Diese Arbeit wurde finanziell von Swiss National Science Foundation (- Smart Materials NFP 62) unterstützt. Auch anerkannt ist die Unterstützung von B. Hanselmann, K. Kehl, U. Schütz und B. Leuthold.

1. Plasma-initiierte Polymerisation <ol><li> Vorbereiten der Monomer-Lösung. <ol><li> Auflösen HEMA (100 ml, 0,718 mol) in 200 ml Wasser und wasche 3x mit Hexan (100 ml) in einen Scheidetrichter. Sättigt die wässrige Phase mit Natriumchlorid und extrahiert die HEMA mit Diethylether (50 ml). Trocknen der organischen Phase über MgSO 4 getrocknet und das Lösemittel wird im Vakuum (100 mbar, 40 ° C). Destillation des HEMA unter vermindertem Druck (15 mbar, 99 ° C). </li><li> Bereiten Sie eine 0,62 M methanolischen Lösung der in Abschnitt 1.1.1 hergestellt Inhibitor-freie HEMA. Gießen von 30 ml der Lösung in einen Einhalskolben Sauerstoff zu eliminieren und durch Einblasen von Ar durch die Lösung für 1 Stunde. </li></ol></li><li> Oberflächeninduzierte Polymerisation. <ol><li> Position zwei Polycarbonatmembranen nebeneinander in die Plasmakammer (2). Zeigen die glänzende Seite der Membran in Richtung der Gasphase. </li><li> Schließen Sie die Plasmakammer ahoch Vakuum (20 mbar) für 5 min. Schließen Sie das Ventil zur Vakuum und öffnen Sie das andere Ventil, das Argon und Sauerstoff verbunden ist und reinigen die Kammer mit dieser Mischung für 2 h mit 15 sccm Argon und 2,5 sccm Sauerstoff. </li><li> Initiieren Sie die Plasma-und die Macht, die gewünschte Leistung (für Polycarbonat-Membran: 12 W) zu reduzieren und zu behandeln, die die Membranen für 4 min mit dem Plasma. Schließen Sie das Monomer-Lösung mit der Kammer durch Öffnen des entsprechenden Ventils. Schalten Sie das Plasma und die Kammer zu evakuieren. </li><li> Schließen Sie das Monomer-Lösung mit der Kammer durch Öffnen des entsprechenden Ventils und gießen Sie die Lösung in die Kammer. Sicherzustellen, dass die Membranen mit der Monomerlösung behandelt. Öffnen des Ventils in die Argon verbunden und speichert die Reaktionsmischung für 12 h bei 20 ° C (Raumanlage). </li><li> Entfernen Sie die Monomer-Lösung. Waschen der Membranen mit Methanol in einem Ultraschallbad 5 min. Wiederholen Sie den Waschvorgang mit Wasser. </li><li> Trocknen der Membran inVakuum über Molekularsieben für 2 Stunden. </li></ol></li></ol> 2. Postmodification von beschichteten Membranen <ol><li> Eine Lösung aus Spirobenzopyran 1 (Fig. 1) (100 mg, 0,27 mmol), N, N-Dicyclohexylcarbodiimid (DCC) (55 mg, 0,27 mmol) und