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

REMARQUE: Les placentas doivent être collectées auprès des grossesses saines uniques, avec une préférence pour les césariennes électives terme. Écrit, le consentement éclairé doit être donné pour la collecte de leur placenta. Votre comité d&#39;éthique de la recherche sur les humains pertinent devrait approuver toute la collection et l&#39;utilisation de tissus humains. 1. Isolement des cellules épithéliales Amnion <ol style="margin-left: 40px;"><li> Placez le placenta sur une surface stérile dans une enceinte de sécurité biologique de classe II. </li><li> Utilisation écheveaux stériles solution saline équilibrée (HBSS), laver autant de sang que possible de la surface de placenta et les membranes. </li><li> Avec le placenta placé avec le cordon ombilical vers le haut, séparer mécaniquement le bord externe de l&#39;amnios et chorion membranes. </li><li> Une fois séparé, enlever manuellement la membrane amniotique de la membrane chorion du placenta, de travail autour du bord de la membrane, vers le cordon ombilical. NOTE: Soyez CAREFul pour enlever les morceaux de contaminer membrane chorion de l&#39;amnios. </li><li> Avec des ciseaux stériles, couper la membrane amniotique d&#39;environ 2 cm de la base du cordon ombilical et le placer dans un récipient fermé 500 ml contenant 250 ml de HBSS. </li><li> Laver soigneusement en agitant la bouteille scellée contenant la membrane amniotique. </li><li> Retirer la membrane amniotique du récipient de collection à l&#39;aide de pinces stériles, et les placer dans un plat 15 cm Petri. </li><li> Couper la membrane amniotique en morceaux d&#39;environ 5 cm de long (lorsqu&#39;on la tient verticalement avec une pince), en écartant morceaux sanglants ou déchirés. </li><li> Placer dans un récipient scellé 500 ml et laver avec 250 ml de HBSS environ 3-5 fois ou jusqu&#39;à ce que la membrane amniotique devient translucide sans contaminer le sang. NOTE: Toute présence de sang peut réduire l&#39;efficacité de la solution enzymatique digérer. </li><li> Transférer tous les morceaux de membrane amniotique dans un récipient contenant 50 ml de la solution enzymatique digérer, en prenant soin deminimiser report de HBSS. </li><li> Incuber les pièces amnios dans enzymatique digèrent solution dans un bain d&#39;eau ou salle chaude à 37 ° C pendant 15 min en agitant doucement pour enlever tout reste de sang. </li><li> Retirez les morceaux de membrane amnios de la solution enzymatique digérer et placer dans un nouveau récipient stérile. </li><li> Ajouter 100 ml de solution enzymatique frais digérer à la membrane amniotique et incuber à 37 ° C pendant 60 min dans un bain d&#39;eau ou salle chaude avec agitation douce (première digestion). </li><li> Retirez les morceaux de membrane amnios et le placer dans une nouvelle prise de conteneurs soins pour permettre à la digestion enzymatique solution se écouler depuis chaque pièce de la membrane. NOTE: On peut y parvenir en faisant glisser les pièces amnios le long du bord intérieur du récipient. </li><li> Ajouter 100 ml de solution enzymatique frais digérer les morceaux de membrane amnios et incuber 60 min dans un bain d&#39;eau ou salle chaude à 37 ° C avec agitation douce (seconde digestion). </li><li> Passez le suspensi cellulaireà partir de l&#39;étape à travers un filtre 1,14 um et 70 centrifuger à 1000 g pendant 10 min. </li><li> Retirer et jeter le surnageant et remettre en suspension le culot cellulaire dans 4 ml de HBSS contenant un inhibiteur à base de soja 1 mg / ml enzyme. Pipeter doucement plusieurs fois avec une pipette de 1 ml pour obtenir une suspension cellulaire unique. NOTE: A ce stade, les cellules peuvent être laissées sur la glace jusqu&#39;à ce que les cellules de la seconde digestion sont au même stade de transformation. </li><li> Retirez les morceaux de membrane amnios et le placer dans une nouvelle prise de conteneurs soins pour permettre à la digestion enzymatique solution se écouler depuis chaque pièce de la membrane. NOTE: On peut y parvenir en faisant glisser les pièces amnios le long du bord intérieur du récipient. </li><li> Faire passer la suspension de cellules provenant de l&#39;étape à travers un filtre 1,18 um et 70 centrifuger à 1000 g pendant 10 min. </li><li> Retirer et jeter le surnageant et remettre en suspension le culot cellulaire dans 4 ml de HBSS contenant un inhibiteur à base de soja 1 mg / ml enzyme. Pipette doucement à plusieurs reprises avec un 1 ml de pipette pour obtenir une suspension cellulaire unique. </li><li> Piscine cellules des deux recueils ou encore les garder digère cellulaires séparés qu&#39;après le nombre de cellules. NOTE: Cela empêche le mélange des cellules de viabilité potentiellement faibles avec des cellules de haute viabilité. </li><li> Ajouter HBSS à un volume final de 10 à 20 ml et effectuer le nombre de cellules en utilisant du bleu trypan pour mesurer la viabilité. </li><li> Confirmez le phénotype des cellules épithéliales par cytométrie de flux. Compléter à l&#39;aide des cocktails d&#39;anticorps appropriés anti-EpCAM-PE (dilution 1: 2) anti-CD90-PECy5 (1: 250) et anti-CD105-APC (1: 100) dans du HBSS. </li><li> Remettre en suspension un sous-ensemble de cellules de l&#39;une ou des deux produits de digestion dans le cocktail d&#39;anticorps à une concentration de 25 x 10 6 cellules / ml. </li><li> Pour la cytométrie de flux, une tache x 10 6 cellules avec des cocktails d&#39;anticorps contenant contrôles isotypiques (ex. Contrôle-PE IgG + 1 isotype anti-CD90-PECy5 + anti-CD105-APC). Gardez ~ 1 x 10 6 cellules non colorées pour cytomètre de flux set-up. </li><li> Incuber les cellules à un anticorpscocktails à 4 ° C pendant 60 min. </li><li> Laver les cellules 3x dans HBSS et remettre en suspension de 5 à 10 millions de cellules par ml pour cytométrie de flux. </li><li> Effectuer cytométrie de flux et les résultats de note. La pureté de l&#39;isolât de cellule (le pourcentage d&#39;EpCAM positif et CD90 / CD105 cellules négatives) devrait être de 90 à 95% et &lt;1%, respectivement. </li><li> Mettez de côté un nombre approprié de cellules pour tester les critères de contrôle de la qualité ou de libération appropriées tel que déterminé par application finale. NOTE: Cela peut inclure la maladie / dépistage viral, ou autre sécurité ou des critères biologiques. </li><li> Pour les cellules restantes, suivre le protocole de cryoconservation, ou encore placer directement dans la culture (directe à la culture étape 3.6). </li></ol> 2. Cryoconservation de HAECs <ol style="margin-left: 40px;"><li> Déterminer le nombre et la viabilité cellulaire en utilisant la cytométrie de flux de données ou de comptage manuel avec exclusion au bleu trypan. </li><li> Centrifuger à 700 g pendant 5 min. </li><li> Remettre en suspension cellulaires entre 1-10000000 cellules par ml chez l&#39;animal médias de cryoconservation produit-libre. </li><li> Pipeter un volume approprié de cellules dans des flacons de cryoconservation O-anneaux et placer dans un appareil de congélation à vitesse contrôlée dans lequel le refroidissement se produit à environ 1 ° C / min jusqu&#39;à -80 ° C soit atteinte. </li><li> Transfert flacons dans l&#39;azote liquide pour le stockage à long terme. </li></ol> 3. La décongélation et la culture de Cyropreserved HAECs <ol style="margin-left: 40px;"><li> Retirer flacons de cryoconservation de l&#39;azote liquide et placer sur la glace. NOTE: Pour le transfert seulement - procéder rapidement à l&#39;étape suivante. </li><li> Décongeler les flacons de cryoconservation rapidement à 37 ° C jusqu&#39;à ce que seulement un petit morceau (~ 5 mm x 5 mm) de glace reste. NOTE: Il est important que la solution de cellules ne réchauffe pas pour réduire les effets toxiques de DMSO dans les médias de cryoconservation. </li><li> Diluer médias de cryoconservation 10 fois avec (~ 4 ° C) les médias sans sérum froid, goutte à goutte dans une taille appropriée ajoutéetube d ou conteneur. </li><li> Centrifuger à 700 g pendant 5 min à température ambiante. </li><li> Remettre en suspension dans du milieu exempt de sérum, à une concentration approximative de 1 million de cellules par ml. Déterminer le nombre et la viabilité cellulaire en utilisant un comptage automatique ou manuel avec exclusion au bleu trypan. Prévue viabilité post-dégel sera 5-10% de moins que pré-cryoconservation viabilité. </li><li> cellules de la plaque sur la culture traitée articles en plastique cellulaire standard, ou encore utiliser collagène de type I sur les surfaces de culture de manteau. </li><li> Pour collagène de type I revêtement suivre le protocole ci-dessous <ol><li> Préparer acide soluble collagène de type I à partir de placenta humain (15 mg de collagène avec 25 ml d&#39;eau désionisée et 50 ul d&#39;acide acétique glacial). </li><li> Couvrir bécher avec Parafilm et remuez modérément à 37 ° C jusqu&#39;à brins de collagène sont dissous. </li><li> Solution de collagène filtre stérile en utilisant un filtre de 0,22 um. </li><li> Diluer le collagène filtrée stocks 1:10 avec de l&#39;eau déminéralisée. Ce stock est diluée la conce travailntration solution pour les surfaces en matière plastique et la membrane de revêtement. </li><li> Collagène couche plastique, les surfaces en verre et la membrane pendant 2 heures à la température ambiante à 37 ° C, ou O / N à 2-8 ° C. </li><li> Retirer l&#39;excès de liquide de la surface revêtue, et laisser sécher O / N. </li><li> Rincer à l&#39;eau stérile de qualité culture de tissu ou HBSS avant d&#39;introduire cellules et le milieu. </li></ol></li><li> HAECs Plate à une densité de 25 000 cellules / cm2 dans un milieu sans sérum, et permettent l&#39;attachement à se produire pour 72-96 h. </li><li> À la suite de l&#39;attachement, changer de support par aspiration avec précaution et le remplacer par un volume de milieu sans sérum approprié tous les 2-3 jours. </li><li> Passage cellules utilisant le protocole ci-dessous <ol><li> Aspirer milieux de culture de cellules et laver avec 1x volume de HBSS stérile </li><li> Ajouter le volume 1x solution de digestion enzymatique et incuber pendant 5 à 15 min, ou jusqu&#39;à ce que les cellules sont observées détachant de la surface de culture cellulaire. </li><li> Récupérer enzymatique digérer solution contenant des cellules, assurant CElls sont détachées de la surface de culture de cellules en pipetant plusieurs fois la solution de digestion enzymatique sur la surface de culture cellulaire </li><li> Centrifuger à 700 g pendant 5 min. </li><li> Remettre en suspension le culot cellulaire dans un volume de HBSS contenant un inhibiteur approprié à base de soja 1 mg / ml de l&#39;enzyme. Pipeter doucement plusieurs fois avec une pipette de 1 ml pour obtenir une suspension cellulaire unique. </li><li> Ajouter du milieu sans sérum à une concentration approximative de 1 million de cellules / ml. Déterminer le nombre et la viabilité cellulaire en utilisant un comptage automatique ou manuel avec exclusion au bleu trypan. </li><li> HAECs Plate à une densité de 25 000 cellules / cm2 dans un milieu sans sérum, et continuent avec les méthodes de culture standard. </li></ol></li></ol>

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The authors have nothing to disclose.

Il ya plusieurs paramètres critiques qui peuvent avoir un impact important dans le succès de cette méthodologie. Stockage du placenta ou amnios jusqu&#39;à 3 heures avant l&#39;isolement du HAECs peut être souhaitable à des fins logistiques ou d&#39;horaires, il est toutefois recommandé que le tissu est traité dès que possible. Si le tissu doit être stocké, il est recommandé que le stockage est effectué après dissection et de lavage de la membrane amniotique. Amnios peut être stocké dans HBSS contenant d...

Cellules dérivées de sources périnatales, comme le placenta, les membranes placentaires, le cordon ombilical et le liquide amniotique ont attiré l&#39;attention des chercheurs et des cliniciens comme une source potentielle de cellules pour la médecine régénérative 1,2. La raison de cet intérêt est que ces types de cellules possèdent toutes un certain degré de plasticité et immunomodulateur capacité 3, propriétés qui sont fondamentales à leurs applications thérapeutiques potentielles. HAECs sont une population hétérogène qui épithéliale peut être dérivé de terme ou avant terme membrane amniotique 4, fournissant une source potentielle abondante de matériel cellulaire régénérative. Les propriétés qui rendent HAECs faisant appel en tant que thérapie cellulaire comprennent leur multipotence, une faible immunogénicité, et des propriétés anti-inflammatoires. HAECs se sont révélés être très multipotentes in vitro et in vivo, capable de se différencier en lignées mésodermiques (cardiomyocytes, myocytes, ostéocytes, adipocytes), lignées endodermiques (cellules pancréatiques, les cellules hépatiques, les cellules pulmonaires) et lignages ectodermiques (cheveux, peau, cellules neurales et astrocytes) 5-10. Rassurant, malgré leurs HAECs de multipotence ne semblent pas soit de former des tumeurs ou de promouvoir le développement de la tumeur in vivo. En outre, sont également HAECs immunitaire privilégié, exprimant de faibles niveaux de classe II antigènes de leucocytes humains courante (AcVC) 8. Cette propriété sous-tend probablement leur capacité à échapper rejet immunitaire après transplantation allogénique et xénogénique, comme l&#39;a démontré dans des études sur des singes immunitaires compétentes, lapins, cobayes, rats et cochons 11-13. HAECs afficher immunomodulateur puissant et des propriétés immunosuppressives et offrent ainsi des avantages pratiques importants pour les applications cliniques potentielles dans la thérapie de la maladie auto-immune. HAECs sont soupçonnés d&#39;exercer des fonctions immunomodulatrices sur les deux systèmes immunitaires innées et adaptatives. Sure des mécanismes suggérés, est par la sécrétion de facteurs immunomodulateur 14. Les applications actuelles de HAECs dans les modèles de maladies animales précliniques comprennent le traitement des accidents vasculaires cérébraux, la sclérose en plaques, une maladie du foie, le diabète et les maladies pulmonaires chroniques et aiguës. Les chercheurs ont montré intérêt à utiliser pour traiter HAECs post-AVC inflammation du cerveau en raison de leurs propriétés uniques. Il est prouvé que HAECs peut traverser la barrière hémato-encéphalique où ils peuvent se greffer, survivre jusqu&#39;à 60 jours, se différencier en neurones, diminuer l&#39;inflammation et favoriser la régénération des tissus du système nerveux central endommagé dans des modèles animaux de maladies neurologiques 15. HAECs offrent la possibilité de cibler de multiples voies et inverser pathologiques qui contribuent au développement et à la progression de la sclérose en plaques. Par exemple, les résultats provenant d&#39;études animales précliniques suggèrent que HAECs sont fortement immunosuppressive etpeut potentiellement induire une tolérance immunitaire périphérique et inverser les réponses inflammatoires en cours. HAECs se sont également avérés avoir la capacité de se différencier en cellules neurales in vivo et améliorer neurorégénération endogène par la sécrétion d&#39;une vaste gamme de facteurs neurotrophiques 16. Les cellules épithéliales amniotiques humaines et de rongeurs ont déjà prouvé leur efficacité thérapeutique pour le traitement de maladie du foie chez des modèles animaux. Dans un modèle de maladie du foie, la transplantation d&#39;haec conduisent à la prise de greffe de HAECs viables dans le foie, accompagnée d&#39;une réduction de l&#39;apoptose des hépatocytes dommages induction tétrachlorure de carbone, et une diminution de l&#39;inflammation et de la fibrose hépatique 17. HAECs peut être stimulée à des facteurs pancréatiques exprimées notamment insuline et de glucose transporteurs. Plusieurs études ont étudié le potentiel de HAECs pour rétablir les niveaux de glucose dans le sang chez les souris diabétiques 18. Chez les souris recevantHAECs, deux poids et la glycémie niveaux du corps des animaux a diminué à des niveaux normaux après l&#39;injection des cellules. Ces études présentent un dossier solide pour l&#39;utilisation de HAECs pour le traitement du diabète sucré. HAECs ont un rôle avéré dans la prévention et la réparation de lésion pulmonaire aiguë et chronique expérimentale dans les deux adultes et néonatales 19 modèles. Ces études ont révélé que HAECs différencient in vitro dans des cellules epitheliales pulmonaires fonctionnel exprimant de multiples protéines pulmonaires associées, y compris Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), le canal ionique qui est muté chez les patients atteints de fibrose kystique 20. En outre, lorsque HAECs sont livrés à l&#39;adulte lésée et du poumon néonatal, ils exercent leurs effets par l&#39;intermédiaire de la modulation de réparation des cellules immunitaires de l&#39;hôte, ce qui réduit le recrutement des leucocytes pulmonaire, y compris les neutrophiles, les macrophages et les lymphocytes 21-23. Compte tenu de leur abondance,dossier de sécurité, et les applications cliniques éprouvées pour plusieurs maladies, les essais cliniques utilisant HAECs est inévitable. Dans le but d&#39;accélérer l&#39;application des thérapies haec en cliniques-essais, nous avons développé des méthodes pour isoler, la cryoconservation et HAECs de la culture d&#39;une manière appropriée pour les essais cliniques, en utilisant des réactifs de libre-produits d&#39;animaux conformément aux pratiques de fabrication actuelles bonnes (cGMP) des lignes directrices . Nous avons fondé ce protocole un protocole déjà publié que nous utilisions avec succès pour isoler HAECs utilisant des réactifs d&#39;origine animale 6. Nous avons modifié le protocole original pour remplacer les produits d&#39;origine animale avec des réactifs de libre-produits animaux, et l&#39;optimisation ultérieure a été effectuée pour optimiser le rendement de la cellule, la viabilité et la pureté. Notre objectif était de développer un protocole qui serait conforme aux normes réglementaires pour la fabrication de cellules pour des essais cliniques humains.

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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;">Collection Kit</td>
			<td style="width:119px;"></td>
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			<td></td>
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			<td height="21" style="height:21px;width:220px;">Stripping tray</td>
			<td style="width:119px;">Fisher Scientific</td>
			<td style="width:119px;">13-361B</td>
			<td></td>
		</tr>
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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>
			<td></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>
			<td></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></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>
		</tr>
		<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>
		</tr>
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			<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>
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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="21" style="height:21px;">Media and Reagents</td>
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			<td style="width:119px;"></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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TrypZean(animal product&#8211;free recombinant trypsin)</td>
			<td style="width:119px;">Sigma Aldrich</td>
			<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>
		</tr>
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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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		</tr>
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			<td height="21" style="height:21px;">anti-EpCam-PE</td>
			<td style="width:119px;">Miltenyi Biotec</td>
			<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>
			<td></td>
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			<td height="21" style="height:21px;">anti-CD105-APC</td>
			<td style="width:119px;">BD Pharmingen</td>
			<td align="right" style="width:119px;">562408</td>
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		<tr height="21">
			<td height="21" style="height:21px;">APC-isotype control</td>
			<td style="width:119px;">BD Pharmingen</td>
			<td align="right" style="width:119px;">340754</td>
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			<td style="width:119px;">Sigma Aldrich</td>
			<td>C7521</td>
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			<td height="21" style="height:21px;">50mL graduated pipette</td>
			<td style="width:119px;">BD/Falcon</td>
			<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>
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		<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;"></td>
			<td style="width:119px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;"></td>
			<td style="width:119px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<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.

Lorsque cette procédure est correctement suivie, un rendement moyen de 120 millions de HAECs devrait se attendre, avec une gamme typique de 80 à 160.000.000 cellules. De ces rendements, une viabilité moyenne de 83 ± 4% peut être attendue. Le rendement augmenté et la viabilité moyenne légèrement inférieure à la méthode clinique peut être due à l&#39;activité de la trypsine élevé que le produit d&#39;origine animale, et peut-être également en raison de l&#39;absence de protéines sériques. HAECs isolé...

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.

Isolement, cryoconservation et de la culture des droits de cellules épithéliales Amnion pour des applications cliniques

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

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13.8K vues.  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. 

Vidéo: 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.

Différenciation isolement, caractérisation et comparative de l&#39;homme dentaires cellules souches de pulpe Dérivé de dents permanentes à l&#39;aide de deux méthodes différentes

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

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Médecine

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).

Patient Culture cellulaire dérivée et isolement de CD133<sup&gt; +</sup&gt; Putatifs cellules souches du cancer du mélanome

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.

L&#39;isolement, la culture, et de l&#39;imagerie de l&#39;homme causés par des amas de cellules pancréatiques

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.

Adaptation des semi-automatique de circulation des cellules tumorales (CTC) Essais pour les applications cliniques et précliniques de recherche

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.

Isolement et culture Expansion des cellules endothéliales spécifique de la tumeur

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.

Tridimensionnelle Alginate-perle culture de cellules humaines adénome hypophysaire

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.

Isolement et culture de cellules endothéliales primaires de Artères Canine et Veins

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.

Une méthode d’isolement roman de Grade clinique pour les cellules stromales de rein humain périvasculaires

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

Culture and Imaging of Human Nasal Epithelial Organoids

Individualized therapy for cystic fibrosis (CF) patients can be achieved with an in vitro disease model to understand baseline Cystic Fibrosis Transmembrane conductance Regulator (CFTR) activity and restoration from small molecule compounds. Our group recently focused on establishing a well-differentiated organoid model directly derived from primary human nasal epithelial cells (HNE). Histology of sectioned organoids, whole-mount immunofluorescent staining, and imaging (using confocal microscopy, immunofluorescent microscopy, and bright field) are essential to characterize organoids and confirm epithelial differentiation in preparation for functional assays. Furthermore, HNE organoids produce lumens of varying sizes that correlate with CFTR activity, distinguishing between CF and non-CF organoids. In this manuscript, the methodology for culturing HNE organoids are described in detail, focusing on the assessment of differentiation using the imaging modalities, including the measurement of baseline lumen area (a method of CFTR activity measurement in organoids that any laboratory with a microscope can employ) as well as the developed automated approach to a functional assay (which requires more specialized equipment).

A detailed protocol is presented here to describe an in vitro organoid model from human nasal epithelial cells. The protocol has options for measurements requiring standard laboratory equipment, with additional possibilities for specialized equipment and software.

Culture et imagerie des organoïdes épithéliaux nasaux humains

Individualized therapy for cystic fibrosis (CF) patients can be achieved with an in vitro disease model to understand baseline Cystic Fibrosis ...

Isolation and Culture of Human Adipose-Derived Stem Cells With an Innovative Xenogeneic-Free Method for Human Therapy

Considering the increasing impact of stem cell therapy, biosafety concerns have been raised regarding potential contamination or infection transmission due to the introduction of animal-derived products during in vitro manipulation. The xenogeneic components, such as collagenase or fetal bovine serum, commonly used during the cell isolation and expansion steps could be associated with the potential risks of immune reactivity or viral, bacterial, and prion infection in the receiving patients. Following good manufacturing practice guidelines, chemical tissue dissociation should be avoided, while fetal bovine serum (FBS) can be substituted with xenogeneic-free supplements. Moreover, to ensure the safety of cell products, the definition of more reliable and reproducible methods is important. We have developed an innovative, completely xenogeneic-free method for the isolation and in vitro expansion of human adipose-derived stem cells without altering their properties compared to collagenase FBS-cultured standard protocols. Here, human adipose-derived stem cells (hASCs) were isolated from abdominal adipose tissue. The sample was mechanically minced with scissors/a scalpel, micro-dissected and mechanically dispersed in a 10 cm Petri dish, and prepared with scalpel incisions to facilitate the attachment of the tissue fragments and the migration of hASCs. Following the washing steps, hASCs were selected due to their plastic adherence without enzymatic digestion. The isolated hASCs were cultured with medium supplemented with 5% heparin-free human platelet lysate and detached with an animal-free trypsin substitute. Following good manufacturing practice (GMP) directions on the production of cell products intended for human therapy, no antibiotics were used in any culture media.

Xenogeneic (chemical or animal-derived) products introduced in the cell therapy preparation/manipulation steps are associated with an increased risk of immune reactivity and pathogenic transmission in host patients. Here, a complete xenogeneic-free method for the isolation and in vitro expansion of human adipose-derived stem cells is described.

Isolement et culture de cellules souches humaines dérivées du tissu adipeux à l’aide d’une méthode innovante sans xénogène pour la thérapie humaine

Considering the increasing impact of stem cell therapy, biosafety concerns have been raised regarding potential contamination or infection transmission ...