Questo lavoro è stato supportato dal NIH / NHLBI concessione R01 HL082689 a Scott I. Simon e Anthony G. Passerini.

1. Di coltura cellulare e Preparazione del substrato <ol><li> Tagliare supporti circolari da 3 pollici da una x 100 20 piatto di coltura tissutale mm (BD Falcon) usando un tornio. Sterilizzare substrati mediante immersione in etanolo al 70%. Posto in una capsula di Petri e mantello con 4 ml di collagene di tipo I (100 pg / ml) per 1 ora a temperatura ambiente, poi lavare con 4 ml 1 x PBS. </li><li> Sospendere Le cellule endoteliali aortiche (HAEC, passaggio 4-6) a 6.5x10 5 cellule / ml e di sementi mediante l&#39;applicazione di 1 ml direttamente al substrato. Posto a 37 ° C, 5% CO 2 incubatore e permettere alle cellule di aderire al substrato per 1 h. </li><li> Aggiungere 9 ml di crescita endoteliale Medio-2 (EGM-2) integrato con 1x antibiotico-antimicotico soluzione alle cellule. Modificare i media ogni 2 giorni fino a quando le cellule raggiungere il 90% di confluenza. </li><li> Mantenere cellule THP-1 in mezzo RPMI 1640 integrato con 10% FBS e 1x antibiotico-antimicotico soluzione. Cellule Passage quando raggiungono una densità di 10 6 cellule / ml. </li></ol> 2. Cella Shearing protocollo Le cellule sono condizionati in un costume cono-and-piastra di dispositivo a cella Shearing (CSD). Dettagli del dispositivo e specifiche di progetto sono documentati nella Tabella 1 e Figura 4. <ol><li> Preparare il CSD. Sterilizzare l&#39;alloggiamento camera con etanolo e risciacquare con PBS sterile. Riscaldare l&#39;alloggiamento a 37 ° C e livello per assicurare che il cono è perpendicolare al monostrato cellulare. Assemblare il substrato con le cellule dal fondo del contenitore e impostare il cono all&#39;altezza desiderata. </li><li> Utilizzare Leibovitz-15 (L-15) mezzo supplementato con BulletKit Endothelial come mezzo di taglio per mantenere il pH corretto in assenza di CO 2. Per stimolare infiammazione, aggiungere fattore di necrosi tumorale-α (TNF-α, 0,5 ng / ml) al mezzo di taglio e iniettare 6 ml nel dispositivo tramite una siringa collegata ad una porta di ingresso, avendo cura di non introdurre ariabolle nella camera. </li><li> Cellule di taglio ad un elevato sforzo di taglio grandezza costante (HSS, 15 dyne / cm 2) o una sollecitazione di taglio oscillatoria (OSS, 0 ± 5 dyne / cm 2, 1 Hz) per 4 ore. La SS desiderato si ottiene ruotando il cono di sopra delle celle, che è controllato con precisione con un micro-motore a passo, come precedentemente descritto 6 La parete SS in superficie (τ w) è approssimata da.: <img src="/files/ftp_upload/4169/4169eq1.jpg" alt="Equazione 1" /> dove μ è la viscosità del mezzo, ω è la velocità angolare del cono, e α è l&#39;angolo del cono (0,5 °). L&#39;allineamento cono con il substrato viene regolata entro 0,05 ° attraverso il livellamento di precisione e l&#39;altezza gap fissato a 20 ± 10 micron utilizzando un calibro di profondità per ridurre al minimo variazione circonferenziale e radiale in τ w. Il dispositivo è stato convalidato per garantire caratteristiche di flusso laminare. Rimuovere cells dal dispositivo e preparare l&#39;analisi di adesione dei monociti. </li></ol> 3. Fabbricazione di PDMS Microfluidic Camera <ol><li> Ottenere stampo master per numero di canale desiderato microfluidica e l&#39;altezza del canale a seconda dell&#39;applicazione. Le caratteristiche del disegno specifico utilizzato in questo protocollo sono dettagliati nella Tabella 1. Il metodo per creare questi è già ben documentata in letteratura. 1 In breve, la rete di canali è progettato utilizzando software CAD (Autodesk Inventor) e stampato a 5000 dpi su una trasparenza. Fotoresist negativo (SU8) viene filata su un wafer di silicio ad uno spessore di 100 um. La trasparenza è sovrapposta ed esposto alla luce UV. Non polimerizzato photoresist viene rimosso per generare la matrice positiva replica. </li><li> Per rendere la soluzione base di silicone PDMS mix con silicone agente di indurimento a 10:01 in peso in una barca a pesare e mescolare con una pipetta 5 ml sierologico. Fare attenzione a non grattare le paretidel peso della barca per evitare di aggiungere fiocchi di plastica per la soluzione. </li><li> Posizionare il wafer master in un piatto 100x15mm Petri e versare la soluzione PDMS nel piatto. </li><li> Coprire il piatto e metterla in un contenitore vuoto per 15 minuti per rimuovere le bolle. Dopo 15 minuti prendere la piastra di Petri e rimuovere le bolle in eccesso delicatamente soffiando aria sulla superficie. </li><li> Porre la capsula Petri in un forno per 1 ora a 70 ° C. Rimuovere il piatto e tagliare le camere microfluidici utilizzando una lama affilata. </li></ol> 4. Montaggio del dispositivo sul microscopio <ol><li> Accendere sorgente di luce al microscopio invertito e impostare riscaldamento a 37 ° C. Collegare il tubo di