The authors acknowledge financial support from the Victorian Government&#8217;s Operational Infrastructure Support Program.

NOTA: placenta dovrebbe essere raccolto da gravidanze sane Singleton, con una preferenza per tagli cesarei elettivi termine. Scritto, consenso informato deve essere dato per la raccolta dei loro placenta. Il tuo comitati etici di ricerca umani dovrebbero approvazione tutta la linea e l&#39;utilizzo di tessuti umani. 1. Isolamento di cellule epiteliali Amnion <ol style="margin-left: 40px;"><li> Posizionare la placenta su una superficie sterile all&#39;interno di una cappa di sicurezza biologica di classe II. </li><li> Utilizzando matasse sterili soluzione salina bilanciata (HBSS), lavare il più sangue possibile dalla superficie della placenta e le membrane. </li><li> Con la placenta disposto con il cordone ombelicale rivolto verso l&#39;alto, separare meccanicamente il bordo esterno delle membrane amnion e corion. </li><li> Una volta separati, striscia manualmente la membrana amniotica dalla membrana corion della placenta, lavorando intorno al bordo della membrana, verso il cordone ombelicale. NOTA: Essere Careful per rimuovere eventuali pezzi di contaminanti membrana chorion dalla membrana amniotica. </li><li> Usando forbici sterili, tagliare la membrana amniotica 2 cm circa dalla base del cordone ombelicale e posto in un contenitore sigillato 500 ml contenente 250 ml HBSS. </li><li> Lavare accuratamente agitando la bottiglia sigillata contenente la membrana amniotica. </li><li> Rimuovere la membrana amniotica dal contenitore di raccolta con pinza sterile, e posto in una capsula di Petri 15 cm. </li><li> Tagliare la membrana amniotica in pezzi lunghi circa 5 cm (se tenuto in verticale con le pinze), scartando i pezzi sanguinosi o strappati. </li><li> Mettere in un contenitore sigillato da 500 ml e lavare con 250 ml HBSS circa 3-5 volte o fino a quando la membrana amniotica diventa traslucida con niente sangue contaminante. NOTA: Qualsiasi sangue presente può ridurre l&#39;efficienza della enzimatica soluzione di analisi. </li><li> Trasferire tutti i pezzi di membrana amniotica in un recipiente contenente 50 ml della soluzione enzimatica digerire, avendo cura diridurre al minimo riporto di HBSS. </li><li> Incubare i pezzi amnion in enzimatica digeriscono soluzione in un bagno d&#39;acqua o camera calda a 37 ° C per 15 minuti con agitazione per rimuovere il sangue residuo. </li><li> Togliere i pezzi di membrana amnion dalla enzimatica digerire soluzione in un nuovo contenitore sterile. </li><li> Aggiungere 100 ml di soluzione enzimatica fresco digerire alla membrana amniotica e incubare a 37 ° C per 60 minuti in un bagno d&#39;acqua o una stanza calda con agitando delicatamente (prima digestione). </li><li> Togliere i pezzi di membrana amnios e inserire in un nuovo contenitore cura presa per consentire alla enzimatica digerire soluzione per drenare da ogni pezzo di membrana. NOTA: Ciò può essere ottenuto trascinando pezzi amnion lungo il bordo interno del contenitore. </li><li> Aggiungere 100 ml di enzimatica fresco digeriscono soluzione ai pezzi di membrana amnios e incubare 60 minuti in un bagno di acqua o camera calda a 37 ° C con un leggero scuotimento (seconda digestione). </li><li> Passare il suspensi cellulareon dal punto 1.14 attraverso un filtro 70 micron e centrifugare a 1000 xg per 10 min. </li><li> Rimuovere e scartare il surnatante e risospendere il pellet cellulare in 4 ml HBSS contenente 1 mg / inibitore basato soia-ml. Pipettare delicatamente ripetutamente con una pipetta 1 ml per ottenere una sospensione di cellule singole. NOTA: In questa fase le cellule possono essere lasciati in ghiaccio fino cellule dal secondo digest sono nella stessa fase di lavorazione. </li><li> Togliere i pezzi di membrana amnios e inserire in un nuovo contenitore cura presa per consentire alla enzimatica digerire soluzione per drenare da ogni pezzo di membrana. NOTA: Ciò può essere ottenuto trascinando pezzi amnion lungo il bordo interno del contenitore. </li><li> Passare la sospensione cellulare dal punto 1.18 attraverso un filtro 70 micron e centrifugare a 1000 xg per 10 min. </li><li> Rimuovere e scartare il surnatante e risospendere il pellet cellulare in 4 ml HBSS contenente 1 mg / inibitore basato soia-ml. Pipettare delicatamente ripetutamente con un 1 ml pipette per ottenere una sospensione di cellule singole. </li><li> Cellule piscina dal due digest o in alternativa mantenere digest cellule separate fino a dopo il conteggio delle cellule. NOTA: Questo impedisce la miscelazione di cellule viabilità potenzialmente bassi con cellule di alta redditività. </li><li> Aggiungere HBSS ad un volume finale di 10-20 ml ed eseguire la conta delle cellule utilizzando trypan blu per misurare la vitalità. </li><li> Confermare il fenotipo delle cellule epiteliali mediante citometria a flusso. Make up adeguati cocktail di anticorpi con anti-EpCAM-PE (diluizione 1: 2) anti-CD90-PeCy5 (1: 250) e anti-CD105-APC (1: 100) in HBSS. </li><li> Risospendere un sottoinsieme di cellule da uno o entrambi i digest del cocktail di anticorpi ad una concentrazione di 25 x 10 6 cellule / ml. </li><li> Per la citometria a flusso, macchia 1 x 10 6 cellule con un cocktail di anticorpi contenenti controlli isotipici (ad es. Controllo-PE IgG 1 isotipo + anti-CD90-PeCy5 + anti-CD105-APC). Mantenere ~ 1 x 10 6 cellule senza macchia per citofluorimetro set-up. </li><li> Incubare cellule anticorpococktail a 4 ° C per 60 min. </li><li> Lavare le cellule 3x in HBSS e risospendere tra i 5 ei 10 milioni di cellule per ml di citometria a flusso. </li><li> Eseguire citometria di flusso e risultati di nota. La purezza del campione isolato cella (la percentuale di EpCAM positive e CD90 / CD105 cellule negative) dovrebbe essere rispettivamente 90-95% e &lt;1%. </li><li> Mettere da parte un adeguato numero di cellule per test di criteri di controllo della qualità o di rilascio appropriate come determinato dall&#39;applicazione finale. NOTA: Questo può includere malattia / di screening virale, o altro di sicurezza o criteri biologici. </li><li> Per le cellule rimanenti, seguire il protocollo di crioconservazione, o in alternativa, inserire direttamente nella cultura (diretta alla cultura passo 3.6). </li></ol> 2. Crioconservazione di hAECs <ol style="margin-left: 40px;"><li> Determinare numero di cellulare e la vitalità con citometria a flusso di dati o di conteggio manuale con trypan esclusione blu. </li><li> Centrifugare a 700 xg per 5 min. </li><li> Cell Risospenderes tra 1-10000000 cellule per ml in animali crioconservazione multimediale gratuito prodotto. </li><li> Pipettare un volume adeguato di cellule in vials crioconservazione O-anelli e posto in un apparato velocità di congelamento controllato dove avviene il raffreddamento a circa 1 ° C / min fino a -80 ° C si ottiene. </li><li> Trasferire fiale in azoto liquido per la conservazione a lungo termine. </li></ol> 3. scongelamento e Cultura Cyropreserved hAECs <ol style="margin-left: 40px;"><li> Rimuovere le fiale di crioconservazione di azoto liquido e disporli sul ghiaccio. NOTA: solo per il trasferimento - procedere rapidamente alla fase successiva. </li><li> Scongelare le fiale crioconservazione rapidamente a 37 ° C fino a che solo una piccola parte (~ 5 mm x 5 mm) del ghiaccio rimane. NOTA: È importante che la soluzione cella non riscalda per ridurre gli effetti tossici di DMSO nei media crioconservazione. </li><li> Diluire mezzi crioconservazione 10 volte con il freddo (~ 4 ° C), i media senza siero, goccia a goccia in un formato adatto aggiuntod tubo o contenitore. </li><li> Centrifugare a 700 xg per 5 minuti a temperatura ambiente. </li><li> Risospendere in mezzo privo di siero, ad una concentrazione di circa 1 milione di cellule per ml. Determinare numero di cellulare e la vitalità con conteggio automatico o manuale con trypan esclusione blu. Previsto vitalità post-scongelamento sarà 5-10% in meno rispetto redditività pre-crioconservazione. </li><li> Cellule piastra su standard di coltura cellulare trattati e plastico, o in alternativa utilizzare collagene di tipo I per le superfici di cultura cappotto. </li><li> Per tipo I rivestimento collagene seguire il protocollo di seguito <ol><li> Preparare l&#39;acido tipo solubile I collagene dalla placenta umana (15 mg di collagene con 25 ml di acqua deionizzata e 50 ml di acido acetico glaciale). </li><li> Coprire bicchiere con Parafilm e agitazione moderata a 37 ° C fino a filamenti di collagene sono sciolte. </li><li> Soluzione filtro collagene sterile utilizzando un filtro di 0,22 micron. </li><li> Diluire il collagene magazzino 01:10 filtrata con acqua deionizzata. Questa azione diluito è la conce di lavoroSoluzione ntration per superfici di rivestimento in plastica e membrana. </li><li> Collagene plastica cappotto, superfici di vetro e membrana per 2 ore a temperatura ambiente a 37 ° C, o O / N a 2-8 ° C. </li><li> Rimuovere il liquido in eccesso dalla superficie rivestita, e lasciarlo asciugare O / N. </li><li> Risciacquare con acqua di grado colture di tessuti sterili o HBSS prima di introdurre cellule e mezzo. </li></ol></li><li> HAECs piastra a una densità di 25.000 cellule / cm 2 in terreno privo di siero, e consentono il collegamento a verificarsi per 72-96 ore. </li><li> Dopo allegato, cambiare supporto con attenzione aspirare e sostituirli con un opportuno volume di mezzo privo di siero ogni 2-3 giorni. </li><li> Cellule Passage che utilizzano il protocollo di seguito <ol><li> Mezzi di coltura cellulare Aspirare e lavare con il volume di 1x sterile HBSS </li><li> Aggiungere il volume 1x enzimatica digerire soluzione e incubare per 5-15 minuti, o fino a quando le cellule sono osservati staccano dalla superficie di coltura cellulare. </li><li> Raccogliere enzimatica digerire soluzione contenente le cellule, garantendo cells sono staccati dalla superficie coltura cellulare pipettando ripetutamente il enzimatica digerire soluzione contro la superficie di coltura cellulare </li><li> Centrifugare a 700 xg per 5 min. </li><li> Risospendere il pellet di cellule in un volume adeguato di HBSS contenente 1 mg / inibitore enzimatico a base di soia-ml. Pipettare delicatamente ripetutamente con una pipetta 1 ml per ottenere una sospensione di cellule singole. </li><li> Aggiungere mezzo privo di siero ad una concentrazione approssimativa di 1 milione di cellule / ml. Determinare numero di cellulare e la vitalità con conteggio automatico o manuale con trypan esclusione blu. </li><li> HAECs piastra a una densità di 25.000 cellule / cm 2 in terreno privo di siero, e continuare con i metodi di coltura standard. </li></ol></li></ol>

