Les auteurs tiennent à remercier le financement du ministère de l&#39;Éducation ACRF Tier 1 (RG41) et démarrer bourse de l&#39;Université technologique de Nanyang. Low et Tjin sont financés par la bourse de recherche pour étudiants (RSS) de Nanyang Technological University, Singapour.

1. Clonage de plasmides recombinants Encodage pour AECM Protéines <ol><li> Conception de la séquence d&#39;acides aminés du domaine fonctionnel (par exemple, le domaine de liaison aux cellules et d&#39;élastine répétitions analogues). les sites de restriction flanquant design les extrémités des domaines fonctionnels pour faciliter la sous-clonage en utilisant le logiciel libre selon l&#39;une des instructions de logiciel (par exemple, http://biologylabs.utah.edu/jorgensen/wayned/ape/). Ici, sélectionner des sites de restriction uniques qui ne sont pas présents dans le domaine fonctionnel de limiter la digestion des sites destinés. Choisissez sites de restriction qui apparaissent dans le site de clonage multiple (MCS) du vecteur hôte (par exemple, pET22b (+)) pour le sous-clonage. </li><li> Inverser traduire la séquence d&#39;acides aminés dans la séquence de nucleotides en utilisant des sites libres selon les instructions du logiciel. (Par exemple, http://www.bioinformatics.org/sms2/rev_trans.html). Assurer codons sont optimisés pour E. coli hôtes. </li><li> Gène d&#39;intérêt peut être Purchasoligonucléotides échoués ed commercialement comme simples (par exemple, si les oligonucléotides &lt;100 paires de bases (pb)) et effectuer l&#39;ADN recuit (voir l&#39;étape 1.4) afin de réduire le coût. Sinon, pour les gènes de plus de 100 pb, ils pourraient être achetés par les sociétés commerciales. </li><li> ADN annelage des oligonucleotides <ol><li> Recuire les oligomères d&#39;ADN pour obtenir la séquence de gène souhaité. Dissoudre oligonucleotides d&#39;ADN dans un tampon d&#39;ADN oligomère (Tris 10 mM: 2-amino-2- (hydroxyméthyl) -propane-1,3-diol, pH 8,0, filtré) jusqu&#39;à une concentration finale de 1 pg / pl. </li><li> Ajouter 4 ul de chaque oligomère à 32 pi de tampon d&#39;hybridation d&#39;ADN (Tris 10 mM, NaCl 100 mM et MgCl2 100 nM) afin d&#39;obtenir un mélange total de 40 ul. </li><li> Faire bouillir un bécher d&#39;eau en utilisant une plaque de cuisson et plongez le mélange pendant 5 min, 95 ° C. Retirer le bécher et refroidir progressivement l&#39;ensemble mis en place dans une boîte en polystyrène O / N. Les oligomères ont été recuit et sont prêts pour la digestion. </li></ol></li&gt; <li> Ajouter les enzymes de restriction correspondantes pour digérer les oligomères recuites (ie, dénommée insert) et vecteur hôte (par exemple, pET22b (+)) séparément. Utiliser la recette suivante (1-2 ug d&#39;ADN, 2 ul de chaque enzyme de restriction, 5 ul de tampon de l&#39;enzyme de restriction 10x, et de l&#39;eau jusqu&#39;à un mélange total 50 ul) pour 3-4 heures à 37 ° C. NOTE: Le pET22b (+) vecteur plasmidique est antibiotique ampicilline-résistant et contient une 6x-His à l&#39;extrémité C-terminale. Le 6x-His présent dans pET22b (+) nous permet d&#39;utiliser ses anticorps marqués pour identifier la protéine cible par western blot à l&#39;étape 7. </li><li> Ajouter 6x colorant de chargement de chaque mélange de digestion. Exécuter les mélanges de digestion comprenant séparément une échelle d&#39;ADN de 1,2% de gels d&#39;ADN d&#39;agarose contenant un colorant d&#39;ADN fluorescent aux UV pendant 1 heure à 100 V. Visualiser le gel d&#39;agarose d&#39;ADN de 1,2% avec un illuminateur UV. </li><li> Trancher le gel pour extraire les produits de ADN digéré en utilisant des kits de purification de gel commerciale. Elute en utilisant le volume minimum de la colonne pour obtenir une concentration minimum d&#39;ADN de 50-100 ng / ul. </li><li> Moissonneuse le gène d&#39;intérêt séquentiellement par ligature l&#39;insert d&#39;ADN digéré (par exemple, des répétitions d&#39;élastine ou des domaines de liaison de cellules obtenues par l&#39;ADN recuit) dans le vecteur plasmidique en utilisant la ligase de T4 en utilisant la recette suivante: (2 pi de vecteur, 1 pi de ligase de T4, 1,5 pi de tampon de ligature T4, x ul de l&#39;insert, 10,5 pi x-eau pour un mélange total 15 ul). Incuber le mélange de ligature à la température ambiante pendant 2 heures. NOTE: concentration molaire de vecteur à insérer doit être modifiée pour optimiser l&#39;efficacité de ligature. Volume de l&#39;insert comme décrit dans la recette x dépend de la concentration de l&#39;ADN élué à l&#39;étape 1.7. </li><li> Thaw E. coli DH5a cellules chimiquement compétentes (ou toute souche de clonage) sur de la glace. Des plaques de gélose YT 2x chaud (tableau 1) contenant de l&#39;ampicilline (25 ug / ml) à 37 ° C. </li><li> Transformer les cellules en utilisantchoc thermique: <ol><li> Aliquote de 50 pi de cellules compétentes dans des tubes micro-centrifugeuse propres, pré-réfrigérés. Introduire à la pipette 5 ul (entre 100 pg à 100 ng) du mélange de ligation dans les cellules, le pipetage de haut en bas doucement pour mélanger. Laisser le mélange sur de la glace pendant 20 min. </li><li> Immerger le tube de micro-centrifugation contenant le mélange de cellules dans un bain d&#39;eau à 42 ° C pendant 2 min et est retourné à la glace pendant 2 min. Chronométrer le temps d&#39;immersion pour minimiser les dommages de la chaleur vers les cellules. </li><li> Ajouter 500 ul de milieu SOC (tableau 1) dans le tube de micro-centrifugeuse et incuber à 37 ° C avec agitation par secousses pendant 1 heure. </li><li> Diffuser 50 500 ul du mélange de cellules / ligation sur une plaque de gélose YT 2x qui a été pré-chauffé à la température ambiante, contenant de l&#39;ampicilline (25 ug / ml) et on incube les plaques à l&#39;envers à 37 ° CO / N (12-16 h) . </li></ol></li><li> Le lendemain, ramasser colonies d&#39;ADN provenant de la plaque de gélose en utilisant des embouts de pipette propres. Développer les colonies dans 5 ml de milieu 2YT (tableau1) contenant de l&#39;ampicilline (25 pg / ml) O / N à 37 ° CO / N (12 à 16 h) sous agitation (225 rpm). </li><li> Le jour suivant, utiliser un kit d&#39;isolement de plasmide pour extraire les plasmides d&#39;ADN de chaque colonie choisi selon le protocole du