Questo lavoro è stato sostenuto dal National Institutes of Health statunitensi concedere NIH / NICHD # HD051998 (per CAM).

1. Collagene Bead Preparazione <ol><li> Prima di EVTS di carico per 3-D di coltura cellulare, si ha la necessità di preparare il Cytodex-3 perle microcarrier: <ol><li> Pesare la quantità appropriata di Cytodex-3 perline richieste per l&#39;esperimento. Questo protocollo è stato adattato per la nave 10ml RCC, in cui 0.05g di perline sono necessari. Per una nave da 50 ml RCC, scala di conseguenza. In un tubo 50mL autoclavabile conica, miscelare 250 mg Cytodex-3 perle con fosfato 12mL soluzione tamponata Dulbecco (DPBS). Questa quantità è sufficiente per 5 vasi RCC. </li></ol></li><li> Garantire un adeguato volume è presente nel tubo conico, come il processo in autoclave si tradurrà in evaporazione. Liberamente tappo del tubo e autoclave per 10 min a 110 ° C. </li><li> Rimuovere il tubo conico al completamento del ciclo di autoclave e permettere al Cytodex-3 tallone soluzione per il raffreddamento. </li><li> Dopo che il tubo conico è raffreddata a temperatura ambiente, utilizzare una tecnica sterile per portare il volume complessivo a 12.5mL usando 1X DPBS. </li><li> Cap e conservare il preparato Cytodex-3 perline a temperatura ambiente. Lasciare le perline pronti a gonfiarsi e raffreddare a temperatura ambiente prima dell&#39;uso. La preparazione di sopra di Cytodex-3 perle fornirà per cinque vasi da 10 ml RCC. Abbiamo osservato che lo stoccaggio prolungato di Cytodex-3 perline a temperatura ambiente si tradurrà in un peggioramento delle prestazioni, come il collagene destabilizzerà dopo circa un mese. Cytodex-3 perle variano nel formato 133-215 micron. </li></ol> 2. Preparazione dei media <ol><li> Preparare 1L di RCC ottimizzato GTSF-2 media (adattato da 22), che consiste di 40% MEM alfa, oltre a integratori e il 60% L-15 Leibovitz media (adattato da 22). In primo luogo, preparare 400mL nel volume totale di MEM alfa integrato con: Bicarbonato di sodio 21.2mM 0,06% peptone Fruttosio 0,7 mm Galattosio 1,4 mm Glucosio 5,6 mm 1% HEPES 1% di L-glutammina 0,5% ITS 10% FBS </li><li> Per preparare una soluzione madre di ITS, si sciolgono in 5 ml sterili acidificato H 2 O preparato con l&#39;aggiunta di acido acetico glaciale (circa 0,05 ml). Swirl per sciogliere, seguire con 45ml di acqua sterile. </li><li> Pesare abbastanza L-15 Leibovitz polvere per 600ml di media sciogliendo in tessuto-cultura di grado H 2 O. </li><li> Portare il volume totale di terreno di coltura cellulare per 1L con L-15 di media Leibovitz. Filtro-sterilizzare e conservare a 4 ° C al buio. Aggiungere 1% penicillina-streptomicina singolarmente a ciascuna aliquota del supporto utilizzato. </li><li> Molte formulazioni supporti diversi GTSF-2 media sono stati testati con successo nel RCC. I singoli laboratori devono decidere da soli quale mezzo è ottimale per l&#39;esperimento in corso. </li></ol> 3. Cellule e incubazione Bead <ol><li> Propagare la EVTS al ~ 80% confluenza, trypsinize e contare utilizzando pratiche accettate colture cellulari. </li><li> Sospendere 1x10 6 EVTS in 4 ml di caldoed i media. </li><li> Mescolare delicatamente preparato Cytodex-3 perline. Usando tecniche sterili, rimuovere 2,5 ml di perline preparati con una punta larga, 10mL sierologici pipetta e trasferire ad un tubo di 15 ml conica inutilizzato. Si noti che ci può essere una piccola perdita di perle come essi attribuiscono alla pipetta. </li><li> Lasciare che il Cytodex-3 perline a depositarsi sul fondo