Questo studio è stato eseguito nel programma di ricerca dei materiali biomedici (BMM) dell&#39;istituto. BMM è cofinanziato dal ministero olandese degli Affari economici, dell&#39;agricoltura e dell&#39;innovazione. Il contributo finanziario della Hartstichting Nederlandse Si ringrazia.

1. Cultura di umani Vena safena Cells <ol><li> Isolare le cellule dalla vena safena magna, acquisita da un donatore in conformità alle linee guida per l&#39;uso materiale secondario, secondo il protocollo di Schnell et al. 9 e memorizzare questi in azoto liquido. Dalla parte della vena safena magna da un donatore pezzi tagliati di 2 x 2 mm a coltura in una piastra a sei pozzetti. Usare 2 pezzi per pozzetto. Generalmente sufficienti cellule possono essere ottenute per riempire circa 3 flaconcini con 0,25 x 10 6 cellule in azoto liquido. HVSCs sono caratterizzati come miofibroblasti, mostrando espressione di vimentina, nessuna espressione di desmin e una sottopopolazione che esprime muscolo liscio α actina 10. Successiva avviare il protocollo di scongelare le cellule dal azoto liquido per aumentare il numero di cellule. </li><li> Posizionare le cellule da una fiala in un pallone di coltura tissutale T75 e aggiungere mezzo di crescita (GM), costituito Avanzato DMEM, 50 ml di siero bovino fetale (FBS), 5 ml di penicillina /streptomicina e 5 ml di L-glutamax. Cambiare media ogni 2-3 giorni. </li><li> Cellule crescono confluente dopo circa 14 giorni. Negozio 0.5 x 10 6 cellule in un flaconcino in azoto liquido, indicato come passaggio 1. </li></ol> Nota: Congelamento delle cellule non è necessario quando la raccolta HVSCs, ma viene esclusivamente utilizzato per l&#39;archiviazione. <ol start="4"><li> Posizionare i HVSCs da una fiala in un pallone di coltura tissutale T175 e aggiungere GM. Cambiare media ogni 2-3 giorni. Cellule crescono confluente dopo circa 7 giorni. Negozio 3 x 10 6 cellule in un flaconcino in azoto liquido, indicato come passaggio 2. </li><li> Mettere le cellule da una fiala in un rollerbottle e aggiungere GM. Cambiare media ogni 2-3 giorni. Cellule crescono confluenti circa dopo 8 giorni. Uno rollerbottle contiene circa 20-30 x 10 6 cellule. Cellule di coltura fino al passaggio 6. Non utilizzare cellule di un passaggio superiore passaggio 9, poiché fenotipo cellulare può cambiare. </li></ol&gt; 2. Ingegneria di costrutti tissutali a base di fibrina <ol><li> Preparare colla siliconica miscelando l&#39;elastomero con il catalizzatore (10:1). Tagliare 7 x 3 mm segmenti rettangolari di velcro. Incollare il velcro in una piastra di coltura 6 bene, con membrane flessibili per formare una croce, e di lasciare uno spazio quadrato di 3 mm tra le strisce di velcro. </li></ol> Note: usare il lato morbido del velcro solo e questo lato verso l&#39;alto. Per incollare il velcro, coprono solo il velcro con colla al silicone, non si diffondono colla tutto il bene. Poiché le piastre di coltura hanno fondo membrana di silicone, usare qualcosa sotto la piastra bene per rinforzare le membrane flessibili, per garantire il facile incollaggio alla piastra. <ol start="2"><li> Asciugare la colla siliconica durante la notte in un forno a 60 ° C per garantire indurimento della colla. Sterilizzare con l&#39;aggiunta di 70% EtOH ai pozzetti ed incubare per 30 min. Risciacquare 3 volte con PBS e rimuovere tutti PBS dal welLS e il velcro. Mettere sotto UV per 30 minuti e mantenere sterile fino al momento dell&#39;uso. </li><li> Preparare Tissue Ingegneria medie (TM), costituito da GM e 130 mg di acido L-ascorbico 2-fosfato. </li><li> Aggiungere 1 mg / ml di acido caproico ε-Amino (ACA) alla TM. ACA è usato per i primi 7 giorni di coltura per impedire la fibrina dalla degradante 11. Alternativamente, aprotinina può essere utilizzato. </li><li> Per prevenire la formazione di ciuffo, fibrinogeno permettono di raggiungere la temperatura ambiente prima dell&#39;apertura. Senza ciuffi fibrinogeno si dissolverà più facilmente. Sciogliere fibrinogeno ad una concentrazione di 10 mg effettivo proteina / ml TM 7 integrato con ACA. Filtro sterile la soluzione di fibrinogeno, è necessaria la più filtri. Conservare in ghiaccio fino al momento dell&#39;uso. </li></ol> Nota: Per sciogliere mix fibrinogeno delicatamente per evitare la formazione di schiuma eccessiva. <ol start="6"><li> Sciogliere trombina ad una concentrazione di 10 UI / ml TM 7 integrato con ACA. Conservare in ghiaccio fino al momento dell&#39;uso. </li> &lt;/ Ol&gt; Nota: Stoccaggio in ghiaccio è necessaria per evitare la gelificazione precoce di trombina e fibrinogeno. <ol start="7"><li> Trypsinize le cellule, risospendere in GM e conteggio. </li><li> Utilizzare 15 x 10 6 cellule / ml per semina i costrutti tissutali fibrina-based (concentrazione basato su metodi di ingegneria tissutale valvole cardiache 7). Un unico gel consiste di 100 pl gof el, e quindi di 1,5 x 10 6 