Dimethylaminopyridin (DMAP) (33 mg, 0,27 mmol) in tert-butylmetylether (TBME) (12 ml). </li><li> Legen Sie eine Schutz Rührstab und einem Schutzgitter in einen Rundkolben. Trocknen Sie die Kolben und Flut der Kolben mit Argon. </li><li> Gießen der Lösung in den Kolben gegeben, gefolgt von der beschichteten Membran. </li><li> Rühren Sie vorsichtig bei Raumtemperatur für 12 Stunden. </li><li> Entfernen der Lösung und Waschen der Membran mit tert-butylmetylether in einem Ultraschallbad 5 min. Diesen Vorgang mit Ethanol und Wasser. </li><li> Trocknen der Membran unter Vakuum über Molekularsieben für 2 Stunden. </li></ol> 3. Oberflächenspannungsmessungen <ol><li&gt; Fix die Membran mit einer Standard-Band auf einem Metall-O-Ring. Positionieren einen Tropfen nanoreinem Wasser (3,3 ul) auf diesem Teil der Membranoberfläche, die in Kontakt mit dem O-Ring nicht. Messung des Kontaktwinkels (CA) an 5 verschiedenen Stellen der Membran. </li><li> Für die Prüfung der Langzeitstabilität der Proben zu messen, die Kontaktwinkel an drei verschiedenen Stellen der Membranen nach 0, 1, 2, 3, 7, 14 und 21 Tagen. </li></ol> 4. Durchlässigkeit Tests der Photochrome Membranen <ol><li> Füllen Sie den Rezeptor Kammer des Franz-Diffusionszelle mit Wasser (12 ml). </li><li> Befestigen Sie die Membran in einer Franz-Diffusionszelle. Sicherzustellen, dass die Membran in Kontakt mit dem Wasser der Aufnahmekammer. Füllen des Spenders Kammer (die Kammer auf der Membran) mit einer wässrigen Coffeinlösung (20 mM, 3,0 ml). Bestrahlen der Membran von der Oberseite des Spenderkammer mit Weißlicht (Abbildung 3). Die Proben (200 ul) aus der Empfängerzelle, for Spur kantige Polycarbonatmembranen mit einem Porendurchmesser von 200 nm, sammeln Proben alle 10 min. </li><li> Wiederholen Sie den Versuch wie in Schritt 4.2 beschrieben. aber bestrahlen die Membran mit UV-Licht (366 nm, 80 W / m 2) während des gesamten Durchlässigkeitstest. </li><li> Bestimmung der Koffeinkonzentration der gesammelten Proben. <ol><li> Eichkurve mit 15 verschiedenen Koffein-Konzentrationen (zwischen 0,05 mg / ml und 1,5 mM / L) unter Verwendung eines UV / Vis-Spektrometer. Kalibrieren bei 293 nm auf. </li><li> Bestimmung der Konzentration von jeder der entnommenen Proben unter Verwendung der Eichkurve. </li><li> Zeichnen Sie die ermittelte Konzentration vs. die Zeit der gesammelten Proben. Machen Sie eine lineare Anpassung durch die Punkte und bestimmen, &amp;Dgr; c aus der Steigung. </li></ol></li></ol>

