Name: engineered 3d silk-collagen-based model of polarized neural tissue
Uploaded: 2015-10-23T14:00:00
Duration: 6 min 17 s
Description: Watch this Scientific Journal Video about Engineered 3D Silk-collagen-based Model of Polarized Neural Tissue at JoVE.com

We thank the laboratory of Dr. Stephen Moss for providing embryonic rat brain tissues. M.D.T. designed the original protocol. This work was funded by National Institutes of Health P41 Tissue Engineering Resource Center Grant EB002520. K.C. was supported with Postdoctoral Fellowship from German Research Foundation (DFG) (CH 1400/2-1).

Il protocollo di isolamento tessuto cerebrale è stato approvato dal Istituzionale Cura e Comitato uso degli animali Tufts University ed è conforme con il National Institutes of Health Guide per la cura e l&#39;uso di animali da laboratorio (Institutional Animal Care and Use Committee B2011-45). 1. Silk Scaffold Preparazione <ol><li> Preparazione della soluzione da seta Bombyx mori bozzoli, come descritto in precedenza 19,20. <ol><li> Tagliare ogni bozzolo in 8 pezzi uguali con le forbici. Usare circa 11 bozzoli per 5 g di bozzoli frammentati. (15 min) </li><li> Preparare 2 L di 0,02 M Na 2 CO 3 soluzione e portarla a bollore con un piatto caldo. (15 min) </li><li> Pesare 5 g di bozzoli frammentate e lessateli in Na 2 CO 3 soluzione per 30 minuti. Mescolare la seta bollente ogni paio di minuti con una spatola. Questa fase, detta anche de-gommatura, purifica fibroina di seta da proteine ​​idrofili, sericins. (30 min) </li><li&gt; Strizzare fibroina a mano e sciacquarlo con acqua distillata almeno tre volte per lavare qualsiasi sericina e prodotti chimici rimanente. (5 minuti) </li><li> Posizionare la fibroina bagnato su una capsula di Petri e asciugare l&#39;estratto fibroina nella cappa a flusso O / N. </li><li> Il giorno dopo pesare la massa fibroina secca e posizionare il fibroina in un bicchiere di vetro. (15 min) </li><li> Per sciogliere la fibroina in soluzione 9,3 M LiBr, calcolare il volume necessario (ml) di LiBr moltiplicando la massa della fibroina secca per 4. Versare lentamente soluzione LiBr sul fibroina di seta e immergere tutte le fibre di fibroina utilizzando una spatola. Coprire il becher per evitare l&#39;evaporazione e posizionarlo a 60 ° C per 4 ore per consentire alle fibre di sciogliere. (15 min) </li><li> Usando la siringa, raccogliere la soluzione fibroina dal bicchiere e caricarlo nei MWCO 3.500 cassette di dialisi. Eseguire la dialisi contro acqua distillata per 48 ore. Cambiare l&#39;acqua ogni due ore. </li><li> Utilizzando la siringa, raccogliere la soluzione da fibroinale cassette in 50 ml provette coniche e centrifughi due volte a 9.000 giri (~ 12,700 xg) a 4 ° C per 20 min. Versare il surnatante in una nuova provetta dopo ogni fase di centrifugazione e scartare il pellet. (40 min) </li><li> Misurare la concentrazione della fibroina stimando il peso secco. Versare 1 ml di soluzione di seta in una barca pesare. Essiccare il campione in stufa a 60 ° C per 2 ore. Pesare il fibroina di seta secca e calcolare la concentrazione della soluzione di fibroina di seta moltiplicando il peso ottenuto per 100. La concentrazione previsto di soluzione seta è 6-9% (w / v). </li><li> Regolare la concentrazione di seta al 6% (w / v) diluendo in acqua distillata. Fermata: La fibroina seta liquida può essere conservato a 4 ° C fino ad un mese in un contenitore chiuso. </li></ol></li><li> Preparazione di scaffold porosi di soluzione di seta. <ol><li> Setacciare la granulari NaCl per separare i granuli di dimensioni 500-600 micron, che verrà utilizzato nei passaggi successivi. Eliminare il granulos dimensioni di sotto di 500 micron e sopra 600 micron. (15 min) </li><li> Versare 30 ml di soluzione di seta 6% nello stampo politetrafluoroetilene (PTFE) (Figura 1). Spargere delicatamente 60 g di NaCl setacciata sulla seta. Premere il contenitore per ottenere uno strato uniforme di sale. Incubare 48 ore a RT per polimerizzare la seta. </li><li> Collocare lo stampo PTFE contenente scaffold in forno a 60 ° C per 1 ora per completare la polimerizzazione e far evaporare il liquido in eccesso. </li><li> Posizionare il contenuto dello stampo PTFE in un bicchiere contenente 2 L di acqua distillata per 48 ore a percolare il sale. Cambiare l&#39;acqua 2-3 volte al giorno. Rimuovere le impalcature spugna dagli stampi quando il sale è completamente colato fuori del punto finale:. Le spugne possono essere conservati immersi in acqua a 4 ° C in un contenitore chiuso per impedire scaffold disidratazione. </li><li> Quando si è pronti, tagliare i ponteggi con diametro 5 mm biopsia. Pre-tagliare le impalcature per raggiungere circa 2 mm di altezza. PuNCH ​​il centro del ponteggio con il punzone di diametro biopsia 2 mm (Figura 2A). (1 ora) </li><li> Autoclavare i ponteggi immersi in acqua per sterilizzarli (ciclo bagnato, 121 ° C, 20 min) del punto finale:. Le spugne possono essere conservati immersi in acqua a 4 ° C in un contenitore chiuso per impedire scaffold disidratazione. </li><li> Prima della semina cellulare programmata, immergere i ponteggi in soluzione sterile 0,1 mg / ml di poli-D-lisina (PDL). Incubare per 1 ora a 37 ° C. </li><li> Lavare i ponteggi 3x con tampone fosfato salino (PBS) per rimuovere PDL non vincolati. (30 min) </li></ol></li></ol> 2. Isolamento di ratto neuroni corticali <ol><li> Sezionare cortecce da giorno embrionale 18 ratti (E18) Sprague-Dawley come precedentemente descritto da Pacifici e Peruzzi 27 dopo l&#39;ottenimento del protocollo animale approvato. (2 ore) </li><li> Incubare 10 cortecce in 5 ml di 0,25% tripsina con 0.3% DNasi I (da pancreas bovino) per 20 min a 37 ° C. </li><li> Inattivare i tripsina aggiungendo uguale volume di 1 mg / ml di proteine ​​di soia. </li><li> Triturare le cortecce con 10 ml pipetta