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The authors have nothing to disclose.

Ci sono diversi parametri critici che possono avere un impatto significativo per il successo di questa metodologia. Conservazione della placenta o amnion fino a 3 ore prima isolamento di hAECs può essere desiderabile per scopi logistici o di programmazione, ma si raccomanda che il tessuto viene elaborata appena possibile. Se il tessuto deve essere memorizzato, si raccomanda di memorizzazione eseguito in seguito dissezione e lavaggio della membrana amnion. Amnion può essere immagazzinato in HBSS sterili contenenti anti...

Le cellule derivate da fonti perinatali, come la placenta, membrane placentari, cordone ombelicale e liquido amniotico hanno attirato l&#39;attenzione dei ricercatori e clinici come potenziale fonte di cellule per la medicina rigenerativa 1,2. La ragione di questo interesse è che questi tipi di cellule tutti possiedono un certo grado di plasticità e immunomodulatorie capacità 3, proprietà che sono fondamentali per loro potenziali applicazioni terapeutiche. hAECs sono una popolazione eterogenea epiteliale che può derivare dal termine o pre-termine membrana amniotica 4, fornendo una abbondante fonte potenziale di materiale cellulare rigenerativa. Le proprietà che rendono hAECs accattivanti come terapia cellulare comprendono la multipotenza, bassa immunogenicità, e le proprietà anti-infiammatorie. hAECs sono stati trovati per essere altamente multipotenti sia in vitro che in vivo, in grado di differenziarsi in linee mesodermici (cardiomyocytes, miociti, osteociti, adipociti), lignaggi endodermici (cellule pancreatiche, cellule epatiche, cellule polmonari) e lignaggi ectodermiche (capelli, pelle, cellule neurali e astrociti) 5-10. Rassicurante, nonostante le loro hAECs multipotenza non sembrano né formare tumori o promuovere lo sviluppo del tumore in vivo. Inoltre, hAECs sono immuni privilegiata, esprimendo bassi livelli di classe II antigeni leucociti umani (HLA) 8. La struttura, probabilmente alla base la capacità di eludere rigetto immunitario dopo il trapianto allogenico e xenogenica, come dimostrato in studi che utilizzano scimmie immunitarie competenti, conigli, cavie, topi e maiali 11-13. hAECs visualizzare potenti immunomodulante proprietà immunosoppressive e quindi offrono notevoli vantaggi pratici per le potenziali applicazioni cliniche nella terapia delle malattie autoimmuni. hAECs si ritiene di esercitare le funzioni di immunomodulatori sia sul sistema immunitario innato e adattativo. Sue dei meccanismi proposti, è attraverso la secrezione di fattori immunomodulante 14. Le attuali applicazioni di hAECs in modelli di malattia degli animali pre-clinici comprendono il trattamento di ictus, sclerosi multipla, malattia del fegato, diabete e malattie polmonari croniche e acute. I ricercatori hanno mostrato interesse ad utilizzare hAECs per il trattamento di post-ictus infiammazione del cervello a causa delle loro proprietà uniche. Ci sono prove che hAECs può attraversare la barriera emato-encefalica, dove possono innestare, sopravvivere fino a 60 giorni, differenziarsi in neuroni, diminuire l&#39;infiammazione e promuovere la rigenerazione del tessuto danneggiato del sistema nervoso centrale in modelli animali di malattie