fabricant. Eluer l&#39;ADN avec 50 ul d&#39;eau. </li><li> Effectuez un test digérer avec des enzymes de restriction en utilisant la recette suivante: (ADN 5 pi, 0,2 pi de chaque enzyme de restriction, tampons de l&#39;enzyme 1 ul 10x de restriction et l&#39;appoint d&#39;eau pour un mélange total de 10 pi), incuber pendant 2 heures à 37 ° C à l&#39;écran pour les colonies qui contiennent probablement l&#39;insert et sur un gel de l&#39;ADN d&#39;agarose à 1,2% comme dans l&#39;étape 1.6. REMARQUE: Lors de la digestion, d&#39;une ligature réussie devrait les résultats dans deux bandes, une pour chaque vecteur et insert qui correspondent à leur poids moléculaire respectif (Figure 1). </li><li> Envoyer les colonies probables pour le séquençage de l&#39;ADN en utilisant le promoteur T7 amorces sens et antisens à séquenceurs commerciales. </li></ol><ol><li> A partir du résultat de séquençage, sélectionner une colonie qui a été ligaturé avec succès et en utilisant le plasmide d&#39;ADN pour la transformation dans un E. hôte d&#39;expression coli. </li><li> Thaw E. coli souche d&#39;expression (BL21 (DE3) pLysS) sur de la glace. Pendant ce temps, des plaques d&#39;agar contenant de l&#39;ampicilline chaudes (25 ug / ml) et du chloramphenicol (34 ug / ml) à TA. NOTE: Le plasmide pLysS présent dans les bactéries souche BL21 (DE3) pLysS contient un gène de résistance au chloramphénicol. Le plasmide pLysS contient un gène de répresseur T7 qui est exprimé de manière constitutive à limiter l&#39;expression de la protéine qui fuit AECM. Le chloramphénicol est nécessaire de sélectionner les cellules bactériennes qui contiennent pLysS au cours de la culture. </li><li> Répétez l&#39;étape 1.10 pour obtenir transformé E. cellules de E. coli qui sont prêts à exprimer les protéines artificielles. Enveloppez plaques d&#39;agar dans Parafilm et stocker à l&#39;envers dans 4 6; C pendant jusqu&#39;à un mois. </li></ol> 3. Expression bactérienne de l&#39;AECM Protéines <ol><li> Reportez-vous à la figure 2, choisir une colonie à partir de la plaque de gélose transformée avec une pointe de pipette et l&#39;inoculer dans 10 ml de bouillon Terrific (TB) milieux stériles (tableau 1) contenant de l&#39;ampicilline et les antibiotiques de chloramphénicol dans un tube à essai. Incuber cette culture de départ à 37 ° CO / N (12-16 h) avec agitation à 225 rpm. </li><li> Transfert 10 ml de la culture de départ dans 1 litre du milieu frais et stériles TB supplémenté avec les mêmes antibiotiques dans un flacon Erlenmeyer de 3 litres. Incuber la culture à 37 ° C sous agitation à 225 tours par minute pendant 2-3 heures et observer la densité optique de la culture (OD 600) atteint 0,6-0,8 par pipetage 1 ml de la culture dans une cuvette vide pour la lecture. Enregistrer 1 ml de culture avant l&#39;induction pour SDS-PAGE caractérisation. <ol><li> Pour mesurer DO 600 de la culture cellulaire, préparer 1 flacon de médias de la tuberculose dans une cuvettepour une mesure à blanc. Séparément, transférer 1 ml de la culture de la fiole de culture dans une nouvelle cuvette vide. Mesurer la densité optique de la culture en utilisant un spectrophotomètre par rapport au témoin à blanc à une absorbance de 600 nm. </li><li> Économisez de l&#39;échantillon (pour analyse ultérieure SDS-PAGE) en transférant les 1 ml d&#39;échantillon de la culture dans un tube de 1,5 ml, centrifuger à 12 000 g pendant 2 min et décanter le surnageant. </li></ol></li><li> Induire la culture avec isopropyle β-D-1-thiogalactopyranoside (IPTG) à une concentration finale de 1 mM et on incube à 37 ° C sous agitation à 225 tours par minute pendant 4 h un autre. Enregistrer 1 ml de la culture à la fin de l&#39;induction à 4 h pour SDS-PAGE caractérisation. </li><li> Récolter les cellules par transfert de la culture à 1 L flacons de centrifugation et centrifuger à 12 000 xg pendant 30 min à 4 ° C. Jeter le surnageant, peser les culots de cellules et remettre en suspension dans un tampon TEN (1 M de Tris, 0,01 M d&#39;EDTA, 0,1 M de NaCl, pH = 8,0) à 0,5 g / ml. </li></ol> 4. lyse des cultures bactériennes <ol><li> Geler la culture de cellules remises en suspension à -80 ° CO / N. Décongeler la culture de cellules congelées dans un bain d&#39;eau à température ambiante ou sur de la glace pour lyser les cellules. Ajouter 10 ug / ml de désoxyribonucléase I (DNase I), 10 ug / ml de ribonucléase A (RNase I), et 50 ug / ml de fluorure de phénylméthylsulfonyle (PMSF) pendant la décongélation et homogénéiser la solution sous agitation lente. </li><li> Une fois que toutes les cellules remises en suspension sont décongelés, ajuster la solution à un pH de 9,0 à augmenter la solubilité de la protéine dans de l&#39;eau 12. Ajouter NaOH 6 N goutte à goutte sous agitation sur de la glace pour obtenir une consistance homogène. Lyse par la perturbation ultrasons pendant 20 min sur la glace en utilisant un diamètre pointe plate de 2 mm, 5 impulsion de sec. </li><li> Centrifuger la solution de cellules à 12.000 g pendant 30 min à 4 ° C. Transférer le surnageant, une bouteille et un magasin vide propre à 4 ° C pour la purification plus tard. </li><li> Pendant ce temps, la remise en suspension de nouveau le culot cellulaire avec un tampon TEN et re-congélation à -80 ° C. Àlyse cellulaire complète, répétez gel / dégel et sonication processus jusqu&#39;à trois fois. Économisez 20 lysat cellulaire pi pour SDS-PAGE caractérisation. </li></ol> 5. Purification de protéines en utilisant l&#39;AECM inverse transition vélo <ol><li> Rassembler le lysat cellulaire de l&#39;étape 4.3 et passez à purifier les protéines AECM utilisant ITC, similaire à celle utilisée pour la purification de protéines à base d&#39;élastine 21. Cycles seront effectuées avec les différentes températures qui sont de 4 ° C (appelés «à froid») et 37 ° C (appelés &quot;chaudes&quot;) cycles, respectivement. </li><li> Diviser le lysat cellulaire dans 50 ml des flacons de centrifugation, et centrifuger à 40 000 g pendant 2 h à 4 ° C. L&#39;apparition de la pastille cellulaire devrait être brun foncé et de regarder qui coule. </li><li> Eliminer le surnageant par pipetage pour obtenir une séparation nette du culot. Récupérer le surnageant dans des flacons à centrifuger propre et ajouter NaCl