The support from ERC through HealInSynergy (306990) and the FPU program AP2009-3626 are acknowledged.

1. Produzione di dorsali Substrati <ol><li> Produzione di un film piano di PLLA per colata solvente. <ol><li> Lavorare in una cappa aspirante per preparare una soluzione di 2% (w / v) PLLA in cloroformio (2 g PLLA in 100 ml di cloroformio) agitando a temperatura ambiente (RT) fino a completa dissoluzione (3 ore circa). </li><li> Mettere le rondelle su una piastra di Petri di vetro. Nota: Usare rondelle in acciaio inossidabile con un diametro interno di 10,5 mm (leggermente inferiore al diametro del campione ventrale) in modo che la rondella è posto sulla parte superiore del substrato ventrale. Altri materiali quali politetrafluoroetilene (PTFE) o lavabicchieri possono essere usati pure. </li><li> Aggiungere 200 microlitri della soluzione di PLLA nella lavatrice e far evaporare lentamente per 30 minuti a RT. Per consentire lenta evaporazione del solvente, chiudere la piastra di Petri con un foglio di alluminio con alcuni fori. </li><li> Riscaldare i campioni a 120 ° C per 5 minuti in modo da far evaporare tracce di solvente. </li><li> Lasciate che i campioni fresca fannown a RT. </li><li> Una volta che i campioni si sono raffreddati (30 min circa), coprire con 18,2 MW · cm di acqua nella scatola di Petri e incubare per 8 minuti a temperatura ambiente. Nota: Se i campioni non sono completamente raffreddate, possono richiedere fino 18,2 MW · cm di acqua e adottare uno stato gommoso. Inoltre, incubando a 18,2 M · cm di acqua per meno di 8 min può provocare il distacco improprio, e per più di 8 min nel distacco dalla lavatrice. </li><li> Con una lama di rasoio, leva le rondelle di toglierle la piastra di Petri. </li><li> Rimuovere eventuali imperfezioni dal bordo della rondella con le pinzette e lasciare che i campioni di aria secca. </li><li> Conservare i campioni in un essiccatore sotto vuoto (80 mbar) fino al giorno dell&#39;esperimento perché PLLA è degradabile per idrolisi. </li></ol></li><li> Produzione di fibre PLLA da electrospinning. <ol><li> Lavorare in una cappa aspirante per preparare una soluzione di 8% (w / v) PLLA in esafluoroisopropanolo (8 g PLLA in 100 ml esafluoroisopropanolo) da stirring a RT fino a completa dissoluzione (3 ore circa). </li><li> Posizionare il politetrafluoroetilene (PTFE) rondelle su un foglio di alluminio o su un tamburo per la electrospinning per ottenere fibre casuale o allineate rispettivamente. Nota: Utilizzare politetrafluoroetilene (PTFE) rondelle con diametro interno di 10,5 mm (leggermente inferiore al diametro del campione ventrale) in modo che la rondella è posto sulla parte superiore del substrato ventrale. Altri materiali quali rondelle in acciaio inossidabile o di vetro possono essere usati pure. Il tamburo deve ruotare ad una velocità angolare di 160,7 sec -1 (raggio = 7 cm) in modo da ottenere fibre allineate. Una velocità più bassa può non innescare allineamento delle fibre e di velocità comporta fibre rotte. </li><li> Caricare la siringa con la soluzione di polimero e posizionarlo sulla pompa a siringa. </li><li> Electrospin soluzione PLLA utilizzando una portata 0,9 ml / h con una tensione di 30 kV e una distanza di collettore di 12 cm. </li><li> Arrestare il processo electrospinning poppaer 20-30 min, una volta una buona densità di fibra è ottenuta (poiché le cellule devono interagire con più fibre contemporaneamente). </li><li> Riscaldare i campioni a 120 ° C per 5 minuti in modo da far evaporare tracce di solvente. </li><li> Conservare i campioni in un essiccatore sotto vuoto (80 mbar) fino al giorno dell&#39;esperimento perché PLLA è degradabile per idrolisi. </li></ol></li></ol> Culture 2. Sandwich <ol><li> Montare la cultura sandwich volta che le cellule sono rispettate supporto 2D ventrale. <ol><li> Lavorare in una cappa coltura per garantire condizioni di sterilità e sterilizzare tutti i materiali (vetrini, substrati dorsali, pinzette, ecc) in caso di esposizione ai raggi UV per 30 min. </li><li> Cappotto il substrato ventrale e dorsale con fibronectina per dirigere specifiche adesione cellulare di proteine. Per fare questo, incubare i campioni in 20 mg / ml di fibronectina disciolti in Dulbecco Phosphate-Buffered Saline (DPBS) contenente Ca 2+ e Mg 2+ (200 pl / sampio) per 1 ora. Nota: A causa del processo di fabbricazione, substrati dorsali hanno uno designato lati superiore e inferiore perché le fibre film e elettrofilate solvente