Questo lavoro è stato condotto utilizzando le strutture del Dipartimento di Ingegneria Biomedica presso la Florida International University.

1. Setup Electrospinning Collector <ol><li> Tagliare il foglio di alluminio in un pezzo rettangolare. </li><li> Piegare il pezzo rettangolare in una striscia rettangolare, e allegarlo perpendicolare ad una piastra metallica piana con nastro (Figura 1). Nota: La dimensione del tappeto di fibre dipende dalla lunghezza e la larghezza della striscia. Pertanto, le dimensioni strip possono essere regolate come necessario. </li></ol> 2. Polimerico Solution Preparazione <ol><li> Mescolare glicerolo e acido dodecanodioico (DDA) in rapporto 1:1 molare in un becher a 120 ° C per 100 ore per ottenere polimero PGD. </li><li> Sciogliere poli (ossido di etilene) (PEO) e gelatina in 65% di etanolo, con un rapporto in peso di 1.5:3:95.5 in una provetta da 15 ml, serrare il tappo e scaldare la miscela in un forno a 60 ° C per 1 ora sotto agitazione fino ad ottenere una soluzione omogenea (soluzione Basal). </li><li> Per elettrofilatura, mescolare il polimero PGD e la soluzione basale in 04:06 rapporto di peso. Nota: concentrazione PGD ha un big effetto sul diametro delle fibre. 30% -50% per cento PGD è accettabile per la produzione di fibre di lunghezza superiore a 5 cm, con un aumento dei diametri delle fibre. </li><li> Aggiungere 0,1% riboflavina alla soluzione polimerica e mescolare bene. </li></ol> 3. Electrospinning <ol><li> Alimentare la soluzione polimerica in una siringa da 5 ml di serie con un 18 G smussati ago in acciaio inossidabile. </li><li> Inserire la siringa nella pompa a siringa. </li><li> Fissare il cavo di messa a terra di una sorgente di alimentazione ad alta tensione per la piastra metallica e il conduttore carica positiva all&#39;ago. </li><li> Regolare la distanza tra l&#39;ago e la striscia di foglio di alluminio a 15 cm. </li><li> Posizionare la pompa a siringa in un angolo di circa 15 ° con l&#39;orizzontale per prevenire l&#39;aggregazione delle fibre nella parte anteriore della striscia. </li><li> Accendere la pompa a siringa e regolare la portata della pompa di 0,6 ml / hr. </li><li> Accendere la fonte di alimentazione ad alta tensione e impostare la tensione di esercizio a 14,6 kV. </li></ol> 4.Fiber Processing <ol><li> Dopo la collezione è completa, esporre il tappeto di fibre di luce UV per 60 minuti per la reticolazione. </li><li> Trasferire il tappeto di fibre dalla striscia di foglio di alluminio per una capsula di Petri 100 mm, ed esporlo alla luce UV per altri 20 minuti per la sterilizzazione. </li><li> In un armadio biosicurezza, tagliare il tappeto di fibre in pezzi rotondi della stessa dimensione con una lama chirurgica e disporre i pezzi in una piastra da 24 pozzetti. </li><li> Per cellula di trattamento pre-semina, immergere campioni di fibre in 1 ml di tampone fosfato (PBS) e incubare a 37 ° C per una notte. </li><li> Il giorno seguente, aspirare PBS con attenzione, aggiungere 1 ml di terreno di differenziamento (DMEM/F12, N2, e FGF2) (vedi ricetta in tabella materiali) in ciascun pozzetto ed incubare a 37 ° C per 3 ore. </li><li> Aspirare il terreno di differenziamento accuratamente, aggiungere 0,2 ml di matrigel a ciascun campione fibra e incubare a 37 ° C per 30 min. Nota: laminina può essere utilizzato anche per il rivestimento di fibra. Aggiungere 0,2 ml di 20 mg / ml lamininaalla fibra e incubare a temperatura ambiente per 3 ore. </li><li> Rimuovere l&#39;eccesso di matrigel da ogni pozzetto. Lavare con 2 ml di terreno di differenziazione volta. Nota: I campioni di fibre sono pronti per la coltura cellulare. </li></ol> 5. Semina delle cellule su fibre <ol><li> Aggiungere 1 ml di Accutase alla cella piatto cultura MES e incubare per 10 min a 37 ° C. </li><li> Dopo 10 minuti, le cellule MES sono staccate dalla piastra di coltura. Aggiungere 4 ml di terreno di differenziamento alla piastra. Raccogliere le cellule MES galleggianti in una provetta da 15 ml e le cellule pipetta su e giù per rompere colonie (circa 15x). </li><li> Centrifugare a 400 xg per 5 min e risospendere le cellule in 4 ml di terreno di differenziamento. </li><li> Contare le cellule utilizzando un emocitometro. Trasferire 200 microlitri della sospensione cellulare in una provetta da 15 ml e diluire 10x con terreno di differenziamento. Trasferire 15 ml di sospensione cellulare diluita ad una camera sul emocitometro con una copertura antiscivolo in posto. Contarele celle nel centro di piazza 1 mm e le quattro piazze d&#39;angolo del emocitometro sotto il microscopio. Nota: Cell per ml = conteggio medio per quadrato x fattore di diluizione x 10 4 </li><li> Porre circa 5 x 10 4 cellule MES su ogni bene. Lentamente cadere la sospensione cellulare in mezzo ai campioni di fibre, invece di scivolare fuori al lato del pozzo, per evitare che la soluzione di drenaggio tappeto di fibre. </li><li> Aggiungere 1 ml di terreno di differenziamento in ciascun pozzetto, e mantenere la piastra in un incubatore a 37 ° C e 5% di CO 2 per consentire cellule attaccamento, crescita e differenziazione. </li><li> Aspirare il vecchio mezzo da ciascun pozzetto e sostituirlo con 1 ml di terreno di differenziamento fresco ogni altro giorno. </li></ol> 6. Vitalità cellulare <ol><li> Aspirare il vecchio mezzo da ogni pozzetto. </li><li> Mescolare 1/10 del volume di resazurina reagente fluorescenza con terreno di coltura e aggiungere 1 ml in ogni pozzetto. </li><li> Incubmangiato a 37 ° C e 5% di CO 2 per 4 ore, al riparo dalla luce diretta. </li><li> Dopo 4 ore, trasferire 100 microlitri 3x del reagente da ciascun pozzetto di una piastra da 96 pozzetti, e poi i campioni sono pronti per essere misurata mediante uno spettrofotometro a fluorescenza usando impostazioni di filtro 560EX nm/590EM. </li></ol> 7. Real Time PCR <ol><li> Dopo 14 giorni di coltura, aggiungere tampone di lisi per campioni e isolare l&#39;RNA totale da cellule sui campioni di fibre. </li><li> Utilizzare 4 pl di RNA totale per sintetizzare cDNA in 20 microlitri di reazione scale mediante trascrizione inversa. </li><li> Preparare la miscela di reazione PCR in ciascun tubo ottico: 10 microlitri Master Mix (2x), 0,2 ml di colorante, 8 microlitri di acqua priva di nucleasi, 1 ml cDNA, e 1 ml di fondo (primer utilizzati in questo studio sono elencati nella Tabella 1). </li><li> Impostare il programma PCR: a. 95 ° C 2:20 min, 1 ciclo b. 95 ° C 3 sec → 60 ° C 1 min, 40 ciclo c. 95 ° C 15 sec, 1 ciclo d. 60 ° C 1min, 1 ciclo e. 95 ° C 15 sec, 1 ciclo. Scegli il metodo comparativo C T per determinare i livelli di espressione genica relativa. </li><li> Dopo la PCR è finito, analizzare i real-time PCR risultati con il software StepOne ed esportare i risultati in Excel. </li></ol>