una siringa da 10 ml e riempirlo con acqua distillata in modo tale che non ci siano bolle. Posizionare la siringa nella pompa a siringa e portata impostata per indurre una parete di SS 1 dyne / cm 2. La sollecitazione di taglio parete (τ w) generata lungo la mezzeria del canale è approssimata utilizzando lo standardequazione per un parallelo-plate camera di flusso: <img src="/files/ftp_upload/4169/4169eq2.jpg" alt="Equazione 2" /> dove μ è la viscosità del mezzo, Q è la portata volumetrica, h è l&#39;altezza del canale, e w è la larghezza del canale. Nota: poiché i canali sono di larghezza finita effettivo τ w varia con w seconda del canale rapporto di aspetto, e le misurazioni vengono evitati al 25% più vicino alle pareti laterali. 1,7 </li><li> Posizionare PDMS camera in acqua e utilizzare una micropipetta 100 microlitri per rimuovere aria dai singoli canali. Collegare l&#39;alimentatore alla camera a vuoto facendo attenzione che la valvola è disattivata. </li><li> Porre Petri con monostrato HAEC rispetto all&#39;obiettivo. Posizionare la camera di PDMS sulla piastra di Petri e cautela impostato sul monostrato HAEC, assicurando che non si formano bolle d&#39;aria tra il dispositivo e il monostrato. Ruotare la valvola in modo che il vuoto è acceso e la camera è fissato saldamente. </li&gt; <li> Tagliare 19 aghi calibro smorzare tale che ci sia solo 5 mm di sinistra metallo. Riempire il serbatoio di plastica con il tampone e mettere in ingresso PDMS camera. Ripetere per appropriato numero di canali da utilizzare. Collegare il tubo dalle pompe siringa per PDMS uscite canale. </li></ol> 5. Adesione Assay monociti sotto stress Shear <ol><li> Sospensione cellule THP-1 a 2x10 6 cellule / ml in HBSS + Ca 2 + / Mg 2 + + HSA 0,1%. Attivazione cellule THP-1 mediante incubazione con stromale fattore-1 derivato (SDF-1, 50 ug / ml) per 15 min a temperatura ambiente. </li><li> Accendere pompa a siringa per stabilire un flusso, quindi si aggiungono 50 microlitri di sospensione cellulare al serbatoio senza introdurre bolle d&#39;aria. Quando il flusso è completamente sviluppato, iniziare l&#39;acquisizione dei dati. </li><li> Eseguire per 5 min a velocità di flusso del caso, aggiungendo tampone al serbatoio se la soluzione diventa bassa. Continuare tampone scorre nei canali per lavare le cellule non legate nella corrente di flusso. </li></ol> 6. Acquisizione dati e analisi <ol><li> Acquisire sequenze di immagini digitali a 3 fotogrammi / s per 2 min a seguito del flusso pienamente sviluppata lungo l&#39;asse longitudinale del canale a 3 posizioni nel canale, facendo attenzione a non registrare immediatamente dopo l&#39;ingresso o prima o porte di uscita 25% in più vicina al lato pareti. </li><li> Contare il numero di cellule arrestate per campo identificando loro fase aspetto brillante nello stesso piano focale del monostrato. </li><li> Cell arresto impresa è definita dal movimento &lt;cella diametro ½ in 10 s. Dati di una media di 3 campi aleatori per canale e quantificare utilizzando il software ImageJ. </li></ol> 7. Risultati rappresentativi Anche se l&#39;endotelio vascolare è uniformemente esposta agli agonisti che contribuiscono a infiammazione sistemica, l&#39;aterosclerosi è una malattia focale che si sviluppa preferenzialmente ai siti di disturbo del flusso nelle arterie, implicando eterogeneità spaziale in funzione endoteliale. 8 </sup&gt; mediatori circolanti infiammatorie come il TNF-α sono elevati nel siero umano in condizioni di stress metabolico e correlare con compromissione della funzione endoteliale. 9, 10, TNF-α induce una risposta ben caratterizzato in CE che include up-regolazione di molecole di adesione cellulare (CAM : ad esempio, VCAM-1, ICAM-1, E-selectina) e chemochine che reclutano monociti dal circolo, un segno distintivo della lesione aterosclerotica primi 11 emodinamica è un importante regolatore del fenotipo endoteliale infiammatoria 12 Alta tensione di taglio (HSS. magnitudo.; ad esempio 15 dyne / cm 2) o sollecitazione di taglio pulsatile (PSS, ad esempio 15 ± 5 dyne / cm 2) associati a siti di resistenza a aterosclerosi in arterie down-regolano TNF-α-risposte indotte. Al contrario, lo stress shear a bassa magnitudo (LSS: ad esempio, 2 dyne / cm 2) o di taglio oscillatoria (OSS, ad esempio 0 ± 5 dyne / cm 2), caratteristiche dei siti sensibili in vivo, esacerbare cytokine-indotta infiammazione. 