fusa attaccati ad uno dei lati della rondella. Questo lato è quindi quello rivolto verso le cellule (Figura 3). <ol><li> Dopo il rivestimento proteico, lavare i campioni due volte con DPBS per rimuovere proteine ​​in eccesso. Poi incubare campioni in DPBS fino al loro utilizzo per evitare di ottenere campioni secco poiché questo provoca proteina denaturalizzazione. Utilizzare DPBS contenenti Ca 2+ e Mg 2+ da cationi bivalenti regolano ripiegamento delle proteine. </li></ol></li><li> Cellule seme sul vetro coprioggetto 13. Nota: Siccome la cultura sandwich si basa su substrati 2D, semina cellulare sarà effettuata allo stesso modo come su un campione 2D. Ad esempio, C2C12 mioblasti vengono seminate a 17.500 cellule cm -1 per esperimenti di differenziazione e NIH3T3 fibroblasti vengono seminate a 7000 cells cm -1 per le culture isolate per studiare la morfologia delle cellule e l&#39;adesione. </li><li> Porre i campioni in un incubatore a 37 ° C con 5% di CO 2 e consentono alle cellule di aderire al substrato ventrale per 3 ore. </li><li> Posizionare il substrato seminato ventrale in un nuovo pozzo di una piastra 12 multi-pozzo (dove le rondelle). Nota: Assemblaggio cultura sandwich in un nuovo pozzo è un modo per eliminare le cellule che hanno aderito al fondo del pozzo dopo la semina poiché possono consumare nutrienti e secernere fattori di crescita e rifiuti che influenzano le cellule coltivate all&#39;interno sandwich sistema. Questo è anche un must per estrazioni cellulari per lisare solo le cellule coltivate all&#39;interno dell&#39;ambiente sandwich. </li><li> Attenzione e con l&#39;aiuto di un paio di pinzette, sovrapporre il substrato dorsale sulle cellule. Riempire con prodotto (1 ml) e incubare a 37 ° C con 5% di CO 2. Nota: Il peso della rondella impedirà il sub dorsalistrare da galleggiante. </li><li> Cambia il mezzo due volte alla settimana, come nelle culture 2D standard. Non spostare il substrato dorsale in quanto ciò potrebbe causare danni alle cellule. </li></ol></li><li> Montare la cultura sandwich appena dopo la semina delle cellule. <ol><li> Lavorare in una cappa coltura per garantire condizioni di sterilità e sterilizzare tutti i materiali (vetrini, substrati dorsali, pinzette, ecc) in caso di esposizione ai raggi UV per 30 min. </li><li> Cappotto il substrato ventrale e dorsale con fibronectina per dirigere specifiche adesione cellulare di proteine. Per fare questo, incubare i campioni in 20 ug / ml di fibronectina disciolti in DPBS contenenti Ca 2+ e Mg 2+ (200 microlitri / campione) per 1 ora. <ol><li> Dopo il rivestimento proteico, lavare i campioni due volte con DPBS per rimuovere proteine ​​in eccesso. Poi incubare campioni in DPBS fino al loro utilizzo per evitare di ottenere campioni secco poiché questo provoca proteina denaturalizzazione. Utilizzare DPBS contenenti Ca 2+ e Mg <sup&gt; 2+ da cationi bivalenti regolano ripiegamento delle proteine. </li></ol></li><li> Cellule seme su vetrini in un pozzetto di una piastra 12 multi-pozzo (dove rondelle adatte). Per fare questo, utilizzare una sospensione cellulare altamente concentrato per evitare perdita di cellule dopo la posa del substrato dorsale. </li><li> Attenzione e con l&#39;aiuto di un paio di pinzette, sovrapporre il substrato dorsale sulle cellule. Riempire con prodotto (1 ml) e incubare a 37 ° C con 5% di CO 2. Nota: Il peso della rondella impedirà substrato dorsale dal galleggiante. </li><li> Cambia il mezzo due volte alla settimana, come nelle culture 2D standard. Nota: non spostare il substrato dorsale poiché ciò potrebbe causare danni alle cellule. </li></ol></li></ol> 3. Analisi Nota: le culture a sandwich si basano su substrati 2D, e quindi può essere comunemente analizzati con procedure già sviluppate per le culture 2D standard. Ad esempio, poiché PLLA è trasparente e cells sono costretti a spostarsi all&#39;interno del piano xy, microscopia avviene come su substrati 2D. La migrazione cellulare può essere quindi analizzato come per culture 2D, senza la necessità di tracciare cellule in asse z come per culture 3D, che semplifica l&#39;analisi dell&#39;esperimento e l&#39;immagine. Per studiare il saggio guarigione della ferita da un saggio di zero seguire questo protocollo: <ol><li> La migrazione di studio delle cellule (ferita saggio di guarigione), all&#39;interno della cultura sandwich. <ol><li> Lavorare in una cappa coltura per garantire condizioni di sterilità e sterilizzare tutti i materiali (vetrini, substrati dorsali, pinzette, ecc) in caso di esposizione ai raggi UV per 30 min. </li><li> Cappotto il substrato ventrale e dorsale con fibronectina per dirigere specifiche adesione cellulare di proteine. Per fare questo, incubare i campioni in 20 mg / ml di fibronectina disciolti in Dulbecco Phosphate-Buffered Saline (DPBS) contenente Ca 2+ e Mg 2+ per 1 ora. <ol><li> Dopo il rivestimento proteico, lavare i campioni due volte con DPBSal fine di rimuovere la proteina in eccesso. Poi incubare campioni in DPBS fino al loro utilizzo per evitare di ottenere campioni secco poiché questo potrebbe causare proteine ​​denaturalizzazione. Utilizzare DPBS contenenti Ca 2+ e Mg 2+ da cationi bivalenti regolano ripiegamento delle proteine. </li></ol></li><li> Cellule seme sui vetrini in una alta densità 13. </li><li> Porre i campioni in un incubatore a 37 ° C con 5% di CO 2 e consentono alle cellule di raggiungere confluenza. </li><li> Utilizzare un puntale a graffiare il monostrato cellulare, al fine di ottenere 2 popolazioni cellulari separati da uno spazio. </li><li> Posizionare il substrato seminato ventrale in un nuovo pozzo adatto per l&#39;acquisizione time-lapse e dove i substrati dorsali in forma (cioè 12 piastra multi-bene). </li><li> Attenzione e con l&#39;aiuto di un paio di pinzette, sovrapporre il substrato dorsale sulle cellule e poi riempire con terreno (1 ml per una piastra 12 multi-pozzo). Nota: Il peso della rondella impedirà tegli dorsale supporto a partire da galleggiante. </li><li> Porre il campione nel microscopio time-lapse (impostato a 37 ° C e 5% di CO 2) e prendere le immagini della chiusura del gap ogni 20 min. </li><li> Analizzare la chiusura del gap utilizzando un software specifico come il MiToBo plugin dal ImageJ. 14 </li></ol></li></ol> Nota: Proteine ​​e estrazione degli acidi nucleici avviene allo stesso modo come su substrati 2D. C&#39;è solo un ulteriore passaggio che consiste nello smontare la coltura a sandwich per aggiungere il tampone di lisi direttamente sulle cellule al fine di aumentare l&#39;efficienza di estrazione. Ad esempio, per l&#39;estrazione di mRNA: <ol start="2"><li> Estratto mRNA da culture sandwich <ol><li> Preparare tutti i buffer dal kit commerciale secondo le istruzioni del produttore. </li><li> Prendere la cultura sandwich fuori dal termostato. </li><li> Rimuovere il terreno di coltura dai campioni. </li><li> Lavare i campioni una volta con DPBS contenente Ca2+ e Mg 2+ (1 ml) e rimuovere tutta la soluzione. </li><li> Con l&#39;aiuto di un paio di pinzette, rimuovere il substrato dorsale e aggiungere la soluzione tampone di lisi (350 microlitri) al substrato ventrale. Sovrapporre nuovo substrato dorsale sul substrato ventrale per lisare le cellule anche aderito al substrato dorsale. </li><li> Rimuovere il substrato dorsale per isolare la soluzione di lisi. </li><li> Seguire le istruzioni del produttore per ottenere il mRNA. </li></ol></li></ol> Nota: immunorilevazione delle proteine ​​può essere eseguita anche su substrati come 2D. Dal momento che le culture sandwich potevano ostacolare la corretta diffusione degli anticorpi e buffer, tempi di incubazione dovrebbero essere aumentati. Inoltre, il sandwich può essere smontato prima di avviare il protocollo di colorazione, ma in quest&#39;ultimo caso alcune cellule rimarrà attaccato al substrato dorsale e alcuni al substrato ventrale. <ol start="3"><li> Proteine ​​Immunodetect all&#39;interno di una cultura sandwich <ol&gt; <li> Lavare il campione una volta con DPBS e fissare con formaldeide al 4% in PBS per 20 minuti a 4 ° C. Nota: substrato dorsale può essere rimosso durante il processo di fissazione in modo da evitare problemi di diffusione. </li><li> Risciacquare con DPBS (1 ml) e quindi permeabilize con 0,5% Triton X-100 in DPBS (1 ml) per 5 minuti a temperatura ambiente. </li><li> Bloccare non specifici siti di legame con 1% di albumina in DPBS (1 ml) per 30 minuti a RT. </li><li> Incubare con l&#39;anticorpo primario (2 mg / ml; 1 ml) in soluzione di bloccaggio per 3 ore a temperatura ambiente. </li><li> Lavare 3 volte per 10 min con DPBS / 0,5% Tween 20 (1 ml). </li><li> Incubare 2 hr con anticorpi secondari (2 mg / ml) e 1 mg / ml DAPI in una soluzione bloccante (1 ml) a temperatura ambiente. </li><li> Lavare 3 volte per 10 minuti in DPBS / 0,5% Tween 20 (1 ml). </li><li> Lavare in DPBS. 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The authors have nothing to disclose.