della provetta. Dopo la sedimentazione, utilizzare una pipetta per rimuovere lo strato superiore della DPBS senza disturbare il Cytodex-3 perline. </li><li> Mescolare la 1x10 6 EVTS preparato in media con il preparato Cytodex-3 perline. </li><li> Incubare le cellule tallone miscela a temperatura ambiente per 30 min. Periodicamente mescolare delicatamente. Incubare per altri 30 minuti a 37 ° C e 5% di CO 2. Periodicamente mescolare delicatamente. </li></ol> 4. Caricamento del RWV <ol><li> In una cappa a flusso laminare, rimuovere un RCC 10 ml dalla confezione e collocarlo in una sterile, 6-ben piastra di coltura per la stabilità. Fare riferimento alla Figura 1 per un diagramma etichetta della RCCS. </li><li> Togliere il tappo di grandi dimensioni dal porto. </li><li> Portare il volume totale del incubando cellule tallone miscela di 10 ml con i media riscaldato. </li><li> Caricare il cellulare / tallone miscela nel RCC attraverso il grande porto. Il RCC dovrebbe essere inclinato a 45 ° (fino grande porto) durante il caricamento per favorire l&#39;eliminazione delle bolle d&#39;aria potenziale. Evitare l&#39;accumulo di pressione positiva compilando la nave lentamente e costantemente. </li><li> Sostituire il fermo nel porto di grandi dimensioni. </li><li> Rimuovere i pistoni dal 3-ml siringhe e le siringhe vuoto sul piccoli porti. Aggiungere 1-3 ml di media per ogni siringa. Aprire lentamente le due valvole. Sostituire i pistoni siringa sul siringhe delicatamente. Aggiungere i media da una siringa fino a quando tutte le bolle vengono rimossi dalla camera interna. </li><li> Sollevare il RCC e picchiettare delicatamente sul lato mentre ruotando di fronte a voi per verificare le bolle. Se ci sono delle bolle, è necessario uscire dalla camera, come bolle d&#39;aria si interferisce con la formazione of aggregati cellulari e introdurre forze di taglio. Eliminare le bolle ruotando il RCC fino a quando le bolle sono sotto il piccolo porto. Quindi spingere delicatamente verso il basso la siringa sul lato opposto per costringere le bolle nella porta e fuori della camera. </li><li> Chiudere una porta laterale. Premere delicatamente il pistone siringa altro di introdurre una piccola quantità di pressione positiva nel vaso, che impedisce la formazione di bolle. Chiudere la valvola secondo. </li><li> Caricare il RCC sul rotore. Avviare la rotazione a 19rpm a 37 ° C CO 2 incubatore. </li></ol> 5. Modifica della media <ol><li> Cambiare il supporto a giorni alterni per i primi tre giorni, poi ogni giorno successivo. </li><li> Spegnere il rotore e rimuovere il RCC. Tirare i pistoni fino a creare qualche aspirazione, rimuovere le siringhe da ogni piccolo porto e posizionare il RCC in un angolo in modo che le perline risolvere opposto del porto grande. </li><li> Dopo le perle hanno risolta, aperta una delle valvole piccole epermettere ai media di flusso fuori del RCC e in un contenitore dei rifiuti. Rimuovere 2 / 3 dei media in questo modo. Essere sicuri di non disturbare le perline e scartare qualsiasi non aggregati. </li><li> Chiudere la valvola di piccole e aprire la porta di grandi dimensioni. Aggiungi supporti indietro nel RCC attraverso il grande porto. Sostituire il fermo nel porto di grandi dimensioni. </li><li> Ripetere i passaggi 4,6-4,9. </li><li> Come gli aggregati aumento delle dimensioni, è necessario aumentare la velocità di rotazione per tenerli in sospensione in ogni momento, idealmente in un pattern circolatorio piccolo a velocità ottimale. In generale, aumentare la velocità