cellule. Mettere 1,5 x 10 6 cellule in una provetta da centrifuga, risultante in altrettanti tubi da centrifuga come il numero di gel che verranno fatte. </li><li> Centrifugare le cellule a 350 xg per 7 minuti e scartare il surnatante. Risospendere le cellule in 50 ml di trombina. Aggiungere 4 ml di blu fluorescente microsfere di polistirolo a questa sospensione. Mettere 50 ml di fibrinogeno in un flaconcino. Utilizzare una pipetta a prendere il 50 trombina microlitri con le cellule. Aumentare il volume della pipetta 100 microlitri. Pipettare la soluzione 50 ml di trombina concellule nel flaconcino con il fibrinogeno per mescolare la trombina e fibrinogeno, e occupano la miscela di 100 ml. </li></ol> Nota: La fluorescente microsfere di polistirolo vengono utilizzate come marcatori di riferimento interni per l&#39;analisi dell&#39;immagine. Quando trombina e fibrinogeno miscelazione, impediscono la formazione di bolle d&#39;aria pipettando accuratamente la miscela. Bolle d&#39;aria si tradurrà in fori nel gel di fibrina. <ol start="10"><li> Pipettare il composto in gel e tra le strisce di velcro. Il tipico tempo di gelificazione per i gel di fibrina, una volta che i componenti sono miscelati, è dell&#39;ordine di 20 sec. </li></ol> Note: Fate questo il più rapidamente possibile per evitare la gelificazione prima che la miscela viene pipettato nella piastra bene. Pratica prima di utilizzare le cellule e perline. <ol start="11"><li> Incubare sospensioni per 30 min a 37 ° C in un umidificata 95% aria / 5% CO 2 incubatore per consentire gelificazione prima di aggiungere cultura TM con ACA. </li><li> Sostituire il TM con ACA ogni 2-3 giorni per i primi 7 giorni. I gel sono abbastanza stabili dopo una settimana per essere coltivato senza ACA. Dopo la prima settimana TM sostituire ogni 2-3 giorni. Aggiungere 6 ml di TM per pozzetto. Il giorno 12 celle hanno prodotto abbastanza collagene per la visualizzazione. </li></ol> 3. Applicazione Strain e indurre cambiamenti in Strain e Vincoli <ol><li> Deformazione statica viene applicato direttamente dalle cellule, dovute alla trazione cella e compattazione 12. Per indurre il collagene di riorganizzazione, ha tagliato il tessuto costruire sciolto da due strisce di velcro in una sola direzione. Ciò si traduce in vincoli unidirezionali (Figura 1A). Eseguire questo il giorno 12, dal momento che il costrutto è quindi abbastanza stabile e il collagene è stato depositato. </li><li> Applicare deformazione ciclica applicando un vuoto al fondo delle piastre di coltura con membrane flessibili, con l&#39;utilizzo del sistema FX4000T Flexcell. Posizionare le piastre su una piastra di base, sulla parte superiore del post carico (che sono una parte del secolodispositivo di carico ciclico e). La pompa si applica un vuoto sulla membrana e tira in tal modo la membrana sopra il post rettangolare giace sotto. Dovuto alle allegati sagomate trasversali del tessuto costruisce alla membrana (attraverso il velcro) e il post rettangolare il ceppo ciclico applicato è monoassiale (Figura 1B). </li><li> Inizialmente, utilizzare la cultura statica per 5 giorni per raggiungere l&#39;integrità meccanica iniziale, prima dell&#39;applicazione di sforzo ciclico a partire dal giorno 6. Programma un protocollo macchia ciclico nel regolatore per la pompa del vuoto. Ad esempio, utilizzare un protocollo ceppo ciclica intermittente precedentemente stabilito, con la direzione monoassiale 13. Questo consiste in un ceppo intermittente di un&#39;onda sinusoidale con 1 Hz, tendendo da 0 a 5% di deformazione, per periodi di 3 ore, alternate con periodi di riposo 3 ore. Eseguire questo protocollo sforzo ciclico per 7 giorni, inducendo una organizzazione collagene allineate. </li><li> In genere dopo 12 giorni HVSCs hanno prodotto un organizatio collagene allineaten. Dopo aver raggiunto una organizzazione collagene allineata, cambiare la direzione di deformazione ciclica uniassiale, per essere perpendicolare alla direzione ceppo originale. Fare questo girando i posti rettangolari di 90 °. </li></ol> 4. Visualizzare cellule e collagene <ol><li> Per visualizzare attivo, in tempo reale rimodellamento del collagene, i campioni di etichette con le sonde che non interferiscono con la vitalità delle cellule o la formazione di collagene. Utilizzare sonde a macchiare fluorescently citoplasma della cellula e collagene. </li><li> In alternativa generazione di seconda armonica con microscopio confocale a scansione laser può essere usato per visualizzare le strutture di collagene mediante autofluorescenza 14, con eccitazione a 780 nm e il rilevamento tra 500-550 nm. </li><li> Rimuovere le piastre di coltura dalla configurazione e l&#39;incubatore, per i campioni ciclicamente tese durante il periodo di riposo di 3 ore, e trasportarli in un microscopio confocale a scansione laser per visualizzare le cellule e collagene. </li></ol>

