chemically defined medium with enzymatic single-cell passaging.</a> Methods Mol.Biol. 767, 137-146 (2011).</li><li>Yoshida, Y., Takahashi, K., Okita, K., Ichisaka, T., Yamanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia+enhances+the+generation+of+induced+pluripotent+stem+cells.">Hypoxia enhances the generation of induced pluripotent stem cells.</a> Cell Stem Cell. 5 (3), 237-241 (2009).</li></ol>

The authors declare that they have no competing financial interests.

Le cellule coltivate nei CPF vedono alcun cambiamento nelle concentrazioni di ossigeno o di anidride carbonica mentre si spostano da incubatrice al trattamento da camera al microscopio da camera e indietro. È fondamentale che le condizioni in ciascuna camera sono abbinati al particolare incubatore in cui le cellule sono mantenute prima che le cellule vengono rimosse dal termostato. L&#39;atmosfera all&#39;interno dell&#39;apparato è continuamente HEPA filtrata ed è personalizzabile in materia di concentrazioni di ossigeno e anidride carbonica. Le cellule possono essere coltivate in concentrazioni standard per PSC o NSC, 5% e il 9%, rispettivamente; o concentrazioni alternative possono essere scelti per diversi tipi di cellule o per esperimenti specifici. Pertanto, l&#39;apparato è fornito con fonti costanti di grado medico ossigeno, anidride carbonica e azoto (Figura 4). Tutti e tre di questi gas sono forniti da sistemi molteplici specifiche di gas che garantiscono forniture costanti. L&#39;apparecchio è fornito con una miscela di gas di taratura rappresentati10% (± 0,01%) di anidride carbonica in ossigeno. I sistemi di collettore si trovano al di fuori dello stabilimento di produzione delle cellule e i gas vengono convogliati nella struttura attraverso il soffitto. Il gas di taratura è alloggiato all&#39;interno della struttura. L&#39;apparecchio è inoltre fornito con il vuoto casa, anche attraverso il soffitto. L&#39;utilizzo di un sistema di monitoraggio elettronico e wireless unità invio, le pressioni di uscita di tutti i collettori sono costantemente monitorati. Nel caso in cui qualsiasi pressione scende fuori campo, i gestori di impianti di produzione di celle vengono automaticamente telefonato e notificate in modo tale che l&#39;azione appropriata può essere adottata. I requisiti di alimentazione degli apparecchi sono soddisfatte da sei dedicati 120 V circuiti che scendono dal soffitto e collegate a generatori di back-up dell&#39;ospedale al fine di garantire un costante rifornimento. Il funzionamento dell&#39;apparecchiatura è controllata via software su un computer basato su PC alimentato tramite un gruppo di continuità. Queste disposizioni di potenza e computergarantire che il sistema funzioni continuamente anche in caso di guasto del sistema di alimentazione pubblica. Il software che controlla l&#39;apparecchio ha un&#39;interfaccia grafica user-friendly (figura 1) che consente il controllo delle concentrazioni di ossigeno e anidride carbonica e pressioni di temperatura, umidità, e da camera. I valori di tutti questi parametri vengono registrate in continuo per fornire una registrazione parziale di tutti i parametri dell&#39;apparato. Questi dati viene eseguito il backup su un server remoto ogni notte per proteggere la loro integrità. Il computer e il software è possibile accedere in remoto dagli utenti amministrativi per valutare e / o modificare qualsiasi parametro. Inoltre, il computer e il software è possibile accedere in remoto, consentendo la valutazione interattiva dei parametri di apparecchi e la risoluzione dei problemi con gli utenti locali. Un ulteriore unità di invio allarme è collegato all&#39;apparato tale che gli operatori degli impianti di produzione di celle vengono informati di qualsiasi condizione out-of-range dell&#39;apparecchiatura. L&#39;accesso a distanza capabilities permettono il login e la valutazione delle specificità della condizione out-of-range. L&#39;apparecchio è progettato come un sistema modulare sia in una macro e micro senso. I singoli moduli di coltura cellulare, quali incubatori e camere di lavorazione, possono essere personalizzati in vista delle loro dimensioni e requisiti nonché nella loro disposizione rispetto all&#39;altro. Inoltre, la maggior parte delle funzioni di controllo dei singoli moduli sono essi stessi modulare tale che singoli regolatori gas atmosferici, per esempio, può essere facilmente sostituito senza notevoli disagi al sistema. camere di elaborazione specializzati, ad esempio uno per visualizzazione microscopica e manipolazione di colture cellulari, possono essere facilmente adattati al sistema. Sia a contrasto di fase e microscopio a fluorescenza sono all&#39;interno del sistema (figura 6) in modo che le cellule possono essere vivo macchiati, e le colonie possono essere sezionati nelle stesse condizioni atmosferiche come all&#39;interno tegli incubatrici. Routing dei cavi attraverso occhielli sigillati nelle pareti laterali della camera di trattamento consente apparecchiature come alimentatori e computer per essere tenuti al di fuori dell&#39;apparato, di solito su un carrello (Figura 6). Le camere di elaborazione nella struttura di produzione di cellule hanno un modello diverso da quello del flusso d&#39;aria BSC convenzionali. In BSC convenzionali, il flusso d&#39;aria scorre verso il basso da una bocca di scarico centrale e si divide in due flussi separati, che vengono poi ripresi da due differenti prese d&#39;aria nella parte anteriore ed a poppa del pavimento del cabinet. Per contro, il CPF ha un unico sfogo nella porzione anteriore del soffitto. L&#39;aria fluisce verso il basso e verso la parte posteriore della camera, dove viene quindi trascinato verso l&#39;alto in una bocchetta di aspirazione. Sebbene il CPF è intrinsecamente molto pulito, questo campo di flusso unico significa che i tecnici devono modificare leggermente la tecnica per ridurre il rischio di contaminazione. Come con un BSC convenzionale, un tecnico di laboratorio should evitare di mettere le mani monte di piastre di coltura di cellule aperte e bottiglie di media. Tuttavia, la direzione che è a monte è stato alterato nel CPF L&#39;impianto di laboratorio di produzione cellula stessa è abbastanza standard ed è dotato di un congelatore C -20 °, a -80 ° C congelatore, un frigorifero C 4 °, una centrifuga, e un bagno di acqua. Il laboratorio ha anche un lavandino con i comandi a pedale per il funzionamento conveniente a mani libere. Affinché questo laboratorio per diventare un impianto di produzione di celle funzionale clinica, tuttavia, devono ancora essere effettuate diverse modifiche aggiuntive. In primo luogo, l&#39;apparato stesso deve essere aggiornato per avere la capacità di monitorare i composti organici volatili, particolato, e le concentrazioni di biossido di cloro che viene utilizzato per la decontaminazione. In secondo luogo, una camera di trattamento contenente una macchina FACS può essere alloggiato e collegato al resto del dispositivo tramite un modulo buffer. Ciò consentirà di selezione cellulare e purificazione di trpopolazioni di cellule ansplantable alle condizioni ambientali adeguate. Infine, l&#39;intero apparato deve essere alloggiata all&#39;interno di una stanza pulita muro morbido. Ciò fornisce un International Organization for Standardization (ISO) Classe 8 ambiente per l&#39;apparato 5. L&#39;elevata sterilità e la natura controllato dal computer del CPF lo rende un sistema ideale per le applicazioni future con la terapia a base di cellule e buoni processi di fabbricazione. Il rischio di contaminazione è notevolmente mitigato, ma ancora più importante, le condizioni di espansione delle cellule vengono automaticamente registrati e archiviati dal sistema informatico. Deviazioni in concentrazioni di gas, temperatura, umidità, e tutti gli eventi di accesso al sistema sono rigorosamente documentati. Questo può essere di grande aiuto quando si studiano i problemi di qualità del prodotto. Tuttavia, vi sono ancora limiti. L&#39;utilizzo di qualsiasi e tutti i reagenti e materiali di consumo (ad esempio, i componenti multimediali, pipette, piastre) deve essere documentata separatamente. Aggiungereitionally, ci sono una moltitudine di potenziali problemi (tra cui molte forme di errore umano) che possono sorgere che sono completamente estranei alle variabili documentate dal sistema di monitoraggio del CPF. Pertanto, la necessità di personale altamente qualificato e documentazione dettagliato manuale di compiti rimane al suo posto.

