Grazie alla Anseth Kristi laboratorio della University of Colorado, Boulder generosamente per fornire MIN6 cellule. Il finanziamento per questo progetto è stato fornito dalla NSF.

1. PEGDM sintesi e PEGDM fotoattivi preparazione macromer soluzione <ol><li> Pesare 2 g di PEG (lineari, 10.000 Da) in un flacone di vetro 40ml con tappo in plastica rigida. </li><li> Aggiungere circa 308μL di anidride metacrilico e vagamente tappo del flaconcino. </li><li> Microonde la fiala in alto per 2 minuti in un forno a microonde standard ad uso privato. Indossare guanti resistenti al calore, completamente vortice il flacone, poi in alto a microonde per altri 5 minuti. </li><li> Brevemente il flacone permettono di raffreddare abbastanza da essere maneggiato con i guanti. Stappare e aggiungere lentamente il cloruro di metilene, mentre agitando il flaconcino. Aggiungi sufficiente in modo che la soluzione appare chiara ed omogenea, vortex il campione, se necessario. Il volume totale del cloruro di metilene utilizzato deve essere 1,5 - 2 ml. </li><li> PEGDM precipitato in 200 ml di freddo, agitato dietiletere aggiungendo goccia a goccia la soluzione. </li><li> Raccogliere il PEGDM filtrazione sotto vuoto e lasciarlo asciugare. </li><li> Sciogliere il PEGDM in quantità adeguatadi acqua deionizzata (tipicamente 100 -150 ml). </li><li> Dializzare la soluzione contro l&#39;acqua deionizzata per 5 giorni in tubi di dialisi, con un peso molecolare di cut-off di 1.000 Da. Sostituire l&#39;acqua una volta al giorno. </li><li> Aliquota della soluzione in appositi contenitori e congelare a -80 ° C durante la notte. Liofilizzare la soluzione per 4 giorni fino ad ottenere una polvere bianca PEGDM purificata. Conservare la polvere a 4 ° C, quando non in uso. </li><li> Per preparare una soluzione PEGDM fotoattivi, prima preparare una percentuale 0,5 Peso soluzione fotoiniziatore magazzino sciogliendo Irgacure 2959 in acqua deionizzata. Vortex la soluzione e conservarla nel buio. </li><li> Preparare un 10% in peso PEGDM e 0,025% in peso Irgacure 2959 soluzione in soluzione salina bilanciata di Hank (HBSS). Vortex a fondo per garantire lo scioglimento del PEGDM. Noi di solito preparare 1 ml di questa soluzione in un momento. </li><li> Sterilizzare la soluzione filtrandola attraverso un filtro 0,2 micron siringa in una provetta per microcentrifuga da 1,8 ml. Avvolgere il tubo in alluminioe conservare a 4 ° C </li></ol> 2. Cultura, l&#39;infezione e l&#39;aggregazione di cellule MIN6 <ol><li> MIN6 cellule sono coltivate in RPMI 1640 medium supplementato con 10% FBS, glucosio 7mm, 100 unità / mL penicillina / streptomicina, e 0,5 mg / ml amphotercin B in un ambiente umidificato a 37 ° C e 5% di CO 2. </li><li> Per rilasciare le cellule dalla superficie coltura trattata fiasco, aspirare il terreno, lavare le cellule con 37 ° C di calcio / magnesio senza HBSS, aspirare il HBSS, poi aggiungere 2 ml di 37 ° C tripsina-EDTA soluzione e permettono alle cellule di incubare per 3 minuti. </li><li> Saldamente vaso sul lato del pallone per battere il celle libere, quindi aggiungere 8 mL di 37 ° C di media e vigorosamente pipetta la sospensione cellulare su e giù facendo attenzione a non introdurre bolle. </li><li> Pipettare le cellule in una provetta sterile 15 ml centrifuga ed effettuare un conteggio delle cellule utilizzando un emocitometro o una cella di altri procedura di conteggio. </li><li> Nella preparazione di infettare le cellule con il produttorevirus, in primo luogo determinare il numero totale delle cellule di essere infetti e calcolare il volume di sospensione di virus necessari per raggiungere il desiderato molteplicità di infezione (MOI) (da 50 a 100 particelle infettive per cella). </li><li> Diluire il virus in HBSS con attenzione pipeting la giusta quantità di sospensione di virus in un pre-aliquotati volume di HBSS in una provetta da microcentrifuga da 1,8 ml. 50 ml di HBSS per pozzetto di cellule aggregazione è appropriato. </li><li> Disperdere le cellule pipeting su e giù nel loro tubo, poi pipettare il volume necessario per raggiungere 400.000 cellule per pozzetto nei pozzetti di un 6-pozzetti sospensione cultura. </li><li> Aggiungere il volume appropriato di virus diluito in ciascun pozzetto per ottenere il desiderato MOI. </li><li> Aggiungi medio in ciascun pozzetto per portare il volume totale fino a 1,7 ml. </li><li> Posizionare la piastra su un agitatore orbitale all&#39;interno dell&#39;incubatore. Impostare l&#39;agitatore in un ambiente basso in modo che il mezzo lava delicatamente intorno ai pozzi (~ 100 rpm). Permettono alle cellule di aggregazionete per 24-36 ore. Aggregati con un diametro approssimativo di 75-175 micron dovrebbe essere formato. </li></ol> 3. Incapsulamento delle celle a PEGDM <ol><li> Le cellule possono essere incapsulati come una dispersione (passo 2.4) o l&#39;aggregazione seguente (punto 2.10). Dal momento che il precursore soluzione idrogel PEGDM è un liquido, le cellule possono tendere a risolvere prima di reticolazione e la formazione di idrogel. Se il regolamento dovrebbe avvenire rapidamente, come nel caso di grandi aggregati, può essere utile per produrre l&#39;idrogel in due fasi in modo che le cellule risolvere su un piano centrale del gel. Un one-step procedura di sintesi idrogel appropriata per le cellule disperse è mostrato (Figura 2) e la procedura in due fasi è mostrato così (Figura 3) e di dettaglio di seguito (3,2-3,9). </li><li> Creare un vaso in cui l&#39;idrogel sarà formata da taglio la punta affusolata off di una siringa da 1 ml di plastica e l&#39;immissione aprirlo finire in un rack. </li><li> Pipettare 20 ml di PEGDM fotoattivi cosìluzione in l&#39;estremità aperta della siringa sul pistone assicurandosi il volume copre l&#39;intera superficie