The authors would like to acknowledge the help of Walter Scholdei (Max-Planck-Institute for Polymer Research, Mainz, Germany.

1. Herstellung von Natriumnaphthalenid Aktivierungslösung und Oberflächenaktivierung Hinweis: Führen Sie die Reaktionen in einem gut belüfteten Abzug durchführen. Die allgemeinen Regeln für den Umgang mit hoch brennbaren Lösungsmitteln und korrosiven Metalle wie metallisches Natrium. Naphtalin hat einen sehr unangenehmen Geruch (mothball), auch in sehr kleinen Mengen! Wenn nicht anders angegeben Reaktionen werden bei Raumtemperatur durchgeführt. Natriumazid ist hochgiftig! THF (99,9%, siehe Materialliste) wurde über etwa 20% (bezogen auf das Volumen) Molekularsieb gelagert. Destillieren THF mit einem merklichen Wassergehalt über Natrium. Die Bildung von Natriumnaphthalenid tritt nicht auf, wenn Spurenmengen von Wasser vorhanden sind. <ol><li> Zu einer Lösung von 1,4 g (10,9 mmol) Naphthalin in 20 ml Tetrahydrofuran (THF, getrocknet über 3 Å Molekularsieb), Zugabe von 0,25 g (10,9 mmol) Natriummetall in einem Schraubverschluss 100 ml Glasflasche mit einem PTFE- ausgestattet beschichteten magnetischen Rührstab. Hinweis: dissolu(- 40 ° C 35) tion kann durch Schneiden des Natrium in kleine Stücke und bescheiden Erwärmung stark verbessert werden. Die endgültige Lösung hat eine dunkle, leicht grünliche Farbe und kann unter streng trocken gelagert werden. </li><li> Punch-Out-PTFE-Scheiben von 12 mm Durchmesser von 0,5 mm dicken Folienmaterial. Markieren Sie eine Seite (zB mit einem Brief Stempel Stempel) und sauber und mit iso-Propanol. </li><li> Inkubieren PTFE-Proben einzeln in der Aktivierungslösung (Schritt 1.1) mit 1 - 2 Minuten mit einer Pinzette. Hinweis: Der Farbumschlag von weiß bis dunkelbraun erfolgreiche Behandlung zeigt. </li><li> Anschließend spülen zweimal mit THF und dann mit Isopropanol. Hinweis: Die Naphthalenid ist erschöpft, wenn seine Farbe ändert sich zu einem wässrig hellbraun. Nicht verwendete Naphthalenid Lösung (möglicherweise metallisches Natrium enthält) muss durch langsam zerlegt werden, indem Isopropanol vor der Entsorgung. </li><li> Oxidieren behandelten Proben in Wasserstoffperoxid (30%) 20% (w / v) Trichloressigsäure für 3 Stunden. Waschenmit Wasser und trocken. Die Oberfläche hat jetzt eine leichte bräunliche Aussehen. Hinweis: Die Aktivierung und Oxidation führt zu einer drastisch erhöhten Benetzbarkeit der Oberfläche. Diese Feststellung wurde im Detail zuvor 14 untersucht. </li><li> Behandeln Sie oxidierte Scheiben mit 50% (v / v) Hexamethylendiisocyanat (HMDI) in trockenem THF 2 h gründlich mit THF und trocknen lassen. </li><li> Hydrolysieren Isocyanat Lagerproben in Wasser für 2 - 3 Stunden und trocken. Anmerkung: Die endgültige aminofunktionalisierte PTFE-Oberfläche als die Isocyanat-tragenden Proben wesentlich stabiler ist und ist kompatibel mit vielen Standard-Vernetzer für die weitere Kupplung. </li></ol> 2. Peptide Immobilisation <ol><li> Eine Lösung aus 20% (v / v) Diethylenglycol-diglycidylether (Diepoxid) in 50 mM Carbonatpuffer, pH 9. </li><li> Legen Sie die aminierte Scheiben mit der markierten Seite nach oben einzeln in eine 24-Well-Platte und fügen Sie 1,5 ml der Epoxid-Lösung. Achten Sie darauf, eine vollständige Abdeckung der Proben. Inkubieren für 2 Stunden und waschen zweimals mit Wasser und einmal mit Carbonatpuffer. </li><li> Zugabe von 50 ul einer 0,5 mg / ml Peptid (zB REDV) in 50 mM Carbonatpuffer, pH 9 , enthaltend 0,01% NaN 3, auf den Boden der einzelnen Vertiefungen einer frischen 24 - Well - Platte und legen sorgfältig die epoxy-funktionalisierten Scheiben upside -down (dh markierte Seite nach unten) auf den Abfall der Peptidlösung. Stellen Sie sicher, dass der Raum zwischen dem Boden des Bohrlochs und der PTFE Scheibe Kapillarwirkung aufgrund vollständig benetzt wird. </li><li> Inkubieren in einer feuchten Kammer (zB jede verschließbaren Plastikkasten mit einem dicht schließendem Deckel) mit befeuchteten Atmosphäre (durch nasse Seidenpapier auf dem Boden platzieren) für mindestens 3 Stunden oder über Nacht. Hinweis: Wenn auch das REDV- Motiv als gut charakterisiert, kann ein Rühr- oder Reverse Aminosäuresequenz angesehen werden kann als zusätzliche Negativkontrolle einbezogen werden. </li><li> Waschen dreimal mit Wasser und Sterilisieren in 50% iso-Propanol / Wasser für mindestens 30 min. Vor der Aussaat zu Zelle, rinse die Proben in steriler Phosphat-gepufferter Salzlösung (PBS). </li></ol> 3. Zell Seeding <ol><li> Wachsen HUVECs unter Verwendung von Standardzellkulturverfahren 17,18. </li><li> Legen Sie die Peptid-konjugierten Proben mit der modifizierten Seite nach oben in einer 24-Well-Platten. Verwenden Sie unbehandelten PTFE-Discs als Kontrolle. </li><li> Seed 5 x 10 4 HUVECs in 2 ml Medium pro Vertiefung und Inkubation für 4 h bei 37 ° C und 5% CO 2 in einem Inkubator. </li><li> Entfernen Sie die Scheiben gründlich sorgfältig ungebunden Zellen zu entfernen und in ein frisches 24-Well-Platte. 2 ml frisches Medium und Inkubation für den gewünschten Zeitraum (zB 24 Stunden, 1 w, etc.). Ändern Sie das Medium alle zwei Tage. </li><li> Bereiten Sie eine Stammlösung von 1 mg / ml Calcein-AM in Dimethylsulfoxid (DMSO). </li><li> Nach dem Züchten der Zellen des Mediums, Waschen mit PBS entfernen und PBS , das 1 &amp; mgr; l / ml Calcein-AM - Färbung (beispielsweise 1 &amp; mgr; l einer Stammlösung pro ml PBS) hinzuzufügen. Vorsichtig geschwenkt und INCUBATe bei 37 ° C für 45 min im Dunkeln. </li><li> Waschen mit PBS und sofort nehmen mikroskopische Aufnahmen auf einem Fluoreszenzmikroskop mit Standard-FITC-Filter ausgestattet (Ex 488 nm, Em 515 nm). Verwenden Sie das 100-fache Vergrößerung. Führen Sie Triplikaten von unmodifizierten sowie behandelten Proben. </li><li> Mit ImageJ Software bestimmen den Bereich von den Zellen besiedelt. zählen Alternativ können die Zellen manuell oder ImageJ verwenden. Beide Verfahren geben ähnliche Informationen über die Anhaftung und das Wachstum von Zellen auf den jeweiligen Oberflächen. </li></ol>