13, 14 I nostri dispositivi microfluidici PDMS fornire una piattaforma per la realizzazione di studi meccanicistici per verificare ipotesi relative alla sovrapposizione di stress metabolico con i fattori idrodinamici nella regolazione della funzione endoteliale e la patologia. L&#39;utilità e la versatilità del metodo è ampiamente illustrata attraverso i seguenti risultati rappresentativi. <img src="/files/ftp_upload/4169/4169fig1.jpg" alt="Figura 1" /> Figura 1. Shear stress modula differenziale THP-1 di adesione delle cellule al TNF-alfa monostrati infiammate endoteliali. Questi dati sono stati acquisiti utilizzando il protocollo dettagliato descritto in precedenza e documentato nel video. Monostrati HAEC sono stati esposti contemporaneamente a una bassa dose di citochina infiammatoria TNF-α (0,5 ng / ml, ~ EC 50 per VCAM-1 up-regulation) e sia HSS (15 dyne / cm 2) o OSS (0 ± 5 dynes / cm 2, 1 Hz) in un CSD personalizzato per 4 ore come descriletto nel protocollo dettagliato. THP-1 arresto cellulare è stata quantificata sotto flusso di taglio in una camera di flusso PDMS. Questa linea cellulare che imita il fenotipo di monociti umani è stato scelto per la sua riproducibilità e facilità d&#39;uso. HSS attenuato la risposta infiammatoria rispetto agli OSS, con conseguente diminuzione THP-1 arresto cellulare. I dati sono stati analizzati mediante Student t-test e sono rappresentati come media ± SEM di 3 esperimenti indipendenti. Adattato con il permesso di DeVerse et al. 5 I dati che seguono dimostrano la versatilità del nostro approccio con esempi dal nostro lavoro pubblicato. Partenze dal protocollo di cui sopra sono indicati, se del caso. <img src="/files/ftp_upload/4169/4169fig2.jpg" alt="Figura 2" /> Figura 2. Modulazione di citochine indotta VCAM-1 e reclutamento dei monociti in un gradiente di sollecitazione di taglio. A) HAEC monostrati sono state stimolate con TNF-α (0,3 ng / ml) per 4 ore sotto flow in uno VMMC progettato sulla base Hele-Shaw teoria del flusso. 15 A decremento lineare in grandezza SS lungo l&#39;asse del canale è stato ottenuto progettando le pareti laterali della camera di coincidere con la ottimizza di un flusso due stagnazione dimensionali e di forma alla fine del canale per abbinare i iso-potenziali linee. La sollecitazione di taglio parete (τ w) generata lungo la mezzeria di questo canale ad una distanza assiale dall&#39;ingresso (x) è dato da: <img src="/files/ftp_upload/4169/4169eq3.jpg" alt="Equazione 3" /> dove μ è la viscosità del mezzo, Q è la portata volumetrica, h è l&#39;altezza del canale, w 1 è la larghezza del canale di ingresso, ed L è la lunghezza totale del canale. Q è stato scelto per generare un gradiente di grandezza da 16 SS dyne / cm 2 a monte che a 0 al punto di stagnazione &quot;a&quot; appena prossimale al outlet. B)-C), VCAM-1 espressione è stata misurata in situ tramite microscopia immunofluorescenza usando anticorpi coniugati punti quantici e presentati come percentuale di intensità di fluorescenza media (MFI) di controllo non stimolato statico. SS non modulare il livello basale di VCAM-1, che è basso in HAEC non stimolate (triangoli grigi). Tuttavia, espressione di VCAM-1 è aumentato rispetto al TNF-α a grandezze LSS, con un picco a ~ 2 dyne / cm 2 e tornare al TNF-α basale o inferiore a grandezze di taglio di 5 dyne / cm 2 (quadrati neri). D) monociti (10 6 / ml) sono stati perfusi a 2 dyne / cm 2 per 5 min in canali di flusso paralleli su monostrati precondizionati come in A). Reclutamento dei monociti all&#39;endotelio quantitativamente correlato con l&#39;espressione di VCAM-1 in un ampiezza di taglio dato stress. In particolare, la capacità di sondare espressione di VCAM-1 e il reclutamento dei monociti con fine risoluzione spaziale in un monolayer rivelato cambiamenti significativi su un range limitato in grandezza SS che non dovrebbe essere apprezzato dagli applicando valori discreti poche di taglio in esperimenti separati. I dati sono stati analizzati mediante ANOVA misure ripetute e valutate differenze di Newman-Keuls posttest, * P &lt;0,05 da TNF-α in condizioni statiche. I dati sono media ± SEM di 3-6 esperimenti indipendenti. Adattato con il permesso di Tsou et al. 2 <img src="/files/ftp_upload/4169/4169fig3.jpg" alt="Figura 3" /> Figura 3. Lipoproteine ​​ricche di trigliceridi (TGRL) modulare citochine indotta infiammazione in proporzione a livelli di trigliceridi di siero e VCAM-1 espressione. HAEC monostrati sono state stimolate con TNF-α (0,3 ng / ml) per 4 ore simultaneamente con TGRL (10 mg / dl ApoB) isolato da soggetti umani dopo un pasto ricco di grassi nella cultura statica. A) espressione di VCAM-1 è stato misurato separatamente in sospeso CE mediante citometria a flussoe adesione monociti quantificato utilizzando un VMMC come descritto nel protocollo dettagliato. I dati sono presentati come variazione% di TNF-α. Arresto Monocyte varia in proporzione diretta alla endoteliale espressione di VCAM-1, che ha aumentato direttamente con il livello di trigliceridi di siero. B) Monocyte arresto per un sottoinsieme di pro-infiammatorie donatori è abrogata in presenza di un anticorpo VCAM-1 blocco. La significatività è stata determinata la Student t-test accoppiato, significa ± SEM di 3-5 esperimenti indipendenti. Adattato con il permesso di Wang et al. 4