dello stantuffo. Regolare lo stantuffo in piccoli incrementi per ottenere una superficie piana fluido. </li><li> Posizionare la pipetta nel rack e la posizione del rack sotto una lampada UV (365nm, ~ 7 mW / cm 2) per circa 8 minuti per consentire la reticolazione idrogel. Durante questo tempo, la preparazione delle cellule può essere iniziata </li><li> Pipettare un volume contenente il numero desiderato di cellule / aggregati in un tubo di 1,8 ml microcentrifuga, berretto, e centrifugare la provetta a 130 g per 5 minuti. </li><li> Utilizzando un micropipettor, rimuovere con attenzione i media senza disturbare il pellet di cellule sulla punta del tubo. Come capo dei media si avvicina il pellet, può essere utile utilizzare una più fine, 10 punta della pipetta microlitri. </li><li> Delicatamente risospendere il pellet cellulare in 20 ml di soluzione fotoattivi PEGDM (usare una bocca larga punta della pipetta se si lavora con aggregati) e aggiungere la sospensione cellulare alla siringa sulla parte superiore del secoloe precedentemente reticolato porzione idrogel. </li><li> Esporre la siringa per ulteriori 8 minuti di luce UV per completare la costruzione del idrogel. Al termine, le cellule devono essere completamente incapsulato all&#39;interno del idrogel cilindrico (Figura 3). </li><li> Espellere l&#39;idrogel dalla siringa e in 1 ml di HBSS nel pozzo di un 24-pozzetti per lavare brevemente il gel. Trasferire il gel per 1 ml di media nel pozzo di un 24-pozzetti e cultura sotto le condizioni desiderate. </li></ol> 4. Monitoraggio ipossia <ol><li> In qualsiasi momento durante l&#39;immagine della cultura, le cellule usando la microscopia a fluorescenza per monitorare l&#39;avvio di segnalazione ipossiche. A seconda di cosa viene esposta si può immagine nel multi-pozzetti o trasferire il gel di un vetrino da microscopio prima del posizionamento sul palco microscopio. Eccitazione picco di Ds Rosso è raggiunta a 556nm e picco di emissione avviene a 586nm. Emissione è abbastanza intenso, e quindi photobleaching non è stato osservatoried per essere problematico. Intervallo di tempo tra l&#39;inizio della produzione di proteine ​​e il rilevamento primo segnale è di circa 12 ore. Il segnale è sufficientemente specifica da identificare le singole cellule di segnalazione, ma la risoluzione può essere inadeguato per determinare la localizzazione del segnale intracellulare. Nelle cellule di segnalazione, il segnale si osserva tipicamente in modo uniforme in tutto il citoplasma. Intensità del segnale può anche aumentare con l&#39;esposizione estesa a ipossia, anche se non abbiamo ancora pienamente determinato se questo è dovuto alla maggiore espressione della proteina, accumulo di proteine ​​nel complesso, o ritardata maturazione delle proteine. </li></ol> 5. Rappresentante risultati Un esempio rappresentativo di MIN6 aggregati incapsulato in un idrogel PEG è mostrato in Figura 4. Il gel reticolato sarà solido in tutto, prendendo la forma del recipiente in cui è stata eseguita la reazione. Un gel con lisce superfici esterne è preferibile per l&#39;impianto di aiuto nella prevenzione di un nuovo corpo estraneosponse. All&#39;interno il gel, aggregati di cellule dovrebbe essere completamente chiusa nella matrice e omogeneamente distribuita in modo da migliorare il trasporto dei nutrienti. Immagini rappresentative di segnalazione ipossiche in MIN6 aggregati sono illustrati nella Figura 5. Identico MIN6 aggregati infettati con il virus marcatore sono state coltivate in entrambe il 20% O 2 (5a) o 1% O 2 (5b) per 44 ore prima della cattura dell&#39;immagine. <img alt="Figura 1" src="/files/ftp_upload/3521/3521fig1.jpg" /> Figura 1. Schematica delle attività del nostro sistema marcatore ipossia. Inserimento adenovirali della Red Ds DR gene a monte e promotore HRE permette di ipossia indotta la produzione della proteina fluorescente sotto il controllo di HIF-1. <img alt="Figura 2" src="/files/ftp_upload/3521/3521fig2.jpg" /> Figura 2. Diagramma di flusso procedurale per l&#39;unico passaggio incapsulamento di dispersi MIN6 celle di una crosslinked idrogel PEGDM. Per ogni gel, le cellule disperse sono sospese in 40μL della soluzione macromer fotoattivi PEGDM. Questo è posto in una siringa 1 ml e decapitato l&#39;idrogel è formata sotto la luce UV 365nm dopo 10-12 minuti. Al termine, il disco idrogel è rimosso dalla siringa, lavato e messo in un mezzo pieno di pozzetti per l&#39;incubazione. <img alt="Figura 3" src="/files/ftp_upload/3521/3521fig3.jpg" /> Figura 3. Diagramma di flusso procedurale per il doppio passo incapsulamento di aggregati MIN6 cellule in un idrogel PEGDM reticolato. Per ogni gel, un mezzo-gel è il primo formato da reticolazione UV 20μL della soluzione macromer PEGDM fotoattivi per 8 minuti. MIN6 aggregati sono accuratamente sospesi in un 20μL addizionale di soluzione fotoattivi macromer che viene aggiunto in cima alla pre-formata a metà gel. Il gel completo è formato da un ulteriore 8 minuti di esposizione ai raggi UV con aggregati completamente incapsulato nel medialepiano del gel. <img alt="Figura 4" src="/files/ftp_upload/3521/3521fig4.jpg" /> Figura 4. Immagine di un idrogel 40μL sotto ingrandimento 20X. MIN6 aggregati (~ 400.000 cellule totali) si vedono chiaramente all&#39;interno del gel. Diametro idrogel è di circa 6mm (bar = 1mm). <img alt="Figura 5" src="/files/ftp_upload/3521/3521fig5.jpg" /> Ipossia Figura 5. Fluorescente di segnalazione in forma aggregata MIN6 cellule in un idrogel PEGDM. Le cellule che sono state incapsulate e poi messe in incubazione a 20% O 2 per 44 ore non vengono visualizzati segnalazione ipossia (a), mentre le cellule che sono state incapsulate poi incubate in 2 O 2% per 44 ore visualizzare segnale chiaro e onnipresente. (Bar = 100μm) (b).