<ol>
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The authors have nothing to disclose.

Die detaillierte Beschreibung der Oberflächenmodifikationsprotokoll PTFE besteht aus aufeinanderfolgenden Stufen unter Abspaltung von Fluor aus dem Polymergerüst in 6 als dargestellt zu starten. Als Ergebnis wird eine Schicht gebildet, die eine reichliche Menge an konjugierten Kohlenstoff-Kohlenstoff - Doppelbindungen enthält , in gemäß der dunkelbraune Farbe, die auf Naphthalenid Behandlung entwickelt. Standard Oxidation mit sauren Wasserstoffperoxid ergibt eine hydroxylierte Oberfläche durch Auf...

Verschiedene Polymere in der Medizin eingesetzt , die seit einiger Zeit zugelassen wurden keine verbesserte Biokompatibilität aufweisen, das heißt, der Mangel an Zell-Haft, Induktion von fibrotischen Verkapselung und Thrombogenität, um nur einige zu nennen. Wechselwirkungen zwischen dem Biomaterial und dem biologischen System erfolgt hauptsächlich an der Oberfläche des Implantats. Als Folge hat sich die Forschung auf die Oberflächenmodifikation konzentriert, um geeignete Eigenschaften für eine gewünschte Anwendung zu schaffen, während die Volumeneigenschaften des Materials unbeeinflusst bleibt. Polytetrafluorethylen (PTFE) als ein physiologisch inertes Polymer wird in vielen medizinischen Gebieten, wie Hernie chirurgisches Netz 1, medizinische Anschlüsse 2 verwendet und, am wichtigsten, vaskuläre Transplantate 3. Insbesondere in Blut in Kontakt Situationen die hydrophobe Natur von PTFE verursacht unspezifische Adsorption von Plasmabestandteilen und als Folge Thrombozytenadhäsion, oft in thromboti ergebc Ereignisse und Okklusion des Transplantats 4. Weiterhin PTFE, wie die meisten Polymere nicht unterstützt Zelladhäsion und Abdeckung , die ein wünschenswertes Merkmal 5 wäre die Bildung einer nützlichen Schicht von Endothelzellen (ECs) auf der inneren (luminale) Oberfläche des vaskulären Transplantats zu induzieren. Eine biomimetische Endothel wird erwartet , dass viele der Funktionen seiner natürlichen Äquivalent zu erfüllen, vor allem seine antithrombogenen Eigenschaften 6. Eine allgemeine biomimetischen Modifikationsstrategie basiert auf dem Konzept der ausschließlich das Material mit Zellhaftfähigkeit verleihen, während die Eigenschaften Materialien bulk verlassen unberührt. Zusätzlich kann Thrombozytenadhäsion durch Einarbeiten antiadhäsive (anti-fouling) Attribute 7 reduziert werden. Verschiedene Peptide - meist aus Proteinen der extrazellulären Matrix abgeleitet ist - wurden beschrieben , die stark durch die Bindung an zelluläre Rezeptoren gehört zu der Klasse von Integrinen 8 Zelladhäsion verbessern. Das Seinst bekanntes Beispiel in dieser Hinsicht ist das Peptid Arg-Gly-Asp (RGD), die mit den meisten Zelltypen interagiert. Andere Aminosäuresequenzen werden durch Integrine exprimiert ausschließlich auf spezifische Zellen erkannt. Zum Beispiel Arg-Glu-Asp-Val (REDV-) und Tyr-Ile-Gly-Ser-Arg (YIGSR-) wurden ECs zu binden , in einer bestimmten Art und Weise 9 gefunden. Kovalente Immobilisierung solcher Peptide wurde auch von Natur aus nicht klebenden Materialien durchgeführt auf einer Vielzahl von Metallen und Polymeren 10,11. Poröse PTFE, genauer aus expandiertem PTFE (ePTFE) - zusammen mit Polyethylenterephthalat (PET) - ist das wichtigste Material für die Herstellung von vaskulären Transplantaten 12. Etablierte physikalische Techniken für entsprechende Behandlungen, wie Plasmamodifizierung 13 oder durch photochemische Verfahren 14 werden durch die Tatsache behindert , daß poröse und / oder röhrenförmige Strukturen sind nicht leicht behandelbar in den Poren oder dem Lumen verbunden. naßchemischenauf PTFE ist eine schwierige Aufgabe aufgrund der stark inert Natur des fluorin enthaltenden Polymer , das die meisten chemischen Angriffen 15 widersteht . In diesem Papier beschreiben wir ein vergleichsweise einfaches Verfahren für eine kovalente Modifikation Strategie. Angepasst von einem Verfahren PTFE bindungsfähig zu machen, funktionelle Gruppen wurden auf der Materialoberfläche geschaffen, die für eine weitere Konjugation von biologisch aktiven Molekülen als Ankerpunkte dienen.

<table border="0" cellpadding="0" cellspacing="0" width="700">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:323px;">PTFE foil 0.5 mm</td>
			<td style="width:119px;">Cadillac Plastic&#160;</td>
			<td style="width:145px;">n/a</td>
			<td style="width:115px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">REDV peptide</td>
			<td>Genecust</td>
			<td>n/a</td>
			<td>custom synthesis &#62;95 % purity</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">iso-propanol</td>
			<td>Sigma Aldrich</td>
			<td>34965</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">tetrahydrofurane (THF)</td>
			<td>Sigma Aldrich</td>
			<td>401757</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">dimethylsulfoxide</td>
			<td>Sigma Aldrich</td>
			<td>D8418</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">molecular sieve 3&#197;</td>
			<td>Sigma Aldrich</td>
			<td>208574</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">sodium metal</td>
			<td>Sigma Aldrich</td>
			<td>483745</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">phosphate buffered saline (PBS)</td>
			<td>Sigma Aldrich</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">naphthalene</td>
			<td>Sigma Aldrich</td>
			<td>147141</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">hydrogen peroxide 30 %</td>
			<td>Sigma Aldrich</td>
			<td>95321</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">trichloroacetic acid</td>
			<td>Sigma Aldrich</td>
			<td>T6399&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">diethylene glycol diglycidyl ether</td>
			<td>Sigma Aldrich</td>
			<td>17741</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">hexamethylene diisocyanate (HMDI)</td>
			<td>Sigma Aldrich</td>
			<td>52650</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Calcein-AM</td>
			<td>Sigma Aldrich</td>
			<td>56496</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">sodium bicarbonate</td>
			<td>Sigma Aldrich</td>
			<td>S6014&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">sodium azide</td>
			<td>Sigma Aldrich</td>
			<td>71290</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">24 well plates</td>
			<td>Greiner-Bio-One</td>
			<td>662 160</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ATR-FTIR spectrophotometer Nicolet Magna-IR 850</td>
			<td>&#160;Nicolet</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">fluorescence microscope Olympus X-70</td>
			<td>Olympus</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">humbilical vein endothelial cells (HUVECs)</td>
			<td>Lonza</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ePTFE vascular graft</td>
			<td>Gore</td>
			<td>n/a</td>
			<td></td>
		</tr>
	</tbody>
</table>

wet chemistry and peptide immobilization on polytetrafluoroethylene for improved cell-adhesion

Stiften Materialien Oberfläche mit zellKlebeEigenschaften ist eine gemeinsame Strategie in Biomaterial Forschung und Tissue Engineering. Dies ist besonders interessant für bereits zugelassene Polymere, die eine langjährige Verwendung in der Medizin haben, weil diese Materialien sind gut charakterisiert und rechtliche Fragen bei der Einführung im Zusammenhang von neu synthetisierten Polymere vermieden werden können. Polytetrafluorethylen (PTFE) ist eine der am häufigsten verwendeten Materialien für die Herstellung von Gefäßprothesen, aber das Polymer fehlt Förderung Merkmale Zelladhäsion. Endothelialisierung, dh eine vollständige Abdeckung der Transplantate Innenfläche mit einer konfluenten Schicht von Endothelzellen ist der Schlüssel für eine optimale Leistung angesehen, vor allem durch Thrombogenität der künstlichen Schnittstelle reduziert wird . Diese Studie untersucht, das Wachstum von Endothelzellen auf Peptid-modifiziertem PTFE und vergleicht diese Ergebnisse mit denen auf unmodifizierte Substrat erhalten. Kupplung mit demendotheliale Zellen-Haftungs-Peptid Arg-Glu-Asp-Val (REDV) über die Aktivierung des fluorin enthaltenden Polymers unter Verwendung des Reagenzes Natriumnaphthalenid, gefolgt durch nachfolgende Konjugation Schritten durchgeführt. Zellkulturen Zellen (HUVECs) und eine hervorragende Zellwachstum auf Peptid-immobilisierte Material unter Verwendung von humanen Umbilical Vein Endothelial erreicht wird, ein Zeitraum von zwei Wochen gezeigt über.

Die Ergebnisse der entscheidenden chemischen Reaktionsschritte wurden durch IR - Spektroskopie (Figur 1) überwacht. Die anfängliche Aktivierung mit Natriumnaphthalenid erzeugt Doppelbindungen - und in geringerem Umfang - OH-Funktionalitäten. Das Signal, das anzeigt C = C-Bindungen verschwinden bei der Oxidation, eine Oberfläche ergibt sich fast ausschließlich Hydroxyl-Gruppen tragen. Analyse weiterer Standard Konjugation Schritte sind hier nicht dargestellt. Die Far...