neurologiche 15. hAECs offrono la possibilità di individuare e invertire molteplici percorsi patologici che contribuiscono allo sviluppo e la progressione della sclerosi multipla. Ad esempio, i risultati di studi su animali pre-clinici suggeriscono che hAECs fortemente immunosoppressiva epuò potenzialmente indurre tolleranza immunitaria periferica e invertire risposte infiammatorie in corso. hAECs hanno anche dimostrato di avere la capacità di differenziarsi in cellule neurali in vivo e migliorare neurorigenerazione endogena attraverso la secrezione di una vasta gamma di fattori neurotrofici 16. Cellule epiteliali umane e roditori amniotiche hanno già dimostrato la loro efficacia terapeutica per il trattamento della malattia epatica in modelli animali. In un modello di tetracloruro di carbonio induzione danni delle malattie del fegato, il trapianto haec portare ad attecchimento di hAECs vitali nel fegato, accompagnato con una ridotta epatociti apoptosi, e diminuito l&#39;infiammazione epatica e fibrosi 17. hAECs può essere stimolato da fattori pancreatiche espresse compreso insulina e glucosio trasportatori. Diversi studi hanno indagato la possibilità di hAECs per ripristinare i livelli di glucosio nel sangue in topi diabetici 18. Nei topi che ricevehAECs, sia di peso e di glucosio nel sangue i livelli del corpo animale è sceso a livelli normali dopo l&#39;iniezione di cellule. Questi studi presentano un caso forte per l&#39;uso di hAECs per il trattamento del diabete mellito. hAECs hanno un ruolo provata nella prevenzione e riparazione di sperimentale danno polmonare acuto e cronico in modelli neonatali 19 adulti e. Questi studi hanno trovato che hAECs differenziano in vitro in cellule epiteliali polmonari funzionale che esprimono più proteine ​​polmonari associate, tra cui la fibrosi cistica regolatore della conduttanza transmembrana (CFTR), il canale ionico che è mutato in pazienti affetti da fibrosi cistica 20. Inoltre, quando hAECs vengono consegnati l&#39;adulto ferito e polmonare neonatale, che esercitano i loro effetti riparatori tramite la modulazione delle cellule immunitarie dell&#39;ospite, riducendo il reclutamento dei leucociti polmonare, tra cui neutrofili, macrofagi e linfociti 21-23. Data la loro abbondanza,record di sicurezza, e le applicazioni cliniche collaudate per più malattie, studi clinici utilizzando hAECs è inevitabile. Con l&#39;obiettivo di accelerare la traduzione di terapie HAEC in sperimentazioni cliniche, abbiamo sviluppato metodi per isolare, criopreservare e hAECs cultura in un modo adatto per la sperimentazione clinica, utilizzando reagenti senza prodotti di origine animale in conformità alle buone pratiche di fabbricazione (cGMP) le linee guida . Abbiamo basato questo protocollo un protocollo precedentemente pubblicato che stavamo usando con successo per isolare hAECs utilizzando reagenti di origine animale 6. Abbiamo modificato il protocollo originale di sostituire i prodotti di origine animale con reagenti privi di prodotti animali, e la successiva ottimizzazione è stata eseguita per ottimizzare il rendimento delle cellule, la vitalità e purezza. Il nostro obiettivo era quello di sviluppare un protocollo che rispettare le norme regolamentari per la produzione di celle per test clinici sull&#39;uomo.