Al giorno d&#39;oggi, la cultura 3D è un tema importante per l&#39;industria farmaceutica e biotecnologica, nonché la ricerca in biologia cellulare, tra cui il cancro e le cellule staminali. Di conseguenza sono stati sviluppati diversi sistemi di coltura 3D. Purtroppo, le differenze tra i sistemi 3D di solito determinano il comportamento cellulare diverso, ostacolando la comprensione del destino delle cellule. Inoltre, le procedure sperimentali di solito non sono così semplice come per sistemi di coltura 2D. Quindi l...

Tradizionalmente, coltura cellulare è stata effettuata su (2D) substrati bi-dimensionali, anche se la maggior parte dei microambienti cellulari in vivo hanno un (3D) natura tridimensionale. Questo ambiente 2D innaturale provoca cambiamenti nel comportamento delle cellule, come un modo di auto-adattamento ad un mondo piatto, che influenza direttamente il destino della cellula 1,2. Pertanto, i risultati ottenuti su colture cellulari 2D non sono sempre riproducibili in vivo. Ciò ha favorito lo sviluppo di nuovi sistemi di coltura interessati cercano di fornire condizioni più fisiologiche simili per ottenere ulteriori approfondimenti qualsiasi meccanismo biologico 3,4 dimensione-dipendente. Una delle principali differenze tra la cultura 2D e 3D in ambiente vivo è la distribuzione dei recettori cellulari ancorate alla matrice extracellulare (ECM): considerando che il 2D substrati cellule aderiscono ventralmente, la maggior parte delle cellule in vivo sono completamente circondato dal ECM e quindi cell adesione avviene attraverso una distribuzione 3D dei recettori. Questo innesca diverse vie di segnalazione di adesione delle cellule che modulano in tal modo i processi importanti, come la crescita cellulare, la differenziazione cellulare e l&#39;espressione genica. Nel corso degli ultimi decenni, molti diversi sistemi di coltura 3D sono stati stabiliti 5-8, anche se la loro variabilità e complessità ostacolano la loro standardizzazione delle procedure di coltura delle cellule comune. Inoltre sistemi 3D di solito non sono facili da gestire e le attuali procedure sperimentali su supporti 2D non possono essere facilmente stabiliti per le culture 3D. Inoltre, la letteratura a confronto raramente culture 3D con la condizione 2D equivalente o altri sistemi 3D, ostacolando la corretta comprensione del comportamento delle cellule in questi modelli. Una volta che le cellule aventi aderito su un substrato 2D, l&#39;eccitazione dei recettori dorsali - sovrapponendo una pellicola di un nuovo materiale (coltura a sandwich) - può innescare risposte cellulari simili ambienti 3D. La reafiglio di questo è l&#39;attivazione simultanea di entrambi dorsale e ventrale recettori di aderire e diffondere all&#39;interno dell&#39;ambiente panino (Figura 1) 9,10. Di conseguenza, le cellule subiscono cambiamenti rilevanti rispetto alle colture 2D 11,12. Così, il destino della cellula è determinata durante il montaggio a causa della cultura panino, poiché la stimolazione dorsale provoca variazioni percorsi cellulari chiave. Pertanto, il destino cellulare è fortemente determinato dal tempo in cui la coltura a sandwich è assemblato 11. A causa della natura del sistema, una cultura sandwich è uno strumento semplice e versatile che consente lo studio dei parametri differenti nelle interazioni cellula / materiale come rivestimenti chimica, topografia, rigidità e proteine ​​ad entrambi i lati ventrale e dorsale. Ciò offre un maggior grado di flessibilità rispetto ad altri sistemi 3D (Figura 2) a causa dorsale indipendente e combinazione ventrale un&#39;ampia variety delle condizioni della superficie. Inoltre, diverse linee cellulari e tempi diversi per assemblare la coltura a sandwich possono essere studiati, aumentando l&#39;ampio spettro di possibilità. Un protocollo standard della cultura sandwich è di seguito utilizzando poli-L-lattico (PLLA) fibre elettrofilate o film come substrati dorsali, vetrino di vetro come substrato ventrale e fibronectina come rivestimento proteico. Culture sandwich sono stati assemblati subito dopo la semina delle cellule o dopo 3 ore di coltura 2D. Si noti tuttavia che potrebbero essere utilizzati altri sistemi di materiali e proteine; analogamente la cultura sandwich può essere montato a tempi diversi.

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			<td>1,1,1,3,3,3-Hexafluoro-2-propanol (HFIP)</td>
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			<td>Hucoa-Herl&#246;s</td>
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			<td>Human Fibronectin</td>
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			<td>F2006</td>
			<td>Reagent</td>
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			<td>RNeasy Micro Kit</td>
			<td>Qiagen</td>
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			<td>Reagent</td>
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			<td>Inverted microscope</td>
			<td>Leica Microsystems</td>
			<td>DMI 6000</td>
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sandwich-like microenvironments to harness cell/material interactions

