Questo lavoro è stato finanziato dalla Swiss National Science Foundation (PNR 62 - Smart Materials). Anche riconosciuto è il supporto di B. Hanselmann, K. Kehl, U. Schütz e B. Leuthold.

1. Polimerizzazione al plasma avviata <ol><li> Preparazione della soluzione di monomero. <ol><li> Sciogliere HEMA (100 ml, 0,718 moli) in 200 ml di acqua e lavare 3x con esano (100 ml) in un imbuto separatore. Saturare la fase acquosa con cloruro di sodio ed estrarre il HEMA con etere etilico (50 ml). Essiccare la fase organica su MgSO4 e rimuovere il solvente sotto vuoto (100 mbar, 40 ° C). Distillare l&#39;HEMA a pressione ridotta (15 mbar; 99 ° C). </li><li> Preparare una soluzione 0,62 M metanolica di HEMA libero-inibitore prodotta in sezione 1.1.1. Versare 30 ml della soluzione in un matraccio da un collo ed eliminare facendovi gorgogliare Ar attraverso la soluzione per 1 ora. </li></ol></li><li> Polimerizzazione-Surface indotta. <ol><li> Posizione due membrane di policarbonato accanto all&#39;altro nella camera di plasma (Figura 2). Posizionare il lato lucido della membrana rivolta verso la fase gassosa. </li><li> Collegare la camera di plasma ahIGH vuoto (20 mbar) per 5 min. Chiudere la valvola al vuoto e aprire l&#39;altra valvola, che è collegato al argon e ossigeno gassoso e spurgare la camera con questa miscela per 2 ore con 15 sccm di argon e 2,5 sccm ossigeno. </li><li> Avviare il plasma e ridurre la potenza alla potenza desiderata (per membrana di policarbonato: 12 W) e trattare le membrane per 4 min con il plasma. Collegare la soluzione di monomero con la camera aprendo la valvola corrispondente. Spegnere il plasma ed evacuare la camera. </li><li> Collegare la soluzione di monomero con la camera aprendo la valvola corrispondente e versare la soluzione nella camera. Assicurarsi che le membrane sono coperti con la soluzione di monomero. Aprire la valvola collegata al argon e conservare la miscela di reazione per 12 ore a 20 ° C (ambiente condizionato). </li><li> Rimuovere la soluzione di monomero. Lavare le membrane con metanolo in un bagno ad ultrasuoni per 5 min. Ripetere la procedura di lavaggio con acqua. </li><li> Essiccare la membrana invuoto su setacci molecolari per 2 ore. </li></ol></li></ol> 2. Postmodifica di membrane rivestite <ol><li> Preparare una soluzione di spirobenzopyran 1 (Figura 1) (100 mg, 0,27 mmol), N, N-dicicloesilcarbodiimmide (DCC) (55 mg, 0,27 mmol) e dimetil aminopiridina (DMAP) (33 mg, 0,27 mmol) in terz-butylmetylether (TBME) (12 ml). </li><li> Posizionare un bar agitatore protezione e una griglia di protezione in un pallone a fondo rotondo. Asciugare il pallone e inondare il pallone con argon. </li><li> Versare la soluzione nel pallone, seguita dalla membrana rivestita. </li><li> Mescolare delicatamente a temperatura ambiente per 12 ore. </li><li> Rimuovere la soluzione e lavare la membrana con tert-butylmetylether in un bagno ad ultrasuoni per 5 min. Ripetere la procedura di lavaggio con etanolo e acqua. </li><li> Essiccare la membrana sotto vuoto su setacci molecolari per 2 hr. </li></ol> 3. Misure Surface Tension <ol><li&gt; Fix la membrana con un nastro standard su un O-ring di metallo. Posizionare una goccia d&#39;acqua Nanopure (3,3 ml) su quella parte della superficie della membrana, che non è in contatto con l&#39;O-ring. Misurare l&#39;angolo di contatto (CA) in 5 punti diversi della membrana. </li><li> Per testare la stabilità a lungo termine dei campioni, misurare gli angoli di contatto in tre punti diversi delle membrane dopo 0, 1, 2, 3, 7, 14 e 21 giorni. </li></ol> 4. Prove di permeabilità delle membrane fotocromatiche <ol><li> Riempire la camera recettore della cellula diffusione Franz con acqua (12 ml). </li><li> Fissare la membrana in una cella di diffusione Franz. Assicurarsi che la membrana è in contatto con l&#39;acqua della camera di recettore. Riempire la camera donatore (camera di sopra della membrana) con una soluzione acquosa di caffeina (20 mM; 3,0 ml). Irradiare la membrana dalla parte superiore della camera di donatore con luce bianca (Figura 3). Raccogliere campioni (200 microlitri) dalla cella recettore; for membrane in policarbonato taglio pista con un diametro dei pori di 200 nm, raccogliere campioni ogni 10 min. </li><li> Ripetere l&#39;esperimento come descritto al punto 4.2. ma irradiare la membrana con luce UV (366 nm, 80 W / m 2) durante l&#39;intero test di permeabilità. </li><li> Determinazione delle concentrazioni di caffeina dei campioni raccolti. <ol><li> Tracciare la curva di taratura con 15 differenti concentrazioni di caffeina (tra 0,05 mg / ml e 1,5 mM / L) utilizzando un UV / Vis spettrometro. Calibrare a 293 nm. </li><li> Determinare la concentrazione di ciascuno dei campioni raccolti utilizzando la curva di calibrazione. </li><li> Tracciare la concentrazione determinata vs. il tempo dei campioni raccolti. Fare un fit lineare attraverso i punti e determinare Δc dalla pista. </li></ol></li></ol>