Pasteur pipettando su e giù per 20 volte fino a quando viene generato una sospensione singola cellula. Sii gentile e evitare la formazione di bolle d&#39;aria. (10 min) </li><li> Centrifugare la sospensione cellulare a 127 xg per 5 min. </li><li> Risospendere il pellet cellulare in 10 ml di terreno di coltura (Neurobasal medio, 1x B27 supplemento, 1x Glutamax, 1% di penicillina / streptomicina). Contare le cellule. La concentrazione cellulare è di circa 2x10 previsto 7 / ml. (20 min) </li></ol> 3. Costruire Montaggio e Cultura <ol><li> Ponteggio semina con le cellule. <ol><li> Spostare i ponteggi sterili e tutti gli utensili necessari all&#39;interno di una cappa di coltura cellulare. Con pinza sterile posto i ponteggi in un piatto di coltura cellulare a 96 pozzetti attribuzione di un ponteggio per pozzetto. (10 min) </li><li> Immergere i ponteggi in terreno di coltura cellulare per equilibrare loro prima di logge dei semiing. Incubare per 1 ora a 37 ° C. (10 min) </li><li> Aspirare l&#39;eccesso del mezzo dalle impalcature. </li><li> Applicare 100 ml di sospensione cellulare / patibolo. (10 min) </li><li> Incubare le cellule a 37 ° CO / N per permettere l&#39;adesione delle cellule al ponteggio. </li><li> La mattina seguente aspirare le cellule non iscritti e applicare 200 pl / pozzetto di terreno di coltura fresco. (10 min) </li></ol></li><li> Impalcatura embedding con matrice di collagene. (2 ore) <ol><li> Mettere 10x PBS, acqua, 1 N NaOH e coda di topo collagene sul ghiaccio. Preparare una soluzione di lavoro di collagene secondo le istruzioni del produttore. Mantenere in ghiaccio fino alle costruzioni cellulari-seeded sono pronti (fino a 1 ora). </li><li> Rimuovere i costrutti di seta cellulari testa di serie dal termostato e aspirare l&#39;eccesso di mezzo. </li><li> Con pinza sterile trasferire i ponteggi nei pozzetti vuoti sulla piastra pozzo e immergono ogni impalcatura in 100 ml di 3 mg / ml soluzione di collagene. Posizionare la piastra di coltura tissutale indietroin incubatrice per 30 min per permettere la polimerizzazione del collagene. </li><li> Applicare 100 ml di pre-riscaldato terreno di coltura cellulare / bene. Cultura i costrutti per uno settimana cambiando il mezzo ogni giorno sostituendo solo la metà del volume del mezzo. </li></ol></li></ol> 4. Microscopia Analisi <ol><li> Vivo colorazione / morti (1 ora) <ol><li> Preparare soluzione stock di ioduro di propidio (PI) 1 mg / ml (1,5 mm) in acqua distillata. </li><li> Preparare soluzione stock di diacetato fluoresceina (FDA) 2 mg / ml in acetone. </li><li> Preparare la soluzione di lavoro contenente 10 micron e 0,15 micron PI FDA diluito in PBS. Pre-riscaldare la soluzione a 37 ° C in un bagno d&#39;acqua. </li><li> Lavare le cellule con PBS preriscaldata a rimuovere le esterasi siero. Aspirare il PBS. </li><li> Applicare la soluzione preriscaldata lavorando sulle cellule per 2 minuti e lavare con PBS. </li><li> Utilizzare microscopio epifluorescente all&#39;immagine delle cellule (PI ex λ = 490 nm, em λ = 570 nm; FDA ex λ = 490 nm, em λ = 514 nm). La macchia rimane stabile nel 40 min. Colorazione prolungata può causare macchie aspecifica risultante da PI captazione da parte delle cellule e co-localizzazione di PI / FDA in cellule. </li></ol></li><li> Immunostaining <ol><li> Al punto di tempo desiderato della cultura fissare le cellule con 4% paraformaldeide (PFA) per 30 minuti a RT. A causa della tossicità dei fumi PFA la fase di fissaggio deve essere eseguito nella cappa. </li><li> Lavare 3x ponteggi con PBS Fermata:. I costrutti PFA-fisse possono essere memorizzati in PBS a 4 ° C per diversi giorni prima di procedere con ulteriori passi. </li><li> Incubare i ponteggi a 0,2% Triton, 0,25% di albumina sierica bovina (BSA) in PBS contenenti anticorpo primario O / N a 4 ° C. </li><li> Il giorno seguente scartare la soluzione colorante e lavare i ponteggi 3 x 30 minuti con PBS per rimuovere l&#39;anticorpo primario non legato. </li><li> Incubare i campioni con anticorpo secondario diluito in 0,2% Triton, 0,25% BSA in PBS per 2ora a RT. </li><li> Rimuovere la soluzione colorante e lavare i ponteggi 3 x 30 minuti con PBS per rimuovere l&#39;anticorpo secondario non legato. </li><li> Immagine i ponteggi utilizzando un microscopio confocale con obiettivo 20X prendendo 100 micron z-sezioni del campione con 1 micron passo. Per visualizzare le neuriti sottili la risoluzione dell&#39;immagine dovrebbe essere di almeno 1.024 x 1.024 pixel. </li></ol></li><li> RNA, DNA e isolamento delle proteine Nota: RNA, DNA e l&#39;isolamento delle proteine ​​viene eseguita utilizzando un commerciale DNA AllPrep / RNA / proteine ​​Mini Kit secondo le istruzioni del produttore. <ol><li> Con pinza sterile trasferire i ponteggi di piastra di coltura di una provetta sterile 2 ml. Mettere una impalcatura per provetta. </li><li> Aggiungere 200 ml di tampone RLT a ciascuna provetta. </li><li> Interrompere il costrutto frammentando con microscissors sterili. </li><li> Per omogeneizzare il tessuto perturbato, trasferire il contenuto del tubo alla colonna di spin. Spin per 2 minuti a tutta velocità in una microcentrifuga.Il lisato viene omogeneizzata che passa attraverso la colonna di spin. </li><li> Raccogliere il flusso attraverso ed utilizzare immediatamente per isolare l&#39;RNA, DNA e proteine ​​seguendo il protocollo del produttore. </li><li> Quantificare la resa degli acidi nucleici qualsiasi altro metodo compatibile (ad esempio, NanoDrop). </li><li> Quantificare la resa proteica tramite BCA Protein Assay secondo le istruzioni del produttore. Misurare i risultati di test con lettore di micropiastre alla lunghezza d&#39;onda 562 nm. Nota: La resa prevista di acidi nucleici e proteine ​​per costrutto in relazione alla densità cellulare è mostrato in Figura 4. </li></ol></li></ol>