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			<td height="21" style="height:21px;width:220px;">Name of Material/ Equipment</td>
			<td style="width:119px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:184px;">Comments/Description</td>
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			<td height="21" style="height:21px;width:220px;">Stripping tray</td>
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			<td style="width:119px;">13-361B</td>
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			<td height="34" style="height:34px;width:220px;">Liberty Dressing Forcep, pointed 13cm</td>
			<td style="width:119px;">Fisher Scientific</td>
			<td>S17329&#160;</td>
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			<td height="21" style="height:21px;width:220px;">Scissors - Sharp/Blunt Straight</td>
			<td style="width:119px;">Fisher Scientific</td>
			<td>NC0562592&#160;</td>
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			<td height="21" style="height:21px;">Sterile latex gloves&#160;</td>
			<td style="width:119px;">Fisher Scientific</td>
			<td>19-014-643&#160;</td>
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			<td height="21" style="height:21px;width:220px;">Protective Apparel (Gown)</td>
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			<td style="width:119px;">S-15374-M</td>
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			<td height="21" style="height:21px;">Protective Apparel</td>
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			<td height="21" style="height:21px;">Isolation gowns</td>
			<td style="width:119px;">U-line</td>
			<td style="width:119px;">S-15374-M</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Sterile latex gloves&#160;</td>
			<td style="width:119px;">Fisher Scientific</td>
			<td>19-014-643&#160;</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">General purpose face mask</td>
			<td style="width:119px;">Cardinal Health</td>
			<td style="width:119px;">AT7511</td>
			<td></td>
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			<td height="21" style="height:21px;">Bonnets</td>
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			<td style="width:119px;">CRI1001</td>
			<td></td>
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			<td height="21" style="height:21px;">Shoe covers</td>
			<td style="width:119px;">U-line</td>
			<td style="width:119px;">S-7873W</td>
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			<td height="33" style="height:33px;">Hanks&#8217; Balanced Salt Solution (HBSS)</td>
			<td style="width:119px;">Life Technologies</td>
			<td align="right" style="width:119px;">14175095</td>
			<td>without calcium or magnesium</td>
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		<tr height="21">
			<td height="21" style="height:21px;">TrypZean(animal product&#8211;free recombinant trypsin)</td>
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			<td style="width:119px;">T3449</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Soybean Trypsin Inhibitor&#160; 1g/50mL</td>
			<td style="width:119px;">Sigma Aldrich</td>
			<td style="width:119px;">T6522</td>
			<td></td>
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			<td height="21" style="height:21px;">Cryostor CS5</td>
			<td style="width:119px;">BioLife Solutions</td>
			<td align="right" style="width:119px;">205102</td>
			<td></td>
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			<td height="23" style="height:23px;">Trypan blue reagent</td>
			<td style="width:119px;">Life Technologies</td>
			<td style="width:119px;">15250-061</td>
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			<td height="21" style="height:21px;">anti-EpCam-PE</td>
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			<td style="width:119px;">130 - 091-253</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">PE-isotype control</td>
			<td style="width:119px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-098-845</td>
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			<td height="21" style="height:21px;">anti-CD90-PeCy5</td>
			<td style="width:119px;">BD Pharmingen</td>
			<td align="right" style="width:119px;">555597</td>
			<td></td>
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			<td height="21" style="height:21px;">PeCy5-isotype control</td>
			<td style="width:119px;">BD Pharmingen</td>
			<td align="right" style="width:119px;">557224</td>
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			<td style="width:119px;">BD Pharmingen</td>
			<td align="right" style="width:119px;">562408</td>
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			<td align="right" style="width:119px;">340754</td>
			<td></td>
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			<td height="21" style="height:21px;">Collagen Type VI</td>
			<td style="width:119px;">Sigma Aldrich</td>
			<td>C7521</td>
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			<td height="21" style="height:21px;">Consumables</td>
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			<td height="21" style="height:21px;">50mL graduated pipette</td>
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			<td>356550</td>
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			<td height="21" style="height:21px;">10mL graduated pipette</td>
			<td style="width:119px;">BD/Falcon</td>
			<td>356551</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">5mL graduated pipette</td>
			<td style="width:119px;">BD/Falcon</td>
			<td>356543</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">50mL falcon tubes</td>
			<td style="width:119px;">BD/Falcon</td>
			<td>352070</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">15mL falcon tubes</td>
			<td style="width:119px;">BD/Falcon</td>
			<td>352096</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">15-cm petri dishes</td>
			<td style="width:119px;">Corning</td>
			<td align="right" style="width:119px;">351058</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">70-&#956;m filters</td>
			<td style="width:119px;">BD/Falcon</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.22-&#956;m filters</td>
			<td style="width:119px;">Millipore</td>
			<td>SLGV033RS</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">1ml Pipette tips</td>
			<td style="width:119px;">Fisherbrand</td>
			<td style="width:119px;">02-707-401</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">200ul Pipette tips</td>
			<td style="width:119px;">Fisherbrand</td>
			<td style="width:119px;">02-707-409</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">20ml Syringe</td>
			<td style="width:119px;">BD/Medical</td>
			<td align="right" style="width:119px;">309661</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Plastic spatula</td>
			<td style="width:119px;">Fisher Scientific</td>
			<td>14-245-97&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Plastic weighing boat</td>
			<td style="width:119px;">Fisher Scientific</td>
			<td>02-202-102&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Cryo vials</td>
			<td>Nunc</td>
			<td>377267</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:220px;"></td>
			<td style="width:119px;"></td>
			<td style="width:119px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:220px;"></td>
			<td style="width:119px;"></td>
			<td style="width:119px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Equipment</td>
			<td style="width:119px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Mr Frosty</td>
			<td style="width:119px;">Fisher Scientific</td>
			<td>A451-4&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Biohazard Cabinet</td>
			<td style="width:119px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation, cryopreservation and culture of human amnion epithelial cells for clinical applications

