Name: polymeric microneedle array fabrication by photolithography
Uploaded: 2015-11-17T13:00:00.000+00:00
Duration: 8 min 15 s
Description: Watch this Scientific Journal Video about Polymeric Microneedle Array Fabrication by Photolithography at JoVE.com

This study was supported by a Singapore National Research Foundation (NRF) Grant NRF2012NRF-POC001-043.

1. Photomask Fabrication <ol><li> Pulire un wafer 4 &quot;vetro con soluzione Piranha (H 2 SO 4 / H 2 O 2 in rapporto 2: 1) per 20 minuti a 120 ° C per immersione in una vasca di quarzo. </li><li> Depositare uno strato di cromo / oro (30 nm di Cr / 1 micron di Au) strato sul wafer di vetro usando un e-beam evaporatore 7 (Figura 1A). <ol><li> Posizionare i wafer in un evaporatore e-beam. Una volta che il vuoto raggiunge 5 x 10 -6 Torr, accendere la sorgente ad alta tensione (10 kV). Controllare lo spessore dalla centrale del monitor. </li><li> Pre-pulita ogni materiale per 30 secondi utilizzando la pistola e-beam, mantenendo l&#39;otturatore &quot;OFF&quot; (per evitare la deposizione sui wafer). </li></ol></li><li> Generare un Cr / Au strato di resina fotosensibile di mascheratura per una profonda incisione umido del vetro. <ol><li> Applicare un photoresist spessore 2 micron filando 5 ml di soluzione per 30 secondi a 3000 rpm utilizzando un sistema di rivestimento spin-spruzzo.Precottura fotoresist su una piastra riscaldante a 100 ° C per 1,5 min. </li><li> Esporre e duro cuocere il fotoresist a 120 ° C per 30 minuti su una piastra calda. È fondamentale per generare una superficie idrofobica e una forte adesione del fotoresist allo strato metallico. Motivo dello strato di Cr / Au utilizzando Cr e Au soluzioni di attacco attraverso il photoresist maschera 8,9. </li></ol></li><li> Per la protezione della superficie del vetro non-modellato, legare temporaneamente il wafer vetro per un wafer di silicio manichino. 9 <ol><li> Posizionare il wafer di vetro su una piastra riscaldante a 110 ° C e sciogliere la cera sul lato opposto del wafer di vetro (in modo tale che l&#39;intera superficie del wafer è coperto di cera). </li><li> Collocare un wafer di silicio manichino in contatto con il wafer di vetro e premere per rimuovere l&#39;eccesso di cera. Per evitare fuoriuscite di cera, posizionare una carta velina camera pulita sulla piastra. </li></ol></li><li> Eseguire attacco isotropo della lente utilizzando acido fluoridrico ottimizzato (49% v / v)soluzione di acido cloridrico (37% v / v) (in un rapporto volumetrico di 10: 1). con un agitatore magnetico per 10 min 8.5 Presenza di HCl è critico per il raggiungimento di una buona qualità superficiale di lenti generati. <ol><li> Assicurarsi che la velocità di attacco è di 7 micron / min; utilizzando un volume totale di 200 ml di soluzione di attacco. Eseguire incisione in un contenitore di plastica e prendere precauzioni di sicurezza per questa fase di lavorazione. </li><li> Pulire il wafer in (DI) acqua deionizzata da risciacquo e asciugatura ulteriormente a temperatura ambiente. </li></ol></li><li> Dopo il completamento della procedura, separare il wafer di vetro dal wafer di silicio manichino e riscaldare la cera con una piastra calda a 100 ° C per 15 sec. Come la cera si scioglie a questa temperatura, staccare il wafer di vetro dal wafer di silicio manichino. </li><li> Rimuovere rimanente cera, il photoresist e gli strati sovrastanti Cr / Au ai bordi delle lenti con ultrasuoni per 1 ora usando N-metil-2-pirrolidone come solvente a 80 ° C in un bagno di ultrasuoni.</li><li> Creare uno stampo replica PDMS delle microlenti fabbricati sui fotomaschere 11. </li><li> Caratterizzare le dimensioni fotomaschere (lunghezza e larghezza) e lo stampo microlenti PDMS (profondità e diametro) repliche utilizzando rispettivamente un microscopio elettronico a scansione e stereomicroscopio. 12-14 </li></ol> 2. Alberi MN Fabrication <ol><li> Creare una cavità di 2,5 cm × 0,9 cm utilizzando i vetrini montate su entrambi i lati di un vetro. Il numero dei vetrini impilati su ciascun lato determinerà l&#39;altezza della cavità noto come lo spessore del distanziale (Figura 1B). </li><li> Fissare ogni strato del vetrino applicando uno strato sottile della soluzione prepolimero contenente poli (glicole etilenico) diacrilato (PEGDA, MW = 258 Da) con 0,5% w / w 2-idrossi-2-metil-propiofenone (HMP) sulla vetrino seguito da irradiazione della luce impostato con l&#39;ultravioletta ad alta intensità (UV) per 2 sec. </li><li> Posizionare il photomask (precedentemente fabbricato) con le superfici Cr / Au rivestite rivolta verso l&#39;interno della cavità. Assicurarsi che i lati delle pareti della cavità non sono oscurando le lenti incorporate nel fotomaschere. </li><li> Riempire la cavità con la soluzione di prepolimero finché la superficie rivestita Cr / Au è in contatto con la soluzione senza bolle visibili. </li><li> Irradiare la configurazione con luce UV ad alta intensità di intensità desiderata per 1 sec ad una distanza di 3,5 cm dalla sorgente UV tramite la stazione di trattamento UV con una gamma filtro UV di 320-500 nm. Utilizzare un adattatore di collimazione con la sonda luce UV. </li><li> Misurare l&#39;intensità della luce UV utilizzata con un radiometro. </li><li> A seguito di esposizione ai raggi UV, rimuovere il fotomaschera con l&#39;array di MNs. Versare la soluzione di prepolimero eccesso che non polimerizzate nel processo nel suo recipiente originale per il riutilizzo. </li><li> Quantificare la lunghezza e punta il diametro delle MNs utilizzando uno stereomicroscopio secondo le istruzioni del produttore. </li></ol><pclass = &quot;jove_title&quot;&gt; 3. MN Backing strato Fabrication <ol><li> Con pinze, collocare i MNs (precedentemente fabbricato) attaccati sopra fotomaschera in un pozzetto di una piastra da 24 pozzetti, come mostrato nella Figura 1C. </li><li> Aggiungere un volume specificato (300 - 400 ml) della soluzione di prepolimero nel pozzo fino gli aghi sono sommersi all&#39;altezza desiderata. Questo volume determina lo spessore dello strato di supporto risultante. </li><li> Irradiare la configurazione con luce ad alta intensità UV (15,1 W / cm 2), 10,5 cm di distanza dalla sorgente UV per una durata di 1 sec. </li><li> Separare lo strato di supporto sulla matrice MN dalla fotomaschera utilizzando una lama affilata. </li><li> Quantificare la lunghezza, diametro punta e base diametro delle MNs con lo strato di supporto utilizzando uno stereomicroscopio secondo le istruzioni del produttore. </li></ol>