<ol>
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T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrin+degradation+enhances+vascular+smooth+muscle+cell+proliferation+and+matrix+deposition+in+fibrin-based+tissue+constructs+fabricated+in+vitro.">Fibrin degradation enhances vascular smooth muscle cell proliferation and matrix deposition in fibrin-based tissue constructs fabricated in vitro.</a> Tissue Eng. Part A. 16, 3261-3270 (2010).</li><li>John, J., Quinlan, A. T., Silvestri, C., Billiar, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Boundary+stiffness+regulates+fibroblast+behavior+in+collagen+gels.">Boundary stiffness regulates fibroblast behavior in collagen gels.</a> Ann. Biomed. Eng. 38, 658-673 (2010).</li><li>Rubbens, M. 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V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strain-induced+Collagen+Organization+at+the+Micro-level+in+Fibrin-based+Engineered+Tissue+Constructs.">Strain-induced Collagen Organization at the Micro-level in Fibrin-based Engineered Tissue Constructs.</a> Ann. Biomed. Eng. 41, 763-774 (2013).</li><li>Merryman, W. 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=Correlation+between+heart+valve+interstitial+cell+stiffness+and+transvalvular+pressure:+implications+for+collagen+synthesis.">Correlation between heart valve interstitial cell stiffness and transvalvular pressure: implications for collagen synthesis.</a> Am. J. Physiol. 290, (2006).</li><li>Ingber, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+cellular+mechanotransduction+to+biologically+inspired+engineering:+2009+Pritzker+Award+Lecture,+BMES+Annual+Meeting+October+10,+2009.">From cellular mechanotransduction to biologically inspired engineering: 2009 Pritzker Award Lecture, BMES Annual Meeting October 10, 2009.</a> Ann. Biomed. Eng. 38, 1148-1161 (2009).</li><li>Sander, E. A., Barocas, V. H., Tranquillo, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initial+fiber+alignment+pattern+alters+extracellular+matrix+synthesis+in+fibroblast-populated+fibrin+gel+cruciforms+and+correlates+with+predicted+tension.">Initial fiber alignment pattern alters extracellular matrix synthesis in fibroblast-populated fibrin gel cruciforms and correlates with predicted tension.</a> Ann. Biomed. Eng. 39, 714-729 (2010).</li><li>van der Schaft, D. 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=Engineering+Skeletal+Muscle+Tissues+from+Murine+Myoblast+Progenitor+Cells+and+Application+of+Electrical+Stimulation.">Engineering Skeletal Muscle Tissues from Murine Myoblast Progenitor Cells and Application of Electrical Stimulation.</a> J. Vis. Exp. (73), e4267 (2013).</li></ol>

Gli autori dichiarano di non avere interessi finanziari in competizione.

Il sistema modello descritto costrutti di fibrina cellula-popolate ha un grande potenziale per lo studio della cellula e collagene (ri) organizzazione (de Jonge et al. 15), ad esempio da utilizzare per scopi di ingegneria tissutale. Utilizzando fibrina come il vettore cellula iniziale, dopo la degradazione della fibrina, un tessuto è creato con cellule e unica matrice endogena. In questo modo, le cellule sono stimolate a reagire a ceppo, o statico o ciclico in natura, applicando forze contr...