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Il processo plasma produce un gas viola, che è causato da argon ionizzato. Un colore arancione denota la presenza di azoto indesiderato da una perdita. Il processo al plasma non solo formare radicali sulla superficie, ma anche incide la membrana 7,12. Troppo incisione può cambiare il diametro dei pori significativamente, che influenzerebbe la permeabilità della membrana. Le condizioni di reazione controllate della configurazione presentata permettono migliorare la riproducibilità del processo di innesto p...

Modifica plasma dei materiali è diventato un processo importante in molti campi industriali. Pulizia delle superfici e funzionalizzazione di superfici senza cambiare la proprietà maggior parte del materiale è fatto il trattamento al plasma un processo essenziale nella scienza delle superfici 1-8. Il trattamento al plasma dei polimeri si traduce in omolitico legame scissione. Questo porta ad un bordo del materiale polimerico e alla formazione di superfici ricche radicali. Utilizzando plasma contenente molecole di ossigeno, la superficie diventa ossigenato e quindi più idrofilo 9-11. Tuttavia, l&#39;idrofilia delle superfici non è stabile nel tempo 12. Al fine di migliorare la stabilità a lungo termine, la superficie plasma trattato può essere modificato chimicamente dopo o durante il processo al plasma 13-15. Questo trattamento viene normalmente eseguito aggiungendo un reattivo specie monomeriche nella fase gassosa durante il processo al plasma; questi monomeri poi polimerizzanodai radicali creati della superficie del polimero. Se il trattamento chimico viene eseguita con un monomero non volatile, l&#39;innesto polimero deve avvenire dopo la modifica plasma. Al fine di effettuare un innesto controllata dopo i radicali si formano sulla superficie, una configurazione plasma è descritto, che consente la polimerizzazione superficie indotta plasma avviati dalla superficie in soluzione in condizioni controllate 12,16. La presentazione si concentra sulla modifica delle membrane polimeriche taglio track 12,17. Modificando la tensione superficiale di queste membrane, il tasso di permeabilità può essere variata 12. Questo processo pulito e veloce permette la creazione di strati molto sottili (&lt;5 nm), che coprono l&#39;intera superficie della membrana senza modificare la struttura grosso della membrana polimerica. A causa del bordo durante il processo al plasma, i diametri dei pori delle membrane taglio pista aumentano leggermente 12. Il tasso di bordo è depending sul polimero e ha un comportamento lineare. Quando si utilizzano monomeri con gruppi funzionali reattivi, i polimeri innestati possono essere ulteriormente funzionalizzati. Ciò è dimostrato dal postmodifica di una membrana PHEMA innestata con un acido carbossilico spiropyran funzionalizzato. Ciò si traduce in una superficie fotocromatica, poiché spiropyran è noto per trasformare in una specie merocyanine quando irradiato con luce UV. Il modulo spiropyran può essere ristabilita irradiando la forma merocyanine con luce visibile (Figura 1) 18,19. Poiché la forma merocyanine è più polare stato spiropyran, la tensione superficiale del rivestimento può essere attivato con la luce 20. La variazione di tensione superficiale influenza la resistenza permeabilità della membrana verso soluzioni acquose. Il set-up come eseguire le prove di permeabilità di queste membrane di luce-reattiva verrà mostrato e il cambiamento significativo nella resistenza permeabilità (diminuire in Resistenza permeabilità del 97%) è dimostrata. Tale membrana può essere integrato in una configurazione di somministrazione di farmaci o in sistemi intelligenti di rilevamento. <img alt="Figura 1" fo:content-width="5in" fo:src="/files/ftp_upload/51680/51680fig1highres.jpg" src="/files/ftp_upload/51680/51680fig1.jpg" /> Figura 1. Fotoisomerizzazione di composto spirobenzopyran 1.

