Ringraziamo Mike Vahey dal Fletcher Lab presso l&#39;Università della California, Berkeley per consigli sui parametri microjetting. Questo lavoro è stato sponsorizzato dal NIH concedere DP2 HL117748-01.

1. Infinity Camera Fabrication <ol><li> Progettare la camera infinito (chiamato per la sua forma), utilizzando un software di progettazione assistita (CAD), e salvare il file in modo che sia compatibile con un cutter laser. Per creare questa forma, distinte due cerchi del diametro di 0.183 in una distanza da centro a centro di 0.15 dentro Tagliare la camera da 1/8- 3/16 in acrilico trasparente con il taglio laser. La forma dell&#39;infinito facilita interfaccia gocciolina formazione doppio strato e stabilità. </li><li> Praticare un 1/16 nel foro attraverso il bordo della camera acrilico al pozzo a forma di sfioro. Ripetere sul lato opposto. Tagliare un piccolo rettangolo da una lastra acrilica 0,2 millimetri con le forbici o un cutter laser per servire come fondo ai pozzetti. </li><li> Applicare uno strato sottile di adesivo ma completa asciugatura rapida ad un lato della acrilico 0,2 mm di colla al fondo della camera. Tenere il acrilica 0,2 millimetri saldamente in posizione contro il fondo della camera ed erogarela colla all&#39;interfaccia per consentire la colla per creare una tenuta ma evitare che copre l&#39;area di visualizzazione. Assicurarsi di allineare l&#39;acrilico in modo che il suo bordo è a filo con il bordo della parete della camera e copre completamente il ritaglio camera di sfioro. Ciò consentirà sufficiente penetrazione del getto e di evitare perdite del pozzo. </li><li> Tagliate due piccoli pezzi di gomma naturale per coprire i fori. Applicare l&#39;adesivo ad asciugatura rapida attorno al foro. Posizionare la gomma sopra il foro e premere su tutte le aree con un paio di pinze per fissare. Ripetere l&#39;operazione per entrambi i fori. Assicurarsi che tutte le connessioni incollati guarnizioni complete in modo da evitare eventuali perdite. </li><li> Utilizzando un 23 G, 1 in ago, fare un buco nella gomma naturale su entrambi i lati della camera di facilitare l&#39;inserimento della punta a getto d&#39;inchiostro piezoelettrica. </li></ol> 2. Preparazione Sperimentale Conservare la soluzione magazzino lipidi in cloroformio in un -20 ° C freezer. Per questo studio, o 1,2-diphytanoyl-sn-glycero-3-fosfocolina (DPhPC) o 1,2-dioleoyl-sn-glycero-3-fosfocolina (DOPC) è stato utilizzato. 2 ml di 25 mg / ml soluzione lipidica è tipicamente preparato in questo protocollo e durerà per due mesi se conservato a -20 ° C. <ol><li> Per risospendere il lipide n-decano, prima trasferirlo da uno stock flacone in un piccolo barattolo di vetro, e asciugare delicatamente sotto argon o azoto. Tenere il vaso in un angolo durante l&#39;asciugatura per esporre più area al gas, permettendo il lipide asciugare più velocemente. </li><li> Con il tappo del vaso solo leggermente avvitato, lasciare che la soluzione essiccata per sedersi sotto vuoto in essiccatore per 1-2 ore. Poi aggiungere n-decano a resolubilize lipide a 25 mg / ml. Vortex la soluzione brevemente lipidico e sonicare in un bagno sonicatore per 15 min. Conservare il lipide ricostituita in n-decano a -20 ° C. </li><li> Preparare una soluzione madre di saccarosio. In una provetta da 1,5 ml, aggiungere 900 ml di saccarosio mm 300 e 100 ml1% di metilcellulosa (MC). Viene aggiunta metilcellulosa per aumentare la viscosità della soluzione, che aiuta a getto. Passaggio facoltativo: Aggiungi un optional 1 ml di colore scuro colorante alimentare o perline fluorescenti per dare più contrasto o fluorescenza per immagini, rispettivamente. Questa soluzione è una soluzione di 300 mOsm saccarosio progettato per abbinare osmolarità cellulare e per fornire contrasto durante microjetting. </li><li> Aspirare la soluzione in una siringa monouso di plastica 1 ml. Tenendo la siringa con la punta rivolta verso l&#39;alto, sfogliare l&#39;albero ripetutamente per espellere eventuali bolle verso la punta, e spingere lo stantuffo per espellere l&#39;aria intrappolata. Assicurati di evacuare tutta l&#39;aria dalla siringa prima di procedere, in quanto essa possa interferire con la corretta contrazione piezoelettrico responsabile di getto. </li><li> Installare un filtro da 0,22 micron sull&#39;estremità della siringa. Per evitare che l&#39;aria venga intrappolata nel filtro, tenere la siringa verticale e spingere lo stantuffo finché si forma una gocciolina sopra la punta. Nota: A33 millimetri di diametro unità filtro a siringa ha operato meglio, ma filtri alternativi piccolo come un filtro a siringa da 3 mm di diametro può essere utilizzato per ridurre il volume morto. </li><li> Svitare l&#39;adattatore Luer femmina del getto di inchiostro, e premere saldamente in posizione sopra l&#39;estremità del filtro. Anche in questo caso, espellere il fluido per evitare di intrappolare aria. Avvitare la parte superiore del getto di inchiostro. Fluido deve recarsi presso la punta del getto di inchiostro dopo che è stato fissato completamente. </li></ol> 3. Preparazione dei componenti <ol><li> Utilizzando un v-clamp, montare il gruppo siringa sul palco microscopio. Collegare il filo dal getto d&#39;inchiostro al controller a getto d&#39;inchiostro. Nota: Una fase usanza è stata costruita per questo protocollo, mentre la scenografia può essere determinato dall&#39;utente, è fondamentale avere il controllo xyz indipendente della siringa e del controllo xy del supporto del campione. </li><li> Determinare l&#39;ingrandimento e la combinazione obiettivo necessario per raggiungere la rappresentazione desiderata. Qui vengono utilizzati un obiettivo 10X e 10X oculare. </li&gt; <li> Utilizzare una telecamera ad alta velocità (≥ 1.000 fps) per visualizzare il getto e la generazione di vescicole. Prima di imaging, effettuare la calibrazione fotocamera necessario. Utilizzare auto-trigger basato su immagini all&#39;interno del software della fotocamera per avviare la cattura delle immagini. </li><li> Montare la camera di sfioro sul palco microscopio. Fissare la camera con nastro adesivo in posizione sul palcoscenico. </li><li> Allineare con cura la punta a getto d&#39;inchiostro con il foro perforato in gomma naturale (cfr. figura 1b). A tale scopo, portare il getto d&#39;inchiostro vicino alla camera e regolare il posizionamento a occhio, poi effettuare regolazioni più precise attraverso la lente microscopio. Procedere con cautela, in quanto i movimenti grossolani possono danneggiare la punta a getto d&#39;inchiostro. </li><li> Una volta che l&#39;inchiostro è allineato, eseguire il montaggio siringa lontano dalla camera per evitare danni al getto di inchiostro durante il caricamento dei pozzetti. Assicurarsi che il moto del getto di inchiostro è unidirezionale in modo che rimanga allineato con il foro nella membrana. </li><li> Premere delicatamente sulla plunger del gruppo siringa fino a piccole forme gocciolina presso l&#39;ugello a getto d&#39;inchiostro. Questo fornisce alcune contropressione iniziale. </li><li> Inserire i parametri di getto. Per un&#39;onda bipolare trapezoidale, utilizzare i seguenti parametri: frequenza di 20 kHz impulso, 3 tempo msec luogo, durata 35 msec impulso, 3 msec di tempo di caduta, 65 V di tensione applicata (ampiezza di impulso), e 100 impulsi al getto di innesco (numero di impulsi) . Tuttavia, la tensione applicata (ampiezza di impulso) e numero di impulsi (impulsi jet per scatto) possono essere variate per controllare le dimensioni delle vescicole. </li></ol> 4. Vesicle Generation <ol><li> Aggiungere 25-30 ml soluzione lipidica sospesa in n-decano alla camera all&#39;infinito, che copre l&#39;intera superficie di entrambi i pozzetti. </li><li> Aggiungere 25 ml di glucosio (osmolarità della stessa come la soluzione di saccarosio) al bordo più esterno di un pozzo, pipettaggio lentamente ed uniformemente. Su deposizione, una goccia di glucosio dovrebbe costituire, perché la soluzione di glucosio e lipidi non si mescolano. Aggiungi un altro 251; l di glucosio al pozzo opposto, che farà un&#39;altra goccia e formare la membrana a doppio strato lipidico nel centro della camera entro 5-10 min. </li><li> Inserire l&#39;inchiostro attraverso la gomma naturale, e guidare con attenzione verso il doppio strato di interfaccia gocciolina. Avvicinatevi al doppio strato lentamente, come il getto d&#39;inchiostro introdotto sposti volume e può rompersi il doppio strato. </li><li> Quando l&#39;inchiostro è a ~ 200 micron, applicare il getto con le impostazioni descritte al punto 3.8. La distanza dal doppio strato può variare principalmente in funzione del numero di impulsi di tensione e, tra gli altri parametri. Questo protocollo raccomanda aumentando lentamente impostazioni (tensione e numero di impulsi) e osservando doppio strato deformazione. </li></ol> 5. Pulizia della Equipaggiamento <ol><li> Staccare il gruppo siringa dal palco microscopio, e smaltire la siringa in plastica da 1 ml e filtrare. </li><li> Per pulire il getto di inchiostro, aspirare le seguenti soluzioni in ordine immergendo la punta della solution 7-10x ciascuno: il 70% di etanolo, 2% soluzione Neutrad in acqua calda, il 70% di etanolo, e DDH 2 O. Se il getto d&#39;inchiostro non si adatta in modo sicuro sul pipetta di aspirazione, tagliare un puntale per formare un adattatore. </li><li> Asciugare la camera con il tessuto. Posizionare la camera di infinito in un bicchiere da 250 ml con il 2% v / v Neutrad in acqua calda, e sonicare per 5-10 min. Dopo sonicazione, asciugare accuratamente i pozzetti sotto aria compressa. L&#39;umidità nei pozzetti può compromettere la stabilità della membrana lipidica a due strati, per cui si raccomanda inoltre che le camere sono poste in un forno a 60 ° C per 15 min. </li></ol>

<ol>
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Nessun conflitto di interessi dichiarati.

Sono state sviluppate molte tecniche per la generazione di vescicole, compresi electroformation, emulsione, e generazione gocciolina 14-16. Tuttavia, nuove tecniche sperimentali sono necessari per consentire la progettazione di sistemi biologici con crescente somiglianza ai sistemi viventi. Metodi di microfluidica, in particolare, hanno offerto un aumento del livello di controllo che regola unilamellarity membrana, monodispersity di dimensioni e contenuti interni 17,18, portando i modelli vescicole...

<html> E &#39;diventato sempre più chiaro che la biologia delle cellule è un problema multi-scala che coinvolge integrando la nostra comprensione dalle molecole alle cellule. Di conseguenza, sapere esattamente come le molecole lavorano individualmente non è sufficiente per capire i comportamenti cellulari complesse. Ciò è in parte dovuto all&#39;esistenza di comportamenti emergenti dei sistemi a più componenti, come esemplificato dalla ricostituzione di interazione rete actina con vescicole doppio strato lipidico 4, del fuso mitotico in Xenopus estrarre 5, e le dinamiche spaziali della batteriche macchinari divisione cellulare 6. Un modo per integrare l&#39;approccio riduzionista della dissezione dei processi molecolari dei sistemi viventi è quello di prendere l&#39;approccio opposto di ricostituire comportamenti cellulari utilizzando un set minimo di componenti biologiche. Una parte importante di questo approccio comporta l&#39;incapsulamento affidabile di biomolecole in un volume confinato, una caratteristica fondamentale di una cella. e_content &quot;&gt; Esistono diverse strategie per incapsulare biomolecole per studiare sistemi biomimetici. Il sistema più biologicamente rilevante è membrane bistrato lipidico, che imitano i vincoli fisici e biochimici imposti dalla membrana plasmatica della cellula. Formazione di vescicole unilamellari giganti (GUVs) da electroformation 7, una delle tecniche più utilizzate per la generazione GUV 14, tipicamente ha una resa di incapsulamento povero a causa della sua incompatibilità con tampone di sali 8. Electroformation richiede anche grandi volumi di campione (&gt; 100 ml), che potrebbe essere un problema per lavorare con proteine ​​purificate , e incorpora inefficiente grandi molecole causa di difficoltà di diffusione tra strati lipidici ravvicinati. Sono stati sviluppati diversi approcci per la generazione microfluidici vescicole lipidiche. I metodi a doppio emulsione, che passano attraverso due componenti interfacce tra strati acqua-olio-acqua (W / O / W), si basa sul evaporazione di un volatile solvente per guidare la formazione di doppio strato lipidico 9. Altri hanno usato una linea di assemblaggio microfluidica che produce un flusso continuo di lipidi vescicole a doppio strato 10 o in due fasi indipendenti 11. Abbiamo sviluppato una tecnica alternativa basata su rapidamente applicare impulsi di fluido contro un bistrato un&#39;interfaccia gocciolina 12 per produrre GUVs di dimensione controllata, composizione e incapsulamento. Il nostro approccio, noto come getto microfluidica, offre i vantaggi combinati di diverse tecniche di generazione vescicola esistenti, fornendo un metodo per la creazione di sistemi biomolecolari funzionali per investigare una varietà di problemi biologici.

<table border="0" cellpadding="0" cellspacing="0" style="width:633px;" width="633">
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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:153px;">Name</td>
			<td style="width:163px;">Company</td>
			<td style="width:157px;">Catalog Number</td>
			<td style="width:160px;">Comments</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:153px;">Piezoelectric Inkjet</td>
			<td style="width:163px;">MicroFab Technologies</td>
			<td style="width:157px;">MJ-AL-01-xxx</td>
			<td style="width:160px;">xxx denotes orifice diameter in microns</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:153px;">Jet Drive III Controller</td>
			<td style="width:163px;">MicroFab Technologies</td>
			<td style="width:157px;">CT-M3-02</td>
			<td style="width:160px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:153px;">High-speed camera</td>
			<td style="width:163px;">Vision Research</td>
			<td style="width:157px;">MiroEX2</td>
			<td style="width:160px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:153px;">DPhPC lipid in chloroform</td>
			<td style="width:163px;">Avanti</td>
			<td style="width:157px;">850356C</td>
			<td style="width:160px;">Ordered in small aliquots in vials</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:153px;">33mm PVDF filters, 0.2 &#181;m</td>
			<td style="width:163px;">Fisher Scientific</td>
			<td style="width:157px;">SLGV033RS</td>
			<td style="width:160px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:153px;">1ml syringes</td>
			<td style="width:163px;">Fisher Scientific</td>
			<td style="width:157px;">14823434</td>
			<td style="width:160px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:153px;">n-Decane</td>
			<td style="width:163px;">Acros Organics</td>
			<td style="width:157px;">111871000</td>
			<td style="width:160px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:153px;">Glucose</td>
			<td style="width:163px;">Acros Organics</td>
			<td style="width:157px;">410950010</td>
			<td style="width:160px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:153px;">Sucrose</td>
			<td style="width:163px;">Sigma-Aldrich</td>
			<td style="width:157px;">S7903-1KG</td>
			<td style="width:160px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:153px;">Methylcellulose</td>
			<td style="width:163px;">Fisher Scientific</td>
			<td style="width:157px;">NC9084958</td>
			<td style="width:160px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:153px;">1/8&#34; Acrylic</td>
			<td style="width:163px;">McMaster Carr</td>
			<td style="width:157px;">8560K239</td>
			<td style="width:160px;">CAD designs for the infinity-shaped chamber are available upon request</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:153px;">0.2 mm Acrylic</td>
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			<td height="63" style="height:63px;width:153px;">Loctite 3494 UV Strengthening Adhesive</td>
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			<td height="42" style="height:42px;width:153px;">Custom stage</td>
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			<td style="width:160px;">CAD designs are available upon request</td>
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</table>

lipid bilayer vesicle generation using microfluidic jetting

Bottom-up biologia sintetica presenta un nuovo approccio per lo studio e la ricostituzione di sistemi biochimici e, potenzialmente, organismi minime. Questo settore emergente coinvolge ingegneri, chimici, biologi e fisici per progettare e assemblare componenti biologiche di base nei complessi sistemi funzionanti dal basso verso l&#39;alto. Tali sistemi bottom-up potrebbe portare allo sviluppo di cellule artificiali per le indagini biologiche fondamentali e terapie innovative 1,2. Vescicole unilamellari giganti (GUVs) possono fungere da piattaforma modello per la biologia sintetica a causa della loro struttura della membrana cellulare-like e dimensioni. Getto microfluidica, o microjetting, è una tecnica che consente la generazione di GUVs di dimensione controllata, la composizione della membrana, proteine ​​transmembrana incorporazione, e l&#39;incapsulamento 3. Il principio di base di questo metodo è l&#39;uso di impulsi multipli, fluidi ad alta frequenza generati da un dispositivo a getto d&#39;inchiostro piezo-azionato per deformare un l sospesabistrato IPID in un GUV. Il processo è simile a soffiando bolle di sapone da una pellicola di sapone. Variando la composizione della soluzione idromassaggio, la composizione della soluzione comprende, e / o le componenti inclusi nel doppio strato, i ricercatori possono applicare questa tecnica per creare vescicole personalizzate. Questo documento descrive la procedura per generare vescicole semplici da un bistrato un&#39;interfaccia gocciolina da microjetting.

Abbiamo messo insieme un setup getto microfluidica su un microscopio a fluorescenza convenzionale invertito con una fase personalizzata assemblato da parti lavorate e micrometri manuali (Figura 1). Caratterizzazione del getto di inchiostro permette di comprendere meglio il processo di generazione delle vescicole. Variando la distanza tra l&#39;ugello a getto d&#39;inchiostro e doppio strato lipidico colpisce la forza applicata per causare la deformazione della membrana. Vicinanza al doppio strato concen...

Watch this Scientific Journal Video about Lipid Bilayer Vesicle Generation Using Microfluidic Jetting at JoVE.com

Lipid Bilayer Vesicle Generation Using Microfluidic Jetting

Getto microfluidica contro un doppio strato lipidico interfaccia gocciolina fornisce un modo affidabile per generare vescicole con controllo sulla asimmetria membrana, incorporazione di proteine ​​transmembrana, e incapsulamento del materiale. Questa tecnica può essere applicata a studiare una varietà di sistemi biologici in cui sono determinanti biomolecole compartimenti.

Doppio strato lipidico delle vescicole Generation Uso Microfluidic getto

Bottom-up synthetic biology presents a novel approach for investigating and reconstituting biochemical systems and, potentially, minimal organisms. This ...

lipid-bilayer-vesicle-generation-using-microfluidic-jetting

Research

JoVE Journal

Bioengineering

14.7K Views.  University of Michigan.  Getto microfluidica contro un doppio strato lipidico interfaccia gocciolina fornisce un modo affidabile per generare vescicole con controllo sulla asimmetria membrana, incorporazione di proteine ​​transmembrana, e incapsulamento del materiale. Questa tecnica può essere applicata a studiare una varietà di sistemi biologici in cui sono determinanti biomolecole compartimenti. 

Video: Lipid Bilayer Vesicle Generation Using Microfluidic Jetting

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer (SALB) Method

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related processes while conferring biocompatibility and biofunctionality to solid substrates. The spontaneous adsorption and rupture of phospholipid vesicles is the most commonly used method to form SLBs. However, under physiological conditions, vesicle fusion (VF) is limited to only a subset of lipid compositions and solid supports. Here, we describe a one-step general procedure called the solvent-assisted lipid bilayer (SALB) formation method in order to form SLBs which does not require vesicles. The SALB method involves the deposition of lipid molecules onto a solid surface in the presence of water-miscible organic solvents (e.g., isopropanol) and subsequent solvent-exchange with aqueous buffer solution in order to trigger SLB formation. The continuous solvent exchange step enables application of the method in a flow-through configuration suitable for monitoring bilayer formation and subsequent alterations using a wide range of surface-sensitive biosensors. The SALB method can be used to fabricate SLBs on a wide range of hydrophilic solid surfaces, including those which are intractable to vesicle fusion. In addition, it enables fabrication of SLBs composed of lipid compositions which cannot be prepared using the vesicle fusion method. Herein, we compare results obtained with the SALB and conventional vesicle fusion methods on two illustrative hydrophilic surfaces, silicon dioxide and gold. To optimize the experimental conditions for preparation of high quality bilayers prepared via the SALB method, the effect of various parameters, including the type of organic solvent in the deposition step, the rate of solvent exchange, and the lipid concentration is discussed along with troubleshooting tips. Formation of supported membranes containing high fractions of cholesterol is also demonstrated with the SALB method, highlighting the technical capabilities of the SALB technique for a wide range of membrane configurations.

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer SALB Method

We present an experimental protocol to form a supported lipid bilayer on solid substrates without using lipid vesicles. We demonstrate a one-step method to form a lipid bilayer on silicon dioxide and gold as well as supported membranes with cholesterol-enriched domain for various biological applications.

Biomembrane Fabrication dal solvente assistita doppio strato lipidico (SALB) Metodo

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related ...

Ricerca

Bioingegneria

Automated Lipid Bilayer Membrane Formation Using a Polydimethylsiloxane Thin Film

An artificial lipid bilayer, or black lipid membrane (BLM), is a powerful tool for studying ion channels and protein interactions, as well as for biosensor applications. However, conventional BLM formation techniques have several drawbacks and they often require specific expertise and laborious processes. In particular, conventional BLMs suffer from low formation success rates and inconsistent membrane formation time. Here, we demonstrate a storable and transportable BLM formation system with controlled thinning-out time and enhanced BLM formation rate by replacing conventionally used films (polytetrafluoroethylene, polyoxymethylene, polystyrene) to polydimethylsiloxane (PDMS). In this experiment, a porous-structured polymer such as PDMS thin film is used. In addition, as opposed to conventionally used solvents with low viscosity, the use of squalene permitted a controlled thinning-out time via slow solvent absorption by PDMS, prolonging membrane lifetime. In addition, by using a mixture of squalene and hexadecane, the freezing point of the lipid solution was increased (~16 &#176;C), in addition, membrane precursors were produced that can be indefinitely stored and readily transported. These membrane precursors have reduced BLM formation time of &#60; 1 hr and achieved a BLM formation rate of ~80%. Moreover, ion channel experiments with gramicidin A demonstrated the feasibility of the membrane system.

We demonstrate a storable, transportable lipid bilayer formation system. A lipid bilayer membrane can be formed within 1 hr with over 80% success rate when a frozen membrane precursor is brought to ambient temperature. This system will reduce laborious processes and expertise associated with ion channels.

Formazione automatizzato doppio strato lipidico della membrana Utilizzando un Polidimetilsilossano Thin Film

An artificial lipid bilayer, or black lipid membrane (BLM), is a powerful tool for studying ion channels and protein interactions, as well as for ...

Ligand Nano-cluster Arrays in a Supported Lipid Bilayer

Currently there is considerable interest in creating ordered arrays of adhesive protein islands in a sea of passivated surface for cell biological studies. In the past years, it has become increasingly clear that living cells respond, not only to the biochemical nature of the molecules presented to them but also to the way these molecules are presented. Creating protein micro-patterns is therefore now standard in many biology laboratories; nano-patterns are also more accessible. However, in the context of cell-cell interactions, there is a need to pattern not only proteins but also lipid bilayers. Such dual proteo-lipidic patterning has so far not been easily accessible. We offer a facile technique to create protein nano-dots supported on glass and propose a method to backfill the inter-dot space with a supported lipid bilayer (SLB). From photo-bleaching of tracer fluorescent lipids included in the SLB, we demonstrate that the bilayer exhibits considerable in-plane fluidity. Functionalizing the protein dots with fluorescent groups allows us to image them and to show that they are ordered in a regular hexagonal lattice. The typical dot size is about 800 nm and the spacing demonstrated here is 2 microns. These substrates are expected to serve as useful platforms for cell adhesion, migration and mechano-sensing studies.

We present a protocol to functionalize glass with nanometric protein patches surrounded by a fluid lipid bilayer. These substrates are compatible with advanced optical microscopy and are expected to serve as platform for cell adhesion and migration studies.

Array Ligand Nano-cluster in un doppio strato lipidico supportati

Currently there is considerable interest in creating ordered arrays of adhesive protein islands in a sea of passivated surface for cell biological ...

Multi-step Variable Height Photolithography for Valved Multilayer Microfluidic Devices

Microfluidic systems have enabled powerful new approaches to high-throughput biochemical and biological analysis. However, there remains a barrier to entry for non-specialists who would benefit greatly from the ability to develop their own microfluidic devices to address research questions. Particularly lacking has been the open dissemination of protocols related to photolithography, a key step in the development of a replica mold for the manufacture of polydimethylsiloxane (PDMS) devices. While the fabrication of single height silicon masters has been explored extensively in literature, fabrication steps for more complicated photolithography features necessary for many interesting device functionalities (such as feature rounding to make valve structures, multi-height single-mold patterning, or high aspect ratio definition) are often not explicitly outlined. 
Here, we provide a complete protocol for making multilayer microfluidic devices with valves and complex multi-height geometries, tunable for any application. These fabrication procedures are presented in the context of a microfluidic hydrogel bead synthesizer and demonstrate the production of droplets containing polyethylene glycol (PEG diacrylate) and a photoinitiator that can be polymerized into solid beads. This protocol and accompanying discussion provide a foundation of design principles and fabrication methods that enables development of a wide variety of microfluidic devices. The details included here should allow non-specialists to design and fabricate novel devices, thereby bringing a host of recently developed technologies to their most exciting applications in biological laboratories.

Multilayer microfluidic devices often involve the fabrication of master molds with complex geometries for functionality. This article presents a complete protocol for multi-step photolithography with valves and variable height features tunable to any application. As a demonstration, we fabricate a microfluidic droplet generator capable of producing hydrogel beads.

Multi-step altezza variabile fotolitografia per Valvolati multistrato microfluidici Devices

Microfluidic systems have enabled powerful new approaches to high-throughput biochemical and biological analysis. However, there remains a barrier to ...

Generation of Dynamical Environmental Conditions using a High-Throughput Microfluidic Device

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and organs are either highly expensive, like robotics, or lack nanoliter volume and millisecond time accuracy in liquid manipulation. We herein present the design and fabrication of a microfluidic system, which consists of 1,500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. To demonstrate the capacities of the microfluidic device, neural stem cell (NSC) spheres are maintained in the proposed system. We observed that when the NSC sphere is exposed to CXCL in day 1 and EGF in day 2, the round-shaped conformation is well maintained. Variation in the input order of 6 drugs causes morphological changes to the NSC sphere and the expression level representative marker for NSC stemness (i.e., Hes5 and Dcx). These results indicate that dynamic and complex environmental conditions have great effects on NSC differentiation and self-renewal, and the proposed microfluidic device is a suitable platform for high throughput studies on the complex life machinery.

We present a microfluidic system for high throughput studies on complex life machinery, which consists of 1500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. The microfluidic chip allows for the analysis of the highly complex and dynamic micro-environmental conditions in vivo.

Generazione di condizioni ambientali dinamiche utilizzando un dispositivo microfluidico ad alta produttività

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and ...

Formulating and Characterizing Lipid Nanoparticles for Gene Delivery using a Microfluidic Mixing Platform

Lipid-based drug carriers have been used for clinically and commercially available delivery systems due to their small size, biocompatibility, and high encapsulation efficiency. Use of lipid nanoparticles (LNPs) to encapsulate nucleic acids is advantageous to protect the RNA or DNA from degradation, while also promoting cellular uptake. LNPs often contain multiple lipid components including an ionizable lipid, helper lipid, cholesterol, and polyethylene glycol (PEG) conjugated lipid. LNPs can readily encapsulate nucleic acids due to the ionizable lipid presence, which at low pH is cationic and allows for complexation with negatively charged RNA or DNA. Here LNPs are formed by encapsulating messenger RNA (mRNA) or plasmid DNA (pDNA) using rapid mixing of the lipid components in an organic phase and the nucleic acid component in an aqueous phase. This mixing is performed using a precise microfluidic mixing platform, allowing for nanoparticle self-assembly while maintaining laminar flow. The hydrodynamic size and polydispersity are measured using dynamic light scattering (DLS). The effective surface charge on the LNP is determined by measuring the zeta potential. The encapsulation efficiency is characterized using a fluorescent dye to quantify entrapped nucleic acid. Representative results demonstrate the reproducibility of this method and the influence that different formulation and process parameters have on the developed LNPs.

Lipid nanoparticles are developed using a microfluidic mixing platform approach for mRNA and DNA encapsulation.

Formulazione e caratterizzazione di nanoparticelle lipidiche per la somministrazione genica utilizzando una piattaforma di miscelazione microfluidica

Lipid-based drug carriers have been used for clinically and commercially available delivery systems due to their small size, biocompatibility, and high ...

Generation of a Simplified Three-Dimensional Skin-on-a-chip Model in a Micromachined Microfluidic Platform

This work presents a new, cost-effective, and reliable microfluidic platform with the potential to generate complex multilayered tissues. As a proof of concept, a simplified and undifferentiated human skin containing a dermal (stromal) and an epidermal (epithelial) compartment has been modelled. To accomplish this, a versatile and robust, vinyl-based device divided into two chambers has been developed, overcoming some of the drawbacks present in microfluidic devices based on polydimethylsiloxane (PDMS) for biomedical applications, such as the use of expensive and specialized equipment or the absorption of small, hydrophobic molecules and proteins. Moreover, a new method based on parallel flow was developed, enabling the in situ deposition of both the dermal and epidermal compartments. The skin construct consists of a fibrin matrix containing human primary fibroblasts and a monolayer of immortalized keratinocytes seeded on top, which is subsequently maintained under dynamic culture conditions. This new microfluidic platform opens the possibility to model human skin diseases and extrapolate the method to generate other complex tissues.

Here, we present a protocol to generate a three-dimensional simplified and undifferentiated skin model using a micromachined microfluidic platform. A parallel flow approach allows the in situ deposition of a dermal compartment for the seeding of epithelial cells on top, all controlled by syringe pumps.

Generazione di un modello skin-on-a-chip tridimensionale semplificato in una piattaforma microfluidica microlavorazione

This work presents a new, cost-effective, and reliable microfluidic platform with the potential to generate complex multilayered tissues. As a proof of ...

Assembly of Cell Mimicking Supported and Suspended Lipid Bilayer Models for the Study of Molecular Interactions

Model cell membranes are a useful screening tool with applications ranging from early drug discovery to toxicity studies. The cell membrane is a crucial protective barrier for all cell types, separating the internal cellular components from the extracellular environment. These membranes are composed largely of a lipid bilayer, which contains outer hydrophilic head groups and inner hydrophobic tail groups, along with various proteins and cholesterol. The composition and structure of the lipids themselves play a crucial role in regulating biological function, including interactions between cells and the cellular microenvironment, which may contain pharmaceuticals, biological toxins, and environmental toxicants. In this study, methods to formulate uni-lipid and multi-lipid supported and suspended cell mimicking lipid bilayers are described. Previously, uni-lipid phosphatidylcholine (PC) lipid bilayers as well as multi-lipid placental trophoblast-inspired lipid bilayers were developed for use in understanding molecular interactions. Here, methods for achieving both types of bilayer models will be presented. For cell mimicking multi-lipid bilayers, the desired lipid composition is first determined via lipid extraction from primary cells or cell lines followed by liquid chromatography-mass spectrometry (LC-MS). Using this composition, lipid vesicles are fabricated using a thin-film hydration and extrusion method and their hydrodynamic diameter and zeta potential are characterized. Supported and suspended lipid bilayers can then be formed using quartz crystal microbalance with dissipation monitoring (QCM-D) and on a porous membrane for use in a parallel artificial membrane permeability assay (PAMPA), respectively. The representative results highlight the reproducibility and versatility of in vitro cell membrane lipid bilayer models. The methods presented can aid in rapid, facile assessment of the interaction mechanisms, such as permeation, adsorption, and embedment, of various molecules and macromolecules with a cell membrane, helping in the screening of drug candidates and prediction of potential cellular toxicity.

This protocol describes the formation of cell mimicking uni-lipid and multi-lipid vesicles, supported lipid bilayers, and suspended lipid bilayers. These in vitro models can be adapted to incorporate a variety of lipid types and can be used to investigate various molecule and macromolecule interactions.

Assemblaggio di modelli di doppio strato lipidico supportati e sospesi per lo studio delle interazioni molecolari

Model cell membranes are a useful screening tool with applications ranging from early drug discovery to toxicity studies. The cell membrane is a crucial ...

Testing the In Vitro and In Vivo Efficiency of mRNA-Lipid Nanoparticles Formulated by Microfluidic Mixing

Lipid nanoparticles (LNPs) have attracted widespread attention recently with the successful development of the COVID-19 mRNA vaccines by Moderna and Pfizer/BioNTech. These vaccines have demonstrated the efficacy of mRNA-LNP therapeutics and opened the door for future clinical applications. In mRNA-LNP systems, the LNPs serve as delivery platforms that protect the mRNA cargo from degradation by nucleases and mediate their intracellular delivery. The LNPs are typically composed of four components: an ionizable lipid, a phospholipid, cholesterol, and a lipid-anchored polyethylene glycol (PEG) conjugate (lipid-PEG). Here, LNPs encapsulating mRNA encoding firefly luciferase are formulated by microfluidic mixing of the organic phase containing LNP lipid components and the aqueous phase containing mRNA. These mRNA-LNPs are then tested in vitro to evaluate their transfection efficiency in HepG2 cells using a bioluminescent plate-based assay. Additionally, mRNA-LNPs are evaluated in vivo in C57BL/6 mice following an intravenous injection via the lateral tail vein. Whole-body bioluminescence imaging is performed by using an in vivo imaging system. Representative results are shown for the mRNA-LNP characteristics, their transfection efficiency in HepG2 cells, and the total luminescent flux in C57BL/6 mice.

Testing the In Vitro and In Vivo Efficiency of mRNA-Lipid Nanoparticles Formulated by Microfluidic Mixing

Here, a protocol for formulating lipid nanoparticles (LNPs) that encapsulate mRNA encoding firefly luciferase is presented. These LNPs were tested for their potency in vitro in HepG2 cells and in vivo in C57BL/6 mice.

Test dell'efficienza in vitro e in vivo di nanoparticelle mRNA-lipidi formulate mediante miscelazione microfluidica

Lipid nanoparticles (LNPs) have attracted widespread attention recently with the successful development of the COVID-19 mRNA vaccines by Moderna and ...

Configurazione di un dispositivo microfluidico per la produzione di tappi combinatoriali

Bilayer Microfluidic Device for Combinatorial Plug Production

Droplet microfluidics is a versatile tool that allows the execution of a large number of reactions in chemically distinct nanoliter compartments. Such systems have been used to encapsulate a variety of biochemical reactions - from incubation of single cells to implementation of PCR reactions, from genomics to chemical synthesis. Coupling the microfluidic channels with regulatory valves allows control over their opening and closing, thereby enabling the rapid production of large-scale combinatorial libraries consisting of a population of droplets with unique compositions. In this paper, protocols for the fabrication and operation of a pressure-driven, PDMS-based bilayer microfluidic device that can be utilized to generate combinatorial libraries of water-in-oil emulsions called plugs are presented. By incorporating software programs and microfluidic hardware, the flow of desired fluids in the device can be controlled and manipulated to generate combinatorial plug libraries and to control the composition and quantity of constituent plug populations. These protocols will expedite the process of generating combinatorial screens, particularly to study drug response in cells from cancer patient biopsies.

Autore in primo piano: Integrazione di approcci computazionali e sperimentali nell'oncologia di precisione

The fabrication of a polydimethylsiloxane (PDMS)-based bilayer device for the production of combinatorial libraries in water-in-oil emulsions (plugs) is presented here. The necessary hardware and software required to automate plug production are detailed in the protocol, and the production of a quantitative library of fluorescent plugs is also demonstrated.

Dispositivo microfluidico a doppio strato per la produzione di tappi combinatori

Droplet microfluidics is a versatile tool that allows the execution of a large number of reactions in chemically distinct nanoliter compartments. Such ...