Tessuti cardiovascolari hanno una funzione di primo piano portante. In particolare, i contenuti e l&#39;organizzazione delle fibre di collagene nella matrice extracellulare contribuire alle proprietà portanti e dominare la forza del tessuto complessivo 1. In ingegneria dei tessuti è utilizzato condizionata meccanica del costrutto - costituiti in genere da (ciclico) regimi sforzare - per migliorare l&#39;organizzazione dei tessuti e le proprietà meccaniche 2,3. Piena comprensione di organizzazione collagene deformazione indotta in geometrie complesse dei tessuti per creare tessuti con architettura collagene predefinito non è stato ancora raggiunto. Ciò è dovuto principalmente alla nostra conoscenza limitata del rimodellamento del collagene nei tessuti sviluppo. Modelli esistenti forniscono principalmente informazioni sul risultato netto finale di rimodellamento del collagene con l&#39;utilizzo di sforzo statico 4-6. Qui forniamo un sistema modello altamente sintonizzabile che consente lo studio del collagene (ri) organizzazione in un modo tempo reale, in 3D, sotto influenzadi deformazione statica o ciclico. I costrutti tissutali sono basati fibrina, assicurando che tutta collagene nel costrutto è endogena. Organizzazione cellulare e collagene nei costrutti viene visualizzato, e un sistema di riferimento interno ci permette di trasferire le cellule e le strutture di collagene per l&#39;analisi time-lapse. In questo protocollo verrà descritto l&#39;uso del sistema modello per umani Vena safena Cells (HVSCs), poiché queste cellule sono noti per la loro maggiore produzione di matrice extracellulare e la capacità di rimodellare la matrice e il nostro uso stabilita in ingegnerizzati tessuti cardiovascolari 7, basato sul lavoro di de Jonge et al. 8

<table border="1">
	<tbody>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Culture plastic</td>
			<td>Greiner</td>
			<td></td>
			<td>Includes culture flasks and pipettes</td>
		</tr>
		<tr>
			<td>Advanced DMEM</td>
			<td>Gibco</td>
			<td>12491</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Greiner</td>
			<td>758075</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>Gibco</td>
			<td>10378016</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMax</td>
			<td>Gibco</td>
			<td>35050-079</td>
			<td></td>
		</tr>
		<tr>
			<td>Elastomer and curing agent</td>
			<td>Dow Corning Corporation</td>
			<td>3097358-1004</td>
			<td>Silastic MDX 4-4210#</td>
		</tr>
		<tr>
			<td>Velcro</td>
			<td>Regular store</td>
			<td></td>
			<td>You can buy this at a regular store, only use the soft side</td>
		</tr>
		<tr>
			<td>Bioflex culture plates</td>
			<td>Flexcell Int</td>
			<td>BF-3001U</td>
			<td>Untreated</td>
		</tr>
		<tr>
			<td>L-Ascorbic Acid 2-phosphatase</td>
			<td>Sigma</td>
			<td>A8960</td>
			<td></td>
		</tr>
		<tr>
			<td>&#949;-Amino Caproic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>D7754</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine thrombin</td>
			<td>Sigma</td>
			<td>T4648</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine fibrinogen</td>
			<td>Sigma</td>
			<td>F8630</td>
			<td></td>
		</tr>
		<tr>
			<td>0.45 syringe filter</td>
			<td>Whatmann (Schleicher and Scheul)</td>
			<td>10462100</td>
			<td></td>
		</tr>
		<tr>
			<td>Polystyrene microspheres</td>
			<td>Invitrogen</td>
			<td>F-8829</td>
			<td>Blue fluorescent, 10 &#956;m diameter</td>
		</tr>
		<tr>
			<td>Flexcell FX-4000T</td>
			<td>Flexcell Int</td>
			<td></td>
			<td>Includes rectangular loading posts</td>
		</tr>
		<tr>
			<td>Cell Tracker Orange</td>
			<td>Invitrogen Molecular Probes</td>
			<td>C2927</td>
			<td></td>
		</tr>
		<tr>
			<td>CNA35-OG488</td>
			<td></td>
			<td></td>
			<td>Cordially provided by the Laboratory for Macromolecular and Organic Chemistry, Department of Biomedical Engineering, Eindhoven University of Technology</td>
		</tr>
		<tr>
			<td>Confocal laser scanning microscope</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td>LSM 510 Meta laser scanning microscope and Two-Photon-LSM mode</td>
		</tr>
		<tr>
			<td>Amphotericin</td>
			<td>Gibco</td>
			<td>15290-018</td>
			<td>Needed for cell isolation</td>
		</tr>
	</tbody>
</table>

engineering fibrin-based tissue constructs from myofibroblasts and application of constraints and strain to induce cell and collagen reorganization