<ol>
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H. y. p. o. x. i. a. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=hypoxia-inducible+factors+(HIF),+HIF+hydroxylases+and+oxygen+sensing.">hypoxia-inducible factors (HIF), HIF hydroxylases and oxygen sensing.</a> Cell. Mol. Life Sci. 66, 3539-3554 (2009).</li></ol>

Nessun conflitto di interessi dichiarati.

Il metodo presentato qui offre una tecnica rapida e semplice per l&#39;incapsulamento delle cellule in un idrogel PEG con il minimo uso di non-fisiologiche condizioni. PEG rappresenta un materiale di incapsulamento molto utile per la sua biocompatibilità e la facilità di modifica. Semplice variazione di percentuale PEG nella soluzione fotoattivi, per esempio, possono essere utilizzati per regolare le proprietà meccaniche, come modulo di compressione, e proprietà di trasporto attraverso la dimensione dei pori. Inoltre, PEG è facilmente modificabile con l&#39;aggiunta di catene laterali. Idrogel PEG, quindi, rappresentano sia un dispositivo promettente clinico e una piattaforma flessibile per la ricerca in vitro Un metodo per il monitoraggio ipossia nel PEG-incapsulato cellule è stato presentato. Questo metodo è utile per la semplicità di rilevazione ipossia e per evitare la necessità di sacrificare le cellule di interesse. La tecnica può essere applicata ad una varietà di tipi di cellule in una varietà di condizioni che rendono le sue noiefulness ampio. Per esempio, l&#39;ipossia come spunto per la differenziazione delle cellule staminali possono essere rintracciati in colture di cellule staminali Micromass. Tuttavia, questo metodo può essere applicato solo per disperdere i sistemi cellulari o sistema in cui le cellule sono disperse in seguito aggregate. Inoltre, il rilevamento del segnale fluorescente può essere difficile nei tessuti più grandi o più dense.

