Ce travail a été soutenu par les Instituts nationaux américains de la Santé subvention du NIH / NICHD # HD051998 (au CAM).

1. Collagène talon Préparation <ol><li> Avant de TTE chargement pour 3-D de culture cellulaire, on a besoin pour préparer les perles microporteur Cytodex-3: <ol><li> Peser la quantité appropriée de Cytodex-3 perles nécessaires pour l&#39;expérience. Ce protocole est adapté pour le navire 10ml CRCT, dans lequel 0.05g de perles sont nécessaires. Pour un navire de 50ml CRCT, l&#39;échelle en conséquence. Dans un tube conique de 50 ml autoclavable, mélanger 250 mg Cytodex-3 perles avec 12 mL de tampon phosphate de Dulbecco solution (DPBS). Ce montant est suffisant pour les navires CRCT 5. </li></ol></li><li> Assurer un volume suffisant est présent dans le tube conique, comme le procédé autoclave se traduira par évaporation. Librement bouchon du tube et autoclaver pendant 10 min à 110 ° C. </li><li> Retirez le tube conique à l&#39;achèvement du cycle de l&#39;autoclave et laisser la solution Cytodex-3 billes pour refroidir. </li><li> Après le tube conique a refroidi à température ambiante, utilisez une technique stérile pour porter le volume total à 12,5 ml en utilisant 1X DPBS. </li><li> Cap et de stocker les prêts Cytodex-3 perles à température ambiante. Autoriser les billes préparées à gonfler et refroidir à température ambiante avant utilisation. La préparation ci-dessus de Cytodex-3 perles fournira pendant cinq vaisseaux CRCT 10mL. Nous avons observé que le stockage prolongé des Cytodex-3 perles à la température ambiante se traduira par des performances diminuées, comme le collagène va déstabiliser après environ un mois. Cytodex-3 perles varient en taille de 133 à 215 um. </li></ol> 2. Préparation des médias <ol><li> Préparer 1L de CRCT optimisé GTSF-2 supports (adapté de 22), qui se compose de 40% MEM alpha, plus les suppléments et 60% de L-15 des médias Leibovitz (adapté de 22). D&#39;abord, préparer 400 ml de volume total de MEM alpha complété par: Bicarbonate de sodium 21.2mM Peptonée 0,06% Le fructose 0,7 mm Galactose 1.4mm Glucose 5.6mm HEPES 1% 1% de L-glutamine 0,5% DE SON F 10%BS </li><li> Pour préparer une solution stock de SES, se dissoudre dans 5 ml stériles acidifiés H 2 O préparé par addition d&#39;acide acétique glacial (environ 0,05 ml). Swirl de dissoudre, suivent avec 45 ml d&#39;eau stérile. </li><li> Peser assez de L-15 de poudre Leibovitz pour 600ml de milieu par dissolution dans la culture de tissus de qualité H 2 O. </li><li> Porter le volume total de milieu de culture cellulaire à 1L avec milieu L-15 Leibovitz. Filtre-stériliser et conserver à 4 ° C dans l&#39;obscurité. Ajouter 1% de pénicilline-streptomycine individuellement à chaque aliquote de support utilisé. </li><li> Beaucoup de formulations de milieux autres que GTSF-2 médias ont été testés avec succès dans le CRCT. Les laboratoires doivent décider eux-mêmes moyennes est optimal pour l&#39;expérience étant réalisée. </li></ol> 3. Les cellules et incubation de billes <ol><li> Propager les TTE à ~ 80% de confluence, trypsiniser et compter suivant des pratiques de culture cellulaire. </li><li> Suspendre 1x10 6 TTE dans 4 ml d&#39;eau tièdeed médias. </li><li> Mélanger délicatement préparés Cytodex-3 perles. En utilisant des techniques stériles, retirer 2,5 ml de billes préparées en utilisant une pointe de large, 10 ml pipette sérologique et le transfert à un tube conique de 15 ml inutilisées. Notez qu&#39;il peut y avoir une petite perte de perles qu&#39;ils attachent à la pipette. </li><li> Laisser le Cytodex-3 perles de s&#39;installer sur le fond du tube. Après la sédimentation, l&#39;utilisation d&#39;une pipette pour enlever la couche supérieure de DPBS sans déranger les perles Cytodex-3. </li><li> Mélanger les 6 1x10 TTE préparés dans les médias avec le prêt Cytodex-3 perles. </li><li> Incuber le mélange de cellules-perles à température ambiante pendant 30 min. Périodiquement mélanger doucement. Incuber pendant 30 min supplémentaires à 37 ° C et 5% de CO 2. Périodiquement mélanger doucement. </li></ol> 4. Chargement de la RWV <ol><li> Dans une hotte à flux laminaire, supprimer un CRCT 10mL de l&#39;emballage et le placer dans une solution stérile, plaque de culture à 6 puits pour la stabilité. Reportez-vous à la Figure 1 pour un diagramme étiqueté de la RCCS. </li><li> Retirez le bouchon grands du port. </li><li> Porter le volume total de l&#39;incubation de cellules-perles mélange à 10 ml avec les médias réchauffé. </li><li> Chargez la cellule / perle-mélange dans le CRCT à travers le grand port. Le CRCT devrait être incliné à un angle de 45 ° (jusqu&#39;à grand port) lors du chargement d&#39;aide à l&#39;élimination des bulles d&#39;air éventuelles. Empêcher l&#39;accumulation de pression positive en remplissant la cuve lentement et régulièrement. </li><li> Remplacer le bouchon dans le grand port. </li><li> Retirez les pistons des seringues de 3 ml et les seringues vides sur les petits ports. Ajouter 3 ml de 1-médias pour chaque seringue. Ouvrez lentement les deux valves. Remplacer les pistons des seringues sur les seringues doucement. Ajouter le média d&#39;une seringue jusqu&#39;à ce que toutes les bulles sont retirés de la chambre à l&#39;intérieur. </li><li> Ramassez le CRCT et tapotez délicatement le côté tout en le tournant en face de vous pour vérifier bulles. S&#39;il ya des bulles, vous devez les faire sortir de la chambre, comme les bulles d&#39;air vont interférer avec la formation