<ol>
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Die Autoren haben nichts zu offenbaren.

Die Plasma-Verfahren erzeugt eine lila Gas, das von ionisierten Argon verursacht wird. Eine orange Farbe würde das Vorhandensein von unerwünschten Stickstoff aus einem Leck anzuzeigen. Die Plasma-Prozess nicht nur zu bilden Radikale auf der Oberfläche, sondern auch ätzt die Membran 7,12. Zu viel Ätzen können die Porendurchmesser erheblich ändern, wodurch die Durchlässigkeit der Membran beeinflussen würde. Die kontrollierten Reaktionsbedingungen der dargestellten Einrichtung ermöglichen die Verbesser...

Plasmamodifizierung von Materialien ist ein wichtiges Verfahren in vielen Industriebereichen. Reinigung von Oberflächen und Funktionalisierung von Oberflächen, ohne die Fließeigenschaften des Materials der Plasmabehandlung ein wesentlicher Prozess in Surface Science 1-8 hergestellt. Die Plasmabehandlung von Polymeren ergibt homolytische Bindungsspaltung. Dies führt zu einem Rand aus dem Polymermaterial und die Bildung von Radikalreichen Oberflächen. Durch die Verwendung von Sauerstoff-Plasma-Moleküle enthält, wird die Oberfläche sauerstoffreichen und somit hydrophiler 9-11. Jedoch ist die Hydrophilie der Oberfläche 12 nicht mit der Zeit stabil. Um die langfristige Stabilität zu erhöhen, kann das Plasma behandelten Oberfläche chemisch nach oder während des Plasmaprozesses 13-15 geändert werden. Diese Behandlung wird in der Regel durch Zugabe einer reaktiven Monomer-Spezies in der Gasphase während der Plasmaprozeß durchgeführt wird, wobei diese Monomere zu polymerisieren, dannaus den erzeugten Reste der Polymeroberfläche. Wenn man die chemische Behandlung mit einem nicht-flüchtigen Monomers durchgeführt wird, hat das Polymer Pfropfung stattfindet, nachdem das Plasma Modifikation nehmen. Um eine kontrollierte Pfropfen durchzuführen, nachdem die Radikale auf der Oberfläche gebildet wird, wird ein Plasma Einrichtung beschrieben, die das Plasma initiiert oberflächeninduzierte Polymerisation von der Oberfläche in der Lösung unter kontrollierten Bedingungen 12,16 ermöglicht. Die Präsentation konzentriert sich auf die Änderung der Spurkantige Polymermembranen 12,17. Durch Modifizieren der Oberflächenspannung dieser Membranen kann die Durchlässigkeitsrate 12 variiert werden. Diese saubere und schnelle Verfahren ermöglicht die Erzeugung von sehr dünnen Schichten (&lt;5 nm), die die gesamte Membranoberfläche ohne Veränderung der Fließeigenschaften der Polymermembran abzudecken. Aufgrund der Kanten während des Plasmaprozesses, die Porendurchmesser der Spurkantigen Membranen 12 leicht zu erhöhen. Das Abschluss Rate depending auf dem Polymer und hat ein lineares Verhalten. Bei der Verwendung von Monomeren mit reaktiven funktionellen Gruppen, können die gepfropften Polymere weiter funktionalisiert werden. Dies wird durch die postmodification eines PHEMA-Membran gepfropft mit einer Carbonsäure funktionalisiert Spiropyran gezeigt. Dies führt zu einer photochromen Oberflächen, da Spiropyran ist bekannt, in einem Merocyanin-Spezies zu transformieren, wenn sie mit UV-Licht bestrahlt. Die Spiropyranform kann durch Bestrahlung der Merocyaninform mit sichtbarem Licht (Abbildung 1) 18,19 wiederhergestellt werden. Da die Merocyaninform polarer ist als das Spiropyran Zustand kann die Oberflächenspannung der Beschichtung mit Licht 20 ausgelöst werden. Die Änderung der Oberflächenspannung beeinflusst die Durchlässigkeit Widerstand der Membran gegenüber wässrigen Lösungen. Das Set-up, wie man die Durchlässigkeit dieser Tests auf Licht reagieren, Membranen durchführen wird gezeigt werden, und die wesentliche Änderung der Durchlässigkeit Widerstand (i verringernn Durchlässigkeit Widerstand um 97%) nachgewiesen wird. Eine solche Membran in einer Drug-Delivery-Installation oder in Smart Sensing-Systeme integriert werden. <img alt="Figur 1" fo:content-width="5in" fo:src="/files/ftp_upload/51680/51680fig1highres.jpg" src="/files/ftp_upload/51680/51680fig1.jpg" /> Abbildung 1. Photoisomerisierung von Spirobenzopyran Verbindung ein.

<table border="0" cellpadding="0" cellspacing="0" style="width:943px;" width="943">
	<colgroup>
		<col />
		<col />
		<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