<ol>
	<li>Migneco, F., Huang, Y. -. C., Birla, R. K., Hollister, 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=Poly+(glycerol-dodecanoate),+a+biodegradable+polyester+for+medical+devices+and+tissue+engineering+scaffolds.">Poly (glycerol-dodecanoate), a biodegradable polyester for medical devices and tissue engineering scaffolds.</a> Biomaterials. 30, 6479-6484 (2009).</li><li>Reneker, D. H., Yarin, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospinning+jets+and+polymer+nanofibers.">Electrospinning jets and polymer nanofibers.</a> Polymer. 49, 2387-2425 (2008).</li><li>Wang, Y., Ameer, G. A., Sheppard, B. J., 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=A+tough+biodegradable+elastomer.">A tough biodegradable elastomer.</a> Nature biotechnology. 20, 602-606 (2002).</li><li>Panunzi, S., De Gaetano, A., Mingrone, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Approximate+linear+confidence+and+curvature+of+a+kinetic+model+of+dodecanedioic+acid+in+humans.">Approximate linear confidence and curvature of a kinetic model of dodecanedioic acid in humans.</a> American Journal of Physiology-Endocrinology And Metabolism. 289, (2005).</li><li>Park, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apparatus+for+preparing+electrospun+nanofibers:+designing+an+electrospinning+process+for+nanofiber+fabrication.">Apparatus for preparing electrospun nanofibers: designing an electrospinning process for nanofiber fabrication.</a> Polymer Internationa l. 56, 1361-1366 (2007).</li><li>Barnes, C. P., Sell, S. A., Boland, E. D., Simpson, D. G., Bowlin, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanofiber+technology:+designing+the+next+generation+of+tissue+engineering+scaffolds.">Nanofiber technology: designing the next generation of tissue engineering scaffolds.</a> Advanced drug delivery reviews. 59, 1413-1433 (2007).</li><li>Li, W. -. J., Mauck, R. L., Tuan, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospun+nanofibrous+scaffolds:+production,+characterization,+and+applications+for+tissue+engineering+and+drug+delivery.">Electrospun nanofibrous scaffolds: production, characterization, and applications for tissue engineering and drug delivery.</a> Journal of Biomedical Nanotechnology. 1, 259-275 (2005).</li><li>Pham, Q. P., Sharma, U., Mikos, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospinning+of+polymeric+nanofibers+for+tissue+engineering+applications:+a+review.">Electrospinning of polymeric nanofibers for tissue engineering applications: a review.</a> Tissue engineering. 12, 1197-1211 (2006).</li><li>Lim, S. H., Mao, H. -. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospun+scaffolds+for+stem+cell+engineering.">Electrospun scaffolds for stem cell engineering.</a> Advanced drug delivery reviews. 61, 1084-1096 (2009).</li><li>Lowery, J. L., Datta, N., Rutledge, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+fiber+diameter,+pore+size+and+seeding+method+on+growth+of+human+dermal+fibroblasts+in+electrospun+poly+(epsilon-caprolactone)+fibrous+mats.">Effect of fiber diameter, pore size and seeding method on growth of human dermal fibroblasts in electrospun poly (epsilon-caprolactone) fibrous mats.</a> Biomaterials. 31, 491-504 (2010).</li><li>Tillman, B. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+in+vivo+stability+of+electrospun+polycaprolactone-collagen+scaffolds+in+vascular+reconstruction.">The in vivo stability of electrospun polycaprolactone-collagen scaffolds in vascular reconstruction.</a> Biomaterials. 30, 583-588 (2009).</li><li>Ju, Y. M., Choi, J. S., Atala, A., Yoo, J. J., Lee, 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=Bilayered+scaffold+for+engineering+cellularized+blood+vessels.">Bilayered scaffold for engineering cellularized blood vessels.</a> Biomaterials. 31, 4313-4321 (2010).</li><li>McCullen, S. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+collagen+polymerization+of+layered+cell-seeded+electrospun+scaffolds+for+bone+tissue+engineering+applications.">In situ collagen polymerization of layered cell-seeded electrospun scaffolds for bone tissue engineering applications.</a> Tissue Engineering Part C: Methods. 16, 1095-1105 (2010).</li></ol>