à une concentration finale de 1 M (pour un maximum de 3 M). Chaleureuxla solution à 37 ° C pendant 2 heures sous agitation à 225 tours par minute. NOTE: L&#39;addition de NaCl va déclencher la transition de la composante de l&#39;élastine provoquant l&#39;agrégation AECM. Le surnageant deviendra trouble. Si la concentration en protéine est suffisamment élevée, la protéine de blanc mousseux peut être vu sur les côtés de la bouteille de centrifugeuse. </li><li> Centrifuger le surnageant de l&#39;étape 5.3 à 40.000 g pendant 2 heures à 37 ° C. Décanter le surnageant. Écraser la pastille à l&#39;aide d&#39;une spatule métallique et remettre les morceaux de boulettes dans l&#39;autoclave de l&#39;eau distillée glacée (50 mg / ml) avec agitation vigoureuse O / N à 4 ° C en utilisant une barre d&#39;agitation magnétique et la plaque. Le culot doit être complètement dissous. </li><li> Répéter les étapes 5.2 à 5.4 pendant trois à cinq fois pour obtenir une plus grande pureté de la protéine. </li><li> Après le cycle final de purification, dessaler la solution de protéine par dialyse contre de l&#39;eau distillée à 4 ° C. Dialyser solution de protéines contre l&#39;eau pendant 2-3 jours avec des changements dans l&#39;eau tous les 4h ou 8 h pour O / N. Économisez 20 pi de la protéine purifiée pour SDS-PAGE et lyophiliser le reste de la protéine purifiée et conserver à -80 ° C jusqu&#39;à utilisation ultérieure. </li></ol> 6. Caractérisation des protéines en utilisant l&#39;AECM Electrophorèse SDS-PAGE <ol><li> Préparer un gel SDS-PAGE 12% (si le poids moléculaire est grande, utiliser 8% de gels de SDS-PAGE pour une meilleure séparation). Ajouter tampon 2x de chargement SDS à chaque échantillon, chauffer les échantillons pendant 10 min à 100 ° C et analyser des échantillons sur le gel pendant 1 heure à 100 V ou jusqu&#39;à ce que l&#39;échelle de la protéine correspondant au poids moléculaire de la protéine AECM atteint le milieu de la gel. </li><li> Récupérer le gel de la SDS-PAGE mettre en place et ajouter du bleu de Coomassie tache (tableau 2) avec un volume de submerger le gel pendant 1 h dans une boîte de Petri. Rincer gel dans une solution de décoloration (tableau 2) pendant 5 minutes sur une bascule. Changer la solution de décoloration, et de continuer à décolorer le gel avec bascule jusqu&#39;à ce que le fond de la til devient clair gel. Les bandes de protéines doivent être clairement visibles. </li><li> Comparer la position de la protéine cible contre l&#39;échelle de la protéine pour déterminer le poids moléculaire de la protéine cible. </li><li> Pour confirmer en outre la présence de la protéine cible, effectuer un transfert de Western comme à l&#39;étape 7. </li></ol> 7. Caractérisation des protéines en utilisant l&#39;AECM Western Blot <ol><li> Exécutez les échantillons sur un gel SDS-PAGE à 12%, comme dans l&#39;article 6, sans taches. </li><li> Couper une membrane de nitrocellulose à une taille légèrement plus grande que le gel SDS-PAGE. Coupez quatre morceaux de papier filtre à la même taille. </li><li> Wet un morceau de papier filtre avec transfert Western tampon (20% v / v de méthanol, Tris 25 mM, glycine 190, pH 8,3), placer le gel SDS-PAGE sur le dessus du papier filtre, suivi par un autre morceau de papier filtre , puis la membrane de nitrocellulose et une finale autres deux morceaux de papier-filtre. Assurer l&#39;ensemble set-up est submergé avec un tampon de transfert ouest suffisante. NOTE: Le gel et la membrane ne doivent pas être en contact à ce point du temps que les protéines pourraient se lier à la membrane au contact. </li><li> Transférer les protéines sur la membrane de nitrocellulose en plaçant deux papiers-filtres dans une unité de transfert semi-sec occidentale, suivie par la membrane de nitrocellulose, et placer soigneusement le gel SDS-PAGE à l&#39;intérieur et sur la membrane, placer enfin les deux derniers papier filtre humide sur le gel SDS-PAGE. Exécutez le transfert ouest à 45 mA, 30 min. Ajouter tampon si la tension est supérieure à 30 V. REMARQUE: De préférence, placer le gel SDS-PAGE sur la membrane avec une tentative et éviter de déplacer inutilement le gel sur la membrane sous forme de protéines peuvent se lier à la membrane au moment du contact. </li><li> Récupérer et bloquer la membrane de nitrocellulose avec une solution de blocage (5% de lait écrémé dans du PBS pH 7,4, filtré avec un papier filtre) pendant 2 heures à la température ambiante. Éliminer tampon bloquant et ajouter PBS pour rincer. NOTE: Changer pour de nouveaux gants pour éviter la contamination de la membrane avec prote indésirablesins. </li><li> Incuber primaire anticorps anti-His avec dilution dans du PBS à 1: 1 000 à température ambiante pendant 1 h avec bascule. Préparer un mélange dans un volume qui peut submerger l&#39;ensemble membrane, par exemple, avec un rapport de dilution de 1: 1000, ajouter 1 pl d&#39;anticorps (concentration de valeurs: 1 mg / ml) à 999 ul de PBS pour un 1 ml mélange total. </li><li> Rincer la membrane avec 5 ml de PBST (PBS avec 0,1% de Tween 20). </li><li> Incuber avec l&#39;anticorps secondaire conjugué à la peroxydase de raifort (HRP) avec une dilution dans du PBS à 1: 5,000 à TA pendant 1 h à bascule. Préparer un mélange dans un volume qui pourrait plonger l&#39;ensemble membrane, par exemple, avec un rapport de dilution de 1: 5000, ajouter 1 pl de l&#39;anticorps (concentration du stock: 1 mg / ml) à 4999 pi de PBS pour un 5 ml mélange total. </li><li> Laver la membrane avec 5 ml de PBST et répéter deux fois. </li><li> Appliquer le substrat chimioluminescent à la membrane avec un volume pour couvrir la membrane et incuber en suivant les instructions pour le substrat utilisé. NOTE: La sensibilité du substrat à la détection de protéines peut varier, nous vous conseillons un substrat avec une sensibilité maximale pour les signaux très faibles dans le cas augmentation de la concentration d&#39;anticorps ne pas augmenter signaux. </li><li> Capturez les signaux chimiluminescents en utilisant un imageur caméra CCD. Ajustez le taux d&#39;anticorps dilution primaire et secondaire si les signaux sont faibles et non spécifique. Incuber plus dans la solution de blocage si le signal de fond est élevé. </li></ol>

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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Designing+ECM-mimetic+materials+using+protein+engineering.">Designing ECM-mimetic materials using protein engineering.</a> Acta Biomater. 10 (4), 1751-1760 (2014).</li><li>Annabi, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elastomeric+recombinant+protein-based+biomaterials.">Elastomeric recombinant protein-based biomaterials.</a> Biochemical Engineering Journal. 77, 110-118 (2013).</li><li>Heilshorn, S. C., DiZio, K. A., Welsh, E. R., Tirrell, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+cell+adhesion+to+the+fibronectin+CS5+domain+in+artificial+extracellular+matrix+proteins.">Endothelial cell adhesion to the fibronectin CS5 domain in artificial extracellular matrix proteins.</a> Biomaterials. 24 (23), 4245-4252 (2003).</li><li>Simnick, A. J., Lim, D. W., Chow, D., Chilkoti, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomedical+and+Biotechnological+Applications+of+Elastin-Like+Polypeptides.">Biomedical and Biotechnological Applications of Elastin-Like Polypeptides.</a> Polymer Reviews. 47 (1), 121-154 (2007).</li><li>Tjin, M. S., Chua, A. W. C., Ma, D. R., Lee, S. T., Fong, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Epidermal+Keratinocyte+Cell+Response+on+Integrin-Specific+Artificial+Extracellular+Matrix+Proteins.">Human Epidermal Keratinocyte Cell Response on Integrin-Specific Artificial Extracellular Matrix Proteins.</a> Macromolecular Bioscience. 14 (8), 1125-1134 (2014).</li><li>MacNeil, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+and+opportunities+for+tissue-engineered+skin.">Progress and opportunities for tissue-engineered skin.</a> Nature. 445 (7130), 874-880 (2007).</li><li>Kariya, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+regulation+of+cellular+adhesion+and+migration+by+recombinant+laminin-5+forms+with+partial+deletion+or+mutation+within+the+G3+domain+of+&#945;3+chain.">Differential regulation of cellular adhesion and migration by recombinant laminin-5 forms with partial deletion or mutation within the G3 domain of &#945;3 chain.</a> J Cell Biochem. 88 (3), 506-520 (2003).</li><li>Shang, M., Koshikawa, N., Schenk, S., Quaranta, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+LG3+module+of+laminin-5+harbors+a+binding+site+for+integrin+&#945;3&#914;1+that+promotes+cell+adhesion,+spreading,+and+migration.">The LG3 module of laminin-5 harbors a binding site for integrin &#945;3&#914;1 that promotes cell adhesion, spreading, and migration.</a> J Biol Chem. 276 (35), 33045-33053 (2001).</li><li>Hassouneh, W., Christensen, T., Chilkoti, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elastin-like+polypeptides+as+a+purification+tag+for+recombinant+proteins.">Elastin-like polypeptides as a purification tag for recombinant proteins.</a> Current Protocols in Protein Science. , 6.11.1-6.11.16 (2010).</li><li>Keeley, F., Mecham, R., Keeley, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ch.+4.">Ch. 4.</a> Evolution of Extracellular Matrix.Biology of Extracellular Matrix. , 73-119 (2013).</li><li>Le, D. H. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-Assembly+of+Elastin-Mimetic+Double+Hydrophobic+Polypeptides.">Self-Assembly of Elastin-Mimetic Double Hydrophobic Polypeptides.</a> Biomacromolecules. 14 (4), 1028-1034 (2013).</li><li>MacEwan, S. R., Hassouneh, W., Chilkoti, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-chromatographic+Purification+of+Recombinant+Elastin-like+Polypeptides+and+their+Fusions+with+Peptides+and+Proteins+from+Escherichia+coli.">Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli.</a> Journal of visualized experiments: JoVE. (88), e51583 (2014).</li><li>Meyer, D. E., Chilkoti, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetically+encoded+synthesis+of+protein-based+polymers+with+precisely+specified+molecular+weight+and+sequence+by+recursive+directional+ligation:+examples+from+the+elastin-like+polypeptide+system.">Genetically encoded synthesis of protein-based polymers with precisely specified molecular weight and sequence by recursive directional ligation: examples from the elastin-like polypeptide system.</a> Biomacromolecules. 3 (2), 357-367 (2002).</li></ol>

Les auteurs déclarent qu&#39;ils ont aucun intérêt financier concurrentes.

Recombinant ingénierie des protéines est une technique polyvalent pour créer de nouveaux matériaux de protéines en utilisant une approche bottom-up. Les matériaux à base de protéines peuvent être conçues pour avoir de multiples fonctionnalités, adaptées en fonction de l&#39;application d&#39;intérêts. En raison de l&#39;augmentation progrès dans les technologies de clonage et d&#39;expression de protéine, il est devenu relativement simple (et rentable) pour créer une variété de protéines artificielle...

For several decades, both synthetic and natural materials have been explored for use as scaffolds in tissue engineering1,2. While synthetic materials such as polymers offer excellent structural integrity and tunable mechanical properties, they often have insufficient bioactivity to promote growth and infiltration of tissues. On the other hand, natural materials such as extracellular matrix (ECM) proteins have excellent biological activity, but have limitations such as batch-to-batch variability, rapid degradation and immunogenicity issues. As such, recombinant proteins are desired, since they can be designed to mimic only the desirable properties of native proteins3,4.
Recombinant protein engineering has garnered widespread interests as a versatile platform for the design and production of novel artificial protein biopolymers. By controlling the genetic sequence, the functionalities of the artificial proteins can be tailored for a wide variety of applications5,6. In particular, artificial extracellular matrix (aECM) proteins can be tailored to have multiple functionalities for applications in tissue engineering, regeneration and wound repair2,7. More importantly, advances in cloning and purification technologies have increased scalability and reduced the cost of manufacturing recombinant proteins tremendously. It is possible to produce large quantities of recombinant proteins at low production costs which are economic for use in the clinic5. 
Artificial extracellular matrix proteins have been developed for tissue engineering applications8-11. For instance, Tirrell et al. designed a small diameter vascular graft using artificial proteins containing fibronectin CS5 sequence and elastin-like repeats (ELP-CS5). They showed that human umbilical vein endothelial cells (HUVECs) were able to adhere and grow on these materials12. Others have also incorporated short bioactive sequences taken from fibronectin, collagen, laminin, fibrinogen and vitronectin as well as structural domains that mimic elastin, spider silk and collagens to create a variety of fusion proteins10. Bulk cross-linked films made out of elastin-based aECM proteins also exhibited mechanical properties similar to that of native elastin (elastic moduli ranges between 0.3-0.6 MPa)13. Subsequently, aECM proteins containing longer fibronectin fragments were also reported to accelerate wound healing in vitro due to increased integrin binding affinities8. 
Recently, integrin-specific artificial ECM proteins have been developed by Tjin and coworkers14. Each aECM protein contains a bioactive cell-binding domain taken from ECM components of native human skin2,7,15, such as laminin-5, collagen-IV and fibronectin. For example, the integrin &#945;3&#914;1 has been shown to bind the PPFLMLLKGSTR sequence found in the laminin-5 alpha-3 chain globular domain 3 (LG3)16,17. In their report, they showed that primary human skin epidermal keratinocytes preferentially engage different integrins for binding to each of the aECM proteins, depending on the type of cell binding domain present.
The aECM proteins discussed in the work by Tjin et al. contain flanking elastin-like domains {(VPGIG)2VPGKG(VPGIG)2}8 that confer elasticity which mimics the mechanical properties of human skin. In addition, the incorporation of lysine residues within the elastin-like repeats also increases the overall protein solubility in aqueous solvents. In addition, the lysine residues also serve as crosslinking sites to facilitate the formation of crosslinked aECM films12. Inclusion of elastin-like repeats within the aECM protein sequence allow the proteins to be readily purified via Inverse Transition Cycling (ITC)14. Elastins undergo a sharp and reversible phase transition at a specific temperature known as the lower critical solution temperature (LCST) or the inverse transition temperature (Tt)18-20. Elastins and elastin-like repeats adopt hydrophilic random coil conformations below their LCST and become soluble in water, whereas above their LCST, elastins aggregate rapidly into micron-size particles. Such phase transitions are reversible and hence, can be exploited to allow elastin-based aECM proteins to be readily purified via the ITC technique21.
In this work, we report a generalized procedure to design, construct and purify artificial ECM proteins containing bioactive cell-binding domains, fused to elastin-like repeats. The process to design and clone the plasmids that encode for the amino acid sequences for the aECM proteins is described. The steps involved to purify the aECM proteins using ITC are outlined. Finally, the methods to determine the purity of the aECM proteins obtained using SDS-PAGE electrophoresis and Western Blotting are discussed.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>pET22b (+)</td>
			<td>Novagen</td>
			<td>69744</td>
			<td>T7 expression vectors with resistance to ampicillin&#160;</td>
		</tr>
		<tr>
			<td>BL21(DE3)pLysS&#160;</td>
			<td>Invitrogen</td>
			<td>C6060-03</td>
			<td>additional antibiotics - chloramphenicol</td>
		</tr>
		<tr>
			<td>Isopropyl-beta-D-thiogalactoside (IPTG)</td>
			<td>Gold Biotechnology</td>
			<td>I2481C</td>
			<td>1M stock solution with autoclaved water, make fresh prior to induction.</td>
		</tr>
		<tr>
			<td>QIAprep Spin Miniprep Kit</td>
			<td>Qiagen</td>
			<td>27106</td>
			<td>plasmid isolation kit</td>
		</tr>
		<tr>
			<td>T4 ligase</td>
			<td>New England Biolabs</td>
			<td>M0202S</td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>Affymetrix</td>
			<td>11259</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloramphenicol</td>
			<td>Affymetrix</td>
			<td>23660</td>
			<td></td>
		</tr>
		<tr>
			<td>Zymoclean&#8482; gel DNA recovery kit</td>
			<td>Zymo Research</td>
			<td>D4001</td>
			<td></td>
		</tr>
		<tr>
			<td>XL10-gold strain</td>
			<td>Agilent Technologies</td>
			<td>200315</td>
			<td></td>
		</tr>
	</tbody>
</table>

design and construction of artificial extracellular matrix (aecm) proteins from escherichia coli for skin tissue engineering

Recombinant technology is a versatile platform to create novel artificial proteins with tunable properties. For the last decade, many artificial proteins that have incorporated functional domains derived from nature (or created de novo) have been reported. In particular, artificial extracellular matrix (aECM) proteins have been developed; these aECM proteins consist of biological domains taken from fibronectin, laminins and collagens and are combined with structural domains including elastin-like repeats, silk and collagen repeats. To date, aECM proteins have been widely investigated for applications in tissue engineering and wound repair. Recently, Tjin and coworkers developed integrin-specific aECM proteins designed for promoting human skin keratinocyte attachment and propagation. In their work, the aECM proteins incorporate cell binding domains taken from fibronectin, laminin-5 and collagen IV, as well as flanking elastin-like repeats. They demonstrated that the aECM proteins developed in their work were promising candidates for use as substrates in artificial skin. Here, we outline the design and construction of such aECM proteins as well as their purification process using the thermo-responsive characteristics of elastin.

Lors de la conception des protéines de fusion contenant des séquences répétées en élastine analogue, il est important de maintenir une teneur en élastine dans l&#39;ensemble, assez grande fraction de la protéine de fusion 18. Ceci afin d&#39;assurer que la construction de la protéine de fusion conserve ses caractéristiques de type élastine, afin d&#39;utiliser l&#39;ITC pour la purification. La conception et les séquences décrites dans cette section protéines AECM ont été spécifiquement pris ...

Watch this Scientific Journal Video about Design and Construction of Artificial Extracellular Matrix aECM Proteins from Escherichia coli for Skin Tissue Engineering at JoVE.com

Design and Construction of Artificial Extracellular Matrix aECM Proteins from Escherichia coli for Skin Tissue Engineering

Recombinant technologies have enabled material designers to create novel artificial proteins with customized functionalities for tissue engineering applications. For example, artificial extracellular matrix proteins can be designed to incorporate structural and biological domains derived from native ECMs. Here, we describe the construction and purification of aECM proteins containing elastin-like repeats.

Conception et construction de la matrice extracellulaire artificielle (AECM) Protéines de<em&gt; Escherichia coli</em&gt; Pour l&#39;ingénierie tissulaire de la peau

Recombinant technology is a versatile platform to create novel artificial proteins with tunable properties. For the last decade, many artificial proteins ...

design-construction-artificial-extracellular-matrix-aecm-proteins

Research

JoVE Journal

Bioengineering

Design and Construction of Artificial Extracellular Matrix (aECM) Proteins from Escherichia coli for Skin Tissue Engineering

8.8K vues.  Nanyang Technological University. Recombinant technologies have enabled material designers to create novel artificial proteins with customized functionalities for tissue engineering applications. For example, artificial extracellular matrix proteins can be designed to incorporate structural and biological domains derived from native ECMs. Here, we describe the construction and purification of aECM proteins containing elastin-like repeats. 

Vidéo: Design and Construction of Artificial Extracellular Matrix aECM Proteins from Escherichia coli for Skin Tissue Engineering

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

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

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

Sans contact, sans étiquette de surveillance des cellules et la matrice extracellulaire en utilisant la spectroscopie Raman

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

Recherche

Bio-ingénierie

Design of a Biaxial Mechanical Loading Bioreactor for Tissue Engineering

We designed a loading device that is capable of applying uniaxial or biaxial mechanical strain to a tissue engineered biocomposites fabricated for transplantation. While the device primarily functions as a bioreactor that mimics the native mechanical strains, it is also outfitted with a load cell for providing force feedback or mechanical testing of the constructs. The device subjects engineered cartilage constructs to biaxial mechanical loading with great precision of loading dose (amplitude and frequency) and is compact enough to fit inside a standard tissue culture incubator. It loads samples directly in a tissue culture plate, and multiple plate sizes are compatible with the system. The device has been designed using components manufactured for precision-guided laser applications. Bi-axial loading is accomplished by two orthogonal stages. The stages have a 50 mm travel range and are driven independently by stepper motor actuators, controlled by a closed-loop stepper motor driver that features micro-stepping capabilities, enabling step sizes of less than 50 nm. A polysulfone loading platen is coupled to the bi-axial moving platform. Movements of the stages are controlled by Thor-labs Advanced Positioning Technology (APT) software. The stepper motor driver is used with the software to adjust load parameters of frequency and amplitude of both shear and compression independently and simultaneously. Positional feedback is provided by linear optical encoders that have a bidirectional repeatability of 0.1 &mu;m and a resolution of 20 nm, translating to a positional accuracy of less than 3 &mu;m over the full 50 mm of travel. These encoders provide the necessary position feedback to the drive electronics to ensure true nanopositioning capabilities. In order to provide the force feedback to detect contact and evaluate loading responses, a precision miniature load cell is positioned between the loading platen and the moving platform. The load cell has high accuracies of 0.15% to 0.25% full scale.

We designed a novel mechanical loading bioreactor that can apply uniaxial or biaxial mechanical strain to a cartilage biocomposite prior to transplantation into an articular cartilage defect.

Conception d&#39;un bioréacteur de chargement mécanique biaxial pour l&#39;ingénierie tissulaire

We  designed a loading device that is capable of applying uniaxial or biaxial  mechanical strain to a tissue engineered biocomposites fabricated for  ...

Tissue Engineering: Construction of a Multicellular 3D Scaffold for the Delivery of Layered Cell Sheets

Many tissues, such as the adult human hearts, are unable to adequately regenerate after damage.2,3 Strategies in tissue engineering propose innovations to assist the body in recovery and repair. For example, TE approaches may be able to attenuate heart remodeling after myocardial infarction (MI) and possibly increase total heart function to a near normal pre-MI level.4 As with any functional tissue, successful regeneration of cardiac tissue involves the proper delivery of multiple cell types with environmental cues favoring integration and survival of the implanted cell/tissue graft. Engineered tissues should address multiple parameters including: soluble signals, cell-to-cell interactions, and matrix materials evaluated as delivery vehicles, their effects on cell survival, material strength, and facilitation of cell-to-tissue organization. Studies employing the direct injection of graft cells only ignore these essential elements.2,5,6 A tissue design combining these ingredients has yet to be developed. Here, we present an example of integrated designs using layering of patterned cell sheets with two distinct types of biological-derived materials containing the target organ cell type and endothelial cells for enhancing new vessels formation in the &#8220;tissue&#8221;. Although these studies focus on the generation of heart-like tissue, this tissue design can be applied to many organs other than heart with minimal design and material changes, and is meant to be an off-the-shelf product for regenerative therapies. The protocol contains five detailed steps. A temperature sensitive Poly(N-isopropylacrylamide) (pNIPAAM) is used to coat tissue culture dishes. Then, tissue specific cells are cultured on the surface of the coated plates/micropattern surfaces to form cell sheets with strong lateral adhesions. Thirdly, a base matrix is created for the tissue by combining porous matrix with neovascular permissive hydrogels and endothelial cells. Finally, the cell sheets are lifted from the pNIPAAM coated dishes and transferred to the base element, making the complete construct.

For creation of highly organized structures of complex tissue, one must assemble multiple material and cell types into an integrated composite. This combinatorial design incorporates organ-specific layered cell sheets with two distinct biologically-derived materials containing a strong fibrous matrix base, and endothelial cells for enhancing new vessels formation.

Ingénierie tissulaire: Construction d&#39;un échafaudage 3D multicellulaire pour la livraison de stratifiés feuilles cellulaires

Many tissues, such as the adult human hearts, are unable to adequately regenerate after damage.2,3 Strategies in tissue engineering propose innovations to ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

Génération d&#39;une épaisseur en trois dimensions équivalent de peau et automatisé Blessant

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

Epithelial Cell Repopulation and Preparation of Rodent Extracellular Matrix Scaffolds for Renal Tissue Development

This protocol details the generation of acellular, yet biofunctional, renal extracellular matrix (ECM) scaffolds that are useful as small-scale model substrates for organ-scale tissue development. Sprague Dawley rat kidneys are cannulated by inserting a catheter into the renal artery and perfused with a series of low-concentration detergents (Triton X-100 and sodium dodecyl sulfate (SDS)) over 26 hr to derive intact, whole-kidney scaffolds with intact perfusable vasculature, glomeruli, and renal tubules. Following decellularization, the renal scaffold is placed inside a custom-designed perfusion bioreactor vessel, and the catheterized renal artery is connected to a perfusion circuit consisting of: a peristaltic pump; tubing; and optional probes for pH, dissolved oxygen, and pressure. After sterilizing the scaffold with peracetic acid and ethanol, and balancing the pH (7.4), the kidney scaffold is prepared for seeding via perfusion of culture medium within a large-capacity incubator maintained at 37 &#176;C and 5% CO2. Forty million renal cortical tubular epithelial (RCTE) cells are injected through the renal artery, and rapidly perfused through the scaffold under high flow (25 ml/min) and pressure (~230 mmHg) for 15 min before reducing the flow to a physiological rate (4 ml/min). RCTE cells primarily populate the tubular ECM niche within the renal cortex, proliferate, and form tubular epithelial structures over seven days of perfusion culture. A 44 &#181;M resazurin solution in culture medium is perfused through the kidney for 1 hr during medium exchanges to provide a fluorometric, redox-based metabolic assessment of cell viability and proliferation during tubulogenesis. The kidney perfusion bioreactor permits non-invasive sampling of medium for biochemical assessment, and multiple inlet ports allow alternative retrograde seeding through the renal vein or ureter. These protocols can be used to recellularize kidney scaffolds with a variety of cell types, including vascular endothelial, tubular epithelial, and stromal fibroblasts, for rapid evaluation within this system.

This protocol describes decellularization of Sprague Dawley rat kidneys by antegrade perfusion of detergents through the vasculature, producing acellular renal extracellular matrices that serve as templates for repopulation with human renal epithelial cells. Recellularization and use of the resazurin perfusion assay to monitor growth is performed within specially-designed perfusion bioreactors.

Épithéliale repeuplement cellulaire et préparation des rongeurs matrice extracellulaire échafaudages pour le développement de tissu rénal

This protocol details the generation of acellular, yet biofunctional, renal extracellular matrix (ECM) scaffolds that are useful as small-scale model ...

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, which is regulated by a variety of soluble and physical signals in the microenvironment. Described here is a method created to study the process of branching morphogenesis by forming engineered three-dimensional (3D) epithelial tissues of defined shape and size that are completely embedded within an extracellular matrix (ECM). This method enables the formation of arrays of identical tissues and enables the control of a variety of environmental factors, including tissue geometry, spacing, and ECM composition. This method can also be combined with widely used techniques such as traction force microscopy (TFM) to gain more information about the interactions between cells and their surrounding ECM. The protocol can be used to investigate a variety of cell and tissue processes beyond branching morphogenesis, including cancer invasion.

This manuscript describes a soft lithography-based technique to engineer uniform arrays of three-dimensional (3D) epithelial tissues of defined geometry surrounded by extracellular matrix. This method is amenable to a wide variety of cell types and experimental contexts and allows for high-throughput screening of identical replicates.

Ingénierie en trois dimensions des tissus épithéliaux incorporés au sein de la matrice extracellulaire

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, ...

Preparation of Extracellular Matrix Protein Fibers for Brillouin Spectroscopy

Brillouin spectroscopy is an emerging technique in the biomedical field. It probes the mechanical properties of a sample through the interaction of visible light with thermally induced acoustic waves or phonons propagating at a speed of a few km/sec. Information on the elasticity and structure of the material is obtained in a nondestructive contactless manner, hence opening the way to in vivo applications and potential diagnosis of pathology. This work describes the application of Brillouin spectroscopy to the study of biomechanics in elastin and trypsin-digested type I collagen fibers of the extracellular matrix. Fibrous proteins of the extracellular matrix are the building blocks of biological tissues and investigating their mechanical and physical behavior is key to establishing structure-function relationships in normal tissues and the changes which occur in disease. The procedures of sample preparation followed by measurement of Brillouin spectra using a reflective substrate are presented together with details of the optical system and methods of spectral data analysis.

We present a protocol for the application of Brillouin light scattering spectroscopy to elastin and trypsin-purified type I collagen fibers of the extracellular matrix to extract their full elastic properties.

Préparation de la matrice extracellulaire des fibres de protéines pour Brillouin Spectroscopy

Brillouin spectroscopy is an emerging technique in the biomedical field. It probes the mechanical properties of a sample through the interaction of ...

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung tissue from porcine, rat, or mouse, the tissue is perfused and submerged in subsequent chemical detergents to remove the cellular debris. Histological comparison of the tissue before and after processing confirms removal of over 95% of double stranded DNA and alpha galactosidase staining suggests the majority of cellular debris is removed. After decellularization, the tissue is lyophilized and then cryomilled into a powder. The matrix powder is digested for 48 hr in an acidic pepsin digestion solution and then neutralized to form the pregel solution. Gelation of the pregel solution can be induced by incubation at 37 &#176;C and can be used immediately following neutralization or stored at 4 &#176;C for up to two weeks. Coatings can be formed using the pregel solution on a non-treated plate for cell attachment. Cells can be suspended in the pregel prior to self-assembly to achieve a 3D culture, plated on the surface of a formed gel from which the cells can migrate through the scaffold, or plated on the coatings. Alterations to the strategy presented can impact gelation temperature, strength, or protein fragment sizes. Beyond hydrogel formation, the hydrogel stiffness may be increased using genipin.

This is a method to create a 3-dimensional cell culture scaffold from pulmonary extracellular matrix. Intact lung is processed into hydrogels that can support the growth of cells in three-dimensions.

Accordable hydrogels de matrice extracellulaire pulmonaire pour la 3D Culture Cellulaire

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung ...

Fabrication of Extracellular Matrix-derived Foams and Microcarriers as Tissue-specific Cell Culture and Delivery Platforms

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus of this article is on the methods for fabricating ECM-derived porous foams and microcarriers for use as biologically-relevant substrates in advanced 3D in vitro cell culture models or as pro-regenerative scaffolds and cell delivery systems for tissue engineering and regenerative medicine. Using decellularized tissues or purified insoluble collagen as a starting material, the techniques can be applied to synthesize a broad array of tissue-specific bioscaffolds with customizable geometries. The approach involves mechanical processing and mild enzymatic digestion to yield an ECM suspension that is used to fabricate the three-dimensional foams or microcarriers through controlled freezing and lyophilization procedures. These pure ECM-derived scaffolds are highly porous, yet stable without the need for chemical crosslinking agents or other additives that may negatively impact cell function. The scaffold properties can be tuned to some extent by varying factors such as the ECM suspension concentration, mechanical processing methods, or synthesis conditions. In general, the scaffolds are robust and easy to handle, and can be processed as tissues for most standard biological assays, providing a versatile and user-friendly 3D cell culture platform that mimics the native ECM composition. Overall, these straightforward methods for fabricating customized ECM-derived foams and microcarriers may be of interest to both biologists and biomedical engineers as tissue-specific cell-instructive platforms for in vitro and in vivo applications.

The tissue-specific extracellular matrix (ECM) is a key mediator of cell function. This article describes methods for synthesizing pure ECM-derived foams and microcarriers that are stable in culture without the need for chemical crosslinking for applications in advanced 3D in vitro cell culture models or as pro-regenerative bioscaffolds.

Fabrication de Mousses et microsupports dérivés Matrice extracellulaires culture cellulaire spécifique de tissu et plates-formes de livraison

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus ...

Fabricating a Kidney Cortex Extracellular Matrix-Derived Hydrogel

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

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

Fabrication d’un Hydrogel de dérivés de matrice extracellulaire rein Cortex

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