of agrégats de cellules et d&#39;introduire des forces de cisaillement. Retirer les bulles en tournant le CRCT jusqu&#39;à ce que les bulles sont sous le petit port. Puis poussez doucement la seringue sur le côté opposé à la force des bulles dans le port et hors de la chambre. </li><li> Fermer un port côté. Appuyez doucement sur le piston de la seringue d&#39;autres d&#39;introduire une petite quantité de pression positive dans la cuve, ce qui empêche la formation de bulles. Fermer le robinet secondes. </li><li> Chargez le CRCT sur le rotor. Démarrer la rotation à 19rpm dans un 37 ° C incubateur à CO 2. </li></ol> 5. Changer les médias <ol><li> Changer les médias chaque jour pendant les trois premiers jours, puis tous les jours par la suite. </li><li> Eteignez le rotor et enlevez le CRCT. Tirez sur le piston pour créer une certaine aspiration, enlever les seringues de chaque petit port et la place du CRCT sur un angle de telle sorte que les billes de s&#39;installer en face du grand port. </li><li> Après les perles ont tous installés, ouvrez l&#39;une des valves petites etpermettre aux médias de s&#39;écouler hors du CRCT et dans un conteneur à déchets. Retirer les 2 / 3 de la presse de cette manière. Soyez sûr de ne pas perturber les perles et de ne pas jeter tout agrégats. </li><li> Fermez le robinet et ouvrir la petite grand port. Ajouter le média de nouveau dans le CRCT à travers le grand port. Remplacer le bouchon dans le grand port. </li><li> Répétez les étapes 4.6 à 4.9. </li><li> Comme l&#39;augmentation de la taille des agrégats, dont vous avez besoin pour augmenter la vitesse de rotation pour les maintenir en suspension, en tout temps, idéalement dans un schéma de circulation petite vitesse optimale. En règle générale, augmenter la vitesse de 0.3 à 0,7 entre les tours après chaque tétée une fois les agrégats commencent à croître visiblement. </li></ol> 6. Collecte Propagé Cellules <ol><li> Retirez le CRCT par le rotor. Retirer les seringues de chaque petit port et la place du CRCT sur un angle de telle sorte que les billes peuvent s&#39;installer en face du grand port. </li><li> Après les perles ont tous installés, ouvrez l&#39;une des valves petites et permettre aux médias de coulerde l&#39;CRCT et dans un conteneur à déchets. Retirer 1 / 3 de la presse de cette manière. </li><li> Fermez le robinet et ouvrir la petite grand port. Agiter doucement la cuve pour disperser les agrégats dans la solution. Videz le mélange liquide dans un tube conique 50ml stériles. Autoriser les agrégats recueillis à s&#39;installer dans le tube conique avant d&#39;utiliser le surnageant de laver à fond du récipient de culture pour optimiser la récupération des agrégats. Les agrégats peuvent être utilisés pour les essais en aval immédiatement, comme la cytométrie de flux, des dosages d&#39;invasion, d&#39;immunofluorescence et d&#39;autres. </li></ol> 7. Les résultats représentatifs Un exemple de EVT-comme des cellules (SGHPL-4 lignée cellulaire trophoblastique) cultivés dans le CRCT sur Cytodex-3 perles est montré dans la figure 2. La lignée cellulaire EVT-like affiche projections s&#39;étendant loin de l&#39;amas principal et attachant aux clusters voisins. Beaucoup de perles sont entièrement recouvertes de cellules multiplication. Une fois sorti du CRCT et PLATed sur une matrice extracellulaire, l&#39;EVT-like 3-D des cellules cultivées envahissent agressivement et / ou migrer (figure 3). RT-PCR données confirment l&#39;augmentation de l&#39;expression de MMP vu en 3-D par opposition aux agrégats traditionnels monocouches de culture cellulaire (figure 4). Fait intéressant, les gènes ne pas associée à l&#39;invasion sont également surexprimés dans le CRCT (tableau 1) et peut aider à délimiter des domaines tels que les interactions immunitaires avec l&#39;invasion des cellules trophoblastes. <img alt="Figure 1" src="/files/ftp_upload/3367/3367fig1.jpg" /> Figure 1. Portrait caricatural du système de culture cellulaire de rotation (CRCT). <img alt="Figure 2" src="/files/ftp_upload/3367/3367fig2.jpg" /> Figure 2. Micrographie par contraste de phase d&#39;un agrégat représentant après une croissance de 5 jours dans un CRCT. Les flèches indiquent les cellules envahissant et * indique les perles microporteur Cytodex-3. <imgalt = &quot;Figure 3&quot; src = &quot;/ files/ftp_upload/3367/3367fig3.jpg&quot; /&gt; Figure 3. Agrégats CRCT Grown ont été incorporés dans des gels de fibrine. Invasion par le gel de fibrine a été observée dès le (A) 24 heures après la fibrine intégration et (B) a continué pendant 48 heures. Les flèches indiquent les cellules envahissant et * indique les perles Cytodex-3 microporteur (Adapté, avec permission, de Réf. 7). <img alt="Figure 4" src="/files/ftp_upload/3367/3367fig4.jpg" /> Figure 4. Comparative zymogramme gélatine de monocouche et CRCT propagé SGHPL-4 EVT-comme les cellules. Pro-formes et active de la MMP-2 et MMP-9 ont été sécrétés par des cellules CRCT le trophoblaste grandi (Adapté, avec permission, de Réf. 7). <img alt="Tableau 1" src="/files/ftp_upload/3367/3367table1.jpg" /> Tableau 1. Sommaire des résultats de puces à ADN SGHPL-4 des cellules cultivées dans un CRCT (Adapté, avec permission, de Réf. 7). <ol class="lalpha"><liL&#39;induction&gt; Replier les agrégats en 3-D a été calculée comme suit: normalisé en 3-D du gène de valeur / valeur normalisée du gène monlayer. </li><li> Indique les gènes qui ont été en dessous de la limite de détection dans les monocouches. </li></ol>