Colture cellulari è stata tradizionalmente effettuata su substrati bi-dimensionali (2D) in cui le cellule aderiscono con recettori ventrali alla superficie biomateriale. Tuttavia in vivo, la maggior parte delle cellule sono completamente circondati dalla matrice extracellulare (ECM), risultando in un tridimensionale (3D) distribuzione dei recettori. Questo può innescare differenze nella outside-in vie di segnalazione e quindi nel comportamento delle cellule. Questo articolo dimostra che la stimolazione dei recettori dorsali di cellule già aderito ad un substrato 2D sovrapponendo una pellicola di un nuovo materiale (una coltura a sandwich) innesca modifiche importanti rispetto a culture 2D standard. Inoltre, l&#39;eccitazione contemporanea dei recettori ventrale e dorsale sposta comportamento delle cellule più vicina a quella che si trova in ambienti 3D. Inoltre, a causa della natura del sistema, una cultura sandwich è uno strumento versatile che permette lo studio di diversi parametri di cella / materiale interactions, ad esempio, la topografia, la rigidità e diversi rivestimenti di proteine ​​su entrambi i lati ventrale e dorsale. Infine, dal momento che le culture sandwich si basano su substrati 2D, diverse procedure di analisi già sviluppati per le culture 2D standard, può essere utilizzato normalmente, superando le procedure più complesse necessarie per i sistemi 3D.

La stimolazione dei recettori dorsali all&#39;interno della cultura sandwich innesca cambiamenti nella morfologia cellulare, adesione cellulare e vie di segnalazione intracellulare (ad es chinasi di adesione focale, FAK) 10-12. Come esempio, colture di fibroblasti nel sistema sandwich overexpressed la α 5 integrina subunità rispetto al 2D, come osservato per altre culture 3D 15,16. Destino cellulare è strettamente dipendente dal tempo in cui i rec...

Watch this Scientific Journal Video about Sandwich-like Microenvironments to Harness Cell/Material Interactions at JoVE.com

Sandwich-like Microenvironments to Harness Cell/Material Interactions

The following protocol describes the procedure to assemble sandwich-like cultures to be used as an intermediate stage between bi-dimensional (2D) and three-dimensional (3D) cellular environments. The engineered systems can have applications in microscopy, biomechanics, biochemistry and cell biology assays.

Microambienti sandwich di sfruttare Cellulari / Materiale Interazioni

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. ...

sandwich-like-microenvironments-to-harness-cellmaterial-interactions

Research

JoVE Journal

Bioengineering

7.5K Views.  Universitat Politècnica de València. The following protocol describes the procedure to assemble sandwich-like cultures to be used as an intermediate stage between bi-dimensional (2D) and three-dimensional (3D) cellular environments. The engineered systems can have applications in microscopy, biomechanics, biochemistry and cell biology assays.

Video: Sandwich-like Microenvironments to Harness Cell/Material Interactions

Micropatterned Surfaces to Study Hyaluronic Acid Interactions with Cancer Cells

Cancer invasion and progression involves a motile cell phenotype, which is under complex regulation by growth factors/cytokines and extracellular matrix (ECM) components within the tumor microenvironment. Hyaluronic acid (HA) is one stromal ECM component that is known to facilitate tumor progression by enhancing invasion, growth, and angiogenesis1. Interaction of HA with its cell surface receptor CD44 induces signaling events that promote tumor cell growth, survival, and migration, thereby increasing metastatic spread2-3. HA is an anionic, nonsulfated glycosaminoglycan composed of repeating units of D-glucuronic acid and D-N-acetylglucosamine. Due to the presence of carboxyl and hydroxyl groups on repeating disaccharide units, native HA is largely hydrophilic and amenable to chemical modifications that introduce sulfate groups for photoreative immobilization 4-5. Previous studies involving the immobilizations of HA onto surfaces utilize the bioresistant behavior of HA and its sulfated derivative to control cell adhesion onto surfaces6-7. In these studies cell adhesion preferentially occurs on non-HA patterned regions.
To analyze cellular interactions with exogenous HA, we have developed patterned functionalized surfaces that enable a controllable study and high-resolution visualization of cancer cell interactions with HA. We utilized microcontact printing (uCP) to define discrete patterned regions of HA on glass surfaces. A "tethering" approach that applies carbodiimide linking chemistry to immobilize HA was used 8. Glass surfaces were microcontact printed with an aminosilane and reacted with a HA solution of optimized ratios of EDC and NHS to enable HA immobilization in patterned arrays. Incorporating carbodiimide chemistry with mCP enabled the immobilization of HA to defined regions, creating surfaces suitable for in vitro applications. Both colon cancer cells and breast cancer cells implicitly interacted with the HA micropatterned surfaces. Cancer cell adhesion occurred within 24 hours with proliferation by 48 hours. Using HA micropatterned surfaces, we demonstrated that cancer cell adhesion occurs through the HA receptor CD44. Furthermore, HA patterned surfaces were compatible with scanning electron microscopy (SEM) and allowed high resolution imaging of cancer cell adhesive protrusions and spreading on HA patterns to analyze cancer cell motility on exogenous HA.

A novel approach that allows the high-resolution analysis of cancer cell interactions with exogenous hyaluronic acid (HA) is described. Patterned surfaces are fabricated by combining carbodiimide chemistry and microcontact printing.

Superfici Micropatterned per studiare le interazioni acido ialuronico con le cellule tumorali

Cancer invasion and progression involves a motile cell phenotype, which is under complex regulation by growth factors/cytokines and extracellular matrix ...

Ricerca

Bioingegneria

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

Progettazione di un bioreattore per la pressione ciclica Ex Vivo Studio delle valvole cardiache aortica

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Endothelialized Microfluidics for Studying Microvascular Interactions in Hematologic Diseases

Advances in microfabrication techniques have enabled the production of inexpensive and reproducible microfluidic systems for conducting biological and biochemical experiments at the micro- and nanoscales 1,2. In addition, microfluidics have also been specifically used to quantitatively analyze hematologic and microvascular processes, because of their ability to easily control the dynamic fluidic environment and biological conditions3-6. As such, researchers have more recently used microfluidic systems to study blood cell deformability, blood cell aggregation, microvascular blood flow, and blood cell-endothelial cell interactions6-13.However, these microfluidic systems either did not include cultured endothelial cells or were larger than the sizescale relevant to microvascular pathologic processes. A microfluidic platform with cultured endothelial cells that accurately recapitulates the cellular, physical, and hemodynamic environment of the microcirculation is needed to further our understanding of the underlying biophysical pathophysiology of hematologic diseases that involve the microvasculature.
Here, we report a method to create an "endothelialized" in vitro model of the microvasculature, using a simple, single mask microfabrication process in conjunction with standard endothelial cell culture techniques, to study pathologic biophysical microvascular interactions that occur in hematologic disease. This "microvasculature-on-a-chip" provides the researcher with a robust assay that tightly controls biological as well as biophysical conditions and is operated using a standard syringe pump and brightfield/fluorescence microscopy. Parameters such as microcirculatory hemodynamic conditions, endothelial cell type, blood cell type(s) and concentration(s), drug/inhibitory concentration etc., can all be easily controlled. As such, our microsystem provides a method to quantitatively investigate disease processes in which microvascular flow is impaired due to alterations in cell adhesion, aggregation, and deformability, a capability unavailable with existing assays.

A method to culture an endothelial cell monolayer throughout the entire inner 3D surface of a microfluidic device with microvascular-sized channels (&lt;30 &mu;m) is described. This in vitro microvasculature model enables the study of biophysical interactions between blood cells, endothelial cells, and soluble factors in hematologic diseases.

Microfluidica Endothelialized per lo studio delle interazioni microvascolari nelle patologie ematologiche

Advances in  microfabrication techniques have enabled the production of inexpensive and  reproducible microfluidic systems for conducting biological and ...

Correlative Microscopy for 3D Structural Analysis of Dynamic Interactions

Cryo-electron tomography (cryoET) allows 3D visualization of cellular structures at molecular resolution in a close-to-physiological state1. However, direct visualization of individual viral complexes in their host cellular environment with cryoET is challenging2, due to the infrequent and dynamic nature of viral entry, particularly in the case of HIV-1. While time-lapse live-cell imaging has yielded a great deal of information about many aspects of the life cycle of HIV-13-7, the resolution afforded by live-cell microscopy is limited (~ 200 nm). Our work was aimed at developing a correlation method that permits direct visualization of early events of HIV-1 infection by combining live-cell fluorescent light microscopy, cryo-fluorescent microscopy, and cryoET. In this manner, live-cell and cryo-fluorescent signals can be used to accurately guide the sampling in cryoET. Furthermore, structural information obtained from cryoET can be complemented with the dynamic functional data gained through live-cell imaging of fluorescent labeled target.
In this video article, we provide detailed methods and protocols for structural investigation of HIV-1 and host-cell interactions using 3D correlative high-speed live-cell imaging and high-resolution cryoET structural analysis. HeLa cells infected with HIV-1 particles were characterized first by confocal live-cell microscopy, and the region containing the same viral particle was then analyzed by cryo-electron tomography for 3D structural details. The correlation between two sets of imaging data, optical imaging and electron imaging, was achieved using a home-built cryo-fluorescence light microscopy stage. The approach detailed here will be valuable, not only for study of virus-host cell interactions, but also for broader applications in cell biology, such as cell signaling, membrane receptor trafficking, and many other dynamic cellular processes.

We describe a correlative microscopy method that combines high-speed 3D live-cell fluorescent light microscopy and high-resolution cryo-electron tomography. We demonstrate the capability of the correlative method by imaging dynamic, small HIV-1 particles interacting with host HeLa cells.

Microscopia correlativa per l&#39;analisi strutturale 3D di Interazioni Dinamiche

Cryo-electron tomography (cryoET) allows 3D visualization of cellular structures at molecular resolution in a close-to-physiological state1. However, ...

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced birth defects and many later studies afterwards proved the opposite. Today several harmful xenobiotics like nicotine, heroin, methadone or drugs as well as environmental pollutants were described to overcome this barrier. With the growing use of nanotechnology, the placenta is likely to come into contact with novel nanoparticles either accidentally through exposure or intentionally in the case of potential nanomedical applications. Data from animal experiments cannot be extrapolated to humans because the placenta is the most species-specific mammalian organ 1. Therefore, the ex vivo dual recirculating human placental perfusion, developed by Panigel et al. in 1967 2 and continuously modified by Schneider et al. in 1972 3, can serve as an excellent model to study the transfer of xenobiotics or particles.
Here, we focus on the ex vivo dual recirculating human placental perfusion protocol and its further development to acquire reproducible results.
The placentae were obtained after informed consent of the mothers from uncomplicated term pregnancies undergoing caesarean delivery. The fetal and maternal vessels of an intact cotyledon were cannulated and perfused at least for five hours. As a model particle fluorescently labelled polystyrene particles with sizes of 80 and 500 nm in diameter were added to the maternal circuit. The 80 nm particles were able to cross the placental barrier and provide a perfect example for a substance which is transferred across the placenta to the fetus while the 500 nm particles were retained in the placental tissue or maternal circuit. The ex vivo human placental perfusion model is one of few models providing reliable information about the transport behavior of xenobiotics at an important tissue barrier which delivers predictive and clinical relevant data.