<table border="0" cellpadding="0" cellspacing="0" style="width:943px;" width="943">
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		<col />
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		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Name of Reagent/ Equipment</td>
			<td style="width:284px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">2-Hydroxyethyl methacrylate, 97%</td>
			<td style="width:284px;">Sigma-Aldrich</td>
			<td style="width:119px;">128635</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Hexane 99%</td>
			<td style="width:284px;">Biosolve</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Magnesium sulfate (MgSO4, anhydrous)</td>
			<td style="width:284px;">Sigma-Aldrich</td>
			<td>M7506</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Methanol, 99%&#160;</td>
			<td style="width:284px;">Sigma-Aldrich</td>
			<td style="width:119px;">14262</td>
			<td>dried over molecular sieves</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">N,N-Dicylcohexylcarbodiimide, 99%</td>
			<td style="width:284px;">Sigma-Aldrich</td>
			<td style="width:119px;">D8002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Dimethyl aminopyridine, 99%</td>
			<td style="width:284px;">Sigma-Aldrich</td>
			<td style="width:119px;">107700</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Tert-butylmethylether, 98%</td>
			<td style="width:284px;">Fluka</td>
			<td style="width:119px;">306975</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Polycarbonate membrane</td>
			<td style="width:284px;">Whatman</td>
			<td style="width:119px;"></td>
			<td>Nanopore Track Etched (TE) (1.0 &#956;m, 0.2 &#956;m, 0.1 &#956;m, 50 nm, 30 nm and 15 nm pore diameter; 47 mm or 25 mm membrane diameter)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Caffeine (reagent plus)</td>
			<td style="width:284px;">Sigma-Aldrich</td>
			<td style="width:119px;">C0750&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Franz diffusion cell (12 ml)</td>
			<td style="width:284px;">SES-Analysesysteme</td>
			<td style="width:119px;">6C010015</td>
			<td>15mm unjacheted Franz Cell, 12 ml Receptor volume, Flat ground, clear glass, stirbat and clamp</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">UV-Lamp</td>
			<td style="width:284px;"></td>
			<td style="width:119px;"></td>
			<td>UV irradiation (366 nm, 15 W/m2)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">White light lamp</td>
			<td style="width:284px;"></td>
			<td style="width:119px;"></td>
			<td>White light irradiation (500 W bulb)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">UV/Vis spectrophotometer</td>
			<td style="width:284px;">Varian 50Bio/50MPR</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:373px;">Polyester membranes</td>
			<td style="width:284px;">Sterlitech</td>
			<td style="width:119px;">PET0225100</td>
			<td>Polyester Membrane Filters, 0.2 &#956;m pore diameter, 25 mmm diameter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Polyvinylidene fluoride membranes</td>
			<td style="width:284px;">Millipore</td>
			<td style="width:119px;"></td>
			<td>PVDF Membranes Durapore (0.22 &#956;m pore diameter; 47 mm membrane diameter)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Argon (99.9995%)</td>
			<td style="width:284px;">Alphagaz</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Dressler Cesar RF Power Generator</td>
			<td style="width:284px;"></td>
			<td style="width:119px;"></td>
			<td>Plasma chamber setup</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">MKS Multi Gas Controller 647C</td>
			<td style="width:284px;"></td>
			<td style="width:119px;"></td>
			<td>Plasma chamber setup</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">MKS Mass-Flow controllers</td>
			<td style="width:284px;"></td>
			<td style="width:119px;"></td>
			<td>Plasma chamber setup</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Vacuubrand RE 2.5 rotary vane vacuum pump</td>
			<td style="width:284px;"></td>
			<td style="width:119px;"></td>
			<td>Plasma chamber setup</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Contact angle measurement device Kr&#252;ss G10</td>
			<td style="width:284px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Balances Mettler Toledo AB204-S and Mettler ME30</td>
			<td style="width:284px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation of light-responsive membranes by a combined surface grafting and postmodification process