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Nous n&#39;avons rien à révéler.

La technique de la culture présentées ici fournit aux enquêteurs hautement invasive EVT-comme les cellules. Il a maintenant été reconnu que la perte de la différenciation se produit dans les monocouches due à l&#39;inhibition des réponses cellulaires aux signaux chimiques et moléculaires dans les trois dimensions (surface des cellules apicales, basales et latérales) 10,13. Cette technique reflète les caractéristiques noté in utero sur l&#39;invasion des cellules TEV. Comme la procédure ...

<table border="1"><tbody><tr><td> Nom du réactif </td><td> Société </td><td> Numéro de catalogue </td><td> Commentaires </td></tr><tr><td> Cytodex perles microporteur </td><td> Sigma-Aldrich </td><td> C3275 </td><td></td></tr><tr><td> Rotation système de culture cellulaire (CRCT) </td><td> Synthecon </td><td> CRCT-D </td><td> Comprend le rotor d&#39;alimentation de base, quatre unités jetables CRCT </td></tr><tr><td> Unités CRCT jetables </td><td> Synthecon </td><td> Contactez Synthecon </td><td></td></tr><tr><td> Seringue de 3 ml embout Luer-Lock </td><td> BD </td><td> 309585 </td><td></td></tr><tr><td> 10ml à large pointe de pipette sérologique </td><td> BD </td><td> 357504 </td><td></td></tr><tr><td> MEM Alpha </td><td> Invitrogen </td><td> 12561-072 </td><td></td></tr><tr><td> L-15 Leibovitz moyennes, poudre </td><td> Invitrogen </td><td> 41300-039 </td><td></td></tr><tr><td> H 2 O, endotoxines gratuitement </td><td> Fisher </td><td> MT-25-055-CM </td><td></td></tr><tr><td> Bicarbonate de sodium </td><td> Sigma-Aldrich </td><td> S-7795 </td><td></td></tr><tr><td> Peptone </td><td> Fisher Scientific </td><td> BP1420-100 </td><td></td></tr><tr><td> Fructose </td><td> Sigma-Aldrich </td><td> F3510-100 </td><td></td></tr><tr><td> Galactose </td><td> Sigma-Aldrich </td><td> G5388-100 </td><td></td></tr><tr><td> Le glucose </td><td> Sigma-Aldrich </td><td> G7528-250 </td><td></td></tr><tr><td> HEPES </td><td> Invitrogen </td><td> 15630-080 </td><td></td></tr><tr><td> L-glutamine </td><td> Invitrogen </td><td> 25030 </td><td></td></tr><tr><td> Insulin-transferrine-sélénite de sodium (SES) </td><td> Sigma-Aldrich </td><td> I1884 </td><td></td></tr><tr><td> FBS </td><td> Invitrogen </td><td> 10437 </td><td></td></tr><tr><td> Pénicilline-streptomycine </td><td> Invitrogen </td><td> 15140 </td><td></td></tr></tbody></table>

