Dieses Projekt wurde von der Deshpande Center for Technological Innovation am MIT (RK und JMK) und der NSF CAREER Award 0952493 durch die chemischen und biologischen Separations Programm RK unterstützt. Wir danken dem Institute for Soldier Nanotechnologies (ISN) und der Microsystems Technology Laboratory (MTL) am MIT für die Nutzung ihrer Einrichtungen.

1. HL60 Zellrollen 1.1. Herstellung von gemusterten Substrate. <ol><li> Mit Mikrokontaktdrucken (mCP) 4-7 zu machen abwechselnd selbstorganisierten Monoschichten (SAMs) von PEG-Moleküle auf der gold-beschichtete Glasträger: Fertigen Mikrokontaktdrucken Polydimethylsiloxan (PDMS) Briefmarken, dass der Rezeptor-Muster mit einem Neigungswinkel von α = 10 definiert ° durch eine SU-8 Formverfahren. Reinigen Sie die Goldoberfläche mit Piranha-Lösung (3:1 Mischung von Schwefelsäure zu 30% Wasserstoffperoxid) für 20 Minuten und dann spülen Sie die Oberfläche mit reichlich VE-Wasser bei 24,5 ° C vor dem Gebrauch. Ink die PDMS-Stempel mit 5 mM PEG-Lösung in Ethanol. Trocknen Sie die Stempel in Luft für 20 Minuten. Vorsichtig legte der Stempel auf der Goldoberfläche für 40 sec und stellen Sie sicher, gibt es einen guten Kontakt zwischen der Goldoberfläche und der Stempel. Kein Überdruck erforderlich ist. Spülen Sie die Oberfläche mit Ethanol und trocknen Sie es unter einem Strom von N 2. </li><li> Inkubieren des Substrats in P-Selektin-Lösung (15 pg / mL in DPBS) mit einem Perfusionskammer (Electron Microscopy Sciences) für 3 Stunden bei 24,5 ° C um Muster die verbleibenden Bereiche mit P-Selektin. Spülen Sie die Oberfläche mit reichlich DPBS. </li><li> Backfill die Oberfläche mit BSA (1 mg / mL in DPBS) für 1 h zu blockieren unspezifische Wechselwirkungen. Spülen Sie die Oberfläche mit reichlich DPBS. </li></ol> 1.2. Zellrollen Experimente in einer Flusskammer. <ol><li> Durchfluss einer Suspension von HL60-Zellen (~ 10 5 Zellen / ml) über den gemusterten Oberflächen in einem rechteckigen Flusskammer (Glycotech, Inc; Breite w = 1,0 cm, Länge = 6 cm, Höhe h = 0,0127 cm) bei 24,5 ° C. Verwenden Sie eine Spritzenpumpe (World Precision Instruments, SP230IW) auf Durchfluss von 75 ul / min erzeugen, mit entsprechenden Schubspannung rund 0,5 dyn / cm 2 (~ 0,05 Pa). Berechnen Schubspannung τ mit dem Flugzeug Poiseuille-Strömung Gleichung τ = 6μQ/wh 2, wobei μ der kinematischen Viskosität (0,001002 Pa s), Q Volumenstrom ist, ist w Breite der Flusskammer und h ist die Höhe der Flusskammer. </li><li> Verwenden Sie einen inversen Mikroskop (Nikon TE2000-U) mit einer montierten Kamera (Andor iXon 885) zu HL60 Zellen rollenden Interaktionen mit Klebstoff P-Selektin Substrate mit einem 4 × Rekord Ziel, in der Regel mit einer Rate von 1 Bild pro Sekunde für eine Dauer von 300 s. Führen Sie drei unabhängigen Experimenten für jede Schubspannung Größe und Muster Neigungswinkel. Die vorliegenden Daten als Mittelwerte und Standardabweichung der Mittelwerte aus jedem Experiment erhalten. </li><li> Data Analysis. Analysieren Sie die Bildsequenzen in einem individuellen Matlab (Mathworks, Inc.) Programm, das eine particle tracking freeware 8 bis Tracks entlang der gemusterten line Kanten erzeugen genutzt. Tracks Verlängerung bis zum Ende der P-Selektin-Band ausgewählt und mit zwei geraden Liniensegmenten - eins mit der Strömung ausgerichtet ist, die andere mit dem Muster Rand ausgerichtet. Diese beiden Segmente werden dann verwendet, um den Rand Tracking Länge zu berechnen, Abrollgeschwindigkeit auf der Kante und Abrollgeschwindigkeit auf der Ebene Region. </li></ol> 2. Repräsentative Ergebnisse: <img src="/files/ftp_upload/2640/2640fig1.jpg" alt="Abbildung 1" /> Abbildung (A) zeigt eine mikroskopische Bilder aus dem Video von HL60 rollenden Interaktionen mit Klebstoff P-Selektin Substrate mit einem 4 × Ziel umgewandelt. Helle und dunkle Regionen auf P-Selektin-Rezeptors und PEG Regionen entsprechen jeweils. Abbildung (B) zeigt die Spuren unter Verwendung eines kundenspezifischen Matlab-Programm. Die Kante Neigungswinkel betrug 10 ° und die Schubspannung betrug 0,5 dyn / cm 2. Die Kante Tracking Länge l e, Vertreibung, d, und das rollende Geschwindigkeiten auf dem Rand und innerhalb der Bänder, V e und V p, bzw., wie in Abbildung beschrieben sind (C-1). Abbildung (C-2) zeigt die Verteilung (die Anzahl der aufgenommenen Zellen) der Kante geschnitten wird. Einschübe zeigen die durchschnittlichen Wert von l e und die rollenden Geschwindigkeit auf der Kante (V e) und innerhalb der Bänder (V p) bei der Neigungswinkel α = 10 ° und die Flüssigkeit Schubspannung Größenordnung von rund 0,5 dyn / cm 2. Fehlerbalken stellen eine Standardabweichung, n = 3 wiederholten Experimenten (3 replizieren Oberflächen) für jede Bedingung.

<ol>
	<li>Karnik, R., Hong, S., Zhang, H., Mei, Y., Anderson, D. G., Karp, J. M., Langer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanomechanical+control+of+cell+rolling+in+two+dimensions+through+surface+Patterning+of+receptors.">Nanomechanical control of cell rolling in two dimensions through surface Patterning of receptors.</a> Nano Lett. 8 (4), 1153-1158 (2008).</li><li>Lee, C. H., Bose, S., Van Vliet, K. J., Karp, J. M., Karnik, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Examining+Lateral+Displacement+of+HL60+Cells+Rolling+on+Asymmetric+P-selectin+Patterns.">Examining Lateral Displacement of HL60 Cells Rolling on Asymmetric P-selectin Patterns.</a> Langmuir. 27 (1), 240-249 (2011).</li><li>Norman, K. E., Moore, K. L., McEver, R. P., Ley, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leukocyte+rolling+in-vivo+is+mediated+by+p-selectin+glycoprotein+ligand-1.">Leukocyte rolling in-vivo is mediated by p-selectin glycoprotein ligand-1.</a> Blood. 86 (12), 4417-4421 (1995).</li><li>Bernard, A., Delamarche, E., Schmid, H., Michel, B., Bosshard, H. R., Biebuyck, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Printing+patterns+of+proteins.">Printing patterns of proteins.</a> Langmuir. 14 (9), 2225-2229 (1998).</li><li>James, C. D., Davis, R. C., Kam, L., Craighead, H. G., Isaacson, M., Turner, J. N., Shain, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterned+protein+layers+on+solid+substrates+by+thin+stamp+microcontact+printing.">Patterned protein layers on solid substrates by thin stamp microcontact printing.</a> Langmuir. 14 (4), 741-744 (1998).</li><li>Mrksich, M., Whitesides, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+self-assembled+monolayers+to+understand+the+interactions+of+man-made+surfaces+with+proteins+and+cells.">Using self-assembled monolayers to understand the interactions of man-made surfaces with proteins and cells.</a> Annual Review of Biophysics and Biomolecular Structure. 25, 55-78 (1996).</li><li>Tan, J. L., Tien, J., Chen, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microcontact+printing+of+proteins+on+mixed+self-assembled+monolayers.">Microcontact printing of proteins on mixed self-assembled monolayers.</a> Langmuir. 18 (2), 519-523 (2002).</li><li>Lee, D., King, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microcontact+Printing+of+P-Selectin+Increases+the+Rate+of+Neutrophil+Recruitment+Under+Shear+Flow.">Microcontact Printing of P-Selectin Increases the Rate of Neutrophil Recruitment Under Shear Flow.</a> Biotechnology Progress. 24 (5), 1052-1059 .</li><li>Greenberg, A. W., Hammer, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+separation+mediated+by+differential+rolling+adhesion.">Cell separation mediated by differential rolling adhesion.</a> Biotechnol. Bioeng. 73 (2), 111-124 (2001).</li><li>Higuchi, A., Tsukamoto, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+separation+of+hepatocytes+and+fibroblasts+through+surface-modified+polyurethane+membranes.">Cell separation of hepatocytes and fibroblasts through surface-modified polyurethane membranes.</a> J. Biomed. Mater. Res. Part A. 71A (3), 470-479 (2004).</li><li>Alexeev, A., Verberg, R., Balazs, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterned+surfaces+segregate+compliant+microcapsules.">Patterned surfaces segregate compliant microcapsules.</a> Langmuir. 23 (3), 983-987 (2007).</li></ol>