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The authors declare no conflict of Interest.

Il protocollo descritto sopra per la fabbricazione della matrice MNs è stata presentata per fabbricare la matrice MNs di ~ 1 cm 2. Gli array possono essere scalati da creare una grande cavità dimensioni e utilizzando un fotomaschere grande. La maggiore dimensione della cavità può essere creata aumentando la larghezza tra i distanziatori su entrambi i lati. Anche se ogni passo per fabbricare gli array MN nel protocollo era importante, i passi più importanti sono stati: il posizionamento fotomaschera, il riempimento della soluzione di prepolimero, e irradiazione della messa a punto. Posizionamento della fotomaschera dovrebbe essere in modo tale che le superfici Cr / Au rivestite affrontano l&#39;interno della cavità ed i lati delle pareti della cavità oscurare le lenti incorporate nel fotomaschere. Quando si riempie lo stampo con la soluzione di prepolimero, in modo che l&#39;aria bolle non sono intrappolate che possono comunque portare a un array di MN forza deformata e bassa. Le bolle d&#39;aria possono essere prevenute attraverso un&#39;azione assorbimento controllato aggiungendo lentamente l&#39;prepolymesoluzione R e garantire che non vi siano bolle d&#39;aria presenti nella soluzione prepolimero. Posizionamento del setup per irraggiamento dovrebbe essere fatto in modo guidato per garantire l&#39;esposizione UV uniforme. Prima della esposizione alla luce UV, la configurazione è stato allineato e posizionato entro la demarcazione sul supporto di base. I MNs formati usando fotolitografia notevolmente è stato influenzato dalla presenza delle microlenti come obiettivo determinato polimerizzazione in un percorso convergente, che ha portato alla formazione delle MNs nitide rispetto alle MNs cilindrici formati usando il fotomaschere planare. Nel fotomaschera planare, la luce UV attraversa con poca deviazione (quasi dritto) con conseguente formazione delle MNs cilindrici con punte affilate meno. Mentre nella fotomaschera microlenti-embedded, la luce UV che passa attraverso le lenti sottoposto rifrazione e convergenti, causando la formazione di MNs capovolgibili taglienti. Equazione del produttore dell&#39;obiettivo che è stato usato come un predictiVE modello per approssimare la lunghezza del MN correlando con la lunghezza focale della microlente ha dato una previsione della lunghezza tre volte inferiore a quello reale. La discrepanza può essere dovuta alla superficie convessa appiattita delle microlenti, che non permettono la rifrazione della luce come la lente convessa convenzionale. 13 Un altro fattore per la geometria MN era l&#39;intensità della luce UV. L&#39;intensità di 6.44 W / cm 2 è stato scelto perché gli aghi prodotte a questa intensità possedevano sufficiente resistenza meccanica per le applicazioni di pelle. Un&#39;altra scoperta correlate all&#39;intensità era che ogni incremento dell&#39;intensità UV, la lunghezza del MN aumenta. Questo potrebbe essere attribuito alla cima piatta delle microlenti, che ha consentito alcuni dei raggi di luce per viaggiare oltre il punto focale. 14,15 Inoltre, l&#39;estensione della polimerizzazione ha il suo limite, secondo la legge dell&#39;inverso del quadrato della luce, cioè, la luce perde energia come la distanzadalle sorgenti aumenta. 16 I protocolli qui descritti hanno il vantaggio di fabbricazione senza stampo in un breve periodo di tempo. Ma non possiamo prevedere quanto lontano può essere preso ulteriormente per la produzione di massa. MNS array sono state fatte da un polimero biocompatibile a basso costo. Si può potenzialmente essere utilizzato come dispositivo di somministrazione di farmaci transdermica per applicazioni farmaceutiche e cosmetiche. Più interessante, può essere utilizzato come una combinazione del sistema di erogazione e del dispositivo, come i composti terapeutici possono essere incapsulati (miscelando o solubilizzante in soluzione prepolimero) durante la fabbricazione di MNs. 5,6,17 La solubilità del composto aggiunto all&#39;interno della soluzione di prepolimero dovrebbe essere preso in considerazione le caratteristiche MN, per esempio, la forza MN, può cambiare 17.