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a potential source of cells for regenerative medicine. The reason for this interest is evidence that these cells have highly multipotent differentiation ability, low immunogenicity, and anti-inflammatory functions. These properties have prompted researchers to investigate the potential of hAECs to be used to treat a variety of diseases and disorders in pre-clinical animal studies with much success.
hAECs have found widespread application for the treatment of a range of diseases and disorders. Potential clinical applications of hAECs include the treatment of stroke, multiple sclerosis, liver disease, diabetes and chronic and acute lung diseases. Progressing from pre-clinical animal studies into clinical trials requires a higher standard of quality control and safety for cell therapy products. For safety and quality control considerations, it is preferred that cell isolation protocols use animal product-free reagents. 
We have developed protocols to allow researchers to isolate, cryopreserve and culture hAECs using animal product-free reagents. The advantage of this method is that these cells can be isolated, characterized, cryopreserved and cultured without the risk of delivering potentially harmful animal pathogens to humans, while maintaining suitable cell yields, viabilities and growth potential. For researchers moving from pre-clinical animal studies to clinical trials, these methodologies will greatly accelerate regulatory approval, decrease risks and improve the quality of their therapeutic cell population.

Quando questa procedura è seguita correttamente, una resa media di 120 milioni di hAECs dovrebbe essere previsto, con un raggio di 80-160,000,000 cellule. Da queste rese, una redditività media di 83 ± 4% può essere previsto. La maggiore resa media e leggermente inferiore redditività nel metodo clinica possono essere dovuti all&#39;attività tripsina superiore prodotto di origine animale, e forse anche a causa della mancanza di proteine ​​del siero. Isolati hAECs hanno un profilo medio della superficie cellulare...

Watch this Scientific Journal Video about Isolation, Cryopreservation and Culture of Human Amnion Epithelial Cells for Clinical Applications at JoVE.com

Isolation, Cryopreservation and Culture of Human Amnion Epithelial Cells for Clinical Applications

We describe a protocol to isolate and culture human amnion epithelial cells (hAECs) using animal product-free reagents in accordance with current good manufacturing practices (cGMP) guidelines.

Isolamento, Crioconservazione e Cultura umani Cellule epiteliali Amnion per le applicazioni cliniche

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a ...

isolation-cryopreservation-culture-human-amnion-epithelial-cells-for

Research

JoVE Journal

Medicine

13.8K Views.  Wake Forest University Health Sciences. We describe a protocol to isolate and culture human amnion epithelial cells (hAECs) using animal product-free reagents in accordance with current good manufacturing practices (cGMP) guidelines. 

Video: Isolation, Cryopreservation and Culture of Human Amnion Epithelial Cells for Clinical Applications

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

Differenziazione Isolamento, caratterizzazione e comparata delle cellule umane Dental Pulp staminali derivate da denti permanenti mediante due diversi metodi

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Ricerca

Medicina

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

Paziente coltura cellulare derivato e isolamento di CD133<sup&gt; +</sup&gt; Putativi staminali alle cellule tumorali di melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

Isolation, Culture, and Imaging of Human Fetal Pancreatic Cell Clusters

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning endocrine cells, as evidenced by a significant increase in circulating human C-peptide following glucose stimulation1-9. However in vitro, genesis of insulin producing cells from human fetal ICCs is low10; results reminiscent of recent experiments performed with human embryonic stem cells (hESC), a renewable source of cells that hold great promise as a potential therapeutic treatment for type 1 diabetes. Like ICCs, transplantation of partially differentiated hESC generate glucose responsive, insulin producing cells, but in vitro genesis of insulin producing cells from hESC is much less robust11-17. A complete understanding of the factors that influence the growth and differentiation of endocrine precursor cells will likely require data generated from both ICCs and hESC. While a number of protocols exist to generate insulin producing cells from hESC in vitro11-22, far fewer exist for ICCs10,23,24. Part of that discrepancy likely comes from the difficulty of working with human fetal pancreas. Towards that end, we have continued to build upon existing methods to isolate fetal islets from human pancreases with gestational ages ranging from 12 to 23 weeks, grow the cells as a monolayer or in suspension, and image for cell proliferation, pancreatic markers and human hormones including glucagon and C-peptide. ICCs generated by the protocol described below result in C-peptide release after transplantation under the kidney capsule of nude mice that are similar to C-peptide levels obtained by transplantation of fresh tissue6. Although the examples presented here focus upon the pancreatic endoderm proliferation and &#946;&#160;cell genesis, the protocol can be employed to study other aspects of pancreatic development, including exocrine, ductal, and other hormone producing cells.

A protocol to isolate, culture, and image islet cell clusters (ICCs) derived from human fetal pancreatic cells is described.&#160; The method details the steps necessary to generate ICCs from tissue, culture as monolayers or in suspension as aggregates, and image for markers of proliferation and pancreatic cell fate decisions.

Isolamento, Cultura e Imaging di Human Fetal pancreatici gruppi di cellule

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning ...

Adaptation of Semiautomated Circulating Tumor Cell (CTC) Assays for Clinical and Preclinical Research Applications

The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the presence of circulating tumor cells (CTCs). These rare cells have been demonstrated to be of clinical significance in metastatic breast, prostate, and colorectal cancers. The current gold standard in clinical CTC detection and enumeration is the FDA-cleared CellSearch system (CSS). This manuscript outlines the standard protocol utilized by this platform as well as two&#160;additional adapted protocols that describe the detailed process of user-defined marker optimization for protein characterization of patient CTCs and a comparable protocol for CTC capture in very low volumes of blood, using standard CSS reagents, for studying in vivo preclinical mouse models of metastasis. In addition, differences in CTC quality between healthy donor blood spiked with cells from tissue culture versus patient blood samples are highlighted. Finally, several commonly discrepant items that can lead to CTC misclassification errors are outlined. Taken together, these protocols will provide a useful resource for users of this platform interested in preclinical and clinical research pertaining to metastasis and CTCs.

Adaptation of Semiautomated Circulating Tumor Cell CTC Assays for Clinical and Preclinical Research Applications

Circulating tumor cells (CTCs) are prognostic in several metastatic cancers. This manuscript describes the gold standard CellSearch system (CSS) CTC enumeration platform and highlights common misclassification errors. In addition, two&#160;adapted protocols are described for user-defined marker characterization of CTCs and CTC enumeration in preclinical mouse models of metastasis using this technology.