<table border="1"><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti (opzionale </td></tr><tr><td> PEG </td><td> Sigma-Aldrich </td><td> 309028-500G </td><td></td></tr><tr><td> Anidride metacrilico </td><td> Sigma-Aldrich </td><td> 276685-100ML </td><td></td></tr><tr><td> Microonda </td><td> Emerson </td><td> MW8784SB </td><td></td></tr><tr><td> Vortex </td><td> Settori scientifico </td><td> SI-A236 </td><td></td></tr><tr><td> Cloruro di metilene </td><td> Sigma-Aldrich </td><td> D65100-1L </td><td></td></tr><tr><td> Etere dietilico </td><td> Sigma-Aldrich </td><td> 346136-1L </td><td></td></tr><tr><td> Dialisi Tubi </td><td> Spettro </td><td> 132640 </td><td></td></tr><tr><td></td><td> Laboratori </td><td></td><td></td></tr><tr><td> Congelatore </td><td></td><td></td><td></td></tr><tr><td&gt; Liofilizzatore </td><td> Labconco </td><td> 7670521 </td><td></td></tr><tr><td> Pompa del vuoto </td><td> Welch </td><td> 8917Z-01 </td><td></td></tr><tr><td> Irgacure 2959 </td><td> Ciba-Geigy </td><td> 029891301PS04 </td><td></td></tr><tr><td> HBSS </td><td> Mediatech </td><td> 21-022-CV </td><td></td></tr><tr><td> Filtro siringa </td><td> VWR </td><td> 28145-477 </td><td></td></tr><tr><td> RPMI 1640 </td><td> Mediatech </td><td> * </td><td> * Formulazione personalizzati </td></tr><tr><td> FBS </td><td> PAA Laboratories </td><td> A15-351 </td><td></td></tr><tr><td> Penicillina-Streptomicina </td><td> Mediatech </td><td> 30-002-CI </td><td></td></tr><tr><td> Amfotericina B </td><td> Mediatech </td><td> 30-003-CF </td><td></td></tr><tr><td> Incubatrice </td><td> Thermo Scientific </td><td> 3597 </td><td> NAPCO Serie 8000 WJ w / O2 soppressione </td></tr><tr><td> Tripsina EDTA </td><td> Mediatech </td><td> 25-052-CI </td><td></td></tr><tr><td> Shaker orbitale</td><td> VWR </td><td> 12620-926 </td><td></td></tr><tr><td> Lampada UV </td><td> Sanyo Denri </td><td> FLR40SBLB / M </td><td> Tiene due 40W, lampade UV 365nm blu blacklight </td></tr><tr><td> Centrifuga </td><td> Eppendorf </td><td> 5811 000.010 * </td><td> * Numero d&#39;ordine. Modello 5810 R </td></tr><tr><td> Microscopio </td><td> Nikon </td><td> TI-ND6-PFS </td><td> Con filterset di eccitazione 556nm / 586nm emissione </td></tr></table>