Somministrazione di farmaci transdermica offre un approccio alternativo attraente per la somministrazione del farmaco, soprattutto per le biomolecole, che sono gestiti quasi esclusivamente da iniezioni ipodermiche. Tuttavia, la pelle, in particolare lo strato superiore (strato corneo), è una formidabile barriera impedendo molecole esogene di entrare nel corpo umano. Di recente, i dispositivi MN sono emersi come consentire strumenti per fornire farmaci attraverso la pelle. I dispositivi MN creano pori temporanei all&#39;interno dello strato corneo per consentire il passaggio di molecole di farmaco per ottenere l&#39;attività fisiologica desiderata con migliorata compliance del paziente e convenienza 1-3. Vari metodi di fabbricazione sono stati adottati per fabbricare MNs polimerici 4. Tuttavia, di solito coinvolgono processi step complicati e più che richiedono tempi lunghi e / o alte temperature per fabbricare array MNS. 4 Per semplificare il processo di fabbricazione, un unico processo senza muffa passo conun photomask stato sviluppato recentemente 5,6. Tuttavia, con questo metodo, fabbricato MNs avevano punte smussate aghi, come era alcun meccanismo in atto per modificare il percorso ultravioletta (UV) coinvolti nella fotolitografia. In questo studio, microlenti incorporati nella fotomaschera sono stati proposti per definire la geometria delle MNs. Il protocollo per fabbricare fotomaschere costituiti da microlenti integrate e successivamente Mn fabbricazione con punte affilate utilizzando fotomaschera sono segnalati.

<table><tbody><tr><td>Poly(ethylene glycol) diacrylate (PEGDA Mn=258)</td><td>SIGMA&amp;nbsp;</td><td>475629-500ML</td><td></td></tr><tr><td>2-hydroxy-2-methyl-propiophenone (HMP)</td><td>SIGMA&amp;nbsp;</td><td>405655-50ML</td><td></td></tr><tr><td>Bovine collagen type 1, FITC conjugate&amp;nbsp;</td><td>SIGMA&amp;nbsp;</td><td>C4361</td><td></td></tr><tr><td>UV curing station&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>EXFO Photonic Solutions Inc., Canada</td><td>OmniCure S2000-XL</td><td></td></tr><tr><td>Collimating Adaptor&amp;nbsp;</td><td>EXFO Photonic Solutions Inc., Canada</td><td>EXFO 810-00042</td><td></td></tr><tr><td>24-well plate</td><td>Thermo Fisher Scientific, USA</td><td></td><td></td></tr><tr><td>Nikon SMZ 1500 stereomicroscope&amp;nbsp;</td><td>Nikon, Japan</td><td></td><td></td></tr><tr><td>Dillon GL-500 digital force gauge&amp;nbsp;</td><td>Dillon, USA</td><td></td><td></td></tr><tr><td>A-1R confocal microscope&amp;nbsp;</td><td>Nikon, Japan</td><td></td><td></td></tr></tbody></table>

polymeric microneedle array fabrication by photolithography

Questo manoscritto descrive la fabbricazione di microago polimerico (MN) array per fotolitografia. Si tratta di un semplice processo senza stampo utilizzando una fotomaschera costituito da incorporati microlenti. Embedded microlenti sono stati trovati per influenzare MN geometria (nitidezza). Array MN Robusti con diametri compresi tra 41,5 punta micron ± 8.4 micron e 71,6 micron ± 13,7 micron, con due diverse lunghezze (1336 micron ± 193 micron e 957 micron ± 171 micron) sono stati fabbricati. Questi array MN possono fornire potenziali applicazioni nella fornitura di agenti terapeutici a basso molecolare e macromolecolare attraverso la pelle.