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Here we described the guidelines to assemble a compartmentalized 3D brain-like tissue model. The model is characterized by dense polarized culture of neurons resulting in the development of two morphologically different compartments: one containing densely packed cell bodies, second containing pure axonal networks. Overall, the scaffold architecture and growth pattern mimic brain cortical tissue including the six laminar layers and white matter tracts21.
The donut-shaped silk protein-based tissue model allows for modifications and tuning of mechanical properties, versatile structural forms, hydrogel fillers and cellular components. Thus, this tissue model establishes a base platform for a wide range of studies. Silk is a favorable candidate for biomaterial platforms due to its biocompatibility, aqueous-based processing, and robust mechanical and degradation properties22. The silk matrix also serves as a stable anchor to reduce collagen gel contraction over time in culture. The properties such as silk concentration and porosity of the scaffold used in this model have been previously adjusted to achieve optimal cell growth and mechanical properties resulting in brain-like tissue elasticity18,23,24. We suggest to keep these parameters constant to ensure the successful outcome and reproducibility of the experiments.
The 3D neuronal network formation was achieved by combining two types of biomaterials with different mechanical properties: a stiff scaffold to provide neuronal anchoring and a softer gel matrix to permit axon penetration and connectivity in 3D. Selective material preferences of the silk scaffold and soft hydrogel provide the underlying principle for compartmentalizing the neurons from the axonal connections. Due to the inert nature of the silk scaffold, functionalization with poly-D-lysine is required for neuronal attachment. However, other cell adhesion promoting factors can be applied such as RGD25 or fibronectin26. To achieve the 3D network formation the silk scaffold needs to be filled with hydrogel in a timely manner as soon as neurons are attached to the scaffold. Likewise, the collagen matrix filler can be replaced by other hydrogels, thus allowing the study of the influence of 3D extracellular environments on axonal network formation to serve as a platform to evaluate novel hydrogels in terms of neuronal compatibility. Additionally, apart from rat cortical neurons other neuronal sources such as hippocampal neurons or induced pluripotent cell (iPSc)-derived neurons can be utilized. Moreover, the tissue model can be used to study heterocellular interactions by including glial cells along with neurons and to build more complex brain-like tissue.
As shown previously, our model can be utilized to evaluate neuronal functionality in 3D microenvironment with a variety of assays such as cell viability, gene expression, LC-MS/MS and electrophysiology, thus demonstrating physiological relevance of the model18. Other methods, which are frequently utilized to evaluate 3D tissue-engineered constructs, such as immunostaining and microscopy8,9,11 can also be used to assess cell distribution and extent of axonal network formation. However, it has to be noted, that due to the high density of the collagen matrix, the penetration of antibodies and depth of the imaging is limited to few hundred micrometers. Moreover, the signal to noise ratio may be affected by nonspecific background fluorescence. This can be overcome by using lipophilic cell tracers and genetically expressed fluorescent proteins which diminish the need for immunostaining11. Alternatively, the imaging can be performed with 2-photon microscopy instead of usual one-photon technique, which may reduce the signal loss, photobleaching, and can be extended to several hundred micrometers of depth.
Summarizing, the silk and collagen-based brain-like tissue offers a robust platform to study neuronal networks in 3D and is a starting point for the development of more advanced models of neurological disorders in the future. Independent from the mode of evaluation, the relative simplicity of this tissue model supports its utility, success and reproducibility.