Contenuto di collagene e di organizzazione per lo sviluppo di tessuti di collagene possono essere influenzati dai ceppi dei tessuti locali e di vincolo dei tessuti. Ingegneri dei tessuti hanno lo scopo di utilizzare questi principi per creare tessuti con architetture collagene predefiniti. Una piena comprensione delle esatte sottostanti processi di rimodellamento del collagene per controllare l&#39;architettura del tessuto finale, però, è carente. In particolare, si conosce poco l&#39;orientamento (ri) delle fibre di collagene in risposta ai cambiamenti nelle condizioni di carico meccanico del tessuto. Abbiamo sviluppato un sistema modello in vitro, composto da bi-costretti myofibroblast testa di serie fibrina costrutti, per chiarire ulteriormente il collagene (ri) orientamento in risposta ad i), tornando biassiale a condizioni di carico statico monoassiali e ii) ciclica monoassiale carico del bi-vincolato costrutti prima e dopo un cambio di direzione del carico, con l&#39;uso del dispositivo di caricamento Flexcell FX4000T. Time-lapse imaging confocale viene utilizzato a visualize collagene orientamento (ri) in modo non distruttivo. Organizzazione cellulare e collagene nei costrutti possono essere visualizzati in tempo reale, e di un sistema di riferimento interno ci permette di trasferire le cellule e le strutture di collagene per l&#39;analisi time-lapse. Vari aspetti del sistema modello può essere regolata, come fonte cellulare o l&#39;uso di cellule sane e malate. Additivi possono essere utilizzati per chiarire ulteriormente i meccanismi sottostanti rimodellamento del collagene, per esempio con l&#39;aggiunta di MMPs o blocco integrine. Forma e dimensioni del costrutto possono essere facilmente adattati alle esigenze specifiche, risultante in un sistema modello per lo studio altamente sintonizzabile organizzazione cellulare e collagene (ri).

Questo sistema consente di effettuare la coltura myofibroblast teste di serie gel di fibrina. Figura 1A mostra un tessuto coltivato prima sotto vincoli biassiali statiche. Vincoli dei tessuti vengono rilasciati tagliando il gel di fibrina da due vincoli, per creare vincoli statici monoassiali e compatta del tessuto e rimodella dopo (Figura 1A). Per deformazione ciclica, il tessuto viene coltivato sotto vincoli biassiali statiche pure. Dopo 5 giorni deformazione uniassiale ciclico può e...

Watch this Scientific Journal Video about Engineering Fibrin-based Tissue Constructs from Myofibroblasts and Application of Constraints and Strain to Induce Cell and Collagen Reorganization at JoVE.co

Engineering Fibrin-based Tissue Constructs from Myofibroblasts and Application of Constraints and Strain to Induce Cell and Collagen Reorganization

Questo sistema modello parte da un gel di fibrina myofibroblast popolata che può essere usato per studiare collagene endogeno (ri) organizzazione in tempo reale in modo non distruttivo. Il sistema modello è molto sintonizzabile, in quanto può essere usato con sorgenti cellulari differenti, additivi medie, e può essere facilmente adattato alle esigenze specifiche.

Ingegneria fibrina a base di tessuto costrutti da miofibroblasti e applicazione di vincoli e la tensione per indurre Organizzazione cellulare e collagene (Re)

Collagen content and organization in developing collagenous tissues can be influenced by local tissue strains and tissue constraint. Tissue engineers aim ...

engineering-fibrin-based-tissue-constructs-from-myofibroblasts

Research

JoVE Journal

Bioengineering

10.8K Views.  Eindhoven University of Technology.  Questo sistema modello parte da un gel di fibrina myofibroblast popolata che può essere usato per studiare collagene endogeno (ri) organizzazione in tempo reale in modo non distruttivo. Il sistema modello è molto sintonizzabile, in quanto può essere usato con sorgenti cellulari differenti, additivi medie, e può essere facilmente adattato alle esigenze specifiche. 

Video: Engineering Fibrin-based Tissue Constructs from Myofibroblasts and Application of Constraints and Strain to Induce Cell and Collagen Reorganization

Directed Cellular Self-Assembly to Fabricate Cell-Derived Tissue Rings for Biomechanical Analysis and Tissue Engineering

Each year, hundreds of thousands of patients undergo coronary artery bypass surgery in the United States.1 Approximately one third of these patients do not have suitable autologous donor vessels due to disease progression or previous harvest. The aim of vascular tissue engineering is to develop a suitable alternative source for these bypass grafts. In addition, engineered vascular tissue may prove valuable as living vascular models to study cardiovascular diseases. Several promising approaches to engineering blood vessels have been explored, with many recent studies focusing on development and analysis of cell-based methods.2-5 Herein, we present a method to rapidly self-assemble cells into 3D tissue rings that can be used in vitro to model vascular tissues.
To do this, suspensions of smooth muscle cells are seeded into round-bottomed annular agarose wells. The non-adhesive properties of the agarose allow the cells to settle, aggregate and contract around a post at the center of the well to form a cohesive tissue ring.6,7 These rings can be cultured for several days prior to harvesting for mechanical, physiological, biochemical, or histological analysis. We have shown that these cell-derived tissue rings yield at 100-500 kPa ultimate tensile strength8 which exceeds the value reported for other tissue engineered vascular constructs cultured for similar durations (&lt;30 kPa).9,10 Our results demonstrate that robust cell-derived vascular tissue ring generation can be achieved within a short time period, and offers the opportunity for direct and quantitative assessment of the contributions of cells and cell-derived matrix (CDM) to vascular tissue structure and function.

