Questo lavoro è stato sostenuto dal Fondo di Ricerca sul Cancro ovarico [concedere LT/UIC/01.2011], il Centro UIC per la scienza clinica e traslazionale, il Cancer Center UIC, e il National Institutes of Health concedere C06RR15482. Gli esperimenti sugli animali sono stati eseguiti in conformità con le linee guida e dei regolamenti previsti dal AAALAC sotto protocolli animali approvati cura dalla cura degli animali e del Comitato Usa a UIC. Animali per questo progetto sono alloggiati in camere barriera nel Laboratorio risorse biologiche (BRL) presso l&#39;Università dell&#39;Illinois a Chicago. Il BRL dispone di uno staff completamente veterinario che controlla gli animali, almeno due volte al giorno e fornisce consigli sulla cura degli animali.

1. Ovarico dissezione e isolamento dei tessuti <ol><li> Preparare una soluzione allo 0,5% (w / v) di alginato di sodio, preparare da una versione modificata del protocollo descritto in precedenza 5, utilizzando PBS sterile e calore a 37 ° C. Miscelare invertendo o dondolo, ma non vortice. Disegnare la alginato in una siringa da 1 ml con un ago da 25 G e mantenere a 37 ° C. </li><li> Preparare 10mM sterile CaCl 2 soluzione per la reticolazione alginato su incapsulamento dei tessuti e calda a 37 ° C. </li><li> Preparare 1 ml di terreno di coltura (αMEM + 5 unità di penicillina e 5 mg / ml di streptomicina) per bene in un 24 pozzetti per ciascun esperimento. Comprendono diversi farmaci, peptidi, o virus nel terreno di coltura. Per la trasfezione prima di incapsulamento organo, incubare il tessuto con Lipofectamine il 2000 e il DNA plasmidico per quattro ore a 37 ° C. </li><li> Sezionare gli organi dall&#39;animale, rimuovere eventuali residui di tessuto adiposo o, staccare delicatamente la membrana che circonda la borsa ovarica, e tagliare l&#39;ovaio o ovidotto in quattro pezzi da bisecando l&#39;organo. Gli animali sono stati sacrificati dalla CO 2 asfissia seguita da dislocazione cervicale. Per l&#39;ovidotto, delicatamente srotolare il tessuto tirando fuori le membrane di collegamento ed estendendo le estremità del tubo in direzioni opposte con pinze prima di tagliare. </li><li> Inserire una singola goccia alginato (microlitri ~ 1-3) su una rete in fibra di sterilizzato. </li><li> Utilizzando pinza sterile, spostare un pezzo di organo nella goccia alginato e il centro della goccia con pinza o un ago sterile. </li><li> Invertire la goccia che contiene il pezzo d&#39;organo (organoide) afferrando la rete in fibra con una pinza sterile e si rovescia per consentire la gravità di forzare l&#39;alginato in una goccia. </li><li> Toccare la pinza sul bordo di un piatto di 10 centimetri di Petri contenente 10mM CaCl 2 soluzione in modo che la goccia cade in soluzione. </li><li> Incubare per 2 minuti, rimuovere con un cucchiaio di sterili contenenti pori di rilasciare supplementare 10mM CaCl 2, e trasferire nel terreno di coltura. Fino a quattro gel alginato può essere trasferito in un singolo pozzo di una piastra 24 pozzetti contenenti il ​​terreno di coltura. Cultura per un periodo di tempo definito. </li></ol> 2. Degrado di alginato e la raccolta di cellule <ol><li> Per la solubilizzazione del gel alginato, incubare le culture in alginato liasi (3,4 mg / ml) disciolto in L-15 Leibovitz dei media a 37 ° C per 30 minuti o fino a quando il gel è completamente sciolto. </li><li> Rimuovere il tessuto dal alginato liasi. </li><li> Per la raccolta delle OSE, incubare il organoide con 500 unità di collagenasi in Leibovitz L-15 supporti per 1 ora 8. Vortex in 2 impulsi secondo (2 secondi e 2 