rotating cell culture systems for human cell culture: human trophoblast cells as a model

Le domaine de la recherche trophoblaste humain aide à comprendre l&#39;environnement complexe créé pendant la placentation. En raison de la nature de ces études, l&#39;expérimentation in vivo humaine est impossible. Une combinaison de cultures primaires, des cultures d&#39;explants et des lignées cellulaires trophoblastiques une aide notre compréhension de l&#39;invasion de la paroi utérine 2 et le remodelage des artères utérines 3,4 spirale par les cellules trophoblastiques extravilleux (TTE), qui est nécessaire à l&#39;établissement réussi de la grossesse. Malgré la richesse des connaissances glanées ces modèles, il est admis que dans les modèles de culture cellulaire in vitro utilisant des lignées cellulaires EVT-comme l&#39;affichage altéré les propriétés cellulaires par rapport à leurs homologues de 5,6 vivo. Les cellules cultivées dans le système de culture cellulaire en rotation (CRCT) afficher les propriétés morphologiques, phénotypiques et fonctionnelles des lignées cellulaires EVT-like qui imitent plus étroitement de différenciation dans uTTE tero, avec une expression accrue des gènes de l&#39;invasion de médiation (par exemple, les métalloprotéases matricielles (MMP)) et 7,8,9 de différenciation du trophoblaste. L&#39;Hôpital Saint-Georges de cellules placentaires Ligne-4 ​​(SGHPL-4) (fournie gracieusement par le Dr Guy Whitley et la Dre Judith Cartwright) est une lignée cellulaire EVT-like qui a été utilisé pour les essais dans le CRCT. La conception du récipient de culture CRCT est basé sur le principe que les organes et tissus de fonction dans une des trois dimensions (3-D) environnement. En raison des conditions de culture dynamique dans le navire, y compris des conditions de cisaillement physiologiquement pertinents, les cellules cultivées en trois dimensions forment des agrégats basés sur les affinités naturelles cellulaire et se différencier en tissus organotypiques-like assemblées 10,11,12. Le maintien d&#39;une orbite de liquide fournit une faible cisaillement, faible turbulence environnement similaire aux conditions rencontrées in vivo. Sédimentation des cellules cultivées est contré par l&#39;ajustement de la rotationvitesse de la CRCT pour assurer une constante chute libre des cellules. Les échanges gazeux se produit à travers une membrane hydrophobe perméable située à l&#39;arrière du bioréacteur. Comme leurs parents tissus in vivo, CRCT cellules cultivées sont capables de répondre aux gradients chimiques et moléculaires en trois dimensions (c&#39;est à dire à leurs surfaces apicale, basale et latérale), parce qu&#39;ils sont cultivés sur la surface de billes de microsupport poreux. Lorsqu&#39;il est cultivé comme deux dimensions monocouches sur des surfaces imperméables comme le plastique, les cellules sont privés de cette communication importante à leur surface basale. Par conséquent, les contraintes spatiales imposées par l&#39;environnement affectent profondément la manière dont le sens et les signaux des cellules du microenvironnement décoder environnantes, ce qui implique un rôle important pour le milieu en 3-D 13. Nous avons utilisé le CRCT à l&#39;ingénieur biologiquement significative modèles 3-D des différents tissus épithéliaux humains 7,14,15,16. En effet, de nombreux rapports précédents ont demonstrated que les cellules cultivées dans le CRCT peut supposer phénotypes physiologiquement pertinents qui n&#39;ont pas été possibles avec d&#39;autres modèles 10,17-21. En résumé, la culture dans le CRCT représente un moyen facile, reproductibles, à haut débit plateforme qui fournit un grand nombre de cellules différenciées qui se prêtent à une variété de manipulations expérimentales. Dans le protocole suivant, en utilisant TTE comme un exemple, nous décrivent clairement les étapes nécessaires à la culture de cellules en trois dimensions adhérente à la CRCT.