La geometria delle MNs può essere significativamente influenzata dalle caratteristiche fotomaschere e microlenti integrate. Il grado di rifrazione influenza il percorso di trasmissione dei raggi UV, che hanno influenzato la geometria MN (Figura 2A). Ogni microlenti è stato trovato ad avere un diametro di 350 micron, a 130 micron appiattite superficie convessa, e un 62,3 micron di profondità (Figura 2B-D). Utilizzando il teorema di Pitagora, il raggio di curvatura della prima superficie è risultata 272,89 micron. La lunghezza focale è stata calcolata in 509,28 micron (considerando n vetro = 1,53627; n = 1.000 aria; λ = 365 nm) tramite l&#39;equazione della lente creatore 12 come indicato di seguito: 1 / f = (n 1 / n m -1) * (1 / r 1 -1 / r 2) Dove n 1 è l&#39;indice di rifrazione del materiale della lente, n m è l&#39;indice di rifrazione del mezzo ambiente, R 1 è il radius di curvatura della prima superficie, ed R 2 è il raggio di curvatura della seconda superficie. L&#39;effetto di intensità UV alla lunghezza MN, nitidezza e deformazione strutturale è stato studiato da intensità della luce UV da 3,14 variando al 15,1 W / cm 2 a una lunghezza focale costante e leggera distanza di sorgente. Si è constatato che la lunghezza media MN aumentato significativamente (p &lt;0,05) con intensità crescente da 3.14 al 9.58 W / cm 2 (Figura 3A). Ulteriori aumenti di intensità fino a 15,1 W / cm 2 non hanno prodotto significative variazioni nella lunghezza. Il diametro della punta (misura di nitidezza) e la struttura di punta MN sono stati trovati a variare con incrementi di intensità (Figura 3B). I neuroni specchio con una forma regolare e senza alcuna deformazione strutturale sono stati osservati a 6,4 W / cm 2. Lo strato di supporto è stato fabbricato per consentire la rimozione di MN in forma di una patche per rendere il riutilizzabile fotomaschera. Ha inoltre fornito resistenza agli alberi MN. Pertanto, è stato studiato l&#39;effetto del volume strato di supporto (il volume delle soluzioni prepolimero per formare lo strato posteriore). Con un diametro di punta inalterati, i neuroni specchio con una gamma di lunghezza (1.336 ± 193 micron per 300 ml e 957 ± 171 micron per 400 ml) sono stati osservati dopo l&#39;esposizione ai raggi UV (Figura 4). <img alt="Figura 1" src="/files/ftp_upload/52914/52914fig1highres.jpg" width="700" /> Figura 1. (A) Rappresentazione schematica del processo di fabbricazione di lenti incorporate fotomaschere. (1) 4 &quot;wafer vetro. (2) Cr / Au strato depositato usando un evaporatore e-beam. (3) L&#39;esposizione di Cr / Au / fotoresist livello di mascheratura ai raggi UV con fotomaschere. (4) Formazione di pattern sul livello utilizzando Cr / Au mordenzante. (5) incollaggio temporaneo di vetro su un wafer di silicio manichino. (6) - (7) Incisione Wet (isotropo) processo utilizzando HF / mordenzanti HCl seguiti da ultrasuoni. (8) Debonding di wafer di silicio manichino e la rimozione dello strato di fotoresist. (B) Rappresentazione schematica del processo di fabbricazione di aghi. Photomask rivestito di cromo (9 x 9 array), è disposto sopra una cavità contenente soluzione di prepolimero ed esposto a UV. (C) Rappresentazione schematica del processo di fabbricazione dello strato di supporto. Photomask, con microaghi allegati, è posto in un pozzo pieno di pre-polimero e esposti a raggi UV. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52914/52914fig1large.jpg" target="_blank">Cliccate qui per vedere una versione più grande di questa figura.</a> <img alt="Figura 2" src="/files/ftp_upload/52914/52914fig2highres.jpg" width="450" /> Figura 2. Caratterizzazione di fotomaschere. (A) l&#39;esposizione ai raggi UV si concentra la luce in una conica<html> percorso l, producendo MNs conici. (B) e (C) Una immagine SEM di una microlente. (D) Una porzione di una matrice di stampo PDMS repliche copiati dalle microlenti, mostrando la superficie convessa appiattita, sotto uno stereomicroscopio. (E) una fotomaschera mostrando il modello. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52914/52914fig2large.jpg" target="_blank">Cliccate qui per vedere una versione più grande di questa figura.</a> <img alt="Figura 3" src="/files/ftp_upload/52914/52914fig3highres.jpg" width="450" /> Figura 3. Effetto dei parametri UV sulla geometria microneedle. Effetto di (A) Intensità e (B) Spessore distanziale alla durata microneedle. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52914/52914fig3large.jpg" target="_blank">Cliccate qui per vedere una versione più grande di questa figura.</a> tenda &quot;fo: keep-together.within-page =&quot; always &quot;&gt; <img alt="Figura 4" src="/files/ftp_upload/52914/52914fig4highres.jpg" width="700" /> Figura 4. Effetto di volume variabile pre-polimero utilizzato per lo strato di supporto fabbricazione. (A - B) immagini a volume diverso, con durata media MN per brevi (957 micron) e lunghi (1.336 micron) MNs. (C - D) Le immagini corrispondenti a (AB) dopo la prova forza frattura. (E) Diminuzione di lunghezza MN con aumento del volume utilizzato per la fabbricazione strato di supporto. (F) MN forza frattura tra i due volumi pre-polimeri utilizzati per fabbricare strato di supporto (BL). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52914/52914fig4large.jpg" target="_blank">Cliccate qui per vedere una versione più grande di questa figura.</a>

Watch this Scientific Journal Video about Polymeric Microneedle Array Fabrication by Photolithography at JoVE.com

Polymeric Microneedle Array Fabrication by Photolithography

Here, we present a protocol describing a mold-free fabrication process of the polymeric microneedles by photolithography.