Watch this Scientific Journal Video about Wet Chemistry and Peptide Immobilization on Polytetrafluoroethylene for Improved Cell-adhesion at JoVE.com

Wet Chemistry and Peptide Immobilization on Polytetrafluoroethylene for Improved Cell-adhesion

Cell-adhesiveness is key to many approaches in biomaterial research and tissue engineering. A step-by-step technique is presented using wet-chemistry for the surface modification of the important polymer PTFE with peptides.

Nasschemie und Peptid Immobilisation auf Polytetrafluoroethylen für verbesserte Zellhaftung

Endowing materials surface with cell-adhesive properties is a common strategy in biomaterial research and tissue engineering. This is particularly ...

wet-chemistry-peptide-immobilization-on-polytetrafluoroethylene-for

Research

JoVE Journal

Bioengineering

7.6K Aufrufe.  Sidra Cardiovascular Research. Cell-adhesiveness is key to many approaches in biomaterial research and tissue engineering. A step-by-step technique is presented using wet-chemistry for the surface modification of the important polymer PTFE with peptides.

Serie: Wet Chemistry and Peptide Immobilization on Polytetrafluoroethylene for Improved Cell-adhesion

Bacterial Immobilization for Imaging by Atomic Force Microscopy

AFM is a high-resolution (nm scale) imaging tool that mechanically probes a surface. It has the ability to image cells and biomolecules, in a liquid environment, without the need to chemically treat the sample. In order to accomplish this goal, the sample must sufficiently adhere to the mounting surface to prevent removal by forces exerted by the scanning AFM cantilever tip. In many instances, successful imaging depends on immobilization of the sample to the mounting surface. Optimally, immobilization should be minimally invasive to the sample such that metabolic processes and functional attributes are not compromised. By coating freshly cleaved mica surfaces with porcine (pig) gelatin, negatively charged bacteria can be immobilized on the surface and imaged in liquid by AFM. Immobilization of bacterial cells on gelatin-coated mica is most likely due to electrostatic interaction between the negatively charged bacteria and the positively charged gelatin. Several factors can interfere with bacterial immobilization, including chemical constituents of the liquid in which the bacteria are suspended, the incubation time of the bacteria on the gelatin coated mica, surface characteristics of the bacterial strain and the medium in which the bacteria are imaged. Overall, the use of gelatin-coated mica is found to be generally applicable for imaging microbial cells.

Live Gram-negative and Gram-positive bacteria can be immobilized on gelatin-coated mica and imaged in liquid using Atomic Force Microscopy (AFM).

Bakterielle Immobilisierung für Imaging von Atomic Force Microscopy

AFM is a high-resolution (nm scale) imaging tool that mechanically probes a surface.  It has the ability to image cells and biomolecules, in a liquid ...

Forschung

Biotechnik

Hydrophobic Salt-modified Nafion for Enzyme Immobilization and Stabilization

Over the last decade, there has been a wealth of application for immobilized and stabilized enzymes including biocatalysis, biosensors, and biofuel cells.1-3 In most bioelectrochemical applications, enzymes or organelles are immobilized onto an electrode surface with the use of some type of polymer matrix. This polymer scaffold should keep the enzymes stable and allow for the facile diffusion of molecules and ions in and out of the matrix. Most polymers used for this type of immobilization are based on polyamines or polyalcohols - polymers that mimic the natural environment of the enzymes that they encapsulate and stabilize the enzyme through hydrogen or ionic bonding. Another method for stabilizing enzymes involves the use of micelles, which contain hydrophobic regions that can encapsulate and stabilize enzymes.4,5 In particular, the Minteer group has developed a micellar polymer based on commercially available Nafion.6,7 Nafion itself is a micellar polymer that allows for the channel-assisted diffusion of protons and other small cations, but the micelles and channels are extremely small and the polymer is very acidic due to sulfonic acid side chains, which is unfavorable for enzyme immobilization. However, when Nafion is mixed with an excess of hydrophobic alkyl ammonium salts such as tetrabutylammonium bromide (TBAB), the quaternary ammonium cations replace the protons and become the counter ions to the sulfonate groups on the polymer side chains (Figure 1). This results in larger micelles and channels within the polymer that allow for the diffusion of large substrates and ions that are necessary for enzymatic function such as nicotinamide adenine dinucleotide (NAD). This modified Nafion polymer has been used to immobilize many different types of enzymes as well as mitochondria for use in biosensors and biofuel cells.8-12 This paper describes a novel procedure for making this micellar polymer enzyme immobilization membrane that can stabilize enzymes. The synthesis of the micellar enzyme immobilization membrane, the procedure for immobilizing enzymes within the membrane, and the assays for studying enzymatic specific activity of the immobilized enzyme are detailed below.

This article will describe the procedure for synthesizing a hydrophobically modified Nafion enzyme immobilization membrane and how to immobilize proteins and/or enzymes within the membrane and test their specific activity.

Hydrophobe Salz-modifizierte Nafion zur Enzymimmobilisierung und Stabilisierung

Over the last decade, there has been a wealth of application for immobilized and stabilized enzymes including biocatalysis, biosensors, and biofuel ...

An Improved Method for the Preparation of Type I Collagen From Skin

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. Clinically, this protein is widely used for cosmetic surgery, dermal injections, bone grafting, and reconstructive surgery. The sources of COL1 for these procedures are commonly nonhuman, which increases the potential for inflammation and rejection as well as xenobiotic disease transmission. In view of this, a method to efficiently and quickly purify COL1 from limited quantities of autologously-derived tissues would circumvent many of these issues; however, standard isolation protocols are lengthy and often require large quantities of collagenous tissues. Here, we demonstrate an efficient COL1 extraction method that reduces the time needed to isolate and purify this protein from about 10 days to less than 3 hr. We chose the dermis as our tissue source because of its availability during many surgical procedures. This method uses traditional extraction buffers combined with forceful agitation and centrifugal filtration to obtain highly-pure, soluble COL1 from small amounts of corium. Briefly, dermal biopsies are washed thoroughly in ice-cold dH2O after removing fat, connective tissue, and hair. The skin samples are stripped of noncollagenous proteins and polysaccharides using 0.5 M sodium acetate and a high speed bench-top homogenizer. Collagen from residual solids is subsequently extracted with a 0.075 M sodium citrate buffer using the homogenizer. These extracts are purified using 100,000 MW cut-off centrifugal filters that yield COL1 preparations of comparable or superior quality to commercial products or those obtained using traditional procedures. We anticipate this method will facilitate the utilization of autologously-derived COL1 for a multitude of research and clinical applications.

Traditional procedures for the isolation of soluble type 1 collagen (COL1) require about 10 days from start to finish because of lengthy buffer incubations and laborious resuspensions of fibrils. Here, we describe a means to purify COL1 from small dermal biopsies in less than 3 hr.

Ein verbessertes Verfahren zur Herstellung von Typ I-Kollagen aus der Haut

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. ...

Bead Aggregation Assays for the Characterization of Putative Cell Adhesion Molecules

Cell-cell adhesion is fundamental to multicellular life and is mediated by a diverse array of cell surface proteins. However, the adhesive interactions for many of these proteins are poorly understood. Here we present a simple, rapid method for characterizing the adhesive properties of putative homophilic cell adhesion molecules. Cultured HEK293 cells are transfected with DNA plasmid encoding a secreted, epitope-tagged ectodomain of a cell surface protein. Using functionalized beads specific for the epitope tag, the soluble, secreted fusion protein is captured from the culture medium. The coated beads can then be used directly in bead aggregation assays or in fluorescent bead sorting assays to test for homophilic adhesion. If desired, mutagenesis can then be used to elucidate the specific amino acids or domains required for adhesion. This assay requires only small amounts of expressed protein, does not require the production of stable cell lines, and can be accomplished in 4 days.

Here we present a simple, rapid method for characterizing the intrinsic adhesive properties of putative cell adhesion molecules. The secreted, epitope-tagged ectodomain of a cell adhesion molecule is captured from the culture medium on small, uniform functionalized beads. These beads can then be used immediately in simple bead aggregation assays.

Wulst Aggregationsassays zur Charakterisierung von putativen Zelladhäsionsmoleküle

Cell-cell adhesion is fundamental to multicellular life and is mediated by a diverse array of cell surface proteins. However, the adhesive interactions ...

Circulating MicroRNA Quantification Using DNA-binding Dye Chemistry and Droplet Digital PCR

Circulating (of cell-free) microRNAs (miRNAs) are released from cells into the blood stream. The amount of specific microRNAs in the circulation has been linked to a disease state and has the potential to be used as disease biomarker. A sensitive and accurate method for circulating microRNA quantification using a dye-based chemistry and droplet digital PCR technology has been recently developed. Specifically, using Locked Nucleic Acid (LNA)-based miRNA-specific primers with a green fluorescent DNA-binding dye in a compatible droplet digital PCR system it is possible to obtain the absolute quantification of specific miRNAs. Here, we describe how performing this technique to assess miRNA amount in biological fluids, such as plasma and serum, is both feasible and effective.

A sensitive and accurate method for cell-free microRNAs quantification using a dye-based chemistry and droplet digital PCR technology is described.

Zirkulierende MicroRNA Quantifizierung der Basis von DNA-bindenden Farbstoff Chemie und Droplet Digital-PCR

Circulating (of cell-free) microRNAs (miRNAs) are released from cells into the blood stream. The amount of specific microRNAs in the circulation has been ...

Long-term Live Imaging Device for Improved Experimental Manipulation of Zebrafish Larvae

The zebrafish larva is an important model organism for both developmental biology and wound healing. Further, the zebrafish larva is a valuable system for live high-resolution microscopic imaging of dynamic biological phenomena in space and time with cellular resolution. However, the traditional method of agarose encapsulation for live imaging can impede larval development and tissue regrowth. Therefore, this manuscript describes the zWEDGI (zebrafish Wounding and Entrapment Device for Growth and Imaging), which was designed and fabricated as a functionally compartmentalized device to orient larvae for high-resolution microscopy while permitting caudal fin transection within the device and subsequent unrestrained tail development and re-growth. This device allows for wounding and long-term imaging while maintaining viability. Given that the zWEDGI mold is 3D printed, the customizability of its geometries make it easily modified for diverse zebrafish imaging applications. Furthermore, the zWEDGI offers numerous benefits, such as access to the larva during experimentation for wounding or for the application of reagents, paralleled orientation of multiple larvae for streamlined imaging, and reusability of the device.

This manuscript describes the zWEDGI (zebrafish Wounding and Entrapment Device for Growth and Imaging), which is a compartmentalized device designed to orient and restrain zebrafish larvae. The design permits tail transection and long-term collection of high-resolution fluorescent microscopy images of wound healing and regeneration.

Langfristige Live Imaging-Gerät für verbesserte experimentelle Manipulation von Zebrafischlarven

The zebrafish larva is an important model organism for both developmental biology and wound healing. Further, the zebrafish larva is a valuable system for ...

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation

In the central nervous system, numerous acute injuries and neurodegenerative disorders, as well as implanted devices or biomaterials engineered to enhance function result in the same outcome: excess inflammation leads to gliosis, cytotoxicity, and/or formation of a glial scar that collectively exacerbate injury or prevent healthy recovery. With the intent of creating a system to model glial scar formation and study inflammatory processes, we have generated a 3D cell scaffold capable of housing primary cultured glial cells: microglia that regulate the foreign body response and initiate the inflammatory event, astrocytes that respond to form a fibrous scar, and oligodendrocytes that are typically vulnerable to inflammatory injury. The present work provides a detailed step-by-step method for the fabrication, culture, and microscopic characterization of a hyaluronic acid-based 3D hydrogel scaffold with encapsulated rat brain-derived glial cells. Further, protocols for characterization of cell encapsulation and the hydrogel scaffold by confocal immunofluorescence and scanning electron microscopy are demonstrated, as well as the capacity to modify the scaffold with bioactive substrates, with incorporation of a commercial basal lamina mixture to improved cell integration.

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation

Herein, we present a protocol for the 3D culture of rat brain-derived glia cells, including astrocytes, microglia, and oligodendrocytes. We demonstrate primary cell culture, methacrylated hyaluronic acid (HAMA) hydrogel synthesis, HAMAphoto-polymerization and cell encapsulation, and sample processing for confocal and scanning electron microscopic imaging.

3D Hydrogel Kulturen von primären Gliazellen für In-vitro- Modellierung von Neuroinflammation verbessert

In the central nervous system, numerous acute injuries and neurodegenerative disorders, as well as implanted devices or biomaterials engineered to enhance ...

Site-Directed Immobilization of Bone Morphogenetic Protein 2 to Solid Surfaces by Click Chemistry

Different therapeutic strategies for the treatment of non-healing long bone defects have been intensively investigated. Currently used treatments present several limitations that have led to the use of biomaterials in combination with osteogenic growth factors, such as bone morphogenetic proteins (BMPs). Commonly used absorption or encapsulation methods require supra-physiological amounts of BMP2, typically resulting in a so-called initial burst release effect that provokes several severe adverse side effects. A possible strategy to overcome these problems would be to covalently couple the protein to the scaffold. Moreover, coupling should be performed in a site-specific manner in order to guarantee a reproducible product outcome. Therefore, we created a BMP2 variant, in which an artificial amino acid (propargyl-L-lysine) was introduced into the mature part of the BMP2 protein by codon usage expansion (BMP2-K3Plk). BMP2-K3Plk was coupled to functionalized beads through copper catalyzed azide-alkyne cycloaddition (CuAAC). The biological activity of the coupled BMP2-K3Plk was proven in vitro and the osteogenic activity of the BMP2-K3Plk-functionalized beads was proven in cell based assays. The functionalized beads in contact with C2C12 cells were able to induce alkaline phosphatase (ALP) expression in locally restricted proximity of the bead. Thus, by this technique, functionalized scaffolds can be produced that can trigger cell differentiation towards an osteogenic lineage. Additionally, lower BMP2 doses are sufficient due to the controlled orientation of site-directed coupled BMP2. With this method, BMPs are always exposed to their receptors on the cell surface in the appropriate orientation, which is not the case if the factors are coupled via non-site-directed coupling techniques. The product outcome is highly controllable and, thus, results in materials with homogeneous properties, improving their applicability for the repair of critical size bone defects.

Biomaterials doped with Bone Morphogenetic Protein 2 (BMP2) have been used as a new therapeutic strategy to heal non-union bone fractures. To overcome side effects resulting from an uncontrollable release of the factor, we propose a new strategy to site-directly immobilize the factor, thus creating materials with improved osteogenic capabilities.

Site-verwiesene Immobilisierung von Knochen morphogenetische Protein 2 an feste Oberflächen durch Click-Chemie

Different therapeutic strategies for the treatment of non-healing long bone defects have been intensively investigated. Currently used treatments present ...

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 ...

Immobilization of Live Caenorhabditis elegans Individuals Using an Ultra-thin Polydimethylsiloxane Microfluidic Chip with Water Retention

Radiation is widely used for biological applications and for ion-beam breeding, and among these methods, microbeam irradiation represents a powerful means of identifying radiosensitive sites in living organisms. This paper describes a series of on-chip immobilization methods developed for the targeted microbeam irradiation of live individuals of Caenorhabditis elegans. Notably, the treatment of the polydimethylsiloxane (PDMS) microfluidic chips that we previously developed to immobilize C. elegans individuals without the need for anesthesia is explained in detail. This chip, referred to as a worm sheet, is resilient to allow the microfluidic channels to be expanded, and the elasticity allows animals to be enveloped gently. Also, owing to the self-adsorption capacity of the PDMS, animals can be sealed in the channels by covering the surface of the worm sheet with a thin cover film, in which animals are not pushed into the channels for enclosure. By turning the cover film over, we can easily collect the animals. Furthermore, the worm sheet shows water retention and allows C. elegans individuals to be subjected to microscopic observation for long periods under live conditions. In addition, the sheet is only 300 &#181;m thick, allowing heavy ions such as carbon ions to pass through the sheet enclosing the animals, thus allowing the ion particles to be detected and the applied radiation dose to be measured accurately. Because selection of the cover films used for enclosing the animals is very important for successful long-term immobilization, we conducted the selection of the suitable cover films and showed a recommended one among some films. As an application example of the chip, we introduced imaging observation of muscular activities of animals enclosing the microfluidic channel of the worm sheet, as well as the microbeam irradiation. These examples indicate that the worm sheets have greatly expanded the possibilities for biological experiments.

Immobilization of Live Caenorhabditis elegans Individuals Using an Ultra-thin Polydimethylsiloxane Microfluidic Chip with Water Retention

A series of immobilization methods has been established to allow the targeted irradiation of live Caenorhabditis elegans individuals using a recently developed ultra-thin polydimethylsiloxane microfluidic chip with water retention. This novel on-chip immobilization is also adequate for imaging observations. The detailed treatment and application examples of the chip are explained.

Immobilisierung von Live Caenorhabditis Elegans Einzelpersonen, die eine ultraflache Polydimethylsiloxan Mikrofluidik-Chip mit Wassereinlagerungen

Radiation is widely used for biological applications and for ion-beam breeding, and among these methods, microbeam irradiation represents a powerful means ...