Watch this Scientific Journal Video about Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model at JoVE.com

Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model

Traditionnel, en deux dimensions techniques de culture cellulaire entraînent souvent des caractéristiques modifiées par rapport aux marqueurs de différenciation, de cytokines et de facteurs de croissance. Culture cellulaire en trois dimensions dans le système de culture cellulaire en rotation (CRCT) rétablit l&#39;expression de plusieurs de ces facteurs comme montré ici avec une lignée de cellules trophoblastiques extravilleux.

Rotation systèmes de culture cellulaire de la culture de cellules humaines: les cellules du trophoblaste humain comme modèle

The field of human trophoblast research aids in  understanding the complex environment established during placentation. Due to the  nature of these ...

rotating-cell-culture-systems-for-human-cell-culture-human

Research

JoVE Journal

Bioengineering

16.4K vues.  Tulane University Medical School. Traditionnel, en deux dimensions techniques de culture cellulaire entraînent souvent des caractéristiques modifiées par rapport aux marqueurs de différenciation, de cytokines et de facteurs de croissance. Culture cellulaire en trois dimensions dans le système de culture cellulaire en rotation (CRCT) rétablit l'expression de plusieurs de ces facteurs comme montré ici avec une lignée de cellules trophoblastiques extravilleux.

Vidéo: Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model

Microfabricated Platforms for Mechanically Dynamic Cell Culture

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or fundamental cell biology studies is limited by current bioreactor technologies, which cannot simultaneously apply a variety of mechanical stimuli to cultured cells. In order to address this issue, we have developed a series of microfabricated platforms designed to screen for the effects of mechanical stimuli in a high-throughput format. In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and further demonstrate how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

Plates-formes pour la culture cellulaire microfabriqués mécanique dynamique

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or ...

Recherche

Bio-ingénierie

Shape Memory Polymers for Active Cell Culture

Shape memory polymers (SMPs) are a class of "smart" materials that have the ability to change from a fixed, temporary shape to a pre-determined permanent shape upon the application of a stimulus such as heat1-5. In a typical shape memory cycle, the SMP is first deformed at an elevated temperature that is higher than its transition temperature, Ttrans [either the melting temperature (Tm) or the glass transition temperature (Tg)]. The deformation is elastic in nature and mainly leads to a reduction in conformational entropy of the constituent network chains (following the rubber elasticity theory). The deformed SMP is then cooled to a temperature below its Ttrans while maintaining the external strain or stress constant. During cooling, the material transitions to a more rigid state (semi-crystalline or glassy), which kinetically traps or "freezes" the material in this low-entropy state leading to macroscopic shape fixing. Shape recovery is triggered by continuously heating the material through Ttrans under a stress-free (unconstrained) condition. By allowing the network chains (with regained mobility) to relax to their thermodynamically favored, maximal-entropy state, the material changes from the temporary shape to the permanent shape.
Cells are capable of surveying the mechanical properties of their surrounding environment6. The mechanisms through which mechanical interactions between cells and their physical environment control cell behavior are areas of active research. Substrates of defined topography have emerged as powerful tools in the investigation of these mechanisms. Mesoscale, microscale, and nanoscale patterns of substrate topography have been shown to direct cell alignment, cell adhesion, and cell traction forces7-14. These findings have underscored the potential for substrate topography to control and assay the mechanical interactions between cells and their physical environment during cell culture, but the substrates used to date have generally been passive and could not be programmed to change significantly during culture. This physical stasis has limited the potential of topographic substrates to control cells in culture.
Here, active cell culture (ACC) SMP substrates are introduced that employ surface shape memory to provide programmed control of substrate topography and deformation. These substrates demonstrate the ability to transition from a temporary grooved topography to a second, nearly flat memorized topography. This change in topography can be used to control cell behavior under standard cell culture conditions.

A method for developing cell culture substrates with the ability to change topography during culture is described. The method makes use of smart materials known as shape memory polymers that have the ability to memorize a permanent shape. This concept is adaptable to a wide range of materials and applications.

Polymères à mémoire de forme pour la Culture cellule active

Shape memory polymers (SMPs) are a class of "smart" materials that have the ability to change from a fixed, temporary shape to a pre-determined permanent ...