Polimerici Microneedle Array Fabrication per Fotolitografia

This manuscript describes the fabrication of polymeric microneedle (MN) arrays by photolithography. It involves a simple mold-free process by using a ...

polymeric-microneedle-array-fabrication-by-photolithography

Nanyang Technological University

Research

JoVE Journal

Bioengineering

12.0K Views.  National University of Singapore. Here, we present a protocol describing a mold-free fabrication process of the polymeric microneedles by photolithography. 

Video: Polymeric Microneedle Array Fabrication by Photolithography

Micropunching Lithography for Generating Micro- and Submicron-patterns on Polymer Substrates

Conducting polymers have attracted great attention since the discovery of high conductivity in doped polyacetylene in 19771. They offer the advantages of low weight, easy tailoring of properties and a wide spectrum of applications2,3. Due to sensitivity of conducting polymers to environmental conditions (e.g., air, oxygen, moisture, high temperature and chemical solutions), lithographic techniques present significant technical challenges when working with these materials4. For example, current photolithographic methods, such as ultra-violet (UV), are unsuitable for patterning the conducting polymers due to the involvement of wet and/or dry etching processes in these methods. In addition, current micro/nanosystems mainly have a planar form5,6. One layer of structures is built on the top surfaces of another layer of fabricated features. Multiple layers of these structures are stacked together to form numerous devices on a common substrate. The sidewall surfaces of the microstructures have not been used in constructing devices. On the other hand, sidewall patterns could be used, for example, to build 3-D circuits, modify fluidic channels and direct horizontal growth of nanowires and nanotubes.
A macropunching method has been applied in the manufacturing industry to create macropatterns in a sheet metal for over a hundred years. Motivated by this approach, we have developed a micropunching lithography method (MPL) to overcome the obstacles of patterning conducting polymers and generating sidewall patterns. Like the macropunching method, the MPL also includes two operations (Fig. 1): (i) cutting; and (ii) drawing. The "cutting" operation was applied to pattern three conducting polymers4, polypyrrole (PPy), Poly(3,4-ethylenedioxythiophen)-poly(4-styrenesulphonate) (PEDOT) and polyaniline (PANI). It was also employed to create Al microstructures7. The fabricated microstructures of conducting polymers have been used as humidity8, chemical8, and glucose sensors9. Combined microstructures of Al and conducting polymers have been employed to fabricate capacitors and various heterojunctions9,10,11. The "cutting" operation was also applied to generate submicron-patterns, such as 100- and 500-nm-wide PPy lines as well as 100-nm-wide Au wires. The "drawing" operation was employed for two applications: (i) produce Au sidewall patterns on high density polyethylene (HDPE) channels which could be used for building 3D microsystems12,13,14, and (ii) fabricate polydimethylsiloxane (PDMS) micropillars on HDPE substrates to increase the contact angle of the channel15.

A micropunching lithography approach is developed to generate micro- and submicron-patterns on top, sidewall and bottom surfaces of polymer substrates. It overcomes the obstacles of patterning conducting polymers and generating sidewall patterns. This method allows rapid fabrication of multiple features and is free of aggressive chemistry.

Micropunching litografia per la generazione di Micro-e submicroniche modelli sul Polymer Substrati

Conducting polymers have attracted great attention since the discovery of high conductivity in doped polyacetylene in 19771. They offer the advantages of ...

Ricerca

Ingegneria

Fabrication of Micropatterned Hydrogels for Neural Culture Systems using Dynamic Mask Projection Photolithography

Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the need for 3D environments to represent in vitro experiments which better mimic in vivo qualities 1-3. Studies in fields such as cancer research 4, neural engineering 5, cardiac physiology 6, and cell-matrix interaction7,8have shown cell behavior differs substantially between traditional monolayer cultures and 3D constructs.
Hydrogels are used as 3D environments because of their variety, versatility and ability to tailor molecular composition through functionalization 9-12. Numerous techniques exist for creation of constructs as cell-supportive matrices, including electrospinning13, elastomer stamps14, inkjet printing15, additive photopatterning16, static photomask projection-lithography17, and dynamic mask microstereolithography18. Unfortunately, these methods involve multiple production steps and/or equipment not readily adaptable to conventional cell and tissue culture methods. The technique employed in this protocol adapts the latter two methods, using a digital micromirror device (DMD) to create dynamic photomasks for crosslinking geometrically specific poly-(ethylene glycol) (PEG) hydrogels, induced through UV initiated free radical polymerization. The resulting "2.5D" structures provide a constrained 3D environment for neural growth. We employ a dual-hydrogel approach, where PEG serves as a cell-restrictive region supplying structure to an otherwise shapeless but cell-permissive self-assembling gel made from either Puramatrix or agarose. The process is a quick simple one step fabrication which is highly reproducible and easily adapted for use with conventional cell culture methods and substrates.
Whole tissue explants, such as embryonic dorsal root ganglia (DRG), can be incorporated into the dual hydrogel constructs for experimental assays such as neurite outgrowth. Additionally, dissociated cells can be encapsulated in the photocrosslinkable or self polymerizing hydrogel, or selectively adhered to the permeable support membrane using cell-restrictive photopatterning. Using the DMD, we created hydrogel constructs up to ~1mm thick, but thin film (&lt;200 &mu;m) PEG structures were limited by oxygen quenching of the free radical polymerization reaction. We subsequently developed a technique utilizing a layer of oil above the polymerization liquid which allowed thin PEG structure polymerization.
In this protocol, we describe the expeditious creation of 3D hydrogel systems for production of microfabricated neural cell and tissue cultures. The dual hydrogel constructs demonstrated herein represent versatile in vitro models that may prove useful for studies in neuroscience involving cell survival, migration, and/or neurite growth and guidance. Moreover, as the protocol can work for many types of hydrogels and cells, the potential applications are both varied and vast.

