Ringraziamo T. Hunt, M. Rosenbluth, e il Laboratorio Lam per i loro consigli e le discussioni utili. Noi riconosciamo il sostegno G. Spinner e l&#39;Istituto di Elettronica e Nanotecnologia presso il Georgia Institute of Technology. Il sostegno finanziario per questo lavoro è stato fornito dal NIH una sovvenzione di K08-HL093360, UCSF premio REAC, uno nanomedicina NIH Award PN2EY018244 Development Center, e il finanziamento del Centro di Biologia delle cellule endoteliali Healthcare dei bambini di Atlanta.

1. Realizzazione della Microdevice Endothelial <ol><li> Creare un fotomaschera presentando un Computer Assisted Design (CAD) disegno del dispositivo a microfluidi a un fornitore esterno la maschera. La maschera utilizzato è composto da uno strato di cromo su vetro soda lime. In questo caso la larghezza del canale microfluidico era 30 pm. </li><li> Pulire un wafer di silicio nudo con Piranha (10:1 rapporto di acido solforico e perossido di idrogeno) per 15 minuti e immerse in acido fluoridrico per 30 secondi. Risciacquare con acqua deionizzata (DI) di acqua per circa 10 secondi. </li><li> Utilizzando un rivestitore a rotazione, rotazione MicroChem SU-8 2025 fotoresist sul wafer per una altezza di 30 pm. Per SU-8 2025, una velocità di rotazione di 3000 rpm è raccomandato. Altre viscosità di SU-8 sono disponibili che realizzerà questa altezza. Le istruzioni complete per il SU-8 l&#39;uso sono disponibili presso <a href="http://www.microchem.com" target="_blank">www.microchem.com</a> </li><li> Posizionare il wafer con SU-8 su una piastra riscaldante a 95 ° C per5 minuti per eliminare l&#39;eccesso di solvente. Nota: Un forno si secca il wafer diverso rispetto a un piano di cottura e non è raccomandato. </li><li> Posizionare una maschera con la forma funzione desiderata sul wafer, ed esporre alla luce UV (160 mJ / cm 2 misurata a 365 nm) in un allineatore maschera (Karl Suss, MA-6). Questa croce collega il fotoresist. </li><li> Posizionare il wafer posteriore su una piastra riscaldante a 95 ° C per altri 5 minuti per accelerare ulteriormente la polimerizzazione di SU-8. </li><li> Immergere il wafer in SU-8 Developer, composta principalmente da PGMEA (acetato di metile glicole propilenico etere) per 4 minuti per rimuovere il non-cross-linked SU-8. </li><li> Lavare il wafer di nuova concezione con il 100% di alcool isopropilico (IPA) per 10 s. Il wafer può essere essiccato con azoto pressurizzato o consentendo l&#39;evaporazione del solvente dal wafer in una cappa aspirante pulito per diversi minuti. </li><li> Nastro i bordi del wafer secco con modellato SU-8 in una capsula Petri di presfiatare movimento. </li><li> Applicare 1 mL di Sigmacote al wafer utilizzando una pipetta, coprire con la parte superiore della capsula di Petri, wafer e agitare per assicurare rivestimento completo della fetta, rimuovere la copertura, e consentire wafer asciugare per alcuni minuti fino a quando tutto il solvente è evaporato. </li></ol> 2. PDMS (Polidimetilsilossano) Preparazione <ol><li> Mix PDMS polimero e l&#39;agente di indurimento in un rapporto 10:1 (w / w) e rimuovere le bolle d&#39;aria utilizzando un essiccatore a vuoto. La lunghezza del tempo necessario per degassare il PDMS varia sulla forza del sistema a vuoto disponibile, ma varia tipicamente da alcuni minuti a un&#39;ora. Per un periodo di sei pollici capsula petri senza precedentemente versata PDMS, un volume totale di 60 ml di polimero è raccomandato. </li><li> Versare il composto sul wafer, circa 5 mm di spessore. Inoltre versare su un piatto fondo piatto per creare un sottile foglio di PDMS, circa 1 mm di spessore. Polimerizzazione a 60 ° C in un forno per una notte. </li><li> Usando un coltello o bisturi, tagliare intorno alla crato dispositivo PDMS e rimuoverlo dal wafer. Inoltre, tagliare un foglio sottile di PDMS leggermente più grande del dispositivo. </li><li> Creare fori di ingresso e di uscita del dispositivo utilizzando un PDMS foratura 1,0 mm. Ciò può essere realizzato utilizzando un perno morsa, dispositivo Harris Uni-core, o simili. </li><li> Pulire le superfici del dispositivo e il foglio con del nastro adesivo. </li><li> Utilizzo di un pulitore plasma, esporre le superfici del dispositivo PDMS e foglio PDMS a plasma di ossigeno per 30 s. Il plasma di ossigeno crea specie reattive sulla superficie esposta del PDMS che legame quando entrano in contatto fisico. </li><li> Collegare tubi di piccolo ad una piccola lunghezza (alcuni centimetri) di tubo grande, che a sua volta è collegato ad una siringa con un ago appuntito-punto, riempito con 50 pg / ml di fibronectina da plasma umano in PBS. Il tubo e l&#39;ago sono dimensionati in modo tale che un accoppiamento ad attrito si crea tra il tubo. Inserire piccolo tubo nella insenatura di PDMS Microdevice e unione pressione alla siringa per riempire i canali completamente con la soluzione fibronectina e per creare una piccola goccia 100 pl a luce di uscita, per garantire che i canali rimanere umida. Incubare il dispositivo a 37 ° C per 40-60 minuti. </li><li> Collegare una siringa fresca ripiena con PBS per l&#39;ago punta smussata. Applicare una pressione alla siringa per sciacquare il dispositivo con PBS. </li></ol> 3. Semina il dispositivo Microfluidic con le cellule endoteliali <ol><li> Preparare 1.000.000 cellule / ml di cellule endoteliali della vena ombelicale umana (HUVEC) in terreni di crescita endoteliali con 8% destrano. Una gamma di 500.000 a 2.000.000 cellule / ml è stato usato con successo. L&#39;aggiunta di destrano al mezzo cella di carico aumenta la viscosità del fluido, che diminuisce la velocità di cellule endoteliali che entrano la fibronectina rivestita sistema microfluidica. Ciò, a sua volta, aumenta la probabilità che le cellule aderiscono e cultura con successo all&#39;interno del microdispositivo. </li><li> Collegare la siringa nuova e il tubo come indicato al punto 2.7. ma con un tubo più lungo (circa 1 metro di lunghezza). </li><li> Per ottimizzare le prestazioni di questo sistema, è critico a questo punto, per evitare fuoriuscite o bolle nel sistema di perfusione intero; qualsiasi fuoriuscita di soluzione o di presenza di bolle nei media cambierà il flusso e prevenire successo semina di cellule endoteliali. Pertanto, aderente del tubo è essenziale per eliminare le perdite tra i collegamenti. Bolle nella siringa e / o tubi devono essere eliminati prima cellule vengono introdotte nella microdispositivo e l&#39;intero sistema di perfusione devono essere trattate con la soluzione cella prima di collegare il tubo per la microfluidica per evitare l&#39;introduzione di bolle d&#39;aria. </li><li> Utilizzando una pompa a siringa, infonda sospensione cellulare nel dispositivo di PDMS a portata volumetrica di 1,23 microlitri / min per 2 ore a 37 ° C e 5% CO 2. Risultati ottimali sono stati ottenuti quando la pompa a siringa era al<html> stessa altezza del dispositivo PDMS. Per l&#39;ingresso, il tubo più lungo, che può essere avvolto entro l&#39;incubatore, assicura che le cellule ei supporti adeguatamente riscaldata prima di venire in contatto con il dispositivo. Nel nostro sistema, la portata volumetrica di 1,23 microlitri / min approssima una velocità midstream di circa 1 mm / s nei minimi microcanali, corrispondenti ad una sforzi di taglio di circa 1 dyne / cm 2 a quei canali utilizzando sangue intero. </li><li> Utilizzando la stessa pompa siringa e tubo lungo, profumato terreno di coltura fresco per 2-8 giorni al rapporto di 1,23 pl / min. Un dispositivo di successo avrà un monostrato di cellule endoteliali che crescono all&#39;interno del dispositivo in 24-48 ore. Esperimenti precedenti hanno mostrato che monostrati confluenti opportunamente esprimere VE-caderina a cellula-cellula giunzioni tutto il dispositivo 14. </li><li> Iniettare sangue o sospensione di cellule nel sistema per la sperimentazione. </li></ol> 4. Risultati rappresentativi _content &quot;&gt; Usando questo protocollo, le normali tecniche di microfabbricazione litografica sono utilizzati per creare lo stampo necessario per produrre i canali microfluidici fisiologicamente che imitano la sizescale del microcircolo (Figura 1A). Usando una tecnica ottimizzata perfusione, cellule endoteliali e quindi sementi confluently cultura intera superficie interna del sistema microfluidico entro 24-48 ore dalla semina cellulare (figura 1B). Poiché il sistema microfluidico è trasparente, il microdispositivo intero può essere collocato su un campo chiaro / fase microscopio a fluorescenza per l&#39;imaging e la raccolta dei dati. Il sistema può quindi essere applicato studiare le patologie che coinvolgono ematologiche alterati proprietà biofisiche, quali anemia falciforme, in cui la maggiore rigidità di eritrociti falciformi e aberrante adesione endoteliale dei leucociti e contribuiscono ad ostruzione microvascolare. Un farmaco clinicamente approvato, idrossiurea, migliora i sintomi, ma il suo direct effetto sul flusso microvascolare è sconosciuto. Il saggio prende in considerazione sia la rigidità e l&#39;adesione delle cellule, e dimostra che idrossiurea migliora significativamente il flusso in malattia a cellule falciformi (Figura 2). Anemia falciforme è solo un esempio di applicazione per la microvascolarizzazione-on-a-chip, in quanto questo sistema è ideale per studiare ematologica qualsiasi processo in cui i globuli interagiscono con ciascuno altre cellule endoteliali e del microcircolo. Altre applicazioni includono clinicamente rilevanti disturbi infiammatori, sepsi / polmone lesioni, microangiopatie trombotiche, la malaria e le metastasi del cancro, mentre altre applicazioni di base includono la biologia dei leucociti e la biologia delle cellule staminali ematopoietiche, tra molti altri. <img src="/files/ftp_upload/3958/3958fig1.jpg" alt="Figura 1"/> Figura 1. A) iniziale PDMS dispositivo microfluidico prima endotelializzazione. Qui, il Microdevice viene iniettato with colorante alimentare per illustrare progettazione sizescale e globale del sistema. B) Microscopia a campo chiaro mostra il sistema microfluidico è completamente endothelialized entro 48 ore dalla semina cella utilizzando il protocollo descritto qui. <img src="/files/ftp_upload/3958/3958fig2.jpg" alt="Figura 2"/> Figura 2. A) Il microcircolo-on-a-chip con microcanali approssima la dimensione del venule post-capillari (30 micron), il sito della maggior parte degli eventi ostruttivi delle cellule falciformi microvascolari. Sangue intero da pazienti con anemia falciforme che ricevono idrossiurea e da pazienti non trattati con idrossiurea viene fatto fluire attraverso due differenti microcircolo-on-a-chip di dispositivi. B) di sangue intero da pazienti con anemia falciforme che ricevono flussi di idrossiurea con relativa facilità all&#39;interno dei microcanali endothelialized. C) Alle stesse condizioni emodinamiche, sangue intero da pazienti con anemia falciforme che non ricevono idrossiurea, tuttavia, mostra il flusso molto più lento con microchAnnel ostruzione. Il canale inferiore è completamente ostruita con assenza di flusso e le velocità di flusso nei microcanali altri endothelialized sono notevolmente inferiori nella condizione idrossiurea. La barra della scala in tutte e tre le immagini è di 30 micron.

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Non ci sono conflitti di interesse dichiarati.

Il nostro sistema Microdevice endothelialized è più adatto se usato in combinazione con gli esperimenti in vivo, e il suo approccio riduzionista può aiutare a chiarire i meccanismi dei processi biofisici ematologiche che si osservano negli esseri umani e modelli animali. Inoltre, il nostro sistema non è senza limiti. Per esempio, i nostri canali microfluidici sono quadrati in sezione trasversale. Anche se tecnicamente microcanali circolari possono essere fabbricate 10,11, abbiamo scelto di utiliz...

<table border="1"><tbody><tr><td> Nome del reattivo </td><td> Azienda </td><td> Numero di catalogo </td><td> Comments </td></tr><tr><td> punta dell&#39;ago smussata </td><td> OK International </td><td> 920050-TE </td><td> Precision TE ago 20 Gauge x 1/2 &quot;, rosa </td></tr><tr><td> destrano </td><td> Sigma-Aldrich </td><td> 31392 </td><td></td></tr><tr><td> Fibronectina </td><td> Sigma-Aldrich </td><td> F0895 </td><td></td></tr><tr><td> Hole puncher (morsa pin) </td><td> Innovazioni tecniche </td><td></td><td></td></tr><tr><td> Cellule di cordone ombelicale umane endoteliali (HUVEC) </td><td> Lonza </td><td> CC-2519 </td><td></td></tr><tr><td> Plasma cleaner </td><td> Plasma </td><td> PDC-326 </td><td></td></tr><tr><td>Polidimetilsilossano (PDMS) </td><td> Fisher Scientific </td><td> NC9285739 </td><td> Sylgard 184 KIT silicone elastomero </td></tr><tr><td> Sigmacote </td><td> Sigma-Aldrich </td><td> SL2 </td><td></td></tr><tr><td> SU-8 2025 </td><td> MicroChem </td><td> Y111069 </td><td></td></tr><tr><td> SU-8 Developer </td><td> MicroChem </td><td> Y020100 </td><td></td></tr><tr><td> Pompa a siringa </td><td> Harvard Apparatus </td><td> 70-3008 </td><td> PHD-ULTRA </td></tr><tr><td> tubi (più grande) </td><td> Cole-Parmer Instrument Società </td><td> 06418-02 </td><td> Tygonreg microbore tubi, 0.020 &quot;ID x 0.060&quot; OD </td></tr><tr><td> tubi (più piccolo) </td><td> Cole-Parmer Instrument Società </td><td> 06417-11 </td><td> PTFE microbore tubi, 0.012 &quot;ID x 0.030&quot; OD </td></tr></tbody></table>

endothelialized microfluidics for studying microvascular interactions in hematologic diseases

I progressi nelle tecniche di microfabbricazione hanno permesso la produzione di sistemi microfluidici economici e riproducibili per lo svolgimento di esperimenti biologici e biochimici alla micro-scala nanometrica e 1,2. Inoltre, microfluidica sono stati specificamente utilizzato per analizzare quantitativamente processi ematologiche e microvascolari, a causa della loro capacità di controllare facilmente il ambiente dinamico fluidico condizioni biologiche e 3-6. Come tale, i ricercatori hanno più recentemente usati sistemi microfluidici per studiare deformabilità delle cellule del sangue, cellule aggregazione e flusso sanguigno microvascolare, e globuli-endoteliali interazioni cellulari 6-13. Tuttavia, questi sistemi microfluidici o non includere una coltura di cellule endoteliali o erano più grandi più pertinenti sizescale microvascolari di processi patologici. Una piattaforma microfluidica con colture di cellule endoteliali che riassume con precisione il cellulare, fisico e hemodynambiente Amic della microcircolazione è necessaria per migliorare la nostra comprensione della fisiopatologia sottostante biofisico di malattie ematologiche che coinvolgono il microcircolo. Qui riportiamo un metodo per creare un &quot;endothelialized&quot; modello in vitro del microcircolo, con un semplice, singolo processo di microfabbricazione maschera in combinazione con tecniche di celle standard endoteliali coltura, per studiare le interazioni patologiche microvascolari biofisiche che si verificano nella malattia ematologica. Questo &quot;microcircolo-on-a-chip&quot; fornisce il ricercatore con un saggio robusto che controlla strettamente biologica nonché delle condizioni biofisiche e viene gestito tramite una pompa standard siringa e brightfield / microscopia a fluorescenza. Parametri quali le condizioni emodinamiche microcircolazione, il tipo di cellule endoteliali, le cellule del sangue di tipo (s) e di concentrazione (s), la droga / concentrazione inibitoria, ecc, possono essere facilmente controllati. Come tale, il microsistema fornisceun metodo per indagare quantitativamente processi patologici in cui viene compromessa flusso microvascolare causa di alterazioni nella adesione cellulare, aggregazione, e deformabilità, una capacità disponibile con saggi esistenti.

Watch this Scientific Journal Video about Endothelialized Microfluidics for Studying Microvascular Interactions in Hematologic Diseases at JoVE.com

Endothelialized Microfluidics for Studying Microvascular Interactions in Hematologic Diseases

Un metodo per la cultura un monostrato di cellule endoteliali durante l&#39;intera superficie interna 3D di un dispositivo a microfluidi con microvascolare canali di dimensioni (&lt;30 micron) è descritta. Questo<em&gt; In vitro</em&gt; Modello microvascolarizzazione consente lo studio di interazioni tra le cellule del sangue biofisiche, cellule endoteliali, e fattori solubili in malattie ematologiche.

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 ...

endothelialized-microfluidics-for-studying-microvascular-interactions

Research

JoVE Journal

Bioengineering

15.8K Views.  Emory University School of Medicine. Un metodo per la cultura un monostrato di cellule endoteliali durante l'intera superficie interna 3D di un dispositivo a microfluidi con microvascolare canali di dimensioni (

Video: Endothelialized Microfluidics for Studying Microvascular Interactions in Hematologic Diseases

Microfluidic Mixers for Studying Protein Folding

The process by which a protein folds into its native conformation is highly relevant to biology and human health yet still poorly understood. One reason for this is that folding takes place over a wide range of timescales, from nanoseconds to seconds or longer, depending on the protein1. Conventional stopped-flow mixers have allowed measurement of folding kinetics starting at about 1 ms. We have recently developed a microfluidic mixer that dilutes denaturant ~100-fold in ~8 &mu;s2. Unlike a stopped-flow mixer, this mixer operates in the laminar flow regime in which turbulence does not occur. The absence of turbulence allows precise numeric simulation of all flows within the mixer with excellent agreement to experiment3-4.
Laminar flow is achieved for Reynolds numbers Re &le;100. For aqueous solutions, this requires micron scale geometries. We use a hard substrate, such as silicon or fused silica, to make channels 5-10 &mu;m wide and 10 &mu;m deep (See Figure 1). The smallest dimensions, at the entrance to the mixing region, are on the order of 1 &mu;m in size. The chip is sealed with a thin glass or fused silica coverslip for optical access. Typical total linear flow rates are ~1 m/s, yielding Re~10, but the protein consumption is only ~0.5 nL/s or 1.8 &mu;L/hr. Protein concentration depends on the detection method: For tryptophan fluorescence the typical concentration is 100 &mu;M (for 1 Trp/protein) and for FRET the typical concentration is ~100 nM.
The folding process is initiated by rapid dilution of denaturant from 6 M to 0.06 M guanidine hydrochloride. The protein in high denaturant flows down a central channel and is met on either side at the mixing region by buffer without denaturant moving ~100 times faster (see Figure 2). This geometry causes rapid constriction of the protein flow into a narrow jet ~100 nm wide. Diffusion of the light denaturant molecules is very rapid, while diffusion of the heavy protein molecules is much slower, diffusing less than 1 &mu;m in 1 ms. The difference in diffusion constant of the denaturant and the protein results in rapid dilution of the denaturant from the protein stream, reducing the effective concentration of the denaturant around the protein. The protein jet flows at a constant rate down the observation channel and fluorescence of the protein during folding can be observed using a scanning confocal microscope5.

In this work we explain the fabrication and use of a microfluidic mixer capable of mixing two solutions in ~8 &mu;s. We also demonstrate the use of these mixers with spectroscopic detection using UV fluorescence and fluorescence resonance energy transfer (FRET).

Miscelatori per lo studio microfluidici Protein Folding

The process by which a protein folds into its native conformation is highly relevant to biology and human health yet still poorly understood. One reason ...

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Bioingegneria

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 ...

Procedure for the Development of Multi-depth Circular Cross-sectional Endothelialized Microchannels-on-a-chip

Efforts have been focused on developing in vitro assays for the study of microvessels because in vivo animal studies are more time-consuming, expensive, and observation and quantification are very challenging. However, conventional in vitro microvessel assays have limitations when representing in vivo microvessels with respect to three-dimensional (3D) geometry and providing continuous fluid flow. Using a combination of photolithographic reflowable photoresist technique, soft lithography, and microfluidics, we have developed a multi-depth circular cross-sectional endothelialized microchannels-on-a-chip, which mimics the 3D geometry of in vivo microvessels and runs under controlled continuous perfusion flow. A positive reflowable photoresist was used to fabricate a master mold with a semicircular cross-sectional microchannel network. By the alignment and bonding of the two polydimethylsiloxane (PDMS) microchannels replicated from the master mold, a cylindrical microchannel network was created. The diameters of the microchannels can be well controlled. In addition, primary human umbilical vein endothelial cells (HUVECs) seeded inside the chip showed that the cells lined the inner surface of the microchannels under controlled perfusion lasting for a time period between 4 days to 2 weeks.

A microchannels-on-a-chip platform was developed by the combination of photolithographic reflowable photoresist technique, soft lithography, and microfluidics. The endothelialized microchannels platform mimics the three-dimensional (3D) geometry of in vivo microvessels, runs under controlled continuous perfusion flow, allows for high-quality and real-time imaging&#160;and can be applied for microvascular research.

Procedura per lo sviluppo di multi-profondità circolare a sezione trasversale Endothelialized microcanali-on-a-chip

Efforts have been focused on developing in vitro assays for the study of microvessels because in vivo animal studies are more time-consuming, expensive, ...

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae

Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of the Drosophila larva makes it an attractive model organism for live imaging studies. However, an important challenge for live imaging techniques is to noninvasively immobilize and position an animal on the microscope. This protocol presents a simple and easy to use method for immobilizing and imaging Drosophila larvae on a polydimethylsiloxane (PDMS) microfluidic device, which we call the 'larva chip'. The larva chip is comprised of a snug-fitting PDMS microchamber that is attached to a thin glass coverslip, which, upon application of a vacuum via a syringe, immobilizes the animal and brings ventral structures such as the nerve cord, segmental nerves, and body wall muscles, within close proximity to the coverslip. This allows for high-resolution imaging, and importantly, avoids the use of anesthetics and chemicals, which facilitates the study of a broad range of physiological processes. Since larvae recover easily from the immobilization, they can be readily subjected to multiple imaging sessions. This allows for longitudinal studies over time courses ranging from hours to days. This protocol describes step-by-step how to prepare the chip and how to utilize the chip for live imaging of neuronal events in 3rd instar larvae. These events include the rapid transport of organelles in axons, calcium responses to injury, and time-lapse studies of the trafficking of photo-convertible proteins over long distances and time scales. Another application of the chip is to study regenerative and degenerative responses to axonal injury, so the second part of this protocol describes a new and simple procedure for injuring axons within peripheral nerves by a segmental nerve crush.

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae

Drosophila larvae are an attractive model system for live imaging due to their translucent cuticle and powerful genetics. This protocol describes how to utilize a single-layer PDMS device, called the &#39;larva chip&#39; for live imaging of cellular processes within neurons of 3rd instar Drosophila larvae.

Utilizzando Chips microfluidica per l&#39;imaging dal vivo e studio delle risposte lesioni in<em&gt; Drosophila</em&gt; Larve

Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of ...

The Submerged Printing of Cells onto a Modified Surface Using a Continuous Flow Microspotter

The printing of cells for microarray applications possesses significant challenges including the problem of maintaining physiologically relevant cell phenotype after printing, poor organization and distribution of desired cells, and the inability to deliver drugs and/or nutrients to targeted areas in the array. Our 3D microfluidic printing technology is uniquely capable of sealing and printing arrays of cells onto submerged surfaces in an automated and multiplexed manner. The design of the microfluidic cell array (MFCA) 3D fluidics enables the printhead tip to be lowered into a liquid-filled well or dish and compressed against a surface to form a seal. The soft silicone tip of the printhead behaves like a gasket and is able to form a reversible seal by applying pressure or backing away. Other cells printing technologies such as pin or ink-jet printers are unable to print in submerged applications. Submerged surface printing is essential to maintain phenotypes of cells and to monitor these cells on a surface without disturbing the material surface characteristics. By printing onto submerged surfaces, cell microarrays are produced that allow for drug screening and cytotoxicity assessment in a multitude of areas including cancer, diabetes, inflammation, infections, and cardiovascular disease.

This 3D microfluidic printing technology prints arrays of cells onto submerged surfaces. We describe how arrays of cells are delivered microfluidically in 3D flow cells onto submerged surfaces. By printing onto submerged surfaces, cell microarrays were produced that allow for drug screening and cytotoxicity assessment in a multitude of areas.

La stampa Sommerso di celle su una superficie modificata utilizzando un flusso di Microspotter continua

The printing of cells for microarray applications possesses significant challenges including the problem of maintaining physiologically relevant cell ...

Sandwich-like Microenvironments to Harness Cell/Material Interactions

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. However in vivo, most of the cells are completely surrounded by the extracellular matrix (ECM), resulting in a three-dimensional (3D) distribution of receptors. This may trigger differences in the outside-in signaling pathways and thus in cell behavior.
This article shows that stimulating the dorsal receptors of cells already adhered to a 2D substrate by overlaying a film of a new material (a sandwich-like culture) triggers important changes with respect to standard 2D cultures. Furthermore, the simultaneous excitation of ventral and dorsal receptors shifts cell behavior closer to that found in 3D environments. Additionally, due to the nature of the system, a sandwich-like culture is a versatile tool that allows the study of different parameters in cell/material interactions, e.g., topography, stiffness and different protein coatings at both the ventral and dorsal sides. Finally, since sandwich-like cultures are based on 2D substrates, several analysis procedures already developed for standard 2D cultures can be used normally, overcoming more complex procedures needed for 3D systems.

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. ...

Multicolor Fluorescence Detection for Droplet Microfluidics Using Optical Fibers

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as multiplex assays, enzyme evolution, and molecular biology enhanced cell sorting require the detection of two or more colors of fluorescence. Standard multicolor detection systems that couple free space lasers to epifluorescence microscopes are bulky, expensive, and difficult to maintain. In this paper, we describe a scheme to perform multicolor detection by exciting discrete regions of a microfluidic channel with lasers coupled to optical fibers. Emitted light is collected by an optical fiber coupled to a single photodetector. Because the excitation occurs at different spatial locations, the identity of emitted light can be encoded as a temporal shift, eliminating the need for more complicated light filtering schemes. The system has been used to detect droplet populations containing four unique combinations of dyes and to detect sub-nanomolar concentrations of fluorescein.

Multicolor fluorescence detection in droplet microfluidics typically involves bulky and complex epifluorescence microscope-based detection systems. Here we describe a compact and modular multicolor detection scheme that utilizes an array of optical fibers to temporally encode multicolor data collected by a single photodetector.

Multicolore fluorescenza Detection per Droplet microfluidica tramite fibre ottiche

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as ...

Isolation of Murine Adipose Tissue-derived Microvascular Fragments as Vascularization Units for Tissue Engineering

A functional microvascular network is of pivotal importance for the survival and integration of engineered tissue constructs. For this purpose, several angiogenic and prevascularization strategies have been established. However, most cell-based approaches include time-consuming in vitro steps for the formation of a microvascular network. Hence, they are not suitable for intraoperative one-step procedures. Adipose tissue-derived microvascular fragments (ad-MVF) represent promising vascularization units. They can be easily isolated from fat tissue and exhibit a functional microvessel morphology. Moreover, they rapidly reassemble into new microvascular networks after in vivo implantation. In addition, ad-MVF have been shown to induce lymphangiogenesis. Finally, they are a rich source of mesenchymal stem cells, which may further contribute to their high vascularization potential. In previous studies we have demonstrated the remarkable vascularization capacity of ad-MVF in engineered bone and skin substitutes. In the present study, we report on a standardized protocol for the enzymatic isolation of ad-MVF from murine fat tissue.

We present a protocol to isolate adipose tissue-derived microvascular fragments that represent promising vascularization units. They can be rapidly isolated, do not require in vitro processing and, thus, may be used for one-step prevascularization in different fields of tissue engineering.

L&#39;isolamento di Murine adiposo derivate dal tessuto microvascolare Frammenti come vascolarizzazione Unità per Tissue Engineering

A functional microvascular network is of pivotal importance for the survival and integration of engineered tissue constructs. For this purpose, several ...

Interfacing Microfluidics with Microelectrode Arrays for Studying Neuronal Communication and Axonal Signal Propagation

Microelectrode arrays (MEAs) are widely used to study neuronal function in vitro. These devices allow concurrent non-invasive recording/stimulation of electrophysiological activity for long periods. However, the property of sensing signals from all sources around every microelectrode can become unfavorable when trying to understand communication and signal propagation in neuronal circuits. In a neuronal network, several neurons can be simultaneously activated and can generate overlapping action potentials, making it difficult to discriminate and track signal propagation. Considering this limitation, we have established an in vitro setup focused on assessing electrophysiological communication, which is able to isolate and amplify axonal signals with high spatial and temporal resolution. By interfacing microfluidic devices and MEAs, we are able to compartmentalize neuronal cultures with a well-controlled alignment of the axons and microelectrodes. This setup allows recordings of spike propagation with a high signal-to-noise ratio over the course of several weeks. Combined with specialized data analysis algorithms, it provides detailed quantification of several communication related properties such as propagation velocity, conduction failure, firing rate, anterograde spikes, and coding mechanisms.
This protocol demonstrates how to create a compartmentalized neuronal culture setup over substrate-integrated MEAs, how to culture neurons in this setup, and how to successfully record, analyze and interpret the results from such experiments. Here, we show how the established setup simplifies the understanding of neuronal communication and axonal signal propagation. These platforms pave the way for new in vitro models with engineered and controllable neuronal network topographies. They can be used in the context of homogeneous neuronal cultures, or with co-culture configurations where, for example, communication between sensory neurons and other cell types is monitored and assessed. This setup provides very interesting conditions to study, for example, neurodevelopment, neuronal circuits, information coding, neurodegeneration and neuroregeneration approaches.

This protocol aims to demonstrate how to combine in vitro microelectrode arrays with microfluidic devices for studying action potential transmission in neuronal cultures. Data analysis, namely the detection and characterization of propagating action potentials, is performed using a new advanced, yet user-friendly and freely available, computational tool.

Microfluidica di interfacciamento con microelettrodi per studiare comunicazione neuronale e propagazione del segnale assonale

Microelectrode arrays (MEAs) are widely used to study neuronal function in vitro. These devices allow concurrent non-invasive recording/stimulation of ...

Human Neural Organoids for Studying Brain Cancer and Neurodegenerative Diseases

The lack of relevant in vitro neural models is an important obstacle on medical progress for neuropathologies. Establishment of relevant cellular models is crucial both to better understand the pathological mechanisms of these diseases and identify new therapeutic targets and strategies. To be pertinent, an in vitro model must reproduce the pathological features of a human disease. However, in the context of neurodegenerative disease, a relevant in vitro model should provide neural cell replacement as a valuable therapeutic opportunity. 
Such a model would not only allow screening of therapeutic molecules but also can be used to optimize neural protocol differentiation [for example, in the context of transplantation in Parkinson's disease (PD)]. This study describes two in vitro protocols of 1) human glioblastoma development within a human neural organoids (NO) and 2) neuron dopaminergic (DA) differentiation generating a three-dimensional (3D) organoid. For this purpose, a well-standardized protocol was established that allows the production of size-calibrated neurospheres derived from human embryonic stem cell (hESC) differentiation. The first model can be used to reveal molecular and cellular events occurring during in glioblastoma development within the neural organoid, while the DA organoid not only represents a suitable source of DA neurons for cell therapy in Parkinson's disease but also can be used for drug testing.

This study introduces and describes protocols to derive two specific human neural organoids&#160;as a relevant and accurate model for studying 1) human glioblastoma development within human neural organoids exclusively in humans and 2) neuron dopaminergic differentiation generating a three-dimensional organoid.

Organidi neurali umani per lo studio del cancro al cervello e delle malattie neurodegenerative

The lack of relevant in vitro neural models is an important obstacle on medical progress for neuropathologies. Establishment of relevant cellular models ...