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells (hESC) to Different Lineages

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture indefinitely and differentiated to any cell types in the body. Various types of biomaterials have also been used in stem cell cultures to provide a microenvironment mimicking the stem cell niche1-3. The latter is important for promoting cell-to-cell interaction, cell proliferation, and differentiation into specific lineages as well as tissue organization by providing a three-dimensional (3D) environment4 such as encapsulation. The principle of cell encapsulation involves entrapment of living cells within the confines of semi-permeable membranes in 3D cultures2. These membranes allow for the exchange of nutrients, oxygen and stimuli across the membranes, whereas antibodies and immune cells from the host that are larger than the capsule pore size are excluded5. Here, we present an approach to culture and differentiate hESC DA neurons in a 3D microenvironment using alginate microcapsules. We have modified the culture conditions2 to enhance the viability of encapsulated hESC. We have previously shown that the addition of p160-Rho-associated coiled-coil kinase (ROCK) inhibitor, Y-27632 and human fetal fibroblast-conditioned serum replacement medium (hFF-CM) to the 3D platform significantly enhanced the viability of encapsulated hESC in which the cells expressed definitive endoderm marker genes1. We have now used this 3D platform for the propagation of hESC and efficient differentiation to DA neurons. Protein and gene expression analyses after the final stage of DA neuronal differentiation showed an increased expression of tyrosine hydroxylase (TH), a marker for DA neurons, &gt;100 folds after 2 weeks. We hypothesized that our 3D platform using alginate microcapsules may be useful to study the proliferation and directed differentiation of hESC to various lineages. This 3D system also allows the separation of feeder cells from hESC during the process of differentiation and also has potential for immune-isolation during transplantation in the future.

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells hESC to Different Lineages

We have optimized a microencapsulation technique as an effective 3D platform for propagation and differentiation of embryonic stem cells to endoderm and dopaminergic (DA) neurons. It also provides an opportunity for immune-isolation of cells from the host during transplantation. This platform can be adapted for other cell types.

Microcapsules d&#39;alginate comme une plate-forme 3D pour la Propagation et différenciation des cellules souches embryonnaires humaines (CSEh) à différentes lignées

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture ...

Three-dimensional Cell Culture Model for Measuring the Effects of Interstitial Fluid Flow on Tumor Cell Invasion

The growth and progression of most solid tumors depend on the initial transformation of the cancer cells and their response to stroma-associated signaling in the tumor microenvironment 1. Previously, research on the tumor microenvironment has focused primarily on tumor-stromal interactions 1-2. However, the tumor microenvironment also includes a variety of biophysical forces, whose effects remain poorly understood. These forces are biomechanical consequences of tumor growth that lead to changes in gene expression, cell division, differentiation and invasion3. Matrix density 4, stiffness 5-6, and structure 6-7, interstitial fluid pressure 8, and interstitial fluid flow 8 are all altered during cancer progression.
Interstitial fluid flow in particular is higher in tumors compared to normal tissues 8-10. The estimated interstitial fluid flow velocities were measured and found to be in the range of 0.1-3 &mu;m s-1, depending on tumor size and differentiation 9, 11. This is due to elevated interstitial fluid pressure caused by tumor-induced angiogenesis and increased vascular permeability 12. Interstitial fluid flow has been shown to increase invasion of cancer cells 13-14, vascular fibroblasts and smooth muscle cells 15. This invasion may be due to autologous chemotactic gradients created around cells in 3-D 16 or increased matrix metalloproteinase (MMP) expression 15, chemokine secretion and cell adhesion molecule expression 17. However, the mechanism by which cells sense fluid flow is not well understood. In addition to altering tumor cell behavior, interstitial fluid flow modulates the activity of other cells in the tumor microenvironment. It is associated with (a) driving differentiation of fibroblasts into tumor-promoting myofibroblasts 18, (b) transporting of antigens and other soluble factors to lymph nodes 19, and (c) modulating lymphatic endothelial cell morphogenesis 20.
The technique presented here imposes interstitial fluid flow on cells in vitro and quantifies its effects on invasion (Figure 1). This method has been published in multiple studies to measure the effects of fluid flow on stromal and cancer cell invasion 13-15, 17. By changing the matrix composition, cell type, and cell concentration, this method can be applied to other diseases and physiological systems to study the effects of interstitial flow on cellular processes such as invasion, differentiation, proliferation, and gene expression.

Interstitial fluid flow is elevated in solid tumors and can modulate tumor cell invasion. Here we describe a technique to apply interstitial fluid flow to cells embedded in a matrix and then measure its effects on cell invasion. This technique can be easily adapted to study other systems.

Tridimensionnelle modèle de culture cellulaire pour mesurer les effets de l&#39;écoulement des fluides interstitiels sur l&#39;invasion tumorale

The growth and progression of most solid tumors depend on the initial transformation of the cancer cells and their response to stroma-associated signaling ...