Il sistema nervoso centrale (CNS) può essere influenzata da una varietà di disturbi che coinvolgono vascolare, strutturale, funzionale, infettiva o degenerativa. Si stima che 6,8 milioni di persone muoiono ogni anno a causa di disturbi neurologici, che rappresenta un crescente onere socioeconomico in tutto il mondo 1. Tuttavia, solo alcuni dei disturbi sono trattamenti disponibili. Pertanto, vi è una necessità critica per strategie terapeutiche innovative per i pazienti affetti da disturbi neurologici. Purtroppo, molte terapie CNS-mirate falliscono durante gli studi clinici, in parte a causa dell&#39;utilizzo di modelli inadeguati di ricerca pre-clinica, che non consentono la valutazione degli impatti acuti e cronici con fisiologicamente rilevanti letture funzionali. Nonostante i significativi sforzi di ricerca nel corso degli ultimi decenni, vi è una grande quantità sconosciuto circa la struttura e la funzione del sistema nervoso centrale. Al fine di acquisire maggiori conoscenze, i modelli animali sono spesso noied al modellare stati patologici, come i traumi cerebrali (TBI) o demenza, soprattutto in studi pre-clinici. Tuttavia, gli animali differiscono significativamente dagli esseri umani sia in anatomia del sistema nervoso centrale, nonché in funzione, l&#39;espressione genica e metabolismo 2-4. D&#39;altra parte, culture 2D in vitro sono il metodo comune per indagare la biologia cellulare e sono abitualmente utilizzati per la scoperta di farmaci. Tuttavia, le colture cellulari 2D mancano della complessità e rilevanza fisiologica rispetto al cervello umano 5-7. Mentre non vi è alcun sostituto per il basso costo e la semplicità di studi su colture cellulari in 2D o la complessità fornito da modelli animali, l&#39;ingegneria dei tessuti 3D potrebbe generare modelli di ricerca migliorati per colmare il divario che esiste tra il 2D in vitro e in vivo approcci. Ingegneria dei tessuti 3D fornisce più fisiologicamente rilevanti condizioni sperimentali ottenuti da interazioni cellula-cellula 3D e segnali extracellulari forniti dalle impalcature biomateriale. Despite le prove significative dietro il valore delle culture in 3D, ci sono attualmente solo pochi modelli di tessuto 3D del sistema nervoso centrale, come staminali derivati ​​dalle cellule culture organoide 8-10, neurospheroids 11 e dispersi culture idrogel 12,13. Modalità tecniche avanzate che includono multilayer litografia 14, e la stampa 3D 15 sono stati utilizzati per lo studio del polmone, del fegato e tessuto renale. Tuttavia, vi è la mancanza di modelli 3D SNC che permettono la crescita neuronale compartimentato, come mimando l&#39;architettura corticale e biologia. Separato crescita dei neuriti da corpi cellulari neuronali è stato precedentemente dimostrato nelle culture 2D utilizzando microfabbricazione 16,17 permettendo lo studio di tracciato del tratto assoni, flusso di calcio, l&#39;architettura di rete e attività. Questa idea ci ha ispirato a sviluppare un tessuto neurale polarizzata 3D in cui i corpi cellulari e tratti assonali sono situati in diversi comparti che mimano l&#39;architettura a strati del cervello 18 </sup&gt;. Il nostro approccio è basato sull&#39;uso di design unico scaffold seta alloggiante alta densità di cellule in un volume confinato e permette conseguenza di rete assonale fitta in un gel di collagene. Qui mostriamo l&#39;intera procedura di assemblaggio del tessuto cerebrale simile tra cui la fabbricazione patibolo e la cultura neuronale.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Na2CO3</td>
			<td>Sigma-Aldrich</td>
			<td>223530</td>
			<td>-</td>
		</tr>
		<tr>
			<td>LiBr</td>
			<td>Sigma-Aldrich</td>
			<td>213225</td>
			<td>-</td>
		</tr>
		<tr>
			<td>MWCO 3500 dialysis cassettes&#160;</td>
			<td>Thermo Fisher</td>
			<td>66110</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Heating plate</td>
			<td>Fisher Scientific</td>
			<td>Isotemp</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Centrifuge 5804 R</td>
			<td>Eppendorf</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Sieve</td>
			<td>Fisher Scientific</td>
			<td>-</td>
			<td>No. 270, No. 35, No. 30</td>
		</tr>
		<tr>
			<td>PTFE mold</td>
			<td>-</td>
			<td>-</td>
			<td>made in house (Figure 1) 10 cm diameter, 2 cm height</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>71382</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Biopsy Punch 5mm</td>
			<td>World Precision Instrument</td>
			<td>501909</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Biopsy Punch 2mm</td>
			<td>World Precision Instrument</td>
			<td>501908</td>
			<td>-</td>
		</tr>
		<tr>
			<td>poly-D-lysine</td>
			<td>Sigma-Aldrich</td>
			<td>P6407-5MG</td>
			<td>final concentration 10 ug/ml, dissolved in water</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Sigma-Aldrich</td>
			<td>D1283-500ML</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Sigma-Aldrich</td>
			<td>T4049-500ML</td>
			<td>-</td>
		</tr>
		<tr>
			<td>DNase</td>
			<td>Roche</td>
			<td>10104159001</td>
			<td>final concentration 0.3%</td>
		</tr>
		<tr>
			<td>Soybean protein</td>
			<td>Sigma-Aldrich</td>
			<td>T6522-100MG</td>
			<td>final concentration 1 mg/ml</td>
		</tr>
		<tr>
			<td>Neurobasal medium</td>
			<td>Gibco</td>
			<td>21103049</td>
			<td>warm up to 37&#176;C before use</td>
		</tr>
		<tr>
			<td>B27 supplement 50x</td>
			<td>Gibco</td>
			<td>17504-044</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Glutamax</td>
			<td>Gibco</td>
			<td>35050-061</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Penicilin Streptomycin</td>
			<td>Corning</td>
			<td>30-002-CI</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Collagen I, rat tail, 100 mg</td>
			<td>Corning</td>
			<td>354236</td>
			<td>final concentration 3 mg/ml</td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Sigma-Aldrich</td>
			<td>S2770</td>
			<td>corrosive</td>
		</tr>
		<tr>
			<td>Propidium Iodide</td>
			<td>Sigma-Aldrich</td>
			<td>P4170-10MG</td>
			<td>toxic</td>
		</tr>
		<tr>
			<td>Fluorescein Diacetate</td>
			<td>Sigma-Aldrich</td>
			<td>F7378-5G</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Olympus MVX10</td>
			<td>Olympus</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>P6148</td>
			<td>toxic, final concentration 4%</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A7906-10G</td>
			<td>-</td>
		</tr>
		<tr>
			<td>anti-&#946;IIITubulin antibody&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>T2200</td>
			<td>rabbit 1:500</td>
		</tr>
		<tr>
			<td>anti-rabbit Alexa-546</td>
			<td>Molecular Probes</td>
			<td>A11010</td>
			<td>goat 1:250</td>
		</tr>
		<tr>
			<td>goat serum</td>
			<td>Sigma-Aldrich</td>
			<td>G9023-5ML</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Leica SP2 confocal microscope</td>
			<td>Leica</td>
			<td>-</td>
			<td>objective 20x</td>
		</tr>
		<tr>
			<td>QIAshredder</td>
			<td>Qiagen</td>
			<td>79654</td>
			<td>-</td>
		</tr>
		<tr>
			<td>AllPrep DNA/RNA/Protein Mini Kit</td>
			<td>Qiagen</td>
			<td>80004</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Ethyl alcohol, Pure</td>
			<td>Sigma-Aldrich</td>
			<td>E7023&#160;</td>
			<td>-</td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Sigma-Aldrich</td>
			<td>63689</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoDrop 2000c/2000 UV-Vis Spectrophotometer</td>
			<td>Thermo Scientific</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>BCA protein assay</td>
			<td>Thermo Scientific</td>
			<td>23225</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Plate reader Spectramax M2</td>
			<td>Molecular Devices</td>
			<td>-</td>
			<td>absorbance 562 nm</td>
		</tr>
		<tr>
			<td>Micro Scissors, Economy, Vannas-type</td>
			<td>TedPella</td>
			<td>1346</td>
			<td>-</td>
		</tr>
	</tbody>
</table>

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.

Solid spugne seta pretagliato alla forma di ciambella rappresentano un&#39;idea unica e semplice da realizzare un&#39;architettura compartmentalized simile tessuto neurale (Figura 2A). La struttura altamente porosa del ponteggio con la dimensione dei pori di circa 500 micron risultati in eccezionalmente elevata area superficiale permettono la semina e la crescita di alta densità cellulare in un piccolo volume (2x10 7 / ml) (Figura 2B). Inoltre, l&#39;elevata porosità del patibolo permette per la diffusione illimitata di sostanze nutritive e di rifiuti con conseguente redditività superiore di colture cellulari dense un lasso di tempo prolungato. Dopo la semina i neuroni rapidamente ed uniformemente fissarle con la superficie dei pori ponteggio. Dopo l&#39;adesione cellulare è completa e le cellule sono in equilibrio sul substrato di seta, l&#39;impalcatura spugna è riempito con la matrice di collagene morbido per fornire un ambiente 3D per la formazione della rete assonale (Figura 3 </strong&gt;). In assenza della matrice di collagene la compartimentazione di assoni e corpi cellulari non è possibile in quanto i neuroni crescono estensioni solo sulla superficie dei pori con conseguente reti miste come trovata con superfici 2D (Figura 3B). Al contrario, il gel di collagene fornisce un reticolo che supporta una vasta crescita degli assoni in 3D, mentre i corpi cellulari neuronali rimangono attaccate al ponteggio seta (Figura 3C). Questo modello di crescita porta a compartimentazione dei corpi cellulari e reti assonale puri (Figura 3D), che ricorda la materia grigia e bianca della corteccia del cervello. Oltre microscopia, i costrutti possono essere valutati con una varietà di altri metodi 18, a seconda della domanda dietro l&#39;esperimento. Ad esempio, l&#39;isolamento di DNA, RNA e di proteine ​​dei costrutti può essere facilmente realizzato utilizzando kit commerciali (Figura 4). La resa degli acidi nucleici e proteine ​​per costrutto dipende dal numero di cellule inizialmente seminate su scaffold seta. Più cellule seminate maggiore la resa di DNA, RNA e proteine. <img alt="Figura 1" src="/files/ftp_upload/52970/52970fig1.jpg" /> Figura 1. stampo in PTFE utilizzati per la preparazione di spugna di seta patibolo le dimensioni:.. 10 cm di diametro, 2 cm altezza <a href="https://www.jove.com/files/ftp_upload/52970/52970fig1large.jpg" target="_blank">Cliccate qui per vedere una versione più grande di questa figura.</a> <img alt="Figura 2" src="/files/ftp_upload/52970/52970fig2.jpg" /> Figura 2. Modello 3D tessuto cerebrale-like. (A) poroso spugna seta patibolo. (B) Risultati in diretta colorazione / morti di neuroni al giorno 1 su semina sull&#39;impalcatura di seta prima di collagene embedding (cellule verdi-live, le cellule rosse-morti). I pannelli sul lato destro mostrano la magnification di area catturata in cornici rosse. <a href="https://www.jove.com/files/ftp_upload/52970/52970fig2large.jpg" target="_blank">Cliccate qui per vedere una versione più grande di questa figura.</a> <img alt="Figura 3" src="/files/ftp_upload/52970/52970fig3.jpg" /> Figura 3. neuronale escrescenza compartimenti stagni nel modello di tessuto cerebrale 3D-like (A) scompartimenti impalcature:. Red-cell vano corpo, vano Blu-assonale. (B) modello di crescita neuronale al giorno 1 su semina prima collagene incorporamento. (C). Escrescenza neuronale al giorno 7 su seminare nel vano corpo cellulare. (D) escrescenza neuronale al giorno 7 su semina nel compartimento assonale. Verde -. ΒIIITubulin <a href="https://www.jove.com/files/ftp_upload/52970/52970fig3large.jpg" target="_blank">Cliccate qui per vedere una versione più grande di questo</a>figura. <img alt="Figura 4" src="/files/ftp_upload/52970/52970fig4.jpg" /> Figura 4. Rendimento atteso di (A) RNA e DNA, e (B) proteine ​​per costrutto in relazione alla quantità di cellule seminati per ponteggio (± SD, n = 5). <a href="https://www.jove.com/files/ftp_upload/52970/52970fig4large.jpg" target="_blank">Cliccate qui per vedere una versione più grande di questa figura .</a>

Watch this Scientific Journal Video about Engineered 3D Silk-collagen-based Model of Polarized Neural Tissue at JoVE.com

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

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 ...

engineered-3d-silk-collagen-based-model-of-polarized-neural-tissue

Research

JoVE Journal

Bioengineering

Electric Field-controlled Directed Migration of Neural Progenitor Cells in 2D and 3D Environments

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the central nervous system1,2. These endogenous EFs are generated by cellular regulation of ionic transport combined with the electrical resistance of cells and tissues. It has been reported that applied EF treatment can promote functional repair of spinal cord injuries in animals and humans3,4. In particular, EF-directed cell migration has been demonstrated in a wide variety of cell types5,6, including neural progenitor cells (NPCs)7,8. Application of direct current (DC) EFs is not a commonly available technique in most laboratories. We have described detailed protocols for the application of DC EFs to cell and tissue cultures previously5,11. Here we present a video demonstration of standard methods based on a calculated field strength to set up 2D and 3D environments for NPCs, and to investigate cellular responses to EF stimulation in both single cell growth conditions in 2D, and the organotypic spinal cord slice in 3D. The spinal cordslice is an ideal recipient tissue for studying NPC ex vivo behaviours, post-transplantation, because the cytoarchitectonic tissue organization is well preserved within these cultures9,10. Additionally, this ex vivo model also allows procedures that are not technically feasible to track cells in vivo using time-lapse recording at the single cell level. It is critically essential to evaluate cell behaviours in not only a 2D environment, but also in a 3D organotypic condition which mimicks the in vivo environment. This system will allow high-resolution imaging using cover glass-based dishes in tissue or organ culture with 3D tracking of single cell migration in vitro and ex vivo and can be an intermediate step before moving onto in vivo paradigms.

This protocol demonstrates methods used to establish 2D and 3D environments in custom-designed electrotactic chambers, which can track cells in vivo/ex vivo using time-lapse recording at the single cell level, in order to investigate galvanotaxis/electrotaxis and other cellular responses to direct current (DC) electric fields (EFs).

Di campo elettrico controllato migrazione diretta di cellule progenitrici neurali in ambienti 2D e 3D

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the ...

Ricerca

Bioingegneria

Magnetic Resonance Elastography Methodology for the Evaluation of Tissue Engineered Construct Growth

Traditional mechanical testing often results in the destruction of the sample, and in the case of long term tissue engineered construct studies, the use of destructive assessment is not acceptable. A proposed alternative is the use of an imaging process called magnetic resonance elastography. Elastography is a nondestructive method for determining the engineered outcome by measuring local mechanical property values (i.e., complex shear modulus), which are essential markers for identifying the structure and functionality of a tissue. As a noninvasive means for evaluation, the monitoring of engineered constructs with imaging modalities such as magnetic resonance imaging (MRI) has seen increasing interest in the past decade1. For example, the magnetic resonance (MR) techniques of diffusion and relaxometry have been able to characterize the changes in chemical and physical properties during engineered tissue development2. The method proposed in the following protocol uses microscopic magnetic resonance elastography (&mu;MRE) as a noninvasive MR based technique for measuring the mechanical properties of small soft tissues3. MRE is achieved by coupling a sonic mechanical actuator with the tissue of interest and recording the shear wave propagation with an MR scanner4. Recently, &mu;MRE has been applied in tissue engineering to acquire essential growth information that is traditionally measured using destructive mechanical macroscopic techniques5. In the following procedure, elastography is achieved through the imaging of engineered constructs with a modified Hahn spin-echo sequence coupled with a mechanical actuator. As shown in Figure 1, the modified sequence synchronizes image acquisition with the transmission of external shear waves; subsequently, the motion is sensitized through the use of oscillating bipolar pairs. Following collection of images with positive and negative motion sensitization, complex division of the data produce a shear wave image. Then, the image is assessed using an inversion algorithm to generate a shear stiffness map6. The resulting measurements at each voxel have been shown to strongly correlate (R2&gt;0.9914) with data collected using dynamic mechanical analysis7. In this study, elastography is integrated into the tissue development process for monitoring human mesenchymal stem cell (hMSC) differentiation into adipogenic and osteogenic constructs as shown in Figure 2.

The procedure demonstrates the methodology of magnetic resonance elastography for monitoring the engineered outcome of adipose and osteogenic tissue engineered constructs through noninvasive local assessment of the mechanical properties using microscopic magnetic resonance elastography (&mu;MRE).

Metodologia Elastografia risonanza magnetica per la valutazione dei Tissue Engineered Construct crescita

Traditional mechanical testing often results in the destruction of the sample, and in the case of long term tissue engineered construct studies, the use ...

Protocol for Relative Hydrodynamic Assessment of Tri-leaflet Polymer Valves

Limitations of currently available prosthetic valves, xenografts, and homografts have prompted a recent resurgence of developments in the area of tri-leaflet polymer valve prostheses. However, identification of a protocol for initial assessment of polymer valve hydrodynamic functionality is paramount during the early stages of the design process. Traditional in vitro pulse duplicator systems are not configured to accommodate flexible tri-leaflet materials; in addition, assessment of polymer valve functionality needs to be made in a relative context to native and prosthetic heart valves under identical test conditions so that variability in measurements from different instruments can be avoided. Accordingly, we conducted hydrodynamic assessment of i) native (n = 4, mean diameter, D = 20 mm), ii) bi-leaflet mechanical (n= 2, D = 23 mm) and iii) polymer valves (n = 5, D = 22 mm) via the use of a commercially available pulse duplicator system (ViVitro Labs Inc, Victoria, BC) that was modified to accommodate tri-leaflet valve geometries. Tri-leaflet silicone valves developed at the University of Florida comprised the polymer valve group. A mixture in the ratio of 35:65 glycerin to water was used to mimic blood physical properties. Instantaneous flow rate was measured at the interface of the left ventricle and aortic units while pressure was recorded at the ventricular and aortic positions. Bi-leaflet and native valve data from the literature was used to validate flow and pressure readings. The following hydrodynamic metrics were reported: forward flow pressure drop, aortic root mean square forward flow rate, aortic closing, leakage&#160;and regurgitant volume, transaortic closing, leakage, and total energy losses. Representative results indicated that hydrodynamic metrics from the three valve groups could be successfully obtained by incorporating a custom-built assembly into a commercially available pulse duplicator system and subsequently, objectively compared to provide insights on functional aspects of polymer valve design.

There has been renewed interest in developing polymer valves. Here, the objectives are to demonstrate the feasibility of modifying a commercial pulse duplicator to accommodate tri-leaflet geometries and to define a protocol to present polymer valve hydrodynamic data in comparison to native and prosthetic valve data collected under near-identical conditions.

Protocollo per la relativa valutazione idrodinamica del Tri-volantino Polymer Valvole

Limitations of currently available prosthetic valves, xenografts, and homografts have prompted a recent resurgence of developments in the area of ...

Tissue Engineering of a Human 3D in vitro Tumor Test System

Cancer is one of the leading causes of death worldwide. Current therapeutic strategies are predominantly developed in 2D culture systems, which inadequately reflect physiological conditions in vivo. Biological 3D matrices provide cells an environment in which cells can self-organize, allowing the study of tissue organization and cell differentiation. Such scaffolds can be seeded with a mixture of different cell types to study direct 3D cell-cell-interactions. To mimic the 3D complexity of cancer tumors, our group has developed a 3D in vitro tumor test system.	
	Our 3D tissue test system models the in vivo situation of malignant peripheral nerve sheath tumors (MPNSTs), which we established with our decellularized porcine jejunal segment derived biological vascularized scaffold (BioVaSc). In our model, we reseeded a modified BioVaSc matrix with primary fibroblasts, microvascular endothelial cells (mvECs) and the S462 tumor cell line. For static culture, the vascular structure of the BioVaSc is removed and the remaining scaffold is cut open on one side (Small Intestinal Submucosa SIS-Muc). The resulting matrix is then fixed between two metal rings (cell crowns).	
	Another option is to culture the cell-seeded SIS-Muc in a flow bioreactor system that exposes the cells to shear stress. Here, the bioreactor is connected to a peristaltic pump in a self-constructed incubator. A computer regulates the arterial oxygen and nutrient supply via parameters such as blood pressure, temperature, and flow rate. This setup allows for a dynamic culture with either pressure-regulated pulsatile or constant flow.	
	In this study, we could successfully establish both a static and dynamic 3D culture system for MPNSTs. The ability to model cancer tumors in a more natural 3D environment will enable the discovery, testing, and validation of future pharmaceuticals in a human-like model.

Tissue Engineering of a Human 3D in vitro Tumor Test System

Methods to create human 3D tumor tissues as test systems are described. These technologies are based on a decellularized Biological Vascularized Scaffold (BioVaSc), primary human cells and a tumor cell line, which can be cultured under static as well as under dynamic conditions in a flow bioreactor.

Ingegneria dei tessuti di un Umano 3D<em&gt; In vitro</em&gt; Sistema Tumore test

Cancer is one of the leading causes of death worldwide.  Current therapeutic strategies are predominantly developed in 2D culture  systems, which ...

Generation and Grafting of Tissue-engineered Vessels in a Mouse Model

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. The current protocol presents an efficient and reproducible strategy in which functional tissue engineered vessel grafts can be generated using partially induced pluripotent stem cell (PiPSC) from human fibroblasts. We designed a decellularized vessel scaffold bioreactor, which closely mimics the matrix protein structure and blood flow that exists within a native vessel, for seeding of PiPSC-endothelial cells or smooth muscle cells prior to grafting into mice. This approach was demonstrated to be advantageous because immune-deficient mice engrafted with the PiPSC-derived grafts presented with markedly increased survival rate 3 weeks after surgery. This protocol represents a valuable tool for regenerative medicine, tissue engineering and potentially patient-specific cell-therapy in the near future.

Here, we present a protocol to generate tissue engineered vessel grafts that are functional for grafting into mice by double seeding partially induced pluripotent stem cell (PiPSC) - derived smooth muscle cells and PiPSC - derived endothelial cells on a decellularized vessel scaffold bioreactor.

Generazione e innesto delle navi tissutale in un modello di topo

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. ...

Production, Characterization and Potential Uses of a 3D Tissue-engineered Human Esophageal Mucosal Model

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore esophageal adenocarcinoma generally has a poor prognosis, with little improvement in survival rates in recent years. These are difficult conditions to study and there has been a lack of suitable experimental platforms to investigate disorders of the esophageal mucosa.
A model of the human esophageal mucosa has been developed in the MacNeil laboratory which, unlike conventional 2D cell culture systems, recapitulates the cell-cell and cell-matrix interactions present in vivo and produces a mature, stratified epithelium similar to that of the normal human esophagus. Briefly, the model utilizes non-transformed normal primary human esophageal fibroblasts and epithelial cells grown within a porcine-derived acellular esophageal scaffold. Immunohistochemical characterization of this model by CK4, CK14, Ki67 and involucrin staining demonstrates appropriate recapitulation of the histology of the normal human esophageal mucosa.
This model provides a robust, biologically relevant experimental model of the human esophageal mucosa. It can easily be manipulated to investigate a number of research questions including the effectiveness of pharmacological agents and the impact of exposure to environmental factors such as alcohol, toxins, high temperature or gastro-esophageal refluxate components. The model also facilitates extended culture periods not achievable with conventional 2D cell culture, enabling, inter alia, the study of the impact of repeated exposure of a mature epithelium to the agent of interest for up to 20 days. Furthermore, a variety of cell lines, such as those derived from esophageal tumors or Barrett&#8217;s Metaplasia, can be incorporated into the model to investigate processes such as tumor invasion and drug responsiveness in a more biologically relevant environment.

This manuscript describes the production, characterization and potential uses of a tissue engineered 3D esophageal construct prepared from normal primary human esophageal fibroblast and squamous epithelial cells seeded within a de-cellularized porcine scaffold. The results demonstrate the formation of a mature stratified epithelium similar to the normal human esophagus.

Produzione, caratterizzazione e possibilità di utilizzo di un 3D tissutale umano esofageo mucose Modello

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore ...

A Combined 3D Tissue Engineered In Vitro/In Silico Lung Tumor Model for Predicting Drug Effectiveness in Specific Mutational Backgrounds

In the present study, we combined an in vitro 3D lung tumor model with an in silico model to optimize predictions of drug response based on a specific mutational background. The model is generated on a decellularized porcine scaffold that reproduces tissue-specific characteristics regarding extracellular matrix composition and architecture including the basement membrane. We standardized a protocol that allows artificial tumor tissue generation within 14 days including three days of drug treatment. Our article provides several detailed descriptions of 3D read-out screening techniques like the determination of the proliferation index Ki67 staining&#39;s, apoptosis from supernatants by M30-ELISA and assessment of epithelial to mesenchymal transition (EMT), which are helpful tools for evaluating the effectiveness of therapeutic compounds. We could show compared to 2D culture a reduction of proliferation in our 3D tumor model that is related to the clinical situation. Despite of this lower proliferation, the model predicted EGFR-targeted drug responses correctly according to the biomarker status as shown by comparison of the lung carcinoma cell lines HCC827 (EGFR -mutated, KRAS wild-type) and A549 (EGFR wild-type, KRAS-mutated) treated with the tyrosine-kinase inhibitor (TKI) gefitinib. To investigate drug responses of more advanced tumor cells, we induced EMT by long-term treatment with TGF-beta-1 as assessed by vimentin/pan-cytokeratin immunofluorescence staining. A flow-bioreactor was employed to adjust culture to physiological conditions, which improved tissue generation. Furthermore, we show the integration of drug responses upon gefitinib treatment or TGF-beta-1 stimulation - apoptosis, proliferation index and EMT - into a Boolean in silico model. Additionally, we explain how drug responses of tumor cells with a specific mutational background and counterstrategies against resistance can be predicted. We are confident that our 3D in vitro approach especially with its in silico expansion provides an additional value for preclinical drug testing in more realistic conditions than in 2D cell culture.

A Combined 3D Tissue Engineered In Vitro/In Silico Lung Tumor Model for Predicting Drug Effectiveness in Specific Mutational Backgrounds

We present a three-dimensional (3D) lung cancer model based on a biological collagen scaffold to study sensitivity towards non-small-cell-lung-cancer-(NSCLC)-targeted therapies. We demonstrate different read-out techniques to determine the proliferation index, apoptosis and epithelial-mesenchymal transition (EMT) status. Collected data are integrated into an in silico model for prediction of drug sensitivity.

Un Tessuto 3D combinata Engineered<em&gt; In Vitro</em&gt; /<em&gt; In Silico</em&gt; Polmone tumore per predire l&#39;efficacia della droga in specifici Sfondi mutazionali

In the present study, we combined an in vitro 3D lung tumor model with an in silico model to optimize predictions of drug response based on a specific ...

Bioprinting of Cartilage and Skin Tissue Analogs Utilizing a Novel Passive Mixing Unit Technique for Bioink Precellularization

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both cartilage and skin analogs were fabricated after bioink pre-cellularization utilizing a novel passive mixing unit technique. This technique was developed with the aim to simplify the steps involved in the mixing of a cell suspension into a highly viscous bioink. The resolution of filaments deposited through bioprinting necessitates the assurance of uniformity in cell distribution prior to printing to avoid the deposition of regions without cells or retention of large cell clumps that can clog the needle. We demonstrate the ability to rapidly blend a cell suspension with a bioink prior to bioprinting of both cartilage and skin analogs. Both tissue analogs could be cultured for up to 4 weeks. Histological analysis demonstrated both cell viability and deposition of tissue specific extracellular matrix (ECM) markers such as glycosaminoglycans (GAGs) and collagen I respectively.

Cartilage and skin analogs were bioprinted using a nanocellulose-alginate based bioink. The bioinks were cellularized prior to printing via a single step passive mixing unit. The constructs were demonstrated to be uniformly cellularized, have high viability, and exhibit favorable markers of differentiation.

Multimateriali di cartilagine e pelle tessuto analoghi utilizzando un romanzo passivo unità tecnica di miscelazione per Bioink Precellularization

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both ...

Seeding and Implantation of a Biosynthetic Tissue-engineered Tracheal Graft in a Mouse Model

Treatment options for congenital or secondary long segment tracheal defects have historically been limited due to an inability to replace functional tissue. Tissue engineering holds great promise as a potential solution with its ability to integrate cells and signaling molecules into a 3-dimensional scaffold. Recent work with tissue engineered tracheal grafts (TETGs) has seen some success but their translation has been limited by graft stenosis, graft collapse, and delayed epithelialization. In order to investigate the mechanisms driving these issues, we have developed a mouse model for tissue engineered tracheal graft implantation. TETGs were constructed using electrospun polymers polyethylene terephthalate (PET) and polyurethane (PU) in a mixture of PET and PU (20:80 percent weight). Scaffolds were then seeded using bone marrow mononuclear cells isolated from 6-8 week-old C57BL/6 mice by gradient centrifugation. Ten million cells per graft were seeded onto the lumen of the scaffold and allowed to incubate overnight before implantation between the third and seventh tracheal rings. These grafts were able to recapitulate the findings of stenosis and delayed epithelialization as demonstrated by histological analysis and lack of Keratin 5 and Keratin 14 basal epithelial cells on immunofluorescence. This model will serve as a tool for investigating cellular and molecular mechanisms involved in host remodeling.

Graft stenosis poses a critical obstacle in tissue engineered airway replacement. To investigate cellular mechanisms underlying stenosis, we utilize a murine model of tissue engineered tracheal replacement with seeded bone marrow mononuclear cells (BM-MNC). Here, we detail our protocol, including scaffold manufacturing, BM-MNC isolation, graft seeding, and implantation.

Semina e impianto di un innesto trachea tessutale biosintetico in un modello murino

Treatment options for congenital or secondary long segment tracheal defects have historically been limited due to an inability to replace functional ...

Tissue-Engineered Graft for Circumferential Esophageal Reconstruction in Rats

The use of biocompatible materials for circumferential esophageal reconstruction is a technically challenging task in rats and requires an optimal implant technique with nutritional support. Recently, there have been many attempts at esophageal tissue engineering, but the success rate has been limited due to difficulty in early epithelization in the special environment of peristalsis. Here, we developed an artificial esophagus that can improve the regeneration of the esophageal mucosa and muscle layers through a two-layered tubular scaffold, a mesenchymal stem cell-based bioreactor system, and a bypass feeding technique with modified gastrostomy. The scaffold is made of polyurethane (PU) nanofibers in a cylindrical shape with a three-dimensional (3D) printed polycaprolactone strand wrapped around the outer wall. Prior to transplantation, human-derived mesenchymal stem cells were seeded into the lumen of the scaffold, and bioreactor cultivation was performed to enhance cellular reactivity. We improved the graft survival rate by applying surgical anastomosis and covering the implanted prosthesis with a thyroid gland flap, followed by temporary nonoral gastrostomy feeding. These grafts were able to recapitulate the findings of initial epithelialization and muscle regeneration around the implanted sites, as demonstrated by histological analysis. In addition, increased elastin fibers and neovascularization were observed in the periphery of the graft. Therefore, this model presents a potential new technique for circumferential esophageal reconstruction.

Esophageal reconstruction is a challenging procedure, and development of a tissue-engineered esophagus that enables regeneration of esophageal mucosa and muscle and that can be implanted as an artificial graft is necessary. Here, we present our protocol to generate an artificial esophagus, including scaffold manufacturing, bioreactor cultivation, and various surgical techniques.

Innesto ingegnerizzato per la ricostruzione esofagea circonferenza nei ratti

The use of biocompatible materials for circumferential esophageal reconstruction is a technically challenging task in rats and requires an optimal implant ...