This article outlines a versatile method to create cell-derived tissue rings by cellular self-assembly. Smooth muscle cells seeded into ring-shaped agarose wells aggregate and contract to form robust three-dimensional (3D) tissues within 7 days. Millimeter-scale tissue rings are conducive to mechanical testing and serve as building blocks for tissue assembly.

Diretto Cellular Self-Assembly di fabbricare Cell-Derived Anelli tessuto per l&#39;analisi biomeccanica e di ingegneria dei tessuti

Each year, hundreds of thousands of patients undergo coronary artery bypass surgery in the United States.1 Approximately one third of these patients do ...

Ricerca

Bioingegneria

Non-contact, Label-free Monitoring of Cells and Extracellular Matrix using Raman Spectroscopy

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, currently available routine methods require processing steps and alter sample integrity. Raman spectroscopy is a fast method that enables the measurement of biological samples without the need for further processing steps. This laser-based technology detects the inelastic scattering of monochromatic light.6 As every chemical vibration is assigned to a specific Raman band (wavenumber in cm-1), each biological sample features a typical spectral pattern due to their inherent biochemical composition.7-9 Within Raman spectra, the peak intensities correlate with the amount of the present molecular bonds.1 Similarities and differences of the spectral data sets can be detected by employing a multivariate analysis (e.g. principal component analysis (PCA)).10
Here, we perform Raman spectroscopy of living cells and native tissues. Cells are either seeded on glass bottom dishes or kept in suspension under normal cell culture conditions (37 &deg;C, 5% CO2) before measurement. Native tissues are dissected and stored in phosphate buffered saline (PBS) at 4 &deg;C prior measurements. Depending on our experimental set up, we then either focused on the cell nucleus or extracellular matrix (ECM) proteins such as elastin and collagen. For all studies, a minimum of 30 cells or 30 random points of interest within the ECM are measured. Data processing steps included background subtraction and normalization.

Raman spectroscopy is a suitable technique for the non-contact, label-free analysis of living cells, tissue-engineered constructs and native tissues. Source-specific spectral fingerprints can be generated and analyzed using multivariate analysis.

Senza contatto, Label-free monitoraggio delle cellule e matrice extracellulare mediante Spettroscopia Raman

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, ...

Engineering Skeletal Muscle Tissues from Murine Myoblast Progenitor Cells and Application of Electrical Stimulation

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to study pressure ulcers, for regenerative medicine and as a meat alternative 1. The first reported 3D muscle constructs have been made many years ago and pioneers in the field are Vandenburgh and colleagues 2,3. Advances made in muscle tissue engineering are not only the result from the vast gain in knowledge of biochemical factors, stem cells and progenitor cells, but are in particular based on insights gained by researchers that physical factors play essential roles in the control of cell behavior and tissue development. State-of-the-art engineered muscle constructs currently consist of cell-populated hydrogel constructs. In our lab these generally consist of murine myoblast progenitor cells, isolated from murine hind limb muscles or a murine myoblast cell line C2C12, mixed with a mixture of collagen/Matrigel and plated between two anchoring points, mimicking the muscle ligaments. Other cells may be considered as well, e.g. alternative cell lines such as L6 rat myoblasts 4, neonatal muscle derived progenitor cells 5, cells derived from adult muscle tissues from other species such as human 6 or even induced pluripotent stem cells (iPS cells) 7. Cell contractility causes alignment of the cells along the long axis of the construct 8,9 and differentiation of the muscle progenitor cells after approximately one week of culture. Moreover, the application of electrical stimulation can enhance the process of differentiation to some extent 8. Because of its limited size (8 x 2 x 0.5 mm) the complete tissue can be analyzed using confocal microscopy to monitor e.g. viability, differentiation and cell alignment. Depending on the specific application the requirements for the engineered muscle tissue will vary; e.g. use for regenerative medicine requires the up scaling of tissue size and vascularization, while to serve as a meat alternative translation to other species is necessary.

Engineered muscle tissue has great potential in regenerative medicine, as disease model and also as an alternative source for meat. Here we describe the engineering of a muscle construct, in this case from mouse myoblast progenitor cells, and the stimulation by electrical pulses.

Ingegneria tessuti muscolo scheletrico di cellule murine progenitrici mioblasti e di applicazione di stimolazione elettrica

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to ...