Standard stem cell culture techniques suffer from several environmental constraints that place undue stresses on the cells and expose the cells to unacceptable risks of contamination. Among the stresses that cells may endure under standard cell culture conditions are precipitous changes in the levels of carbon dioxide and oxygen concentrations3,4. This occurs when the cells are moved from the incubator to the biosafety cabinet and/or microscope which may not be optimal for the cells. Previous studies have confirmed the advantages of culturing both pluripotent and neural stem cells in hypoxic conditions4,11, and for best results, these conditions need to be continuous. Moreover, risks of cellular contamination are higher as the laboratory environment and personnel impinge upon the cells at almost every step of their culture and manipulation. Traditional clean rooms comprise one effective method to greatly decrease contamination risks but they are expensive, have a large footprint and fail to address stressors related to carbon dioxide and oxygen concentrations. 
A cell production facility that can address both contamination risks and gas concentrations and that can be qualified to meet cGMP criteria9 provides high quality cells for basic science research as well as clinical applications1,6,7. Such a cell production facility consists, at a minimum, of the following components: a process chamber, which acts as a heated workspace for the feeding and manipulation of cell cultures; a laminar flow hood, for the initial sterilization of reagents, tubes, and tools; two buffering airlock chambers in between the hood and the process chamber; two cell culture incubators accessible from the process chamber; a microscope chamber adjacent to the process chamber; and finally, computer software to set and monitor the conditions within these modules. Using this basic infrastructure, a wide variety of tasks can be performed, such as standard feeding and passaging of pluripotent stem cells and multipotent neural stem cells, as well as more specialized methods like Sendai virus-based reprogramming, in vitro migration studies, and differentiation of neural stem cells for electrophysiological characterization.

<table><tbody><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;i&gt;Xvivo System&lt;/i&gt;</td><td>Biospherix</td><td>custom made</td><td></td></tr><tr><td>&lt;i&gt;Xvivo&lt;/i&gt; Software</td><td>Biospherix</td><td>version i.o.2.1.2.1</td><td></td></tr><tr><td>O&lt;sub&gt;2&lt;/sub&gt; Manifold</td><td>Amico</td><td>P-M2H-C3-S-U-OXY</td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; Manifold</td><td>Amico</td><td>M2H-C3-D-U-CO2</td><td></td></tr><tr><td>N&lt;sub&gt;2&lt;/sub&gt; Manifold</td><td>Western Innovator</td><td>CTM75-7-2-2-BM</td><td></td></tr><tr><td>Microscope with DP21 camera and fluorescence</td><td>Olympus Corporation</td><td>CKX41</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>DMEM/F12 Glutamax</td><td>Life Technologies</td><td>10565-018</td><td></td></tr><tr><td>StemPro hESC Supplement</td><td>Life Technologies</td><td>A100006-01</td><td></td></tr><tr><td>Accutase</td><td>Millipore</td><td>SCR005</td><td></td></tr><tr><td>Phosphate-Buffered Sodium</td><td>Hyclone</td><td>9236</td><td></td></tr><tr><td>Fibroblast Growth Factor 2</td><td>R&amp;amp;D Systems</td><td>AFL233</td><td></td></tr><tr><td>Dimethyl sulfoxide</td><td>Protide</td><td>PP1130</td><td></td></tr><tr><td>Hank&#039;s-based Cell dissociation Buffer</td><td>Life Technologies</td><td>13150-016</td><td></td></tr><tr><td>2-Mercaptoethanol</td><td>Life Technologies</td><td>21985-023</td><td></td></tr><tr><td>Epidermal Growth Factor</td><td>R&amp;amp;D Systems</td><td>AFL236</td><td></td></tr><tr><td>Oct-3/4 Antibody</td><td>Millipore</td><td>AB3209</td><td></td></tr><tr><td>TRA-1-60 Antibody</td><td>Millipore</td><td>MAB4260</td><td></td></tr><tr><td>SSEA4 Antibody</td><td>Millipore</td><td>MAB4304</td><td></td></tr><tr><td>BIT-9500 Serum Supplement</td><td>Stemcell Technologies</td><td>9500</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Consumable Supplies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>2 ml Serological pipet</td><td>VWR</td><td>89130-894</td><td></td></tr><tr><td>5 ml Serological pipet</td><td>Olympus Plastics</td><td>12-102</td><td></td></tr><tr><td>10 ml Serological pipet</td><td>Olympus Plastics</td><td>12-104</td><td></td></tr><tr><td>25 ml Serological pipet</td><td>Olympus Plastics</td><td>12-106</td><td></td></tr><tr><td>50 ml Serological pipet</td><td>Olympus Plastics</td><td>12-107</td><td></td></tr><tr><td>6-well plate</td><td>Corning</td><td>353046</td><td></td></tr><tr><td>12-well plate</td><td>Corning</td><td>353043</td><td></td></tr><tr><td>T25 flask</td><td>TPP</td><td>90026</td><td></td></tr><tr><td>T-75 flask</td><td>TPP</td><td>90076</td><td></td></tr><tr><td>20 &amp;micro;l pipet tips</td><td>Eppendorf</td><td>22491130</td><td></td></tr><tr><td>200 &amp;micro;l pipet tips</td><td>Eppendorf</td><td>22491148</td><td></td></tr><tr><td>1,000 pipet tips</td><td>Eppendorf</td><td>22491156</td><td></td></tr><tr><td>Cryovials</td><td>Thermo Scientific</td><td>5000.102</td><td></td></tr><tr><td>70% ethanol</td><td>BDH</td><td>BDH1164-4LP</td><td></td></tr><tr><td>Sanimaster 4</td><td>Ecolab</td><td>65332960</td><td></td></tr><tr><td>Bleach</td><td>Clorox</td><td>A714239</td><td></td></tr></tbody></table>

culturing human pluripotent and neural stem cells in an enclosed cell culture system for basic and preclinical research