tra 0,3-0,7 giri dopo ogni poppata, una volta aggregati iniziano a crescere visibilmente. </li></ol> 6. Raccolta di propagazione delle cellule <ol><li> Rimuovere il RCC dal rotore. Rimuovere le siringhe da ogni piccolo porto e posizionare il RCC in un angolo in modo che le perline può risolvere opposto del porto grande. </li><li> Dopo le perle sono tutti sistemati, aprire una delle piccole valvole e consentire ai media di defluiredel RCC e in un contenitore dei rifiuti. Togliere 1 / 3 della media in questo modo. </li><li> Chiudere la valvola di piccole e aprire la porta di grandi dimensioni. Agitare delicatamente la nave per disperdere gli aggregati riportare in soluzione. Vuoto miscela liquida in una sterile da 50ml tubo conico. Lasciare che il aggregati raccolti a stabilirsi nel tubo conica prima di utilizzare il surnatante per lavare a fondo la nave cultura a massimizzare il recupero degli inerti. Gli aggregati possono essere utilizzati per le analisi immediatamente a valle, come la citometria a flusso, saggi di invasione, immunofluorescenza e altri. </li></ol> 7. Rappresentante Risultati Un esempio di come le cellule-EVT (SGHPL-4 linea cellulare trofoblasto) coltivate nella RCC di Cytodex-3 perline è mostrato in Figura 2. La EVT-come linea cellulare mostra le proiezioni si estende lontano dal cluster principale e allegando ai cluster vicini. Molte delle perle sono completamente coperte con le cellule di propagazione. Una volta tolto dal RCC e plaTed su una matrice extracellulare, la EVT-come le cellule coltivate in 3-D aggressivamente invadere e / o migrare (Figura 3). RT-PCR dati confermano l&#39;aumentata espressione di MMP visto in 3-D aggregati al contrario di monostrati tradizionali colture cellulari (Figura 4). È interessante notare che i geni non sono associati con invasione anche sovraregolati in RCC (Tabella 1) e può aiutare a delineare aree come le interazioni con l&#39;invasione delle cellule immunitarie trophoblasts. <img alt="Figura 1" src="/files/ftp_upload/3367/3367fig1.jpg" /> Figura 1. Cartoon rappresentazione del Cell System Rotating Cultura (CCR). <img alt="Figura 2" src="/files/ftp_upload/3367/3367fig2.jpg" /> Figura 2. Micrografia contrasto di fase di un aggregato rappresentante crescita dopo 5 giorni in un RCC. Le frecce indicano le cellule invadono e * denota la Cytodex-3 perline microcarrier. <imgalt = &quot;Figura 3&quot; src = &quot;/ files/ftp_upload/3367/3367fig3.jpg&quot; /&gt; Figura 3. Aggregati RCC Grown sono stati incorporati in gel di fibrina. Invasione attraverso il gel di fibrina è stato osservato già nel (A) 24 ore dopo la fibrina embedding e (B) ha continuato per 48 ore. Le frecce indicano le cellule invadono e * denota la Cytodex-3 perle microcarrier (Adattato, con il permesso, dal rif. 7). <img alt="Figura 4" src="/files/ftp_upload/3367/3367fig4.jpg" /> Figura 4. Comparativa zymogram gelatina di monostrato e RCC propagato SGHPL EVT-4-come le cellule. Forme e pro-attiva di MMP-2 e MMP-9 sono stati secreti dalle cellule del trofoblasto cresciuto RCC (Adattato, con il permesso, dal rif. 7). <img alt="Tabella 1" src="/files/ftp_upload/3367/3367table1.jpg" /> Tabella 1. Sintesi dei risultati da microarray SGHPL-4 cellule coltivate in RCC (Adattato, con il permesso, dal rif. 7). <ol class="lalpha"><liInduzione&gt; Piegare in 3-D aggregati è stato calcolato come segue: normalizzato 3-D gene valore / valore normalizzato gene monlayer. </li><li> Indica geni che erano al di sotto del limite di rilevamento in monostrati. </li></ol>