Cell Co-culture Patterning Using Aqueous Two-phase Systems

Cell patterning technologies that are fast, easy to use and affordable will be required for the future development of high throughput cell assays, platforms for studying cell-cell interactions and tissue engineered systems. This detailed protocol describes a method for generating co-cultures of cells using biocompatible solutions of dextran (DEX) and polyethylene glycol (PEG) that phase-separate when combined above threshold concentrations. Cells can be patterned in a variety of configurations using this method. Cell exclusion patterning can be performed by printing droplets of DEX on a substrate and covering them with a solution of PEG containing cells. The interfacial tension formed between the two polymer solutions causes cells to fall around the outside of the DEX droplet and form a circular clearing that can be used for migration assays. Cell islands can be patterned by dispensing a cell-rich DEX phase into a PEG solution or by covering the DEX droplet with a solution of PEG. Co-cultures can be formed directly by combining cell exclusion with DEX island patterning. These methods are compatible with a variety of liquid handling approaches, including manual micropipetting, and can be used with virtually any adherent cell type.

Aqueous two-phase systems were used to simultaneously pattern multiple populations of cells. This fast and easy method for cell patterning takes advantage of the phase separation of aqueous solutions of dextran and polyethylene glycol and the interfacial tension that exists between the two polymer solutions.

La co-culture de cellules utilisant une solution aqueuse Modélisation des systèmes biphasiques

Cell patterning  technologies that are fast, easy to use and affordable will be required for the  future development of high throughput cell assays, ...

In vitro Cell Culture Model for Toxic Inhaled Chemical Testing

Cell cultures are indispensable to develop and study efficacy of therapeutic agents, prior to their use in animal models. We have the unique ability to model well differentiated human airway epithelium and heart muscle cells. This could be an invaluable tool to study the deleterious effects of toxic inhaled chemicals, such as chlorine, that can normally interact with the cell surfaces, and form various byproducts upon reacting with water, and limiting their effects in submerged cultures. Our model using well differentiated human airway epithelial cell cultures at air-liqiuid interface circumvents this limitation as well as provides an opportunity to evaluate critical mechanisms of toxicity of potential poisonous inhaled chemicals. We describe enhanced loss of membrane integrity, caspase release and death upon toxic inhaled chemical such as chlorine exposure. In this article, we propose methods to model chlorine exposure in mammalian heart and airway epithelial cells in culture and simple tests to evaluate its effect on these cell types.

In vitro Cell Culture Model for Toxic Inhaled Chemical Testing

This protocol is designed to demonstrate exposure method of cell cultures to inhaled toxic chemicals. Exposure of differentiated air-liquid&#160;interface (ALI) cultures of airway epithelial cells provides a unique model of airway exposure to toxic gases such as chlorine. In this manuscript we describe effect of chlorine exposure on air-liquid&#160;interface cultures of epithelial cells and submerged culture of cardiomyocytes. In vitro exposure systems allow important mechanistic studies to evaluate pathways that could then be utilized to develop novel therapeutic agents.

Cellulaire in vitro modèle de culture pour le contrôle des produits chimiques toxiques inhalés

Cell cultures are indispensable to develop and study efficacy of therapeutic agents, prior to their use in animal models. We have the unique ability to ...

Ultrathin Porated Elastic Hydrogels As a Biomimetic Basement Membrane for Dual Cell Culture

The basement membrane is a critical component of cellular bilayers that can vary in stiffness, composition, architecture, and porosity. In vitro studies of endothelial-epithelial bilayers have traditionally relied on permeable support models that enable bilayer culture, but permeable supports are limited in their ability to replicate the diversity of human basement membranes. In contrast, hydrogel models that require chemical synthesis are highly tunable and allow for modifications of both the material stiffness and the biochemical composition via incorporation of biomimetic peptides or proteins. However, traditional hydrogel models are limited in functionality because they lack pores for cell-cell contacts and functional in vitro migration studies. Additionally, due to the thickness of traditional hydrogels, incorporation of pores that span the entire thickness of hydrogels has been challenging. In the present study, we use poly-(ethylene-glycol) (PEG) hydrogels and a novel zinc oxide templating method to address the previous shortcomings of biomimetic hydrogels. As a result, we present an ultrathin, basement membrane-like hydrogel that permits the culture of confluent cellular bilayers on a customizable scaffold with variable pore architectures, mechanical properties, and biochemical composition.

Current bilayer culture models do not allow for functional in vitro studies that mimic in vivo microenvironments. Using polyethylene glycol and a zinc oxide templating method, this protocol describes the development of an ultrathin biomimetic basement membrane with tunable stiffness, porosity, and biochemical composition that closely mimics in vivo extracellular matrices.