Nessun conflitto di interessi dichiarati.

Le limitazioni di collettori semplici o la complessità di collettori che sono attualmente utilizzati per elettrospinning aumentare la restrizione di ottenere la lunghezza desiderata e dimensione del tappeto di fibre per alcune applicazioni rotanti. Inoltre, il trasferimento delle fibre dal collettore di terra alla piastra di coltura o altri substrati è una sfida 5. In questo rapporto, un collezionista di nuova concezione, realizzato semplicemente collegando una striscia di foglio di alluminio al collettore ...

Electrospinning è uno dei metodi di trattamento efficaci per produrre scaffold fibre dimensioni micro-to-nanometri. Il principio di base di elettrospinning comporta un cono di Taylor soluzione che viene tenuta in corrispondenza dell&#39;orifizio di un ago applicando alta tensione tra la punta dell&#39;ago e un collettore a terra. Quando la repulsione elettrostatica nella soluzione supera la tensione superficiale, un getto di fluido caricato viene espulso dalla punta dell&#39;ago, viaggia attraverso l&#39;aria con evaporazione del solvente, ed è infine depositato sul collettore a terra. La pompa a siringa fornisce un flusso continuo di soluzione che emerge dalla filiera e quindi più copie delle fibre elettrofilate può essere fabbricato in un breve periodo di tempo. Nel corso del lasciando la filiera per arrivare al collettore, il getto carica subirà stretching e frustate secondo un certo numero di parametri che includono la viscosità e la tensione superficiale della soluzione polimerica, i electrostatic forza nella soluzione, e l&#39;interazione del campo elettrico esterno, ecc 2. Nel processo electrospinning, un collettore funge da substrato conduttore dove si possono depositare le fibre micro-to-nanometri. In questo studio, un nuovo tipo di collettore fibra creata avere stuoie in fibra con la dimensione desiderata (lunghezza x larghezza). Tradizionalmente, un foglio di alluminio è usato come collettore ma è difficile trasferire le fibre dalla superficie piana per un altro substrato. La difficoltà di raccolta un feltro di fibre intatte da un collettore tradizionale è dovuto principalmente al fatto che le fibre elettrofilate attaccano fortemente alla superficie del collettore. Pertanto, abbiamo modificato il collettore piegando un pezzo di foglio di alluminio in una striscia rettangolare e legandolo perpendicolare ad una piastra metallica piana. Le fibre sono tese elettrofilate tutta l&#39;area tra la punta della striscia e la piastra metallica, che può essere facilmente trasferito ad un altro Substrate. L&#39;interesse per polimeri elastomerici termicamente reticolati è in rapida crescita a causa del lavoro pionieristico del gruppo di Robert Langer, che ha introdotto poli (glicerolo sebacate) (PGS), un poliestere che è analoga alla gomma vulcanizzata, nel 2002 3. Simile a PGS, abbiamo sviluppato con successo poli (glicerolo-dodecanoato) (PGD) per condensazione termica di glicerolo e acidi dodecanodioico e ha dimostrato la sua unica proprietà di memoria di forma 1. A differenza più rigido materiali sintetici poli (butirrato ossidrile) o poli (L-lattide) (moduli di Young di 250 MPa e 660 MPa rispettivamente), PGD esibisce proprietà elastomeriche come gomma, con un modulo di Young di 1,08 MPa quando la temperatura è superiore a 37 ° C, che è una stretta corrispondenza al nervo periferico in-situ (0,45 MPa). Inoltre, PGD è biodegradabile e il tempo di degradazione può essere messo a punto variando il rapporto di glicerolo e acidi dodecanodioico. L&#39;acido dodecanodioico è un sub dodici carbonioposizione con due gruppi carbossilici terminali, HOOC (CH 2) 10 COOH. Acidi bicarbossilici Anche numerati come l&#39;acido sebacico e acido dodecanodioico possono essere metabolizzati ad acetil-CoA e inserire il acidi tricarbossilici (TCA) / (acido citrico) ciclo. Il prodotto metabolico di acidi bicarbossilici, succinil-CoA, è un precursore gluconeogenetic e intermedio del ciclo di TCA 4. Così, alcuni studi hanno suggerito che possano essere utilizzate come substrato combustibile alternativo per la nutrizione enterale e parenterale, specialmente nelle condizioni patologiche. Inoltre, PGD presenta memoria di forma unica perché la sua temperatura di transizione vetrosa è 31 ° C, quindi si vede distinte proprietà meccaniche a temperatura ambiente e alla temperatura corporea. In sintesi, la PGD è biodegradabile, biocompatibile, esibendo uniche proprietà elastiche con proprietà meccaniche simili ai tessuti nervosi; quindi, è un materiale adatto per applicazioni di ingegneria del tessuto nervoso. In questo protocollo, il electrospunfibre lunghe che coprono una vasta area di deposito sono stati realizzati attraverso il collettore di nuova concezione da PGD. I ponteggi in fibra in grado di supportare la crescita delle cellule staminali pluripotenti di topo e la differenziazione.

<table><tbody><tr><td>Glycerol</td><td>Sigma-Aldrich</td><td>G7757</td><td></td></tr><tr><td>Dodecanedioic acid</td><td>Sigma-Aldrich</td><td>D1009</td><td></td></tr><tr><td>Gelatin</td><td>Sigma-Aldrich</td><td>D1890</td><td></td></tr><tr><td>Poly(ethylene oxide) (PEO)</td><td>Sigma-Aldrich</td><td>182028</td><td></td></tr><tr><td>Riboflavin</td><td>Sigma-Aldrich</td><td>132350250</td><td>0.10%</td></tr><tr><td>Mouse embryonic stem cells</td><td>GlobalStem</td><td>GSC-5002</td><td></td></tr><tr><td>Matrigel</td><td>Becton Dickinson</td><td>356234</td><td></td></tr><tr><td>DMEM/F12</td><td>Thermo Scientific</td><td>SH30272.02</td><td></td></tr><tr><td>N2 supplement</td><td>Invitrogen</td><td>17502048</td><td>1%</td></tr><tr><td>FGF2</td><td>Stemgent</td><td>03-0002</td><td>10 ng/ml</td></tr><tr><td>Accutase</td><td>Invitrogen</td><td>A11105-01</td><td></td></tr><tr><td>Phosphate buffered saline (PBS)</td><td>Invitrogen</td><td>10010-031</td><td></td></tr><tr><td>Resazurin fluorescence dye</td><td>Sigma-Aldrich</td><td>62758-13-8</td><td></td></tr><tr><td>SV Total RNA Isolation System</td><td>Promega</td><td>Z3100</td><td></td></tr><tr><td>GoScript Reverse Transcription System</td><td>Promega</td><td>A5000</td><td></td></tr><tr><td>GoTaq qPCR Master Mix</td><td>Promega</td><td>A6001</td><td></td></tr><tr><td>Syringe pump&amp;nbsp;</td><td>Fisher scientific</td><td>14-831-200</td><td></td></tr><tr><td>High voltage power source&amp;nbsp;</td><td>Spellman High Voltage Electronics Corporation</td><td></td><td>SL30</td></tr><tr><td>UV light</td><td>Philips</td><td>308643</td><td>15W/G15T8</td></tr><tr><td>Synergy HT Multi-Mode Microplate Reader</td><td>BioTek</td><td></td><td></td></tr><tr><td>Perkin Elmer GeneAmp PCR System 9600</td><td>Perkin Elmer</td><td>8488</td><td></td></tr><tr><td>StepOne Real-time PCR System</td><td>Applied Biosystems</td><td>4376357</td><td></td></tr></tbody></table>

electrospun fibrous scaffolds of poly(glycerol-dodecanedioate) for engineering neural tissues from mouse embryonic stem cells

Per applicazioni di ingegneria tissutale, la preparazione di supporti biodegradabili e biocompatibili è il compito più desiderabile ma impegnativo. Tra i vari metodi di fabbricazione, elettrospinning è il più attraente uno per la sua semplicità e versatilità. Inoltre, nanofibre elettrofilate imitano la dimensione della matrice extracellulare naturale, garantendo un ulteriore sostegno per la sopravvivenza e la crescita delle cellule. Questo studio ha dimostrato la fattibilità della fabbricazione di fibre lunghe che coprono un&#39;area più grande deposito per un romanzo polimero biodegradabile e biocompatibile chiamato poli (glicerolo-dodecanoato) (PGD) 1 utilizzando un collettore di nuova concezione per electrospinning. PGD ​​presenta proprietà uniche elastici con proprietà meccaniche simili a tessuti nervosi, quindi è adatto per applicazioni di ingegneria tissutale neurali. La sintesi e la fabbricazione di set-up per la fabbricazione di materiali ponteggi fibrosi era semplice, altamente riproducibile e poco costoso. In biocompatibilitàtest, cellule derivate da cellule staminali embrionali di topo potrebbero aderire e crescere sulle fibre PGD elettrofilate. In sintesi, questo protocollo ha fornito un metodo di fabbricazione versatile per la fabbricazione di fibre PGD elettrofilate per sostenere la crescita di topo cellule staminali embrionali derivate cellule neurali lignaggio.

I componenti principali del elettrospinning sono mostrati in Figura 1. Velo di taglia grande era tipicamente ottenuta attraverso la striscia di foglio di alluminio perpendicolarmente divisoria e una piastra metallica piana. Figura 2 mostra la struttura del collettore e il tappeto di fibre elettrospinning. La larghezza e la lunghezza può essere regolata per diverse applicazioni. La lunghezza della fibra a base di polimero PGD e miscela soluzione basale è fino a 10 cm. La morfologia del...

Watch this Scientific Journal Video about Electrospun Fibrous Scaffolds of Polyglycerol-dodecanedioate for Engineering Neural Tissues From Mouse Embryonic Stem Cells at JoVE.com

Electrospun Fibrous Scaffolds of Polyglycerol-dodecanedioate for Engineering Neural Tissues From Mouse Embryonic Stem Cells

È stata riportata la sintesi e la fabbricazione di fibre lunghe elettrofilate coprono un&#39;area più grande deposito tramite un collettore di nuova concezione da un polimero biodegradabile romanzo chiamato poli (glicerolo-dodecanoato) (PGD). Le fibre sono in grado di sostenere la crescita di cellule derivate da cellule staminali pluripotenti di topo.

Elettrofilate Fibrosa Ponteggi di Poli (glicerolo-dodecanedioate) per l&#39;ingegneria neurale tessuti da cellule staminali embrionali di topo

For tissue engineering applications, the preparation of biodegradable and biocompatible scaffolds is the most desirable but challenging task. &#160;Among ...

electrospun-fibrous-scaffolds-poly-glycerol-dodecanedioate-for

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

Electrospun Fibrous Scaffolds of Poly(glycerol-dodecanedioate) for Engineering Neural Tissues From Mouse Embryonic Stem Cells

10.8K Views.  Florida International University.  È stata riportata la sintesi e la fabbricazione di fibre lunghe elettrofilate coprono un'area più grande deposito tramite un collettore di nuova concezione da un polimero biodegradabile romanzo chiamato poli (glicerolo-dodecanoato) (PGD). Le fibre sono in grado di sostenere la crescita di cellule derivate da cellule staminali pluripotenti di topo. 

Video: Electrospun Fibrous Scaffolds of Polyglycerol-dodecanedioate for Engineering Neural Tissues From Mouse Embryonic Stem Cells

The Culture of Primary Motor and Sensory Neurons in Defined Media on Electrospun Poly-L-lactide Nanofiber Scaffolds

Electrospinning is a technique for producing micro- to nano-scale fibers. Fibers can be electrospun with varying degrees of alignment, from highly aligned to completely random. In addition, fibers can be spun from a variety of materials, including biodegradable polymers such as poly-L-lactic acid (PLLA). These characteristics make electrospun fibers suitable for a variety of scaffolding applications in tissue engineering. Our focus is on the use of aligned electrospun fibers for nerve regeneration. We have previously shown that aligned electrospun PLLA fibers direct the outgrowth of both primary sensory and motor neurons in vitro. We maintain that the use of a primary cell culture system is essential when evaluating biomaterials to model real neurons found in vivo as closely as possible. Here, we describe techniques used in our laboratory to electrospin fibrous scaffolds and culture dorsal root ganglia explants, as well as dissociated sensory and motor neurons, on electrospun scaffolds. However, the electrospinning and/or culture techniques presented here are easily adapted for use in other applications.

Aligned electrospun fibers direct the growth of neurons in vitro and are a potential component of nerve regeneration scaffolds. We describe a procedure for preparing electrospun fiber substrates and the serum-free culture of primary rat E15 sensory (DRG) and motor neurons. Visualization of neurons by immunocytochemistry is also included.

La cultura del motore principale e neuroni sensoriali in Media definiti in elettrofilate Poli-L-lactide Ponteggi nanofibre

Electrospinning is a technique for producing micro- to nano-scale fibers. Fibers can be electrospun with varying degrees of alignment, from highly aligned ...

Ricerca

Neuroscienze

Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications

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 inside of 3D biomaterial scaffolds provides a way to accurately mimic these microenvironments, providing an advantage over traditional 2D culture methods using polystyrene as well as a method for engineering replacement tissues 2. While 2D tissue culture polystrene has been used for the majority of cell culture experiments, 3D biomaterial scaffolds can more closely replicate the microenvironments found in vivo by enabling more accurate establishment of cell polarity in the environment and possessing biochemical and mechanical properties similar to soft tissue.3 A variety of naturally derived and synthetic biomaterial scaffolds have been investigated as 3D environments for supporting stem cell growth. While synthetic scaffolds can be synthesized to have a greater range of mechanical and chemical properties and often have greater reproducibility, natural biomaterials are often composed of proteins and polysaccharides found in the extracelluar matrix and as a result contain binding sites for cell adhesion and readily support cell culture. Fibrin scaffolds, produced by polymerizing the protein fibrinogen obtained from plasma, have been widely investigated for a variety of tissue engineering applications both in vitro and in vivo 4. Such scaffolds can be modified using a variety of methods to incorporate controlled release systems for delivering therapeutic factors 5. Previous work has shown that such scaffolds can be used to successfully culture embryonic stem cells and this scaffold-based culture system can be used to screen the effects of various growth factors on the differentiation of the stem cells seeded inside 6,7.
 This protocol details the process of polymerizing fibrin scaffolds from fibrinogen solutions using the enzymatic activity of thrombin. The process takes 2 days to complete, including an overnight dialysis step for the fibrinogen solution to remove citrates that inhibit polymerization. These detailed methods rely on fibrinogen concentrations determined to be optimal for embryonic and induced pluripotent stem cell culture. Other groups have further investigated fibrin scaffolds for a wide range of cell types and applications - demonstrating the versatility of this approach 8-12.

This work details the preparation of 3D fibrin scaffolds for culturing and differentiating plutipotent stem cells. Such scaffolds can be used to screen the effects of various biological compounds on stem cell behavior as well as modified to contain drug delivery systems.

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 ...

Bioingegneria

Cultivation of Human Neural Progenitor Cells in a 3-dimensional Self-assembling Peptide Hydrogel

The influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally neuronal differentiation is of great interest in order to find new methods for cell-based and standardised therapies in neurological disorders or neurodegenerative diseases. 3D structures are expected to provide an environment much closer to the in vivo situation than 2D cultures. In the context of regenerative medicine, the combination of biomaterial scaffolds with neural stem and progenitor cells holds great promise as a therapeutic tool.1-5 Culture systems emulating a three dimensional environment have been shown to influence proliferation and differentiation in different types of stem and progenitor cells. Herein, the formation and functionalisation of the 3D-microenviroment is important to determine the survival and fate of the embedded cells.6-8 Here we used PuraMatrix9,10 (RADA16, PM), a peptide based hydrogel scaffold, which is well described and used to study the influence of a 3D-environment on different cell types.7,11-14 PuraMatrix can be customised easily and the synthetic fabrication of the nano-fibers provides a 3D-culture system of high reliability, which is in addition xeno-free.
Recently we have studied the influence of the PM-concentration on the formation of the scaffold.13 In this study the used concentrations of PM had a direct impact on the formation of the 3D-structure, which was demonstrated by atomic force microscopy. A subsequent analysis of the survival and differentiation of the hNPCs revealed an influence of the used concentrations of PM on the fate of the embedded cells. However, the analysis of survival or neuronal differentiation by means of immunofluorescence techniques posses some hurdles. To gain reliable data, one has to determine the total number of cells within a matrix to obtain the relative number of e.g. neuronal cells marked by &beta;III-tubulin. This prerequisites a technique to analyse the scaffolds in all 3-dimensions by a confocal microscope or a comparable technique like fluorescence microscopes able to take z-stacks of the specimen. Furthermore this kind of analysis is extremely time consuming. 
Here we demonstrate a method to release cells from the 3D-scaffolds for the later analysis e.g. by flow cytometry. In this protocol human neural progenitor cells (hNPCs) of the ReNcell VM cell line (Millipore USA) were cultured and differentiated in 3D-scaffolds consisting of PuraMatrix (PM) or PuraMatrix supplemented with laminin (PML). In our hands a PM-concentration of 0.25% was optimal for the cultivation of the cells13, however the concentration might be adapted to other cell types.12 The released cells can be used for e.g. immunocytochemical studies and subsequently analysed by flow cytometry. This speeds up the analysis and more over, the obtained data rest upon a wider base, improving the reliability of the data.

Here we describe the use of a self-assembling 3-dimensional scaffold to culture human neural progenitor cells. We present a protocol to release the cells from the scaffolds to be analysed subsequently e.g. by flow cytometry. This protocol might be adapted to other cell types to perform detailed mechanistically studies.

La coltivazione di cellule progenitrici neurali umane in un 3-dimensionale di auto-assemblaggio Peptide Hydrogel

The  influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally  neuronal differentiation is of great interest in order to find new ...

Electrospinning Growth Factor Releasing Microspheres into Fibrous Scaffolds

This procedure describes a method to fabricate a multifaceted substrate to direct nerve cell growth. This system incorporates mechanical, topographical, adhesive and chemical signals. Mechanical properties are controlled by the type of material used to fabricate the electrospun fibers. In this protocol we use 30% methacrylated Hyaluronic Acid (HA), which has a tensile modulus of ~500 Pa, to produce a soft fibrous scaffold. Electrospinning on to a rotating mandrel produces aligned fibers to create a topographical cue. Adhesion is achieved by coating the scaffold with fibronectin. The primary challenge addressed herein is providing a chemical signal throughout the depth of the scaffold for extended periods. This procedure describes fabricating&#160;poly(lactic-co-glycolic acid) (PLGA) microspheres that contain Nerve Growth Factor (NGF) and directly impregnating the scaffold with these microspheres during the electrospinning process. Due to the harsh production environment, including high sheer forces and electrical charges, protein viability is measured after production. The system provides protein release for over 60 days and has been shown to promote primary nerve cell growth.

This protocol combines electrospinning and microspheres to develop tissue engineered scaffolds to direct neurons. Nerve growth factor was encapsulated within PLGA microspheres and electrospun into Hyaluronic Acid (HA) fibrous scaffolds. The protein bioactivity was tested by seeding the scaffolds with primary chick Dorsal Root Ganglia and culturing for 4-6 days.

Electrospinning Growth Factor Releasing Microsfere in Fibrosi Ponteggi

This procedure describes a method to fabricate a multifaceted substrate to direct nerve cell growth.  This system incorporates mechanical, topographical, ...

