Gli autori desiderano ringraziare NSERC Discovery Grant 402462 &quot;scaffolds di ingegneria tessutale indotta per il controllo delle cellule staminali pluripotenti comportamento&quot;.

Note prima di iniziare il protocollo: Il fibrinogeno è una proteina del sangue derivato e formazione sulla sicurezza quindi opportuno deve essere completata prima manipolazione. Questo protocollo richiede 2 giorni per completare così il tempo delle culture staminali desiderati in modo appropriato per assicurare che siano pronti per la semina. In termini di calcolare la quantità di fibrinogeno da pesare, tre piastre da 35 mm di Petri di soluzione salina tamponata Tris (TBS, pH 7,4) contenente 110-130 mg di fibrinogeno sciolto in 3 ml di TBS sarà sufficiente a produrre 1 24 pozzetti contenente 400 ponteggi fibrina pl in ogni pozzetto. 1. Day One: Making soluzioni di fibrinogeno e la dialisi per tutta la notte <ol><li> Prendere fibrinogeno liofilizzato fuori dal frigorifero e lasciare riposare per 20 minuti per permettere di venire a temperatura ambiente. </li><li> Aggiungere 3 mL di soluzione salina tamponata Tris (TBS) a ciascuna capsula di Petri 35 millimetri. Ciascuna piastra di rendimenti fibrinogeno 2 a 3 ml di soluzione di fibrinogeno che vanno in concentrazione 12-20mg / ml e la quantità totale di fibrinogeno necessario può essere calcolato sulla base di queste approssimazioni. </li><li> Pesare circa 100-130 mg di fibrinogeno (Fg) e cospargere sulla superficie del presente TBS in ogni piatto. Attendere 5 minuti per il fibrinogeno per iniziare ad andare in soluzione. Coprire con coperchio capsula di Petri. </li><li> Incubare le piastre di Petri a 37 ° C per 2 ore per permettere il fibrinogeno di andare completamente nella soluzione TBS. </li><li> Dialisi tubo Wet (7000 MW tagliata) con TBS. Fold estremità inferiore della fine tubo e pinza chiusa con una fascetta dialisi. </li><li> Pipettare le soluzioni di fibrinogeno, dai piatti in tubi di dialisi. Ripiegare estremità superiore e bloccare chiuso con una fascetta dialisi. </li><li> Luogo dialisi tubo contenente la soluzione di fibrinogeno in un contenitore 4 di litro di TBS. Mix soluzione con piastra di agitazione impostato a bassa velocità (100 rpm). </li><li> Dializzare soluzione di pernottamento a mescolare piastra per almeno 12 ore. La soluzione TBS non deve essere cambiata in questo periodo di tempo. </li></ol&gt; 2. Giorno 2: Polimerizzazione di ponteggi di fibrina e la semina di cellule staminali in ponteggi Tutte le fasi descritte per tale processo deve essere eseguito in una cappa sterile coltura tissutale come questi scaffold saranno seminate con colture di cellule staminali. <ol><li> Rimuovere la soluzione di fibrinogeno dal tubo di dialisi e posto in un apposito tubo dimensionato conica (o 15 ml o 50 mL). </li><li> Fibrinogeno soluzione filtro con un filtro a siringa da 5,0 um per rimuovere le impurità grandi nella soluzione. A seconda del volume della soluzione, l&#39;utilizzo di più filtri può essere necessario elaborare l&#39;intera soluzione. </li><li> Filtro sterile con la soluzione di fibrinogeno da 0,22 micron filtro in un tubo conico 50mL. Usando una pipetta di dimensioni adeguate, determinare il volume della soluzione filtrata fibrinogeno da utilizzare in seguito quando fare calcoli. A seconda del volume della soluzione, l&#39;utilizzo di più filtri può essere necessario elaborare il entire soluzione. </li><li> Misurare assorbanza della soluzione di fibrinogeno a 280 nm usando uno spettrofotometro. Un fattore di diluizione 50 è spesso necessario per ottenere una assorbanza sotto 1. </li><li> Calcolare la concentrazione di proteine ​​presenti nella soluzione fibrinogeno utilizzando la seguente equazione: [Fibrinogeno in mg / ml] = (A 280 × diluizione Factor) 1,55 ÷ 1,55 dove è il coefficiente di estinzione a 280 A per il fibrinogeno umano 13. Avanti calcolare la quantità di TBS necessaria per diluire la concentrazione della soluzione di 11,1 mg / ml di fibrinogeno in base al volume iniziale. La concentrazione finale di proteine ​​nella ponteggi fibrina sarà di 10 mg / ml dopo la polimerizzazione. </li><li> Ottenere trombina sterile e soluzioni di cloruro di calcio. In primo luogo, aggiungere 15 ul di soluzione di trombina (concentrazione: 40 U / mL - concentrazione finale in scaffold sarà di 2 U / mL) a destra di ciascun pozzetto nella prima riga della piastra a 24 pozzetti. Successivamente, aggiungere 15 microlitri di 50 CHL calcio mMoride soluzione al lato sinistro di ciascun pozzetto. Infine, aggiungere 270 microlitri della soluzione di fibrinogeno alla piastra. Agitare piastra da lato a lato, poi scuotere posteriore a quella anteriore per garantire la miscelazione delle soluzioni. </li><li> Sia la riga contenente la miscela polimerizzare per 5 minuti a temperatura ambiente. Le impalcature diventano più opachi come polimerizzare. </li><li> Ripetere le fasi 2.6 e 2.7 fino al numero desiderato di ponteggi fibrina sono stati polimerizzati. </li><li> Dopo l&#39;ultimo 5 minuti di incubazione, porre le piastre all&#39;interno di un incubatore a 37 ° C per consentire la completa polimerizzazione ponteggi. </li><li> Dopo l&#39;incubazione uno ora è completa, il passo successivo è alle sementi al patibolo con corpi embrionali. Utilizzando un pipettemen 20 microlitri, selezionare un corpo individuale embrioide e posto al centro del patibolo fibrina. Verificare con il microscopio che ogni pozzetto contiene un unico corpo embrioide. </li><li> Aggiungere 5 pl di soluzione di trombina (concentrazione: 40 U / mL) e 5 microlitri di 50 mM cloruro di calciosoluzione su ciascun corpo embrioide seguito da 90 microlitri di soluzione di fibrinogeno ad ogni pozzetto. La soluzione deve formare una bolla che incapsula i corpi embrionali. </li><li> Luogo piastra posteriore in incubatore a 37 ° C per un&#39;ora supplementare per garantire la polimerizzazione. </li><li> Dopo l&#39;incubazione, aggiungere 1 mL dei mezzi appropriati di colture cellulari per ogni piatto ben luogo e indietro nel 37 ° C incubatore. </li></ol> 3. Risultati rappresentativi Figura 1 mostra una schematica vista laterale di un singolo pozzetto contenente il 3D fibrina sistema cultura scaffold seminati con un corpo embrioide. Figura 2A mostra immagini rappresentative di cellule staminali pluripotenti indotte topo in coltura su strati di fibroblasti embrionali di topo feeder. Queste cellule vengono quindi indotte a formare corpi embrionali, aggregati di cellule contenenti progenitori neurali, utilizzando uno 8 giorni retinoico protocollo di trattamento (Figura 2B) come previously descritto 14 mentre la Figura 3 mostra l&#39;aspetto di un IPS-derivato corpo embrioide dopo 3 giorni di coltura all&#39;interno di 3D ponteggi fibrina. Risultati simili sono stati ottenuti in precedenza con cellule staminali embrionali di topo 7. Inoltre, questo metodo è stato utilizzato per la cultura IPS-derivati ​​corpi embrionali prodotte utilizzando un protocollo diverso, con l&#39;acido retinoico e purmorphamine 15 all&#39;interno del 3D ponteggi fibrina, a dimostrazione della versatilità di questo sistema di coltura. <img alt="Figura 1." src="/files/ftp_upload/3641/3641fig1.jpg" /> Figura 1. Schematico che mostra la vista laterale di un singolo pozzetto contenente un 3D fibrina scaffold seminato con un corpo embrioide. <img alt="Figura 2." src="/files/ftp_upload/3641/3641fig2.jpg" /> Figura 2. Mouse staminali pluripotenti indotte (iPS), coltura cellulare e la differenziazione. Per differenziare queste cellule in neurfenotipi al, le cellule iPS sono coltivate in sospensione per la produzione di aggregati di cellule chiamate corpi embrionali (EBS). Questi EBs vengono poi trattate con acido retinoico di indurre il differenziamento neurale e questo protocollo è stato precedentemente usato per indurre il differenziamento neurale delle cellule staminali embrionali. A) indifferenziato topo cellule iPS colonia coltivati ​​su una embrionale strato di fibroblasti topo alimentatore. B) corpi embrionali derivate da cellule iPS topo in coltura in sospensione adottata il giorno 8 dopo trattamento con acido retinoico di indurre il differenziamento neurale. Barra della scala è di 100 micron. <img alt="Figura 3." src="/files/ftp_upload/3641/3641fig3.jpg" /> Risultati dell&#39;esempio Figura 3. Di un IPS topo embryoid corpo dopo 3 giorni di coltura all&#39;interno di uno scaffold 3D fibrina. Le cellule hanno cominciato a migrare e differenziarsi all&#39;interno del patibolo fibrina. Barra della scala è di 500 micron.