Um die Oberflächenspannung der handelsüblichen Spurkantigen Polymer-Membranen zu modifizieren, ist ein Verfahren der Oberflächen initiierte Polymerisation vorgelegt. Die Polymerisation von der Membranoberfläche durch Plasmabehandlung der Membran, gefolgt von Umsetzen der Membranoberfläche mit einer methanolischen Lösung von 2-Hydroxyethylmethacrylat (HEMA) induziert. Besondere Aufmerksamkeit wird auf die Prozessparameter für die Plasmabehandlung vor der Polymerisation auf der Oberfläche gegeben. Zum Beispiel wird der Einfluss der Plasmabehandlung auf verschiedenen Arten von Membranen (z. B. Polyester, Polycarbonat, Polyvinylidenfluorid) untersucht. Weiterhin wird die zeitabhängige Stabilität der Oberfläche gepfropften Membranen durch Kontaktwinkelmessungen gezeigt. Beim Pfropfen von Poly (2-hydroxyethylmethacrylat) (PHEMA), so kann die Oberfläche durch Veresterung der Alkoholanteil des Polymers mit einer Carbonsäurefunktion der gewünschten Substanz modifiziert werden.Diese Reaktionen können daher für die Funktionalisierung der Membranoberfläche verwendet werden. Zum Beispiel kann die Oberflächenspannung der Membran geändert werden oder eine gewünschte Funktionalität wie die vorgelichtReaktions eingefügt werden. Dies wird durch Umsetzen von PHEMA mit einer Carbonsäure funktionalisiert Spirobenzopyran Einheit, die mit einer lichtansprechenden Membran führt demonstriert. Die Wahl des Lösungsmittels spielt eine wesentliche Rolle bei der postmodification Schritt und ist detaillierter in diesem Dokument diskutiert. Die Durchlässigkeitsmessungen von solchen funktionalisierten Membranen werden unter Verwendung einer Franz-Zelle mit einer externen Lichtquelle. Durch Ändern der Wellenlänge des Lichts aus dem sichtbaren UV-Bereich, eine Veränderung der Permeabilität der wässrigen Coffein-Lösungen beobachtet.

Die Ätzrate kann durch Wiegen der Membran nach verschiedenen Zeiten verfolgt werden. Die Ätzrate wie aus Fig. 4 ersichtlich, ergibt sich für Polyester, Polyvinylidenfluorid und Polycarbonat-Membranen eine lineare Ätzrate, die von der Steigung der linearen Korrelation der Ätzzeit gegen Masseverlust festgestellt werden. Wie in Fig. 4 gezeigt ist, die Polycarbonat-Membranen zeigen die niedrigsten Ätzrate aller drei Polymermembranen. Eine Folge des Ätzens ist die Änderung d...

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

Ein Plasma-induzierte Polymerisation Verfahren für die oberflächeninitiierte Polymerisation zu Polymermembranen beschrieben. Weitere postmodification des gepfropften Polymers mit photochromen Substanzen mit einem Protokoll zur Durchführung von Durchlässigkeitsmessungen auf Licht ansprechende Membranen vorgestellt.

Herstellung der lichtreaktions Membranen durch eine Kombinierte Oberflächen-Pfropfen und Postmodification Prozess

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 Aufrufe.  Empa, Swiss Federal Laboratories for Materials Science and Technology.  Ein Plasma-induzierte Polymerisation Verfahren für die oberflächeninitiierte Polymerisation zu Polymermembranen beschrieben. Weitere postmodification des gepfropften Polymers mit photochromen Substanzen mit einem Protokoll zur Durchführung von Durchlässigkeitsmessungen auf Licht ansprechende Membranen vorgestellt. 

Serie: 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.

Die Messung der Aggregate Zusammenhalt durch Gewebeoberfläche Tensiometrie

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

Forschung

Biotechnik

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.

Selbstberichts Gerüste für 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 ...

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.

Generierung und Pfropfen von Tissue Engineering Gefäße in einem Maus-Modell

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.

Anpassung der Elektrospinnverfahren für einen Dreischicht stellen drei einzigartige Umgebungen<em&gt; In-vitro-</em&gt; Modell der Atemwegswand

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.

Qualitative Charakterisierung der wässrigen Fraktion von hydrothermale Verflüssigung von Algen mit Hilfe von 2D-Gaschromatographie mit 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 ...

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.

Automatische Robotic Dosiertechnik für Oberflächen Ausrichtungs- und Bioprinting von Zellen

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.

Synthese von Hydrogelen mit Antifouling-Eigenschaften als Membranen für die Wasserreinigung

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.

Oberflächentechnik von pankreatischen kleinen Inseln mit einer Heparinisierten StarPEG NanoCoatings

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.

Nachweis von Endotoxin im Nano-Rezepturen mit Limulus Amoebocyte Lysate (LAL) Assays

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.

Nass-Spinnen-basierte Spritzgießprozess Gelatine für die Geweberegeneration

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