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

The ex vivo dual recirculating human placental perfusion model can be used to investigate the transfer of xenobiotics and nanoparticles across the human placenta. In this video protocol we describe the equipment and techniques required for a successful execution of a placenta perfusion.

Determinazione del tasso di trasporto di xenobiotici e nanomateriali attraverso la placenta con il<em&gt; Ex vivo</em&gt; Umano Modello perfusione placentare

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced ...

Microfabrication of Nanoporous Gold Patterns for Cell-material Interaction Studies

Nanostructured materials with feature sizes in tens of nanometers have enhanced the performance of several technologies, including fuel cells, biosensors, biomedical device coatings, and drug delivery tools. Nanoporous gold (np-Au), produced by a nano-scale self-assembly process, is a relatively new material that exhibits large effective surface area, high electrical conductivity, and catalytic activity. These properties have made np-Au an attractive material to scientific community. Most studies on np-Au employ macro-scale specimens and focus on fundamental science of the material and its catalytic and sensor applications. The macro-scale specimens limit np-Au's potential in miniaturized systems, including biomedical devices. In order to address these issues, we initially describe two different methods to micropattern np-Au thin films on rigid substrates. The first method employs manually-produced stencil masks for creating millimeter-scale np-Au patterns, while the second method uses lift-off photolithography to pattern sub-millimeter-scale patterns. As the np-Au thin films are obtained by sputter-deposition process, they are compatible with conventional microfabrication techniques, thereby amenable to facile integration into microsystems. These systems include electrically-addressable biosensor platforms that benefit from high effective surface area, electrical conductivity, and gold-thiol-based surface bioconjugation. We describe cell culture, immunostaining, and image processing techniques to quantify np-Au's interaction with mammalian cells, which is an important performance parameter for some biosensors. We expect that the techniques illustrated here will assist the integration of np-Au in platforms at various length-scales and in numerous applications, including biosensors, energy storage systems, and catalysts.

We report on techniques to micropattern nanoporous gold thin films via stencil printing and photolithography, as well as methods to culture cells on the microfabricated patterns. In addition, we describe image analysis methods to characterize morphology of the material and the cultured cells using scanning electron and fluorescence microscopy techniques.

Microfabrication di Patterns Oro nanoporosi per Cell-materiali Interaction Studies

Nanostructured materials with feature sizes in tens of nanometers have enhanced the performance of several technologies, including fuel cells, biosensors, ...

Membrane-SPINE: A Biochemical Tool to Identify Protein-protein Interactions of Membrane Proteins In Vivo

Membrane proteins are essential for cell viability and are therefore important therapeutic targets1-3. Since they function in complexes4, methods to identify and characterize their interactions are necessary5. To this end, we developed the Membrane Strep-protein interaction experiment, called Membrane-SPINE6. This technique combines in vivo cross-linking using the reversible cross-linker formaldehyde with affinity purification of a Strep-tagged membrane bait protein. During the procedure, cross-linked prey proteins are co-purified with the membrane bait protein and subsequently separated by boiling. Hence, two major tasks can be executed when analyzing protein-protein interactions (PPIs) of membrane proteins using Membrane-SPINE: first, the confirmation of a proposed interaction partner by immunoblotting, and second, the identification of new interaction partners by mass spectrometry analysis. Moreover, even low affinity, transient PPIs are detectable by this technique. Finally, Membrane-SPINE is adaptable to almost any cell type, making it applicable as a powerful screening tool to identify PPIs of membrane proteins.

Membrane-SPINE: A Biochemical Tool to Identify Protein-protein Interactions of Membrane Proteins In Vivo

A biochemical approach is described to identify in vivo protein-protein interactions (PPI) of membrane proteins. The method combines protein cross-linking, affinity purification and mass spectrometry, and is adaptable to almost any cell type or organism. With this approach, even the identification of transient PPIs becomes possible.

Membrana-SPINE: uno strumento biochimici per identificare interazioni proteina-proteina di proteine ​​di membrana<em&gt; In Vivo</em

Membrane proteins are essential for cell viability and are therefore important therapeutic targets1-3. Since they function in complexes4, methods to ...