tracking hypoxic signaling within encapsulated cell aggregates

In Diabetes mellitus type 1, autoimmune destruction of the pancreatic &beta;-cells results in loss of insulin production and potentially lethal hyperglycemia. As an alternative treatment option to exogenous insulin injection, transplantation of functional pancreatic tissue has been explored1,2. This approach offers the promise of a more natural, long-term restoration of normoglycemia. Protection of the donor tissue from the host's immune system is required to prevent rejection and encapsulation is a method used to help achieve this aim.
Biologically-derived materials, such as alginate3 and agarose4, have been the traditional choice for capsule construction but may induce inflammation or fibrotic overgrowth5 which can impede nutrient and oxygen transport. Alternatively, synthetic poly(ethylene glycol) (PEG)-based hydrogels are non-degrading, easily functionalized, available at high purity, have controllable pore size, and are extremely biocompatible,6,7,8. As an additional benefit, PEG hydrogels may be formed rapidly in a simple photo-crosslinking reaction that does not require application of non-physiological temperatures6,7. Such a procedure is described here. In the crosslinking reaction, UV degradation of the photoinitiator, 1-[4-(2-Hydroxyethoxy)-phenyl]-2-hydroxy-2-methyl-1-propane-1-one (Irgacure 2959), produces free radicals which attack the vinyl carbon-carbon double bonds of dimethacrylated PEG (PEGDM) inducing crosslinking at the chain ends. Crosslinking can be achieved within 10 minutes. PEG hydrogels constructed in such a manner have been shown to favorably support cells7,9, and the low photoinitiator concentration and brief exposure to UV irradiation is not detrimental to viability and function of the encapsulated tissue10. While we methacrylate our PEG with the method described below, PEGDM can also be directly purchased from vendors such as Sigma.
An inherent consequence of encapsulation is isolation of the cells from a vascular network. Supply of nutrients, notably oxygen, is therefore reduced and limited by diffusion. This reduced oxygen availability may especially impact &beta;-cells whose insulin secretory function is highly dependent on oxygen11-13. Capsule composition and geometry will also impact diffusion rates and lengths for oxygen. Therefore, we also describe a technique for identifying hypoxic cells within our PEG capsules. Infection of the cells with a recombinant adenovirus allows for a fluorescent signal to be produced when intracellular hypoxia-inducible factor (HIF) pathways are activated14. As HIFs are the primary regulators of the transcriptional response to hypoxia, they represent an ideal target marker for detection of hypoxic signaling15. This approach allows for easy and rapid detection of hypoxic cells. Briefly, the adenovirus has the sequence for a red fluorescent protein (Ds Red DR from Clontech) under the control of a hypoxia-responsive element (HRE) trimer. Stabilization of HIF-1 by low oxygen conditions will drive transcription of the fluorescent protein (Figure 1). Additional details on the construction of this virus have been published previously15. The virus is stored in 10% glycerol at -80&deg; C as many 150 &mu;L aliquots in 1.5 mL centrifuge tubes at a concentration of 3.4 x 1010 pfu/mL. 
Previous studies in our lab have shown that MIN6 cells encapsulated as aggregates maintain their viability throughout 4 weeks of culture in 20% oxygen. MIN6 aggregates cultured at 2 or 1% oxygen showed both signs of necrotic cells (still about 85-90% viable) by staining with ethidium bromide as well as morphological changes relative to cells in 20% oxygen. The smooth spherical shape of the aggregates displayed at 20% was lost and aggregates appeared more like disorganized groups of cells. While the low oxygen stress does not cause a pronounced drop in viability, it is clearly impacting MIN6 aggregation and function as measured by glucose-stimulated insulin secretion15. Western blot analysis of encapsulated cells in 20% and 1% oxygen also showed a significant increase in HIF-1&alpha; for cells cultured in the low oxygen conditions which correlates with the expression of the DsRed DR protein.

Watch this Scientific Journal Video about Tracking Hypoxic Signaling within Encapsulated Cell Aggregates at JoVE.com

Tracking Hypoxic Signaling within Encapsulated Cell Aggregates

Un metodo per foto-incapsulamento delle celle in un idrogel PEG reticolato è descritto. Segnalazione ipossiche all&#39;interno incapsulato insulinoma murino (MIN6) aggregati è monitorato attraverso un sistema marcatore fluorescente. Questo sistema permette la visione seriale di cellule all&#39;interno di un idrogel impalcatura e la correlazione di segnalazione ipossia con i cambiamenti nel fenotipo delle cellule.

Tracciamento di segnalazione all&#39;interno di aggregati di cellule ipossiche Encapsulated

In Diabetes mellitus type 1, autoimmune destruction of the pancreatic &beta;-cells results in loss of insulin production and potentially lethal ...

tracking-hypoxic-signaling-within-encapsulated-cell-aggregates

Research

JoVE Journal

Bioengineering

10.5K Views.  University of South Carolina. Un metodo per foto-incapsulamento delle celle in un idrogel PEG reticolato è descritto. Segnalazione ipossiche all'interno incapsulato insulinoma murino (MIN6) aggregati è monitorato attraverso un sistema marcatore fluorescente. Questo sistema permette la visione seriale di cellule all'interno di un idrogel impalcatura e la correlazione di segnalazione ipossia con i cambiamenti nel fenotipo delle cellule.