Tissue Engineering of Tumor Stromal Microenvironment with Application to Cancer Cell Invasion

3D organotypic cultures of epithelial cells on a matrix embedded with mesenchymal cells are widely used to study epithelial cell differentiation and invasion. Rat tail type I collagen and/or matrix derived from Engelbreth-Holm-Swarm mouse sarcoma cells have been traditionally employed as the substrates to model the matrix or stromal microenvironment into which mesenchymal cells (usually fibroblasts) are populated. Although experiments using such matrices are very informative, it can be argued that due to an overriding presence of a single protein (such as in type I Collagen) or a high content of basement membrane components and growth factors (such as in matrix derived from mouse sarcoma cells), these substrates do not best reflect the contribution to matrix composition made by the stromal cells themselves. To study native matrices produced by primary dermal fibroblasts isolated from patients with a tumor prone, genetic blistering disorder (recessive dystrophic epidermolysis bullosa), we have adapted an existing native matrix protocol to study tumor cell invasion. Fibroblasts are induced to produce their own matrix over a prolonged period in culture. This native matrix is then detached from the culture dish and epithelial cells are seeded onto it before the entire coculture is raised to the air-liquid interface. Cellular differentiation and/or invasion can then be assessed over time. This technique provides the ability to assess epithelial-mesenchymal cell interactions in a 3D setting without the need for a synthetic or foreign matrix with the only disadvantage being the prolonged period of time required to produce the native matrix. Here we describe the application of this technique to assess the ability of a single molecule expressed by fibroblasts, type VII collagen, to inhibit tumor cell invasion.

Tissue engineered fibroblast-derived native matrix is an emerging tool to generate a stromal substrate which supports epithelial cell proliferation and differentiation. Here a protocol applying this methodology to assess the impact of different stromal cell types on tumor cell biology is presented.

Ingegneria dei tessuti del tumore stromale Microenvironment con l&#39;applicazione di Invasion Cancer Cell

3D organotypic cultures of epithelial cells on a matrix embedded with mesenchymal cells are widely used to study epithelial cell differentiation and ...

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled.
The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The &#8220;static bioreactor&#8221; provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.

In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described.

Ingegneria 3D Cellularized collagene Gel per Vascolare Rigenerazione Tissutale

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

Sandwich-like Microenvironments to Harness Cell/Material Interactions

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. However in vivo, most of the cells are completely surrounded by the extracellular matrix (ECM), resulting in a three-dimensional (3D) distribution of receptors. This may trigger differences in the outside-in signaling pathways and thus in cell behavior.
This article shows that stimulating the dorsal receptors of cells already adhered to a 2D substrate by overlaying a film of a new material (a sandwich-like culture) triggers important changes with respect to standard 2D cultures. Furthermore, the simultaneous excitation of ventral and dorsal receptors shifts cell behavior closer to that found in 3D environments. Additionally, due to the nature of the system, a sandwich-like culture is a versatile tool that allows the study of different parameters in cell/material interactions, e.g., topography, stiffness and different protein coatings at both the ventral and dorsal sides. Finally, since sandwich-like cultures are based on 2D substrates, several analysis procedures already developed for standard 2D cultures can be used normally, overcoming more complex procedures needed for 3D systems.

The following protocol describes the procedure to assemble sandwich-like cultures to be used as an intermediate stage between bi-dimensional (2D) and three-dimensional (3D) cellular environments. The engineered systems can have applications in microscopy, biomechanics, biochemistry and cell biology assays.

Microambienti sandwich di sfruttare Cellulari / Materiale Interazioni

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. ...

Treatment of Ligament Constructs with Exercise-conditioned Serum: A Translational Tissue Engineering Model

In vitro experiments are essential to understand biological mechanisms; however, the gap between monolayer tissue culture and human physiology is large, and translation of findings is often poor. Thus, there is ample opportunity for alternative experimental approaches. Here we present an approach in which human cells are isolated from human anterior cruciate ligament tissue remnants, expanded in culture, and used to form engineered ligaments. Exercise alters the biochemical milieu in the blood such that the function of many tissues, organs and bodily processes are improved. In this experiment, ligament construct culture media was supplemented with experimental human serum that has been &#39;conditioned&#39; by exercise. Thus the intervention is more biologically relevant since an experimental tissue is exposed to the full endogenous biochemical milieu, including binding proteins and adjunct compounds that may be altered in tandem with the activity of an unknown agent of interest. After treatment, engineered ligaments can be analyzed for mechanical function, collagen content, morphology, and cellular biochemistry. Overall, there are four major advantages versus traditional monolayer culture and animal models, of the physiological model of ligament tissue that is presented here. First, ligament constructs are three-dimensional, allowing for mechanical properties (i.e., function) such as ultimate tensile stress, maximal tensile load, and modulus, to be quantified. Second, the enthesis, the interface between boney and sinew elements, can be examined in detail and within functional context. Third, preparing media with post-exercise serum allows for the effects of the exercise-induced biochemical milieu, which is responsible for the wide range of health benefits of exercise, to be investigated in an unbiased manner. Finally, this experimental model advances scientific research in a humane and ethical manner by replacing the use of animals, a core mandate of the National Institutes of Health, the Center for Disease Control, and the Food and Drug Administration.

We present a model of ligament tissue in which three-dimensional constructs are treated with the human exercise-conditioned serum and analyzed for collagen content, function, and cellular biochemistry.

Trattamento delle strutture ligamentali con serum esercitato-condizionato: un modello di ingegneria tessuto transazionale

In vitro experiments are essential to understand biological mechanisms; however, the gap between monolayer tissue culture and human physiology is large, ...