secondi vortice di riposo) su media potenza per 1 minuto, seguito da 1 impulsi secondo (1 vortice secondo e 1 secondo riposo) per 1 minuto. Lasciate cadere a pezzi organo fondo della provetta e sopranatante pipetta in fresco provetta sterile. Centrifugare 3 minuti a 3000 rpm per epitelio superficie ovarica pellet. </li><li> Per la raccolta dell&#39;epitelio tubarico, tagliare il tubo della lunghezza e del luogo in plastica coltura in DMEM con 5 unità di penicillina e 5 mg / ml streptomicina e il 4% (v / v) di FBS. Più di 3-5 giorni, le cellule si &quot;strisciare&quot; fuori dello stroma tube e crescere in colture di tessuti plastic9. </li></ol> 3. Fissazione di culture organo alginato incapsulato per le sezioni di paraffina <ol><li> Raccogliere l&#39;alginato incapsulato tallone dal terreno di coltura al punto di tempo selezionato. </li><li> Recrosslink l&#39;alginato per irrigidire il gel facendo cadere la cultura in sterili 10mM CaCl 2 soluzione per due minuti. </li><li> Fissare l&#39;organo alginato incapsulato nel 2% paraformaldeide saccarosio contenente 50 mm, 10 mm CaCl 2 e 50 mM di sodio cacodilato per garantire che il gel rimane solido e incapsula completamente il tessuto durante la fissazione. </li><li> A seguito di lavorazione dei tessuti, incorporare l&#39;intero alginato-incapsulato pezzo cultura d&#39;organo in paraffina e sezione con un microtomo per generare 5 sezioni micron. </li><li> L&#39;alginato sarà sciolto e rimosso dalla diapositiva se si esegue l&#39;immunoistochimica a causa del calore di recupero basato antigene. L&#39;alginato resteranno e colorare per eosina in piedi e colorazione ematossilina eosina. </li></ol> 4. Rappresentante dei risultati: Un esempio di tridimensionale cultura organo dell&#39;ovaio e ovidotto è mostrata in Figura 1. Ovaie possono essere tagliati in pezzi di diverse dimensioni, e OSE proliferare e migrare per riparare la superficie che è stato ferito dalla incisione con il bisturi. Durante questo processo, il corretto orientamento della OSE rispetto al sottostante stroma è mantenuto, con l&#39;OSE incapsulare l&#39;ovaio. Come mostrato nella Figura 2, l&#39;OSE di ovaie tagliati a metà incapsulare le ovaie in modo più completo rispetto a ovaie tagliato in quarti o ottavi. La riparazione delle ferite del ovaricosuperficie può essere studiata nel contesto delle ovaie e tutto può far luce su come riparare l&#39;ovulazione e la superficie induce la trasformazione della superficie dell&#39;ovaio. L&#39;endpoint dell&#39;esperimento dipende dalle informazioni che è necessario. Architettura del tessuto, la morfologia cellulare e specifici tassi di crescita cellulare, possono essere facilmente monitorate utilizzando sezioni di paraffina. Ad esempio, un singolo strato di epitelio ovarico superficie si osserva dopo coltura nel siero senza αMEM per 7 giorni (Figura 3A), mentre l&#39;aggiunta di 5 mg / ml di insulina per 7 giorni induce iperproliferazione dell&#39;epitelio superficie ovarica e la formazione di strati multipli di cellule (Figura 3B). Aggiungendo bromodeossiuridina alle culture ad una concentrazione finale di 10 mM 24 ore prima della fissazione, la proliferazione delle cellule può essere misurata (inserto 3A e 3B). Per le culture mantenuto nel siero senza αMEM, circa il 3-5% di OSE subire proliferazione durante il periodo di 24 ore di etichettatura (3A riquadro), mentre una percentuale maggiore di cellule della granulosa proliferano. L&#39;aggiunta di insulina per la cultura dei media aumenta la percentuale di proliferazione OSE a circa il 30% del totale OSE (3B