Al fine di modificare la tensione superficiale delle membrane commerciali disponibili taglio pista polimero, un procedimento di polimerizzazione in superficie avviato è presentato. La polimerizzazione dalla superficie della membrana è indotta dal trattamento al plasma della membrana, seguita facendo reagire la superficie della membrana con una soluzione metanolica di 2-idrossietil metacrilato (HEMA). Particolare attenzione è data ai parametri di processo per il trattamento al plasma prima della polimerizzazione sulla superficie. Ad esempio, l&#39;influenza del plasma-trattamento su diversi tipi di membrane (ad esempio poliestere, policarbonato, fluoruro di polivinilidene) è studiato. Inoltre, la stabilità tempo-dipendente delle membrane superficiali innestato è indicato da misure di angolo di contatto. Quando l&#39;innesto poli (2-idrossietil metacrilato) (PHEMA) in questo modo, la superficie può essere ulteriormente modificata per esterificazione della parte alcoolica del polimero con una funzione di acido carbossilico della sostanza desiderata.Queste reazioni possono pertanto essere utilizzati per la funzionalizzazione della superficie della membrana. Ad esempio, la tensione superficiale della membrana può essere modificata o una funzionalità desiderata come la luce-reattività presentato può essere inserito. Ciò è dimostrato facendo reagire PHEMA con un acido carbossilico funzionalizzato unità spirobenzopyran che porta ad una membrana luce-reattiva. La scelta del solvente gioca un ruolo importante nel passaggio postmodifica e viene discussa in maggior dettaglio in questo documento. Le misure di permeabilità di tali membrane funzionalizzate sono eseguiti utilizzando una cella Franz con una fonte di luce esterna. Modificando la lunghezza d&#39;onda della luce visibile al UV-gamma, si osserva un cambiamento di permeabilità di soluzioni acquose caffeina.

La velocità di incisione può essere seguita pesando la membrana dopo diversi periodi di tempo. Come si può vedere dalla figura 4, la velocità di incisione segue per poliestere, fluoruro di polivinilidene, e policarbonato membrane una velocità di incisione lineare, che può essere determinato dalla pendenza della correlazione lineare del tempo etch contro perdita di massa. Come mostrato in figura 4, le membrane di policarbonato mostrano il tasso di etch basso di tutti i tre...

Watch this Scientific Journal Video about Preparation of Light-responsive Membranes by a Combined Surface Grafting and Postmodification Process at JoVE.com

Preparation of Light-responsive Membranes by a Combined Surface Grafting and Postmodification Process

Una procedura di polimerizzazione indotta dal plasma è descritta per la polimerizzazione superficie avviato su membrane polimeriche. Ulteriori postmodifica del polimero innestato con sostanze fotocromiche è presentato con un protocollo di effettuare le misure di permeabilità delle membrane luce-reattiva.

Preparazione di membrane Light-responsive da un innesto superficie combinata e Postmodifica processo

In order to modify the surface tension of commercial available track-edged polymer membranes, a procedure of surface-initiated polymerization is ...

preparation-light-responsive-membranes-combined-surface-grafting

Research

JoVE Journal

Bioengineering

11.7K Views.  Empa, Swiss Federal Laboratories for Materials Science and Technology.  Una procedura di polimerizzazione indotta dal plasma è descritta per la polimerizzazione superficie avviato su membrane polimeriche. Ulteriori postmodifica del polimero innestato con sostanze fotocromiche è presentato con un protocollo di effettuare le misure di permeabilità delle membrane luce-reattiva. 