Simple techniques are described for the rapid production of microfabricated neural culture systems using a digital micromirror device for dynamic mask projection lithography on regular cell culture substrates. These culture systems may be more representative of natural biological architecture, and the techniques described could be adapted for numerous applications.

Fabbricazione di idrogel Micropatterned per i sistemi Cultura neurali utilizzando Dynamic Fotolitografia proiezione Mask

Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the ...

Bioingegneria

Polymer Microarrays for High Throughput Discovery of Biomaterials

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a combinatorial library of materials to be screened in a parallel, high throughput format1. Herein is described the formation and characterization of a polymer microarray using an on-chip photopolymerization technique 2. This involves mixing monomers at varied ratios to produce a library of monomer solutions, transferring the solution to a glass slide format using a robotic printing device and curing with UV irradiation. This format is readily amenable to many biological assays, including stem cell attachment and proliferation, cell sorting and low bacterial adhesion, allowing the ready identification of 'hit' materials that fulfill a specific biological criterion3-5. Furthermore, the use of high throughput surface characterization (HTSC) allows the biological performance to be correlated with physio-chemical properties, hence elucidating the biological-material interaction6. HTSC makes use of water contact angle (WCA) measurements, atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). In particular, ToF-SIMS provides a chemically rich analysis of the sample that can be used to correlate the cell response with a molecular moiety. In some cases, the biological performance can be predicted from the ToF-SIMS spectra, demonstrating the chemical dependence of a biological-material interaction, and informing the development of hit materials5,3.

A description of the formation of a polymer microarray using an on-chip photopolymerization technique. The high throughput surface characterization using atomic force microscopy, water contact angle measurements, X-ray photoelectron spectroscopy and time of flight secondary ion mass spectrometry and a cell attachment assay is also described.

Microarrays polimerici per Discovery High Throughput di Biomateriali

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a ...

Prescribed 3-D Direct Writing of Suspended Micron/Sub-micron Scale Fiber Structures via a Robotic Dispensing System

A 3-axis dispensing system is utilized to control the initiating and terminating fiber positions and trajectory via the dispensing software. The polymer fiber length and orientation is defined by the spatial positioning of the dispensing system 3-axis stages. The fiber diameter is defined by the prescribed dispense time of the dispensing system valve, the feed rate (the speed at which the stage traverses from an initiating to a terminating position), the gauge diameter of the dispensing tip, the viscosity and surface tension of the polymer solution, and the programmed drawing length. The stage feed rate affects the polymer solution&#8217;s evaporation rate and capillary breakup of the filaments. The dispensing system consists of a pneumatic valve controller, a droplet-dispensing valve and a dispensing tip. Characterization of the direct write process to determine the optimum combination of factors leads to repeatedly acquiring the desired range of fiber diameters. The advantage of this robotic dispensing system is the ease of obtaining a precise range of micron/sub-micron fibers onto a desired, programmed location via automated process control. Here, the discussed self-assembled micron/sub-micron scale 3D structures have been employed to fabricate suspended structures to create micron/sub-micron fluidic devices and bioengineered scaffolds.

Here, we present a protocol to fabricate freely-suspended, micron/sub-micron scale polymer fibers and &#8220;web-like&#8221; structures generated via automated direct writing procedure by means of a 3-axis dispensing system.

Prescritto 3-D diretto Redazione di sospensione Micron / sub-micron scala fibra strutture attraverso un sistema di erogazione robotico

A 3-axis dispensing system is utilized to control the initiating and terminating fiber positions and trajectory via the dispensing software. The polymer ...