Recombinant Collagen I Peptide Microcarriers for Cell Expansion and Their Potential Use As Cell Delivery System in a Bioreactor Model

Tissue engineering is a promising field, focused on developing solutions for the increasing demand on tissues and organs regarding transplantation purposes. The process to generate such tissues is complex, and includes an appropriate combination of specific cell types, scaffolds, and physical or biochemical stimuli to guide cell growth and differentiation. Microcarriers represent an appealing tool to expand cells in a three-dimensional (3D) microenvironment, since they provide higher surface-to volume ratios and mimic more closely the in vivo situation compared to traditional two-dimensional methods. The vascular system, supplying oxygen and nutrients to the cells and ensuring waste removal, constitutes an important building block when generating engineered tissues. In fact, most constructs fail after being implanted due to lacking vascular support. In this study, we present a protocol for endothelial cell expansion on recombinant collagen-based microcarriers under dynamic conditions in spinner flask and bioreactors, and we explain how to determine in this setting cell viability and functionality. In addition, we propose a method for cell delivery for vascularization purposes without additional detachment steps necessary. Furthermore, we provide a strategy to evaluate the cell vascularization potential in a perfusion bioreactor on a decellularized&#160;biological matrix. We believe that the use of the presented methods could lead to the development of new cell-based therapies for a large range of tissue engineering applications in the clinical practice.

We propose a cell expansion protocol on macroporous microcarriers and their use as delivery system in a perfusion bioreactor to seed a decellularized tissue matrix. We also include different techniques to determine cell proliferation and viability of cells cultured on microcarriers. Furthermore, we demonstrate functionality of cells after bioreactor cultures.

Ricombinante collagene Peptide Microcarriers per l'espansione delle cellule e loro potenziale utilizzo come sistema di consegna di cella in un bioreattore modello

Tissue engineering is a promising field, focused on developing solutions for the increasing demand on tissues and organs regarding transplantation ...

A Novel Tenorrhaphy Suture Technique with Tissue Engineered Collagen Graft to Repair Large Tendon Defects

Surgical management of large tendon defects with tendon grafts is challenging, as there are a finite number of sites where donors can be readily identified and used. Currently, this gap is filled with tendon auto-, allo-, xeno-, or artificial grafts, but clinical methods to secure them are not necessarily translatable to animals because of the scale. In order to evaluate new biomaterials or study a tendon graft made up of collagen type 1, we have developed a modified suture technique to help maintain the engineered tendon in alignment with the tendon ends. Mechanical properties of these grafts are inferior to the native tendon. To incorporate engineered tendon into clinically relevant models of loaded repair, a strategy was adopted to offload the tissue engineered tendon graft and allow for the maturation and integration of the engineered tendon in vivo until a mechanically sound neo-tendon was formed. We describe this technique using incorporation of the collagen type 1 tissue engineered tendon construct.

In this paper, we present an in vitro and in situ protocol to repair a tendon gap of up to 1.5 cm by filling it with engineered collagen graft. This was performed by developing a modified suture technique to take the mechanical load until the graft matures into the host tissue.

Una nuova tecnica di sutura Tenorrhaphy con innesto di collagene ingegnerizzato per riparare grandi difetti tendinei

Surgical management of large tendon defects with tendon grafts is challenging, as there are a finite number of sites where donors can be readily ...

Pancreatic Tissue-Derived Extracellular Matrix Bioink for Printing 3D Cell-Laden Pancreatic Tissue Constructs

The transplantation of pancreatic islets is a promising treatment for patients who suffer from type 1 diabetes accompanied by hypoglycemia and secondary complications. However, islet transplantation still has several limitations such as the low viability of transplanted islets due to poor islet engraftment and hostile environments. In addition, the insulin-producing cells differentiated from human pluripotent stem cells have limited ability to secrete sufficient hormones that can regulate the blood glucose level; therefore, improving the maturation by culturing cells with proper microenvironmental cues is strongly required. In this article, we elucidate protocols for preparing a pancreatic tissue-derived decellularized extracellular matrix (pdECM) bioink to provide a beneficial microenvironment that can increase glucose sensitivity of pancreatic islets, followed by describing the processes for generating 3D pancreatic tissue constructs using a microextrusion-based bioprinting technique.

Decellularized extracellular matrix (dECM) can provide suitable microenvironmental cues to recapitulate the inherent functions of target tissues in an engineered construct. This article elucidates the protocols for the decellularization of pancreatic tissue, evaluation of pancreatic tissue-derived dECM bioink, and generation of 3D pancreatic tissue constructs using a bioprinting technique.

Bioinchiostro a matrice extracellulare derivato dal tessuto pancreatico per la stampa di costrutti di tessuto pancreatico 3D Cell-Laden

The transplantation of pancreatic islets is a promising treatment for patients who suffer from type 1 diabetes accompanied by hypoglycemia and secondary ...