Adattamento del semiautomatico circolanti (CTC) Saggi Tumor Cell per applicazioni cliniche e di ricerca preclinica

The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the ...

Isolation and Culture Expansion of Tumor-specific Endothelial Cells

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model for developing new angiogenesis inhibitors for cancer. However, long-term in vitro expansion of murine endothelial cells (EC) is challenging due to phenotypic drift in culture (endothelial-to-mesenchymal transition) and contamination with non-EC. This is especially true for TEC which are readily outcompeted by co-purified fibroblasts or tumor cells in culture. Here, a high fidelity isolation method that takes advantage of immunomagnetic enrichment coupled with colony selection and in vitro expansion is described. This approach generates pure EC fractions that are entirely free of contaminating stromal or tumor cells. It is also shown that lineage-traced Cdh5cre:ZsGreenl/s/l reporter mice, used with the protocol described herein, are a valuable tool to verify cell purity as the isolated EC colonies from these mice show durable and brilliant ZsGreen fluorescence in culture.

We report a reliable method to isolate and culture primary tumor-specific endothelial cells from genetically engineered mouse models.

Isolamento e cultura espansione delle cellule endoteliali tumore-specifici

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model ...

Three-dimensional Alginate-bead Culture of Human Pituitary Adenoma Cells

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from brown sea algae. Briefly, the tumor tissue is cut into small pieces and submitted to an enzymatic digestion with collagenase and trypsin. Next, a cell suspension is obtained. The tumor cell suspension is mixed with 1.2% sodium alginate and dropped into a CaCl2 solution, and the alginate/cell suspension is gelled on contact with the CaCl2 to form spherical beads. The cells embedded in the alginate beads are supplied with nutrients provided by the culture media enriched with 20% FBS. Three-dimensional culture in alginate beads maintains the viability of adenoma cells for long periods of time, up to four months. Moreover, the cells can be liberated from the alginate by washing the beads with sodium citrate and seeded on glass coverslips for further immunocytochemical analyses. The use of a cell culture model allows for the fixation and visualization of the actin cytoskeleton with minimal disorganization. In summary, alginate beads provide a reliable culture system for the maintenance of pituitary adenoma cells.

Three-dimensional culture in alginate beads, due to its simplicity and reproducibility, was chosen to maintain the pituitary adenoma cells. The procedures included an initial enzymatic digestion and mechanical dissociation of the tumor tissue, and the subsequent cell suspension was encapsulated in alginate beads.

Tridimensionale alginato-tallone coltura di cellule umane adenoma ipofisario

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from ...

Isolation and Culture of Primary Endothelial Cells from Canine Arteries and Veins

Cardiovascular disease is studied in both human and veterinary medicine. Endothelial cells have been used extensively as an in vitro model to study vasculogenesis, (tumor) angiogenesis, and atherosclerosis. The current standard for in vitro research on human endothelial cells (ECs) is the use of Human Umbilical Vein Endothelial Cells (HUVECs) and Human Umbilical Artery Endothelial Cells (HUAECs). For canine endothelial research, only one cell line (CnAOEC) is available, which is derived from canine aortic endothelium. Although currently not completely understood, there is a difference between ECs originating from either arteries or veins. For a more direct approach to in vitro functionality studies on ECs, we describe a new method for isolating Canine Primary Endothelial Cells (CaPECs) from a variety of vessels. This technique reduces the chance of contamination with fast-growing cells such as fibroblasts and smooth muscle cells, a problem that is common in standard isolation methods such as flushing the vessel with enzymatic solutions or mincing the vessel prior to digestion of the tissue containing all cells. The technique we describe was optimized for the canine model, but can easily be utilized in other species such as human.

Novel isolation methods of primary endothelial cells from blood vessels are needed. This protocol describes a new technique that completely inverts blood vessels of interest, exposing only the endothelial side to enzymatic digestion. The resulting pure endothelial cell culture can be used to study cardiovascular diseases, disease modelling, and angiogenesis.

Isolamento e coltura di cellule endoteliali primarie dalle arterie e vene Canine

Cardiovascular disease is studied in both human and veterinary medicine. Endothelial cells have been used extensively as an in vitro model to study ...

A Novel Clinical Grade Isolation Method for Human Kidney Perivascular Stromal Cells

Mesenchymal Stromal Cells (MSCs) are tissue homeostatic and immune modulatory cells that have shown beneficial effects in kidney diseases and transplantation. Perivascular Stromal Cells (PSCs) share characteristics with bone marrow MSCs (bmMSCs). However, they also possess, most likely due to local imprinting, tissue-specific properties and play a role in local tissue homeostasis. This tissue specificity may result in tissue specific repair, also within the human kidney. We previously showed that human kidney PSCs (kPSCs) have enhanced kidney epithelial wound healing whereas bmMSCs did not have this potential. Moreover, kPSCs can ameliorate kidney injury in vivo. Therefore, kPSCs constitute an interesting source for cell therapy, particularly for kidney diseases and renal transplantation. Here we show the detailed isolation and culture method for kPSCs from transplant-grade human kidneys based on whole-organ perfusion of digestive enzymes via the renal artery and enrichment for the perivascular marker NG2. In this way, large cell quantities can be obtained that are suitable for cellular therapy.