riquadro). Immunofluorescenza di proteina fluorescente verde (GFP) possono essere visualizzati attraverso il gel alginato con microscopia seguente norma trasfezione del DNA (Figura 3C). Mentre organo cultura alginato offre per la crescita e l&#39;analisi dell&#39;epitelio ovarico superficie e follicoli primari, si deve rilevare che la maturità, i follicoli secondari non riescono a sopravvivere in questo sistema di coltura (Fig. 3D). Cultura dei follicoli singoli maturità all&#39;interno di una matrice idrogel alginato è stata descritta altrove 10. Alterazioni di proteine ​​o RNA può essere analizzata da l&#39;intero organo o da specifici tipi cellulari mediante degradazione enzimatica (Figura 4). Trattamento di organoidi ovarico coltura con collagenasi lascia intatto lo stroma sottostante (Figura 4C), pur consentendo l&#39;arricchimento di OSE nel pellet ottenuto dopo il trattamento, anche se alcune cellule stromali sono anche isolate (fig. 4D). <img alt="Figura 1" src="/files/ftp_upload/2804/2804fig1.jpg" /> Figura 1. Dell&#39;ovaio mouse tridimensionale sistema di coltura utilizzato per propagare OSE dopo il ferimento. A) Sotto il microscopio, la borsa e il cuscinetto di grasso che circonda sono stati rimossi. B) ovaio intatto (O) con tubo oviductal (T) e grassi pad (F). C) ovaio con borsa e cuscinetto adiposo rimosso (a sinistra) e srotolato ovidotto (a destra). D) organoidi ovarica o oviductal sono stati collocati in una gocciolina alginato. E) organoidi alginato racchiusi furono rinviati ad una soluzione di CaCl 2 reticolazione l&#39;alginato di formare un gel. F) alginato incapsulato organoide ovarico. G) alginato incapsulato organoide oviductal. H) organoidi alginato incapsulato incubati in terreno di coltura. <img alt="Figura 2" src="/files/ftp_upload/2804/2804fig2.jpg" /> Figura 2. Superficie rigenerato dalla superficie ovarica misurata con citocheratina 8. Incapsulamento di organoidi ovarico dal OSE dopo essere stato tagliato in ottavi, quarti, e la metà, coltivate in media BSA, e misurato attraverso l&#39;analisi immunoistochimica utilizzando citocheratina 8 (CK8) anticorpi. <img alt="Figura 3" src="/files/ftp_upload/2804/2804fig3.jpg" /> Figura 3. Sistema di coltura alginato permette il passaggio di fattori di crescita e di trasfezione di cDNA nel OSE, ma non supporta una rapida crescita e la maturazione dei follicoli. A) organoide ovarica in coltura per 7 giorni in media basale analizzati per CK8 espressione, che segna l&#39;OSE. Incasso, sezione seriale macchiato per incorporazione di BrdU. B) L&#39;aggiunta di insulina per i terreni di coltura induce iperplasia delle OSE. Incasso, sezione seriale macchiato per incorporazione di BrdU. C) organoide ovarica transfettate con cDNA per esprimere la GFP. D) la cultura del sistema alginato sostiene la crescita dei follicoli primordiali (freccia rossa), ma follicoli secondari non maturi (freccia nera). <img alt="Figura 4" src="/files/ftp_upload/2804/2804fig4.jpg" /> Figura 4. OSE possono essere isolati per la cultura successiva analisi. A) organoide coltivate in media per un massimo di 2 settimane. B) organoide incapsulato è trattato con alginato liasi di lasciare organoide intatto. Dell&#39;ovaio è macchiato con CK8 per segnare OSE. C) organoide è trattato con collagenasi di OSE separato da stroma sottostante. Pezzo rimanente tessuto era macchiata di CK8 per evidenziare la rimozione della superficie dell&#39;ovaio. D) le cellule raccolte dopo il trattamento sono arricchiti per OSE come colorate per CK8 (rosso). Le cellule sono di contrasto con DAPI per marcare i nuclei.

<ol>
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Non c&#39;è nulla da rivelare.

Lo sviluppo di un sistema tridimensionale di organi cultura utilizzando idrogel alginato rappresenta un metodo versatile per l&#39;analisi di molti tipi di tessuto diverso da una grande varietà di organismi. L&#39;uso di colture 3D può essere estesa ai campi della ingegneria dei tessuti e medicina rigenerativa in quanto fornisce una impalcatura su cui le cellule possono crescere 11. Attualmente, il nostro laboratorio è in fase di studio iniziale, usando il tessuto ovarico umano e tube di Falloppio, tuttavi...

<table border="1"><tbody><tr><th> Nome del reagente </th><th> Azienda </th><th> Numero di catalogo </th><th> Commenti (opzionale) </th></tr><tr><td> Leibovitz L-15 di media </td><td> Gibco </td><td> 11415 </td><td></td></tr><tr><td> αMEM media </td><td> Gibco </td><td> 12000-022 </td><td></td></tr><tr><td> Alginato di sodio </td><td> Novamatrix </td><td></td><td></td></tr><tr><td> Sodio cacodilato </td><td> Sigma Aldrich </td><td> C0250 </td><td></td></tr><tr><td> Rete in fibra di polipropilene </td><td> McMaster Carr </td><td></td><td> 45 &quot;di larghezza rotolo; 0,0041&quot; apertura </td></tr><tr><td> Lipofectamine 2000 </td><td> Invitrogen </td><td> 11668 </td><td></td></tr><tr><td> Penicillina / streptomicina </td><td> Gibco </td><td> 15070-063 </td><td></td></tr><tr><td> Alginato liasi </td><td> Sigma Aldrich </td><td> A1603 </td><td></td></tr><tr><td> A1603 </td><td> Sigma Aldrich </td><td> C9697 </td><td> Da Clostridium histolyticum </td></tr><tr><td> Albumina di siero bovino </td><td> MP Biomedicals </td><td> 103700 </td><td></td></tr><tr><td> Siero fetale bovino </td><td> Gibco </td><td> Gibco </td><td></td></tr><tr><td> ITS </td><td> Roche </td><td> 11074547001 </td><td> Insulina / transferrina / sodio selenite </td></tr><tr><td> Citocheratina 8 anticorpi </td><td> Developmental Studies Ibridoma Banca </td><td> Troma-1 </td><td> Uso a 1:200 </td></tr><tr><td> DAPI </td><td> Vector Labs </td><td> H1200 </td><td></td></tr><tr><td> Bromodeossiuridina </td><td> Sigma </td><td> B50002-1G </td><td> Concentrazione finale di 10 micron </td></tr></tbody></table>

alginate hydrogels for three-dimensional organ culture of ovaries and oviducts

Il cancro ovarico è la quinta causa di decessi per cancro nelle donne e ha un tasso di mortalità del 63% negli Stati Uniti 1. Il tipo di cellula d&#39;origine dei tumori ovarici è ancora in discussione e potrebbe essere sia l&#39;epitelio della superficie ovarica (OSE) o l&#39;epitelio distale del fimbrie tube di Falloppio 2,3. Coltura le cellule normali come una coltura primaria in vitro consentirà agli scienziati di modello cambiamenti specifici che potrebbero portare al cancro ovarico nell&#39;epitelio distinti, così definitivamente la determinazione del tipo di cellula di origine. Ciò permetterà lo sviluppo di biomarcatori più precisi, modelli animali con tessuto-specifici cambia gene, e migliori strategie di prevenzione mirate a questa malattia. Il mantenimento di cellule normali in idrogel alginato promuove a breve termine in coltura in vitro di cellule nel loro contesto tridimensionale e permette l&#39;introduzione di DNA plasmidico, siRNA e piccole molecole. Dagli organi coltura in pezzi che sono derivati ​​da tagli strategici con un bisturi, le culture diverse da un singolo organo può essere generato, aumentando il numero di esperimenti da un singolo animale. Questi tagli aspetti del modello di ovulazione porta alla proliferazione di OSE, che è associata alla formazione di cancro ovarico. Tipi di cellule come l&#39;OSE, che non crescono bene su superfici in plastica possono essere coltivate con questo metodo e facilitare l&#39;indagine nei normali processi cellulari o dei primi eventi nella formazione del cancro 4. Idrogel alginato può essere utilizzato per sostenere la crescita di molti tipi di tessuti 5. Alginato è un polisaccaride lineare composto da unità ripetute di β-D-mannuronico acido e α-L-guluronic acido che può essere reticolato con gli ioni calcio, con una conseguente azione gelificante delicata che non danneggiano i tessuti 6,7. Come per le altre cellule matrici tridimensionali cultura come Matrigel, alginato fornisce supporto meccanico per i tessuti, ma le proteine ​​non sono reattivi con la matrice di alginato, e quindi le funzioni alginato come matrice extracellulare sintetico che non avvia segnalazione cellulare 5. L&#39;idrogel alginato galleggia in media standard di coltura cellulare e supporta l&#39;architettura della crescita dei tessuti in vitro. Un metodo viene presentato per la preparazione, la separazione, e l&#39;inclusione di neoplasie ovariche e composizioni per organo oviductal in idrogel alginato, che possono essere mantenute in coltura per un massimo di due settimane. Il rilascio enzimatico delle cellule per l&#39;analisi di campioni di RNA e proteine ​​dalla cultura organo è anche descritto. Infine, la crescita di tipi di cellule primarie è possibile senza immortalizzazione genetica di topi e permette agli investigatori di utilizzare knockout e topi transgenici.