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	<li>Keung, A. J., Healy, K. E., Kumar, S., Schaffer, D. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biophysics+and+dynamics+of+natural+and+engineered+stem+cell+microenvironments.">Biophysics and dynamics of natural and engineered stem cell microenvironments.</a> Wiley Interdiscip. Rev. Syst. Biol. Med. 2, 49-64 (2010).</li><li>Willerth, S. M., Sakiyama-Elbert, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combining+stem+cells+and+biomaterial+scaffolds+for+constructing+tissues+and+cell+delivery.">Combining stem cells and biomaterial scaffolds for constructing tissues and cell delivery.</a> StemBook. , (2008).</li><li>Lee, J., Cuddihy, M. J., Kotov, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+cell+culture+matrices:+state+of+the+art.">Three-dimensional cell culture matrices: state of the art.</a> Tissue Eng. Part B Rev. 14, 61-86 (2008).</li><li>Ahmed, T. A., Dare, E. V., Hincke, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrin:+a+versatile+scaffold+for+tissue+engineering+applications.">Fibrin: a versatile scaffold for tissue engineering applications.</a> Tissue Eng. Part B Rev. 14, 199-215 (2008).</li><li>Breen, A., O'Brien, T., Pandit, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrin+as+a+delivery+system+for+therapeutic+drugs+and+biomolecules.">Fibrin as a delivery system for therapeutic drugs and biomolecules.</a> Tissue Eng. Part B Rev. 15, 201-214 (2009).</li><li>Willerth, S. M., Faxel, T. E., Gottlieb, D. I., Sakiyama-Elbert, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+soluble+growth+factors+on+embryonic+stem+cell+differentiation+inside+of+fibrin+scaffolds.">The effects of soluble growth factors on embryonic stem cell differentiation inside of fibrin scaffolds.</a> Stem Cells. 25, 2235-2244 (2007).</li><li>Willerth, S. M., Arendas, K. J., Gottlieb, D. I., Sakiyama-Elbert, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+fibrin+scaffolds+for+differentiation+of+murine+embryonic+stem+cells+into+neural+lineage+cells.">Optimization of fibrin scaffolds for differentiation of murine embryonic stem cells into neural lineage cells.</a> Biomaterials. 27, 5990-6003 (2006).</li><li>Gorodetsky, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrin+Microbeads+Loaded+with+Mesenchymal+Cells+Support+Their+Long-Term+Survival+While+Sealed+at+Room+Temperature.">Fibrin Microbeads Loaded with Mesenchymal Cells Support Their Long-Term Survival While Sealed at Room Temperature.</a> Tissue Eng. Part C Methods. , (2011).</li><li>Barsotti, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrin+acts+as+biomimetic+niche+inducing+both+differentiation+and+stem+cell+marker+expression+of+early+human+endothelial+progenitor+cells.">Fibrin acts as biomimetic niche inducing both differentiation and stem cell marker expression of early human endothelial progenitor cells.</a> Cell Prolif. 44, 33-48 (2011).</li><li>Park, J. S., Yang, H. N., Woo, D. G., Jeon, S. Y., Park, K. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chondrogenesis+of+human+mesenchymal+stem+cells+in+fibrin+constructs+evaluated+in+vitro+and+in+nude+mouse+and+rabbit+defects+models.">Chondrogenesis of human mesenchymal stem cells in fibrin constructs evaluated in vitro and in nude mouse and rabbit defects models.</a> Biomaterials. 32, 1495-1507 (2011).</li><li>Mooney, R., Tawil, B., Mahoney, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+fibrinogen+and+thrombin+concentrations+promote+neuronal+rather+than+glial+growth+when+primary+neural+cells+are+seeded+within+plasma-derived+fibrin+gels.">Specific fibrinogen and thrombin concentrations promote neuronal rather than glial growth when primary neural cells are seeded within plasma-derived fibrin gels.</a> Tissue Eng. Part A. 16, 1607-1619 (2010).</li><li>Liu, H., Collins, S. F., Suggs, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+culture+for+expansion+and+differentiation+of+mouse+embryonic+stem+cells.">Three-dimensional culture for expansion and differentiation of mouse embryonic stem cells.</a> Biomaterials. 27, 6004-6014 (2006).</li><li>Dellenback, R. J., Chien, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+extinction+coefficient+of+fibrinogen+from+man,+dog,+elephant,+sheep,+and+goat+at+280+mmu.">The extinction coefficient of fibrinogen from man, dog, elephant, sheep, and goat at 280 mmu.</a> Proc. Soc. Exp. Biol. Med. 134, 353-355 (1970).</li><li>Bain, G., Kitchens, D., Yao, M., Huettner, J. E., Gottlieb, D. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+stem+cells+express+neuronal+properties+in+vitro.">Embryonic stem cells express neuronal properties in vitro.</a> Dev. Biol. 168, 342-357 (1995).</li><li>Li, X. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+differentiation+of+ventral+spinal+progenitors+and+motor+neurons+from+human+embryonic+stem+cells+by+small+molecules.">Directed differentiation of ventral spinal progenitors and motor neurons from human embryonic stem cells by small molecules.</a> Stem Cells. 26, 886-893 (2008).</li><li>Lorentz, K. M., Kontos, S., Frey, P., Hubbell, 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=Engineered+aprotinin+for+improved+stability+of+fibrin+biomaterials.">Engineered aprotinin for improved stability of fibrin biomaterials.</a> Biomaterials. 32, 430-438 (2011).</li><li>Willerth, S. M., Rader, A., Sakiyama-Elbert, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+controlled+growth+factor+delivery+on+embryonic+stem+cell+differentiation+inside+fibrin+scaffolds.">The effect of controlled growth factor delivery on embryonic stem cell differentiation inside fibrin scaffolds.</a> Stem Cell Res. 1, 205-218 (2008).</li><li>Chen, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+improvement+of+focal+cerebral+ischemia+injury+by+subdural+transplantation+of+induced+pluripotent+stem+cells+with+fibrin+glue.">Functional improvement of focal cerebral ischemia injury by subdural transplantation of induced pluripotent stem cells with fibrin glue.</a> Stem Cells Dev. 19, 1757-1767 (2010).</li></ol>

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Questo protocollo dettagliato sopra fornisce un metodo per generare 3D ponteggi di fibrina per la coltura di cellule staminali pluripotenti, in particolare per embrionali di topo e cellule staminali pluripotenti indotte. Questo sistema biomateriale cultura 3D basato più accuratamente imita la nicchia delle cellule staminali trovato in vivo e, di conseguenza, può essere usata per selezionare segnali biologici per determinare i loro effetti sulla differenziazione delle cellule staminali 6. Le nostre ...

Attrezzatura necessaria: Bilancia analitica pH-metro Tissue incubatore (37 ° C, 5% CO 2) Mescolare piastra Spettrofotometro Sterile tessuto cappa cultura Tris soluzione salina tamponata (pH 7.4) (Need 4 L plus abbastanza per sciogliere fibrinogeno) 50 mM di soluzione di cloruro di calcio Sterili provette coniche (15 o 50 mL) 35 mm Scatole Petri Dialisi tubi (7.000 MW cutoff) Dialisi clip 5.0 micron filtri per siringa Confezionati singolarmente siringhe sterili 0,22 micron filtri Siringa 24 e piastre di coltura di tessuto <table border="1"><tbody><tr><td> Nome del reattivo </td><td> Azienda </td><td> Numero di catalogo </td><td> Comments </td></tr><tr><td> Fibrinogeno (umano) </td><td> Calbiocorlo </td><td> 341578 </td><td></td></tr><tr><td> Trombina (umana) </td><td> Sigma </td><td> T7009 </td><td></td></tr></tbody></table>

preparation of 3d fibrin scaffolds for stem cell culture applications

Cellule staminali si trovano in natura microambienti 3D in vivo, che sono spesso denominati nicchia cellule staminali 1. Coltura di cellule staminali all&#39;interno di 3D scaffold biomateriale fornisce un modo per riprodurre accuratamente questi microambienti, fornendo un vantaggio rispetto ai tradizionali metodi di coltura 2D utilizzando polistirene nonché un metodo per tessuti di ricambio ingegneria 2. Mentre 2D polistirene coltura tissutale è stato utilizzato per la maggior parte degli esperimenti di coltura cellulare, 3D ponteggi biomateriale può più strettamente replicare i microambienti trovate in vivo consentendo costituzione più accurata di polarità cella nell&#39;ambiente e possiede proprietà biochimiche e meccaniche simili a tessuti molli. 3 Una varietà di derivazione naturale e sintetico ponteggi biomateriale sono stati studiati come ambienti in 3D per sostenere la crescita delle cellule staminali. Mentre ponteggi sintetici possono essere sintetizzati per avere una maggiore range di proprietà meccaniche e chimiche e spesso hanno una maggiore riproducibilità, biomateriali naturali sono spesso composti di proteine ​​e polisaccaridi presenti nella matrice extracellulare e di conseguenza contengono siti di legame per l&#39;adesione cellulare e facilmente sostenere la coltura cellulare. Ponteggi fibrina, prodotto polimerizzando il fibrinogeno proteina ottenuta da plasma, sono stati ampiamente studiati per una varietà di applicazioni di ingegneria tissutale sia in vitro e in vivo 4. Tali ponteggi può essere modificata utilizzando una varietà di metodi per incorporare sistemi a rilascio controllato per fornire fattori terapeutici 5. Studi precedenti hanno dimostrato che tali ponteggi può essere utilizzato con successo per coltura di cellule staminali embrionali e questo scaffold sistema di coltura può essere usata per selezionare gli effetti di vari fattori di crescita sulla differenziazione delle cellule staminali seminate dentro 6,7. Questo protocollo dettagli il processo di polymerizing fibrina ponteggi da soluzioni fibrinogeno utilizzando l&#39;attività enzimatica della trombina. Il processo si fa in 2 giorni per completare, compresa una fase di dialisi per tutta la notte per la soluzione del fibrinogeno per rimuovere citrati che inibiscono la polimerizzazione. Questi metodi dettagliati contare su concentrazioni di fibrinogeno determinati a essere ottimale per la coltura embrionale e indotto cellule staminali pluripotenti. Altri gruppi hanno ulteriormente investigato ponteggi fibrina per una vasta gamma di tipi di cellule e applicazioni - dimostrare la versatilità di questo approccio 8-12.

Watch this Scientific Journal Video about Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications at JoVE.com

Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications

Questi i dettagli del lavoro per la preparazione di fibrina scaffold 3D per la coltura e differenziazione delle cellule staminali plutipotent. Tali ponteggi può essere usata per selezionare gli effetti di vari composti biologici sul comportamento cellule staminali nonché modificata per contenere sistemi di drug delivery.

Preparazione del 3D fibrina ponteggi per le applicazioni delle cellule staminali Cultura

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells ...

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Bioengineering

25.1K Views.  University of Victoria. Questi i dettagli del lavoro per la preparazione di fibrina scaffold 3D per la coltura e differenziazione delle cellule staminali plutipotent. Tali ponteggi può essere usata per selezionare gli effetti di vari composti biologici sul comportamento cellule staminali nonché modificata per contenere sistemi di drug delivery.

Video: Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

Ponteggi self-reporting per coltura cellulare 3-Dimensional

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

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Bioingegneria

Sample Preparation Strategies for Mass Spectrometry Imaging of 3D Cell Culture Models

Three dimensional cell cultures are attractive models for biological research. They combine the flexibility and cost-effectiveness of cell culture with some of the spatial and molecular complexity of tissue. For example, many cell lines form 3D structures given appropriate in vitro conditions. Colon cancer cell lines form 3D cell culture spheroids, in vitro mimics of avascular tumor nodules. While immunohistochemistry and other classical imaging methods are popular for monitoring the distribution of specific analytes, mass spectrometric imaging examines the distribution of classes of molecules in an unbiased fashion. While MALDI mass spectrometric imaging was originally developed to interrogate samples obtained from humans or animal models, this report describes the analysis of in vitro three dimensional cell cultures, including improvements in sample preparation strategies. Herein is described methods for growth, harvesting, sectioning, washing, and analysis of 3D cell cultures via matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) imaging. Using colon carcinoma 3D cell cultures as a model system, this protocol demonstrates the ability to monitor analytes in an unbiased fashion across the 3D cell culture system with MALDI-MSI.

Immortalized cancer cell lines can be grown as 3D cell cultures, a valuable model for biological research. This protocol describes mass spectrometry imaging of 3D cell cultures, including improvements in the sample preparation platform. The goal of this protocol is to instruct users to prepare 3D cell cultures for mass spectrometry imaging analysis.

Campione strategie di preparazione per la Spettrometria di Massa Imaging di 3D Models Cultura cellulare

Three dimensional cell cultures are attractive models for biological research. They combine the flexibility and cost-effectiveness of cell culture with ...

3D Hydrogel Scaffolds for Articular Chondrocyte Culture and Cartilage Generation

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer cartilage tissue offer a promising solution to repair articular cartilage. To select the optimal cell source for tissue repair, it is important to develop an appropriate culture platform to systematically examine the biological and biomechanical differences in the tissue-engineered cartilage by different cell sources. Here we applied a three-dimensional (3D) biomimetic hydrogel culture platform to systematically examine cartilage regeneration potential of juvenile, adult, and osteoarthritic (OA) chondrocytes. The 3D biomimetic hydrogel consisted of synthetic component poly(ethylene glycol) and bioactive component chondroitin sulfate, which provides a physiologically relevant microenvironment for in vitro culture of chondrocytes. In addition, the scaffold may be potentially used for cell delivery for cartilage repair in vivo. Cartilage tissue engineered in the scaffold can be evaluated using quantitative gene expression, immunofluorescence staining, biochemical assays, and mechanical testing. Utilizing these outcomes, we were able to characterize the differential regenerative potential of chondrocytes of varying age, both at the gene expression level and in the biochemical and biomechanical properties of the engineered cartilage tissue. The 3D culture model could be applied to investigate the molecular and functional differences among chondrocytes and progenitor cells from different stages of normal or aberrant development.

Cartilage repair represents an unmet medical challenge and cell-based approaches to engineer human articular cartilage are a promising solution. Here, we describe three-dimensional (3D) biomimetic hydrogels as an ideal tool for the expansion and maturation of human articular chondrocytes.

3D Hydrogel Ponteggi per articolare condrociti, Cultura e Cartilagine Generation

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer ...

Fabrication of Mechanically Tunable and Bioactive Metal Scaffolds for Biomedical Applications

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness and low bioactivity of metals. In this study, we have developed a new method towards fabricating a new type of bioactive and mechanically reliable porous metal scaffolds-densified porous Ti scaffolds. The method consists of two fabrication processes, 1) the fabrication of porous Ti scaffolds by dynamic freeze casting, and 2) coating and densification of the porous scaffolds. The dynamic freeze casting method to fabricate porous Ti scaffolds allowed the densification of porous scaffolds by minimizing the chemical contamination and structural defects. The densification process is distinctive for three reasons. First, the densification process is simple, because it requires a control of only one parameter (degree of densification). Second, it is effective, as it achieves mechanical enhancement and sustainable release of biomolecules from porous scaffolds. Third, it has broad applications, as it is also applicable to the fabrication of functionally graded porous scaffolds by spatially varied strain during densification.

Bioactive and mechanically reliable metal scaffolds have been fabricated through a method which consists of two processes, dynamic freeze casting for the fabrication of porous Ti, and coating and densification of the Ti scaffolds. The densification process is simple, effective and applicable to the fabrication of functionally graded scaffolds.

Fabbricazione di meccanicamente Tunable e Bioactive metallo Ponteggi per applicazioni biomediche

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness ...

Micropatterning and Assembly of 3D Microvessels

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass based substrates, and hydrogel-based tube formation assays. These assays, while informative, do not recapitulate lumen geometry, proper extracellular matrix, and multi-cellular proximity, which play key roles in modulating vascular function. This manuscript describes an injection molding method to generate engineered vessels with diameters on the order of 100 &#181;m. Microvessels are fabricated by seeding endothelial cells in a microfluidic channel embedded within a native type I collagen hydrogel. By incorporating parenchymal cells within the collagen matrix prior to channel formation, specific tissue microenvironments can be modeled and studied. Additional modulations of hydrodynamic properties and media composition allow for control of complex vascular function within the desired microenvironment. This platform allows for the study of perivascular cell recruitment, blood-endothelium interactions, flow response, and tissue-microvascular interactions. Engineered microvessels offer the ability to isolate the influence from individual components of a vascular niche and precisely control its chemical, mechanical, and biological properties to study vascular biology in both health and disease.

This manuscript presents an injection molding method to engineer microvessels that recapitulate physiological properties of endothelium. The microfluidic-based process creates patent 3D vascular networks with tailorable conditions, such as flow, cellular composition, geometry, and biochemical gradients. The fabrication process and examples of potential applications are described.

Micropatterning e montaggio dei microvasi 3D

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass ...

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 ...

Synthesis of Biocompatible Liquid Crystal Elastomer Foams as Cell Scaffolds for 3D Spatial Cell Cultures

Here, we present a step-by-step preparation of a 3D, biodegradable, foam-like cell scaffold. These scaffolds were prepared by cross-linking star block co-polymers featuring cholesterol units as side-chain pendant groups, resulting in smectic-A (SmA) liquid crystal elastomers (LCEs). Foam-like scaffolds, prepared using metal templates, feature interconnected microchannels, making them suitable as 3D cell culture scaffolds. The combined properties of the regular structure of the metal foam and of the elastomer result in a 3D cell scaffold that promotes not only higher cell proliferation compared to conventional porous templated films, but also better management of mass transport (i.e., nutrients, gases, waste, etc.). The nature of the metal template allows for the easy manipulation of foam shapes (i.e., rolls or films) and for the preparation of scaffolds of different pore sizes for different cell studies while preserving the interconnected porous nature of the template. The etching process does not affect the chemistry of the elastomers, preserving their biocompatible and biodegradable nature. We show that these smectic LCEs, when grown for extensive time periods, enable the study of clinically relevant and complex tissue constructs while promoting the growth and proliferation of cells.

This study presents a methodology to prepare 3D, biodegradable, foam-like cell scaffolds based on biocompatible side-chain liquid crystal elastomers (LCEs). Confocal microscopy experiments show that foam-like LCEs allow for cell attachment, proliferation, and the spontaneous alignment of C2C12s myoblasts.

Sintesi di biocompatibili a cristalli liquidi in elastomero Schiume come impalcature cellulari per 3D spaziali colture cellulari

Here, we present a step-by-step preparation of a 3D, biodegradable, foam-like cell scaffold. These scaffolds were prepared by cross-linking star block ...

Construction of a Multilayered Mesenchymal Stem Cell Sheet with a 3D Dynamic Culture System

Stem cell therapy shows a promising future in regenerating injured organ and tissues, and the cell sheet technique has been developed to improve the low cell retention and poor survival within the target zone. However, during the in vitro construction process, a solution for maintaining stem cell bioactivity and increasing the cell amount within the cell sheet is urgently needed. Here, this protocol presents a method for constructing a multilayered cell sheet with favorable stem cell bioactivity and optimal operability. Decellularized porcine pericardium (DPP) is prepared by phospholipase A2 (PLA2) decellularization method as the cell sheet scaffold, and rat bone marrow mesenchymal stem cells (BMSCs) are isolated and expanded as the seeded cells. The temporary multilayered cell sheet structure is constructed by using RAD16-I peptide hydrogel. Finally, the cell sheet is cultured with a dynamic perfusion system to stabilize the three-dimensional (3D) structure, and the cell sheet could be obtained following a 48-hour culture in vitro. This protocol provides an efficient and feasible method for constructing a multilayered stem cell sheet, and the cell sheet could be developed as a favorable stem cell therapy product in the future.

This article provides an efficient and feasible method for constructing multilayered stem cell sheets with favorable stem cell property.

Costruzione di un foglio di multistrato delle cellule staminali mesenchimali con un sistema di cultura dinamica 3D

Stem cell therapy shows a promising future in regenerating injured organ and tissues, and the cell sheet technique has been developed to improve the low ...

A Culture Method to Maintain Quiescent Human Hematopoietic Stem Cells

Human hematopoietic stem cells (HSCs), like other mammalian HSCs, maintain lifelong hematopoiesis in the bone marrow. HSCs remain quiescent in vivo, unlike more differentiated progenitors, and enter the cell cycle rapidly after chemotherapy or irradiation to treat bone marrow injury or in vitro culture. By mimicking the bone marrow microenvironment in the presence of abundant fatty acids, cholesterol, low concentration of cytokines, and hypoxia, human HSCs maintain quiescence in vitro. Here, a detailed protocol for maintaining functional HSCs in the quiescent state in vitro is described. This method will enable studies of the behavior of human HSCs under physiological conditions.

This protocol enables the maintenance of quiescent human hematopoietic stem cells in vitro by mimicking the bone marrow microenvironment using abundant fatty acids, cholesterol, lower concentrations of cytokines, and hypoxia.

Un metodo di coltura per mantenere le cellule staminali ematopoietiche umane quiescenti

Human hematopoietic stem cells (HSCs), like other mammalian HSCs, maintain lifelong hematopoiesis in the bone marrow. HSCs remain quiescent in vivo, ...

Preparation and Characterization of Graphene-Based 3D Biohybrid Hydrogel Bioink for Peripheral Neuroengineering

Peripheral neuropathies can occur as a result of axonal damage, and occasionally due to demyelinating diseases. Peripheral nerve damage is a global problem that occurs in 1.5%-5% of emergency patients and may lead to significant job losses. Today, tissue engineering-based approaches, consisting of scaffolds, appropriate cell lines, and biosignals, have become more applicable with the development of three-dimensional (3D) bioprinting technologies. The combination of various hydrogel biomaterials with stem cells, exosomes, or bio-signaling molecules is frequently studied to overcome the existing problems in peripheral nerve regeneration. Accordingly, the production of injectable systems, such as hydrogels, or implantable conduit structures formed by various bioprinting methods has gained importance in peripheral neuro-engineering. Under normal conditions, stem cells are the regenerative cells of the body, and their number and functions do not decrease with time to protect their populations; these are not specialized cells but can differentiate upon appropriate stimulation in response to injury. The stem cell system is under the influence of its microenvironment, called the stem cell niche. In peripheral nerve injuries, especially in neurotmesis, this microenvironment cannot be fully rescued even after surgically binding severed nerve endings together. The composite biomaterials and combined cellular therapies approach increases the functionality and applicability of materials in terms of various properties such as biodegradability, biocompatibility, and processability. Accordingly, this study aims to demonstrate the preparation and use of graphene-based biohybrid hydrogel patterning and to examine the differentiation efficiency of stem cells into nerve cells, which can be an effective solution in nerve regeneration.

In this manuscript, we demonstrate the preparation of a biohybrid hydrogel bioink containing graphene for use in peripheral tissue engineering. Using this 3D biohybrid material, the neural differentiation protocol of stem cells is performed. This can be an important step in bringing similar biomaterials to the clinic.

Preparazione e caratterizzazione di bioink idrogel bioibrido 3D a base di grafene per la neuroingegneria periferica

Peripheral neuropathies can occur as a result of axonal damage, and occasionally due to demyelinating diseases. Peripheral nerve damage is a global ...