Here we present a novel clinical grade isolation and culture method for kidney Perivascular Stromal Cells (kPSCs) based on whole organ perfusion with digestive enzymes and NG2-cell enrichment. With this method, it is possible to acquire sufficient cell numbers for cellular therapy.

Un metodo di isolamento di romanzo Clinical Grade per rene umano perivascolari cellule stromali

Mesenchymal Stromal Cells (MSCs) are tissue homeostatic and immune modulatory cells that have shown beneficial effects in kidney diseases and ...

Isolation of Adipose Derived Regenerative Cells for the Treatment of Erectile Dysfunction Following Radical Prostatectomy

Stem cells are used in many research areas within regenerative medicine in part because these treatments can be curative rather than symptomatic. Stem cells can be obtained from different tissues and several methods for isolation have been described. The presented method for the isolation of adipose-derived regenerative cells (ADRCs) can be used within many therapeutic areas because the method is a general procedure and, therefore, not limited to erectile dysfunction (ED) therapy. ED is a common and serious side effect to radical prostatectomy (RP) since ED often is not well treated with conventional therapy. Using ADRC&#8217;s as treatment for ED has attracted great interest due to the initial positive results after a single injection of cells into the corpora cavernosum. The method used for the isolation of ADRC&#8217;s is a simple, automated process, that is reproducible and ensures a uniform product. Furthermore, the sterility of the isolated product is ensured because the entire process takes place in a closed system. It is important to minimize the risk of contamination and infection since the stem cells are used for injection in humans. The whole procedure can be done within 2.5-3.5 hours and does not require a classified laboratory which eliminates the need for shipping tissue to an off-site. However, the procedure has some limitations since the minimum amount of drained lipoaspirate for the isolation device to function is 100 g.

Precise disclosure of methods and protocols is crucial for large scale uptake of stem cell therapies. Here, we present a protocol to isolate adipose-derived regenerative cells, used for a single intracavernous injection as treatment of erectile dysfunction (ED) following radical prostatectomy (RP).

Isolamento di cellule rigenerative di derivazione adiposa per il trattamento della disfunzione erettile a seguito di prostatectomia radicale

Stem cells are used in many research areas within regenerative medicine in part because these treatments can be curative rather than symptomatic. Stem ...

Induction and Analysis of Oxidative Stress in Sleeping Beauty Transposon-Transfected Human Retinal Pigment Epithelial Cells

Oxidative stress plays a critical role in several degenerative diseases, including age-related macular degeneration (AMD), a pathology that affects ~30 million patients worldwide. It leads to a decrease in retinal pigment epithelium (RPE)-synthesized neuroprotective factors, e.g., pigment epithelium-derived factor (PEDF) and granulocyte-macrophage colony-stimulating factor (GM-CSF), followed by the loss of RPE cells, and eventually photoreceptor and retinal ganglion cell (RGC) death. We hypothesize that the reconstitution of the neuroprotective and neurogenic retinal environment by the subretinal transplantation of transfected RPE cells overexpressing PEDF and GM-CSF has the potential to prevent retinal degeneration by mitigating the effects of oxidative stress, inhibiting inflammation, and supporting cell survival. Using the Sleeping Beauty transposon system (SB100X) human RPE cells have been transfected with the PEDF and GM-CSF genes and shown stable gene integration, long-term gene expression, and protein secretion using qPCR, western blot, ELISA, and immunofluorescence. To confirm the functionality and the potency of the PEDF and GM-CSF secreted by the transfected RPE cells, we have developed an in vitro assay to quantify the reduction of H2O2-induced oxidative stress on RPE cells in culture. Cell protection was evaluated by analyzing cell morphology, density, intracellular level of glutathione, UCP2 gene expression, and cell viability. Both, transfected RPE cells overexpressing PEDF and/or GM-CSF and cells non-transfected but pretreated with PEDF and/or GM-CSF (commercially available or purified from transfected cells) showed significant antioxidant cell protection compared to non-treated controls. The present H2O2-model is a simple and effective approach to evaluate the antioxidant effect of factors that may be effective to treat AMD or similar neurodegenerative diseases.

Induction and Analysis of Oxidative Stress in Sleeping Beauty Transposon-Transfected Human Retinal Pigment Epithelial Cells

We present a protocol for the development and use ofan oxidative stress-model by treating retinal pigment epithelial cells with H2O2, analyzing cell morphology, viability, density, glutathione, and UCP-2 level. It is a useful model to investigate the antioxidant effect of proteins secreted by transposon-transfected cells to treat neuroretinal degeneration.

Induzione e analisi dello stress ossidativo nelle cellule epiteliali del pigmento retinico umano trasforato dalla bella addormentata nel bosco

Oxidative stress plays a critical role in several degenerative diseases, including age-related macular degeneration (AMD), a pathology that affects ~30 ...