Questo progetto è stato sostenuto dal Centro Deshpande per l&#39;Innovazione Tecnologica del MIT (RK e JMK) e il premio alla carriera NSF 0.952.493 di RK attraverso la chimica e biologica programma Separazioni. Ringraziamo l&#39;Istituto per le Nanotecnologie Soldier (ISN) e il Microsystems Technology Laboratory (MTL) al MIT per l&#39;utilizzo dei loro impianti.

1. HL60 cellula rotolamento 1.1. Fabbricazione di Patterned substrati. <ol><li> Utilizzando stampa microcontact (μCP) 4-7 per rendere alternati monostrati auto-assemblati (SAM) di molecole di PEG sull&#39;oro rivestita vetrini: Realizzare microcontatto stampa polidimetilsilossano (PDMS) francobolli che ha definito i modelli recettore con angolo di inclinazione α = 10 ° con uno SU-8 processo di stampaggio. Pulire la superficie d&#39;oro con soluzione piranha (3:1 miscela di acido solforico al 30% di perossido di idrogeno) per 20 minuti e risciacquare la superficie con abbondante acqua deionizzata a 24,5 ° C prima dell&#39;uso. Dell&#39;inchiostro il timbro PDMS con soluzione PEG 5mM in etanolo. Il timbro a secco in aria per 20 minuti. Gentilmente messo il timbro sulla superficie di oro per 40 sec e assicurarsi che ci sia un buon contatto tra la superficie d&#39;oro e il timbro. Nessuna pressione in eccesso viene richiesto. Lavare la superficie con etanolo e asciugare sotto un flusso di N 2. </li><li> Incubare il substrato all&#39;interno di P-selectina soluzione (15 mg / mL in DPBS) utilizzando una camera di perfusione (Scienze della Microscopia Elettronica) per 3 ore a 24,5 ° C a modello le restanti aree con P-selectina. Lavare la superficie con abbondante DPBS. </li><li> Backfill la superficie con BSA (1 mg / mL in DPBS) per 1 ora per bloccare non specifiche interazioni. Lavare la superficie con abbondante DPBS. </li></ol> 1.2. Cellula di Rolling Esperimenti in una camera di flusso. <ol><li> Flusso di una sospensione di cellule HL60 (~ 10 5 cellule / ml) sulla superficie lavorata in una camera di flusso rettangolare (Glycotech, Inc; larghezza w = cm 1,0; lunghezza = 6 cm, altezza h = 0,0127 centimetri) a 24,5 ° C. Utilizzare una pompa a siringa (Strumenti di precisione mondiale, SP230IW) per generare la portata di 75 microlitri / min, con corrispondente sforzo di taglio di circa 0,5 dyn / cm 2 (~ 0,05 Pa). Calcolare sforzo di taglio τ utilizzando l&#39;aereo equazione del flusso di Poiseuille 6μQ/wh τ = 2, dove μ è la viscosità cinematica (0,001002 Pa s), Q è la portata volumetrica, w è la larghezza della camera di flusso, ed h è l&#39;altezza del camera di flusso. </li><li> Utilizzare un microscopio invertito (Nikon TE2000-U), con una telecamera montata (Andor ixon 885) per registrare le cellule HL60 rotolamento interazioni con adesivo P-selectina substrati con un 4 × obiettivo, di solito ad una velocità di 1 fotogramma al secondo per la durata di 300 s. Eseguire tre esperimenti indipendenti, per ogni grandezza sforzo di taglio e angolo di inclinazione del modello. Presentare i dati come media e deviazione standard dei valori medi ottenuti da ciascun esperimento. </li><li> Analisi dei dati. Analizzare le sequenze di immagini da una personalizzata Matlab (MathWorks, Inc.), programma che utilizzava una particella di tracciamento freeware di 8 a generare binari lungo i bordi linea di fantasia. Tracce di estendere fino alla fine di un P-selectina band sono selezionati e dotati di due segmenti di linea retta - uno allineato con il flusso, l&#39;altro in linea con il bordo del modello. Questi due segmenti sono poi utilizzati per calcolare la lunghezza di tracciamento bordo, velocità di rotazione sul bordo, e la velocità di rotolamento sulla regione pianura. </li></ol> 2. Rappresentante dei risultati: <img src="/files/ftp_upload/2640/2640fig1.jpg" alt="Figura 1" /> Figura (A) mostra una delle immagini al microscopio convertito dal video di HL60 interazioni laminazione con adesivo P-selectina substrati con un 4 × obiettivo. Regioni chiare e scure corrispondenti a P-selectina recettore e regioni PEG, rispettivamente. Figura (B) mostra le tracce ottenute con un programma personalizzato di Matlab. L&#39;angolo di inclinazione limite era 10 ° e la tensione di taglio è stato dello 0,5 dyn / cm 2. La lunghezza di tracciamento bordo, l posta, spostamento, d, e le velocità di rotolamento sul bordo e all&#39;interno della band, V e P e V, rispettivamente, sono descritti nella figura (C-1). Figura (C-2) mostra la distribuzione (il numero di cellule registrato) della lunghezza del bordo di monitoraggio. Riquadri mostrano il valore medio di l e la velocità di rotolamento e sul bordo (V e) e all&#39;interno della band (V p) presso l&#39;angolo di inclinazione α = 10 ° e la portata del fluido sforzo di taglio di circa 0,5 dyn / cm 2. Le barre di errore rappresentano una deviazione standard, dove n = 3 esperimenti di replicare (3 superfici replica) per ogni condizione.