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Si descrivono l&#39;uso di dispositivi microfluidici PDMS per la valutazione on-chip di endoteliale fenotipo infiammatoria attraverso l&#39;imaging in tempo reale di espressione CAM e adesione monociti. Uno dei principali vantaggi del nostro approccio risiede nella capacità di quantificare i risultati associati a disfunzione endoteliale in cellule esposte a mediatori infiammatori come i lipidi alimentari e citochine in condizioni idrodinamiche definiti che rispecchiano shear stress nei vasi aterogene. Il VMMC facilitar...

<table border="1"><tbody><tr><td> Voce </td><td> Azienda </td><td> Numero di catalogo </td></tr><tr><td> 100 x 20mm piastre di Petri </td><td> BD Falcon </td><td> 353003 </td></tr><tr><td> Etanolo 95% </td><td> EMD Chemicals </td><td> EX0290-1 </td></tr><tr><td> DPBS </td><td> Cellgro </td><td> 21-031-CV </td></tr><tr><td> Collagene di tipo I Tail derivata Rat </td><td> Gibco </td><td> A10483-01 </td></tr><tr><td> Cellule endoteliali umane aortica </td><td> Genlantis </td><td> PH30405A </td></tr><tr><td> Antibiotico-antimicotica Solution </td><td> Invitrogen </td><td> 15240-062 </td></tr><tr><td> BulletKit endoteliale </td><td> Lonza </td><td> CC-4176 </td></tr><tr><td> Endoteliali basali Media-2 </td><td> Lonza </td><td> CC-3156 </td></tr><tr><td> Siringhe da 10 ml </td><td> BD Falcon </td><td> 309604 </td></tr><tr><td> Tubi in poliuretano </td><td> Tygon </td><td> ABW0001 </td></tr><tr><td> Leibovitz-15 supporti </td><td> Gibco </td><td> 11415-069 </td></tr><tr><td> Sylgard 184 Base in silicone elastomero </td><td> Dow Corning </td><td> 184 </td></tr><tr><td> Sylgard 184 elastomero siliconico Catalizzatore </td><td> Dow Corning </td><td> 184 </td></tr><tr><td> SU8 Photoresist Maestro Wafer </td><td> UC Davis Pan Lab </td><td> N / A </td></tr><tr><td> Eclipse TE200 microscopio invertito </td><td> Nikon </td><td> Eclipse TE200 </td></tr><tr><td> Pompa a siringa </td><td> Harvard Apparatus </td><td> PHD2000 </td></tr><tr><td> 19 gauge ago ipodermico </td><td> Kendall </td><td> 8881 </td></tr><tr><td> THP-1 linea cellulare monocitica </td><td> ATCC </td><td> TIB-202 </td></tr><tr><td> HBSS (Hanks soluzione salina tamponata) con Ca 2 + / Mg 2 + </td><td&gt; Gibco </td><td> 14025-092 </td></tr><tr><td> Fattore di Necrosi Tumorale alfa (TNF-α) </td><td> R &amp; D Systems </td><td> 210-TA-010 </td></tr><tr><td> Stromal Factor Derivato - 1 (SDF-1) </td><td> R &amp; D Systems </td><td> 350-NS-010 </td></tr><tr><td> RPMI 1640 </td><td> Cellgro </td><td> 10-040-CV </td></tr><tr><td> Albumina serica umana (HSA) </td><td> ZLB Behring </td><td> NDC 0053-7680-32 </td></tr></tbody></table> Tabella 2. Reagenti e attrezzature specifiche.

on-chip endothelial inflammatory phenotyping

Aterogenesi è potenziato da alterazioni metaboliche che contribuiscono ad un elevato stato di infiammazione sistemica con conseguente disfunzione endoteliale. Tuttavia, i primi cambiamenti funzionali dell&#39;endotelio che indicano il livello individuale di rischio non sono direttamente valutato clinicamente per aiutare strategia terapeutica guida. Inoltre, la regolazione di infiammazione emodinamica locali contribuisce alla non casuale distribuzione spaziale di aterosclerosi, ma i meccanismi sono difficili da delineare in vivo. Descriviamo un approccio lab-on-a-chip basato su test di quantitativamente perturbazione metabolica di eventi infiammatori nelle cellule endoteliali umane (CE) e dei monociti in condizioni di flusso precise. Metodi standard di litografia soft sono usati per microfabricate vascolari camere mimetici microfluidica (VMMC), che sono legati direttamente alla coltura monostrati CE. 1 Questi dispositivi hanno il vantaggio di utilizzare piccoli volumi di reagenti, mentrefornendo una piattaforma per l&#39;imaging direttamente gli eventi infiammatori a livello della membrana di CE esposto ad un ben definito campo di taglio. Abbiamo applicato con successo questi dispositivi per indagare citochine, lipidi 2-3, 4 e 5 RAGE indotta infiammazione in umano aortica CE (HAEC). Qui documentare l&#39;uso della cella VMMC di dosaggio monocitiche (THP-1) laminazione e arresto su monostrati HAEC che sono condizionati alle caratteristiche di taglio differenziali e attivato dalla citochina infiammatoria TNF-α. Studi come questi stanno fornendo informazioni meccanicistica in atherosusceptibility in caso di fattori di rischio metabolici.

Watch this Scientific Journal Video about On-Chip Endothelial Inflammatory Phenotyping at JoVE.com

On-Chip Endothelial Inflammatory Phenotyping

Camere di flusso Microfluidic incisi da fotolitografia e fabbricati da PDMS sono applicate per sondare gli esiti funzionali associati alla disfunzione CE e l&#39;infiammazione. In un esperimento rappresentativo, la capacità di sforzo di taglio differenziale di modulare l&#39;adesione cellulare monocitica di citochina attivato CE monostrati è dimostrata.