The Use of a &#946;-lactamase-based Conductimetric Biosensor Assay to Detect Biomolecular Interactions

Biosensors are becoming increasingly important and implemented in various fields such as pathogen detection, molecular diagnosis, environmental monitoring, and food safety control. In this context, we used &#946;-lactamases as efficient reporter enzymes in several protein-protein interaction studies. Furthermore, their ability to accept insertions of peptides or structured proteins/domains strongly encourages the use of these enzymes to generate chimeric proteins. In a recent study, we inserted a single-domain antibody fragment into the Bacillus licheniformis BlaP &#946;-lactamase. These small domains, also called nanobodies, are defined as the antigen-binding domains of single chain antibodies from camelids. Like common double chain antibodies, they show high affinities and specificities for their targets. The resulting chimeric protein exhibited a high affinity against its target while retaining the &#946;-lactamase activity. This suggests that the nanobody and &#946;-lactamase moieties remain functional. In the present work, we report a detailed protocol that combines our hybrid &#946;-lactamase system to the biosensor technology. The specific binding of the nanobody to its target can be detected thanks to a conductimetric measurement of the protons released by the catalytic activity of the enzyme.

The Use of a &amp;#946;-lactamase-based Conductimetric Biosensor Assay to Detect Biomolecular Interactions

In this work, we report a new method to study protein-protein interactions using a conductimetric biosensor based on the hybrid &#946;-lactamase technology. This method relies on release of protons upon hydrolysis of &#946;-lactams.

L'uso di un'analisi di β-lactamase-based conduttimetriche biosensore per rilevare interazioni biomolecolari

Biosensors are becoming increasingly important and implemented in various fields such as pathogen detection, molecular diagnosis, environmental ...

PIP-on-a-chip: A Label-free Study of Protein-phosphoinositide Interactions

Numerous cellular proteins interact with membrane surfaces to affect essential cellular processes. These interactions can be directed towards a specific lipid component within a membrane, as in the case of phosphoinositides (PIPs), to ensure specific subcellular localization and/or activation. PIPs and cellular PIP-binding domains have been studied extensively to better understand their role in cellular physiology. We applied a pH modulation assay on supported lipid bilayers (SLBs) as a tool to study protein-PIP interactions. In these studies, pH sensitive ortho-Sulforhodamine B conjugated phosphatidylethanolamine is used to detect protein-PIP interactions. Upon binding of a protein to a PIP-containing membrane surface, the interfacial potential is modulated (i.e. change in local pH), shifting the protonation state of the probe. A case study of the successful usage of the pH modulation assay is presented by using phospholipase C delta1 Pleckstrin Homology (PLC-&#948;1 PH) domain and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) interaction as an example. The apparent dissociation constant (Kd,app) for this interaction was 0.39 &#177; 0.05 &#181;M, similar to Kd,app values obtained by others. As previously observed, the PLC-&#948;1 PH domain is PI(4,5)P2 specific, shows weaker binding towards phosphatidylinositol 4-phosphate, and no binding to pure phosphatidylcholine SLBs. The PIP-on-a-chip assay is advantageous over traditional PIP-binding assays, including but not limited to low sample volume and no ligand/receptor labeling requirements, the ability to test high- and low-affinity membrane interactions with both small and large molecules, and improved signal to noise ratio. Accordingly, the usage of the PIP-on-a-chip approach will facilitate the elucidation of mechanisms of a wide range of membrane interactions. Furthermore, this method could potentially be used in identifying therapeutics that modulate protein&#39;s capacity to interact with membranes.

Here we present a supported lipid bilayer in the context of a microfluidic platform to study protein-phosphoinositide interactions using a label-free method based on pH modulation.

PIP-on-a-chip: Uno studio privo di etichette delle interazioni proteine-fosfoinositide

Numerous cellular proteins interact with membrane surfaces to affect essential cellular processes. These interactions can be directed towards a specific ...

Fabrication of a Multiplexed Artificial Cellular MicroEnvironment Array

Cellular microenvironments consist of a variety of cues, such as growth factors, extracellular matrices, and intercellular interactions. These cues are well orchestrated and are crucial in regulating cell functions in a living system. Although a number of researchers have attempted to investigate the correlation between environmental factors and desired cellular functions, much remains unknown. This is largely due to the lack of a proper methodology to mimic such environmental cues in vitro, and simultaneously test different environmental cues on cells. Here, we report an integrated platform of microfluidic channels and a nanofiber array, followed by high-content single-cell analysis, to examine stem cell phenotypes altered by distinct environmental factors. To demonstrate the application of this platform, this study focuses on the phenotypes of self-renewing human pluripotent stem cells (hPSCs). Here, we present the preparation procedures for a nanofiber array and the microfluidic structure in the fabrication of a Multiplexed Artificial Cellular MicroEnvironment (MACME) array. Moreover, overall steps of the single-cell profiling, cell staining with multiple fluorescent markers, multiple fluorescence imaging, and statistical analyses, are described.

This article describes the detailed methodology to prepare a Multiplexed Artificial Cellular MicroEnvironment (MACME) array for high-throughput manipulation of physical and chemical cues mimicking in vivo cellular microenvironments and to identify the optimal cellular environment for human pluripotent stem cells (hPSCs) with single-cell profiling.

Fabbricazione di una matrice di multiplex artificiale microambiente cellulare

Cellular microenvironments consist of a variety of cues, such as growth factors, extracellular matrices, and intercellular interactions. These cues are ...