Hollow Microneedle-based Sensor for Multiplexed Transdermal Electrochemical Sensing

The development of a minimally invasive multiplexed monitoring system for rapid analysis of biologically-relevant molecules could offer individuals suffering from chronic medical conditions facile assessment of their immediate physiological state. Furthermore, it could serve as a research tool for analysis of complex, multifactorial medical conditions. In order for such a multianalyte sensor to be realized, it must be minimally invasive, sampling of interstitial fluid must occur without pain or harm to the user, and analysis must be rapid as well as selective.
Initially developed for pain-free drug delivery, microneedles have been used to deliver vaccines and pharmacologic agents (e.g., insulin) through the skin.1-2 Since these devices access the interstitial space, microneedles that are integrated with microelectrodes can be used as transdermal electrochemical sensors. Selective detection of glucose, glutamate, lactate, hydrogen peroxide, and ascorbic acid has been demonstrated using integrated microneedle-electrode devices with carbon fibers, modified carbon pastes, and platinum-coated polymer microneedles serving as transducing elements.3-7,8
This microneedle sensor technology has enabled a novel and sophisticated analytical approach for in situ and simultaneous detection of multiple analytes. Multiplexing offers the possibility of monitoring complex microenvironments, which are otherwise difficult to characterize in a rapid and minimally invasive manner. For example, this technology could be utilized for simultaneous monitoring of extracellular levels of, glucose, lactate and pH,9 which are important metabolic indicators of disease states7,10-14 (e.g., cancer proliferation) and exercise-induced acidosis.15

This article details the construction of a multiplexed microneedle-based sensor. The device is being developed for in situ sampling and electrochemical analysis of multiple analytes in a rapid and selective manner. We envision clinical medicine and biomedical research uses for these microneedle-based sensors.

Hollow microago-based sensore per Multiplexed transdermico elettrochimica Sensing

The development of a minimally invasive multiplexed monitoring system for rapid analysis of biologically-relevant molecules could offer individuals ...

Capillary Force Lithography for Cardiac Tissue Engineering

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical discoveries with the aims of developing functional tissues for cardiac regeneration as well as in vitro screening assays. However, the ability to create high-fidelity models of heart tissue has proven difficult. The heart&#8217;s extracellular matrix (ECM) is a complex structure consisting of both biochemical and biomechanical signals ranging from the micro- to the nanometer scale2. Local mechanical loading conditions and cell-ECM interactions have recently been recognized as vital components in cardiac tissue engineering3-5.
A large portion of the cardiac ECM is composed of aligned collagen fibers with nano-scale diameters that significantly influences tissue architecture and electromechanical coupling2. Unfortunately, few methods have been able to mimic the organization of ECM fibers down to the nanometer scale. Recent advancements in nanofabrication techniques, however, have enabled the design and fabrication of scalable scaffolds that mimic the in vivo structural and substrate stiffness cues of the ECM in the heart6-9.
Here we present the development of two reproducible, cost-effective, and scalable nanopatterning processes for the functional alignment of cardiac cells using the biocompatible polymer poly(lactide-co-glycolide) (PLGA)8 and a polyurethane (PU) based polymer. These anisotropically nanofabricated substrata (ANFS) mimic the underlying ECM of well-organized, aligned tissues and can be used to investigate the role of nanotopography on cell morphology and function10-14.
Using a nanopatterned (NP) silicon master as a template, a polyurethane acrylate (PUA) mold is fabricated. This PUA mold is then used to pattern the PU or PLGA hydrogel via UV-assisted or solvent-mediated capillary force lithography (CFL), respectively15,16. Briefly, PU or PLGA pre-polymer is drop dispensed onto a glass coverslip and the PUA mold is placed on top. For UV-assisted CFL, the PU is then exposed to UV radiation (&#955; = 250-400 nm) for curing. For solvent-mediated CFL, the PLGA is embossed using heat (120 &#176;C) and pressure (100 kPa). After curing, the PUA mold is peeled off, leaving behind an ANFS for cell culture. Primary cells, such as neonatal rat ventricular myocytes, as well as human pluripotent stem cell-derived cardiomyocytes, can be maintained on the ANFS2.

In this protocol, we demonstrate the fabrication of biomimetic cardiac cell culture substrata made from two distinct polymeric materials using capillary force lithography. The described methods provide a scalable, cost-effective technique to engineer the structure and function of macroscopic cardiac tissues for in vitro and in vivo applications.

Capillare Forza litografia per Cardiac Tissue Engineering

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical ...

Simple Lithography-Free Single Cell Micropatterning using Laser-Cut Stencils

Micropatterning techniques have been widely used in cell biology to study effects of controlling cell shape and size on cell fate determination at single cell resolution. Current state-of-the-art single cell micropatterning techniques involve soft lithography and micro-contact printing, which is a powerful technology, but requires trained engineering skills and certain facility support in microfabrication. These limitations require a more accessible technique. Here, we describe a simple alternative lithography-free method: stencil-based single cell patterning. We provide step-by-step procedures including stencil design, polyacrylamide hydrogel fabrication, stencil-based protein incorporation, and cell plating and culture. This simple method can be used to pattern an array of as many as 2,000 cells. We demonstrate the patterning of cardiomyocytes derived from single human induced pluripotent stem cells (hiPSC) with distinct cell shapes, from a 1:1 square to a 7:1 adult cardiomyocyte-like rectangle. This stencil-based single cell patterning is lithography-free, technically robust, convenient, inexpensive, and most importantly accessible to those with a limited bioengineering background.

This protocol introduces a lithography-free micropatterning method that is simple and accessible to those with a limited bioengineering background. This method utilizes customized laser-cut stencils to micropattern extracellular matrix proteins in a shape of interest for modulating cell morphologies. The procedure for micropatterning is demonstrated using induced pluripotent stem cell derived cardiomyocytes.

Micropatterning a cella singola semplice senza litografia con stencil laser-cut

Micropatterning techniques have been widely used in cell biology to study effects of controlling cell shape and size on cell fate determination at single ...

Biologia dello sviluppo

Fabrication of a Multiplexed Artificial Cellular MicroEnvironment Array

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

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

Fabbricazione di una matrice di multiplex artificiale microambiente cellulare

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

Fabrication of Fine Electrodes on the Tip of Hypodermic Needle Using Photoresist Spray Coating and Flexible Photomask for Biomedical Applications

We have introduced a fabrication method for electrical impedance spectroscopy (EIS)-on-a-needle (EoN: EIS-on-a-needle) to locate target tissues in the body by measuring and analyzing differences in the electrical impedance between dissimilar biotissues. This paper describes the fabrication method of fine interdigitated electrodes (IDEs) at the tip of a hypodermic needle using a photoresist spray coating and flexible film photomask in the photolithography process. A polyethylene terephthalate (PET) heat shrink tube (HST) with a wall thickness of 25 &#181;m is employed as the insulation and passivation layer. The PET HST shows a higher mechanical durability compared with poly(p-xylylene) polymers, which have been widely used as a dielectric coating material. Furthermore, the HST shows good chemical resistance to most acids and bases, which is advantageous for limiting chemical damage to the EoN. The use of the EoN is especially preferred for the characterization of chemicals/biomaterials or fabrication using acidic/basic chemicals. The fabricated gap and width of the IDEs are as small as 20 &#181;m, and the overall width and length of the IDEs are 400 &#181;m and 860 &#181;m, respectively. The fabrication margin from the tip (distance between the tip of hypodermic needle and starting point of the IDEs) of the hypodermic needle is as small as 680 &#181;m, which indicates that unnecessarily excessive invasion into biotissues can be avoided during the electrical impedance measurement. The EoN has a high potential for clinical use, such as for thyroid biopsies and anesthesia drug delivery in a spinal space. Further, even in surgery that involves the partial resection of tumors, the EoN can be employed to preserve as much normal tissue as possible by detecting the surgical margin (normal tissue that is removed with the surgical excision of a tumor) between the normal and lesion tissues.

The fabrication method for fine interdigitated electrodes (gap and width: 20 &#181;m) at the tip of a hypodermic needle (diameter: 720 &#181;m) is demonstrated using a spray coating and flexible film photomask in the photolithography process.

Fabbricazione di elettrodi bene sulla punta dell'ago ipodermico utilizzando Photoresist Spray rivestimento e Photomask flessibile per applicazioni biomediche

We have introduced a fabrication method for electrical impedance spectroscopy (EIS)-on-a-needle (EoN: EIS-on-a-needle) to locate target tissues in the ...

Neuroscienze

Applicazioni della stampa 3D per dispositivi di coltura cellulare neuronale su microscala

Two-Photon Polymerization 3D-Printing of Micro-scale Neuronal Cell Culture Devices

Neuronal cultures have been a reference experimental model for several decades. However, 3D cell arrangement, spatial constraints on neurite outgrowth, and realistic synaptic connectivity are missing. The latter limits the study of structure and function in the context of compartmentalization and diminishes the significance of cultures in neuroscience. Approximating ex vivo the structured anatomical arrangement of synaptic connectivity is not trivial, despite being key for the emergence of rhythms, synaptic plasticity, and ultimately, brain pathophysiology. Here, two-photon polymerization (2PP) is employed as a 3D printing technique, enabling the rapid fabrication of polymeric cell culture devices using polydimethyl-siloxane (PDMS) at the micrometer scale. Compared to conventional replica molding techniques based on microphotolitography, 2PP micro-scale printing enables rapid and affordable turnaround of prototypes. This protocol illustrates the design and fabrication of PDMS-based microfluidic devices aimed at culturing modular neuronal networks. As a proof-of-principle, a two-chamber device is presented to physically constrain connectivity. Specifically, an asymmetric axonal outgrowth during ex vivo development is favored and allowed to be directed from one chamber to the other. In order to probe the functional consequences of unidirectional synaptic interactions, commercial microelectrode arrays are chosen to monitor the bioelectrical activity of interconnected neuronal modules. Here, methods to 1) fabricate molds with micrometer precision and 2) perform in vitro multisite extracellular recordings in rat cortical neuronal cultures are illustrated. By decreasing costs and future widespread accessibility of 2PP 3D-printing, this method will become more and more relevant across research labs worldwide. Especially in neurotechnology and high-throughput neural data recording, the ease and rapidity of prototyping simplified in vitro models will improve experimental control and theoretical understanding of in vivo large-scale neural systems.

Autore in primo piano: Reti neuronali modulari per l'analisi delle funzioni cerebrali

3D printing at the micrometer scale enables rapid prototyping of polymeric devices for neuronal cell cultures. As a proof-of-principle, structural connections between neurons were constrained by creating barriers and channels influencing neurite outgrowth, while the functional consequences of such manipulation were observed by extracellular electrophysiology.

Stampa 3D a polimerizzazione a due fotoni di dispositivi di coltura cellulare neuronale su microscala

Neuronal cultures have been a reference experimental model for several decades. However, 3D cell arrangement, spatial constraints on neurite outgrowth, ...