On-Chip Fenotipizzazione Endothelial infiammatoria

Atherogenesis is potentiated by metabolic abnormalities that contribute to a heightened state of systemic inflammation resulting in endothelial ...

on-chip-endothelial-inflammatory-phenotyping

Research

JoVE Journal

Bioengineering

10.8K Views.  University of California, Davis. Camere di flusso Microfluidic incisi da fotolitografia e fabbricati da PDMS sono applicate per sondare gli esiti funzionali associati alla disfunzione CE e l'infiammazione. In un esperimento rappresentativo, la capacità di sforzo di taglio differenziale di modulare l'adesione cellulare monocitica di citochina attivato CE monostrati è dimostrata.

Video: On-Chip Endothelial Inflammatory Phenotyping

Autologous Endothelial Progenitor Cell-Seeding Technology and Biocompatibility Testing For Cardiovascular Devices in Large Animal Model

Implantable cardiovascular devices are manufactured from artificial materials (e.g. titanium (Ti), expanded polytetrafluoroethylene), which pose the risk of thromboemboli formation1,2,3. We have developed a method to line the inside surface of Ti tubes with autologous blood-derived human or porcine endothelial progenitor cells (EPCs)4. By implanting Ti tubes containing a confluent layer of porcine EPCs in the inferior vena cava (IVC) of pigs, we tested the improved biocompatibility of the cell-seeded surface in the prothrombotic environment of a large animal model and compared it to unmodified bare metal surfaces5,6,7 (Figure 1). This method can be used to endothelialize devices within minutes of implantation and test their antithrombotic function in vivo. 
Peripheral blood was obtained from 50 kg Yorkshire swine and its mononuclear cell fraction cultured to isolate EPCs4,8. Ti tubes (9.4 mm ID) were pre-cut into three 4.5 cm longitudinal sections and reassembled with heat-shrink tubing. A seeding device was built, which allows for slow rotation of the Ti tubes. 
We performed a laparotomy on the pigs and externalized the intestine and urinary bladder. Sharp and blunt dissection was used to skeletonize the IVC from its bifurcation distal to the right renal artery proximal. The Ti tubes were then filled with fluorescently-labeled autologous EPC suspension and rotated at 10 RPH x 30 min to achieve uniform cell-coating9. After administration of 100 USP/ kg heparin, both ends of the IVC and a lumbar vein were clamped. A 4 cm veinotomy was performed and the device inserted and filled with phosphate-buffered saline. As the veinotomy was closed with a 4-0 Prolene running suture, one clamp was removed to de-air the IVC. At the end of the procedure, the fascia was approximated with 0-PDS (polydioxanone suture), the subcutaneous space closed with 2-0 Vicryl and the skin stapled closed. 
After 3 - 21 days, pigs were euthanized, the device explanted en-block and fixed. The Ti tubes were disassembled and the inner surfaces imaged with a fluorescent microscope.
We found that the bare metal Ti tubes fully occluded whereas the EPC-seeded tubes remained patent. Further, we were able to demonstrate a confluent layer of EPCs on the inside blood-contacting surface.
Concluding, our technology can be used to endothelialize Ti tubes within minutes of implantation with autologous EPCs to prevent thrombosis of the device. Our surgical method allows for testing the improved biocompatibility of such modified devices with minimal blood loss and EPC-seeded surface disruption.

A method for seeding titanium blood-contacting biomaterials with autologous cells and testing biocompatibility is described. This method uses endothelial progenitor cells and titanium tubes, seeded within minutes of surgical implantation into porcine venae cavae. This technique is adaptable to many other implantable biomedical devices.

Autologo di cellule progenitrici endoteliali-Seeding Tecnologie e test di biocompatibilità per dispositivi cardiovascolari in un modello animale di grandi dimensioni

Implantable cardiovascular devices are manufactured from artificial materials (e.g. titanium (Ti), expanded polytetrafluoroethylene), which pose the risk ...

Ricerca

Bioingegneria

On-chip Isotachophoresis for Separation of Ions and Purification of Nucleic Acids

Electrokinetic techniques are a staple of microscale applications because of their unique ability to perform a variety of fluidic and electrophoretic processes in simple, compact systems with no moving parts. Isotachophoresis (ITP) is a simple and very robust electrokinetic technique that can achieve million-fold preconcentration1,2 and efficient separation and extraction based on ionic mobility.3 For example, we have demonstrated the application of ITP to separation and sensitive detection of unlabeled ionic molecules (e.g. toxins, DNA, rRNA, miRNA) with little or no sample preparation4-8 and to extraction and purification of nucleic acids from complex matrices including cell culture, urine, and blood.9-12
ITP achieves focusing and separation using an applied electric field and two buffers within a fluidic channel system. For anionic analytes, the leading electrolyte (LE) buffer is chosen such that its anions have higher effective electrophoretic mobility than the anions of the trailing electrolyte (TE) buffer (Effective mobility describes the observable drift velocity of an ion and takes into account the ionization state of the ion, as described in detail by Persat et al.13). After establishing an interface between the TE and LE, an electric field is applied such that LE ions move away from the region occupied by TE ions. Sample ions of intermediate effective mobility race ahead of TE ions but cannot overtake LE ions, and so they focus at the LE-TE interface (hereafter called the "ITP interface"). Further, the TE and LE form regions of respectively low and high conductivity, which establish a steep electric field gradient at the ITP interface. This field gradient preconcentrates sample species as they focus. Proper choice of TE and LE results in focusing and purification of target species from other non-focused species and, eventually, separation and segregation of sample species.
We here review the physical principles underlying ITP and discuss two standard modes of operation: "peak" and "plateau" modes. In peak mode, relatively dilute sample ions focus together within overlapping narrow peaks at the ITP interface. In plateau mode, more abundant sample ions reach a steady-state concentration and segregate into adjoining plateau-like zones ordered by their effective mobility. Peak and plateau modes arise out of the same underlying physics, but represent distinct regimes differentiated by the initial analyte concentration and/or the amount of time allotted for sample accumulation.
We first describe in detail a model peak mode experiment and then demonstrate a peak mode assay for the extraction of nucleic acids from E. coli cell culture. We conclude by presenting a plateau mode assay, where we use a non-focusing tracer (NFT) species to visualize the separation and perform quantitation of amino acids.

