Questo lavoro è stato supportato da una Fellowship dell&#39;American Heart Association Predoctoral di JLI e istituti nazionali di Heath concedere R01AR056624.

A. singolo Polymer Electrospinning <ol><li> Prima di preparare la soluzione electrospinning, fare una soluzione di 0,5% in peso del fotoiniziatore, Irgacure 2959 (I2959), in acqua deionizzata, sciogliendo a 37 ° C per diversi giorni. Questo passaggio non è necessario se un polimero fotoreattivo non viene utilizzato. </li><li> Combina methacrylated acido ialuronico (Meha, vedere Burdick et al. Per la sintesi), poli (ossido di etilene) (PEO, 900 kDa), e I2959 in acqua deionizzata per preparare una soluzione con una concentrazione finale del 2% in peso Meha, 3% in peso PEO e 0,05% in peso I2959. Utilizzare un vortice per miscelare la soluzione fino a quando non è chiaro. Il tipo di polimero e la concentrazione, così come l&#39;uso di solventi possono essere modificati in questa fase a seconda delle proprietà desiderate patibolo. </li><li> Trasferire la soluzione in una siringa e collegare un manometro 18, 6 pollici lungo, l&#39;ago senza punta fino alla fine. </li><li> Programma di una pompa a siringa per espellere a una velocità di 1,2 ml / ora. Inserire la siringa e l&#39;ago nel dispositivo. </li><li> Collegare il cavo di messa a terra di una sorgente ad alta tensione per l&#39;apparato di raccolta. Per raccogliere le fibre distribuite in modo casuale utilizzare una piastra piana di metallo, o utilizzare un mandrino ruota a ~ 10 m / s per raccogliere le fibre allineate. Collegare il cavo carica positiva per l&#39;ago. Vedere la Figura 1 per uno schema dell&#39;apparato electrospinning. </li><li> Regolare l&#39;ago o dispositivo di raccolta, in modo tale che vi è un 15 cm di distanza tra i due. </li><li> Avviare il flusso della pompa siringa. Quando il fluido è visualizzato sulla punta dell&#39;ago, accendere la sorgente di alimentazione e impostare la tensione a 22 kV. </li><li> Dopo la raccolta è completa (da alcuni minuti di film sottili, fino a 24 ore per una stuoia spessa), rimuovere l&#39;impalcatura dall&#39;apparato raccolta e conservare sotto vuoto durante la notte per garantire la completa rimozione del solvente. </li><li> Visualizzare la morfologia delle fibre mediante microscopia elettronica a scansione (SEM), un ponteggio rappresentante è mostrata in Figura 1 per entrambe le strutture allineati e non allineati. </li></ol> Nota: La portata del campione, a distanza di dispositivi di raccolta, e la tensione dipendono dalla combinazione di polimeri e solvente e devono essere ottimizzati per ciascun sistema, di solito osservando la morfologia patibolo con SEM. B. Photocrosslinking e Photopatterning <ol><li> Tagliare 5 millimetri campioni x 5mm dal tappetino patibolo. </li><li> Preparati per reticolazione mettendo ogni impalcatura su un foglio coperto vetrino, mettendo il fotomaschera direttamente sul patibolo, coprendo con un vetrino pulito e ritaglio entrambe le estremità con clip legante. Nota: quattro campioni può essere fatto in una sola volta, ed una trasparenza senza uno schema può essere usato per reticolazione se un modello non è desiderato. </li><li> Purge dell&#39;impalcatura in una camera di azoto. E &#39;importante mantenere il costrutto privo di ossigeno, che possono inibire la reticolazione. </li><li> Luogo di installazione ponteggio in camera di azoto sotto ~ 10 mW / cm 2 luce 365 nm con un adattatore di collimazione per 5 minuti. Vedi schema in Figura 2. </li><li> Rimuovere ogni impalcatura e mettere in un piatto ben 12. </li><li> Aggiungere 2 ml di acqua deionizzata in ciascun pozzetto, parafilm la piastra per impedire l&#39;evaporazione dell&#39;acqua e posto a 37 ° C per 24 ore. Cambiare l&#39;acqua tre volte. </li><li> Visualizza formazione di pori sotto microscopio ottico (vedi esempio in Figura 2). Le impalcature sono ora pronti per l&#39;uso. </li></ol> Nota: Photocrosslinking non è necessario per molti tipi di polimeri, tuttavia photopatterning può essere utilizzato solo con polimeri fotoreattivo. C. doppio polimero Electrospinning con visualizzazione fluorescente Fibra <ol><li> Preparare una soluzione al 5% in peso di PEO (200 kDa) nel 90% di etanolo. Mescolate a 700 giri al minuto a 50 ° C per almeno 2 ore prima electrospinning. </li><li> Trasferire la soluzione PEO a una siringa e aggiungere DAPI per una concentrazione finale di 10 mg / mL concentrazione nella soluzione electrospinning. Avvolgere la siringa in un foglio di alluminio per proteggerlo dalla luce. </li><li> Preparare la stessa soluzione descritta in A2 dopo passo, ad eccezione di aggiungere anche methacryloxyethyl thiocarbamoyl rodamina B (MeRho, 683,24 g / mol) per una concentrazione finale di 25 mM nella soluzione electrospinning. Avvolgere la siringa in un foglio di alluminio per proteggerlo dalla luce. </li><li> Usare con attenzione nastro adesivo per fissare coprioggetto di vetro methacrylated (vedi Khademhosseini et al.) Alla superficie del mandrino. Nota: le fibre saranno legati a coprioggetto di vetro methacrylated. </li><li> Collegare un&#39;estremità di un pezzo di 5 mm di tubo in silicone con attacchi luer lock ad una delle siringhe e collegare l&#39;altra estremità ad un ago. Inserire la siringa nella pompa a siringa e inserire l&#39;ago attraverso il foro il contadino. Ripetere con l&#39;altra siringa e coltivatore sul lato opposto del mandrino. Il sventagliatori tradurre il llength del mandrino e vengono utilizzati per garantire un&#39;equa distribuzione di entrambi i polimeri della risultante patibolo. Vedi schema in Figura 3. </li><li> Regolare i parametri descritti nella sezione A appropriato in base alla tabella 1. Assicurarsi che le punte aghi sono centrate sul mandrino. Tabella 1. Doppio polimero Electrospinning Parametri <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> Siringa </td><td> Fanner alla punta dell&#39;ago (cm) </td><td> La punta dell&#39;ago a Mandrino (cm) </td><td> Portata (ml / ora) </td><td> Applicata tensione (kV) </td></tr><tr><td> Meha </td><td> 6 </td><td> 15 </td><td> 1,2 </td><td> 22 </td></tr><tr><td> PEO </td><td> 6 </td><td> 10 </td><td> 1,2 </td><td> 15 </td></tr></tbody></table> </li><li> Una volta che tutto è allineato correttamente, spegnere le luci e accendere il mandrino e le pompe siringa. Rimuovere il foglio di alluminio da siringhe. Quando il fluido è visibile sulla punta dei due aghi, contemporaneamente accendere l&#39;alimentazione e collegare il sventagliatori. </li><li> Quando la raccolta è completa, spegnere l&#39;alimentazione e mandrino e scollegare il sventagliatori. Rimuovere con attenzione i coprioggetti e nastro con una lama di rasoio. </li><li> Visualizza le fibre utilizzando un microscopio a fluorescenza dotato di filtri per la rodamina e DAPI. Vedi esempio in Figura 3. Nota: le fibre fluorescenti sono più chiare per vedere se elettrofilate per brevi periodi di tempo (&lt;3 minuti), tuttavia, questo processo può essere prolungato per un numero illimitato di tempo per creare spesse costrutti, soprattutto senza l&#39;uso del vetro coprioggetto . </li></ol> Cellule Semina il D. Ponteggi <ol><li> Posizionare ogni coprioggetto con elettrofilate polimero allegato in un individuo ben di una piastra di dimensioni appropriate bene. Nota: interazioni cellulari con le fibre sono facilmente visibili da electrospinning sul coprioggetto di vetro methacrylated. Inoltre, MeRho può ancora essere utilizzato per la visualizzazione delle fibre. </li><li> Incubare i ponteggi in PBS durante la notte per garantire la completa rimozione del solvente e qualsiasi rilasciabili possibili sottoprodotti. </li><li> Rimuovere la PBS dai pozzetti. </li><li> Per sterilizzare i ponteggi, li posto sotto una lampada germicida in una cappa a flusso laminare per 30 minuti. Se una completa patibolo è stato seminato, capovolgere il patibolo sopra e posto sotto una lampada germicida per altri 30 minuti. </li><li> Eseguire cultura cella standard e preparare una sospensione concentrata cella la densità cellulare desiderato (ad esempio, 6.000 cellule cm -2). Ad esempio, utilizzare un 100 ml di sospensione cellulare per un mm 22 X 22 coprioggetto mm. Posto in incubatrice per 1 ora. </li><li> Aggiungere la quantità appropriata di terreni di coltura di cellule in ciascun pozzetto. Posizionare i ponteggi nuovamente dentro l&#39;incubatrice. </li><li> Macchia le cellule utilizzando un kit disponibile in commercio Live / Dead. Nota: altri tipi di colorazione delle cellule, come il DAPI (nuclei cellulari) e fluorescente falloidina (fibre di actina di stress) possono essere applicati per visualizzare le cellule. </li><li> Visualizzare le cellule e le fibre utilizzando un microscopio a fluorescenza dotato di filtri per TRITC e FITC. Vedi esempio in Figura 4. </li></ol> Rappresentante dei risultati: <img src="/files/ftp_upload/1589/1589figure1tmb.jpg" border="0" alt="Figura 1" /> Figura 1. Schematica che illustra la configurazione del dispositivo per i non-allineati formazione ponteggio (in alto) e allineato la formazione di ponteggi (in basso). Esempio di microscopia elettronica a scansione di immagini di ogni tipo di ponteggio sono mostrati. Barra di scala = 5 micron. Si prega di <a href="https://www.jove.com/files/ftp_upload/1589/1589figure1.jpg">cliccare qui</a> per vedere una versione più grande della figura 1. <img src="/files/ftp_upload/1589/1589figure2tmb.jpg" border="0" alt="Figura 2" /> Figura 2. Schematica del metodo di patterning. I modelli sono formate mettendo un fotomaschera tra la sorgente di luce e impalcatura durante photocrosslinking e poi lavando via polimero non reagito. Immagine fotomaschera e SEM di ponteggi dopo la formazione dei pori e liofilizzazione. Barra di scala = 100 micron. Si prega di <a href="https://www.jove.com/files/ftp_upload/1589/1589figure2.jpg">cliccare qui</a> per vedere una versione più grande della figura 2. <img src="/files/ftp_upload/1589/1589figure3tmb.jpg" alt="Figura 3" border="0" /> Figura 3 Schema del multi-polimero configurazione elettrofilatura e parametri di lavorazione necessari, così come un&#39;immagine rappresentativa fluorescente di una miscela di due popolazioni di fibre (esempio: rosso e blu Meha PEO). Cui sono stati contemporaneamente elettrofilate per formare un multi-polimero ponteggio . Barra di scala = 100 micron. Si prega di <a href="https://www.jove.com/files/ftp_upload/1589/1589figure3.jpg">cliccare qui</a> per vedere una grande Version di figura 3. <img src="/files/ftp_upload/1589/1589figure4tmb.jpg" border="0" alt="Figura 4" /> Figura 4. Un esempio Live / Dead immagine colorazione delle cellule staminali mesenchimali e le loro interazioni con le fibre elettrofilate. Barra di scala = 100 micron. Si prega di <a href="https://www.jove.com/files/ftp_upload/1589/1589figure4.jpg">cliccare qui</a> per vedere una versione più grande della figura 4.

<ol>
	<li>Burdick, J. A., Chung, C., Jia, X., Randolf, M. A., 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=Controlled+degradation+and+mechanical+behavior+of+photopolymerized+hyaluronic+acid+networks.">Controlled degradation and mechanical behavior of photopolymerized hyaluronic acid networks.</a> Biomacromolecules. 6, 386-391 (2005).</li><li>Baker, B. M., Gee, A. O., Metter, R. B., Nathan, A. S., Marklein, R. A., Burdick, J. A., Mauck, L. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+potential+to+improve+cell+infiltration+in+composite+fiber-aligned+electrospun+scaffolds+by+the+selective+removal+of+sacrificial+fibers.">The potential to improve cell infiltration in composite fiber-aligned electrospun scaffolds by the selective removal of sacrificial fibers.</a> Biomaterials. 29, 2348-2358 (2008).</li><li>Ifkovits, J. L., Burdick, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review:+Photopolymerizable+and+degradable+biomaterials+for+tissue+engineering+applications.">Review: Photopolymerizable and degradable biomaterials for tissue engineering applications.</a> Tissue Engineering. 13, 2369-2385 (2007).</li><li>Khademhosseini, A., Eng, G., Yeh, J., Fukuda, J., Blumling, J., Langer, R., Burdick, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micromolding+of+photocrosslinkable+hyaluronic+acid+for+cell+encapsulation+and+entrapment.">Micromolding of photocrosslinkable hyaluronic acid for cell encapsulation and entrapment.</a> J. Biomed Mater Res A. 79A, 522-532 (2006).</li><li>Mauck, R. L., Baker, B. M., Nerurkar, N. L., Burdick, J. A., Li, W. J., Tuan, R. S., Elliott, D. M., M, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+on+the+Straight+and+Narrow:+The+Mechanics+of+Nanofibrous+Assemblies+for+Fiber-Reinforced+Tissue+Regeneration.">Engineering on the Straight and Narrow: The Mechanics of Nanofibrous Assemblies for Fiber-Reinforced Tissue Regeneration.</a> Tissue Engineering B. 15, 171-193 (2009).</li><li>Sill, T. J., Von Recum, H. a. v. o. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospinning:+applications+in+drug+delivery+and+tissue+engineering.">Electrospinning: applications in drug delivery and tissue engineering.</a> Biomaterials. 29, 1989-2006 (2008).</li><li>Sundararaghavan, H. G., Metter, R. B., Burdick, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospun+fibrous+scaffolds+with+multi-scale+and+photopatterned+porosity.">Electrospun fibrous scaffolds with multi-scale and photopatterned porosity.</a> , (2009).</li></ol>

Electrospinning utilizzata per preparare i ponteggi fibroso da polimeri. Photocrosslinkable ponteggi a base di acido ialuronico sono stati utilizzati a titolo esemplificativo, in cui è necessaria l&#39;esposizione alla luce per la reticolazione. Con l&#39;uso di macromers reattivo, ad esempio Meha, i canali che hanno già dimostrato una maggiore distribuzione cellulare sono stati incorporati nel ponteggi con l&#39;uso di una maschera durante photocrosslinking per formare impalcature macro e micro-porosa. Inoltre, due p...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>DAPI</td>
<td> Reagent</td>
<td>Invitrogen</td>
<td>D1306</td>
<td>&nbsp;</td>
<tr>
<td>I2959</td>
<td>Reagent</td>
<td>Ciba Specialty Chemicals</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>PEO 200 kDa</td>
<td>&nbsp;</td>
<td>Polysciences</td>
<td>17503</td>
<td>&nbsp;</td>
<tr>
<td>PEO 900 kDa</td>
<td>Reagent</td>
<td>Sigma</td>
<td>189456</td>
<td>&nbsp;</td>
<tr>
<td>Methacryloxethyl thiocarbamoyl rhodamine B</td>
<td>Reagent</td>
<td>Polysciences</td>
<td>23591-100</td>
<td>Prepare stock solution in DMSO</td>
</tr>
<tr>
<td>Live/Dead Stain Kit</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>L3224</td>
<td>Contains Calcein (stains live cells green) and ethidium homodime (stains red dead cells)</td>
</tr>
<tr>
<td>Syringe Pump</td>
<td>Equipment</td>
<td>KD Scientific</td>
<td>KDS100</td>
<td>Two are needed for dual polymer spinning</td>
</tr>
<tr>
<td>Power Source</td>
<td>Equipment</td>
<td>Gamma High Voltage</td>
<td>ES30P-5W</td>
<td>Two are needed for dual polymer spinning</td>
</tr>
<tr>
<td>Motor</td>
<td>Equipment</td>
<td>Triem Electric Motors, Inc</td>
<td>0132022-15</td>
<td>Must attach to a custom built mandrel</td>
</tr>
<tr>
<td>Tachometer</td>
<td>Equipment</td>
<td>Network Tool Warehouse</td>
<td>ESI-330</td>
<td>Use to monitor mandrel speed</td>
</tr>
<tr>
<td>Omnicure UV Spot Cure System with collimating adapter</td>
<td>Equipment</td>
<td>Exfo Life Sciences Division</td>
<td>S1000</td>
<td>&nbsp;</td>
<tr>
<td>Silicone Tubing</td>
<td>Equipment</td>
<td>McMaster-Carr</td>
<td>51135K151</td>
<td>&nbsp;</td>
<tr>
<td>Luer Lock Female Adapter</td>
<td>Equipment</td>
<td>McMaster-Carr</td>
<td>51525K293</td>
<td>&nbsp;</td>
<tr>
<td>Luer Lock Male Adapter</td>
<td>Equipment</td>
<td>McMaster-Carr</td>
<td>51525K143</td>
<td>&nbsp;</td>
<tr>
<td>Needles</td>
<td>Equipment</td>
<td>Fisher Scientific</td>
<td>14-825-16H</td>
<td>&nbsp;</td>
<tr>
<td>Coverslips</td>
<td>Equipment</td>
<td>Corning</td>
<td>2875-22</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

electrospinning fibrous polymer scaffolds for tissue engineering and cell culture

Poiché il campo dell&#39;ingegneria tissutale evolve, c&#39;è un&#39;enorme richiesta per produrre materiali più idonei e tecniche di lavorazione al fine di affrontare le esigenze (ad esempio, la meccanica e vascolarizzazione), di organi e tessuti più intricate. Electrospinning è una tecnica popolare per creare ponteggi fibroso che imitano l&#39;architettura e la scala di grandezza della matrice extracellulare nativa. Queste impalcature fibroso sono utili anche come substrato di coltura cellulare in quanto le fibre possono essere utilizzati per dirigere il comportamento cellulare, quali la differenziazione delle cellule staminali (vedi un&#39;esauriente recensione di Mauck<em&gt; Et al.</em&gt; E Sill<em&gt; Et al.</em&gt; Per maggiori informazioni). In questo articolo, si descrive il processo generale di elettrofilatura polimeri e come esempio, electrospin un acido ialuronico reattivo in grado di reticolazione con esposizione alla luce (vedi Ifkovits<em&gt; Et al.</em&gt; Per una rassegna di materiali photocrosslinkable). Abbiamo inoltre introdurre ulteriori capacità di elaborazione, come photopatterning e multi-polimero formazione patibolo. Photopatterning può essere usato per creare impalcature con canali e multi-scala per aumentare la porosità infiltrazione cellulare e distribuzione dei tessuti. Multi-polimero impalcature sono utili per sintonizzare meglio le proprietà (meccanica e del degrado) di un ponteggio, tra cui porosità su misura per infiltrazione cellulare. Inoltre, queste tecniche può essere esteso per includere una vasta gamma di polimeri e macromers reattiva per creare impalcature complesse che forniscono le indicazioni necessarie per lo sviluppo di costrutti di successo tessuto ingegnerizzato.

Watch this Scientific Journal Video about Electrospinning Fibrous Polymer Scaffolds for Tissue Engineering and Cell Culture at JoVE.com

Electrospinning Fibrous Polymer Scaffolds for Tissue Engineering and Cell Culture

Il processo di electrospinning polimeri per l&#39;ingegneria tissutale e colture cellulari è affrontato in questo articolo. In particolare, il electrospinning di macromers fotoreattivo con capacità di elaborazione aggiuntive di photopatterning e multi-polimero electrospinning è descritto.

Electrospinning Ponteggi polimeri fibrosi per l&#39;ingegneria tissutale e colture cellulari

As the field of tissue engineering evolves, there is a tremendous demand to produce more suitable materials and processing techniques in order to address ...

electrospinning-fibrous-polymer-scaffolds-for-tissue-engineering-cell

Research

JoVE Journal

Biology

21.4K Views.  University of Pennsylvania. Il processo di electrospinning polimeri per l'ingegneria tissutale e colture cellulari è affrontato in questo articolo. In particolare, il electrospinning di macromers fotoreattivo con capacità di elaborazione aggiuntive di photopatterning e multi-polimero electrospinning è descritto.

Video: Electrospinning Fibrous Polymer Scaffolds for Tissue Engineering and Cell Culture

Experimental Approaches to Tissue Engineering

Approcci sperimentali di ingegneria dei tessuti

Ricerca

Biologia

Micro-scale Engineering for Cell Biology

Micro-scala di Ingegneria di biologia cellulare

Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates tension and helps maintains cellular shape.  Although the actin cytoskeleton is a rigid structure, it is a dynamic structure that is constantly remodeling.  A number of proteins can bind to the actin cytoskeleton.  The binding of a particular protein to F-actin is often desired to support cell biological observations or to further understand dynamic processes due to remodeling of the actin cytoskeleton. The actin co-sedimentation assay is an in vitro assay routinely used to analyze the binding of specific proteins or protein domains with F-actin.  The basic principles of the assay involve an incubation of the protein of interest (full length or domain of) with F-actin, ultracentrifugation step to pellet F-actin and analysis of the protein co-sedimenting with F-actin.  Actin co-sedimentation assays can be designed accordingly to measure actin binding affinities and in competition assays.

Proteins bind to filamentous actin (F-actin) through distinct actin binding modules. In this video we demonstrate the procedure of actin co-sedimentation, which is an in vitro assay routinely used to analyze proteins or specific domains that bind F-actin.

Actina Co-sedimentazione di analisi, per l&#39;Analisi del Binding Protein a F-Actina

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates ...

Microfabrication of Chip-sized Scaffolds for Three-dimensional Cell cultivation

Using microfabrication technologies is a prerequisite to create scaffolds of reproducible geometry and constant quality for three-dimensional cell cultivation. These technologies offer a wide spectrum of advantages not only for manufacturing but also for different applications. The size and shape of formed cell clusters can be influenced by the exact and reproducible architecture of the microfabricated scaffold and, therefore, the diffusion path length of nutrients and gases can be controlled.1 This is unquestionably a useful tool to prevent apoptosis and necrosis of cells due to an insufficient nutrient and gas supply or removal of cellular metabolites.
Our polymer chip, called CellChip, has the outer dimensions of 2 x 2 cm with a central microstructured area. This area is subdivided into an array of up to 1156 microcontainers with a typical dimension of 300 m edge length for the cubic design (cp- or cf-chip) or of 300 m diameter and depth for the round design (r-chip).2
So far, hot embossing or micro injection moulding (in combination with subsequent laborious machining of the parts) was used for the fabrication of the microstructured chips. Basically, micro injection moulding is one of the only polymer based replication techniques that, up to now, is capable for mass production of polymer microstructures.3 However, both techniques have certain unwanted limitations due to the processing of a viscous polymer melt with the generation of very thin walls or integrated through holes. In case of the CellChip, thin bottom layers are necessary to perforate the polymer and provide small pores of defined size to supply cells with culture medium e.g. by microfluidic perfusion of the containers.
In order to overcome these limitations and to reduce the manufacturing costs we have developed a new microtechnical approach on the basis of a down-scaled thermoforming process. For the manufacturing of highly porous and thin walled polymer chips, we use a combination of heavy ion irradiation, microthermoforming and track etching. In this so called "SMART" process (Substrate Modification And Replication by Thermoforming) thin polymer films are irradiated with energetic heavy projectiles of several hundred MeV introducing so-called "latent tracks" Subsequently, the film in a rubber elastic state is formed into three dimensional parts without modifying or annealing the tracks. After the forming process, selective chemical etching finally converts the tracks into cylindrical pores of adjustable diameter.

We present two processes for the microfabrication of porous polymer chips for three-dimensional cell cultivation. The first one is hot embossing combined with a solvent vapour welding process. The second one uses a recently developed microthermoforming process combined with ion track technology leading to a significant simplification of manufacture.

Microfabbricazione di chip di dimensioni Ponteggi per tridimensionale coltura cellulare

Using microfabrication technologies is a prerequisite to create scaffolds of reproducible geometry and constant quality for three-dimensional cell ...

Interview: Bioreactors and Surfaced-Modified 3D-Scaffolds for Stem Cell Research

A Nature Editorial in 2003 asked the question "Good-bye, flat biology?" What does this question imply? In the past, many in vitro culture systems, mainly monolayer cultures, often suffered from the disadvantage that differentiated primary cells had a relatively short life-span and de-differentiated during culture. As a consequence, most of their organ-specific functions were lost rapidly. Thus, in order to reproduce better conditions for these cells in vitro, modifications and adaptations have been made to conventional monolayer cultures.
The last generation of CellChips -- micro-thermoformed containers -- a specific technology was developed, which offers the additional possibility to modify the whole surface of the 3D formed containers. This allows a surface-patterning on a submicron scale with distinct signalling molecules. Sensors and signal electrodes may be incorporated. Applications range from basic research in cell biology to toxicology and pharmacology. Using biodegradable polymers, clinical applications become a possibility. Furthermore, the last generation of micro-thermoformed chips has been optimized to allow for cheap mass production.

In the past many in vitro culture systems -- mainly monolayer cultures -- often suffered from the disadvantage that differentiated primary cells had a relatively short life-span and de-differentiated during culture. As a consequence, most of their organ-specific functions were lost rapidly. Thus, in order to reproduce better conditions for these cells in vitro, modifications and adaptations have been made to conventional monolayer cultures.

Intervista: Bioreattori e nobilitati-Modified 3D-Ponteggi per la ricerca sulle cellule staminali

A Nature Editorial in 2003 asked the question  "Good-bye, flat biology?"  What does this question imply? In the past, many in vitro culture systems,  ...

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering 

The 3D encapsulation of cells within hydrogels represents an increasingly important and popular technique for culturing cells and towards the development of constructs for tissue engineering. This environment better mimics what cells observe in vivo, compared to standard tissue culture, due to the tissue-like properties and 3D environment. Synthetic polymeric hydrogels are water-swollen networks that can be designed to be stable or to degrade through hydrolysis or proteolysis as new tissue is deposited by encapsulated cells. A wide variety of polymers have been explored for these applications, such as poly(ethylene glycol) and hyaluronic acid. Most commonly, the polymer is functionalized with reactive groups such as methacrylates or acrylates capable of undergoing crosslinking through various mechanisms. In the past decade, much progress has been made in engineering these microenvironments - e.g., via the physical or pendant covalent incorporation of biochemical cues - to improve viability and direct cellular phenotype, including the differentiation of encapsulated stem cells (Burdick et al.).
The following methods for the 3D encapsulation of cells have been optimized in our and other laboratories to maximize cytocompatibility and minimize the number of hydrogel processing steps. In the following protocols (see Figure 1 for an illustration of the procedure), it is assumed that functionalized polymers capable of undergoing crosslinking are already in hand; excellent reviews of polymer chemistry as applied to the field of tissue engineering may be found elsewhere (Burdick et al.) and these methods are compatible with a range of polymer types. Further, the Michael-type addition (see Lutolf et al.) and light-initiated free radical (see Elisseeff et al.) mechanisms focused on here constitute only a small portion of the reported crosslinking techniques. Mixed mode crosslinking, in which a portion of reactive groups is first consumed by addition crosslinking and followed by a radical mechanism, is another commonly used and powerful paradigm for directing the phenotype of encapsulated cells (Khetan et al., Salinas et al.).

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering

We present protocols for the 3-dimensional (3D) encapsulation of cells within synthetic hydrogels. The encapsulation procedure is outlined for two commonly used methods of crosslinking (michael-type addition and light-initiated free radical mechanisms), as well as a number of techniques for assessing encapsulated cell behavior.

Incapsulamento cellulare in idrogel 3D per l&#39;ingegneria tissutale

The 3D encapsulation of cells within hydrogels represents an increasingly important and popular technique for culturing cells and towards the development ...

Engineering Cell-permeable Protein

The protein transduction technique enables the direct delivery of biologically active material into mammalian cells [for review see 1,2]. For this one can make use of the translocating ability of so-called cell penetrating peptides (CPPs), also designated as protein transduction domains (PTDs). The TAT-CPP derived from the human immunodeficiency virus type 1 (HIV-1) Tat (trans-activator of transcription) protein has been widely used. The positively charged TAT promotes cell permeability thereby overcoming the barriers of the cellular membrane by endocytosis or/and direct membrane penetration2. In combination with a nuclear localization signal (NLS) fusion proteins are able to enter the nucleus exhibiting functionality. Our video presentation demonstrates, as an exemplification for the engineering of cell-permeable proteins, the construction, production and application of a cell-permeable version of the DNA-modifying enzyme Cre.
Cre is a site-specific recombinase that is able to recognize and recombine 34 base pair loxP sites in mammalian cells in vitro and in vivo. Therefore the Cre/loxP system is widely used to conditionally induce mutations in the genome of living cells3,4. The delivery of active Cre recombinase to cells, however, represents a limitation.
We describe the pSESAME vector system, which allows a direct insertion of the gene-of-interest and provides a platform to rapidly clone different domains and tags used within the vector in a convenient and standardized manner. Rearranging of the different tags has been shown to modify the biochemical properties of the fusion proteins providing a possibility to achieve higher yield and better solubility. We demonstrate how to express and purify recombinant cell-permeant proteins in and from E. coli. The functionality of the recombinant Cre protein is finally validated in cell culture by assessing its intracellular recombinase activity.

Protein transduction enables the direct delivery of biologically active proteins into cells. In contrast to conventional methods such as DNA transfection or viral transduction this non-invasive paradigm allows highly efficient cellular manipulation in a titratable manner circumventing cellular toxicity and the risk of oncogenic transformation by permanent genetic modification.

Ingegneria Cell-permeabile proteine

The protein transduction technique enables the direct delivery of biologically active material into mammalian cells [for review see 1,2]. For this one can ...

A System for Culturing Iris Pigment Epithelial Cells to Study Lens Regeneration in Newt

Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, heart, the jaw 1. Specifically, newts are unique for its lens regeneration capability. Upon lens removal, IPE cells of the dorsal iris transdifferentiate to lens cells and eventually form a new lens in about a month 2,3. This property of regeneration is never exhibited by the ventral iris cells. The regeneration potential of the iris cells can be studied by making transplants of the in vitro cultured IPE cells. For the culture, the dorsal and ventral iris cells are first isolated from the eye and cultured separately for a time period of 2 weeks (Figure 1). These cultured cells are reaggregated and implanted back to the newt eye. Past studies have shown that the dorsal reaggregate maintains its lens forming capacity whereas the ventral aggregate does not form a lens, recapitulating, thus the in vivo process (Figure 2) 4,5. This system of determining regeneration potential of dorsal and ventral iris cells is very useful in studying the role of genes and proteins involved in lens regeneration.

In newt, the lens regenerates always from the dorsal iris by transdifferentiation of the iris pigmented epithelial cells (IPEs). Here we describe a procedure to culture dorsal and ventral newt IPE cells and their implantation to the newt eye. The implanted cells are then studied by tissue sectioning and immunohistochemistry.

Un sistema per la coltura cellule epiteliali del pigmento Iris a studio Rigenerazione Lens Newt

Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, ...

Transduction of Human Cells with Polymer-complexed Ecotropic Lentivirus for Enhanced Biosafety

Stem and tumor cell biology studies often require viral transduction of human cells with known or suspected oncogenes, raising major safety issues for laboratory personnel. Pantropic lentiviruses, such as the commonly used VSV-G pseudotype, are a valuable tool for studying gene function because they can transduce many cell types, including non-dividing cells. However, researchers may wish to avoid production and centrifugation of pantropic viruses encoding oncogenes due to higher biosafety level handling requirements and safety issues. Several potent oncogenes, including c-Myc and SV40 large T antigen, are known to enhance production of induced pluripotent stem cells (iPSC). All other known iPSC-inducing genetic changes (OCT4, SOX2, KLF4, NANOG, LIN28, and p53 loss of function) also have links to cancer, making them of relatively high safety concern as well. 
While these cancer-related viruses are useful in studying cellular reprogramming and pluripotency, they must be used safely. To address these biosafety issues, we demonstrate a method for transduction of human cells with ecotropic lentivirus, with additional emphasis on reduced cost and convenient handling. We have produced ecotropic lentivirus with sufficiently high titer to transduce greater than 90% of receptor-expressing human cells exposed to the virus, validating the efficacy of this approach. 
Lentivirus is often concentrated by ultracentrifugation; however, this process takes several hours and can produce aerosols infectious to human biomedical researchers. As an alternative, viral particles can be more safely sedimented onto cells by complexation with chondroitin sulfate and polybrene (CS/PB). This technique increases the functional viral titer up to 3-fold in cells stably expressing murine retrovirus receptor, with negligible added time and cost. Transduction of human dermal fibroblasts (HDFs) is maximally enhanced using CS/PB concentrations approximately 4-fold lower than the optimal value previously reported for cancer cell lines, suggesting that polymer concentration should be titrated for the target cell type of interest. We therefore describe the use of methylthiazolyldiphenyl-tetrazolium bromide (MTT) to assay for polymer toxicity in a new cell type. We observe equivalent viability of HDFs after viral transduction using either polymer complexation or the standard dose of polybrene (PB, 6 &mu;g/ml), indicating minimal acute toxicity.
In this protocol, we describe the use of ecotropic lentivirus for overexpression of oncogenes in human cells, reducing biosafety risks and increasing the transduction rate. We also demonstrate the use of polymer complexation to enhance transduction while avoiding aerosol-forming centrifugation of viral particles.

Lentiviruses are a valuable research tool for exploring gene function; however, researchers may wish to avoid production of pantropic lentivirus encoding known or suspected oncogenes. As an alternative, we present a safer protocol for use of ecotropic lentivirus on human cells modified to express the ecotropic receptor mSlc7a1.

Trasduzione di cellule umane con Polymer-complessato Lentivirus Ecotropic per Enhanced biosicurezza

Stem and tumor cell biology studies often require viral transduction of human cells with known or suspected oncogenes, raising major safety issues for ...

Induction and Testing of Hypoxia in Cell Culture

Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells. This decrease of oxygen tension can be due to a reduced supply in oxygen (causes include insufficient blood vessel network, defective blood vessel, and anemia) or to an increased consumption of oxygen relative to the supply (caused by a sudden higher cell proliferation rate). Hypoxia can be physiologic or pathologic such as in solid cancers 1-3, rheumatoid arthritis, atherosclerosis etc&#x2026; Each tissues and cells have a different ability to adapt to this new condition. During hypoxia, hypoxia inducible factor alpha (HIF) is stabilized and regulates various genes such as those involved in angiogenesis or transport of oxygen 4. The stabilization of this protein is a hallmark of hypoxia, therefore detecting HIF is routinely used to screen for hypoxia 5-7. 
In this article, we propose two simple methods to induce hypoxia in mammalian cell cultures and simple tests to evaluate the hypoxic status of these cells.

Here we propose simple methods to induce hypoxia in cell cultures and simple tests to evaluate the hypoxic status of the cultures.

Induzione e test di ipossia in coltura cellulare

Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells.  This decrease of oxygen tension can be due to a reduced supply in ...