Wir haben ein Protokoll beschriebenen Zellrollen Trajektorien auf asymmetrische Rezeptor-gemusterten Oberflächen hergestellt mit Mikrokontaktdrucken 2 zu prüfen. Das optische Mikroskop Bilder von strukturierten Oberfläche zeigt deutliche Kontrast zwischen PEG und P-Selektin Bereichen eingesetzt, um zu bestätigen, ob Stanzen erfolgreich ist. Sharp, geraden Kanten kann beobachtet werden, wenn das Stampfen gut durchgeführt werden. Harte Drücken des Stempels kann Stempel Deformation, die die Präzision der ...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Human promyelocytic leukemia cells</td>
<td>&nbsp;</td>
<td>ATCC</td>
<td>CCL-240</td>
<td>HL60 cells</td>
</tr>
<tr>
<td>Gold-coated glass slides</td>
<td>&nbsp;</td>
<td>EMF</td>
<td>TA134</td>
<td>Gold slides</td>
</tr>
<tr>
<td>(1-Mercaptoundec-11-yl)tetra(ethylene glycol)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>674508</td>
<td>PEG</td>
</tr>
<tr>
<td>Recombinant human P-selectin</td>
<td>&nbsp;</td>
<td>R&amp;D Systems Inc.</td>
<td>ADP3-050</td>
<td>P-selectin</td>
</tr>
<tr>
<td>Bovine serum albumin</td>
<td>&nbsp;</td>
<td>Rockland Immunochemicals, Inc.</td>
<td>BSA-50</td>
<td>BSA</td>
</tr>
<tr>
<td>Dulbecco's phosphate buffered saline </td>
<td>&nbsp;</td>
<td>Mediatech Inc.</td>
<td>21-030</td>
<td>DPBS</td>
</tr>
<tr>
<td>Sulfuric acid </td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>339741</td>
<td>&nbsp;</td>
<tr>
<td>Hydrogen peroxide</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>316989</td>
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studying cell rolling trajectories on asymmetric receptor patterns

Seitliche Verschiebung der Zellen senkrecht zu einer Strömung durch Rollen auf asymmetrische Rezeptor-Muster stellt eine Chance für die Entwicklung neuer Geräte für die Label-Free-Trennung und Analyse von Zellen 1. Solche Geräte können seitliche Verschiebung für Durchlauferhitzer Trennung oder Rezeptor-Muster, die Adhäsion modulieren, um zwischen verschiedenen Zellphänotypen oder Ebenen der Rezeptor-Expression unterscheiden zu verwenden. Das Verständnis der Natur der Zelle rollt Trajektorien auf Rezeptor-gemusterten Substrate notwendig ist für das Engineering der Substrate und Gestaltung von solchen Geräten. Hier zeigen wir ein Protokoll zur Untersuchung von Zell rollenden Bahnen auf asymmetrische Rezeptor Muster, Zellrollen Haftung 2 unterstützen. Klar definierte, um angelegte Muster der P-Selektin-Rezeptoren wurden unter Verwendung Mikrokontaktdrucken auf Gold-beschichtete Folien, die in einer Flusskammer aufgenommen wurden. HL60-Zellen, die PSGL-1-Ligand 3 wurden über ein Feld von gemusterten Linien flossen und visualisiert auf einer invertierten Hellfeld-Mikroskop. Die Zellen gerollt und entlang der schrägen Kanten der Muster verfolgt, was in seitliche Auslenkung 1. Jede Zelle in der Regel für eine bestimmte Strecke entlang der Muster Kanten (definiert als die Kante Tracking Länge) gerollt, losgelöst von der Kante, und wieder an einem nachgeschalteten Muster. Obwohl diese Distanz macht es schwierig, die gesamte Flugbahn einer Zelle vom Eingang bis in die Strömungskammer Ausfahrt track war Partikel-Tracking-Software verwendet werden, um zu analysieren und die Ausbeute der rollenden Bahnen der Zellen während der Zeit, als sie auf einem einzigen Rezeptor bewegten gemusterte Linie. Die Bahnen wurden dann untersucht, um Verteilungen von Zellrollen Geschwindigkeiten und dem Rand Tracking Längen für jede Zelle für verschiedene Muster zu erhalten. Dieses Protokoll ist sinnvoll für die Quantifizierung Zellrollen Trajektorien auf Rezeptor-Muster und über diese Parameter wie Muster Winkel und Schubspannung. Diese Daten werden von Nutzen sein für die Gestaltung von mikrofluidischen Bauteilen für Label-freie Zelle Trennung und Analyse.

Watch this Scientific Journal Video about Studying Cell Rolling Trajectories on Asymmetric Receptor Patterns at JoVE.com

Studying Cell Rolling Trajectories on Asymmetric Receptor Patterns

Wir beschreiben ein Protokoll zu beobachten und zu analysieren Zellrollen Trajektorien auf asymmetrische Rezeptor-gemusterten Substrate. Die resultierenden Daten sind nützlich für das Engineering von Rezeptor-gemusterten Substrate für Label-freie Zelle Trennung und Analyse.

Studieren Zellrollen Trajectories auf Asymmetric Rezeptor Patterns

Lateral displacement of cells orthogonal to a flow stream by rolling on asymmetric receptor patterns presents an opportunity for development of new ...

studying-cell-rolling-trajectories-on-asymmetric-receptor-patterns

Research

JoVE Journal

Bioengineering

9.5K Aufrufe.  MIT - Massachusetts Institute of Technology. Wir beschreiben ein Protokoll zu beobachten und zu analysieren Zellrollen Trajektorien auf asymmetrische Rezeptor-gemusterten Substrate. Die resultierenden Daten sind nützlich für das Engineering von Rezeptor-gemusterten Substrate für Label-freie Zelle Trennung und Analyse.

Serie: Studying Cell Rolling Trajectories on Asymmetric Receptor Patterns

Microfabrication of Nanoporous Gold Patterns for Cell-material Interaction Studies

Nanostructured materials with feature sizes in tens of nanometers have enhanced the performance of several technologies, including fuel cells, biosensors, biomedical device coatings, and drug delivery tools. Nanoporous gold (np-Au), produced by a nano-scale self-assembly process, is a relatively new material that exhibits large effective surface area, high electrical conductivity, and catalytic activity. These properties have made np-Au an attractive material to scientific community. Most studies on np-Au employ macro-scale specimens and focus on fundamental science of the material and its catalytic and sensor applications. The macro-scale specimens limit np-Au's potential in miniaturized systems, including biomedical devices. In order to address these issues, we initially describe two different methods to micropattern np-Au thin films on rigid substrates. The first method employs manually-produced stencil masks for creating millimeter-scale np-Au patterns, while the second method uses lift-off photolithography to pattern sub-millimeter-scale patterns. As the np-Au thin films are obtained by sputter-deposition process, they are compatible with conventional microfabrication techniques, thereby amenable to facile integration into microsystems. These systems include electrically-addressable biosensor platforms that benefit from high effective surface area, electrical conductivity, and gold-thiol-based surface bioconjugation. We describe cell culture, immunostaining, and image processing techniques to quantify np-Au's interaction with mammalian cells, which is an important performance parameter for some biosensors. We expect that the techniques illustrated here will assist the integration of np-Au in platforms at various length-scales and in numerous applications, including biosensors, energy storage systems, and catalysts.

We report on techniques to micropattern nanoporous gold thin films via stencil printing and photolithography, as well as methods to culture cells on the microfabricated patterns. In addition, we describe image analysis methods to characterize morphology of the material and the cultured cells using scanning electron and fluorescence microscopy techniques.

Microfabrication von Nanoporöse Gold-Patterns für Zell-Material-Interaktion Studies

Nanostructured materials with feature sizes in tens of nanometers have enhanced the performance of several technologies, including fuel cells, biosensors, ...

Forschung

Biotechnik

Systematic Analysis of In Vitro Cell Rolling Using a Multi-well Plate Microfluidic System

A major challenge for cell-based therapy is the inability to systemically target a large quantity of viable cells with high efficiency to tissues of interest following intravenous or intraarterial infusion. Consequently, increasing cell homing is currently studied as a strategy to improve cell therapy. Cell rolling on the vascular endothelium is an important step in the process of cell homing and can be probed in-vitro using a parallel plate flow chamber (PPFC). However, this is an extremely tedious, low throughput assay, with poorly controlled flow conditions. Instead, we used a multi-well plate microfluidic system that enables study of cellular rolling properties in a higher throughput under precisely controlled, physiologically relevant shear flow1,2. In this paper, we show how the rolling properties of HL-60 (human promyelocytic leukemia) cells on P- and E-selectin-coated surfaces as well as on cell monolayer-coated surfaces can be readily examined. To better simulate inflammatory conditions, the microfluidic channel surface was coated with endothelial cells (ECs), which were then activated with tumor necrosis factor-&#945; (TNF-&#945;), significantly increasing interactions with HL-60 cells under dynamic conditions. The enhanced throughput and integrated multi-parameter software analysis platform, that permits rapid analysis of parameters such as rolling velocities and rolling path, are important advantages for assessing cell rolling properties in-vitro. Allowing rapid and accurate analysis of engineering approaches designed to impact cell rolling and homing, this platform may help advance exogenous cell-based therapy.

Systematic Analysis of In Vitro Cell Rolling Using a Multi-well Plate Microfluidic System

This study used a multi-well plate microfluidic system, significantly increasing throughput of cell rolling studies under physiologically relevant shear flow. Given the importance of cell rolling in the multi-step cell homing cascade and the importance of cell homing following systemic delivery of exogenous populations of cells in patients, this system offers potential as a screening platform to improve cell-based therapy.

Systematische Analyse von<em&gt; In-vitro-</em&gt; Zellroll Mit einer Multi-Well-Platte mikrofluidischen System

A major challenge for cell-based therapy is the inability to systemically target a large quantity of viable cells with high efficiency to tissues of ...

Electronic Tongue Generating Continuous Recognition Patterns for Protein Analysis

In current protocol, a combinatorial approach has been developed to simplify the design and production of sensing materials for the construction of electronic tongues (eT) for protein analysis. By mixing a small number of simple and easily accessible molecules with different physicochemical properties, used as building blocks (BBs), in varying and controlled proportions and allowing the mixtures to self-assemble on the gold surface of a prism, an array of combinatorial surfaces featuring appropriate properties for protein sensing was created. In this way, a great number of cross-reactive receptors can be rapidly and efficiently obtained. By combining such an array of combinatorial cross-reactive receptors (CoCRRs) with an optical detection system such as surface plasmon resonance imaging (SPRi), the obtained eT can monitor the binding events in real-time and generate continuous recognition patterns including 2D continuous evolution profile (CEP) and 3D continuous evolution landscape (CEL) for samples in liquid. Such an eT system is efficient for discrimination of common purified proteins.

A novel approach is described for construction of electronic tongue (eT), which greatly simplifies the design and production of sensing materials, and allows the eT to generate continuous evolution profiles and landscapes for samples in liquid. The obtained eT is efficient for common protein analysis such as discrimination.

Elektronische Zunge generieren Continuous Erkennungsmuster für die Protein-Analyse

In current protocol, a combinatorial approach has been developed to simplify the design and production of sensing materials for the construction of ...

Fluorescence Biomembrane Force Probe: Concurrent Quantitation of Receptor-ligand Kinetics and Binding-induced Intracellular Signaling on a Single Cell

Membrane receptor-ligand interactions mediate many cellular functions. Binding kinetics and downstream signaling triggered by these molecular interactions are likely affected by the mechanical environment in which binding and signaling take place. A recent study demonstrated that mechanical force can regulate antigen recognition by and triggering of the T-cell receptor (TCR). This was made possible by a new technology we developed and termed fluorescence biomembrane force probe (fBFP), which combines single-molecule force spectroscopy with fluorescence microscopy. Using an ultra-soft human red blood cell as the sensitive force sensor, a high-speed camera and real-time imaging tracking techniques, the fBFP is of ~1 pN (10-12 N), ~3 nm and ~0.5 msec in force, spatial and temporal resolution. With the fBFP, one can precisely measure single receptor-ligand binding kinetics under force regulation and simultaneously image binding-triggered intracellular calcium signaling on a single live cell. This new technology can be used to study other membrane receptor-ligand interaction and signaling in other cells under mechanical regulation.

We describe a technique for concurrently measuring force-regulated single receptor-ligand binding kinetics and real-time imaging of calcium signaling in a single T lymphocyte.

Fluoreszenz Biomembrane Kraft Sonde: Concurrent Quantifizierung von Rezeptor-Ligand-Kinetik und Binding-induzierte intrazelluläre Signaling auf Einzelzell

Membrane receptor-ligand interactions mediate many cellular functions. Binding kinetics and downstream signaling triggered by these molecular interactions ...

Solid Lipid Nanoparticles (SLNs) for Intracellular Targeting Applications

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter cellular signaling and gene expression. In a previous investigation, the synthesis of ultra-small solid lipid nanoparticles (SLNs) for topical drug delivery and biomarker detection applications was demonstrated. SLNs are a well-studied example of a nanoparticle delivery system that has emerged as a promising drug delivery vehicle. In this study, SLNs were loaded with a fluorescent dye and used as a model to investigate particle-cell interactions. The phase inversion temperature (PIT) method was used for the synthesis of ultra-small populations of biocompatible nanoparticles. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylphenyltetrazolium bromide (MTT) assay was utilized in order to establish appropriate dosing levels prior to the nanoparticle-cell interaction studies. Furthermore, primary human dermal fibroblasts and mouse dendritic cells were exposed to dye-loaded SLN over time and the interactions with respect to toxicity and particle uptake were characterized using fluorescence microscopy and flow cytometry. This study demonstrated that ultra-small SLNs, as a nanoparticle delivery system, are suitable for intracellular targeting of different cell types.

Solid Lipid Nanoparticles SLNs for Intracellular Targeting Applications

In this study, a method for synthesizing ultra-small populations of biocompatible nanoparticles was described, as well as several in vitro methods by which to assess their cellular interactions.

Feste Lipidnanopartikel (SLN) für die intrazelluläre Targeting-Anwendungen

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter ...

Studying the Stoichiometry of Epidermal Growth Factor Receptor in Intact Cells using Correlative Microscopy

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative fluorescence microscopy and environmental scanning electron microscopy (ESEM) of whole cells in hydrated state. Fluorescent quantum dots (QDs) were coupled to EGFR via a two-step labeling protocol, providing an efficient and specific protein labeling, while avoiding label-induced clustering of the receptor. Fluorescence microscopy provided overview images of the cellular locations of the EGFR. The scanning transmission electron microscopy (STEM) detector was used to detect the QD labels with nanoscale resolution. The resulting correlative images provide data of the cellular EGFR distribution, and the stoichiometry at the single molecular level in the natural context of the hydrated intact cell. ESEM-STEM images revealed the receptor to be present as monomer, as homodimer, and in small clusters. Labeling with two different QDs, i.e.,&#160;one emitting at 655 nm and at 800 revealed similar characteristic results.

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative light- and electron microscopy of whole cells in hydrated state. The label contained fluorescent quantum dots. The protocol can be used to study the stoichiometry of EGFR at the single molecule level.

Studieren der Stöchiometrie der Epidermal Growth Factor Receptor in intakten Zellen mit korrelative Mikroskopie

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative fluorescence ...

Human Neural Organoids for Studying Brain Cancer and Neurodegenerative Diseases

The lack of relevant in vitro neural models is an important obstacle on medical progress for neuropathologies. Establishment of relevant cellular models is crucial both to better understand the pathological mechanisms of these diseases and identify new therapeutic targets and strategies. To be pertinent, an in vitro model must reproduce the pathological features of a human disease. However, in the context of neurodegenerative disease, a relevant in vitro model should provide neural cell replacement as a valuable therapeutic opportunity. 
Such a model would not only allow screening of therapeutic molecules but also can be used to optimize neural protocol differentiation [for example, in the context of transplantation in Parkinson's disease (PD)]. This study describes two in vitro protocols of 1) human glioblastoma development within a human neural organoids (NO) and 2) neuron dopaminergic (DA) differentiation generating a three-dimensional (3D) organoid. For this purpose, a well-standardized protocol was established that allows the production of size-calibrated neurospheres derived from human embryonic stem cell (hESC) differentiation. The first model can be used to reveal molecular and cellular events occurring during in glioblastoma development within the neural organoid, while the DA organoid not only represents a suitable source of DA neurons for cell therapy in Parkinson's disease but also can be used for drug testing.

This study introduces and describes protocols to derive two specific human neural organoids&#160;as a relevant and accurate model for studying 1) human glioblastoma development within human neural organoids exclusively in humans and 2) neuron dopaminergic differentiation generating a three-dimensional organoid.

Human neurale Organoide zur Untersuchung von Hirntumor und neurodegenerativen Erkrankungen

The lack of relevant in vitro neural models is an important obstacle on medical progress for neuropathologies. Establishment of relevant cellular models ...

A Macrophage Reporter Cell Assay to Examine Toll-Like Receptor-Mediated NF-kB/AP-1 Signaling on Adsorbed Protein Layers on Polymeric Surfaces

The persistent inflammatory host response to an implanted biomaterial, known as the foreign body reaction, is a significant challenge in the development and implementation of biomedical devices and tissue engineering constructs. Macrophages, an innate immune cell, are key players in the foreign body reaction because they remain at the implant site for the lifetime of the device, and are commonly studied to gain an understanding of this detrimental host response. Many biomaterials researchers have shown that adsorbed protein layers on implanted materials influence macrophage behavior, and subsequently impact the host response. The methods in this paper describe an in vitro model using adsorbed protein layers containing cellular damage molecules on polymer biomaterial surfaces to assess macrophage responses. An NF-&#1082;B/AP-1 reporter macrophage cell line and the associated colorimetric alkaline phosphatase assay were used as a rapid method to indirectly examine NF-&#1082;B/AP-1 transcription factor activity in response to complex adsorbed protein layers containing blood proteins and damage-associated molecular patterns, as a model of the complex adsorbed protein layers formed on biomaterial surfaces in vivo.

This protocol provides researchers with a rapid, indirect method of measuring TLR-dependent NF-&#1082;B/AP-1 transcription factor activity in a murine macrophage cell line in response to a variety of polymeric surfaces and adsorbed protein layers that model the biomaterial implant microenvironment.

Ein Makrophagen-Reporter-Zell-Assay zur Untersuchung der toll-like Receptor-vermittelten NF-kB/AP-1-Signalisierung auf adsorbierten Proteinschichten auf polymeren Oberflächen

The persistent inflammatory host response to an implanted biomaterial, known as the foreign body reaction, is a significant challenge in the development ...

A Human 3D Extracellular Matrix-Adipocyte Culture Model for Studying Matrix-Cell Metabolic Crosstalk

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of cellular function. The ECM plays a particularly important role in adipose tissue function in obesity, and alterations in adipose tissue ECM deposition and composition are associated with metabolic disease in mice and humans. Tractable in vitro models that permit dissection of the roles of the ECM and cells in contributing to global tissue phenotype are sparse. We describe a novel 3D in vitro model of human ECM-adipocyte culture that permits study of the specific roles of the ECM and adipocytes in regulating adipose tissue metabolic phenotype. Human adipose tissue is decellularized to isolate ECM, which is subsequently repopulated with preadipocytes that are then differentiated within the ECM into mature adipocytes. This method creates ECM-adipocyte constructs that are metabolically active and retain characteristics of the tissues and patients from which they are derived. We have used this system to demonstrate disease-specific ECM-adipocyte crosstalk in human adipose tissue. This culture model provides a tool for dissecting the roles of the ECM and adipocytes in contributing to global adipose tissue metabolic phenotype and permits study of the role of the ECM in regulating adipose tissue homeostasis.

We describe a 3D human extracellular matrix-adipocyte in vitro culture system that permits dissection of the roles of the matrix and adipocytes in contributing to adipose tissue metabolic phenotype.

Ein menschliches 3D extrazelluläres Matrix-Adipozyten-Kulturmodell zum Studium der Matrix-Zell Metabolischen Crosstalk

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of ...

In Vitro 3D Cell-Cultured Arterial Models for Studying Vascular Drug Targeting Under Flow

The use of three-dimensional (3D) models of human arteries, which are designed with the correct dimensions and anatomy, enables the proper modeling of various important processes in the cardiovascular system. Recently, although several biological studies have been performed using such 3D models of human arteries, they have not been applied to study vascular targeting. This paper presents a new method to fabricate real-sized, reconstructed human arterial models using a 3D printing technique, line them with human endothelial cells (ECs), and study particle targeting under physiological flow. These models have the advantage of replicating the physiological size and conditions of blood vessels in the human body using low-cost components. This technique may serve as a new platform for studying and understanding drug targeting in the cardiovascular system and may improve the design of new injectable nanomedicines. Moreover, the presented approach may provide significant tools for the study of targeted delivery of different agents for cardiovascular diseases under patient-specific flow and physiological conditions.

In Vitro 3D Cell-Cultured Arterial Models for Studying Vascular Drug Targeting Under Flow

Here, we present a new protocol to study and map the targeted deposition of drug carriers to endothelial cells in fabricated, real-sized, three-dimensional human artery models under physiological flow. The presented method may serve as a new platform for targeting drug carriers within the vascular system.

In vitro 3D-zellkulierte arterielle Modelle zur Untersuchung des vaskulären Wirkstoff-Targetings unter Flow

The use of three-dimensional (3D) models of human arteries, which are designed with the correct dimensions and anatomy, enables the proper modeling of ...