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La tecnica di cultura qui presentata fornisce investigatori altamente invasiva EVT-come le cellule. Ora è stato riconosciuto che una perdita di differenziazione avviene in monostrati a causa dell&#39;inibizione della risposta cellulare ad agenti chimici e spunti molecolare in tre dimensioni (superficie delle cellule apicali, basali e laterali), 10,13. Questa tecnica riflette le caratteristiche indicate in utero a invadere le cellule EVT. Poiché la procedura imita convenzionale monostrato tessuto ci...

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti </td></tr><tr><td> Cytodex microcarrier perline </td><td> Sigma-Aldrich </td><td> C3275 </td><td></td></tr><tr><td> Rotazione Cultura Cell System (CCR) </td><td> Synthecon </td><td> RCC-D </td><td> Include rotore di base, alimentatore, 4 unità usa e getta RCC </td></tr><tr><td> Unità RCC monouso </td><td> Synthecon </td><td> Contatta Synthecon </td><td></td></tr><tr><td> 3 ml Luer-Lock siringa </td><td> BD </td><td> 309585 </td><td></td></tr><tr><td> 10ml di ampio punta della pipetta sierologica </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 media, in polvere </td><td> Invitrogen </td><td> 41300-039 </td><td></td></tr><tr><td> H 2 O, endotossine libero </td><td> Pescatore </td><td> MT-25-055-CM </td><td></td></tr><tr><td> Bicarbonato di sodio </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> Fruttosio </td><td> Sigma-Aldrich </td><td> F3510-100 </td><td></td></tr><tr><td> Galattosio </td><td> Sigma-Aldrich </td><td> G5388-100 </td><td></td></tr><tr><td> Glucosio </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-Glutammina </td><td> Invitrogen </td><td> 25030 </td><td></td></tr><tr><td> Insulino-transferrina-Sodio Selenite (ITS) </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> Penicillina-Streptomicina </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

Il campo di ricerca trofoblasto umano aiuta a comprendere il complesso ambiente stabilito durante placentazione. A causa della natura di questi studi, umano nella sperimentazione in vivo è impossibile. Una combinazione di colture primarie, culture espianto e linee cellulari trofoblasto 1 supporto la nostra comprensione di invasione della parete uterina 2 e rimodellamento delle arterie spirali uterine 3,4 da parte delle cellule del trofoblasto extravilloso (EVTS), che è richiesto per la costituzione di successo della gravidanza. Nonostante la ricchezza di conoscenze scaturite da tali modelli, si accetta che in modelli di coltura cellulare in vitro utilizzando EVT-come linee cellulari visualizzare alterato proprietà cellulari rispetto alle loro controparti in vivo 5,6. Cellule in coltura delle cellule nel sistema di rotazione cultura (CCR) visualizzare le proprietà morfologiche, fenotipiche e funzionali di EVT-come linee cellulari che più da vicino imitano differenziare in uEVTS Tero, con aumentata espressione di geni che mediano l&#39;invasione (ad esempio metalloproteinasi della matrice (MMP)) e la differenziazione trofoblasto 7,8,9. Il Saint Georges Hospital cellula placentare Linea-4 (SGHPL-4) (gentilmente donato dal Dr. Guy Whitley e il dottor Judith Cartwright) è un EVT-come linea cellulare che è stato utilizzato per la sperimentazione in RCC. Il progetto della nave cultura RCC si basa sul principio che organi e tessuti funzione in una tridimensionale (3-D) dell&#39;ambiente. A causa delle condizioni di coltura dinamica della nave, comprese le condizioni di taglio fisiologicamente rilevanti, cellule cresciute in tre dimensioni, la forma aggregati sulla base di affinità naturale cellulare e di differenziarsi in tessuti come organotipica assemblee 10,11,12. Il mantenimento di un orbita fluido fornisce un basso taglio, a bassa turbolenza ambiente simile alle condizioni presenti in vivo. Sedimentazione delle cellule in coltura, si contrappone regolando la rotazionevelocità del RCC di assicurare una costante caduta libera delle cellule. Scambio dei gas avviene attraverso una membrana idrofobica permeabile trova sul retro del bioreattore. Come i loro genitori tessuti in vivo, RCC-grown cellule sono in grado di rispondere ad agenti chimici e pendenze molecolare in tre dimensioni (vale a dire la loro superficie apicale, basale e laterale) perché sono colto sulla superficie delle perline microcarrier porosa. Quando coltivate come bidimensionali monostrati su superfici impermeabili come plastica, le celle sono privati ​​di questa importante comunicazione a loro superficie basale. Di conseguenza, i vincoli spaziali imposti dall&#39;ambiente profondamente influenzano il modo in senso cellule e decodificare i segnali dal microambiente circostante, ciò implica un ruolo importante per l&#39;ambiente 3-D 13. Abbiamo usato il RCC di ingegnere biologicamente significativi modelli 3-D dei vari tessuti epiteliali 7,14,15,16. Infatti, molte relazioni precedenti demonstrated che le cellule coltivate in RCC può assumere fenotipi fisiologicamente rilevanti che non sono stati possibili con altri modelli 10,17-21. In sintesi, la cultura nel RCC rappresenta un modo semplice, riproducibile, high-throughput piattaforma che offre un gran numero di cellule differenziate che sono riconducibili a una serie di manipolazioni sperimentali. Nel seguente protocollo, utilizzando EVTS come esempio, si descrivono chiaramente i passaggi necessari per tridimensionalmente cellule aderenti cultura nel RCC.

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

Tradizionale, bidimensionale tecniche di coltura cellulare spesso sfociano in caratteristiche alterate rispetto ai marcatori di differenziazione, citochine e fattori di crescita. Tridimensionale di coltura cellulare nel sistema delle cellule di rotazione cultura (CCR) ristabilisce l&#39;espressione di molti di questi fattori, come illustrato qui con una linea di cellule extravilloso trofoblasto.

Rotazione Cultura sistemi cellulari per colture cellulari umane: cellule del trofoblasto umano come modello

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

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Bioengineering

16.4K Views.  Tulane University Medical School. Tradizionale, bidimensionale tecniche di coltura cellulare spesso sfociano in caratteristiche alterate rispetto ai marcatori di differenziazione, citochine e fattori di crescita. Tridimensionale di coltura cellulare nel sistema delle cellule di rotazione cultura (CCR) ristabilisce l'espressione di molti di questi fattori, come illustrato qui con una linea di cellule extravilloso trofoblasto.

Video: 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.

Piattaforme microfabbricazione per coltura cellulare meccanicamente dinamica

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

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Bioingegneria

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.

Shape Memory Polymers per la Cultura cella attiva

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.

Microcapsule di alginato come piattaforma 3D per la propagazione e la differenziazione delle cellule staminali embrionali umane (hESC) per diverse linee

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.

Tridimensionale Cultura modello cellulare per misurare gli effetti del flusso del fluido interstiziale sulla Invasion cellule tumorali

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.

Cella Co-cultura Patterning Uso acquose due sistemi monofase

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.

In vitro delle cellule Cultura modello per Tossico per inalazione 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 ...

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.

Coltura di cellule di idrogeli ultrasottile Innova elastico come una membrana dello scantinato biomimetici per Dual

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.

Cultura di cellule staminali pluripotenti umane il polivinil alcool-Co-itaconico acido idrogel con diverse rigidità nelle circostanze senza 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 modello di coltura a matrice extracellulare extracellulare -adipocyte umano per lo studio del crosstalk metabolico a matrice

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.

Misurazione del potenziale d'azione ottico a singola cellula in cardiomiociti derivati da cellule staminali pluripotenti indotte umane

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