Hydrogels ultraminces évaluation élastique comme une Membrane de sous-sol biomimétiques pour Dual Culture cellulaire

The basement membrane is a critical component of cellular bilayers that can vary in stiffness, composition, architecture, and porosity. In vitro studies ...

Human Pluripotent Stem Cell Culture on Polyvinyl Alcohol-Co-Itaconic Acid Hydrogels with Varying Stiffness Under Xeno-Free Conditions

The effect of physical cues, such as the stiffness of biomaterials on the proliferation and differentiation of stem cells, has been investigated by several researchers. However, most of these investigators have used polyacrylamide hydrogels for stem cell culture in their studies. Therefore, their results are controversial because those results might originate from the specific characteristics of the polyacrylamide and not from the physical cue (stiffness) of the biomaterials. Here, we describe a protocol for preparing hydrogels, which are not based on polyacrylamide, where various stem, cells including human embryonic stem (ES) cells and human induced pluripotent stem (iPS) cells, can be cultured. Hydrogels with varying stiffness were prepared from bioinert polyvinyl alcohol-co-itaconic acid (P-IA), with stiffness controlled by crosslinking degree by changing crosslinking time. The P-IA hydrogels grafted with and without oligopeptides derived from extracellular matrix were investigated as a future platform for stem cell culture and differentiation. The culture and passage of amniotic fluid stem cells, adipose-derived stem cells, human ES cells, and human iPS cells is described in detail here. The oligopeptide P-IA hydrogels showed superior performances, which were induced by their stiffness properties. This protocol reports the synthesis of the biomaterial, their surface manipulation, along with controlling the stiffness properties and finally, their impact on stem cell fate using xeno-free culture conditions. Based on recent studies, such modified substrates can act as future platforms to support and direct the fate of various stem cells line to different linkages; and further, regenerate and restore the functions of the lost organ or tissue.

A protocol is presented to prepare polyvinyl alcohol-co-itaconic acid hydrogels with varying stiffness, which were grafted with and without oligopeptides, to investigate the effect of the stiffness of biomaterials on the differentiation and proliferation of stem cells. The stiffness of the hydrogels was controlled by the crosslinking time.

Culture de cellules souches pluripotentes humaines sur Polyvinyl alcool-Co-itaconique acides Hydrogels avec raideur variable dans des Conditions sans Xeno

The effect of physical cues, such as the stiffness of biomaterials on the proliferation and differentiation of stem cells, has been investigated by ...

A Human 3D Extracellular Matrix-Adipocyte Culture Model for Studying Matrix-Cell Metabolic Crosstalk

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of cellular function. The ECM plays a particularly important role in adipose tissue function in obesity, and alterations in adipose tissue ECM deposition and composition are associated with metabolic disease in mice and humans. Tractable in vitro models that permit dissection of the roles of the ECM and cells in contributing to global tissue phenotype are sparse. We describe a novel 3D in vitro model of human ECM-adipocyte culture that permits study of the specific roles of the ECM and adipocytes in regulating adipose tissue metabolic phenotype. Human adipose tissue is decellularized to isolate ECM, which is subsequently repopulated with preadipocytes that are then differentiated within the ECM into mature adipocytes. This method creates ECM-adipocyte constructs that are metabolically active and retain characteristics of the tissues and patients from which they are derived. We have used this system to demonstrate disease-specific ECM-adipocyte crosstalk in human adipose tissue. This culture model provides a tool for dissecting the roles of the ECM and adipocytes in contributing to global adipose tissue metabolic phenotype and permits study of the role of the ECM in regulating adipose tissue homeostasis.

We describe a 3D human extracellular matrix-adipocyte in vitro culture system that permits dissection of the roles of the matrix and adipocytes in contributing to adipose tissue metabolic phenotype.

Un modèle de culture matrix-adipocyte extracellulaire humaine 3D pour étudier matrix-Cell Metabolic Crosstalk

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of ...

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically carried out in low throughput. Rapid and ongoing expansion of induced pluripotent stem cell (iPSC) technology presents a new standard in cardiovascular research and alternate methods are now necessary to increase throughput of electrophysiological data at a single cell level. VF2.1Cl is a recently derived voltage sensitive dye which provides a rapid single channel, high magnitude response to fluctuations in membrane potential. It possesses kinetics superior to those of other existing voltage indicators and makes available functional data equivalent to that of traditional microelectrode techniques. Here, we demonstrate simplified, non-invasive action potential characterization in externally paced human iPSC derived cardiomyocytes using a modular and highly affordable photometry system.

Here we describe optical acquisition and characterization of action potentials from induced pluripotent stem cell derived cardiomyocytes using a high-speed modular photometry system.

Mesure du potentiel d’action optique unicellulaire dans les cardiomyocytes dérivés de cellules souches pluripotentes induites par l’homme

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically ...