DOZ è supportata da una sovvenzione concessa dalla Fondazione di Ginevra. SN è stato sostenuto da una borsa post-dottorato dal Initiative Tissue Engineering Pittsburgh.

1. Isolanti derivate da tessuto adiposo cellule staminali (ASC) Nota: Tutte le procedure sono state eseguite a temperatura ambiente salvo diversamente indicato. <ol><li> Isolare adiposo perirenale e epididimale di ratto e lavare con soluzione salina sterile tamponata di Hank (HBSS) contenente 1% siero bovino fetale (FBS) come descritto in precedenza 6. </li><li> Tritare il tessuto e trasferire 1-2 g in 25 ml di HBSS contenente 1% FBS in una provetta da 50 ml e centrifugare a 500 g per 8 minuti a temperatura ambiente. </li><li> Raccogliere il fluttuante strato di tessuto adiposo e trasferimento pallone Erlenmeyer da 125 ml e trattare con 25 ml di collagenasi di tipo II (200 U / mL) in HBSS per 45 min a 37 ° C in un agitatore orbitale (125 rpm). </li><li> Cautela rimuovere la frazione liquida (sotto olio e strato adiposo) e filtrare sequenzialmente attraverso un filtro di nylon da 100 um e 70 um-maglia. Centrifugare il filtrato a 500 g per 10 min a temperatura ambiente, aspirare il supernatant e lavare il pellet due volte con 25 ml di HBSS. </li><li> Risospendere il pellet di cellule in 50 mL di mezzo di crescita (RS MesenPRO medio basale) supplementato con supplemento MesenPRO crescita RS, antibiotico-antimicotico (100 U / mL di penicillina G, 100 pg / mL di streptomicina solfato, e 0,25 mg / ml di anfotericina B) , e 2 mM L-glutammina e cellule pipettare in fiasche T75 2 (25 ml / beuta). </li><li> La cultura il ASC in un 5% CO 2 incubatore umidificato a 37 ° C (Passaggio 2-4 sono utilizzati ASC per tutti gli esperimenti). </li></ol> 2. Preparazione Chitosan Microsfere (CSM) Nota: Tutte le procedure sono state eseguite a temperatura ambiente salvo diversamente indicato. <ol><li> CSM sono preparate da un acqua-in-olio processo di emulsionamento con una tecnica coacervazione ionico usando la nostra precedente protocollo 6. Emulsionare una soluzione acquosa di chitosano (6 mL del 3% w / v chitosano in 0,5 M acido acetico) in 100 ml di una miscela costituita fase olio di soiaolio, n-ottanolo (1:2 v / v) e il 5% sorbitan-monooleato (Span 80) emulsionante, usando testa (1700 rpm) e agitazione magnetica (1000 rpm) simultaneamente, in direzioni opposte. Questo metodo di duplice assicura la miscelazione che micelle formate presto prima reticolazione avviene può rimanere in soluzione e non si depositano sul fondo. Inoltre, l&#39;agitazione magnetica bar aiuti in de-aggregazione chitosano durante la formazione delle micelle e rigidization. </li><li> La miscela viene mantenuta sotto agitazione per circa 1 ora fino uno stabile acqua-in-olio è ottenuto. Reticolazione ionica è iniziata con l&#39;aggiunta di 1,5 ml di 1% w / v di idrossido di potassio in n-ottanolo ogni 15 min per 4 h (24 mL totale). </li><li> Dopo completamento della reazione di reticolazione, lentamente decantare la fase olio della miscela contenente CSM e aggiungere immediatamente le sfere a 100 mL di acetone. Lavare le sfere con acetone finché la fase olio viene completamente rimosso. </li><li> Asciugare le sfere recuperate in un essiccatore a vuoto e analeyze senza ulteriori elaborazioni. La dimensione media delle particelle CSM, superficie per milligrammo e l&#39;unità di volume cubico è stata determinata utilizzando dimensione analizzatore di particelle. </li><li> Per gli esperimenti successivi, lavare CSM tre volte con acqua sterile per eliminare i sali residui e sterilizzare con alcool assoluto. </li></ol> 3. Determinazione del numero di gruppi amminici liberi in CSM Nota: Tutte le procedure sono state eseguite a temperatura ambiente salvo diversamente indicato. <ol><li> Determinare il numero di gruppi amminici liberi presenti nel CSM dopo la reticolazione ionica utilizzando il trinitro benzensolfonico (TNBS) dosaggio di acido Bubins e Ofner 7. Incubare 5 mg di microsfere con 1 mL di soluzione 0,5% TNBS in una provetta di vetro da 50 ml per 4 ore a 40 ° C e idrolizzare con l&#39;aggiunta di 3 ml di HCl 6N a 60 ° C per 2h. </li><li> Raffreddare i campioni a temperatura ambiente, ed estrarre i TNBS libere aggiungendo 5 ml di acqua deionizzata e 10 ml di etiletere. </li><li> Riscaldare Un&#39;aliquota di 5 ml della fase acquosa a 40 ° C a bagnomaria per 15 minuti per evaporare qualsiasi etere residuo, raffreddare a temperatura ambiente, e diluire con 15 ml di acqua. </li><li> Misurare l&#39;assorbanza a 345 nm con uno spettrofotometro con TNBS senza soluzione chitosano come vuoto e il chitosano utilizzata per la preparazione CSM per determinare il numero totale di gruppi amminici. Stimare il numero di gruppi amminici liberi del CSM rispetto al chitosano. </li></ol> 4. Caricamento in ASC CSM Nota: Tutte le procedure sono state eseguite a temperatura ambiente salvo diversamente indicato. <ol><li> Equilibrare 5 mg di CSM sterilizzato dalla sezione 2.5 in HBSS sterili durante la notte e aggiungere ad un inserto di 8 micron dimensione dei pori della membrana piastra di cultura (24 pozzetti). </li><li> Dopo aver risolto il CSM sulla membrana, aspirare accuratamente le HBSS e aggiungere 300 ml di terreno di crescita all&#39;interno dell&#39;inserto e 700 microlitri di terreni di crescita per the fuori dell&#39;inserto. </li><li> Risospendere ASC alla concentrazione adeguata (1 x 10 4 a 4 x 10 4) in 200 pl di mezzo di crescita e seme sulla CSM all&#39;interno dell&#39;inserto piastra di coltura. Il volume finale del mezzo all&#39;interno dell&#39;inserto coltura, dopo la semina, è 500 pl. </li><li> Incubare la testa di serie ASC sul CSM per 24 ore in un 5% di CO 2 incubatore umidificato a 37 ° C. </li></ol> 5. Determinazione della percentuale di caricamento ASC e la vitalità cellulare in CSM Nota: Tutte le procedure sono state eseguite a temperatura ambiente salvo diversamente indicato. <ol><li> Dopo l&#39;incubazione, raccogliere le ASC-caricato CSM in un contenitore sterile da 1,5 ml provetta senza disturbare le cellule che sono migrate nella membrana di inserimento. </li><li> Rimuovere il mezzo residuo e aggiungere 250 microlitri di terreno di crescita fresco al tubo. </li><li> Per aggiungere ciascun tubo 25 pl MTT [3 - (4,5-dimethylthiozole-2-il) -2,5-difeniltetrazolio bromuro]soluzione di (5 mg / mL) e incubare per 4 h in un 5% CO 2 incubatore umidificato a 37 ° C. </li><li> Dopo l&#39;incubazione, rimuovere il supporto, si aggiungano 250 microlitri di dimetilsolfossido e miscelare l&#39;impasto per 2-5 min per solubilizzare il complesso formazan. </li><li> Centrifugare il CSM a 2700 g per 5 min, e determinare l&#39;assorbanza supernatante misurata a 570 nm usando 630 nm come riferimento. </li><li> Determinare il numero di cella associato con il CSM rispetto al valore di assorbanza ottenuto da un numero noto di vitale ASC. </li></ol> 6. Caratterizzare ASC-CSM-Embedded Collagen Gel Nota: Tutte le procedure sono state eseguite a temperatura ambiente salvo diversamente indicato. <ol><li> Mix ASC-caricato CSM (5 mg contenente ≈ 2 x 10 4 cellule) con collagene di tipo 1 (7,5 mg / mL) estratto da tendine coda di ratto secondo il metodo di Bornstein 8 e fibrillano dopo aver regolato il pH a 6,8 con NaOH 2 N. </li&gt; <li> Aggiungere il fibrillato collagene-ASC-CSM miscela a 12-pozzetti e incubare per 30 min in un 5% CO 2 incubatore umidificato a 37 ° C. </li><li> Dopo completa fibrillazione, incubare le collagene-ASC-CSM gel fino a 14 giorni in un 5% CO 2 incubatore umidificato a 37 ° C. </li><li> Rilascio e migrazione di cellule dal CSM nel gel sono state osservate usando tecniche di microscopia standard. </li></ol> 7. Risultati rappresentativi In questo studio, abbiamo sviluppato una strategia in vitro per fornire cellule staminali da microsfere di chitosano (CSM) in un scaffold di collagene gel. CSM poroso di dimensioni uniformi (175-225 micron di diametro) e la composizione sono stati preparati e utilizzati come vettori cellulare (Figura 2). Dopo incubazione con il ASC CSM, le cellule attaccate ad una concentrazione di 2 x 10 4 cells/5mg di CSM. Le cellule erano in grado di diffondere sul microsfere, mentre si estende filopodianelle fessure porose della microsfera (Figura 3). Quando la cellula-caricato CSM sono stati miscelati con il gel di collagene, le cellule immediatamente cominciarono a migrare nei gel (Figura 4). <img alt="Tabella 1" src="/files/ftp_upload/3624/3624table1.jpg" /> Tabella 1. Vantaggi biologici per l&#39;uso di chitosano e collagene in un sistema di erogazione di cellule staminali. <img alt="Figura 1" src="/files/ftp_upload/3624/3624fig1.jpg" /> Figura 1. Schema raffigurante la strategia globale per il doppio uso di ASC-CSM scaffold di collagene caricati. Figure 2, 3 e 4 sono annotati entro lo schema di assistere con l&#39;interpretazione delle immagini. <img alt="Figura 2" src="/files/ftp_upload/3624/3624fig2.jpg" /> Figura 2. Rappresentazione schematica raffigurante il processo di seminare cellule staminali su microsfere di chitosano. Il process comporta co-coltura con ASC CSM in un 8 - micron dimensione dei pori della membrana inserto cultura piatto. Dopo 24 ore, le microsfere vengono rimossi dal dell&#39;inserto e sono pronti per l&#39;incorporamento in una matrice biomateriale. <img alt="Figura 3" src="/files/ftp_upload/3624/3624fig3.jpg" /> Figura 3. Caratterizzazione morfologica dei CSM-caricato con ASC. Pannello A raffigura una micrografia luce di ASC-caricato CSM, mentre il pannello B mostra lo stesso campo di vista con sovrapposta un&#39;immagine ottenuta mediante microscopia confocale a fluorescenza. ASC sono stati precaricato con calceina AM (verde). Pannello C illustra un&#39;immagine di un&#39;immagine SEM di una microsfera scarico, mentre il pannello D mostra cellule caricati sul microsfera (asterischi). L&#39;immagine TEM in E pannello mostra una sezione trasversale di una microsfera scarico. Una moltitudine di pori e fessure si trovano in tutta la microsfera. Pannello F mostra una sezionata microsfere con cellule (frecce) collegato e si estende filopodia nella creazionevizi. Ingrandimenti originali: A &amp; B = 70X; C = 500x, D = 2.000 x; E &amp; F = 2.500 x. <img alt="Figura 4" src="/files/ftp_upload/3624/3624fig4.jpg" /> Figura 4. Migrazione del ASC dal CSM nel tridimensionale collagene scaffold. Pannelli A e B raffigurano il CSM, con cellule che migrano lontano da microsfere e nella matrice di collagene il giorno 3 (A, frecce). Panel B mostra una cultura simile dopo 12 giorni. Microscopia elettronica a trasmissione (TEM) le immagini sono raffigurati in C, D, E. e asterischi in C e D mostrano una microsfera che è stata sezionata con cellule abbandonando la microsfera (frecce). Ingrandimento superiore del pannello D è rappresentato nella E pannello, e mostra filopodia cella attaccati alle fibrille di collagene (inserto). Ingrandimento originale: A &amp; B = 100x, C &amp; D = 6.000 x, x E = 20.000, incasso 150.000 = x. <img alt="Figura 5" src="/files/ftp_upload/3624/3624fig5.jpg" /> Figura 5.Schematico che illustra gli usi vaste CSM nella medicina rigenerativa e la consegna della droga.

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Nessun conflitto di interessi finanziari esistono. Disclaimers I pareri o le affermazioni contenute nel presente documento sono le opinioni personali degli autori e non devono essere interpretate come ufficiale o che riflette le opinioni del Dipartimento della Difesa o il Governo degli Stati Uniti. Gli autori sono dipendenti del governo degli Stati Uniti, e questo lavoro è stato preparato come parte delle loro funzioni ufficiali. Tutto il lavoro è stato sostenuto dalla US Army Medical Research e Materiel Command. Questo studio è stato condotto nell&#39;ambito di un protocollo esaminato e approvato dalla US Army Medical Research e Materiel Command Institutional Review Board, e in conformità con il protocollo approvato.

Uno degli ostacoli principali delle cellule staminali in terapia a base di sta sviluppando metodi efficienti per la consegna delle cellule alle regioni determinate per la riparazione. Grazie al paziente a paziente variabilità, il tipo di tessuto, dimensioni e profondità lesione, la metodologia di cellule staminali che forniscono devono essere determinati caso per caso. Anche se l&#39;incorporamento cellule staminali all&#39;interno di una matrice e di consegnarli al sito della ferita sembra essere un approccio logico ...

<table border="1" ><tbody><tr><td> Nome del reagente / apparecchiatura </td><td> Azienda </td><td> Numero di catalogo </td><td> Comments </td></tr><tr><td> Hanks BalancedSalt Solution (HBSS) </td><td> Gibco </td><td> 14175 </td><td> Consumabili </td></tr><tr><td> Siero fetale bovino </td><td> Hyclone </td><td> SH30071.03 </td><td> Consumabili </td></tr><tr><td> Collagenasi di tipo II </td><td> Sigma-Aldrich </td><td> C6685 </td><td> Consumabili </td></tr><tr><td> 70-micron nylon filtro a rete </td><td> BD Biosciences </td><td> 352350 </td><td> Consumabili </td></tr><tr><td> 100-um nylon filtro a rete </td><td> BD Biosciences </td><td> 352360 </td><td> Consumabili </td></tr><tr><td> MesenPRO crescita media del sistema </td><td> Invitrogen </td><td> 12746-012 </td><td> Consumabili </td></tr><tr><td> L-glutammina </td><td> Gibco </td><td> 25030 </td><td> Consumabili </td></tr><tr><td> T75 Tissue Culture Flask </td><td> BD Biosciences </td><td> 137787 </td><td> Consumabili </td></tr><tr><td> Chitosan </td><td> Sigma-Aldrich </td><td> 448869 </td><td> Consumabili </td></tr><tr><td> Acido acetico </td><td> Sigma-Aldrich </td><td> 320099 </td><td> Consumabili </td></tr><tr><td> N-ottanolo </td><td> Acros Organics </td><td> 150630025 </td><td> Consumabili </td></tr><tr><td> Sorbitan-Mono-oleato </td><td> Sigma-Aldrich </td><td> S6760 </td><td> Consumabili </td></tr><tr><td> Idrossido di potassio </td><td> Sigma-Aldrich </td><td> P1767 </td><td> Consumabili </td></tr><tr><td> Acetone </td><td> Fisher Scientific </td><td> L-4859 </td><td> Consumabili </td></tr><tr><td> Etanolo </td><td> Sigma-Aldrich </td><td> 270741 </td><td> Consumabili </td></tr><tr><td> Trinitro benzensolfonico Acid </td><td> Sigma-Aldrich </td><td> P2297 </td><td> Consumabili </td></tr><tr><td> Acido cloridrico </td><td> Sigma-Aldrich </td><td> 320331 </td><td> Consumabili </td></tr><tr><td> Etere etilico </td><td> Sigma-Aldrich </td><td> 472-484 </td><td> Consumabili </td></tr><tr><td> 8-micron di tessuto coltura in piastra Inserti </td><td> BD Biosciences </td><td> 353097 </td><td> Consumabili </td></tr><tr><td> Microcentrifuga da 1,5 ml Tubes </td><td> Pescatore </td><td> 05-408-129 </td><td> Consumabili </td></tr><tr><td> MTT reagente </td><td> Invitrogen </td><td> M6494 </td><td> Consumabili </td></tr><tr><td> Dimetilsolfossido </td><td> Sigma-Aldrich </td><td> D8779 </td><td> Consumabili </td></tr><tr><td> Qtracker cellulare Labeling Kit (Q Tracker 655) </td><td> Sonde molecolari </td><td> Q2502PMP </td><td> Consumabili </td></tr><tr><td> Collagene di tipo 1 </td><td> Travigen </td><td> 3447-020-01 </td><td> Consumabili </td></tr><tr><td> Di sodio idrossido </td><td> Sigma-Aldrich </td><td> S8045 </td><td> Consumabili </td></tr><tr><td> 12-Well piastre di coltura dei tessuti </td><td> BD Biosciences </td><td> 353043 </td><td> Consumabili </td></tr><tr><td> Centrifuga </td><td> Eppendorf </td><td> 5417R </td><td> Attrezzatura </td></tr><tr><td> Orbital Shaker </td><td> New Brunswick Scienctific </td><td> C24 </td><td> Attrezzatura </td&gt; </tr><tr><td> Incubatore umidificato con Air-5% CO 2 </td><td> Thermo Scientific </td><td> Modello 370 </td><td> Attrezzatura </td></tr><tr><td> Agitatore </td><td> IKA </td><td> Visc6000 </td><td> Attrezzatura </td></tr><tr><td> Agitatore magnetico </td><td> Corning </td><td> PC-210 </td><td> Attrezzatura </td></tr><tr><td> Vacuum Essiccatore </td><td> - </td><td> - </td><td> Attrezzatura </td></tr><tr><td> Particle Size Analyzer </td><td> Malvern </td><td> STP2000 Spraytec </td><td> Attrezzatura </td></tr><tr><td> Water Bath </td><td> Fisher Scientific </td><td> Isotemp210 </td><td> Attrezzatura </td&gt; </tr><tr><td> Spettrofotometro </td><td> Beckman </td><td> Beckman Coulter Spettrofotometro DU800UV/Visible </td><td> Attrezzatura </td></tr><tr><td> Vortice </td><td> Diagger </td><td> 3030a </td><td> Attrezzatura </td></tr><tr><td> Microplate Reader </td><td> Molecular Devices </td><td> Spectramax M2 </td><td> Attrezzatura </td></tr><tr><td> Luce / microscopio a fluorescenza </td><td> Olimpo </td><td> IX71 </td><td> Attrezzatura </td></tr><tr><td> Microscopio confocale </td><td> Olimpo </td><td> FV-500 microscopio confocale a scansione laser </td><td> Attrezzatura </td></tr><tr><td> Microscopio Elettronico a Scansione </td><td> Carl Zeiss Micneuroimaging </td><td> Leo 435 VP </td><td> Attrezzatura </td></tr><tr><td> Microscopio elettronico a trasmissione </td><td> JEOL </td><td> JEOL 1230 </td><td> Attrezzatura </td></tr></tbody></table>

constructing a collagen hydrogel for the delivery of stem cell-loaded chitosan microspheres

Cellule staminali multipotenti hanno dimostrato di essere estremamente utile nel campo della medicina rigenerativa 1-3. Tuttavia, per utilizzare queste cellule efficace per la rigenerazione tissutale, un certo numero di variabili devono essere prese in considerazione. Queste variabili includono: il volume totale e la superficie del sito di impianto, le proprietà meccaniche del tessuto e il microambiente tessuto, che comprende la quantità di vascolarizzazione e dei componenti della matrice extracellulare. Pertanto, i materiali utilizzati per fornire queste cellule deve essere biocompatibile con una composizione chimica definita, pur mantenendo una resistenza meccanica che imita il tessuto ospite. Tali materiali devono anche essere permeabile all&#39;ossigeno e nutrienti per fornire un microambiente favorevole per le cellule per attaccare e proliferare. Il chitosano, un polisaccaride cationico con biocompatibilità, possono essere facilmente modificati chimicamente ed ha una elevata affinità per legare in vivo con macromolecules 4-5. Chitosano imita la porzione glicosaminoglicano della matrice extracellulare, consentendogli di funzionare come un substrato per l&#39;adesione cellulare, la migrazione e la proliferazione. In questo studio si utilizzano chitosano in forma di microsfere per fornire derivate da tessuto adiposo cellule staminali (ASC) in una base di collagene tridimensionale scaffold 6. Un ideale cellula-microsfere rapporto è stato determinato rispetto al tempo di incubazione e la densità delle cellule per ottenere massimo numero di celle che possono essere caricati. Una volta ASC sono seminate su microsfere chitosano (CSM), essi sono incorporati in un ponteggio collagene e possono essere mantenute in coltura per lunghi periodi. In sintesi, questo studio fornisce un metodo per fornire con precisione le cellule staminali all&#39;interno di un biomateriale tridimensionale patibolo.

Watch this Scientific Journal Video about Constructing a Collagen Hydrogel for the Delivery of Stem Cell-loaded Chitosan Microspheres at JoVE.com

Constructing a Collagen Hydrogel for the Delivery of Stem Cell-loaded Chitosan Microspheres

Uno degli ostacoli principali nelle terapie attuali delle cellule staminali sta determinando il metodo più efficace per fornire queste cellule ai tessuti ospitanti. Qui, descriviamo uno a base di chitosano metodo di consegna che sia efficiente e semplice l&#39;approccio, consentendo nel contempo derivate da tessuto adiposo cellule staminali per mantenere il loro multipotenza.

Costruire un idrogel collagene per la fornitura di Stem Cell-loaded Chitosan Microsfere

Multipotent stem cells have been shown to be extremely useful in the field of regenerative medicine1-3. However, in order to use these cells effectively ...

constructing-collagen-hydrogel-for-delivery-stem-cell-loaded-chitosan

Research

JoVE Journal

Bioengineering

16.7K Views.  United States Army Institute of Surgical Research. Uno degli ostacoli principali nelle terapie attuali delle cellule staminali sta determinando il metodo più efficace per fornire queste cellule ai tessuti ospitanti. Qui, descriviamo uno a base di chitosano metodo di consegna che sia efficiente e semplice l'approccio, consentendo nel contempo derivate da tessuto adiposo cellule staminali per mantenere il loro multipotenza.

Video: Constructing a Collagen Hydrogel for the Delivery of Stem Cell-loaded Chitosan Microspheres

Organotypic Collagen I Assay: A Malleable Platform to Assess Cell Behaviour in a 3-Dimensional Context

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and metastasis of tumor cells. Migration has been studied on glass coverslips in order to make cellular dynamics amenable to investigation by light microscopy. However, it has become clear that many aspects of cell migration depend on features of the local environment including its elasticity, protein composition, and pore size, which are not faithfully represented by rigid two dimensional substrates such as glass and plastic 1. Furthermore, interaction with other cell types, including stromal fibroblasts 2 and immune cells 3, has been shown to play a critical role in promoting the invasion of cancer cells. Investigation at the molecular level has increasingly shown that molecular dynamics, including response to drug treatment, of identical cells are significantly different when compared in vitro and in vivo 4. 
Ideally, it would be best to study cell migration in its naturally occurring context in living organisms, however this is not always possible. Intermediate tissue culture systems, such as cell derived matrix, matrigel, organotypic culture (described here) tissue explants, organoids, and xenografts, are therefore important experimental intermediates. These systems approximate certain aspects of an in vivo environment but are more amenable to experimental manipulation such as use of stably transfected cell lines, drug treatment regimes, long term and high-resolution imaging. Such intermediate systems are especially useful as proving grounds to validate probes and establish parameters required to image the dynamic response of cells and fluorescent reporters prior to undertaking imaging in vivo 5. As such, they can serve an important role in reducing the need for experiments on living animals.

A method is described for the preparation of a 3-dimensional matrix consisting of collagen type I and primary human fibroblasts. This organotypic gel serves as a useful substrate to assess invasive cell migration because it mimics basic features of tissue stroma and is amenable to many forms of microscopy.

Organotipiche Collagene I test: una piattaforma malleabile per valutare Comportamento cella in un 3-dimensionale Contesto

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and ...

Ricerca

Bioingegneria

Engineering a Bilayered Hydrogel to Control ASC Differentiation

Natural polymers over the years have gained more importance because of their host biocompatibility and ability to interact with cells in vitro and in vivo. An area of research that holds promise in regenerative medicine is the combinatorial use of novel biomaterials and stem cells. A fundamental strategy in the field of tissue engineering is the use of three-dimensional scaffold (e.g., decellularized extracellular matrix, hydrogels, micro/nano particles) for directing cell function. This technology has evolved from the discovery that cells need a substrate upon which they can adhere, proliferate, and express their differentiated cellular phenotype and function 2-3. More recently, it has also been determined that cells not only use these substrates for adherence, but also interact and take cues from the matrix substrate (e.g., extracellular matrix, ECM)4. Therefore, the cells and scaffolds have a reciprocal connection that serves to control tissue development, organization, and ultimate function. Adipose-derived stem cells (ASCs) are mesenchymal, non-hematopoetic stem cells present in adipose tissue that can exhibit multi-lineage differentiation and serve as a readily available source of cells (i.e. pre-vascular endothelia and pericytes). Our hypothesis is that adipose-derived stem cells can be directed toward differing phenotypes simultaneously by simply co-culturing them in bilayered matrices1. Our laboratory is focused on dermal wound healing. To this end, we created a single composite matrix from the natural biomaterials, fibrin, collagen, and chitosan that can mimic the characteristics and functions of a dermal-specific wound healing ECM environment.

This protocol focuses on utilizing the inherent ability of stem cells to take cue from their surrounding extracellular matrix and be induced to differentiate into multiple phenotypes. This methods manuscript extends our description and characterization of a model utilizing a bilayered hydrogel, composed of PEG-fibrin and collagen, to simultaneously co-differentiate adipose-derived stem cells1.

Progettazione di un Hydrogel doppo strato di controllo differenziazione ASC

Natural polymers over the years have gained more importance because of their host biocompatibility and ability to interact with cells in vitro and in ...

An Improved Method for the Preparation of Type I Collagen From Skin

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. Clinically, this protein is widely used for cosmetic surgery, dermal injections, bone grafting, and reconstructive surgery. The sources of COL1 for these procedures are commonly nonhuman, which increases the potential for inflammation and rejection as well as xenobiotic disease transmission. In view of this, a method to efficiently and quickly purify COL1 from limited quantities of autologously-derived tissues would circumvent many of these issues; however, standard isolation protocols are lengthy and often require large quantities of collagenous tissues. Here, we demonstrate an efficient COL1 extraction method that reduces the time needed to isolate and purify this protein from about 10 days to less than 3 hr. We chose the dermis as our tissue source because of its availability during many surgical procedures. This method uses traditional extraction buffers combined with forceful agitation and centrifugal filtration to obtain highly-pure, soluble COL1 from small amounts of corium. Briefly, dermal biopsies are washed thoroughly in ice-cold dH2O after removing fat, connective tissue, and hair. The skin samples are stripped of noncollagenous proteins and polysaccharides using 0.5 M sodium acetate and a high speed bench-top homogenizer. Collagen from residual solids is subsequently extracted with a 0.075 M sodium citrate buffer using the homogenizer. These extracts are purified using 100,000 MW cut-off centrifugal filters that yield COL1 preparations of comparable or superior quality to commercial products or those obtained using traditional procedures. We anticipate this method will facilitate the utilization of autologously-derived COL1 for a multitude of research and clinical applications.

Traditional procedures for the isolation of soluble type 1 collagen (COL1) require about 10 days from start to finish because of lengthy buffer incubations and laborious resuspensions of fibrils. Here, we describe a means to purify COL1 from small dermal biopsies in less than 3 hr.

Un metodo migliore per la preparazione di tipo I collagene della pelle

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. ...

Imaging Denatured Collagen Strands In vivo and Ex vivo via Photo-triggered Hybridization of Caged Collagen Mimetic Peptides

Collagen is a major structural component of the extracellular matrix that supports tissue formation and maintenance. Although collagen remodeling is an integral part of normal tissue renewal, excessive amount of remodeling activity is involved in tumors, arthritis, and many other pathological conditions. During collagen remodeling, the triple helical structure of collagen molecules is disrupted by proteases in the extracellular environment. In addition, collagens present in many histological tissue samples are partially denatured by the fixation and preservation processes. Therefore, these denatured collagen strands can serve as effective targets for biological imaging. We previously developed a caged collagen mimetic peptide (CMP) that can be photo-triggered to hybridize with denatured collagen strands by forming triple helical structure, which is unique to collagens. The overall goals of this procedure are i)&#160;to image denatured collagen strands resulting from normal remodeling activities in vivo, and ii) to visualize collagens in ex vivo tissue sections using the photo-triggered caged CMPs. To achieve effective hybridization and successful in vivo and ex vivo imaging, fluorescently labeled caged CMPs are either photo-activated immediately before intravenous injection, or are directly activated on tissue sections. Normal skeletal collagen remolding in nude mice and collagens in prefixed mouse cornea tissue sections are imaged in this procedure. The imaging method based on the CMP-collagen hybridization technology presented here could lead to deeper understanding of the tissue remodeling process, as well as allow development of new diagnostics for diseases associated with high collagen remodeling activity.

Imaging Denatured Collagen Strands In vivo and Ex vivo via Photo-triggered Hybridization of Caged Collagen Mimetic Peptides

This procedure demonstrates in vivo near IR fluorescence imaging of collagen remodeling activities in mice as well as ex vivo staining of collagens in tissue sections using caged collagen mimetic peptides that can be photo-triggered to hybridize with denatured collagen strands.

Imaging denaturate collagene fili<em&gt; In vivo</em&gt; E<em&gt; Ex vivo</em&gt; Via Photo-triggered ibridazione di Caged collagene Mimetic peptidi

Collagen is a major structural component of the extracellular matrix that supports tissue formation and maintenance. Although collagen remodeling is an ...

Synthesis of an Intein-mediated Artificial Protein Hydrogel

We present the synthesis of a highly stable protein hydrogel mediated by a split-intein-catalyzed protein trans-splicing reaction. The building blocks of this hydrogel are two protein block-copolymers each containing a subunit of a trimeric protein that serves as a crosslinker and one half of a split intein. A highly hydrophilic random coil is inserted into one of the block-copolymers for water retention. Mixing of the two protein block copolymers triggers an intein trans-splicing reaction, yielding a polypeptide unit with crosslinkers at either end that rapidly self-assembles into a hydrogel. This hydrogel is very stable under both acidic and basic conditions, at temperatures up to 50 &#176;C, and in organic solvents. The hydrogel rapidly reforms after shear-induced rupture. Incorporation of a &#34;docking station peptide&#34; into the hydrogel building block enables convenient incorporation of &#34;docking protein&#34;-tagged target proteins. The hydrogel is compatible with tissue culture growth media, supports the diffusion of 20 kDa molecules, and enables the immobilization of bioactive globular proteins. The application of the intein-mediated protein hydrogel as an organic-solvent-compatible biocatalyst was demonstrated by encapsulating the horseradish peroxidase enzyme and corroborating its activity.

We present the synthesis of a split-intein-mediated protein hydrogel. The building blocks of this hydrogel are two protein copolymers each containing a subunit of a trimeric protein that serves as a crosslinker and one half of a split intein. Mixing of the two protein copolymers triggers an intein trans-splicing reaction, yielding a polypeptide unit that self-assembles into a hydrogel. This hydrogel is highly pH- and temperature-stable, compatible with organic solvents, and easily incorporates functional globular proteins.

Sintesi di una proteina artificiale mediata da inteina Hydrogel

We present the synthesis of a highly stable protein hydrogel mediated by a split-intein-catalyzed protein trans-splicing reaction. The building blocks of ...

Development of Amelogenin-chitosan Hydrogel for In Vitro Enamel Regrowth with a Dense Interface

Biomimetic enamel reconstruction is a significant topic in material science and dentistry as a novel approach for the treatment of dental caries or erosion. Amelogenin has been proven to be a critical protein for controlling the organized growth of apatite crystals. In this paper, we present a detailed protocol for superficial enamel reconstruction by using a novel amelogenin-chitosan hydrogel. Compared to other conventional treatments, such as topical fluoride and mouthwash, this method not only has the potential to prevent the development of dental caries but also promotes significant and durable enamel restoration. The organized enamel-like microstructure regulated by amelogenin assemblies can significantly improve the mechanical properties of etched enamel, while the dense enamel-restoration interface formed by an in situ regrowth of apatite crystals can improve the effectiveness and durability of restorations. Furthermore, chitosan hydrogel is easy to use and can suppress bacterial infection, which is the major risk factor for the occurrence of dental caries. Therefore, this biocompatible and biodegradable amelogenin-chitosan hydrogel shows promise as a biomaterial for the prevention, restoration, and treatment of defective enamel.

Development of Amelogenin-chitosan Hydrogel for In Vitro Enamel Regrowth with a Dense Interface

In this article, we describe a protocol for fabricating an amelogenin-chitosan hydrogel for superficial enamel reconstruction. Organized in situ growth of apatite crystals in the hydrogel formed a dense enamel-restoration interface, which will improve the effectiveness and durability of restorations.

Sviluppo di amelogenina-chitosano Hydrogel per<em&gt; In Vitro</em&gt; Smalto ricrescita con una interfaccia Dense

Biomimetic enamel reconstruction is a significant topic in material science and dentistry as a novel approach for the treatment of dental caries or ...

Microscale Vortex-assisted Electroporator for Sequential Molecular Delivery

Electroporation has received increasing attention in the past years, because it is a very powerful technique for physically introducing non-permeant exogenous molecular probes into cells. This work reports a microfluidic electroporation platform capable of performing multiple molecule delivery to mammalian cells with precise and molecular-dependent parameter control. The system&#8217;s ability to isolate cells with uniform size distribution allows for less variation in electroporation efficiency per given electric field strength; hence enhanced sample viability. Moreover, its process visualization feature allows for observation of the fluorescent molecular uptake process in real-time, which permits prompt molecular delivery parameter adjustments in situ for efficiency enhancement. To show the vast capabilities of the reported platform, macromolecules with different sizes and electrical charges (e.g., Dextran with MW of 3,000 and 70,000 Da) were delivered to metastatic breast cancer cells with high delivery efficiencies (&#62;70%) for all tested molecules. The developed platform has proven its potential for use in the expansion of research fields where on-chip electroporation techniques can be beneficial.

A microfluidic vortex assisted electroporation platform was developed for sequential delivery of multiple molecules into identical cell populations with precise and independent dosage control. The system&#8217;s size based target cell purification step preceding electroporation aided to enhance molecular delivery efficiency and processed cell viability.

Microscala Vortex-assistita Elettroporatore per sequenziale molecolare di consegna

Electroporation has received increasing attention in the past years, because it is a very powerful technique for physically introducing non-permeant ...

An Injectable and Drug-loaded Supramolecular Hydrogel for Local Catheter Injection into the Pig Heart

Regeneration of lost myocardium is an important goal for future therapies because of the increasing occurrence of chronic ischemic heart failure and the limited access to donor hearts. An example of a treatment to recover the function of the heart consists of the local delivery of drugs and bioactives from a hydrogel. In this paper a method is introduced to formulate and inject a drug-loaded hydrogel non-invasively and side-specific into the pig heart using a long, flexible catheter. The use of 3-D electromechanical mapping and injection via a catheter allows side-specific treatment of the myocardium. To provide a hydrogel compatible with this catheter, a supramolecular hydrogel is used because of the convenient switching from a gel to a solution state using environmental triggers. At basic pH this ureido-pyrimidinone modified poly(ethylene glycol) acts as a Newtonian fluid which can be easily injected, but at physiological pH the solution rapidly switches into a gel. These mild switching conditions allow for the incorporation of bioactive drugs and bioactive species, such as growth factors and exosomes as we present here in both in vitro and in vivo experiments. The in vitro experiments give an on forehand indication of the gel stability and drug release, which allows for tuning of the gel and release properties before the subsequent application in vivo. This combination allows for the optimal tuning of the gel to the used bioactive compounds and species, and the injection system.

Supramolecular hydrogelators based on ureido-pyrimidinones allow full control over the macroscopic gel properties and the sol&#8211;gel switching behavior using pH. Here, we present a protocol for formulating and injecting such a supramolecular hydrogelator via a catheter delivery system for local delivery directly in relevant areas in the pig heart.

Un supramolecolare idrogel iniettabile and Drug-caricato per catetere iniezione locale nel cuore di maiale

Regeneration of lost myocardium is an important goal for future therapies because of the increasing occurrence of chronic ischemic heart failure and the ...

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Historically, most cellular processes have been studied in only 2 dimensions. While these studies have been informative about general cell signaling mechanisms, they neglect important cellular cues received from the structural and mechanical properties of the local microenvironment and extracellular matrix (ECM). To understand how cells interact within a physiological ECM, it is important to study them in the context of 3 dimensional assays. Cell migration, cell differentiation, and cell proliferation are only a few processes that have been shown to be impacted by local changes in the mechanical properties of a 3-dimensional ECM. Collagen I, a core fibrillar component of the ECM, is more than a simple structural element of a tissue. Under normal conditions, mechanical cues from the collagen network direct morphogenesis and maintain cellular structures. In diseased microenvironments, such as the tumor microenvironment, the collagen network is often dramatically remodeled, demonstrating altered composition, enhanced deposition and altered fiber organization. In breast cancer, the degree of fiber alignment is important, as an increase in aligned fibers perpendicular to the tumor boundary has been correlated to poorer patient prognosis1. Aligned collagen matrices result in increased dissemination of tumor cells via persistent migration2,3. The following is a simple protocol for embedding cells within a 3-dimensional, fibrillar collagen hydrogel. This protocol is readily adaptable to many platforms, and can reproducibly generate both aligned and random collagen matrices for investigation of cell migration, cell division, and other cellular processes in a tunable, 3-dimensional, physiological microenvironment.

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Collagen is a core component of the ECM, and provides essential cues for several cellular processes ranging from migration to differentiation and proliferation. Provided here is a protocol for embedding cells within 3D collagen hydrogels, and a more advanced technique for generating randomized or aligned collagen matrices using PDMS microchannels.

Preparazione del 3D collagene Gel e microcanali per lo studio delle interazioni 3D<em&gt; In Vivo</em

Historically, most cellular processes have been studied in only 2 dimensions.  While these studies have been informative about general cell signaling ...

Fabricating a Kidney Cortex Extracellular Matrix-Derived Hydrogel

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust mechanical support for in vitro cell culture but lack the necessary protein and ligand composition to elicit physiological behavior from cells. This manuscript describes a fabrication method for a kidney cortex ECM-derived hydrogel with proper mechanical robustness and supportive biochemical composition. The hydrogel is fabricated by mechanically homogenizing and solubilizing decellularized human kidney cortex ECM. The matrix preserves native kidney cortex ECM protein ratios while also enabling gelation to physiological mechanical stiffnesses. The hydrogel serves as a substrate upon which kidney cortex-derived cells can be maintained under physiological conditions. Furthermore, the hydrogel composition can be manipulated to model a diseased environment which enables the future study of kidney diseases.

Here we present a protocol to fabricate a kidney cortex extracellular matrix-derived hydrogel to retain the native kidney extracellular matrix (ECM) structural and biochemical composition. The fabrication process and its applications are described. Finally, a perspective on using this hydrogel to support kidney-specific cellular and tissue regeneration and bioengineering is discussed.

Fabbricando un rene corteccia extracellulare Matrix-derivato idrogel

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust ...

Costruire un idrogel collagene per la fornitura di Stem Cell-loaded Chitosan Microsfere

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