Video: Preparation of Light-responsive Membranes by a Combined Surface Grafting and Postmodification Process

Measurement of Aggregate Cohesion by Tissue Surface Tensiometry

Rigorous measurement of intercellular binding energy can only be made using methods grounded in thermodynamic principles in systems at equilibrium. We have developed tissue surface tensiometry (TST) specifically to measure the surface free energy of interaction between cells. The biophysical concepts underlying TST have been previously described in detail1,2. The method is based on the observation that mutually cohesive cells, if maintained in shaking culture, will spontaneously assemble into clusters. Over time, these clusters will round up to form spheres. This rounding-up behavior mimics the behavior characteristic of liquid systems. Intercellular binding energy is measured by compressing spherical aggregates between parallel plates in a custom-designed tissue surface tensiometer. The same mathematical equation used to measure the surface tension of a liquid droplet is used to measure surface tension of 3D tissue-like spherical aggregates. The cellular equivalent of liquid surface tension is intercellular binding energy, or more generally, tissue cohesivity. Previous studies from our laboratory have shown that tissue surface tension (1) predicts how two groups of embryonic cells will interact with one another1-5, (2) can strongly influence the ability of tissues to interact with biomaterials6, (3) can be altered not only through direct manipulation of cadherin-based intercellular cohesion7, but also by manipulation of key ECM molecules such as FN8-11 and 4) correlates with invasive potential of lung cancer12, fibrosarcoma13, brain tumor14 and prostate tumor cell lines15. In this article we will describe the apparatus, detail the steps required to generate spheroids, to load the spheroids into the tensiometer chamber, to initiate aggregate compression, and to analyze and validate the tissue surface tension measurements generated.

We describe a method of measuring binding energy, expressible as tissue surface tension, between cells within 3D tissue-like aggregates. Differences in tissue surface tension have been demonstrated to correlate with invasiveness of lung, muscle, and brain tumors, and are fundamental determinants of establishing spatial relationships between different cell types.

Misura della coesione aggregata da parte del tessuto superficiale Tensiometry

Rigorous measurement of intercellular binding energy can only be made using methods grounded in thermodynamic principles in systems at equilibrium.  We ...

Ricerca

Bioingegneria

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

Ponteggi self-reporting per coltura cellulare 3-Dimensional

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

Generation and Grafting of Tissue-engineered Vessels in a Mouse Model

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. The current protocol presents an efficient and reproducible strategy in which functional tissue engineered vessel grafts can be generated using partially induced pluripotent stem cell (PiPSC) from human fibroblasts. We designed a decellularized vessel scaffold bioreactor, which closely mimics the matrix protein structure and blood flow that exists within a native vessel, for seeding of PiPSC-endothelial cells or smooth muscle cells prior to grafting into mice. This approach was demonstrated to be advantageous because immune-deficient mice engrafted with the PiPSC-derived grafts presented with markedly increased survival rate 3 weeks after surgery. This protocol represents a valuable tool for regenerative medicine, tissue engineering and potentially patient-specific cell-therapy in the near future.

Here, we present a protocol to generate tissue engineered vessel grafts that are functional for grafting into mice by double seeding partially induced pluripotent stem cell (PiPSC) - derived smooth muscle cells and PiPSC - derived endothelial cells on a decellularized vessel scaffold bioreactor.

Generazione e innesto delle navi tissutale in un modello di topo

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. ...

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to intrinsic parameters within the process allow a high degree of control over scaffold characteristics including fiber diameter, alignment and porosity. By developing scaffolds with similar dimensions and topographies to organ- or tissue-specific extracellular matrices (ECM), micro-environments representative to those that cells are exposed to in situ can be created. 
The airway bronchiole wall, comprised of three main micro-environments, was selected as a model tissue. Using decellularized airway ECM as a guide, we electrospun the non-degradable polymer, polyethylene terephthalate (PET), by three different protocols to produce three individual electrospun scaffolds optimized for epithelial, fibroblast or smooth muscle cell-culture. Using a commercially available bioreactor system, we stably co-cultured the three cell-types to provide an in vitro model of the airway wall over an extended time period.
This model highlights the potential for such methods being employed in in vitro diagnostic studies investigating important inter-cellular cross-talk mechanisms or assessing novel pharmaceutical targets, by providing a relevant platform to allow the culture of fully differentiated adult cells within 3D, tissue-specific environments.

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Advancements in biomaterial technologies enable the development of three-dimensional multi-cell-type constructs. We have developed electrospinning protocols to produce three individual scaffolds to culture the main structural cells of the airway to provide a 3D in vitro model of the airway bronchiole wall.

Adattamento del processo Electrospinning per fornire tre ambienti unici per un Tri-livello<em&gt; In Vitro</em&gt; Modello della parete delle vie aeree

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to ...

Qualitative Characterization of the Aqueous Fraction from Hydrothermal Liquefaction of Algae Using 2D Gas Chromatography with Time-of-flight Mass Spectrometry

Two-dimensional gas chromatography coupled with time-of-flight mass spectrometry is a powerful tool for identifying and quantifying chemical components in complex mixtures. It is often used to analyze gasoline, jet fuel, diesel, bio-diesel and the organic fraction of bio-crude/bio-oil. In most of those analyses, the first dimension of separation is non-polar, followed by a polar separation. The aqueous fractions of bio-crude and other aqueous samples from biofuels production have been examined with similar column combinations. However, sample preparation techniques such as derivatization, solvent extraction, and solid-phase extraction were necessary prior to analysis. In this study, aqueous fractions obtained from the hydrothermal liquefaction of algae were characterized by two-dimensional gas chromatography coupled with time-of-flight mass spectrometry without prior sample preparation techniques using a polar separation in the first dimension followed by a non-polar separation in the second. Two-dimensional plots from this analysis were compared with those obtained from the more traditional column configuration. Results from qualitative characterization of the aqueous fractions of algal bio-crude are discussed in detail. The advantages of using a polar separation followed by a non-polar separation for characterization of organics in aqueous samples by two-dimensional gas chromatography coupled with time-of-flight mass spectrometry are highlighted.

A two-dimensional gas chromatography-time-of-flight mass spectrometry method is described for characterization of the aqueous fraction of bio-crude produced from hydrothermal liquefaction of algae. This protocol can also be employed to analyze the aqueous fraction of liquid products from fast pyrolysis, catalytic fast pyrolysis, catalytic deoxygenation and hydro-treating.

Caratterizzazione qualitativa della frazione acquosa da idrotermale Liquefazione di alghe Utilizzando 2D gascromatografia con tempo di volo spettrometria di massa

Two-dimensional gas chromatography coupled with time-of-flight mass spectrometry is a powerful tool for identifying and quantifying chemical components in ...

Automated Robotic Dispensing Technique for Surface Guidance and Bioprinting of Cells

This manuscript describes the introduction of cell guidance features followed by the direct delivery of cells to these features in a hydrogel bioink using an automated robotic dispensing system. The particular bioink was selected as it allows cells to sediment towards and sense the features. The dispensing system bioprints viable cells in hydrogel bioinks using a backpressure assisted print head. However, by replacing the print head with a sharpened stylus or scalpel, the dispensing system can also be employed to create topographical cues through surface etching. The stylus movement can be programmed in steps of 10 &#181;m in the X, Y and Z directions. The patterned grooves were able to orientate mesenchymal stem cells, influencing them to adopt an elongated morphology in alignment with the grooves&#39; direction. The patterning could be designed using plotting software in straight lines, concentric circles, and sinusoidal waves. In a subsequent procedure, fibroblasts and mesenchymal stem cells were suspended in a 2% gelatin bioink, for bioprinting in a backpressure driven extrusion printhead. The cell bearing bioink was then printed using the same programmed coordinates used for the etching. The bioprinted cells were able to sense and react to the etched features as demonstrated by their elongated orientation along the direction of the etched grooves.

This protocol describes a bioprinting methodology using an automated robotic depositing system that incorporates etched topographical guidance cues with the precision deposition of a cell bearing hydrogel bioink. The printed cells are directly delivered to the etched features and are able to sense and orientate with them.

Tecnica Automated robotica erogazione di orientamento della superficie e Bioprinting di celle

This manuscript describes the introduction of cell guidance features followed by the direct delivery of cells to these features in a hydrogel bioink using ...

Synthesis of Hydrogels with Antifouling Properties As Membranes for Water Purification

Hydrogels have been widely utilized to enhance the surface hydrophilicity of membranes for water purification, increasing the antifouling properties and thus achieving stable water permeability through membranes over time. Here, we report a facile method to prepare hydrogels based on zwitterions for membrane applications. Freestanding films can be prepared from sulfobetaine methacrylate (SBMA) with a crosslinker of poly(ethylene glycol) diacrylate (PEGDA) via photopolymerization. The hydrogels can also be prepared by impregnation into hydrophobic porous supports to enhance the mechanical strength. These films can be characterized by attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) to determine the degree of conversion of the (meth)acrylate groups, using goniometers for hydrophilicity and differential scanning calorimetry (DSC) for polymer chain dynamics. We also report protocols to determine the water permeability in dead-end filtration systems and the effect of foulants (bovine serum albumin, BSA) on membrane performance.

This paper reports practical methods to prepare hydrogels in freestanding films and impregnated membranes and to characterize their physical properties, including water transport properties.

Sintesi di idrogel con proprietà antivegetative come membrane per la purificazione dell&#39;acqua

Hydrogels have been widely utilized to enhance the surface hydrophilicity of membranes for water purification, increasing the antifouling properties and ...

Surface Engineering of Pancreatic Islets with a Heparinized StarPEG Nanocoating

Cell surface engineering can protect implanted cells from host immune attack. It can also reshape cellular landscape to improve graft function and survival post-transplantation. This protocol aims to achieve surface engineering of pancreatic islets using an ultrathin heparin-incorporated starPEG (Hep-PEG) nanocoating. To generate the Hep-PEG nanocoating for pancreatic islet surface engineering, heparin succinimidyl succinate (Heparin-NHS) was first synthesized by modification of its carboxylate groups using N-(3-dimethylamino propyl)-N'-ethyl carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). The Hep-PEG mixture was then formed by crosslinking of the amino end-functionalized eight-armed starPEG (starPEG-(NH2)8) and Heparin-NHS. For islet surface coating, mouse islets were isolated via collagenase digestion and gradient purification using Histopaque. Isolated islets were then treated with ice cold Hep-PEG solution for 10 min to allow covalent binding between NHS and the amine groups of islet cell membrane. Nanocoating with the Hep-PEG incurs minimal alteration to islet size and volume and heparinization of the islets with Hep-PEG may also reduce instant blood-mediated inflammatory reaction during islet transplantation. This "easy-to-adopt" approach is mild enough for surface engineering of living cells without compromising cell viability. Considering that heparin has shown binding affinity to multiple cytokines, the Hep-PEG nanocoating also provides an open platform that enables incorporation of unlimited functional biological mediators and multi-layered surfaces for living cell surface bioengineering.

This protocol aims to achieve surface engineering of pancreatic islets using a heparin-incorporated starPEG nanocoating via pseudo-bioorthogonal chemistry between the N-hydroxysuccinimide groups of the nanocoating and the amine groups of islet cell membrane.

Ingegneria delle superfici degli isolotti pancreatici con un nanorivestimento StarPEG eparinizzata

Cell surface engineering can protect implanted cells from host immune attack. It can also reshape cellular landscape to improve graft function and ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.

Rilevamento dell'endotossina in Nano-formulazioni utilizzando dosaggi di Limulus Amoebocyte Lysate (LAL)

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

Wet-spinning-based Molding Process of Gelatin for Tissue Regeneration

This article presents an inexpensive method to fabricate gelatin, as a natural polymer, into monofilament fibers or other appropriate forms. Through the wet spinning method, gelatin fibers are produced by smooth extrusion in a suitable coagulation medium. To increase the functional surface of these gelatin fibers and their ability to mimic the features of tissues, gelatin can be molded into a tube form by referring to this concept. Examined by in vitro and in vivo tests, the gelatin tubes demonstrate a great potential for application in tissue engineering. Acting as a suitable filling gap material, gelatin tubes can be used to substitute the tissue in the damaged area (e.g., in the nervous or cardiovascular system), as well as to promote regeneration by providing a direct replacement of stem cells and neural circuitry. This protocol provides a detailed procedure for creating a biomaterial based on a natural polymer, and its implementation is expected to greatly benefit the development of correlative natural polymers, which help to realize tissue regeneration strategies.

We developed and describe a protocol based on the wet spinning concept, for the construction of gelatin-based biomaterials used for the application of tissue engineering.

Processo di stampaggio basati su bagnato-filatura di gelatina per la rigenerazione tissutale

This article presents an inexpensive method to fabricate gelatin, as a natural polymer, into monofilament fibers or other appropriate forms. Through the ...