Isotachophoresis (ITP) is a robust electrokinetic separation and preconcentration technique with applications ranging from toxin detection to sample preparation. We review the physical principles of ITP and the methodology of applying this technique to two specific example applications: separation and detection of small molecules and purification of nucleic acids from cell culture lysate.

On-Chip Isotachophoresis per separazione degli ioni e purificazione di acidi nucleici

Electrokinetic techniques are a staple of microscale applications because of their unique ability to perform a variety of fluidic and electrophoretic ...

Lensfree On-chip Tomographic Microscopy Employing Multi-angle Illumination and Pixel Super-resolution

Tomographic imaging has been a widely used tool in medicine as it can provide three-dimensional (3D) structural information regarding objects of different size scales. In micrometer and millimeter scales, optical microscopy modalities find increasing use owing to the non-ionizing nature of visible light, and the availability of a rich set of illumination sources (such as lasers and light-emitting-diodes) and detection elements (such as large format CCD and CMOS detector-arrays). Among the recently developed optical tomographic microscopy modalities, one can include optical coherence tomography, optical diffraction tomography, optical projection tomography and light-sheet microscopy. 1-6 These platforms provide sectional imaging of cells, microorganisms and model animals such as C. elegans, zebrafish and mouse embryos.
Existing 3D optical imagers generally have relatively bulky and complex architectures, limiting the availability of these equipments to advanced laboratories, and impeding their integration with lab-on-a-chip platforms and microfluidic chips. To provide an alternative tomographic microscope, we recently developed lensfree optical tomography (LOT) as a high-throughput, compact and cost-effective optical tomography modality. 7 LOT discards the use of lenses and bulky optical components, and instead relies on multi-angle illumination and digital computation to achieve depth-resolved imaging of micro-objects over a large imaging volume. LOT can image biological specimen at a spatial resolution of &lt;1 &mu;m x &lt;1 &mu;m x &lt;3 &mu;m in the x, y and z dimensions, respectively, over a large imaging volume of 15-100 mm3, and can be particularly useful for lab-on-a-chip platforms.

Lensfree optical tomography is a three-dimensional microscopy technique that offers a spatial resolution of &lt;1 &mu;m &times; &lt;1 &mu;m &times; &lt;3 &mu;m in x, y and z dimensions, respectively, over a large imaging-volume of 15-100 mm3, which can be particularly useful for integration with lab-on-a-chip platforms.

Lensfree On-chip Tomographic Microscopia Utilizzando Multi-angolo di illuminazione e Pixel Super-risoluzione

Tomographic imaging has been a widely used tool in medicine as it can provide three-dimensional (3D) structural information regarding objects of different ...

Quantifying the Mechanical Properties of the Endothelial Glycocalyx with Atomic Force Microscopy

Our understanding of the interaction of leukocytes and the vessel wall during leukocyte capture is limited by an incomplete understanding of the mechanical properties of the endothelial surface layer. It is known that adhesion molecules on leukocytes are distributed non-uniformly relative to surface topography 3, that topography limits adhesive bond formation with other surfaces 9, and that physiological contact forces (&asymp; 5.0 &minus; 10.0 pN per microvillus) can compress the microvilli to as little as a third of their resting length, increasing the accessibility of molecules to the opposing surface 3, 7. We consider the endothelium as a two-layered structure, the relatively rigid cell body, plus the glycocalyx, a soft protective sugar coating on the luminal surface 6. It has been shown that the glycocalyx can act as a barrier to reduce adhesion of leukocytes to the endothelial surface 4. In this report we begin to address the deformability of endothelial surfaces to understand how the endothelial mechanical stiffness might affect bond formation. Endothelial cells grown in static culture do not express a robust glycocalyx, but cells grown under physiological flow conditions begin to approximate the glycocalyx observed in vivo 2. The modulus of the endothelial cell body has been measured using atomic force microscopy (AFM) to be approximately 5 to 20 kPa 5. The thickness and structure of the glycocalyx have been studied using electron microscopy 8, and the modulus of the glycocalyx has been approximated using indirect methods, but to our knowledge, there have been no published reports of a direct measurement of the glycocalyx modulus in living cells. In this study, we present indentation experiments made with a novel AFM probe on cells that have been cultured in conditions to maximize their glycocalyx expression to make direct measurements of the modulus and thickness of the endothelial glycocalyx.

The mechanical characteristics of endothelial glycocalyx were measured by indentation using micron sized spheres on AFM cantilevers. Endothelial cells were cultured in a custom chamber under physiological flow conditions to induce glycocalyx expression. Data were analyzed using a thin film model to determine the glycocalyx thickness and modulus.

Quantificare le proprietà meccaniche del glicocalice endoteliale con microscopia a forza atomica

Our  understanding of the interaction of leukocytes and the vessel wall during  leukocyte capture is limited by an incomplete understanding of the ...

Assessing Leukocyte-endothelial Interactions Under Flow Conditions in an Ex Vivo Autoperfused Microflow Chamber Assay

Leukocyte-endothelial interactions are early and critical events in acute and chronic inflammation and can, when dysregulated, mediate tissue injury leading to permanent pathological damage. Existing conventional assays allow the analysis of leukocyte adhesion molecules only after the extraction of leukocytes from the blood. This requires the blood to undergo several steps before peripheral blood leukocytes (PBLs) can be ready for analysis, which in turn can stimulate PBLs influencing the research findings. The autoperfused micro flow chamber assay, however, allows scientists to study early leukocytes functional dysregulation using the systemic flow of a live mouse while having the freedom of manipulating a coated chamber. Through a disease model, the functional expression of leukocyte adhesion molecules can be assessed and quantified in a micro-glass chamber coated with immobilized endothelial adhesion molecules ex vivo. In this model, the blood flows between the right common carotid artery and left external jugular vein of a live mouse under anesthesia, allowing the interaction of native PBLs in the chamber. Real-time experimental analysis is achieved with the assistance of an intravital microscope as well as a Harvard Apparatus pressure device. The application of a flow regulator at the input point of the glass chamber allows comparable physiological flow conditions amongst the experiments. Leukocyte rolling velocity is the main outcome and is measured using the National Institutes of Health open-access software ImageJ. In summary, the autoperfused micro flow chamber assay provides an optimal physiological environment to study leukocytes endothelial interaction and allows researchers to draw accurate conclusions when studying inflammation.

Assessing Leukocyte-endothelial Interactions Under Flow Conditions in an Ex Vivo Autoperfused Microflow Chamber Assay

Here, we present a protocol that allows the investigator to assess leukocyte recruitment dynamics ex vivo by connecting a chamber coated with endothelial-derived adhesion molecules to the circulatory system of a mouse. This method offers significant advantages since it allows for leukocyte assessment under relative biological conditions.

Valutare interazioni leucociti-endotelio in condizioni di flusso in un<em&gt; Ex Vivo</em&gt; Assay Autoperfused Microflow Camera

Leukocyte-endothelial interactions are early and critical events in acute and chronic inflammation and can, when dysregulated, mediate tissue injury ...

Ferromagnetic Bare Metal Stent for Endothelial Cell Capture and Retention

Rapid endothelialization of cardiovascular stents is needed to reduce stent thrombosis and to avoid anti-platelet therapy which can reduce bleeding risk. The feasibility of using magnetic forces to capture and retain endothelial outgrowth cells (EOC) labeled with super paramagnetic iron oxide nanoparticles (SPION) has been shown previously. But this technique requires the development of a mechanically functional stent from a magnetic and biocompatible material followed by in-vitro and in-vivo testing to prove rapid endothelialization. We developed a weakly ferromagnetic stent from 2205 duplex stainless steel using computer aided design (CAD) and its design was further refined using finite element analysis (FEA). The final design of the stent exhibited a principal strain below the fracture limit of the material during mechanical crimping and expansion. One hundred stents were manufactured and a subset of them was used for mechanical testing, retained magnetic field measurements, in-vitro cell capture studies, and in-vivo implantation studies. Ten stents were tested for deployment to verify if they sustained crimping and expansion cycle without failure. Another 10 stents were magnetized using a strong neodymium magnet and their retained magnetic field was measured. The stents showed that the retained magnetism was sufficient to capture SPION-labeled EOC in our in-vitro studies. SPION-labeled EOC capture and retention was verified in large animal models by implanting 1 magnetized stent and 1 non-magnetized control stent in each of 4 pigs. The stented arteries were explanted after 7 days and analyzed histologically. The weakly magnetic stents developed in this study were capable of attracting and retaining SPION-labeled endothelial cells which can promote rapid healing.

Our goals were to design, manufacture and test ferromagnetic stents for endothelial cell capture. Ten stents were tested for fracture and 10 more stents were tested for retained magnetism. Finally, 10 stents were tested in-vitro and 8 more stents were implanted in 4 pigs to show cell capture and retention.

Bare Metal Stent ferromagnetico per Capture cellule endoteliali e conservazione

Rapid endothelialization of cardiovascular stents is needed to reduce stent thrombosis and to avoid anti-platelet therapy which can reduce bleeding risk. ...

Dry Film Photoresist-based Electrochemical Microfluidic Biosensor Platform: Device Fabrication, On-chip Assay Preparation, and System Operation

In recent years, biomarker diagnostics became an indispensable tool for the diagnosis of human disease, especially for the point-of-care diagnostics. An easy-to-use and low-cost sensor platform is highly desired to measure various types of analytes (e.g., biomarkers, hormones, and drugs) quantitatively and specifically. For this reason, dry film photoresist technology - enabling cheap, facile, and high-throughput fabrication - was used to manufacture the microfluidic biosensor presented here. Depending on the bioassay used afterwards, the versatile platform is capable of detecting various types of biomolecules. For the fabrication of the device, platinum electrodes are structured on a flexible polyimide (PI) foil in the only clean-room process step. The PI foil serves as a substrate for the electrodes, which are insulated with an epoxy-based photoresist. The microfluidic channel is subsequently generated by the development and lamination of dry film photoresist (DFR) foils onto the PI wafer. By using a hydrophobic stopping barrier in the channel, the channel is separated into two specific areas: an immobilization section for the enzyme-linked assay and an electrochemical measurement cell for the amperometric signal readout.
The on-chip bioassay immobilization is performed by the adsorption of the biomolecules to the channel surface. The glucose oxidase enzyme is used as a transducer for electrochemical signal generation. In the presence of the substrate, glucose, hydrogen peroxide is produced, which is detected at the platinum working electrode. The stop-flow technique is applied to obtain signal amplification along with rapid detection. Different biomolecules can quantitatively be measured by means of the introduced microfluidic system, giving an indication of different types of diseases, or, in regard to therapeutic drug monitoring, facilitating a personalized therapy.

A microfluidic biosensor platform was designed and fabricated using low-cost dry film photoresist technology for the rapid and sensitive quantification of various analytes. This single-use system allows for the electrochemical readout of on-chip-immobilized enzyme-linked assays by means of the stop-flow technique.

Film secco basati su Photoresist biosensore elettrochimico Microfluidic Platform: Fabbricazione di dispositivi, saggio su chip preparazione e funzionamento del sistema

In recent years, biomarker diagnostics became an indispensable tool for the diagnosis of human disease, especially for the point-of-care diagnostics. An ...

Fabrication and Validation of an Organ-on-chip System with Integrated Electrodes to Directly Quantify Transendothelial Electrical Resistance

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful research tools for studying human health and disease. To characterize the barrier function of cell layers cultured inside organ-on-chip devices, often transendothelial or transepithelial electrical resistance (TEER) is measured. To this end, electrodes are usually integrated into the chip by micromachining methods to provide more stable measurements than is achieved with manual insertion of electrodes into the inlets of the chip. However, these electrodes frequently hamper visual inspection of the studied cell layer or require expensive cleanroom processes for fabrication. To overcome these limitations, the device described here contains four easily integrated electrodes that are placed and fixed outside of the culture area, making visual inspection possible. Using these four electrodes the resistance of six measurement paths can be quantified, from which the TEER can be directly isolated, independent of the resistance of culture medium-filled microchannels. The blood-brain barrier was replicated in this device and its TEER was monitored to show the device applicability. This chip, the integrated electrodes and the TEER determination method are generally applicable in organs-on-chips, both to mimic other organs or to be incorporated into existing organ-on-chip systems.

This publication describes the fabrication of an organ-on-chip device with integrated electrodes for direct quantification of transendothelial electrical resistance (TEER). For validation, the blood-brain barrier was mimicked inside this microfluidic device and its barrier function was monitored. The presented methods for electrode integration and direct TEER quantification are generally applicable.

Fabbricazione e validazione di un sistema di organo-on-chip con elettrodi integrati direttamente quantificare transendoteliale resistenza elettrica

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful ...

Growth of Human and Sheep Corneal Endothelial Cell Layers on Biomaterial Membranes

Corneal endothelial cell cultures have a tendency to undergo epithelial-to-mesenchymal transition (EMT) after loss of cell-to-cell contact. EMT is deleterious for the cells as it reduces their ability to form a mature and functional layer. Here, we present a method for establishing and subculturing human and sheep corneal endothelial cell cultures that minimizes the loss of cell-to-cell contact. Explants of corneal endothelium/Descemet&#39;s membrane are taken from donor corneas and placed into tissue culture under conditions that allow the cells to collectively migrate onto the culture surface. Once a culture has been established, the explants are transferred to fresh plates to initiate new cultures. Dispase II is used to gently lift clumps of cells off tissue culture plates for subculturing. Corneal endothelial cell cultures that have been established using this protocol are suitable for transferring to biomaterial membranes to produce tissue-engineered cell layers for transplantation in animal trials. A custom-made device for supporting biomaterial membranes during tissue culture is described and an example of a tissue-engineered graft composed of a layer of corneal endothelial cells and a layer of corneal stromal cells on either side of a collagen type I membrane is presented.

This protocol describes the critical steps required to establish and grow corneal endothelial cell cultures from explants of human or sheep tissue. A method for subculturing corneal endothelial cells on membranous biomaterials is also presented.

Crescita degli strati cellulari endoteliali umani e ovini sulle membrane biomateriali

Corneal endothelial cell cultures have a tendency to undergo epithelial-to-mesenchymal transition (EMT) after loss of cell-to-cell contact. EMT is ...

High Throughput Yeast Strain Phenotyping with Droplet-Based RNA Sequencing

The powerful tools available to edit yeast genomes have made this microbe a valuable platform for engineering. While it is now possible to construct libraries of millions of genetically distinct strains, screening for a desired phenotype remains a significant obstacle. With existing screening techniques, there is a tradeoff between information output and throughput, with high-throughput screening typically being performed on one product of interest. Therefore, we present an approach to accelerate strain screening by adapting single cell RNA sequencing to isogenic picoliter colonies of genetically engineered yeast strains. To address the unique challenges of performing RNA sequencing on yeast cells, we culture isogenic yeast colonies within hydrogels and spheroplast prior to performing RNA sequencing. The RNA sequencing data can be used to infer yeast phenotypes and sort out engineered pathways. The scalability of our method addresses a critical obstruction in microbial engineering.

A bottleneck in the &#8216;design-build-test&#8217; cycle of microbial engineering is the speed at which we can perform functional screens of strains. We describe a high-throughput method for strain screening applied to hundreds to thousands of yeast cells per experiment that utilizes droplet-based RNA sequencing.

Phenotipizzazione del ceppo di lievito ad alta velocità effettiva con sequenziamento dell'RNA basato su droplet

The powerful tools available to edit yeast genomes have made this microbe a valuable platform for engineering. While it is now possible to construct ...