<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>

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Abbiamo descritto un protocollo per esaminare le cellule che rotola su traiettorie asimmetriche recettore fantasia superfici fabbricate utilizzando stampa microcontact 2. Le immagini al microscopio ottico di superficie modellata che mostra il contrasto evidente tra PEG e P-selectina aree possono essere usati per confermare se stampaggio è successo. Sharp, bordi dritti si può osservare quando la timbratura viene eseguita bene. Dura pressione del bollo può causare deformazioni timbro che limita la precisione...

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		<th>Catalogue Number</th>
		<th>Comment</th>
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<td>Human promyelocytic leukemia cells</td>
<td>&nbsp;</td>
<td>ATCC</td>
<td>CCL-240</td>
<td>HL60 cells</td>
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<tr>
<td>Gold-coated glass slides</td>
<td>&nbsp;</td>
<td>EMF</td>
<td>TA134</td>
<td>Gold slides</td>
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<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>
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<td>Dulbecco's phosphate buffered saline </td>
<td>&nbsp;</td>
<td>Mediatech Inc.</td>
<td>21-030</td>
<td>DPBS</td>
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<td>Sulfuric acid </td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>339741</td>
<td>&nbsp;</td>
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studying cell rolling trajectories on asymmetric receptor patterns

Spostamento laterale delle cellule ortogonale a un flusso di flusso di laminazione su modelli asimmetrici recettore rappresenta un&#39;opportunità per lo sviluppo di nuovi dispositivi per l&#39;etichetta senza separazione e l&#39;analisi delle cellule 1. Tali dispositivi possono utilizzare spostamento laterale per flusso continuo di separazione, o schemi di recettori che modulano l&#39;adesione di distinguere tra diversi fenotipi cellulari o livelli di espressione del recettore. Capire la natura delle traiettorie delle cellule rotolamento sul recettore modellata substrati è necessario per l&#39;ingegneria dei substrati e progettazione di tali dispositivi. Qui, dimostriamo un protocollo per lo studio delle traiettorie delle cellule che rotola su modelli asimmetrici recettore che sostengono l&#39;adesione cellulare rotolamento 2. Ben definito, micron scala modelli di P-selectina recettori sono stati fabbricati usando stampa microcontact su oro rivestite diapositive che sono state incorporate in una camera di flusso. HL60 cellule che esprimono il PSGL-1 ligando 3 erano scorreva attraverso un campo di linee a motivi geometrici e visualizzati su un microscopio invertito in campo chiaro. Le cellule laminati e tracciati lungo i bordi inclinati dei modelli, con conseguente flessione laterale 1. Ogni cella tipicamente arrotolato per una certa distanza lungo i bordi modello (definito come la lunghezza di tracciamento bordo), staccato dal bordo, e riattaccato a un modello a valle. Anche se questo distacco rende difficile tenere traccia l&#39;intera traiettoria di una cellula dall&#39;entrata all&#39;uscita della camera di flusso, di tracciamento di particelle-software è stato utilizzato per analizzare le traiettorie e la resa rotolamento delle cellule durante il periodo in cui si muovevano su un solo recettore con motivi di linea. Le traiettorie sono stati poi esaminati al fine di ottenere la distribuzione delle velocità di rotolamento delle cellule e le lunghezze di margine di monitoraggio per ogni cella per diversi modelli. Questo protocollo è utile per quantificare le traiettorie delle cellule che rotola su modelli di recettori e relativi a questi parametri di ingegneria come l&#39;angolo pattern e sforzo di taglio. Tali dati saranno utili per la progettazione di dispositivi microfluidici per l&#39;etichetta senza separazione delle cellule e l&#39;analisi.

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

Abbiamo descritto un protocollo per osservare e analizzare le traiettorie delle cellule che rotola su asimmetrici recettore fantasia substrati. I dati risultanti sono utili per l&#39;ingegneria del recettore-fantasia substrati per etichette senza separazione delle cellule e l&#39;analisi.

Studiare Traiettorie cellulare Rolling Patterns Receptor asimmetrica

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

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Bioengineering

9.5K Views.  MIT - Massachusetts Institute of Technology. Abbiamo descritto un protocollo per osservare e analizzare le traiettorie delle cellule che rotola su asimmetrici recettore fantasia substrati. I dati risultanti sono utili per l'ingegneria del recettore-fantasia substrati per etichette senza separazione delle cellule e l'analisi.

Video: 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 di Patterns Oro nanoporosi per Cell-materiali Interaction Studies

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

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Bioingegneria

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.

Analisi sistematica delle<em&gt; In Vitro</em&gt; Cella rotolamento Utilizzando un piatto multi-ben Sistema Microfluidic

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 ...

Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain central aspects of cellular physiology. To easily combine cell patterning with Multi-Electrode Arrays (MEAs) and silicon-based &#8216;lab on a chip&#8217; technologies, a microfabrication-compatible protocol is required. We describe a method that utilizes deposition of the polymer parylene-C on SiO2&#160;wafers. Photolithography enables accurate and reliable patterning of parylene-C at micron-level resolution. Subsequent activation by immersion in fetal bovine serum (or another specific activation solution) results in a substrate in which cultured cells adhere to, or are repulsed by, parylene or SiO2 regions respectively. This technique has allowed patterning of a broad range of cell types (including primary murine hippocampal cells, HEK 293 cell line, human neuron-like teratocarcinoma cell line, primary murine cerebellar granule cells, and primary human glioma-derived stem-like cells). Interestingly, however, the platform is not universal; reflecting the importance of cell-specific adhesion molecules. This cell patterning process is cost effective, reliable, and importantly can be incorporated into standard microfabrication (chip manufacturing) protocols, paving the way for integration of microelectronic technology.

Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

This protocol describes a microfabrication-compatible method for cell patterning on SiO2. A predefined parylene-C design is photolithographically printed on SiO2 wafers. Following incubation with serum (or other activation solution) cells adhere specifically to (and grow according to the conformity of) underlying parylene-C, whilst being repulsed by SiO2 regions.

Patterning cellulare su fotolitograficamente Definito parylene-C: SiO<sub&gt; 2</sub&gt; Substrati

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain ...

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.

Lingua elettronica Generazione di modelli continui di rilevazione delle analisi delle proteine

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.

Fluorescenza Biomembrane Forza Probe: quantificazione simultanea di Cinetica recettore-ligando e Binding-indotta intracellulare segnalazione su una singola cella

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.

Solid Lipid nanoparticelle (linfonodi sentinella) Applicazioni per intracellulare targeting

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.

Studiare la Stechiometria di Epidermal Growth Factor Receptor in cellule intatte con correlativa Microscopia

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.

Organidi neurali umani per lo studio del cancro al cervello e delle malattie neurodegenerative

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.

Un analisi della cella del Macrophage Reporter per esaminare la segnalazione NF-kB/AP-1 mediata da un rata a tollo su superfici polimetiche

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.

Un modello di coltura a matrice extracellulare extracellulare -adipocyte umano per lo studio del crosstalk metabolico a matrice

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