Watch this Scientific Journal Video about Alginate Hydrogels for Three-Dimensional Organ Culture of Ovaries and Oviducts at JoVE.com

Alginate Hydrogels for Three-Dimensional Organ Culture of Ovaries and Oviducts

Coltura di cellule normali in loro contesto tridimensionale rappresenta un metodo alternativo per studiare eventi precoci necessari per la trasformazione cellulare e tumorigenesi. Questo metodo è utilizzato per la coltivazione ovarici normali e cellule oviductal per studiare eventi precoci nella formazione del cancro ovarico.

Idrogeli di alginato per tridimensionale Cultura Organo di ovaie e tube

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ...

alginate-hydrogels-for-three-dimensional-organ-culture-ovaries

Research

JoVE Journal

Bioengineering

21.8K Views.  University of Illinois at Chicago. Coltura di cellule normali in loro contesto tridimensionale rappresenta un metodo alternativo per studiare eventi precoci necessari per la trasformazione cellulare e tumorigenesi. Questo metodo è utilizzato per la coltivazione ovarici normali e cellule oviductal per studiare eventi precoci nella formazione del cancro ovarico.

Video: Alginate Hydrogels for Three-Dimensional Organ Culture of Ovaries and Oviducts

Planar and Three-Dimensional Printing of Conductive Inks

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical devices1-3. In this paper, we focus on one- (1D), two- (2D), and three-dimensional (3D) printing of conductive metallic inks in the form of flexible, stretchable, and spanning microelectrodes.
Direct-write assembly4,5 is a 1-to-3D printing technique that enables the fabrication of features ranging from simple lines to complex structures by the deposition of concentrated inks through fine nozzles (~0.1 - 250 &mu;m). This printing method consists of a computer-controlled 3-axis translation stage, an ink reservoir and nozzle, and 10x telescopic lens for visualization. Unlike inkjet printing, a droplet-based process, direct-write assembly involves the extrusion of ink filaments either in- or out-of-plane. The printed filaments typically conform to the nozzle size. Hence, microscale features (&lt; 1 &mu;m) can be patterned and assembled into larger arrays and multidimensional architectures.
In this paper, we first synthesize a highly concentrated silver nanoparticle ink for planar and 3D printing via direct-write assembly. Next, a standard protocol for printing microelectrodes in multidimensional motifs is demonstrated. Finally, applications of printed microelectrodes for electrically small antennas, solar cells, and light-emitting diodes are highlighted.

Planar and three-dimensional printing of conductive metallic inks is described. Our approach provides new avenues for fabricating printed electronic, optoelectronic, and biomedical devices in unusual layouts at the microscale.

Stampa planare e tridimensionale di inchiostri conduttivi

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical ...

Ricerca

Bioingegneria

Preparation of Intact Bovine Tail Intervertebral Discs for Organ Culture

The intervertebral disc (IVD) is the joint of the spine connecting vertebra to vertebra. It functions to transmit loading of the spine and give flexibility to the spine. It composes of three compartments: the innermost nucleus pulposus (NP) encompassing by the annulus fibrosus (AF), and two cartilaginous endplates connecting the NP and AF to the vertebral body on both sides. Discogenic pain possibly caused by degenerative intervertebral disc disease (DDD) and disc herniations has been identified as a major problem in our modern society. To study possible mechanisms of IVD degeneration, in vitro organ culture systems with live disc cells are highly appealing. The in vitro culture of intact bovine coccygeal IVDs has advanced to a relevant model system, which allows the study of mechano-biological aspects in a well-controlled physiological and mechanical environment. Bovine tail IVDs can be obtained relatively easy in higher numbers and are very similar to the human lumbar IVDs with respect to cell density, cell population and dimensions. However, previous bovine caudal IVD harvesting techniques retaining cartilaginous endplates and bony endplates failed after 1-2 days of culture since the nutrition pathways were obviously blocked by clotted blood. IVDs are the biggest avascular organs, thus, the nutrients to the cells in the NP are solely dependent on diffusion via the capillary buds from the adjacent vertebral body. Presence of bone debris and clotted blood on the endplate surfaces can hinder nutrient diffusion into the center of the disc and compromise cell viability. Our group established a relatively quick protocol to "crack"-out the IVDs from the tail with a low risk for contamination. We are able to permeabilize the freshly-cut bony endplate surfaces by using a surgical jet lavage system, which removes the blood clots and cutting debris and very efficiently reopens the nutrition diffusion pathway to the center of the IVD. The presence of growth plates on both sides of the vertebral bone has to be avoided and to be removed prior to culture. In this video, we outline the crucial steps during preparation and demonstrate the key to a successful organ culture maintaining high cell viability for 14 days under free swelling culture. The culture time could be extended when appropriate mechanical environment can be maintained by using mechanical loading bioreactor. The technique demonstrated here can be extended to other animal species such as porcine, ovine and leporine caudal and lumbar IVD isolation.

This protocol illustrates a harvesting technique for coccygeal bovine intervertebral discs for organ culture for in vitro organ culture.

Preparazione del intatto dischi intervertebrali coda bovina per la Cultura d&#39;organo

The intervertebral disc (IVD) is the joint of the spine connecting  vertebra to vertebra. It functions to transmit loading of the spine and give  ...

Three-dimensional Cell Culture Model for Measuring the Effects of Interstitial Fluid Flow on Tumor Cell Invasion

The growth and progression of most solid tumors depend on the initial transformation of the cancer cells and their response to stroma-associated signaling in the tumor microenvironment 1. Previously, research on the tumor microenvironment has focused primarily on tumor-stromal interactions 1-2. However, the tumor microenvironment also includes a variety of biophysical forces, whose effects remain poorly understood. These forces are biomechanical consequences of tumor growth that lead to changes in gene expression, cell division, differentiation and invasion3. Matrix density 4, stiffness 5-6, and structure 6-7, interstitial fluid pressure 8, and interstitial fluid flow 8 are all altered during cancer progression.
Interstitial fluid flow in particular is higher in tumors compared to normal tissues 8-10. The estimated interstitial fluid flow velocities were measured and found to be in the range of 0.1-3 &mu;m s-1, depending on tumor size and differentiation 9, 11. This is due to elevated interstitial fluid pressure caused by tumor-induced angiogenesis and increased vascular permeability 12. Interstitial fluid flow has been shown to increase invasion of cancer cells 13-14, vascular fibroblasts and smooth muscle cells 15. This invasion may be due to autologous chemotactic gradients created around cells in 3-D 16 or increased matrix metalloproteinase (MMP) expression 15, chemokine secretion and cell adhesion molecule expression 17. However, the mechanism by which cells sense fluid flow is not well understood. In addition to altering tumor cell behavior, interstitial fluid flow modulates the activity of other cells in the tumor microenvironment. It is associated with (a) driving differentiation of fibroblasts into tumor-promoting myofibroblasts 18, (b) transporting of antigens and other soluble factors to lymph nodes 19, and (c) modulating lymphatic endothelial cell morphogenesis 20.
The technique presented here imposes interstitial fluid flow on cells in vitro and quantifies its effects on invasion (Figure 1). This method has been published in multiple studies to measure the effects of fluid flow on stromal and cancer cell invasion 13-15, 17. By changing the matrix composition, cell type, and cell concentration, this method can be applied to other diseases and physiological systems to study the effects of interstitial flow on cellular processes such as invasion, differentiation, proliferation, and gene expression.

Interstitial fluid flow is elevated in solid tumors and can modulate tumor cell invasion. Here we describe a technique to apply interstitial fluid flow to cells embedded in a matrix and then measure its effects on cell invasion. This technique can be easily adapted to study other systems.

Tridimensionale Cultura modello cellulare per misurare gli effetti del flusso del fluido interstiziale sulla Invasion cellule tumorali

The growth and progression of most solid tumors depend on the initial transformation of the cancer cells and their response to stroma-associated signaling ...

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 ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

Generazione di una tridimensionale a tutto spessore della pelle Equivalent e Automated Ferimento

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

Three-dimensional Biomimetic Technology: Novel Biorubber Creates Defined Micro- and Macro-scale Architectures in Collagen Hydrogels

Tissue scaffolds play a crucial role in the tissue regeneration process. The ideal scaffold must fulfill several requirements such as having proper composition, targeted modulus, and well-defined architectural features. Biomaterials that recapitulate the intrinsic architecture of in vivo tissue are vital for studying diseases as well as to facilitate the regeneration of lost and malformed soft tissue. A novel biofabrication technique was developed which combines state of the art imaging, three-dimensional (3D) printing, and selective enzymatic activity to create a new generation of biomaterials for research and clinical application. The developed material, Bovine Serum Albumin rubber, is reaction injected into a mold that upholds specific geometrical features. This sacrificial material allows the adequate transfer of architectural features to a natural scaffold material. The prototype consists of a 3D collagen scaffold with 4 and 3 mm channels that represent a branched architecture. This paper emphasizes the use of this biofabrication technique for the generation of natural constructs. This protocol utilizes a computer-aided software (CAD) to manufacture a solid mold which will be reaction injected with BSA rubber followed by the enzymatic digestion of the rubber, leaving its architectural features within the scaffold material.

An innovative biofabrication technique was developed to engineer three-dimensional constructs that resemble the architectural features, components, and mechanical properties of in vivo tissue. This technique features a newly developed sacrificial material, BSA rubber, which transfers detailed spatial features, reproducing the in vivo architectures of a wide variety of tissues.

Tridimensionale Tecnologia biomimetica: Novel Biorubber Consente di creare Definito micro e macro scala Architetture in collagene idrogel

Tissue scaffolds play a crucial role in the tissue regeneration process. The ideal scaffold must fulfill several requirements such as having proper ...

Fabrication of Inverted Colloidal Crystal Poly(ethylene glycol) Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing drugs targeted at the liver, requires a platform in which cells receive proper biochemical and mechanical cues. Recent liver tissue engineering systems have employed three-dimensional (3D) scaffolds composed of synthetic or natural hydrogels, given their high water retention and their ability to provide the mechanical stimuli needed by the cells. There has been growing interest in the inverted colloidal crystal (ICC) scaffold, a recent development, which allows high spatial organization, homotypic and heterotypic cell interaction, as well as cell-extracellular matrix (ECM) interaction. Herein, we describe a protocol to fabricate the ICC scaffold using poly (ethylene glycol) diacrylate (PEGDA) and the particle leaching method. Briefly, a lattice is made from microsphere particles, after which a pre-polymer solution is added, properly polymerized, and the particles are then removed, or leached, using an organic solvent (e.g., tetrahydrofuran). The dissolution of the lattice results in a highly porous scaffold with controlled pore sizes and interconnectivities that allow media to reach cells more easily. This unique structure allows high surface area for the cells to adhere to as well as easy communication between pores, and the ability to coat the PEGDA ICC scaffold with proteins also shows a marked effect on cell performance. We analyze the morphology of the scaffold as well as the hepatocarcinoma cell (Huh-7.5) behavior in terms of viability and function to explore the effect of ICC structure and ECM coatings. Overall, this paper provides a detailed protocol of an emerging scaffold that has wide applications in tissue engineering, especially liver tissue engineering.

Fabrication of Inverted Colloidal Crystal Polyethylene glycol Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

This manuscript presents a detailed protocol for the fabrication of an emerging three-dimensional hepatocyte culture platform, the inverted colloidal crystal scaffold, and the concomitant techniques to assess hepatocyte behavior. The size-controllable pores, interconnectivity and ability to conjugate extracellular matrix proteins to the poly(ethylene glycol) (PEG) scaffold enhance Huh-7.5 cell performance.

Fabbricazione di invertito colloidale cristallo poli (glicole etilenico) Scaffold: una piattaforma Cell Culture tridimensionale per il fegato Tissue Engineering

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing ...

Layered Alginate Constructs: A Platform for Co-culture of Heterogeneous Cell Populations

Many load bearing tissues possess structurally and functionally distinct regions, typically accompanied by different cell phenotypes with differential mechanosensing characteristics. Engineering and analysis of these tissue types remain a challenge. Layered hydrogel constructs provide an opportunity for investigating the interactions among multiple cell populations within single constructs. Alginate hydrogels are both biocompatible and allow for easy isolation of cells after experimentation. Here, we describe a method for the development of small sized dual layered alginate hydrogel discs. This process maintains high cell viability of human mesenchymal stem cells during the formation process and these layered discs can withstand unconfined cyclic compression, commonly used for stimulation of hMSCs undergoing chondrogenesis. These layered constructs can potentially be scaled up to include additional levels, and also be used to segregate cell populations initially after layering. This dual layer alginate hydrogel culture platform can be used for many different applications including engineering and analysis of cells of load bearing tissues and co-cultures of other cell types.

Engineering and analysis of load bearing tissues with heterogeneous cell populations are still a challenge. Here, we describe a method for creating bi-layered alginate hydrogel discs as a platform for co-culture of diverse cell populations within one construct.

Layered alginato costrutti: Una piattaforma per la cooperazione culturale delle popolazioni cellulari eterogenee

Many load bearing tissues possess structurally and functionally distinct regions, typically accompanied by different cell phenotypes with differential ...

Light-mediated Formation and Patterning of Hydrogels for Cell Culture Applications

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In particular, light-mediated click reactions, such as photoinitiated thiol&#8722;ene and thiol&#8722;yne reactions, afford spatiotemporal control over material properties and allow the design of systems with a high degree of user-directed property control. Fabrication and modification of hydrogel-based biomaterials using the precision afforded by light and the versatility offered by these thiol&#8722;X photoclick chemistries are of growing interest, particularly for the culture of cells within well-defined, biomimetic microenvironments. Here, we describe methods for the photoencapsulation of cells and subsequent photopatterning of biochemical cues within hydrogel matrices using versatile and modular building blocks polymerized by a thiol&#8722;ene photoclick reaction. Specifically, an approach is presented for constructing hydrogels from allyloxycarbonyl (Alloc)-functionalized peptide crosslinks and pendant peptide moieties and thiol-functionalized poly(ethylene glycol) (PEG) that rapidly polymerize in the presence of lithium acylphosphinate photoinitiator and cytocompatible doses of long wavelength ultraviolet (UV) light. Facile techniques to visualize photopatterning and quantify the concentration of peptides added are described. Additionally, methods are established for encapsulating cells, specifically human mesenchymal stem cells, and determining their viability and activity. While the formation and initial patterning of thiol-alloc hydrogels are shown here, these techniques broadly may be applied to a number of other light and radical-initiated material systems (e.g., thiol-norbornene, thiol-acrylate) to generate patterned substrates.

We describe a sequential process for light-mediated formation and subsequent biochemical patterning of synthetic hydrogel matrices for three-dimensional cell culture applications. The construction and modification of hydrogels with cytocompatible photoclick chemistry is demonstrated. Additionally, facile techniques to quantify and observe patterns and determine cell viability within these hydrogels are presented.

Formazione Light-mediata ed il modello di idrogel per colture cellulari

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In ...

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung tissue from porcine, rat, or mouse, the tissue is perfused and submerged in subsequent chemical detergents to remove the cellular debris. Histological comparison of the tissue before and after processing confirms removal of over 95% of double stranded DNA and alpha galactosidase staining suggests the majority of cellular debris is removed. After decellularization, the tissue is lyophilized and then cryomilled into a powder. The matrix powder is digested for 48 hr in an acidic pepsin digestion solution and then neutralized to form the pregel solution. Gelation of the pregel solution can be induced by incubation at 37 &#176;C and can be used immediately following neutralization or stored at 4 &#176;C for up to two weeks. Coatings can be formed using the pregel solution on a non-treated plate for cell attachment. Cells can be suspended in the pregel prior to self-assembly to achieve a 3D culture, plated on the surface of a formed gel from which the cells can migrate through the scaffold, or plated on the coatings. Alterations to the strategy presented can impact gelation temperature, strength, or protein fragment sizes. Beyond hydrogel formation, the hydrogel stiffness may be increased using genipin.

This is a method to create a 3-dimensional cell culture scaffold from pulmonary extracellular matrix. Intact lung is processed into hydrogels that can support the growth of cells in three-dimensions.

Sintonizzabile idrogel da polmonare matrice extracellulare per colture cellulari 3D

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung ...