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).

Le protocole d&#39;isolement de tissu cérébral a été approuvé par le Comité des soins et l&#39;utilisation des animaux institutionnelle de l&#39;Université Tufts et est conforme avec les National Institutes of Health Guide pour le soin et l&#39;utilisation des animaux de laboratoire (Institutional Animal Care et l&#39;utilisation Comité B2011-45). 1. Préparation de la soie Échafaudages <ol><li> Préparation de la solution de soie de Bombyx mori cocons comme décrit précédemment 19,20. <ol><li> Couper chaque cocon en 8 morceaux égaux aide de ciseaux. Utilisez environ 11 cocons pour 5 g de cocons fragmentés. (15 min) </li><li> Préparer 2 L de 0,02 M Na 2 CO 3 et porter à ébullition en utilisant une plaque chauffante. (15 min) </li><li> Peser 5 g de cocons fragmentées et les faire bouillir dans Na 2 CO 3 pendant 30 min. Incorporer la soie bouillante toutes les deux minutes avec une spatule. Cette étape, également appelé de-gommage, purifie fibroïne de soie de protéines hydrophiles, sericins. (30 min) </li><li&gt; Essorez la fibroïne à la main et le rincer à l&#39;eau distillée au moins trois fois pour laver toute séricine restant et les produits chimiques. (5 min) </li><li> Placez la fibroïne humide sur une boîte de Pétri et sécher l&#39;extrait de fibroïne dans la hotte à flux O / N. </li><li> Le lendemain peser la masse de fibroïne sec et placer la fibroïne dans un récipient en verre. (15 min) </li><li> Pour dissoudre la fibroïne de 9,3 M de solution de LiBr, calculer le volume nécessaire (en ml) de LiBr en multipliant la masse de fibroïne sec par 4. Verser lentement solution LiBr sur la fibroïne de soie et plongez toutes les fibres de fibroïne aide d&#39;une spatule. Couvrir le bécher pour éviter l&#39;évaporation et le placer à 60 ° C pendant 4 heures pour permettre aux fibres de se dissoudre. (15 min) </li><li> Utilisation de la seringue, de recueillir la solution de fibroïne du bécher et le charger dans les MWCO 3.500 cassettes de dialyse. Effectuer une dialyse contre de l&#39;eau distillée pendant 48 heures. Changez l&#39;eau toutes les deux heures. </li><li> Utilisation de la seringue, de recueillir la solution de fibroïne deles cassettes dans 50 ml tubes coniques et centrifuger à 9000 rpm deux fois (~ 12 700 x g) à 4 ° C pendant 20 min. Verser le surnageant dans un nouveau tube après chaque étape de centrifugation et jeter le culot. (40 min) </li><li> Mesurer la concentration de la fibroïne en estimant le poids sec. Verser 1 ml de solution de soie dans une barque peser. Sécher l&#39;échantillon à l&#39;étuve à 60 ° C pendant 2 heures. Peser la fibroïne de soie sèche et calculer la concentration de la solution de fibroïne de soie en multipliant le poids obtenu par 100. La concentration de la solution attendue de soie est 6-9% (p / v). </li><li> Ajuster la concentration de soie à 6% (p / v) en le diluant dans l&#39;eau distillée. Point d&#39;arrêt: La fibroïne de soie liquide peut être conservé à 4 ° C pendant jusqu&#39;à un mois dans un récipient fermé. </li></ol></li><li> Préparation des supports poreux de solution de soie. <ol><li> Tamiser les granulés de NaCl pour séparer les granules calibrés de 500 à 600 um, qui sera utilisé dans les étapes ultérieures. Jeter le granulés taille inférieure à 500 um et 600 um ci-dessus. (15 min) </li><li> Verser 30 ml de solution de soie de 6% dans le moule de polytétrafluoroéthylène (PTFE) (Figure 1). Disperser doucement 60 g de NaCl tamisé sur la soie. Appuyez sur le récipient pour obtenir une couche uniforme de sel. Incuber 48 h à température ambiante pour polymériser la soie. </li><li> Placez le moule contenant PTFE d&#39;échafaudage dans le four à 60 ° C pendant 1 heure pour finaliser la polymérisation et évaporer tout liquide restant. </li><li> Placer le contenu du moule en PTFE dans un bêcher contenant 2 litres d&#39;eau distillée pendant 48 heures à lessiver le sel. Changez l&#39;eau 2-3 fois par jour. Retirez les échafaudages d&#39;éponges des moules lorsque le sel est complètement lessivé point d&#39;arrêt:. Les éponges peuvent être stockés immergé dans l&#39;eau à 4 ° C dans un récipient fermé pour éviter les échafaudages de la déshydratation. </li><li> Lorsque vous êtes prêt, découpez les échafaudages avec 5 mm de diamètre biopsie punch. Pré-couper les échafaudages pour atteindre environ 2 mm de hauteur. PuNCH ​​le centre de l&#39;échafaud avec le poinçon 2 mm de diamètre de biopsie (figure 2A). (1 h) </li><li> Autoclaver les échafaudages immergés dans l&#39;eau pour les stériliser (le cycle humide, 121 ° C, 20 min) Point d&#39;arrêt:. Les éponges peuvent être stockés immergé dans l&#39;eau à 4 ° C dans un récipient fermé pour éviter les échafaudages de la déshydratation. </li><li> Avant l&#39;ensemencement des cellules prévu, plonger dans les échafaudages / ml de poly-D-lysine (PDL) de solution stérile de 0,1 mg. Incuber pendant 1 heure à 37 ° C. </li><li> Laver les échafaudages 3x avec du tampon phosphate salin (PBS) pour éliminer PDL non-lié. (30 min) </li></ol></li></ol> 2. Isolement du Rat neurones corticaux <ol><li> On dissèque le cortex de rats de 18 jours embryonnaires (E18) Sprague-Dawley, comme décrit précédemment par Peruzzi Pacifici et 27 après protocole approuvé animal est obtenue. (2 h) </li><li> Incuber 10 cortex dans 5 ml de trypsine à 0,25% à 0,3% de DNase I (provenant de pancréas de bovin) pendant 20 min à 37 ° C. </li><li> Inactiver la trypsine en ajoutant un volume égal de 1 mg / ml de protéine de soja. </li><li> Triturer les cortex à l&#39;aide de 10 ml de pipette Pasteur par aspiration et refoulement 20 fois jusqu&#39;à une suspension cellulaire unique est généré. Soyez doux et éviter la formation de bulles d&#39;air. (10 min) </li><li> Centrifuger la suspension de cellules à 127 xg pendant 5 min. </li><li> Remettre en suspension le culot cellulaire dans 10 ml de milieu de culture (milieu Neurobasal, 1x supplément B27, 1x Glutamax, 1% de pénicilline / streptomycine). Compter les cellules. La concentration cellulaire est d&#39;environ 2x10 prévu 7 / ml. (20 min) </li></ol> 3. Construire Assemblée et de la culture <ol><li> Échafaudage ensemencement avec des cellules. <ol><li> Déplacer les échafaudages stériles et les ustensiles nécessaires tous à l&#39;intérieur d&#39;une hotte de culture cellulaire. Aide de pinces stériles placent les échafaudages dans une plaque de culture cellulaire de 96 puits attribution d&#39;un échafaudage par puits. (10 min) </li><li> Plonger les échafaudages dans un milieu de culture cellulaire pour équilibrer les graines avant de la celluleing. Incuber pendant 1 heure à 37 ° C. (10 min) </li><li> Aspirer l&#39;excédent du milieu des échafaudages. </li><li> Appliquer 100 pi de suspension de cellules / échafaudage. (10 min) </li><li> Incuber les cellules à 37 ° CO / N pour permettre la fixation des cellules à l&#39;échafaud. </li><li> Le lendemain matin, aspirer les cellules non-inscrits et applique 200 pl / puits de milieu de culture frais. (10 min) </li></ol></li><li> Échafaudage intégration avec matrice de collagène. (2 h) <ol><li> Placez 10x PBS, de l&#39;eau, 1 N NaOH et queue de rat de collagène sur la glace. Préparer une solution de travail de collagène selon les instructions du fabricant. Maintenir sur la glace jusqu&#39;à les constructions ensemencées cellules sont prêts (jusqu&#39;à 1 heure). </li><li> Retirer les constructions de soie cellulaires graines de l&#39;incubateur et aspirer l&#39;excès de milieu. </li><li> Aide de pinces stériles transférer les échafaudages dans les puits vides sur la plaque de puits et immergent chaque échafaudage dans 100 pi de 3 mg / ml solution de collagène. Placer la plaque de culture de tissus de retourdans l&#39;incubateur pendant 30 min pour permettre la polymérisation du collagène. </li><li> Appliquer 100 ul de milieu préchauffé de culture de cellules / puits. Culture constructions depuis une semaine changeant le milieu tous les jours en remplaçant seulement la moitié du volume du milieu. </li></ol></li></ol> 4. Microscopie Analyse <ol><li> Direct coloration / morts (1 h) <ol><li> Préparer une solution stock d&#39;iodure de propidium (PI) 1 mg / ml (1,5 mm) dans de l&#39;eau distillée. </li><li> Préparer une solution stock de diacétate de fluorescéine (FDA) 2 mg / ml dans de l&#39;acétone. </li><li> Préparer la solution de travail contenant 10 uM PI et 0,15 uM FDA dilué dans du PBS. Préchauffer la solution à 37 ° C dans un bain d&#39;eau. </li><li> Laver les cellules avec préchauffé PBS pour éliminer les estérases sériques. Aspirer le PBS. </li><li> Appliquer la solution de travail pré-chauffé sur les cellules pendant 2 min et le laver avec du PBS. </li><li> Utilisez microscope à épifluorescence à l&#39;image les cellules (PI ex λ = 490 nm, em λ = 570 nm; FDA ex λ = 490 nm, em λ = 514 nm). La tache reste stable plus de 40 min. Coloration prolongée peut provoquer une coloration non spécifique résultant de PI absorption par les cellules et co-localisation de PI / FDA dans les cellules. </li></ol></li><li> Immunocoloration <ol><li> Au point de temps désiré de la culture fixer les cellules avec 4% de paraformaldehyde (PFA) pendant 30 min à température ambiante. En raison de la toxicité de la PFA vapeurs d&#39;l&#39;étape de fixation doit être réalisée dans la hotte d&#39;aspiration chimique. </li><li> Laver échafaudages avec 3x PBS point d&#39;arrêt:. Les constructions PFA-fixes peuvent être stockés dans PBS à 4 ° C pendant plusieurs jours avant de procéder à de nouvelles mesures. </li><li> Incuber les échafaudages dans 0,2% de Triton, 0,25% d&#39;albumine de sérum bovin (BSA) dans du PBS contenant l&#39;anticorps primaire O / N à 4 ° C. </li><li> Le lendemain, jeter la solution de coloration et laver les échafaudages 3 x 30 min avec du PBS pour éliminer l&#39;anticorps primaire non lié. </li><li> Incuber les échantillons avec l&#39;anticorps secondaire dilué dans 0,2% de Triton, 0,25% de BSA dans du PBS pendant 2heure à température ambiante. </li><li> Retirer la solution de coloration et laver les échafaudages 3 x 30 min avec du PBS pour éliminer l&#39;anticorps secondaire non lié. </li><li> Image les échafaudages en utilisant un microscope confocal à l&#39;objectif 20X en prenant 100 um z-sections de l&#39;échantillon avec 1 um étape. Pour visualiser les neurites minces la résolution de l&#39;image doit être d&#39;un minimum de 1024 x 1024 pixels. </li></ol></li><li> ARN, l&#39;ADN et l&#39;isolement de la protéine Remarque: l&#39;ARN, l&#39;ADN et l&#39;isolement de la protéine est effectuée en utilisant un kit commercial Mini AllPrep ADN / ARN / protéine selon les instructions du fabricant. <ol><li> Utilisation des pinces stériles transférer les échafaudages sur plaque de culture dans un tube stérile de 2 ml. Placez un échafaudage par tube. </li><li> Ajouter 200 ul de tampon RLT à chaque tube. </li><li> Perturber la construction en fragmentant avec microciseaux stériles. </li><li> Pour homogénéiser le tissu perturbé, transférer le contenu du tube à la colonne de centrifugation. Spin pendant 2 min à pleine vitesse dans une microcentrifugeuse.Le lysat est homogénéisé lors de son passage à travers la colonne de centrifugation. </li><li> Recueillir l&#39;écoulement à travers et l&#39;utiliser immédiatement pour isoler l&#39;ARN, ADN et de protéines en suivant le protocole du fabricant. </li><li> Quantifier le rendement en acides nucléiques de toute autre méthode compatible (par exemple, NanoDrop). </li><li> Quantifier le rendement en protéines en utilisant BCA Protein Assay selon les instructions du fabricant. Mesurer les résultats de test utilisant un lecteur de microplaques à longueur d&#39;onde 562 nm. Remarque: Le rendement attendu d&#39;acides nucléiques et des protéines par construction par rapport à la densité de cellules est représenté sur la Figure 4. </li></ol></li></ol>

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La publication de ce vidéo-article est commandité par Corning.

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.

Le système nerveux central (SNC) peut être affectée par une variété de troubles impliquant vasculaire, structurelle et fonctionnelle, infectieux ou dégénératif. On estime que 6,8 millions de personnes meurent chaque année des suites de troubles neurologiques, qui représente un fardeau socio-économique mondiale croissante 1. Cependant, seulement quelques-uns des troubles ont traitements disponibles. Par conséquent, il existe un besoin critique de stratégies thérapeutiques innovantes pour les patients souffrant de troubles neurologiques. Malheureusement, de nombreuses thérapies ciblées SNC-échouent au cours des essais cliniques, en partie due à l&#39;utilisation de modèles de recherche pré-clinique inadéquate, qui ne permettent pas une évaluation des impacts aigus et chroniques avec affichage fonctionnels physiologiquement pertinents. En dépit des efforts de recherche importants au cours des dernières décennies, il ya une grande quantité inconnue sur la structure et la fonction du système nerveux central. Afin d&#39;acquérir plus de connaissances, modèles animaux sont souvent de noused pour modéliser les états pathologiques, comme une lésion cérébrale traumatique (TBI) ou de démence, en particulier dans les études pré-cliniques. Toutefois, les animaux diffèrent de manière significative à la fois chez l&#39;homme dans l&#39;anatomie du système nerveux central, ainsi que de la fonction, et l&#39;expression des gènes du métabolisme de 2-4. D&#39;autre part, 2D cultures in vitro sont la méthode commune pour étudier la biologie cellulaire et sont couramment utilisés pour la découverte de médicaments. Cependant, des cultures de cellules 2D manquent de la complexité et de la pertinence physiologique par rapport au cerveau humain 7.5. Bien qu&#39;il n&#39;y ait pas de substitut pour le faible coût et la simplicité du 2D études de culture cellulaire ou de la complexité fournies par les modèles animaux, l&#39;ingénierie tissulaire 3D pourrait générer des modèles de recherche améliorées pour combler l&#39;écart qui existe entre la 2D in vitro et dans des approches in vivo. Ingénierie tissulaire 3D fournit des conditions expérimentales plus physiologiquement pertinents obtenus par les interactions entre les cellules et les signaux extracellulaires 3D fournis par les échafaudages de biomatériaux. Despite la preuve significative derrière la valeur des cultures 3D, il n&#39;y a actuellement que quelques modèles de tissus 3D SNC tels que souches dérivées de cellules cultures organoïdes 8-10, neurospheroids 11 et dispersés cultures d&#39;hydrogel 12,13. Y compris les méthodes techniques avancées multicouche 14 lithographie, impression en 3D et 15 ont été utilisées pour l&#39;étude du poumon, du foie, du rein et le tissu. Cependant, il existe des modèles 3D absence du SNC qui permettent la croissance neuronale compartimenté, comme imitant l&#39;architecture corticale et de la biologie. Séparé croissance des neurites à partir de corps cellulaires neuronaux a déjà été démontré dans les cultures 2D en utilisant microfabrication 16,17 permettant l&#39;étude de traçage axone des voies, l&#39;afflux de calcium, l&#39;architecture du réseau et des activités. Cette idée nous a inspiré pour développer un tissu neural polarisée 3D où les corps cellulaires et les voies axonales sont situés dans des compartiments différents imitant l&#39;architecture en couches du cerveau 18 </sup&gt;. Notre approche est basée sur l&#39;utilisation de la conception de l&#39;échafaudage de soie unique qui accueille haute densité de cellules dans un volume confiné et permet excroissance de réseau dense axonale dans un gel de collagène. Ici, nous démontrons la procédure de montage complet de tissu cérébral comme y compris la fabrication de l&#39;échafaudage et de la culture 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.

Solide éponges de soie prédécoupé à la forme de beigne représentent une idée unique et simple à réaliser une architecture compartimentée ressemblant le tissu nerveux (figure 2A). La structure très poreuse de l&#39;échafaudage avec la taille de pores d&#39;environ 500 um résultats dans la zone de surface exceptionnellement élevé permettant l&#39;ensemencement et de croissance de forte densité de cellules dans un petit volume (7 2x10 / ml) (figure 2B). En outre, la grande porosité de l&#39;échafaudage permet la diffusion sans restriction de nutriments et de déchets résultant de la viabilité supérieure de cultures cellulaires denses plus de prolongement des délais. Après ensemencement des neurones fixer rapidement et uniformément à la surface des pores de l&#39;échafaudage. Après la fixation des cellules est terminée et les cellules sont équilibrées sur le substrat de soie, l&#39;échafaudage de mousse est rempli avec la matrice de collagène souple afin de fournir un environnement 3D pour la formation du réseau axonale (Figure 3 </strong&gt;). En l&#39;absence de la matrice de collagène de la compartimentation des axones et des corps cellulaires est impossible que les neurones extensions poussent que sur la surface des pores conduisant à des réseaux mixtes que l&#39;on trouve avec des surfaces 2D (figure 3B). Par contre, le gel de collagène fournit un maillage large qui supporte la croissance axonale en 3D, alors que corps cellulaires neuronaux restent attachés au squelette de soie (figure 3C). Ce modèle de croissance conduit à cloisonnement des corps cellulaires et les réseaux axonales purs (Figure 3D), qui ressemble à la matière grise et blanche du cortex dans le cerveau. En dehors de microscopie, les constructions peuvent être évalués avec une variété d&#39;autres procédés de 18, en fonction de la question derrière l&#39;expérience. Par exemple, l&#39;isolement de l&#39;ADN, de l&#39;ARN et de protéines à partir des constructions peut être facilement réalisée en utilisant des kits commerciaux (Figure 4). Le rendement en acides nucléiques et de protéines per construction dépend du nombre de cellules initialement ensemencées sur l&#39;échafaudage de soie. Les cellules ensemencées plus plus le rendement de l&#39;ADN, de l&#39;ARN et des protéines. <img alt="Figure 1" src="/files/ftp_upload/52970/52970fig1.jpg" /> Figure 1. PTFE moule utilisé pour la préparation éponge de soie d&#39;échafaudage Les dimensions:.. 10 cm de diamètre, 2 cm de hauteur <a href="https://www.jove.com/files/ftp_upload/52970/52970fig1large.jpg" target="_blank">S&#39;il vous plaît cliquer ici pour voir une version plus grande de cette figure.</a> <img alt="Figure 2" src="/files/ftp_upload/52970/52970fig2.jpg" /> Figure 2. 3D modèle de tissu du cerveau comme. (A) poreux éponge de soie échafaud. (B) en direct coloration / morts de neurones à jour 1 sur l&#39;ensemencement sur ​​échafaudage de soie avant de l&#39;intégrer collagène (cellules vertes-live, les cellules rouges morts). Panneaux sur le côté droit montrent la MAgnification de zone capturée dans des cadres rouges. <a href="https://www.jove.com/files/ftp_upload/52970/52970fig2large.jpg" target="_blank">S&#39;il vous plaît cliquer ici pour voir une version plus grande de cette figure.</a> <img alt="Figure 3" src="/files/ftp_upload/52970/52970fig3.jpg" /> Figure 3. neuronale de l&#39;excroissance cloisonnée dans le modèle de tissu cérébral comme 3D (A) compartiments d&#39;échafaudage:. Rouge-cellule compartiment du corps, compartiment Blue-axonale. (B) de modèle de croissance neuronale au jour 1 après l&#39;ensemencement avant collagène encastrement. (C). Excroissance neuronale au jour 7 après l&#39;ensemencement dans le compartiment de corps cellulaire. (D) de l&#39;excroissance neuronale au jour 7 après l&#39;ensemencement dans le compartiment axonal. Vert -. ΒIIITubulin <a href="https://www.jove.com/files/ftp_upload/52970/52970fig3large.jpg" target="_blank">S&#39;il vous plaît cliquer ici pour voir une version plus grande de cette</a>figure. <img alt="Figure 4" src="/files/ftp_upload/52970/52970fig4.jpg" /> Figure 4. Rendement prévu de (A) ARN et l&#39;ADN, et (B) protéines par construction par rapport à la quantité de cellules ensemencées par échafaudage (± SD, n = 5). <a href="https://www.jove.com/files/ftp_upload/52970/52970fig4large.jpg" target="_blank">S&#39;il vous plaît cliquer ici pour voir une version plus grande de cette figure .</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.

Modèle de polarisée Neural Tissue base-Silk-collagène ingénierie 3D

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).

Champ électrique contrôlé par les migrations des Réalisé cellules progénitrices neurales dans des environnements 2D et 3D

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

Recherche

Bio-ingénierie

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).

Méthodologie élastographie par résonance magnétique pour l&#39;évaluation des tissus Conçu croissance Construct

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.

Protocole pour l&#39;évaluation hydrodynamique relative de Vannes polymères tri-leaflet

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.

génie tissulaire d&#39;un être humain 3D<em&gt; In vitro</em&gt; Système de tumeur de 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.

Génération et greffage des navires par ingénierie tissulaire dans un modèle de souris

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.

Production, la caractérisation et utilisations potentielles d&#39;un humain de l&#39;oesophage muqueux modèle 3D de l&#39;ingénierie tissulaire

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 tissu combiné 3D Conçu<em&gt; In vitro</em&gt; /<em&gt; In Silico</em&gt; Lung Tumor modèle de prévision de l&#39;efficacité des médicaments dans le Fond mutationnelles spécifiques

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.

Bioprinting du Cartilage et peau tissus analogues utilisant un roman passif unité Technique de mélange pour 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.

Ensemencement et Implantation d’un greffon biosynthétique de trachéale tissulaire dans un modèle murin

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.

Greffe tissulaire pour la reconstruction oesophagielle circonférence chez les rats

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