Video: Tracking Hypoxic Signaling within Encapsulated Cell Aggregates

3D Orbital Tracking in a Modified Two-photon Microscope: An Application to the Tracking of Intracellular Vesicles

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a modified two-photon microscope1. As opposed to conventional approaches (raster scan or wide field based on a stack of frames), the 3D orbital tracking allows to localize and follow with a high spatial (10 nm accuracy) and temporal resolution (50 Hz frequency response) the 3D displacement of a moving fluorescent particle on length-scales of hundreds of microns2. The method is based on a feedback algorithm that controls the hardware of a two-photon laser scanning microscope in order to perform a circular orbit around the object to be tracked: the feedback mechanism will maintain the fluorescent object in the center by controlling the displacement of the scanning beam3-5. To demonstrate the advantages of this technique, we followed a fast moving organelle, the lysosome, within a living cell6,7. Cells were plated according to standard protocols, and stained using a commercially lysosome dye. We discuss briefly the hardware configuration and in more detail the control software, to perform a 3D orbital tracking experiment inside living cells. We discuss in detail the parameters required in order to control the scanning microscope and enable the motion of the beam in a closed orbit around the particle. We conclude by demonstrating how this method can be effectively used to track the fast motion of a labeled lysosome along microtubules in 3D within a live cell. Lysosomes can move with speeds in the range of 0.4-0.5 &#181;m/sec, typically displaying a directed motion along the microtubule network8.

In this video protocol we track - at high speed and in three dimensions - fluorescently labeled lysosomes within living cells, using the orbital tracking method in a modified two-photon microscope.

3D Orbital Inseguimento in una Modified microscopio a due fotoni: un&#39;applicazione per il monitoraggio di intracellulare vescicole

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a ...

Ricerca

Bioingegneria

Analysis of Cell Migration within a Three-dimensional Collagen Matrix

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic development, wound healing, and immune responses. However, cell migration is also a key mechanism in cancer enabling these cancer cells to detach from the primary tumor to start metastatic spreading. Within the past years various cell migration assays have been developed to analyze the migratory behavior of different cell types. Because the locomotory behavior of cells markedly differs between a two-dimensional (2D) and three-dimensional (3D) environment it can be assumed that the analysis of the migration of cells that are embedded within a 3D environment would yield in more significant cell migration data. The advantage of the described 3D collagen matrix migration assay is that cells are embedded within a physiological 3D network of collagen fibers representing the major component of the extracellular matrix. Due to time-lapse video microscopy real cell migration is measured allowing the determination of several migration parameters as well as their alterations in response to pro-migratory factors or inhibitors. Various cell types could be analyzed using this technique, including lymphocytes/leukocytes, stem cells, and tumor cells. Likewise, also cell clusters or spheroids could be embedded within the collagen matrix concomitant with analysis of the emigration of single cells from the cell cluster/ spheroid into the collagen lattice. We conclude that the 3D collagen matrix migration assay is a versatile method to analyze the migration of cells within a physiological-like 3D environment.

Cell migration is a biological phenomenon that is involved in a plethora of physiological, such as wound healing and immune responses, and pathophysiological processes, like cancer. The 3D-collagen matrix migration assay is a versatile tool to analyze the migratory properties of different cell types within in a 3D physiological-like environment.

Analisi della migrazione delle cellule all&#39;interno di una tridimensionale di collagene Matrix

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic ...

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous number of imaging techniques currently available and the progress in instrumentation, there is still a need for highly sensitive probes that are suitable for in vivo imaging. One typical problem of available preclinical fluorescent probes is their rapid clearance in vivo, which reduces their imaging sensitivity. To circumvent rapid clearance, increase number of dye molecules at the target site, and thereby reduce background autofluorescence, encapsulation of the near-infrared fluorescent dye, DY-676-COOH in liposomes and verification of its potential for in vivo imaging of inflammation was done. DY-676 is known for its ability to self-quench at high concentrations. We first determined the concentration suitable for self-quenching, and then encapsulated this quenching concentration into the aqueous interior of PEGylated liposomes. To substantiate the quenching and activation potential of the liposomes we use a harsh freezing method which leads to damage of liposomal membranes without affecting the encapsulated dye. The liposomes characterized by a high level of fluorescence quenching were termed Lip-Q. We show by experiments with different cell lines that uptake of Lip-Q is predominantly by phagocytosis which in turn enabled the characterization of its potential as a tool for in vivo imaging of inflammation in mice models. Furthermore, we use a zymosan-induced edema model in mice to substantiate the potential of Lip-Q in optical imaging of inflammation in vivo. Considering possible uptake due to inflammation-induced enhanced permeability and retention (EPR) effect, an always-on liposome formulation with low, non-quenched concentration of DY-676-COOH (termed Lip-dQ) and the free DY-676-COOH were compared with Lip-Q in animal trials.

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

The use of fluorophores for in vivo imaging can be greatly limited by opsonization, rapid clearance, low detection sensitivity and cytotoxic effects on the host. Encapsulation of fluorophores in liposomes by film hydration and extrusion leads to fluorescence quenching and protection which enables in vivo imaging with high detection sensitivity.

Fluorescenza tempra di un vicino infrarosso Fluoroforo incapsulata all&#39;interno di liposomi come strumento per<em&gt; In Vivo</em&gt; Optical Imaging

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous ...

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 ...

Transport Properties of Ibuprofen Encapsulated in Cyclodextrin Nanosponge Hydrogels: A Proton HR-MAS NMR Spectroscopy Study

The chemical cross-linking of &#946;-cyclodextrin (&#946;-CD) with ethylenediaminetetraacetic dianhydride (EDTA) led to branched polymers referred to as cyclodextrin nanosponges (CDNSEDTA). Two different preparations are described with 1:4 and 1:8 CD-EDTA molar ratios. The corresponding cross-linked polymers were contacted with 0.27 M aqueous solution of ibuprofen sodium salt (IP) leading to homogeneous, colorless, drug loaded hydrogels.
The systems were characterized by high resolution magic angle spinning (HR-MAS) NMR spectroscopy. Pulsed field gradient spin echo (PGSE) NMR spectroscopy was used to determine the mean square displacement (MSD) of IP inside the polymeric gel at different observation times td. The data were further processed in order to study the time dependence of MSD: MSD = f(td). The proposed methodology is useful to characterize the different diffusion regimes that, in principle, the solute may experience inside the hydrogel, namely normal or anomalous diffusion. The full protocols including the polymer preparation and purification, the obtainment of drug-loaded hydrogels, the NMR sample preparation, the measurement of MSD by HR-MAS NMR spectroscopy and the final data processing to achieve the time dependence of MSD are here reported and discussed. The presented experiments represent a paradigmatic case and the data are discussed in terms of innovative approach to the characterization of the transport properties of an encapsulated guest within a polymeric host of potential application for drug delivery.

The motion regimes of ibuprofen encapsulated in &#946;-cyclodextrin nanosponges polymer network are investigated using pulsed-field-gradient spin-echo (PGSE) NMR technique. Synthesis, purification, drug loading, implementation of the NMR pulse sequence and data analysis to work out the mean square displacement of the drug at several observation times are described in detail.

Proprietà di trasporto di ibuprofene Encapsulated in ciclodestrina Nanosponge idrogel: un protone HR-MAS NMR Spettroscopia di studio

The chemical cross-linking of &#946;-cyclodextrin (&#946;-CD) with ethylenediaminetetraacetic dianhydride (EDTA) led to branched polymers referred to as ...

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, which is regulated by a variety of soluble and physical signals in the microenvironment. Described here is a method created to study the process of branching morphogenesis by forming engineered three-dimensional (3D) epithelial tissues of defined shape and size that are completely embedded within an extracellular matrix (ECM). This method enables the formation of arrays of identical tissues and enables the control of a variety of environmental factors, including tissue geometry, spacing, and ECM composition. This method can also be combined with widely used techniques such as traction force microscopy (TFM) to gain more information about the interactions between cells and their surrounding ECM. The protocol can be used to investigate a variety of cell and tissue processes beyond branching morphogenesis, including cancer invasion.

This manuscript describes a soft lithography-based technique to engineer uniform arrays of three-dimensional (3D) epithelial tissues of defined geometry surrounded by extracellular matrix. This method is amenable to a wide variety of cell types and experimental contexts and allows for high-throughput screening of identical replicates.

Tessuti epiteliali Ingegneria tridimensionali integrati all&#39;interno di matrice extracellulare

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, ...

Targeted Plasma Membrane Delivery of a Hydrophobic Cargo Encapsulated in a Liquid Crystal Nanoparticle Carrier

The controlled delivery of drug/imaging agents to cells is critical for the development of therapeutics and for the study of cellular signaling processes. Recently, nanoparticles (NPs) have shown significant promise in the development of such delivery systems. Here, a liquid crystal NP (LCNP)-based delivery system has been employed for the controlled delivery of a water-insoluble dye, 3,3&#8242;-dioctadecyloxacarbocyanine perchlorate (DiO), from within the NP core to the hydrophobic region of a plasma membrane bilayer. During the synthesis of the NPs, the dye was efficiently incorporated into the hydrophobic LCNP core, as confirmed by multiple spectroscopic analyses. Conjugation of a PEGylated cholesterol derivative to the NP surface (DiO-LCNP-PEG-Chol) enabled the binding of the dye-loaded NPs to the plasma membrane in HEK 293T/17 cells. Time-resolved laser scanning confocal microscopy and F&#246;rster resonance energy transfer (FRET) imaging confirmed the passive efflux of DiO from the LCNP core and its insertion into the plasma membrane bilayer. Finally, the delivery of DiO as a LCNP-PEG-Chol attenuated the cytotoxicity of DiO; the NP form of DiO exhibited ~30-40% less toxicity compared to DiOfree delivered from bulk solution. This approach demonstrates the utility of the LCNP platform as an efficient modality for the membrane-specific delivery and modulation of hydrophobic molecular cargos.

A liquid crystal nanoparticle (LCNP) nanocarrier is exploited as a vehicle for the controlled delivery of a hydrophobic cargo to the plasma membrane of living cells.

Targeted membrana plasmatica di consegna di un idrofobo Cargo incapsulato in un cristallo nanoparticelle Carrier Liquid

The controlled delivery of drug/imaging agents to cells is critical for the development of therapeutics and for the study of cellular signaling processes. ...

Assembly and Tracking of Microbial Community Development within a Microwell Array Platform

The development of microbial communities depends on a combination of complex deterministic and stochastic factors that can dramatically alter the spatial distribution and activities of community members. We have developed a microwell array platform that can be used to rapidly assemble and track thousands of bacterial communities in parallel. This protocol highlights the utility of the platform and describes its use for optically monitoring the development of simple, two-member communities within an ensemble of arrays within the platform. This demonstration uses two mutants of Pseudomonas aeruginosa, part of a series of mutants developed to study Type VI secretion pathogenicity. Chromosomal inserts of either mCherry or GFP genes facilitate the constitutive expression of fluorescent proteins with distinct emission wavelengths that can be used to monitor community member abundance and location within each microwell. This protocol describes a detailed method for assembling mixtures of bacteria into the wells of the array and using time-lapse fluorescence imaging and quantitative image analysis to measure the relative growth of each member population over time. The seeding and assembly of the microwell platform, the imaging procedures necessary for the quantitative analysis of microbial communities within the array, and the methods that can be used to reveal interactions between microbial species area all discussed.

The development of microbial communities depends on a combination of factors, including environmental architecture, member abundance, traits, and interactions. This protocol describes a synthetic, microfabricated environment for the simultaneous tracking of thousands of communities contained in femtoliter wells, where key factors such as niche size and confinement can be approximated.

Assemblea e monitoraggio dello sviluppo microbico della comunità all&#39;interno di una piattaforma di array Microwell

The development of microbial communities depends on a combination of complex deterministic and stochastic factors that can dramatically alter the spatial ...

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

Currently, most in vitro models of wound healing, such as well-established scratch assays, involve studying cell migration and wound closure on two-dimensional surfaces. However, the physiological environment in which in vivo wound healing takes place is three-dimensional rather than two-dimensional. It is becoming increasingly clear that cell behavior differs greatly in two-dimensional vs. three-dimensional environments; therefore, there is a need for more physiologically relevant in vitro models for studying cell migration behaviors in wound closure. The method described herein allows for the study of cell migration in a three-dimensional model that better reflects physiological conditions than previously established two-dimensional scratch assays. The purpose of this model is to evaluate cell outgrowth via the examination of cell migration away from a spheroid body embedded within a fibrin matrix in the presence of pro- or anti-migratory factors. Using this method, cell outgrowth from the spheroid body in a three-dimensional matrix can be observed and is easily quantifiable over time via brightfield microscopy and analysis of spheroid body area. The effect of pro-migratory and/or inhibitory factors on cell migration can also be evaluated in this system. This method provides researchers with a simple method of analyzing cell migration in three-dimensional wound associated matrices in vitro, thus increasing the relevance of in vitro cell studies prior to the use of in vivo animal models.

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

The goal of this protocol is to evaluate the effect of pro- and anti-migratory factors on cell migration within a three-dimensional fibrin matrix.

Che caratterizzano la migrazione delle cellule all'interno del tridimensionale In Vitro ferita ambienti

Currently, most in vitro models of wound healing, such as well-established scratch assays, involve studying cell migration and wound closure on ...

Encapsulated Cell Technology for the Delivery of Biologics to the Mouse Eye

Many current therapeutics under development for diseases of the posterior pole of the eye are biologics. These drugs need to be administered frequently, typically via intravitreal injections. Encapsulated cells expressing the biologic of choice are becoming a tool for local protein production and release (e.g., via long-term drug delivery). In addition, encapsulation systems utilize permeable materials that allow diffusion of nutrients, waste, and therapeutic factors into and out of cells. This occurs while masking the cells from the host immune response, avoiding the need for suppression of the host immune system. This protocol describes the use of alginate as a polymer in microencapsulation coupled with the electrospray method as a microencapsulation technique. ARPE-19 cells, a spontaneously arising human RPE cell line, has been used in long-term cell therapy experiments due to its lifetime functionality, and it is used here for encapsulation and delivery of the capsules to mouse eyes. The manuscript summarizes the steps for cell microencapsulation, quality control, and ocular delivery.

Presented here is a protocol for the use of alginate as a polymer in microencapsulation of immortalized cells for long-term delivery of biologics to rodent eyes.

Tecnologia a cellule incapsulate per la consegna dei biologici all'occhio del mouse

Many current therapeutics under development for diseases of the posterior pole of the eye are biologics. These drugs need to be administered frequently, ...