Electrospun Nanofiber Scaffolds with Gradations in Fiber Organization

The goal of this protocol is to report a simple method for generating nanofiber scaffolds with gradations in fiber organization and test their possible applications in controlling cell morphology/orientation. Nanofiber organization is controlled with a new fabrication apparatus that enables the gradual decrease of fiber organization in a scaffold. Changing the alignment of fibers is achieved through decreasing deposition time of random electrospun fibers on a uniaxially aligned fiber mat. By covering the collector with a moving barrier/mask, along the same axis as fiber deposition, the organizational structure is easily controlled. For tissue engineering purposes, adipose-derived stem cells can be seeded to these scaffolds. Stem cells undergo morphological changes as a result of their position on the varied organizational structure, and can potentially differentiate into different cell types depending on their locations. Additionally, the graded organization of fibers enhances the biomimicry of nanofiber scaffolds so they more closely resemble the natural orientations of collagen nanofibers at tendon-to-bone insertion site compared to traditional scaffolds. Through nanoencapsulation, the gradated fibers also afford the possibility to construct chemical gradients in fiber scaffolds, and thereby further strengthen their potential applications in fast screening of cell-materials interaction and interfacial tissue regeneration. This technique enables the production of continuous gradient scaffolds, but it also can potentially produce fibers in discrete steps by controlling the movement of the moving barrier/mask in a discrete fashion.

Here, we present a protocol to fabricate electrospun nanofiber scaffolds with gradated organization of fibers and explore their applications in regulating cell morphology/orientation. Gradients with regard to physical and chemical properties of the nanofiber scaffolds offer a wide variety of applications in the biomedical field.

Elettrofilate nanofibre Ponteggi con gradazioni Organizzazione Fiber

The goal of this protocol is to report a simple method for generating nanofiber scaffolds with gradations in fiber organization and test their possible ...

Engineered 3D Silk-collagen-based Model of Polarized Neural Tissue

Despite huge efforts to decipher the anatomy, composition and function of the brain, it remains the least understood organ of the human body. To gain a deeper comprehension of the neural system scientists aim to simplistically reconstruct the tissue by assembling it in vitro from basic building blocks using a tissue engineering approach. Our group developed a tissue-engineered silk and collagen-based 3D brain-like model resembling the white and gray matter of the cortex. The model consists of silk porous sponge, which is pre-seeded with rat brain-derived neurons, immersed in soft collagen matrix. Polarized neuronal outgrowth and network formation is observed with separate axonal and cell body localization. This compartmental architecture allows for the unique development of niches mimicking native neural tissue, thus enabling research on neuronal network assembly, axonal guidance, cell-cell and cell-matrix interactions and electrical functions.

Insight into the complex actions of the brain requires advanced research tools. Here we demonstrate a novel silk-collagen-based 3D engineered model of neural tissue resembling brain-like architecture. The model can be used to study neuronal network assembly, axonal guidance, cell-cell interactions and electrical activity.

A base di seta-collagene Engineered Modello 3D del tessuto neurale polarizzata

Despite huge efforts to decipher the anatomy, composition and function of the brain, it remains the least understood organ of the human body. To gain a ...

Electrospinning Fibrous Polymer Scaffolds for Tissue Engineering and Cell Culture

As the field of tissue engineering evolves, there is a tremendous demand to produce more suitable materials and processing techniques in order to address the requirements (e.g., mechanics and vascularity) of more intricate organs and tissues. Electrospinning is a popular technique to create fibrous scaffolds that mimic the architecture and size scale of the native extracellular matrix. These fibrous scaffolds are also useful as cell culture substrates since the fibers can be used to direct cellular behavior, including stem cell differentiation (see extensive reviews by Mauck et al. and Sill et al. for more information). In this article, we describe the general process of electrospinning polymers and as an example, electrospin a reactive hyaluronic acid capable of crosslinking with light exposure (see Ifkovits et al. for a review on photocrosslinkable materials). We also introduce further processing capabilities such as photopatterning and multi-polymer scaffold formation. Photopatterning can be used to create scaffolds with channels and multi-scale porosity to increase cellular infiltration and tissue distribution. Multi-polymer scaffolds are useful to better tune the properties (mechanics and degradation) of a scaffold, including tailored porosity for cellular infiltration. Furthermore, these techniques can be extended to include a wide array of polymers and reactive macromers to create complex scaffolds that provide the cues necessary for the development of successful tissue engineered constructs.

The process of electrospinning polymers for tissue engineering and cell culture is addressed in this article. Specifically, the electrospinning of photoreactive macromers with additional processing capabilities of photopatterning and multi-polymer electrospinning is described.

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 ...

Biologia

Electrically Conductive Scaffold to Modulate and Deliver Stem Cells

Stem cell therapy has emerged as an exciting stroke therapeutic, but the optimal delivery method remains unclear. While the technique of microinjection has been used for decades to deliver stem cells in stroke models, this technique is limited by the lack of ability to manipulate the stem cells prior to injection. This paper details a method of using an electrically conductive polymer scaffold for stem cell delivery. Electrical stimulation of stem cells using a conductive polymer scaffold alters the stem cell's genes involved in cell survival, inflammatory response, and synaptic remodeling. After electrical preconditioning, the stem cells on the scaffold are transplanted intracranially in a distal middle cerebral artery occlusion rat model. This protocol describes a powerful technique to manipulate stem cells via a conductive polymer scaffold and creates a new tool to further develop stem cell-based therapy.

This protocol describes fabrication of a cell culture system to allow seeding of stem cells on a conductive polymer scaffold for in vitro electrical stimulation and subsequent in vivo implantation of the stem cell-seeded scaffold using a minimally invasive technique.

Elettricamente conduttivo impalcatura di modulare e fornire cellule staminali

Stem cell therapy has emerged as an exciting stroke therapeutic, but the optimal delivery method remains unclear. While the technique of microinjection ...

Anatomically Inspired Three-dimensional Micro-tissue Engineered Neural Networks for Nervous System Reconstruction, Modulation, and Modeling

Functional recovery rarely occurs&#160;following injury or disease-induced degeneration within the central nervous system (CNS)&#160;due to the inhibitory environment and the limited capacity for neurogenesis. We are developing&#160;a strategy to simultaneously address&#160;neuronal and axonal pathway loss within the damaged CNS. This manuscript presents the fabrication protocol for micro-tissue engineered neural networks (micro-TENNs), implantable constructs consisting of neurons and aligned axonal tracts spanning the extracellular matrix (ECM) lumen of a preformed hydrogel cylinder hundreds of microns in diameter that may extend centimeters in length. Neuronal aggregates are delimited to the extremes of the three-dimensional encasement and are spanned by axonal projections. Micro-TENNs are uniquely poised as a strategy for CNS reconstruction, emulating aspects of brain connectome cytoarchitecture and potentially providing means for network replacement. The neuronal aggregates may synapse with host tissue to form new functional relays to restore and/or modulate missing or damaged circuitry. These constructs may also act as pro-regenerative&#160;&#34;living scaffolds&#34; capable of exploiting developmental mechanisms for cell migration and axonal pathfinding, providing synergistic structural and soluble cues based on the state of regeneration. Micro-TENNs are fabricated by pouring liquid hydrogel into a cylindrical mold containing a longitudinally centered needle. Once the hydrogel has gelled, the needle is removed, leaving a hollow micro-column. An ECM solution is added to the lumen to provide an environment suitable for neuronal adhesion and axonal outgrowth. Dissociated neurons are mechanically aggregated for precise seeding within one or both ends of the micro-column. This methodology reliably produces self-contained miniature constructs with long-projecting axonal tracts that may recapitulate features of brain neuroanatomy. Synaptic immunolabeling and genetically encoded calcium indicators suggest that micro-TENNs possess extensive synaptic distribution and intrinsic electrical activity. Consequently, micro-TENNs represent a promising strategy for targeted neurosurgical reconstruction of brain pathways and may also be applied as biofidelic models to study neurobiological phenomena in vitro.

This manuscript details the fabrication of micro-tissue engineered neural networks: three-dimensional micron-sized constructs comprised of long aligned axonal tracts spanning aggregated neuronal population(s) encased in a tubular hydrogel. These living scaffolds can serve as functional relays to reconstruct or modulate neural circuitry or as biofidelic test-beds mimicking gray-white matter neuroanatomy.

Le reti neurali ricalcolate anatomicamente tridimensionali dei micro-tessuti per la ricostruzione, la modulazione e la modellazione del sistema nervoso

Functional recovery rarely occurs&#160;following injury or disease-induced degeneration within the central nervous system (CNS)&#160;due to the inhibitory ...

Controlling Electrospun Polymer Morphology for Tissue Engineering Demonstrated Using hepG2 Cell Line

Electrospinning affords researchers the opportunity to fabricate reproducible micro to nanoscale polymer fibers. The 3D fibrous architecture of electrospun polymers is regarded as a structural imitation of the extracellular matrix (ECM). Hence, electrospun fibers fabricated from biocompatible polymers have been widely investigated by tissue engineering researchers for their potential role as an artificial ECM for guiding tissue growth both in vitro and in vivo. All cells are acutely sensitive to their mechanical environment. This has been demonstrated by the discovery of multiple mechanotransduction pathways intrinsically linked to the cytoskeletal actin filaments. The cytoskeleton acts as a mechanical sensor that can direct the functionality and differentiation of the host cell depending on the stiffness and morphology of its substrate. Electrospun fibers can be tuned both in terms of fiber size and morphology to easily modulate the mechanical environment within a fibrous polymer scaffold. Here, methods for electrospinning polycaprolactone (PCL) for three distinct morphologies at two different fiber diameters are described. The morphological fiber categories consist of randomly oriented fibers, aligned fibers, and porous cryogenically spun fibers, with 1 &#181;m and 5 &#181;m diameters. The methods detailed within this study are proposed as a platform for investigating the effect of electrospun fiber architecture on tissue generation. Understanding these effects will allow researchers to optimize the mechanical properties of electrospun fibers and demonstrate the potential of this technology more thoroughly.

This method provides the means to test different polycaprolactone fiber morphologies and topographies for the purpose of tissue engineering. Small and large fibers are fabricated with random orientations, aligned orientations, and also porous cryogenically electrospun structures and used as platforms for cell culture.

Elettrofilate Fibrosa Ponteggi di Poli (glicerolo-dodecanedioate) per l'ingegneria neurale tessuti da cellule staminali embrionali di topo

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