This paper describes how to use a custom manufactured, commercially available enclosed cell culture system for basic and preclinical research. Biosafety cabinets (BSCs) and incubators have long been the standard for culturing and expanding cell lines for basic and preclinical research. However, as the focus of many stem cell laboratories shifts from basic research to clinical translation, additional requirements are needed of the cell culturing system. All processes must be well documented and have exceptional requirements for sterility and reproducibility. In traditional incubators, gas concentrations and temperatures widely fluctuate anytime the cells are removed for feeding, passaging, or other manipulations. Such interruptions contribute to an environment that is not the standard for cGMP and GLP guidelines. These interruptions must be minimized especially when cells are utilized for therapeutic purposes. The motivation to move from the standard BSC and incubator system to a closed system is that such interruptions can be made negligible. Closed systems provide a work space to feed and manipulate cell cultures and maintain them in a controlled environment where temperature and gas concentrations are consistent. This way, pluripotent and multipotent stem cells can be maintained at optimum health from the moment of their derivation all the way to their eventual use in therapy.

Questo manoscritto descrive in dettaglio un impianto di produzione di cellule, e gli aspetti peculiari della coltura di cellule in un sistema chiuso. La maggior parte della struttura di produzione di celle e dispone di un apparecchio che ha un ingombro ridotto e può essere facilmente alloggiata all&#39;interno di un 6 x 7,5 m 2 Camera (Figura 2) su misura, chiuso, coltura cellulare. In nessun momento sono le cellule all&#39;interno dell&#39;apparato esposta al personale di laboratorio o ambiente. L&#39;apparecchiatura consiste di diversi moduli: una camera di processo, una cappa a flusso laminare, 2 camere buffer tenuta stagna tra il cappuccio e la camera di processo, due incubatori per colture cellulari, e una camera di microscopio adiacente alla camera di processo (Figura 3). Il sistema è controllato da un software consentendo variabili ambientali di essere controllati e monitorati (Figura 1). Il computer che esegue questo software è in un gruppo di continuità. I gas sono alimentati in the sistema da una serie di collettori per ossigeno, azoto e anidride carbonica (Figura 4). Gas per il dispositivo siano alimentati da sistemi molteplici che hanno due serie di vasche di ciascuno - un set attiva e una serie di riserva. Quando il set attivo viene prelevato, il collettore passa automaticamente ai serbatoi di sostituzione ordine di riserva impostato e del personale. L&#39;alimentazione è presente un sistema generatore di backup. Pluripotenti e cellule staminali neurali possono essere coltivate in condizioni di ipossia in questa struttura utilizzando i protocolli sviluppati in precedenza 9, con le complicazioni aggiunti inerenti alle apparecchiature romanzo. Le cellule staminali pluripotenti possono essere derivate utilizzando virus di Sendai, utilizzando anticorpi specifici pluripotenza per identificare colonie completamente transfettate, che vengono poi isolate ed espanse. (Figura 5A e B), Da queste iPSCs, cellule staminali neurali e neuroni possono essere derivate usando l&#39;inibizione doppia SMAD, e il loro fenotipo USI verificati<html> ng anticorpi NSC-specifici (Figura 5C e D). <img alt="Figura 1" src="/files/ftp_upload/53685/53685fig1.jpg" /> Figura 1. Interfaccia grafica per la produzione di celle strumento. (A) La rappresentazione grafica del CPF è la schermata di default e corrisponde al disegno CAD mostrato in Figura 3. Un clic del mouse sul buffer Modulo 1 (2F nella figura 1) apre il suo schermo di controllo (B), dove sono impostati O 2 valori per abbinare la camera di trattamento (3 nella Figura 3). Allo stesso modo, cliccando sul processo di Camera o Incubator 1 porta le loro rispettive schermate di controllo (C e D, rispettivamente), dove i valori possono essere modificati o controllati a seconda dei casi.<html> rget = &quot;_ blank&quot;&gt; Clicca qui per vedere una versione più grande di questa figura. <img alt="figura 2" src="/files/ftp_upload/53685/53685fig2.jpg" /> Figura 2. The Cell Production Facility. (A) Il sistema come visto da una vista anteriore destra. Notare i collegamenti di alimentazione e di gas nel soffitto e il carrello con gli accessori microscopio e il computer a destra. (B) La cappa a flusso laminare con le porte di accesso ai moduli buffer visti a destra. (C) La camera di processo con i due incubatori (porte nere) visto nella parte posteriore. (D) Una vista attraverso il lato destro del sistema che mostra il microscopio e monitorare con le porte delle camere di tampone visto in lontananza. <a href="https://www.jove.com/files/ftp_upload/53685/53685fig2large.jpg" target="_blank">Clicca qui per vedere una versione più grande di questa figura.</a> t &quot;fo: keep-together.within-page =&quot; 1 &quot;&gt; <img alt="Figura 3" src="/files/ftp_upload/53685/53685fig3.jpg" /> Figura 3. Un disegno CAD della cella Production. Tutti gli elementi che entrano nel dispositivo vengono dapprima pulito con alcool ed aria secca nella cappa a flusso laminare (1). Gli articoli vengono quindi trasferiti nella atmosfera appropriata nel modulo front buffer (2F) prima di passare nel modulo UCPC, o Process sezione (3). Le cellule sono coltivate in due incubatrici (4, 5). Colorazione delle cellule vive, colonia raccolta, valutazione della morfologia cellulare generale e analisi di migrazione avviene nel modulo UPC, o Microscopio Camera (6). Infine, i rifiuti esce dal sistema attraverso il modulo di buffer posteriore (2R). <a href="https://www.jove.com/files/ftp_upload/53685/53685fig3large.jpg" target="_blank">Clicca qui per vedere una versione più grande di questa figura.</a> &lt;<html> / P&gt; <img alt="Figura 4" src="/files/ftp_upload/53685/53685fig4.jpg" /> Figura 4. Fonti di gas e di potere. (A) Il collettore di CO 2 è una configurazione 4 x 4 in quanto fornisce tutti gli incubatori del laboratorio. (B) Il collettore O 2 è un 2 x 2 di installazione e fornisce solo l&#39;impianto di produzione di cellule. Azoto per il sistema è generata in un vaporizzatore (C, appena sotto il segno Exit) collegato ad un collettore azoto liquido (verso destra) che riempie anche automaticamente cryofreezers (in basso a sinistra). Il collettore è una configurazione 2 x 2 è rifornita da due coppie di 160 L serbatoi di azoto liquido. CO 2, O 2 e N 2 sono forniti dal soffitto tramite Shutoffs (D). Casa vuoto viene fornito anche in questa posizione. Visto appena dietro le Shutoffs gas di alimentazione sono una coppia di cavi elettrici che alimentano il sistema.f = &quot;https://www.jove.com/files/ftp_upload/53685/53685fig4large.jpg&quot; target = &quot;_ blank&quot;&gt; Clicca qui per vedere una versione più grande di questa figura. <img alt="Figura 5" src="/files/ftp_upload/53685/53685fig5.jpg" /> Figura 5. cellule staminali pluripotenti derivate in ipossia con il virus di Sendai. (A) SC187-SF4-2I0-E3 iPSCs iPSCs (nomenclatura si trovano in Stover et al. 9), contrasto di fase 10X. (B) SC88.1-UH1-2I0 iPSCs live-macchiato con AF-594-marcato Tra-1-60, diluizione 1: 100 in media. Tutti iPSCs sono state trasdotte nella struttura di produzione di celle a 5% O 2 con il virus di Sendai. (C) contrasto di fase di NSCs SC68.1-UH0-2I0-M0S13-N2G6 iPSC-derivati, cresciuto al 5% O 2 in camera di diapositive. Le cellule sono state poi fissate in paraformaldeide al 4% e colorati con anticorpo primario Nestin anti-umano, e Alex-488 anticorpo secondario. Tutte le barre di scala sono a 1001;. M <a href="https://www.jove.com/files/ftp_upload/53685/53685fig5large.jpg" target="_blank">Clicca qui per vedere una versione più grande di questa figura.</a>

Watch this Scientific Journal Video about Culturing Human Pluripotent and Neural Stem Cells in an Enclosed Cell Culture System for Basic and Preclinical Research at JoVE.com

Culturing Human Pluripotent and Neural Stem Cells in an Enclosed Cell Culture System for Basic and Preclinical Research

Here is a protocol to grow pluripotent stem cells (PSC) and neural stem cells (NSC) in an enclosed cell culture system that permits maximum sterility and reproducibility, replacing the traditional biosafety cabinet and incubator. This equipment meets clinical good manufacturing practice (cGMP) and clinical good lab practice (cGLP) guidelines.

Coltura umane pluripotenti e neurali cellule staminali in un sistema di coltura cellulare chiuso per base e preclinica di ricerca

This paper describes how to use a custom manufactured, commercially available enclosed cell culture system for basic and preclinical research. Biosafety ...

culturing-human-pluripotent-neural-stem-cells-an-enclosed-cell

Research

JoVE Journal

Developmental Biology

10.2K Views.  Childrens Hospital of Orange County Research Institute. Here is a protocol to grow pluripotent stem cells (PSC) and neural stem cells (NSC) in an enclosed cell culture system that permits maximum sterility and reproducibility, replacing the traditional biosafety cabinet and incubator. This equipment meets clinical good manufacturing practice (cGMP) and clinical good lab practice (cGLP) guidelines.

Video: Culturing Human Pluripotent and Neural Stem Cells in an Enclosed Cell Culture System for Basic and Preclinical Research

A Novel Culture Model for Human Pluripotent Stem Cell Propagation on Gelatin in Placenta-conditioned Media

The propagation of human pluripotent stem cells (hPSCs) in conditioned medium derived from human cells in feeder-free culture conditions has been of interest. Nevertheless, an ideal humanized ex vivo feeder-free propagation method for hPSCs has not been developed; currently, additional exogenous substrates including basic fibroblast growth factor (bFGF), a master hPSC-sustaining factor, is added to all of culture media and synthetic substrata such as Matrigel or laminin are used in all feeder-free cultures. Recently, our group developed a simple and efficient protocol for the propagation of hPSCs using only conditioned media derived from the human placenta on a gelatin-coated dish without additional exogenous supplementation or synthetic substrata specific to hPSCs. This protocol has not been reported previously and might enable researchers to propagate hPSCs efficiently in humanized culture conditions. Additionally, this model obviates hPSC contamination risks by animal products such as viruses or unknown proteins. Furthermore, this system facilitates easy mass production of hPSCs using the gelatin coating, which is simple to handle, dramatically decreases the overall costs of ex vivo hPSC maintenance.

This protocol provides a simple and efficient way to propagate human pluripotent stem cells (hPSCs) using only conditioned media derived from the human placenta in a gelatin-coated dish without additional exogenous supplementation or hPSC-specific synthetic substrata.

A Novel cultura Modello pluripotenti staminali umane propagazione delle cellule su Gelatina in Media Placenta condizionata

The propagation of human pluripotent stem cells (hPSCs) in conditioned medium derived from human cells in feeder-free culture conditions has been of ...

Ricerca

Biologia dello sviluppo

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 ...

Biologia

Generation of Neural Stem Cells from Discarded Human Fetal Cortical Tissue

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ultimately give rise to intermediate progenitors and later, the various neuronal and glial cell subtypes that form the cerebral cortex. The capacity to generate and expand human NSCs (so called neurospheres) from discarded normal fetal tissue provides a means with which to directly study the functional aspects of normal human NSC development 1-5. This approach can also be directed toward the generation of NSCs from known neurological disorders, thereby affording the opportunity to identify disease processes that alter progenitor proliferation, migration and differentiation 6-9. We have focused on identifying pathological mechanisms in human Down syndrome NSCs that might contribute to the accelerated Alzheimer's disease phenotype 10,11. Neither in vivo nor in vitro mouse models can replicate the identical repertoire of genes located on human chromosome 21. 
Here we use a simple and reliable method to isolate Down syndrome NSCs from aborted human fetal cortices and grow them in culture. The methodology provides specific aspects of harvesting the tissue, dissection with limited anatomical landmarks, cell sorting, plating and passaging of human NSCs. We also provide some basic protocols for inducing differentiation of human NSCs into more selective cell subtypes.

A simple and reliable method on isolation and culture of neural stem cells from discarded human fetal cortical tissue is described. Cultures derived from known human neurological disorders can be used for characterization of pathological cellular and molecular processes, as well as provide a platform to assess pharmacological efficacy.

Generazione di cellule staminali neurali da scartate tessuto fetale umano corticale

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ...

Neuroscienze

Efficient Derivation of Human Neuronal Progenitors and Neurons from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system (CNS) structure and circuitry in today's healthcare industry. Cell-based therapies hold great promise to restore the lost nerve tissue and function for CNS disorders. However, cell therapies based on CNS-derived neural stem cells have encountered supply restriction and difficulty to use in the clinical setting due to their limited expansion ability in culture and failing plasticity after extensive passaging1-3. Despite some beneficial outcomes, the CNS-derived human neural stem cells (hNSCs) appear to exert their therapeutic effects primarily by their non-neuronal progenies through producing trophic and neuroprotective molecules to rescue the endogenous cells1-3. Alternatively, pluripotent human embryonic stem cells (hESCs) proffer cures for a wide range of neurological disorders by supplying the diversity of human neuronal cell types in the developing CNS for regeneration1,4-7. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity7-10. In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic11-13. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules14 (please see a schematic in Fig. 1). Retinoic acid (RA) does not induce neuronal differentiation of undifferentiated hESCs maintained on feeders1, 14. And unlike mouse ESCs, treating hESC-differentiated embryoid bodies (EBs) only slightly increases the low yield of neurons1, 14, 15. However, after screening a variety of small molecules and growth factors, we found that such defined conditions rendered retinoic acid (RA) sufficient to induce the specification of neuroectoderm direct from pluripotent hESCs that further progressed to neuroblasts that generated human neuronal progenitors and neurons in the developing CNS with high efficiency (Fig. 2). We defined conditions for induction of neuroblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human neuronal cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of neuroblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human neuronal progenitors and neuronal cell types in the developing CNS for neural repair.

Derivazione efficiente di progenitori neuronali umane e Neuroni pluripotenti da cellule staminali embrionali umane con induzione piccole molecole

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system ...

Efficient Neural Differentiation using Single-Cell Culture of Human Embryonic Stem Cells

In vitro differentiation of human embryonic stem cells (hESCs) has transformed the ability to study human development on both biological and molecular levels and provided cells for use in regenerative applications. Standard approaches for hESC culture using colony type culture to maintain undifferentiated hESCs and embryoid body (EB) and rosette formation for differentiation into different germ layers are inefficient and time-consuming. Presented here is a single-cell culture method using hESCs instead of a colony-type culture. This method allows maintenance of the characteristic features of undifferentiated hESCs, including expression of hESC markers at levels comparable to colony type hESCs. In addition, the protocol presents an efficient method for neural progenitor cell (NPC) generation from single-cell type hESCs that produces NPCs within 1 week. These cells highly express several NPC marker genes and can differentiate into various neural cell types, including dopaminergic neurons and astrocytes. This single-cell culture system for hESCs will be useful in investigating the molecular mechanisms of these processes, studies of certain diseases, and drug discovery screens.

Presented here is a protocol for the generation of a single-cell culture of human embryonic stem cells and their subsequent differentiation into neural progenitor cells. The protocol is simple, robust, scalable, and suitable for drug screening and regenerative medicine applications.

Differenziazione neurale efficiente utilizzando la coltura a cella singola di cellule staminali embrionali umane

In vitro differentiation of human embryonic stem cells (hESCs) has transformed the ability to study human development on both biological and molecular ...

Differenziazione indotta di cellule staminali pluripotenti mediante un sistema di coltura automatizzato

An Automated Culture System for Maintaining and Differentiating Human-Induced Pluripotent Stem Cells

Human induced pluripotent stem cells (hiPSCs) with infinite self-proliferating ability have been expected to have applications in numerous fields, including the elucidation of rare disease pathologies, the development of new medicines, and regenerative medicine aiming to restore damaged organs. Despite this, the social implementation of hiPSCs is still limited. This is partly because of the difficulty of reproducing differentiation in culture, even with advanced knowledge and sophisticated technical skills, due to the high sensitivity of iPSCs to minute environmental changes. The application of an automated culture system can solve this issue. Experiments with high reproducibility independent of a researcher's skill can be expected according to a shared procedure across various institutes. Although several automated culture systems that can maintain iPSC cultures and induce differentiation have been developed previously, these systems are heavy, large, and costly because they make use of humanized, multi-articulated robotic arms. To improve on the above issues, we developed a new system using a simple x-y-z axis slide rail system, allowing it to be more compact, lighter, and cheaper. Furthermore, the user can easily modify parameters in the new system to develop new handling tasks. Once a task is established, all the user needs to do is prepare the iPSC, supply the reagents and consumables needed for the desired task in advance, select the task number, and specify the time. We confirmed that the system could maintain iPSCs in an undifferentiated state through several passages without feeder cells and differentiate into various cell types, including cardiomyocytes, hepatocytes, neural progenitors, and keratinocytes. The system will enable highly reproducible experiments across institutions without the need for skilled researchers and will facilitate the social implementation of hiPSCs in a wider range of research fields by diminishing the obstacles for new entries.

Autore in primo piano: Automazione della coltura iPSC per una maggiore riproducibilità 

Here, we present a protocol for an automated cell culture system. This automated culture system reduces labor and benefits the users, including researchers unfamiliar with handling induced pluripotent stem (iPS) cells, from the maintenance of iPS cells to differentiation into various types of cells.

Un sistema di coltura automatizzato per il mantenimento e la differenziazione di cellule staminali pluripotenti indotte dall'uomo

Human induced pluripotent stem cells (hiPSCs) with infinite self-proliferating ability have been expected to have applications in numerous fields, ...

Alternative Cultures for Human Pluripotent Stem Cell Production, Maintenance, and Genetic Analysis

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and efficient expansion of large amounts of clinical-grade hPSCs are critical issues in hPSC-based therapies. Conventionally, hPSCs are propagated as colonies on both feeder and feeder-free culture systems. However, these methods have several major limitations, including low cell yields and generation of heterogeneously differentiated cells. To improve current hPSC culture methods, we have recently developed a new method, which is based on non-colony type monolayer (NCM) culture of dissociated single cells. Here, we present detailed NCM protocols based on the Rho-associated kinase (ROCK) inhibitor Y-27632. We also provide new information regarding NCM culture with different small molecules such as Y-39983 (ROCK I inhibitor), phenylbenzodioxane (ROCK II inhibitor), and thiazovivin (a novel ROCK inhibitor). We further extend our basic protocol to cultivate hPSCs on defined extracellular proteins such as the laminin isoform 521 (LN-521) without the use of ROCK inhibitors. Moreover, based on NCM, we have demonstrated efficient transfection or transduction of plasmid DNAs, lentiviral particles, and oligonucleotide-based microRNAs into hPSCs in order to genetically modify these cells for molecular analyses and drug discovery. The NCM-based methods overcome the major shortcomings of colony-type culture, and thus may be suitable for producing large amounts of homogeneous hPSCs for future clinical therapies, stem cell research, and drug discovery.

Here, we present human pluripotent stem cell (hPSC) culture protocols, based on non-colony type monolayer (NCM) growth of dissociated single cells. This new method, utilizing Rho-associated kinase inhibitors or the laminin isoform 521 (LN-521), is suitable for producing large amounts of homogeneous hPSCs, genetic manipulation, and drug discovery.

Le colture per pluripotenti umane alternative staminali produzione di celle, manutenzione e analisi genetica

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and ...

Colture cellulari miste di cervello di topo embrionale per studiare l'infezione e l'immunità innata

Establishing Mixed Neuronal and Glial Cell Cultures from Embryonic Mouse Brains to Study Infection and Innate Immunity

Models of the central nervous system (CNS) must recapitulate the complex network of interconnected cells found in vivo. The CNS consists primarily of neurons, astrocytes, oligodendrocytes, and microglia. Due to increasing efforts to replace and reduce animal use, a variety of in vitro cell culture systems have been developed to explore innate cell properties, which allow the development of therapeutics for CNS infections and pathologies. Whilst certain research questions can be addressed by human-based cell culture systems, such as (induced) pluripotent stem cells, working with human cells has its own limitations with regard to availability, costs, and ethics. Here, we describe a unique protocol for isolating and culturing cells from embryonic mouse brains. The resulting mixed neural cell cultures mimic several cell populations and interactions found in the brain in vivo. Compared to current equivalent methods, this protocol more closely mimics the characteristics of the brain and also garners more cells, thus allowing for more experimental conditions to be investigated from one pregnant mouse. Further, the protocol is relatively easy and highly reproducible. These cultures have been optimized for use at various scales, including 96-well based high throughput screens, 24-well microscopy analysis, and 6-well cultures for flow cytometry and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis. This culture method is a powerful tool to investigate infection and immunity within the context of some of the complexity of the CNS with the convenience of in vitro methods.

Autore in primo piano: Stabilire colture cellulari neuronali e gliali miste da cervelli embrionali di topo per studiare l'infezione e l'immunità innata  

This protocol presents a unique way of generating central nervous system cell cultures from embryonic day 17 mouse brains for neuro(immuno)logy research. This model can be analyzed using various experimental techniques, including RT-qPCR, microscopy, ELISA, and flow cytometry.

Stabilire colture cellulari miste neuronali e gliali da cervelli di topo embrionali per studiare l'infezione e l'immunità innata

Models of the central nervous system (CNS) must recapitulate the complex network of interconnected cells found in vivo. The CNS consists primarily of ...

Coltura umane pluripotenti e neurali cellule staminali in un sistema di coltura cellulare chiuso per base e preclinica di ricerca

Riepilogo

Esplora Altri Video

PLAYLIST